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| J Biol Chem, 1992 Aug 25, 267(24), 16806 - 11 Selective immunoneutralization of the multiple activities of Escherichia coli DNA polymerase I supports the model for separate active sites and indicates a complex 5' to 3' exonuclease; Ruscitti T et al.; DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease . We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I . Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites . In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease . One of them, for example, inhibited the RNase H activity but not the DNase activity . Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength. Nucleic Acids Res, 1992 Aug 25, 20(16), 4319 - 23 Singlet oxygen induced mutation spectrum in mammalian cells; de Oliveira RC et al.; In order to characterize the molecular nature of singlet oxygen (1O2) induced mutations in mammalian cells, a SV40-based shuttle vector (pi SVPC13) was treated with singlet oxygen arising from the thermal decomposition of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) . After the passage of damaged plasmid through monkey COS7 cells, the vector was shuffled into E . coli cells, allowing the screening of supF mutants . The mutation spectrum analysis shows that single and multiple base substitutions arose in 82.5% of the mutants, the others being rearrangements . The distribution of mutations within the supF gene is not random and some hotspots are evident . Most of the point mutations (98.4%) involve G:C base pairs and G:C to T:A transversion was the most frequent mutation (50.8%), followed by G:C to C:G transversion (32.8%) . These results indicate that mutagenesis in mammalian cells, mediated by 1O2-induced DNA damage, is targeted selectively at guanine residues. FEBS Lett, 1992 Aug 24, 308(3), 261 - 3 Synthesis of the proline analogue {2,3-3H}azetidine-2-carboxylic acid . Uptake and incorporation in Arabidopsis thaliana and Escherichia coli; Verbruggen N et al.; Azetidine-2-carboxylic acid, the 4-membered ring noranalogue of proline, is regularly used in the study of proline metabolism as well as the study of protein conformation . We prepared D,L-{2,3-3H}azetidine-2-carboxylic acid with an optimized 10% yield from commercially available 4-amino-{2,3-3H}butyric acid . Purification was performed by fast-protein liquid chromatography . The biological activity was checked in both Arabidopsis thaliana and Escherichia coli . The obtained specific activity of 10 mCi/mmol was sufficient for most uptake and incorporation studies. FEBS Lett, 1992 Aug 24, 308(3), 235 - 9 Protein engineering of Drosophila alcohol dehydrogenase . The hydroxyl group of Tyr152 is involved in the active site of the enzyme; Albalat R et al.; Drosophila alcohol dehydrogenase is the most studied member of the family of short-chain alcohol dehydrogenases, although its tridimensional structure still remains unknown . We have engineered a Drosophila alcohol dehydrogenase in which tyrosine-152, an invariant residue in all members of the family, has been substituted by phenylalanine . The mutated gene has been expressed in yeast and pure mutant enzyme has been prepared by a one-step FPLC chromatographic procedure . Drosophila alcohol dehydrogenase-phenylalanine-152 shows no enzymatic activity . This result suggests not only that tyrosine-152 could constitute an essential building block of the active site but also that its hydroxyl group is directly involved in the redox reaction catalyzed by the enzyme. FEBS Lett, 1992 Aug 24, 308(3), 231 - 4 Binding of RNA by the alfalfa mosaic virus movement protein is biphasic; Schoumacher F et al.; The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography . The purified protein bound single-stranded RNA cooperatively in a biphasic manner . At protein saturation, RNA/protein complexes (designated 'primary complexes') were detected by a nitrocellulose-retention assay within 1 min of mixing, both at 4 and 22 degrees C . In contrast, an incubation of 30 min at 22 degrees C was necessary to obtain electrophoretically retarded complexes ('stabilized complexes'), containing a large number of protein molecules bound stably to each molecule of RNA . Stabilization did not take place at 4 degrees C . The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a bimolecular interaction . In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules . In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation . The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed. FEBS Lett, 1992 Aug 24, 308(3), 240 - 4 DNA unwinding activity of replication protein A; Georgaki A et al.; Replication protein A (RP-A) is a heterotrimeric complex conserved in eukaryotic cells . It binds to single-stranded DNA and is essential for initiation and elongation of DNA replication . In this communication we give evidence that this protein can unwind DNA independent of magnesium and ATP, two essential cofactors for bona fide DNA helicase activity . RP-A can unwind up to at least 350 basepairs and appears to be required in stoichiometric amounts . The reaction is extremely sensitive to NaCl and MgCl2 . This activity of RF-A is suggestive for a possible unwinding function in initiation of DNA replication in eukaryotes. J Med Chem, 1992 Aug 21, 35(17), 3196 - 201 S-(5'-deoxy-5'-adenosyl)-1-ammonio-4-(methylsulfonio)-2-cyclopentene: A potent, enzyme-activated irreversible inhibitor of S-adenosylmethionine decarboxylase; Wu Y et al.; The compound S-(5'-deoxy-5'-adenosyl)-1-ammonio-4-(methylsulfonio)-2- cyclopentene (AdoMac) was prepared and evaluated as an irreversible inhibitor of S-adenosylmethionine decarboxylase (AdoMet-DC) . AdoMac was shown to inhibit AdoMet-DC in a time-dependent manner with a KI of 18.3 microM and a kinact of 0.133 min-1 . In addition, AdoMet-DC activity could not be restored following extensive dialysis of the enzyme-inhibitor complex, and the enzyme was protected from irreversible inactivation by the known competitive inhibitor methylglyoxal bis(guanylhydrazone) . HPLC analysis of the enzymatic reaction products revealed a time-dependent decrease in the peak coeluting with AdoMac, and a corresponding increase in the peak coeluting with (methylthio)adenosine (MTA), a byproduct of the irreversible binding of AdoMac to the enzyme . Thus, AdoMac appears to function as an enzyme-activated, irreversible inhibitor of AdoMet-DC. Biochim Biophys Acta, 1992 Aug 21, 1122(3), 321 - 6 Proton transfer roles of lysine 64 and glutamic acid 64 replacing histidine 64 in the active site of human carbonic anhydrase II; Engstrand C et al.; The CO2 hydration activities of cloned human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) and variants with Lys, Glu, Gln or Ala replacing His at sequence position 64 have been measured in a variety of different buffers in the pH range 6-9 . The variants with Lys-64, Gln-64 and Ala-64 showed non-Michaelis-Menten behavior under some conditions, apparent substrate inhibition being prominent near pH 9 . However, asymptotic Michaelis-Menten parameters could be estimated for the limit of low substrate concentrations . All variants show distinct buffer specificities, and imidazole derivatives, Ches and phosphate buffers yield higher kcat values that Bicine, Taps and Mops buffers under otherwise similar conditions . These results are interpreted in terms of different pathways for a rate-limiting proton transfer . In unmodified enzyme, the very high catalytic activity depends on His-64 functioning as an efficient proton transfer group, but this pathway is not available in the variants with Gln-64 and Ala-64 . Imidazoles, Ches and phosphate are thought to participate in a metal center-to-buffer proton transfer pathway, whereas Bicine, Taps, Mops and Mes appear to lack this capacity, so that the rate-limiting proton transfer occurs in a metal center-to-bulk water pathway for these variants . The Lys-64 and Glu-64 variants give significantly higher kcat values in Taps, Mops and Mes buffers than the Ala-64 and Gln-64 variants . The pH dependencies of these kcat values are compatible with the hypothesis that Lys-64 and Glu-64 can function as proton transfer groups . Thus, at pH near 9, Lys-64 appears to be only 5-times less efficient than His-64, while Glu-64 is inefficient . At pH 6, Lys-64 is an inefficient proton transfer group, but Glu-64 is only 2-3-times less efficient than His-64 . The data indicate that Lys-64 and Glu-64 have pKa values near 8 and below 6, respectively. J Mol Biol, 1992 Aug 20, 226(4), 979 - 87 Novel Escherichia coli mutant, dnaR, thermosensitive in initiation of chromosome replication; Sakakibara Y; A newly isolated Escherichia coli mutant thermosensitive in DNA synthesis had an allele named dnaR130, which was located at 26.3 minutes on the genetic map . The mutant was defective in initiation of chromosome replication but not in propagation at a high temperature . This mutant was capable of growing in the absence of the rnh function at the high temperature by means of a dnaA-independent replication mechanism . In the mutant exposed to the high temperature, an oriC plasmid was able to replicate, although at a lower rate than at the low temperature . The plasmid replication at the high temperature depended on the dnaA function essential for the initiation of replication from oriC . The mutant lacking the rnh function persistently maintained the oriC plasmid at the high temperature in a dnaA-dependent manner . Thus, the dnaR function was required for initiation of replication of the bacterial chromosome from oriC but not the oriC plasmid . This result reveals that a dnaR-dependent initiation mechanism that is dispensable for oriC plasmid replication operates in the bacterial chromosome replication. J Mol Biol, 1992 Aug 20, 226(4), 959 - 77 Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus; Marczynski GT et al.; Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny . Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur . In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication . The C . crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J . Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs . A plasmid, whose replication relies only on DNA from the C . crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C . crescentus chromosome . This implies that cis-acting replication control elements are closely linked to this origin of replication . This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E . coli-like 13-mer, and an exceptional A + T-rich region . Point mutations in one of the DnaA boxes abolish replication in C . crescentus . This origin also possesses three additional motifs that are unique to the C . crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present . The latter two motifs are implicated in essential C . crescentus replication functions, because they are contained within specific deletions that abolish replication. J Mol Biol, 1992 Aug 20, 226(4), 923 - 9 Protein-ligand energetics assessed using deoxy and fluorodeoxy sugars in equilibrium binding and high resolution crystallographic studies; Vermersch PS et al.; Hydrogen bonding interactions are one of the most important single factors in protein-ligand interactions and molecular recognition . To probe the energetics of the interactions, we have analyzed the binding of 1-deoxy-, 2-deoxy- and 6-fluoro-6-deoxy- analogues of D-galactose (Gal) to a primary high-affinity periplasmic receptor for monosaccharide active transport . Kd values and atomic structures refined at 1.81 to 1.45 A resolution of the complexes have been determined and compared with those of Gal binding . With binding site residues and the bound modified sugars in nearly identical positions as found in the complex with Gal, the binding of 1-deoxy-Gal or 2-deoxy-Gal reflects the overall contribution of 1.8 kcal mol-1 per hydrogen bond (neutral-charge type) to the affinity of Gal . Neglected in these estimates is the contribution of van der Waals' forces that accompany the formation of hydrogen bonds with each sugar hydroxyl . Contrary to expectations, the 6-fluoro-6-deoxy analogue proved to be an inadequate probe of Gal OH6 as a hydrogen bond donor due to the binding of a new water molecule and structural changes arising from the electronegative fluoro group . This study sheds new light on the energetics of protein-ligand interactions and the use of engineered ligands in assessing these interactions. J Mol Biol, 1992 Aug 20, 226(4), 1295 - 9 Primary structure of the second largest subunit of human RNA polymerase II (or B); Acker J et al.; The cDNA of the second largest subunit of RNA polymerase II (or B) from HeLa cells has been cloned and sequenced . A predicted amino acid sequence of 1174 residues (calculated molecular mass of 133,896 Da) was derived from the longest open reading frame and compared to the sequences of homologous subunits of polymerases of eukaryotic, archaeal and bacterial origin . After optimal alignment, about 16% of the residues were found to be conserved throughout evolution, from human to Escherichia coli . About 2/3 of the overall length of the conserved domains delineated by these residues are clustered within the C-terminal half of the human polypeptide, whereas the remaining is spread over its N-terminal half . The putative functional significance of these conserved domains is discussed. J Mol Biol, 1992 Aug 20, 226(4), 1283 - 6 Crystallization and preliminary crystallographic studies of glucosamine-6-phosphate deaminase from Escherichia coli K12; Horjales E et al.; Hexameric glucosamine-6-phosphate deaminase from Escherichia coli has been crystallized isomorphously with both phosphate and ammonium sulphate as precipitants, over a wide pH range (6.0 to 9.0) . The crystals belong to space group R32 and the cell parameters in the hexagonal setting are a = b = 125.9 A and c = 223.2 A . A complete native data set was collected to 2.1 A resolution . Self-rotation function studies suggest that the hexamers sit on the 3-fold axis and have point group symmetry 32, with a non-crystallographic dyad relating two monomers linked by an interchain disulfide bridge . A possible packing for the unit cell is proposed. J Mol Biol, 1992 Aug 20, 226(4), 1279 - 81 Crystallization and preliminary crystallographic characterization of GTP cyclohydrolase I from Escherichia coli; Schmid C et al.; GTP cyclohydrolase I of Escherichia coli has been purified from a recombinant bacterial strain . The enzyme was crystallized from 0.6 M-sodium citrate and from 0.8 M-sodium/potassium phosphate, respectively . Crystals grown in citrate showed X-ray diffraction extending to a resolution better than 3 A . The space group was P2(1) with cell dimensions a = 204.8 A, b = 210.1 A, c = 72.2 A, alpha = gamma = 90 degrees and beta = 95.8 degrees. J Mol Biol, 1992 Aug 20, 226(4), 1257 - 70 Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants; Baumeister R et al.; We have analyzed the DNA binding properties of Tet-repressor mutants with single amino acid residue replacements at eight positions within the alpha-helix-turn-alpha-helix DNA-binding motif . A saturation mutagenesis of Gln38, Pro39, Thr40, Tyr42, Trp43 and His44 in the second alpha-helix was performed; in addition, several substitutions of Thr27 and Arg28 in the first alpha-helix were constructed . The abilities of these mutant repressors to bind a set of 16 operator variants were determined and revealed 23 new binding specificities . All repressor mutants with DNA-binding activity were inducible by tetracycline, while mutants lacking binding activity were trans-dominant over the wild-type . All mutant proteins were present at the same intracellular steady-state concentrations as the wild-type . These results suggest the structural integrity of the mutant repressors . On the basis of the new recognition specificities, five contacts between a repressor monomer and each operator half-site and the chemical nature of these repressor-operator interactions are proposed . We suggest that Arg28 contacts guanine of the G.C base-pair at operator position 2 with two H-bonds, Gln38 binds adenine of the A.T base-pair at position 3 with two H-bonds, and the methyl group of Thr40 participates in a van der Waals' contact with cytosine of the G.C base-pair at position 6 of tet operator . A previously unrecognized type of interaction is proposed for Pro39, which inserts its side-chain between the methyl groups of the thymines of T.A and A.T base-pairs at positions 4 and 5 . Computer modeling of these proposed contacts reveals that they are possible using the canonical structures of the helix-turn-helix motif and B-DNA . These contacts suggest an inverse orientation of the Tet repressor helix-turn-helix with respect to the operator center as compared with non-inducible repressor-operator complexes, and are supported by similar contacts of other repressor-operator complexes. J Mol Biol, 1992 Aug 20, 226(4), 1219 - 35 Thermal motions of surface alpha-helices in the D-galactose chemosensory receptor . Detection by disulfide trapping; Careaga CL et al.; The D-galactose chemosensory receptor of Escherichia coli is a .32 kDa globular protein possessing two distinct structural domains, each organized in an alpha/beta folding motif . Helices I and X lie at adjacent approximately parallel positions on the surface of the N-terminal domain, near the hinge region . In order to analyze the relative thermal motions of these two helices, the present study utilizes a generalizable disulfide trapping approach: first, site-directed mutagenesis is used to place a pair of cysteine residues at locations of interest on the protein surface, then disulfide bond formation is used to trap intramolecular cysteine-cysteine collisions resulting from thermal motions . Specifically, four engineered di-cysteine receptors have been constructed, each possessing one cysteine at position 26 on helix I, and a second cysteine at varying positions on helix X . A fifth control receptor possesses one cysteine at position 26, and a second on the opposite surface of the molecule . These surface cysteine substitutions have little or no effect on the measurable receptor parameters as judged by ligand binding equilibria and kinetics, protein stability, and 19F nuclear magnetic resonance, indicating that the engineered receptors are useful probes of native backbone dynamics . Spatial and kinetic features of backbone motions have been investigated by measuring intramolecular disulfide formation rates for cysteine pairs in the fully liganded receptor . The resulting rates decrease monotonically with increasing distance between cysteines in the crystal structure, while no disulfide formation is observed for the control pair unless the molecule is unfolded . The minimum translational amplitudes of the observed backbone motions range from 4.5 to 15.2 A, and the minimum rotational amplitudes are as large as 35 degrees . For each motion the rate of intramolecular sulfhydryl-sulfhydryl collision has been estimated from the measured rate of disulfide formation: the 4.5 and 15.2 A translations yield approximately 10(4) and approximately 10 collisions s-1 molecule-1, respectively . These collision rates, which are faster than ligand dissociation, likely underestimate the actual motional frequencies since only an undetermined fraction of the total motions yield collisions . The simplest plausible trajectory capable of producing such collisions is a rate-limiting translation of one or both helices along their long axes, coupled with minor helix rotations . When sugar is removed from the receptor, a substantial increase in backbone dynamics is observed, indicating the presence of new long-range backbone trajectories . Overall, the results suggest that internal motions in proteins may have larger amplitudes than previously observed. J Mol Biol, 1992 Aug 20, 226(4), 1131 - 41 Three-dimensional structure in solution of acyl-coenzyme A binding protein from bovine liver; Andersen KV et al.; The three-dimensional structure of acyl-coenzyme A binding protein as encoded by the recombinant gene in Escherichia coli has been determined using nuclear magnetic resonance (n.m.r.) spectroscopy . The structure consists of four alpha-helices A1 (residues 3 to 15), A2 (residues 20 to 36), A3 (residues 51 to 60), and A4 (residues 65 to 85) . A1 and A4, and A2 and A3, run in parallel pairs . A2 runs anti-parallel to A1 and A4 . The three-dimensional structure of the protein is reminiscent of a shallow bowl with a rim . The "rim" is characterized by many polar and charged groups, whereas the inside and outside surface is predominantly hydrophobic with patches of uncharged polar hydroxyl groups of threonyl, serinyl and tyrosyl residues . The inside bottom contains through two epsilon-amino groups of lysine residues (Lys13 and Lys32) suggesting that the binding site for the nucleotide part of the acyl-coenzyme A part of the ligand molecule is at the inside surface of the bowl . The structure determination was done on the basis of measurements of the intensities of nuclear Overhauser effects (NOEs) and coupling constants that were translated into interatom distance restraints for 833 atom pairs, and 87 dihedral angle restraints, of which 23 were in chiral centers . In all, 42 hydrogen bonds were identified by n.m.r . and provided an additional 84 distance restraints . A total of 20 structures were calculated and the structures can be aligned to a root-mean-square deviation of 0.5 A for the backbone atoms of the residues in the four helices . A region of six residues could not be defined by the restraints obtained by n.m.r . The program Pronto was used for the spectrum analysis in general, and especially for the assignment of the individual NOEs, the integration of the cross peaks, and the measurements of the coupling constants . The programs DIANA and X-PLOR have been used in the structure calculations and evaluations. J Mol Biol, 1992 Aug 20, 226(4), 997 - 1008 Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA . Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations; Fitzwater T et al.; Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I . We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro . We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo . Each of these mutations alters a second base in primer RNA near the original mutation . We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro . Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer . Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid . The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation . An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation . These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I. J Mol Biol, 1992 Aug 20, 226(4), 989 - 96 dnaR function of the prs gene of Escherichia coli in initiation of chromosome replication; Sakakibara Y; A new Escherichia coli mutant named dnaR, which was temperature sensitive in initiation of DNA replication, has been characterized through identification of the mutant gene . A 1.65 x 10(3) base-pair chromosomal DNA fragment isolated from wild-type cells, but not the corresponding fragment from the dnaR mutant, exhibited an activity that reversed temperature-sensitive growth of the mutant . The DNA fragment was found to include the entire prs-coding sequence and specify a 34,000 M(r) protein with phosphoribosylpyrophosphate synthetase activity . The dnaR mutation resided within the prs-coding segment and made the synthetase thermolabile . The coding segment for the dnaR product was determined, by introduction of various mutations into the cloned fragment, to be the same as that for the synthetase . The dnaR function of the prs gene product in DNA replication is discussed on the basis of an observation that thermal treatment of the dnaR mutant caused a delay in initiation of chromosome replication after the downshift, despite the presence of the synthetase activity at the preheat level. Biochemistry, 1992 Aug 18, 31(32), 7264 - 73 Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin; McTigue MA et al.; The 2.2-A crystal structure of chicken liver dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a ternary complex with NADP+ and biopterin (a poor substrate) . The space group and unit cell are isomorphous with the previously reported structure of chicken liver DHFR complexed with NADPH and phenyltriazine {Volz, K . W., Matthews, D . A., Alden, R . A., Freer, S . T., Hansch, C., Kaufman, B . T., & Kraut, J . (1982) J . Biol . Chem . 257, 2528-2536} . The structure contains an ordered water molecule hydrogen-bonded to both hydroxyls of the biopterin dihydroxypropyl group as well as to O4 and N5 of the biopterin pteridine ring . This water molecule, not observed in previously determined DHFR structures, is positioned to complete a proposed route for proton transfer from the side-chain carboxylate of E30 to N5 of the pteridine ring . Protonation of N5 is believed to occur during the reduction of dihydropteridine substrates . The positions of the NADP+ nicotinamide and biopterin pteridine rings are quite similar to the nicotinamide and pteridine ring positions in the Escherichia coli DHFR.NADP+.folate complex {Bystroff, C., Oatley, S . J., & Kraut, J . (1990) Biochemistry 29, 3263-3277}, suggesting that the reduction of biopterin and the reduction of folate occur via similar mechanisms, that the binding geometry of the nicotinamide and pteridine rings is conserved between DHFR species, and that the p-aminobenzoylglutamate moiety of folate is not required for correct positioning of the pteridine ring in ground-state ternary complexes . Instead, binding of the p-aminobenzoylglutamate moiety of folate may induce the side chain of residue 31 (tyrosine or phenylalanine) in vertebrate DHFRs to adopt a conformation in which the opening to the pteridine binding site is too narrow to allow the substrate to diffuse away rapidly . A reverse conformational change of residue 31 is proposed to be required for tetrahydrofolate release. Biochemistry, 1992 Aug 18, 31(32), 7211 - 8 Prediction of structural and functional features of protein and nucleic acid sequences by artificial neural networks; Hirst JD et al.; The applications of artificial neural networks to the prediction of structural and functional features of protein and nucleic acid sequences are reviewed . A brief introduction to neural networks is given, including a discussion of learning algorithms and sequence encoding . The protein applications mostly involve the prediction of secondary and tertiary structure from sequence . The problems in nucleic acid analysis tackled by neural networks are the prediction of translation initiation sites in Escherichia coli, the recognition of splice junctions in human mRNA, and the prediction of promoter sites in E . coli . The performance of the approach is compared with other current statistical methods. J Biol Chem, 1992 Aug 15, 267(23), 16712 - 8 Activity of recombinant mitogillin and mitogillin immunoconjugates; Better M et al.; A synthetic gene for the Aspergillus protein toxin mitogillin has been synthesized and expressed in Escherichia coli . The recombinant mitogillin is a potent inhibitor of protein synthesis in vitro with an IC50 of 9.7 pM . Immunoconjugates of recombinant mitogillin derivatized with S-acetylmercaptosuccinic anhydride and 5-methyl-2-iminothiolane modified H65 antibody kill T cell lines and peripheral blood mononuclear cells expressing the human CD5 surface antigen . Native mitogillin contains 4 cysteine residues which form two disulfide pairs (Fernandez-Luna, J . L., Lopez-Otin, C., Soriano, F., and Mendez, E . (1985) Biochemistry 24, 861-867) . Three derivatives of mitogillin have been assembled which substitute alanine residues for cysteine residues 5, 147, or 5 and 147 . Each of these molecules retains the ability to inhibit protein synthesis in vitro with at most a 2-fold reduction in activity . The derivative mitogillinC147A can be conjugated to 5-methyl-2-iminothiolane- modified H65 antibody directly without pretreatment with S-acetylmercaptosuccinic anhydride, and the immunoconjugate is active against HSB2 cells . Genetic manipulation of toxin genes to expose an accessible cysteine residue into a recombinant product can thus be used to generate immunotoxins without initial derivatization by nonspecific cross-linking reagents. J Biol Chem, 1992 Aug 15, 267(23), 16595 - 600 Production and functional analysis of normal and variant recombinant human transthyretin proteins; Murrell JR et al.; The most common form of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy associated with single amino acid changes in the plasma protein, transthyretin . In addition, there are two variants of transthyretin (Ser6 and Thr109) not associated with familial amyloidotic polyneuropathy but with familial euthyroid hyperthyroxinemia, also an autosomal dominant disorder . In these autosomal dominant diseases, most affected individuals are heterozygous and therefore have hybrid forms of the tetrameric plasma transthyretin . In order to study the structure/function relationships of homozygous variant transthyretins, normal human transthyretin and five variant transthyretins (Gly6----Ser, Leu58----His, Thr60----Ala, Ile84----Ser, and Ala109----Thr) were produced in Escherichia coli using the expression vector, pCZ11, and site-directed mutagenesis . These recombinant transthyretin (r-TTR) proteins showed the correct size (14 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis and self-associated into tetramers as determined by size exclusion chromatography . Recombinant normal, Ser6, and Ala60 r-TTRs had an affinity for thyroxine indistinguishable from normal human TTR purified from plasma, whereas His58 and Ser84 r-TTRs had significantly reduced affinity . On the other hand, Thr109 r-TTR had a much higher affinity, probably due to its position within the thyroxine-binding pocket . Expression of mutant transthyretins in E . coli provides the opportunity to study structure/function relationships and amyloid-forming capabilities induced by single amino acid substitutions in the transthyretin molecule. J Biol Chem, 1992 Aug 15, 267(23), 16444 - 9 On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange . II . Four-strand exchanges; Kim JI et al.; RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski et al., 1990), calling into question the role of ATP hydrolysis in this reaction . We demonstrate here that the ATP gamma S-mediated process is restricted to homologous strand exchange reactions involving three strands . In four-strand exchanges between a gapped duplex circle and a second linear duplex, joint molecules are formed in the gap but are not extended into the four-strand region when ATP gamma S is present . This result provides evidence that one function of ATP hydrolysis in the recA system is to facilitate reciprocal DNA strand exchange involving four strands . Implications with respect to the role of four-stranded pairing intermediates and the mechanistic relationship between three- and four-strand exchange reactions are discussed. J Biol Chem, 1992 Aug 15, 267(23), 16438 - 43 On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange . I . Bypassing a short heterologous insert in one DNA substrate; Kim JI et al.; RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski, J.P., Bear, D.G., and Kowalczykowski, S.C . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 21-25), calling into question the role of ATP hydrolysis in the strand exchange reaction . Here, we demonstrate that the ATP gamma S-mediated reaction can go to completion when the duplex DNA substrate is only 1.3 kilobase pairs in length . The ATP gamma S-mediated reaction, however, is completely blocked by a 52-base pair heterologous insertion in either DNA substrate . This same barrier is readily bypassed when ATP replaces ATP gamma S . This indicates that at least one function of recA-mediated ATP hydrolysis is to bypass structural barriers in one or both DNA substrates during strand exchange . This suggests that ATP hydrolysis is directly coupled to the branch migration phase of strand exchange, not to promote strand exchange between homologous DNA substrates during recombination, but instead to facilitate the bypass of structural barriers likely to be encountered during recombinational DNA repair. J Biol Chem, 1992 Aug 15, 267(23), 16244 - 51 The importance of aspartate 327 for catalysis and zinc binding in Escherichia coli alkaline phosphatase; Xu X et al.; In order to investigate the function of Asp-327, a bidentate ligand of one of the zinc atoms in Escherichia coli alkaline phosphatase, and the importance of this zinc atom in catalysis, site-specific mutagenesis was used to convert Asp-327 to either asparagine or alanine . The 10(7)-fold decrease in the kcat/Km ratio observed for the Asp-327----Ala enzyme compared to the wild-type enzyme indicates that the side chain of Asp-327 is important for zinc binding at the M1 site . However, only one of the two carboxyl oxygens of Asp-327 is essential for zinc binding, since the Asp-327----Asn enzyme shows approximately the same hydrolysis activity as the wild-type enzyme . The fact that the enzymatic activity of this mutant enzyme shows a dependence on zinc concentration suggests that the other carboxyl oxygen or the negative charge on the side chain of Asp-327 is important in binding of the zinc at the M1 site . However, the zinc hydroxyl must still be appropriately positioned to attack the phosphoserine in the Asp-327----Asn enzyme; therefore, the negative charge and at least one carboxyl oxygen of the side chain are not directly involved in positioning or deprotonating the zinc hydroxyl . 31P NMR studies indicate that the Asp-327----Asn enzyme exhibits transphosphorylation activity at both pH 8.0 and pH 10.0, but at a reduced level compared to the wild-type enzyme . The biphasic production of 2,4-dinitrophenylate in the pre-steady-state kinetics of the mutant enzymes at pH 5.5 suggests that the breaking of the phosphoenzyme covalent complex is rate-limiting for both mutant enzymes . These results suggest that the main function of the zinc atom at the M1 site in catalysis involves decomposition of the phosphoenzyme covalent complex and that it may be important in helping to stabilize the alcohol leaving group. J Biol Chem, 1992 Aug 15, 267(23), 16135 - 7 The role of the iron-sulfur cluster in Escherichia coli endonuclease III . A resonance Raman study; Fu W et al.; Resonance Raman spectroscopy has been used to investigate the function and properties of the iron-sulfur cluster in Escherichia coli endonuclease III . Resonance Raman spectra in the Fe-S stretching region are indicative of a {4Fe-4S}2+ cluster with complete cysteinyl sulfur coordination, and vibrational assignments are made by analogy with bacterial ferredoxins . Minor changes in the vibrational frequencies of the modes primarily involving Fe-S(Cys) stretching accompany the binding of the inhibitor thymine glycol or an oligonucleotide containing a reduced apyrimidinic site . These changes are consistent with perturbation of the orientation of the ligating cysteinyl residues and rule out the possibility that the {4Fe-4S} cluster is directly involved with substrate or inhibitor binding . It is concluded that a structural role is most likely for the {4Fe-4S} cluster in endonuclease III. J Biol Chem, 1992 Aug 15, 267(23), 16015 - 8 RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells; Kelly KO et al.; RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro . To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains . Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase . Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability . The rph mutation also had dramatic effects on tRNA metabolism . Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases . Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence . These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors. Gene, 1992 Aug 15, 117(2), 265 - 9 Production of human serum transferrin in Escherichia coli; Ikeda RA et al.; Transferrin (Tf) crystals diffract to only medium resolution . The mediocre quality of the crystals may be due to two factors: (1) the genetic variations naturally present in the primary sequence of Tf, and (2) the glycosylation of the protein . To control genetic variations and glycosylation of samples of Tf, it would be desirable to express the Tf gene from a recombinant clone . Additionally, expression of Tf from a clone would allow for manipulation of the structure of Tf . The cDNA encoding Tf has been cloned into the pL-based expression vector, pRE1, and the T7-based expression vectors, pRSETA and pET11A . The Tf expression plasmids, pTF-SSn and pTF-ESn, based on the T7 expression vectors, efficiently produce a 76-kDa protein that is approximately the same size as deglycosylated Tf, cross reacts with anti-Tf antibodies, and matches the deduced N-terminal amino acid sequence . Expression of Tf in Escherichia coli will allow the production of genetically pure, unglycosylated protein. Gene, 1992 Aug 15, 117(2), 259 - 63 Synthesis of a gene encoding bovine substance P precursors and its expression in Escherichia coli; Hrabec E et al.; Using an efficient Escherichia coli expression system, we have been able to obtain the precursor of substance P, alpha-preprotachykinin (alpha PPT) . The alpha PPT protein is produced in E . coli as a fusion to beta-galactosidase, and accumulates in the cytoplasm as insoluble inclusion bodies . We also produced protachykinin (alpha PT), i.e., alpha PPT without a signal peptide . Further purification and characterization of the alpha PPT and alpha PT polypeptides strongly suggest that fully purified products can be obtained using our procedures. Arch Biochem Biophys, 1992 Aug 15, 297(1), 86 - 91 Site-directed mutagenesis of glutathione S-transferase YaYa: functional studies of histidine, cysteine, and tryptophan mutants; Wang RW et al.; The rat cytosolic glutathione S-transferase Ya subunit contains three histidine residues (at positions 8, 143, and 159), two cysteine residues (at positions 18 and 112), and a single tryptophan residue (at position 21) . Histidine, cysteine, and tryptophan have been proposed to be present either near or at the active site of other glutathione S-transferase subunits . The functional role of these amino acids at each of the positions was evaluated by site-directed mutagenesis in which valine or asparagine, alanine, and phenylalanine were substituted for histidine, cysteine, and tryptophan, respectively . Mutant enzymes H8V, H143V, H159N, C112A, and W21F retained either full or better catalytic efficiencies (k(cat)/Km) toward 1-chloro-2,4-dinitrobenzene and glutathione . Lower but significant k(cat)/Km values were observed for H159V and C18A toward 1-chloro-2,4-dinitrobenzene . Some mutants displayed different thermal stabilities and intrinsic fluorescence intensities, but all retained the ability to bind heme . These results indicate that histidine, cysteine, and tryptophan in the glutathione S-transferase Ya subunit are not essential for catalysis nor are they involved in the binding of heme to the YaYa homodimer. FEMS Microbiol Lett, 1992 Aug 15, 74(2-3), 231 - 4 8-Azido-ATP labelling of the FecE protein of the Escherichia coli iron citrate transport system; Schultz-Hauser G et al.; The iron(III) transport system via citrate displays the characteristics of a binding protein-dependent transport mechanism through the cytoplasmic membrane . One of the five transport proteins, FecE, contains the two motifs found in ATP-binding proteins . Cloned overexpressed FecE was photoaffinity-labelled with {32P}8-azido-ATP . Labelling was inhibited by 1 mM ATP, ADP, GTP and less by CTP, demonstrating the specificity of the reaction . It is suggested that ATP is the principal energy source for iron(III) citrate transport through the cytoplasmic membrane of Escherichia coli. FEMS Microbiol Lett, 1992 Aug 15, 74(2-3), 137 - 42 Cold-sensitive growth and decreased GTP-hydrolytic activity from substitution of Pro17 for Val in Era, an essential Escherichia coli GTPase; Lerner CG et al.; A substitution mutation of Pro17 by Val (P17V) was constructed in the guanine nucleotide binding domain of Era, an essential protein in Escherichia coli . The mutation is analogous to the oncogenic activating allele at position 12 in the GTP-binding domain of p21ras . The phenotype of this mutant was analysed in a strain which exclusively expressed the mutant protein (Era-V17) in null allele chromosomal background (era1: :kan) . The strain was found to be cold-sensitive for growth . Mutant Era-V17 purified from the strain was cold-sensitive for GTP-hydrolytic activity, suggesting that the GTPase activity of Era is required for cell growth since the P17V mutation resulted in both cold-sensitive growth of cells and cold-labile GTPase activity of the purified protein. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7737 - 41 Phosphorylation stimulates the transcriptional activity of the human beta 1 thyroid hormone nuclear receptor; Lin KH et al.; The role of phosphorylation on the gene activation activity of the human beta 1 thyroid hormone nuclear receptor (h-TR beta 1) was examined . h-TR beta 1 was found to be a phosphoprotein when expressed in COS-1 cells, with serine, threonine, and tyrosine (85:10:5) as the phosphorylation sites . Okadaic acid (a potent inhibitor of phosphatases 1 and 2A) at 0.1, 0.25, and 0.5 microM increased the phosphorylation of h-TR beta 1 by 3-, 7-, and 11-fold, respectively . The increase in phosphorylation was accompanied by a concomitant increase in phosphorylation was accompanied by a concomitant increase in receptor-mediated transcription in transient transfection assays . h-TR beta 1 purified from Escherichia coli was phosphorylated in vitro by the endogenous kinase from cellular extracts . Serine, threonine, and tyrosine were phosphorylated in a similar ratio to that found in COS-1 cells . The in vitro phosphorylation was stimulated by okadaic acid . Phosphorylation did not affect the binding of h-TR beta 1 to 3,3',5-triiodo-L-thyronine . However, phosphorylation of h-TR beta 1 resulted in an increase of its binding to DNA and conferred on it the ability to bind to nuclear accessory proteins . The results indicate that phosphorylation plays an important role in the transcriptional activity of h-TR beta 1. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7732 - 6 Stable genetic transformation of a beneficial arthropod, Metaseiulus occidentalis (Acari: Phytoseiidae), by a microinjection technique; Presnail JK et al.; A microinjection technique has resulted in stable transformation of the western predatory mite Metaseiulus occidentalis . Early preblastoderm eggs within gravid females were microinjected . The needle was inserted through the cuticle of gravid females into the egg, or the tissue immediately surrounding the egg . This maternal injection method resulted in relatively high levels of survival and transformation . Transformation was achieved without the aid of any transposase-producing helper plasmid . The predatory mite was transformed with a plasmid containing the Escherichia coli beta-galactosidase gene (lacZ) regulated by the Drosophila hsp70 heat-shock promoter . Putatively transformed lines were isolated based on beta-galactosidase activity in first-generation larvae . Transformation was confirmed in the sixth generation by polymerase chain reaction amplification of a region spanning the Drosophila/E . coli sequences . Amplification of a nested region, also spanning the interspecific boundary, provided further evidence for stable transformation . Maternal microinjection may be adaptable to other beneficial arthropods, particularly other phytoseiid mites . Genetic transformation of M . occidentalis may improve its efficiency as a biological control agent as well as provide a method for investigating details of its physiology and ecology. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7576 - 80 Purification and characterization of the diphtheria toxin repressor; Schmitt MP et al.; The diphtheria toxin repressor gene (dtxR) encodes a protein (DtxR) that regulates transcription of the diphtheria toxin gene (tox) by an iron-dependent mechanism . Cloned dtxR was expressed in Escherichia coli from the phage T7 gene 10 promoter, and DtxR was purified . Specific binding of DtxR to the tox+ operator was dependent on reduction of DtxR and the presence of ferrous ions . DtxR protected a sequence of approximately 30 nucleotide pairs, partially overlapping the tox promoter and containing a region of dyad symmetry, from digestion by DNase I . DtxR exhibited very little binding to the mutant tox-201 operator region and failed to bind to the promoter/operator region of the ferric uptake regulation (fur) gene of E . coli. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7501 - 5 The evolutionary selection of DNA base pairs in gene-regulatory binding sites; Berg OG; The DNA base-pair sequences that serve as gene-regulatory sites have been selected during evolution to provide an appropriate functional binding for a particular protein . In most cases, the function depends on the binding probability, which can be influenced both by the binding strength and by the abundance of the protein in the cell . As a consequence, the same function can be achieved with strong binding sites and a small amount of protein as with weak binding sites and a large amount of protein . However, increasing the protein burden will decrease the growth rate of the cells, even when all functions remain the same . Thus, for maximal growth, the protein levels should be as low as possible and the binding correspondingly strong . On the other hand, sequences with a weaker binding can be formed in many more ways and are, therefore, more probable, and random mutations are more likely to produce them . Thus, the selection pressure against an increased protein burden can be balanced against the random mutational drift in the recognition sequences, thereby tying together the statistics of base-pair choice, the binding strength, and the protein burden . In terms of this model, the selection pressure can be estimated from the properties of a gene-regulatory protein and its recognition sites . A key feature is the mutational randomization pressure that appears as a fundamental force shaping the optimal solutions that provide maximal growth . The model is tested on a number of gene-regulatory systems in Escherichia coli . The same principles should hold for all proteins for which overall activity in the cell is proportional to abundance; then the selective pressure to increase the efficiency of an individual protein cannot be larger than the selective pressure to decrease the total protein burden. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7457 - 61 Refolding and oriented insertion of a membrane protein into a lipid bilayer; Surrey T et al.; We have studied the refolding and membrane insertion of the outer membrane protein OmpA of Escherichia coli . The protein was extracted from its native membrane by sonication in the presence of urea and dissolved in the urea/water mixture in unfolded form . In this form it was purified . Upon addition of preformed lipid vesicles, the protein spontaneously refolded and inserted into the vesicle membranes . The vesicles had to be small and the lipids had to be in the fluid state . The insertion occurred in an oriented manner. J Inorg Biochem, 1992 Aug 15-Sep, 47(3-4), 161 - 74 Structure, function, and evolution of ferritins; Andrews SC et al.; The ferritins of animals and plants and the bacterioferritins (BFRs) have a common iron-storage function in spite of differences in cytological location and biosynthetic regulation . The plant ferritins and BFRs are more similar to the H chains of mammals than to mammalian L chains, with respect to primary structure and conservation of ferroxidase center residues . Hence they probably arose from a common H-type ancestor . The recent discovery in E . coli of a second type of iron-storage protein (FTN) resembling ferritin H chains raises the question of what the relative roles of these two proteins are in this organism . Mammalian L ferritins lack ferroxidase centers and form a distinct group . Comparison of the three-dimensional structures of mammalian and invertebrate ferritins, as well as computer modeling of plant ferritins and of BFR, indicate a well conserved molecular framework . The characterisation of numerous ferritin homopolymer variants has allowed the identification of some of the residues involved in iron uptake and an investigation of some of the functional differences between mammalian H and L chains. Biochem J, 1992 Aug 15, 286 ( Pt 1), 187 - 91 A large decrease in heat-shock-induced proteolysis after tryptophan starvation leads to increased expression of phage lambda lysozyme cloned in Escherichia coli; Soumillion P et al.; The R gene coding for phage lambda lysozyme (lambda L), cloned under the control of the PL promoter on a multicopy vector, is expressed in an Escherichia coli strain auxotrophic for tryptophan . Induction by a thermal shift after tryptophan supplementation in a culture initially brought into stationary phase by tryptophan starvation leads to highly increased expression . A thermally unstable mutant protein, difficult to obtain under standard conditions, can be easily produced by post-stationary-phase expression . It is shown that this is due to a drastic decrease in the heat-shock-induced proteolysis normally observed on thermal induction . These data are discussed in relation to our present knowledge of stringent and heat-shock responses. Gene, 1992 Aug 15, 117(2), 169 - 78 A broad bean cDNA clone encoding a DNA-binding protein resembling mammalian CREB in its sequence specificity and DNA methylation sensitivity; Ehrlich KC et al.; A plant cDNA has been cloned that encodes a DNA-binding protein displaying a nucleotide (nt) sequence specificity similar to that of the mammalian cyclic AMP response element-binding protein/activating transcription factor (CREB/ATF) family of mammalian proteins . This cDNA was cloned in Escherichia coli from a broad bean (Vicia faba) cDNA expression library using a recognition site probe . The deduced amino acid (aa) sequence of the recombinant cDNA-encoded protein, called VBP1, has a basic region adjacent to a leucine zipper motif, of the type seen in the DNA-binding domains of many eukaryotic DNA-binding proteins, including mammalian CREB/ATF . Although this aa sequence has homology to regions of deduced aa sequences of other cloned plant cDNAs, it is distinct in both the derived primary structure and in its nt sequence specificity . VBP1, as well as proteins in nuclear extracts of V . faba with similar nt sequence specificity, have their binding to DNA suppressed more than tenfold by cytosine methylation at the CREB/ATF consensus sequence. Biochem J, 1992 Aug 15, 286 ( Pt 1), 281 - 8 Structure and expression of the Drosophila ubiquitin-52-amino-acid fusion-protein gene; Cabrera HL et al.; Ubiquitin belongs to a multigene family . In Drosophila two members of this family have been previously described . We report here the organization and expression of a third member, the DUb52 gene, isolated by screening a Drosophila melanogaster genomic library . This gene encodes an ubiquitin monomer fused to a 52-amino acid extension protein . There are no introns interrupting the coding sequence . Recently, it has been described that this extension encodes a ribosomal protein in Saccharomyces, Dictyostelium, and Arabidopsis . The present results show that the 5' regulatory region of DUb52 shares common features with the ribosomal protein genes of Drosophila, Xenopus and mouse, including GC- and pyrimidine-rich regions . Moreover, sequences similar to the consensus Ribo-box in Neurospora crassa have been identified . Furthermore, a sequence has been found that is similar to the binding site for the TFIIIA distal element factor from Xenopus laevis . The DUb52 gene is transcribed to a 0.9 kb mRNA that is expressed constitutively throughout development and is particularly abundant in ovaries . In addition, the DUb52 gene has been found to be preferentially transcribed in exponentially growing Drosophila cells. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7717 - 21 How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor; Barchan D et al.; The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193 . To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins . We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor . In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins . It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro . cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli . The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX . The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment . Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193 . These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His) . The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain . Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX . In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7546 - 50 Poly(A) RNA in Escherichia coli: nucleotide sequence at the junction of the lpp transcript and the polyadenylate moiety; Cao GJ et al.; Although it has been known for some time that bacterial mRNA molecules carry polyadenylate moieties at their 3' ends, nothing is known about the molecular structure of bacterial poly(A) RNA . To define the polyadenylylation site of a specific bacterial mRNA, we took advantage of the presence of elevated levels of poly(A) RNA in cells of Escherichia coli deficient in exoribonucleases and synthesized DNA complementary to polyadenylylated lipoprotein mRNA, encoded by the lpp gene, by using avian myeloblastosis virus reverse transcriptase and an oligo(dT)-containing primer . The 5'-terminal portion of the cDNA was amplified by the polymerase chain reaction and appropriate oligonucleotide primers, and the amplified DNA was cloned in pUC18 and subjected to nucleotide sequence analysis . Four clones were found to contain the entire 3'-terminal coding region of lpp mRNA, with poly(A) attached to either of two sites in the downstream untranslated region of the transcript . In one type of clone, the polyadenylate moiety was attached at the putative transcription termination site of lpp mRNA, whereas other clones lacked the stem-loop structure of the rho-independent transcription terminator and the polyadenylate moiety was attached to the residue just preceding the terminal stem-loop of the primary transcript . A model for the polyadenylylation of bacterial mRNA is proposed in which poly(A) polymerase and exonucleases compete for the 3' ends of mRNA molecules. J Immunol, 1992 Aug 15, 149(4), 1409 - 15 Antineutrophil cytoplasmic autoantibodies in Wegener's granulomatosis recognize conformational epitope(s) on proteinase 3; Bini P et al.; Although proteinase 3 (PR3) has been identified as a major autoantigen in Wegener's granulomatosis, the precise antibody specificity(ies) and requirements for epitope recognition have not been characterized . We analyzed 11 sera containing antineutrophil cytoplasmic antibodies (cANCA) for binding to azurophilic granule proteins extracted from neutrophils under various conditions and for binding to native or rPR3 . Ten of 11 (91%) of the cANCA sera bound to PR3 extracted by nonionic detergents when tested by immunoprecipitation or by IEF followed by capillary immunoblotting . Antibody binding to PR3 was retained when IEF was performed under dissociating conditions (8 M urea) indicating that PR3 is the major autoantigen in azurophilic granules and that association with other proteins is not required for antigenicity . In contrast, antigenicity was totally destroyed by exposure of PR3 to reducing agents or to low pH (less than 3.0) and was either lost or considerably diminished after boiling in SDS . cANCA sera also showed little or no binding to rPR3 expressed as a fusion protein in Escherichia coli or synthesized by wheat germ ribosomes in vitro . Inasmuch as PR3 enzymatic activity was partially retained after acid extraction, these findings indicate that cANCA bind to a limited number of conformational epitopes on PR3 . In addition, IEF followed by capillary immunoblotting appears to be a sensitive and specific method to detect anti-PR3 antibodies in Wegener's granulomatosis. J Biol Chem, 1992 Aug 15, 267(23), 16283 - 7 Characterization of two high affinity human interleukin-8 receptors; Lee J et al.; Interleukin 8 (IL-8) and melanocyte growth-stimulatory activity/gro (MGSA) are structurally related proinflammatory cytokines that are chemoattractants and activators of neutrophils . Recently, cDNA clones encoding a high affinity IL-8 receptor (IL-8R-A) and a "low affinity" IL-8 receptor (IL-8R-B) have been isolated from human cDNA libraries . These two receptors have 77% amino acid identity and are members of the G protein-coupled superfamily of receptors with seven transmembrane domains . We have expressed these two receptors in mammalian cells and find that in this system both receptors bind IL-8 with high affinity (Kd approximately 2 nM) . The receptor affinities differ for MGSA, however . IL-8R-A binds MGSA with low affinity (Kd approximately 450 nM); IL-8R-B binds MGSA with high affinity (Kd approximately 2 nM) . The transfected cells respond to ligand binding with a transient increase in the intracellular Ca2+ concentration . A Ca2+ response is found for IL-8R-A following the binding of IL-8; no response is found for MGSA . A Ca2+ response for IL-8R-B follows the binding of both ligands . Blot hybridization with oligonucleotide probes specific for the two receptors shows that mRNA for both receptors is present in human neutrophils . Analysis of IL-8 and MGSA binding data on neutrophils as well as Ca2+ response and desensitization data shows that the presence of these two IL-8 receptors on the cell surface can account for the profile of these two ligands on neutrophils. Anal Biochem, 1992 Aug 15, 205(1), 166 - 71 A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate; Slaughter TF et al.; Transglutaminases belong to an important family of enzymes involved in hemostasis, skin formation, and wound healing . We describe a technique for the measurement of transglutaminase activity using polystyrene microtiter plates coated with N,N'-dimethylcasein . The substrate 5-(biotinamido)pentylamine is covalently incorporated into N,N'-dimethylcasein by transglutaminase in a calcium-dependent reaction . The biotinylated product is detected by streptavidin-alkaline phosphatase and quantitated by measuring the absorbance at 405 nm following the addition of p-nitrophenyl phosphate . The assay is sensitive, specific, and linear at plasma factor XIIIa concentrations between 0.08 and 1.25 micrograms/ml and at purified guinea pig liver transglutaminase concentrations between 0.05 and 0.8 microgram/ml . The intra-assay coefficient of variation is less than 8% . The solid-phase assay was used to quantitate the transglutaminase activity in Escherichia coli extracts expressing recombinant factor XIII A-chains and to analyze factor XIIIa inhibitors . This method will facilitate the analysis of structure-function relationships of the transglutaminases using recombinant DNA methods . Furthermore, screening of natural and synthetic factor XIIIa inhibitors will be expedited by this solid-phase microtiter plate assay. FEMS Microbiol Lett, 1992 Aug 15, 74(2-3), 219 - 24 Biochemical and serological characterization of Escherichia coli fimbrial antigen F165(2); Dubreuil JD et al.; Mannose-resistant hemagglutinating fimbrial antigen F165 is produced by Escherichia coli strains associated with septicemia in piglets and calves . A fimbrial component with an M(r) of 17,200 as determined by SDS-PAGE was purified to homogeneity from F165-positive E . coli strain 4787 of serogroup O115 . This fimbrial component of F165 antigen was named F165(2) . Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation . Upon removal of ethanolamine, the fimbrial component reassociated into fimbriae . Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of which 37.3% were hydrophobic . The amino acid composition and the isoelectric point (9.5) were readily distinguishable from those of F1 fimbriae . The amino acid sequence was determined for approximately 40% of the molecule . For the first 33 residues, the F165(2) sequence was identical to that of F1B fimbriae and very similar to that of F1C . Fimbriae F165(2) could nevertheless be differentiated antigenically from F1C fimbriae as demonstrated by the immunodot technique using cross-absorbed antisera. Biochem J, 1992 Aug 15, 286 ( Pt 1), 269 - 73 Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus; Jouanneau Y et al.; The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter . The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E . coli cell-free extracts by anti-FdII serum . The purified recombinant ferredoxin was indistinguishable from R . capsulatus FdII on the basis of its molecular, redox and spectroscopic properties . These results indicate that the {3Fe-4S} and {4Fe-4S} clusters were correctly inserted into the recombinant ferredoxin. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7417 - 21 A human transmembrane protein-tyrosine-phosphatase, PTP zeta, is expressed in brain and has an N-terminal receptor domain homologous to carbonic anhydrases; Krueger NX et al.; Protein-tyrosine-phosphatases (PTPases, EC 3.1.3.48) play a crucial role in the regulation of protein tyrosine phosphorylation . Recently, it was found that the PTPase gene family exhibits a large variety of different functional domains associated with the PTPase catalytic domains . In this paper, we report the complete cDNA sequence of a human transmembrane PTPase, PTP zeta, isolated from fetal brain cDNA libraries . The deduced amino acid sequence of human PTP zeta is composed of a putative signal peptide of 19 amino acids, a very large extracellular domain of 1616 amino acids, a transmembrane peptide of 26 amino acids, and a cytoplasmic domain of 653 amino acids . The extracellular portion of human PTP zeta contains two striking structural features: the N-terminal 280-amino acid sequence that is homologous to carbonic anhydrases (carbonate hydro-lyase, EC 4.2.1.1), and a sequence of 1048 amino acids without a cysteine residue . While it is unlikely that the carbonic anhydrase-like domain of PTP zeta has any carbonic anhydrase activity, its three-dimensional structure may be quite similar to that of carbonic anhydrases, a structure that appears ideal for binding a small soluble ligand . The cytoplasmic portion of human PTP zeta contains two repeated PTPase-like domains, which, when expressed in Escherichia coli, had PTPase activity in vitro . Mutational analyses indicate that only the membrane-proximal PTPase domain is catalytically active . Reverse transcription-polymerase chain reaction analyses indicate that human PTP zeta is highly expressed in a glioblastoma cell line. Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7315 - 9 Role of loop-helix interactions in stabilizing four-helix bundle proteins; Chou KC et al.; One of the critical issues regarding proteins with a four-helix bundle motif is which interactions play the major role in stabilizing this type of folded structure: the interaction among the four alpha-helices or the interaction between the loop and helix segments . To answer this question, an energetic analysis has been carried out for three proteins with a four-helix bundle--namely, methemerythrin, cytochrome b-562, and cytochrome c' . The structures on which the analysis has been made were derived from their respective crystallographic coordinates . All three proteins have long helices (16-26 residues) and most of their loops are short (3-5 residues) . However, it was found in all three proteins that loop-helix interactions were stronger than helix-helix interactions . Moreover, not only the nonbonded component but also the electrostatic component of the interaction energy were dominated by loop-helix interactions rather than by interhelix interactions, although the latter involve favorable helix-dipole interactions due to the antiparallel arrangement of neighboring helices . The results of the energetic analysis indicate that the loop segments, whether they are in a theoretical model or in real proteins, play a significant role in stabilizing proteins with four-helix bundles. J Biol Chem, 1992 Aug 15, 267(23), 16669 - 75 Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Li L et al.; Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K . (1985) J . Biol . Chem . 260, 13995-14002) . However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S . J . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 9642-9646) . In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity . Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme . The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme . However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme . Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant . In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase . The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme . The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme . The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis. J Biol Chem, 1992 Aug 15, 267(23), 16545 - 52 Molecular cloning of human macrophage capping protein cDNA . A unique member of the gelsolin/villin family expressed primarily in macrophages; Dabiri GA et al.; Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments . The human cDNA for MCP has been cloned and sequenced . The derived amino acid sequence predicts a polypeptide of 38.4 kDa . Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments . Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins . Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein . Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively . Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1 . Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function. J Biol Chem, 1992 Aug 15, 267(23), 16497 - 502 Organella-targeted expression of rat liver cytochrome P450c27 in yeast . Genetically engineered alteration of mitochondrial P450 into a microsomal form creates a novel functional electron transport chain; Sakaki T et al.; A modified rat cytochrome P450c27, whose mitochondrial targeting signal had been replaced by a possible microsomal targeting signal of bovine cytochrome P450c17, was expressed in yeast . The modified P450c27 hemoprotein was correctly localized on yeast microsomes and exhibited the P450c27-dependent monooxygenase activity by addition of bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR) . Considering the previous observation that P450c27 with its own mitochondrial targeting signal was imported into yeast mitochondria (Akiyoshi-Shibata, M., Usui, E., Sakaki, T., Yabusaki, Y., Noshiro, M., Okuda, K., and Ohkawa, H . (1991) FEBS Lett . 280, 367-370), it is now suggested that the destination of P450c27 to either mitochondria or microsomes in yeast depends solely on the amino-terminal targeting signal . In addition, the modified P450c27 was simultaneously expressed in yeast with mature forms of bovine ADX and ADR . The recombinant yeast produced the P450 on the microsomes and mature forms of ADX and ADR in the cytoplasm, and showed the monooxygenase activity . Accordingly, a novel type of functional electron transport chain has been established between the cytoplasm and the microsomes in yeast. J Biol Chem, 1992 Aug 15, 267(23), 16430 - 7 The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity; Higman MA et al.; Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli . Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity . Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S . (1990) J . Biol . Chem . 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R . The individual subunits lacked methyltransferase activity when assayed alone . However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits . In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins . These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit . Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme. J Biol Chem, 1992 Aug 15, 267(23), 16390 - 5 Metal thiolate coordination in the E7 proteins of human papilloma virus 16 and cottontail rabbit papilloma virus as expressed in Escherichia coli; Roth EJ et al.; The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli . The proteins as purified from E . coli contain one tightly bound Zn(II) ion per molecule . The metal site shows facile exchange with either Cd(II) or Cu(I) . The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions . The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination . E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively . Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state . Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm . The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions . The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins . The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA . The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions. Gene, 1992 Aug 15, 117(2), 185 - 92 Cloning and inducible synthesis of poliovirus nonstructural proteins; Lama J et al.; The poliovirus nonstructural protein-encoding genes have been cloned and expressed in Escherichia coli using the inducible system described by Studier and Moffat {J . Mol . Biol . 189 (1986) 113-130} and Studier {J . Mol . Biol . 219 (1991) 37-44} . The two genes encoding the poliovirus proteases, 2Apro and 3Cpro, were cloned together with their flanking regions in order to test the ability of the polyprotein precursors synthesized to cause proteolytic cleavage and generate mature forms . Both proteases were synthesized and showed activity upon induction in this system . Previously, it had not been possible to produce the three poliovirus nonstructural proteins, 2B, 2C and 3A, and some of their precursors, 2C3AB, 2C3A and 3AB, at high levels in E . coli cells . We report the cloning of their genes using PCR techniques and their efficient expression from pET vectors upon induction with IPTG (isopropyl-beta-D-thiogalactopyranoside) . Moreover, some of these proteins, e.g., 3AB, 3A and 2B, are quite toxic for E . coli cells and lysed them upon production . Our results demonstrate the usefulness of this inducible system using the pET vectors to express these toxic poliovirus proteins. Biochim Biophys Acta, 1992 Aug 14, 1113(2), 161 - 70 What we can learn from the effects of thiol reagents on transport proteins; van Iwaarden PR et al.; Many secondary membrane transport systems contain reactive sulfhydryl groups . In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed . First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed . An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed . Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems. Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1553 - 9 Synthesis and purification of biologically active rat brain-derived neurotrophic factor from Escherichia coli; Negro A et al.; The cDNA for rat brain-derived neurotrophic factor was cloned as the prepro and mature sequences into two independent expression vectors under control of the T7 promoter . When these vectors were transfected into Escherichia coli the prepro and mature forms of brain-derived neurotrophic factor accounted for about 20% and 25% of total E . coli proteins, and displayed molecular sizes of 26 kDa and 15 kDa, respectively . Mature brain-derived neurotrophic factor was extracted from E . coli inclusion bodies, refolded in the presence of CuCl2 and purified . The resulting protein had an ED50 of 3 ng/ml in supporting survival of cultured embryonic dorsal root ganglion neurons. Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1417 - 22 Interaction of DNA with cationic liposomes: ability of transfecting lentil protoplasts; Maccarrone M et al.; The vesicle made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were multilamellar and rather homogeneous in shape as seen by transmission electron microscopy . Upon addition of circular DNA plasmids of different lengths to the liposomes, the formation of vesicle clusters around the DNA filament was observed, with dimensions dictated by the ratio DNA/lipid . These liposomes were able to transfect lentil (Lens culinaris) protoplasts inside the cells two different reporter genes, chloramphenicol-acetyltransferase and beta-glucuronidase . The activity of these two enzymes could be found in the cell lysates after 24 h from the incubation of protoplasts with the lipid-DNA complexes. Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1339 - 44 Temperature-dependent protein folding in vivo--lower growth temperature increases yield of two genetic variants of Xenopus laevis Cu,Zn superoxide dismutase in Escherichia coli; Battistoni A et al.; Two genetic variants of Xenopus laevis Cu,Zn superoxide dismutase, XSODA and XSODB, have been expressed in Escherichia coli by recombinant DNA techniques . Production of both proteins was obtained, although with different yields, XSODB being more abundant than XSODA in all the conditions tested . Lowering the temperature of growth was found to be a specific factor, decisive in obtaining quantitatively abundant, active Xenopus enzymes . Impaired folding of these proteins in the E.coli cytoplasm was found to parallel their in vitro properties. Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1220 - 6 Reactive oxygen-reducing and protein-refolding activities of adult T cell leukemia-derived factor/human thioredoxin; Mitsui A et al.; Reducing and protein-refolding activities of adult T cell leukemia-derived factor (ADF)/human thioredoxin were studied . Recombinant ADF/human thioredoxin produced by E . coli, which has an insulin-reducing activity as efficient as that of E . coli thioredoxin, also reduced some reactive oxygen species, such as hydrogen peroxide . Furthermore, recombinant ADF/human thioredoxin was found to have protein-refolding activity for scrambled (mispaired disulfide-containing) RNase A . Cys-31 at the active site of ADF/human thioredoxin proved essential for reducing activity, and loss of Cys-31 in ADF/human thioredoxin attenuated the protein-refolding activity . These data suggest a physiological role of ADF/human thioredoxin in protecting living cells from proteotoxicity caused by reactive oxygens in vivo. Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1333 - 8 The cell cycle regulator, human p50weel, is a tyrosine kinase and not a serine/tyrosine kinase; Honda R et al.; The human weel protein, a homologue of the yeast weel protein, was expressed in E . coli and purified to homogeneity . The purified weel protein phosphorylated the tyrosine residue of cdc2 kinase in HeLa cell extracts in the presence of human cyclin B1 . It also phosphorylated the tyrosine but not the threonine residue in the peptide of the amino-terminal of cdc2 kinase, although both these residues have been shown to be phosphorylated in higher eukaryotes in vivo . Furthermore, serine and tyrosine residues of the yeast weel protein are reportedly autophosphorylated in vitro, however the tyrosine residue of the human weel protein was autophosphorylated whereas the serine and threonine residues were not . These data indicate that human p50weel is tyrosine kinase and that it phosphorylated the tyrosine residue of the amino-terminal of cdc2 kinase in the presence of cyclin B1 and that the threonine residue is phosphorylated by another, unknown kinase. Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1463 - 70 Alteration of RNA I metabolism in a temperature-sensitive Escherichia coli rnpA mutant strain; Jung YH et al.; E . coli strain A49 carries the themosensitive mutation in the rnpA gene encoding the protein component of RNase P, a tRNA-processing enzyme . Two small RNAs were highly accumulated in the A49 carrying derivatives of ColE1-type plasmids, at nonpermissive temperature . Characterization of these RNAs showed that they were the processed or degraded products derived from RNA I, which is the negative controller of ColE1-type plasmid replication . These derivatives of RNA I only differ in size at the 5' ends . The data of their degradation and synthesis kinetics suggest that they are intermediates of RNA I metabolism. Biochim Biophys Acta, 1992 Aug 12, 1136(2), 169 - 74 Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine; Franson RC et al.; Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function . We report that sphingosine directly inhibits phospholipases A2 and D . Sphingosine inhibits Ca(2+)-dependent phospholipases A2 from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrophil in a dose-dependent manner with IC50 values ranging from 5-40 microM using {1-14C}oleate-labelled autoclaved E . coli (20 microM) as substrate . Inhibition is comparable using the same concentrations (20 microM) of {1-14C}oleate-labelled C . albicans or E . coli, or aqueous dispersions of 1-acyl-2-{1-14C}linoleoylglycerophosphoethanolamine or -choline . Sphinganine and stearylamine are as inhibitory as sphingosine; monoolein is less inhibitory (IC50 = 70 microM), while octylamine, N-acetylsphingosine, sphingomyelin and ceramide have no effect . Inhibition is relieved by increasing concentrations of substrate phospholipid . The molar ratio of sphingosine to phospholipid required for 50% inhibition ranges from 0.5 to 1.0 with 2-100 microM E . coli phospholipid . In contrast, sphingosine has a biphasic effect on the hydrolysis of E . coli by S . chromofuscus phospholipase D; concentrations less than or equal to 25 microM stimulate activity while concentrations greater than 25 microM are inhibitory . Addition of Triton X-100 eliminates both the stimulatory and inhibitory effects of sphingosine on phospholipase D activity. J Virol, 1992 Aug, 66(8), 4710 - 9 Mutational analysis of the core, spacer, and initiator regions of vaccinia virus intermediate-class promoters; Baldick CJ Jr et al.; Activation of vaccinia virus late gene transcription is dependent on DNA replication and the expression of three genes: A1L, A2L, and G8R (J . G . Keck, C . J . Baldick, Jr., and B . Moss, Cell 61:801-809, 1990) . To fully characterize the promoter elements of these trans-activator genes, we prepared more than 140 plasmid vectors containing natural and mutated DNA segments ligated to the Escherichia coli lacZ or chloramphenicol acetyltransferase reporter gene . Expression of the reporter genes occurred when the plasmids were transfected into vaccinia virus-infected cells and was enhanced when DNA replication was prevented, indicating that the A1L, A2L, and G8R promoters belong to the intermediate regulatory class . Deletional mutagenesis demonstrated that the regulatory elements of all three promoters extended between 20 and 30 nucleotides upstream of their RNA start sites . Single-base substitutions of the G8R promoter revealed two critical elements located from -26 to -13 (the core element) and -1 to +3 (the initiator element) . Mutations in these regions drastically affected expression, as determined by beta-galactosidase and mRNA analyses . Additional mutations defined the TAAA sequence as the critical initiator element . The length, but not the nucleotide sequence, of the segment between the core and initiator regions was crucial . The requirement for the spacer to be 10 or 11 nucleotides was consistent with a single turn of a double helix . The A1L and A2L promoters resembled the G8R promoter, and mutations in the conserved bases had the predicted effects on expression . We concluded that the three intermediate promoters are composed of a 14-bp A+T-rich core sequence separated by one turn of the double helix from the TAAA initiator element. Biochemistry, 1992 Aug 11, 31(31), 7143 - 51 Activity and spectroscopic properties of the Escherichia coli glutamate 1-semialdehyde aminotransferase and the putative active site mutant K265R; Ilag LL et al.; Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,1-aminomutase; EC 5.4.3.8; GSA-AT) catalyzes the transfer of the amino group on carbon 2 of glutamate 1-semialdehyde (GSA) to the neighboring carbon 1 to form delta-aminolevulinic acid (ALA) . To gain insight into the mechanism of this enzyme, possible intermediates were tested with purified enzyme and the reaction sequence was followed spectroscopically . While 4,5-dioxovaleric acid (DOVA) was efficiently converted to ALA by the pyridoxamine 5'-phosphate (PMP) form of the enzyme, 4,5-diaminovaleric acid (DAVA) was a substrate for the pyridoxal 5'-phosphate (PLP) form of GSA-AT . Thus, both substances are reaction intermediates . The purified enzyme showed an absorption spectrum with a peak around 338 nm . Addition of PLP led to increased absorption at 338 nm and a new peak around 438 nm . Incubation of the purified enzyme with PMP resulted in an additional absorption peak at 350 nm . The reaction of the PLP and PMP form of the enzyme with GSA allowed the detection of a series of peaks which varied in their intensities in a time-dependent manner . The most drastic changes to the spectrum that were observed during the reaction sequence were at 495 and 540 nm . Some of the detected absorption bands during GSA-AT catalysis were previously described for several other aminotransferases, indicating the relationship of the mechanisms . The reaction of the PMP form of the enzyme with DOVA resulted in a similar spectrum as described above, while the spectrum for the conversion of DAVA by the PLP form of the enzyme indicated a different mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Aug 11, 31(31), 7134 - 42 Energy transduction during catalysis by Escherichia coli DNA photolyase; Ramsey AJ et al.; Native DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate . Quantum yield and action spectral data for thymine dimer repair were obtained by using a novel multiple turnover approach under aerobic conditions . This method assumes that catalysis proceeds via a (rapid-equilibrium) ordered mechanism with light as the second substrate, as verified in steady state kinetic studies . The action spectrum observed with native enzyme matched its absorption spectrum and an action spectrum simulated based on an energy transfer mechanism where dimer repair is initiated either by direct excitation of FADH2 or by pterin excitation followed by singlet-singlet energy transfer to FADH2 . The quantum yield observed for dimer repair with native enzyme (phi Native = 0.722 +/- 0.0414) is similar to that observed with enzyme containing only FADH2 (phi EFADH2 = 0.655 +/- 0.0256), as expected owing to the high efficiency of energy transfer from the natural pterin to FADH2 {EET = 0.92} . The quantum yield observed for dimer repair decreased (2.1-fold) when the natural pterin was partially (68.8%) replaced with 5,10-CH(+)-H4folate (phi obs = 0.342 +/- 0.0149) . This is consistent with the energy transfer mechanism (phi calc = 0.411 +/- 0.0118) since a 2-fold lower energy transfer efficiency is observed when the natural pterin is replaced with 5,10-CH(+)-H4folate (EET = 0.46) (Lipman & Jorns, 1992) . The action spectrum observed for 5,10-CH(+)-H4folate-supplemented enzyme matched a simulated action spectrum which exhibited a small (5 nm) hypsochromic shift as compared with the absorption spectrum (lambda max = 385 nm).(ABSTRACT TRUNCATED AT 250 WORDS) Nucleic Acids Res, 1992 Aug 11, 20(15), 3873 - 80 A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair; Coverley D et al.; The human single-stranded DNA binding protein (HSSB/RPA) is involved in several processes that maintain the integrity of the genome including DNA replication, homologous recombination, and nucleotide excision repair of damaged DNA . We report studies that analyze the role of HSSB in DNA repair . Specific protein-protein interactions appear to be involved in the repair function of HSSB, since it cannot be replaced by heterologous single-stranded DNA binding proteins . Anti-HSSB antibodies that inhibit the ability of HSSB to stimulate DNA polymerase alpha also inhibit repair synthesis mediated by human cell-free extracts . However, antibodies that neutralize DNA polymerase alpha do not inhibit repair synthesis . Repair is sensitive to aphidicolin, suggesting that DNA polymerase epsilon or delta participates in nucleotide excision repair by cell extracts . HSSB has a role other than generally stimulating synthesis by DNA polymerases, as it does not enhance the residual damage-dependent background synthesis displayed by repair-deficient extracts from xeroderma pigmentosum cells . Significantly, when damaged DNA is incised by the Escherichia coli UvrABC repair enzyme, human cell extracts can carry out repair synthesis even when HSSB has been neutralized with antibodies . This suggests that HSSB functions in an early stage of repair, rather than exclusively in repair synthesis . A model for the role of HSSB in repair is presented. Nucleic Acids Res, 1992 Aug 11, 20(15), 4009 - 13 The yeast homolog of the U1 snRNP protein 70K is encoded by the SNP1 gene; Kao HY et al.; The product of the yeast SNP1 gene has high homology to two domains of the metazoan U1 snRNP protein 70K, which binds to stem/loop I of the U1 RNA . However, the absence of other domains conserved in metazoan 70K and the minimal effect of yeast U1 RNA stem/loop I deletion make the assignment of SNP1 as yeast 70K less clear . To address this question, we have expressed the SNP1 gene as a fusion protein in E . coli and developed a gel shift assay for U1 RNA binding . We show here that the product of the yeast SNP1 gene binds directly and specifically to the first 47 nucleotides of yeast U1 RNA, which include the stem/loop 1 structure . We therefore conclude that the SNP1 gene product is the yeast 70K homolog . This is the first yeast protein to be identified as a homolog of a metazoan snRNP protein. Nucleic Acids Res, 1992 Aug 11, 20(15), 4077 - 81 The mechanism of trans-activation of the Escherichia coli operon thrU(tufB) by the protein FIS . A model; Verbeek H et al.; Transcription of the thrU(tufB) operon is trans-activated by the protein FIS which binds to the promoter upstream activator sequence (UAS) . Deletions of parts of the UAS and insertions show that optimal trans-activation requires occupation by FIS of the two FIS-binding regions on the UAS and specific helical positioning of these regions . On the basis of these and other data, a model for the mechanism of thrU(tufB) trans-activation by FIS is proposed . This model implies that the mechanisms underlying stimulation by FIS of two totally different processes: inversion of viral DNA segments and transcription of stable RNA operons, are essentially the same. Nucleic Acids Res, 1992 Aug 11, 20(15), 3911 - 7 The effects of a unique D-loop structure of a minor tRNA(UUALeu) from Streptomyces on its structural stability and amino acid accepting activity; Ueda Y et al.; Streptomyces bldA gene, which encodes a tRNA corresponding to a very minor leucine codon, UUA, regulates pleiotropic gene expression which is involved in sporulation and secondary metabolism . The unique structural feature of this tRNA is the lack of GG sequence in dihydrouridine loop (D-loop) that generally is conserved in tRNAs involved in cytoplasmic protein biosynthesis . In order to investigate the relationship between the D-loop structure and the stability and leucine accepting activity of this tRNA, the wild and D-loop mutant tRNA transcripts were constructed with T7 RNA polymerase in vitro . The wild type tRNA(UUALeu) showed the structural stability and leucine accepting activity at physiological temperature for Streptomyces . The E.coli type D-loop mutant, which has a larger loop size and contains a GG doublet, exhibited increased thermostability . The kinetical analyses of the aminoacylation reaction of tRNA(UUALeu) with S.lividans and E.coli leucyl-tRNA synthetase (LeuRS) suggest there is a unique recognition mechanism of Streptomyces LeuRS toward tRNA(UUALeu). FEBS Lett, 1992 Aug 10, 308(1), 26 - 9 Coenzyme binding of a folding intermediate of aspartate aminotransferase detected by HPLC fluorescence measurements; Herold M et al.; Equilibrium dissociation and unfolding of dimeric aspartate aminotransferase from Escherichia coli proceeds via two compact monomeric intermediates which have similar hydrodynamic volumes but different fluorescence properties . We probed binding of the coenzyme pyridoxal 5'-phosphate to these intermediates by coupling fluorescence detection to size-exclusion HPLC . This procedure gave additionally an internal conformational probe of the unfolding transitions of the enzyme . It was shown that the first intermediate, M, is able to bind the coenzyme, whereas the second intermediate, M*, is not . It is likely that M is the correctly folded monomer of the protein. J Immunol Methods, 1992 Aug 10, 152(2), 149 - 57 Isolation of sequences from a random-sequence expression library that mimic viral epitopes; Lenstra JA et al.; We describe the use of random peptide sequences for the mapping of antigenic determinants . An oligonucleotide with a completely degenerate sequence of 17 or 23 nucleotides was inserted into a bacterial expression vector . This resulted in an expression library producing random hexa- or octapeptides attached to a beta-galactosidase hybrid protein . Mimotopes, or antigenic sequences that mimic an epitope, were selected by immunoscreening of colonies with monoclonal antibodies, which were specific for antigenic sites on the spike protein of the coronavirus transmissible gastroenteritis virus . We report one mimotope for antigenic site II, eight for site III and one for site IV . The site III and site IV mimotopes were closely similar to the corresponding linear epitopes, localized previously in the amino acid sequence of the S protein . An alignment of the site II mimotope and the sequence of the S protein around Trp97, which is substituted in escape mutants, suggests that this mimotope mimics a conformational epitope located around residues 97-103 . Applications of mimotopes to epitope mapping, serodiagnosis and vaccine development are discussed. FEBS Lett, 1992 Aug 10, 308(1), 97 - 100 Elevated cytosolic concentrations of SecA compensate for a protein translocation defect in Escherichia coli cells with reduced levels of negatively charged phospholipids; Kusters R et al.; Cellular extracts from cells with reduced synthesis of negatively charged phospholipids were found to support in vitro translocation of the precursor of the outer membrane protein PhoE with increased efficiency . Analysis of these extracts revealed that they contain increased levels of SecA . SecA depletion resulted in a loss of the translocation stimulatory activity, which could be restored by re-addition of purified SecA . We conclude that elevated cytosolic levels of SecA counteract the reduction of translocation efficiency due to low levels of negatively charged phospholipids in the inner membrane. Biochemistry, 1992 Jul 28, 31(29), 6848 - 55 Purification of human S-adenosylmethionine decarboxylase expressed in Escherichia coli and use of this protein to investigate the mechanism of inhibition by the irreversible inhibitors, 5'-deoxy-5'-{(3-hydrazinopropyl)methylamino}adenosine and 5'-({(Z)-4-amino-2-butenyl}methylamino)-5'-deoxyadenosine; Shantz LM et al.; Human S-adenosylmethionine decarboxylase (AdoMetDC) was expressed in high yield in Escherichia coli using the pIN-III(lppP-5) expression vector and purified to apparent homogeneity using affinity chromatography on methylglyoxal bis(guanylhydrazone)-Sepharose . The inactivation of the purified enzyme by 5'-deoxy-5'-{(3-hydrazinopropyl)methylamino}adenosine (MHZPA) was accompanied by an increase in absorbance at 260 nm of the large subunit . This increase was equivalent to the addition of 1 molecule of MHZPA . After digestion with the protease Lys-C, a peptide that contained the bound MHZPA was isolated and found to have the amino acid composition consistent with that expected from the amino terminus of the large subunit . These results indicate that MHZPA inactivates AdoMetDC by forming a hydrazone derivative at the pyruvate prosthetic group . Inactivation of AdoMetDC by 5'-({(Z)-4-amino-2-butenyl}methylamino}-5'-deoxyadenosine (AbeAdo) led to the appearance of a new peptide peak in the Lys-C protease digest . This peptide had the sequence ASMFVSK . This agrees with the expected sequence from the amino terminus, which is pyruvoyl-SMFVSK, with the exception that the pyruvate has been converted to alanine . Direct gas-phase sequencing of the large subunit of the enzyme also indicated the presence of alanine at the amino terminus after inactivation with AbeAdo . These results indicate that this inhibitor leads to transamination of the pyruvate prosthetic group . Since the pyruvate is covalently linked to the protein, its replacement by alanine leads to an irreversible inactivation of AdoMetDC. Cell, 1992 Aug 7, 70(3), 491 - 500 Specific sequences and a hairpin structure in the template strand are required for N4 virion RNA polymerase promoter recognition; Glucksmann MA et al.; Coliphage N4 virion-encapsidated, DNA-dependent RNA polymerase (vRNAP) is inactive on double-stranded N4 DNA; however, denatured promoter-containing templates are accurately transcribed . We report that all determinants of vRNAP promoter recognition exist in the template strand, indicating that this enzyme is a site-specific, single-stranded DNA-binding protein . We show that conserved sequences and the integrity of inverted repeats present at the promoters are essential for activity, suggesting the necessity for specific secondary structure . Evidence for such a structure is presented . We propose a model for in vivo utilization of vRNAP promoters in which template negative supercoiling yields single-strandedness at the promoter to reveal the determinants of vRNAP binding . This structure is stabilized by the binding of E . coli single-stranded DNA-binding protein to yield an "activated promoter." Cell, 1992 Aug 7, 70(3), 513 - 22 Translation of the prophage lambda cl transcript; Shean CS et al.; Mutations in rpsB that reduce the levels of the ribosomal protein S2 enhance the translation of cl in lambda lysogens . Two features of the cl transcript are required for enhanced translation: the absence of a leader and the presence of a downstream box, a sequence within the cl coding region that is complementary to the 16S rRNA . 30S ribosomal subunits deficient in S2 form ternary complexes with the cl transcript more efficiently than wild-type subunits . The absence of S2 may change the structure of the 16S rRNA, improving contacts with the cl downstream box. Science, 1992 Aug 7, 257(5071), 771 - 8 Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes; Daniels DL et al.; The DNA sequence of 91.4 kilobases of the Escherichia coli K-12 genome, spanning the region between rrnC at 84.5 minutes and rrnA at 86.5 minutes on the genetic map (85 to 87 percent on the physical map), is described . Analysis of this sequence identified 82 potential coding regions (open reading frames) covering 84 percent of the sequenced interval . The arrangement of these open reading frames, together with the consensus promoter sequences and terminator-like sequences found by computer searches, made it possible to assign them to proposed transcriptional units . More than half the open reading frames correlated with known genes or functions suggested by similarity to other sequences . Those remaining encode still unidentified proteins . The sequenced region also contains several RNA genes and two types of repeated sequence elements were found . Intergenic regions include three "gray holes," 0.6 to 0.8 kilobases, with no recognizable functions. Cell, 1992 Aug 7, 70(3), 477 - 89 Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription; Inostroza JA et al.; We have discovered a protein termed Dr1 that interacts with the TATA-binding protein, TBP . The association of Dr1 with TBP results in repression of both basal and activated levels of transcription . The interaction of Dr1 with TBP precludes the formation of a transcription-competent complex by inhibiting the association of TFIIA and/or TFIIB with TBP . Dr1 activity is associated with a 19 kd protein . A cDNA clone encoding Dr1 was isolated . Dr1 is phosphorylated in vivo and phosphorylation of Dr1 affected its interaction with TBP . Our results suggest a regulatory role for Dr1 in repression of transcription mediated via phosphorylation. Eur J Pharmacol, 1992 Aug 6, 218(2-3), 351 - 4 The role of nitric oxide and platelet-activating factor in the inhibition by endotoxin of pentagastrin-stimulated gastric acid secretion; Martinez-Cuesta MA et al.; Administration of E . coli endotoxin (1 mg/kg i.v.) abolished the acid secretory response induced by a bolus injection of pentagastrin (100 micrograms/kg i.v.) in the continuously perfused stomach of the anaesthetized rat . Endotoxin administration did not modify mean systemic arterial blood pressure . Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 5-20 mg/kg i.v.), but not dexamethasone (5 mg/kg s.c . twice) or indomethacin (5 mg/kg i.m.), substantially restored the secretory responses to pentagastrin . The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.v.), but not by its enantiomer D-arginine (100 mg/kg i.v.) . L-NAME (10 mg/kg i.v.) increased blood pressure but this does not seem to be the mechanism by which endotoxin-induced acid inhibition was prevented, since similar systemic pressor responses induced by noradrenaline (15 micrograms/kg per min i.v.) had no such effect . The platelet-activating factor (PAF) receptor antagonist, WEB 2086 (2 mg/kg), induced a partial reversal of the inhibition by endotoxin of acid responses to pentagastrin . In endotoxin-treated rats, the combined administration of L-NAME (10 mg/kg) and WEB 2086 (2 mg/kg) completely restored the degree of H+ output induced by pentagastrin to levels similar to those of control, vehicle-treated animals . These findings suggest that endotoxin-induced acute inhibition of acid responses to pentagastrin involves NO synthesis and the release of PAF. J Biol Chem, 1992 Aug 5, 267(22), 16000 - 6 Expression of TTK, a novel human protein kinase, is associated with cell proliferation; Mills GB et al.; We have isolated the full-length sequence for a unique human kinase, designated TTK . TTK was initially identified by screening of a T cell expression library with anti-phosphotyrosine antibodies . The kinases most closely related to TTK are the SPK1 serine, threonine and tyrosine kinase, the Pim1, PBS2, and CDC2 serine/threonine kinases, and the TIK kinase which was also identified through screening of an expression library with anti-phosphotyrosine antibodies . However, the relationships are distant with less than 25% identity . Nevertheless, TTK is highly conserved throughout phylogeny with hybridizing sequences being detected in mammals, fish, and yeast . TTK mRNA is present at relatively high levels in testis and thymus, tissues which contain a large number of proliferating cells, but is not detected in most other benign tissues . Freshly isolated cells from most malignant tumors assessed expressed TTK mRNA . As well, all rapidly proliferating cell lines tested expressed TTK mRNA . Escherichia coli expressing the complete kinase domain of TTK contain markedly elevated levels of phosphoserine and phosphothreonine as well as slightly increased levels of phosphotyrosine . Taken together, these findings suggest that expression of TTK, a previously unidentified member of the family of kinases which can phosphorylate serine, threonine, and tyrosine hydroxyamino acids, is associated with cell proliferation. J Biol Chem, 1992 Aug 5, 267(22), 15802 - 7 In vitro specificity of EcoRI DNA methyltransferase; Reich NO et al.; The sequence selectivity of enzyme-DNA interactions was analyzed by comparing discrimination between synthetic oligonucleotides containing the canonical site GAATTC and altered DNA sequences with the EcoRI DNA methyltransferase . The specificities (kcat/KmDNA) are decreased from 5- to 23,000-fold relative to the unmodified site . For several substrates the decrease in kcat makes a disproportionate contribution to the specificity difference, suggesting that discrimination is mediated by the placement of critical catalytic residues rather than binding interactions . This is supported by our observation that specificity changes are generally not followed by changes in the stability of the methyltransferase-DNA complexes . Also, base pair substitutions near the site of methylation result in greater decreases in complex stability, suggesting that recognition and catalytic mechanisms overlap. J Biol Chem, 1992 Aug 5, 267(22), 15563 - 7 Function independence of microhelix aminoacylation from anticodon binding in a class I tRNA synthetase; Kim S et al.; The monomeric form of the class I Escherichia coli methionine tRNA synthetase has a distinct carboxyl-terminal domain with a segment that interacts with the anticodon of methionine tRNA . This interaction is a major determinant of the specificity and efficiency of aminoacylation . The end of this carboxyl-terminal domain interacts with the amino-terminal Rossman fold that forms the site for amino acid activation . Thus, the carboxyl-terminal end may have evolved in part to integrate anticodon recognition with amino acid activation . We show here that internal deletions that disrupt the anticodon interaction have no effect on the kinetic parameters for amino acid activation . Moreover, an internally deleted enzyme can aminoacylate an RNA microhelix, which is based on the acceptor stem of methionine tRNA, with the same efficiency as the native protein . These results suggest that, in this enzyme, amino acid activation and acceptor helix aminoacylation are functionally integrated and are independent of the anticodon-binding site. J Biol Chem, 1992 Aug 5, 267(22), 15406 - 11 Roles of FAD and 8-hydroxy-5-deazaflavin chromophores in photoreactivation by Anacystis nidulans DNA photolyase; Malhotra K et al.; DNA photolyase from the cyanobacterium Anacystis nidulans contains two chromophores, flavin adenine dinucleotide (FADH2) and 8-hydroxy-5-deazaflavin (8-HDF) (Eker, A . P . M., Kooiman, P., Hessels, J . K . C., and Yasui, A . (1990) J . Biol . Chem . 265, 8009-8015) . While evidence exists that the flavin chromophore (in FADH2 form) can catalyze photorepair directly and that the 8-HDF chromophore is the major photosensitizer in photoreactivation it was not known whether 8-HDF splits pyrimidine dimer directly or indirectly through energy transfer to FADH2 at the catalytic center . We constructed a plasmid which over-produces the A . nidulans photolyase in Escherichia coli and purified the enzyme from this organism . Apoenzyme was prepared and enzyme containing stoichiometric amounts of either or both chromophores was reconstituted . The substrate binding and catalytic activities of the apoenzyme (apoE), E-FADH2, E-8-HDF, E-FAD(ox)-8-HDF, and E-FADH2-8-HDF were investigated . We found that FAD is required for substrate binding and catalysis and that 8-HDF is not essential for binding DNA, and participates in catalysis only through energy transfer to FADH2 . The quantum yields of energy transfer from 8-HDF to FADH2 and of electron transfer from FADH2 to thymine dimer are near unity. J Biol Chem, 1992 Aug 5, 267(22), 15334 - 9 Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement; Grimshaw CE et al.; Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined . Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH . Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant . The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant . No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme . However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type . NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes . Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases . This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex. J Mol Biol, 1992 Aug 5, 226(3), 707 - 19 Activation of recA protein . The open helix model for LexA cleavage; DiCapua E et al.; RecA protein is induced by the binding of DNA and ATP to become active in the hydrolysis of ATP and the cleavage of repressors . These reactions appear to depend on the structural state of the protein polymerized along the DNA, i.e . a helical coat of six RecA per turn of 95 to 100 A pitch . In support of this model of the active conformation, it was shown that high concentrations of salt also induce this helical polymerized state as well as the enzymatic activities . Here, we describe that, in vitro and with the non-hydrolyzable analogue ATP gamma S, RNA and heparin can also induce both the structural transition and the enzymatic activation of RecA to LexA cleavage in accordance with the model . RNA and heparin do not support the reaction in the presence of ATP, and they do not induce the hydrolysis of ATP either, suggesting that, in contrast to ATP gamma S, the nucleotide is not bound stably enough, and that the combined affinities of polynucleotide and ATP actually modulate the discrimination of RecA for the various possible inducers in vivo. J Mol Biol, 1992 Aug 5, 226(3), 597 - 608 Culture conditions differentially affect the translation of individual Escherichia coli mRNAs; Jacques N et al.; Our aim is to investigate whether changes in growth conditions can differentially affect the initiation of translation from individual Escherichia coli mRNAs that are not subjected to specific translational control . As a model system, we have constructed a series of point-mutated lacZ genes which differ in their Shine-Dalgarno (SD) sequence, their initiator codon, or the secondary structure around these elements . Alterations in growth conditions produced large (up to 8-fold) changes in the relative expression from these genes, which, we argue, stem from changes in their relative efficiencies of translation initiation . In particular, compared to genes bearing mutations outside the SD or initiator codon, genes mutated in these elements experience a significant decrease in their expression when cells are grown in minimal rather than rich medium; at 42 degrees C rather than 37 degrees C; or under amino acid starvation . We discuss the mechanisms underlying these effects, and evocate their possible generality. J Mol Biol, 1992 Aug 5, 226(3), 581 - 96 Interdependence of translation, transcription and mRNA degradation in the lacZ gene; Yarchuk O et al.; We have constructed a collection of Escherichia coli strains which differ by point mutations in the ribosome binding site (RBS) that drives the translation of the lacZ gene . These mutations affect the Shine-Dalgarno sequence or the initiation codon, or create secondary structures that sequester these elements, and result in a 200-fold variation in beta-galactosidase expression . Surprisingly, these variations of expression are paralleled by nearly equivalent changes in the lacZ mRNA level . The ratio of the beta-galactosidase expression to the mRNA level reflects the average spacing between translating ribosomes: hence, paradoxically, mutations that affect translation initiation do not correspondingly change this spacing . Further analysis of the mRNA level variations shows that they originate from two independent mechanisms . When beta-galactosidase expression exceeds a threshold corresponding roughly to one translation event per transcript, the variations in the efficiency of translation initiation affect largely the chemical and functional lifetimes of the mRNA . We further show that the rate-limiting step in the chemical decay process is an RNase E-dependent cleavage, which is outcompeted by translation initiation . Below this expression threshold, the mRNA lifetime levels out and strain-to-strain variations in mRNA level arise solely from polarity effects . We suggest that, in this activity range, most mRNA molecules that escape polarity are crossed by a single ribosome, and hence are identical from the viewpoint of degradation . Altogether, the tight couplings between translation initiation on one hand, polarity and/or mRNA degradation on the other, result in translation initiation events being closely spaced in time even from inefficient RBS, at the expense of the mRNA level . Finally, we evocate the possible beneficial consequences of a coupling between translation, transcription and mRNA degradation, for the management of cellular resources. J Mol Biol, 1992 Aug 5, 226(3), 681 - 8 Inheritance of the replication complex by one of two daughter copies during lambda plasmid replication in Escherichia coli; Wegrzyn G et al.; Direct measurement of DNA synthesis confirmed that lambda plasmid replication proceeds for several hours in an amino acid-starved relA mutant of Escherichia coli, leading to plasmid amplification; this replication is lambda cro-independent, but requires the function of lambda O initiator in the absence of its synthesis . This suggests that after the assembly of the replication complex (RC) at ori lambda the lambda O protein remains in this structure and the affinity of lambda O to ori lambda is alleviated in the assembled RC allowing its movement along the DNA . During amino acid starvation the lambda plasmid DNA synthesis per bacterial mass occurs at a constant level, as would be expected if the number of functioning RCs remained constant . This favors the idea that under these conditions the next replication round operates due to the activity of the RC inherited from the preceding round . Density shift experiments reveal indeed that, from two daughter plasmid copies synthesized after the onset of amino acid starvation only one is able to enter into the next round of replication . We infer that this is the plasmid copy that inherits the lambda O-enclosing RC from the previous replication round . Moreover, the same results of density shift experiments were obtained for plasmids synthesized before the onset of amino acid starvation . Therefore, we presume that in lambda plasmid-harboring bacteria growing in nutrient medium, every second plasmid circle bears an RC that originates from the preceding round of replication . This structure has to be assembled de novo only on the daughter plasmid copy that does not inherit the parental RC . In the absence of lambda O initiator synthesis in amino acid-starved relA cells this process cannot occur, leaving as the only replication pathway that driven by the parental RC . Our results are discussed in relation to the model of regulation of lambda plasmid replication. J Mol Biol, 1992 Aug 5, 226(3), 675 - 80 Stability of coliphage lambda DNA replication initiator, the lambda O protein; Wegrzyn G et al.; The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation . This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection . We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction . In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable . The extension of the {35S}methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis . Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form . We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases . We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v . light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria . The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis . The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication . Another significance of lambda O rapid degradation is proposed. J Biol Chem, 1992 Aug 5, 267(22), 15549 - 51 Cloning, sequencing, and expression of the Escherichia coli peptide methionine sulfoxide reductase gene; Rahman MA et al.; The gene encoding peptide methionine sulfoxide reductase was cloned from an Escherichia coli genomic library using an oligonucleotide probe based on the amino-terminal sequence of the protein . The nucleotide sequence revealed that the gene codes for a polypeptide of 212 amino acid residues with a calculated molecular weight of 23,314 . The protein has been overexpressed in E . coli and is present as a soluble active species. J Mol Biol, 1992 Aug 5, 226(3), 637 - 49 Mechanism of killer gene activation . Antisense RNA-dependent RNase III cleavage ensures rapid turn-over of the stable hok, srnB and pndA effector messenger RNAs; Gerdes K et al.; The hok/sok, srnB and pnd systems of plasmids R1, F and R438 mediate plasmid maintenance by killing plasmid-free segregants . The systems encode exceptionally stable full-length mRNAs that code for potent cell toxins that kill the cells from within . The systems also produce truncated mRNAs whose appearance is correlated with killing activity . The truncated mRNAs are shortened by 35 to 70 nucleotides in the 3' ends, but have the same 5' ends as the full-length transcripts . Translation of the stable killer mRNAs is regulated by unstable antisense RNAs that are complementary to the leader regions of the full-length and truncated mRNAs . We show here, that both the presence of the antisense RNA and of the host enzyme RNase III is required for rapid cleavage of the truncated mRNAs, and we map the cleavage point in the Hok mRNA in vitro and in vivo to be located between nucleotides +245 and +246 . The RNase III cleavage products of the Hok mRNA were found to be very unstable in vivo . Thus, RNase III cleavage seems to be the initial event leading to decay of the killer mRNAs . In an rnc- strain, the truncated mRNA species were found in steady-state cells . This observation indicates that the truncated mRNAs are formed constitutively and independently of the presence of the antisense RNAs . Thus, the antisense RNAs prevent the accumulation of the truncated mRNAs solely by mediating their rapid hydrolysis by RNase III . Furthermore, the generation of the truncated killer mRNAs in the rnc- host indicate that RNase III is dispensable for induction of the killer gene systems . Based on these and on observations obtained previously, we present a molecular model that explains the activation of the killer mRNAs in plasmid-free segregants and after addition of rifampicin. J Mol Biol, 1992 Aug 5, 226(3), 735 - 45 Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes; Bernard P et al.; In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA . We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein . This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide . Hence, the mutation was called gyrA462 . We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed . This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage . CcdB, in the absence of CcdA, induces the SOS pathway . SOS induction is a biological response to DNA-damaging agents . We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes . In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype . This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent . These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison . This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II. J Mol Biol, 1992 Aug 5, 226(3), 623 - 35 Positive regulation of the expression of the Escherichia coli pts operon . Identification of the regulatory regions; De Reuse H et al.; The pts operon of Escherichia coli is composed of the ptsH, ptsI and crr genes coding for three proteins central to the phosphoenolpyruvate dependent phosphotransferase system (PTS), the HPr, enzyme I and EIIIGlc proteins, respectively . We previously showed that transcription from the promoter region located upstream from the pts operon is regulated by two control circuits, which can occur independently from each other . Transcription of the pts operon is (1) stimulated by the CAP-cAMP complex and (2) enhanced during growth on glucose, a PTS substrate . The DNA regions involved in regulation of the expression of the pts operon have been identified . Two promoters, P0 and P1, separated by 100 bp are located upstream from the pts operon . In these promoter regions, we identified two sequences showing similarity with the consensus of CAP-binding sites, CAPa located near P0 and CAPb located in the -35 region of P1 . In vivo experiments showed that binding of CAP-cAMP at the CAPa site stimulates transcription from the P0 promoter . The binding sites of CAP-cAMP and/or RNA-polymerase on a DNA fragment containing both P0 and P1 promoters as well as both CAPa and CAPb sites were examined by the technique of DNase I footprinting . These in vitro experiments suggested that CAP-cAMP binding at the CAPb site might also play a role in regulation of the pts operon expression . In addition, we showed that the DNA region carrying the CAPa site is important for regulation by glucose . We finally propose that the expression of the pts operon is controlled by two alternative positive regulatory mechanisms, which are designed to allow activation of the pts operon under a great variety of growth conditions. J Biol Chem, 1992 Aug 5, 267(22), 15984 - 92 Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I; Morham SG et al.; Analysis of vaccinia topoisomerase mutants that are impaired in DNA relaxation has allowed the identification of amino acid residues required for the transesterification step of catalysis . Missense mutations of wild-type residues Gly-132----Asp and Arg-223----Gln rendered the protein inert in formation of the covalent enzyme-DNA complex and hence completely inactive in DNA relaxation . Mutations of Thr-147----Ile and Gly-132----Ser caused severe defects in covalent adduct formation that correlated with the extent of inhibition of relaxation . None of these point mutations had an effect on noncovalent DNA binding sufficient to account for the defect in relaxation . Deletion of amino- or carboxyl-terminal portions of the polypeptide abrogated noncovalent DNA binding . Two distinct topoisomerase-DNA complexes were resolved by native gel electrophoresis . One complex, which was unique to those proteins competent in covalent adduct formation, contained topoisomerase bound to the 5'-portion of the incised DNA strand . The 3'-segment of the cleaved strand had dissociated spontaneously . This complex was isolated and shown to catalyze transfer of the covalently bound DNA to a heterologous acceptor oligonucleotide, thereby proving that the covalent adduct between protein and duplex DNA is a true intermediate in strand breakage and reunion . The role of the active site region of eukaryotic topoisomerase in determining sensitivity or resistance to camptothecin was examined by converting the active site region of the resistant vaccinia enzyme (SKRAY274) to that of the drug-sensitive yeast enzyme (SKINY) . The SKINY mutation did not alter the resistance of the vaccinia enzyme to the cleavage-enhancing effects of camptothecin. J Biol Chem, 1992 Aug 5, 267(22), 15932 - 7 Expression of poliovirus nonstructural proteins in Escherichia coli cells . Modification of membrane permeability induced by 2B and 3A; Lama J et al.; The poliovirus nonstructural proteins 2B, 2C, 2C3A, 2C3AB, 3A, and 3AB have been cloned and efficiently expressed in Escherichia coli cells . Each individual protein, or combinations of some of them, were cloned using polymerase chain reaction techniques and correspond to the genuine poliovirus protein plus an additional methionine . The system used to express them uses pET vectors containing the promoter of gene 10 of phage T7 . Expression of protein 2C in BL21 (DE3) pLysS cells, which express the T7 lysozyme, is not toxic, and the bacteria synthesize this protein for several hours after induction . In contrast, the expression of proteins 2B, 3A, or 3AB is not tolerated by BL21 (DE3) pLysS cells which could make them only for a limited period of time . Protein 3AB was particularly toxic and induced a rapid lysis of the recombinant clone after its induction with isopropyl-1-thio-beta-D-galactopyranoside alone or with both isopropyl-1-thio-beta-D-galactopyranoside and rifampicin . Further analyses showed that 3AB induced profound modifications in membrane permeability to o-nitrophenyl-beta-D-galactopyranoside, labeled uridine, and nonpermeant translation inhibitors . Cloning and expression of proteins 2B, 3A, and 3AB in BL21 (DE3) cells that do not contain the T7 lysozyme lead to a more sustained expression of these proteins without detectable cell lysis . Changes in permeability to low molecular weight compounds such as radioactive uridine, o-nitrophenyl-beta-D-galactopyranoside, and hygromycin B readily appeared upon induction of 2B, 3A, and 3AB . Our results indicate that the poliovirus nonstructural polypeptides 2B and 3A (or 3AB) are lytic for the bacteria . In fact, both proteins 2B and 3A contain hydrophobic domains in a potential amphipathic helix; this is one characteristic shared with a number of membrane-active peptides. J Biol Chem, 1992 Aug 5, 267(22), 15816 - 22 The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure; Filatov D et al.; Herpes simplex virus ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, which are required together for activity . Active R2 protein contains a tyrosyl free radical and a binuclear iron center . A truncated form of the R2 subunit, lacking 7 amino acid residues in the carboxyl terminus, was constructed, overexpressed in Escherichia coli and purified to homogeneity . In the presence of ferrous iron and oxygen, the truncated protein readily generated similar amounts of tyrosyl free radical as the intact protein . However, the radical showed differences in EPR characteristics in the truncated protein compared with the normal one, indicating an altered structural arrangement of the radical relative to the iron center . The truncated R2* protein was completely devoid of binding affinity to the R1 protein, demonstrating that the subunit interaction is totally dependent on the 7 outermost carboxyl-terminal amino acids of protein R2. J Biol Chem, 1992 Jul 25, 267(21), 14547 - 50 Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase; O'Sullivan MG et al.; We report here that lipopolysaccharide (LPS) priming of rabbit alveolar macrophages leads to amplified synthesis of prostanoids, at least in part, by induction of a novel prostaglandin H synthase (PGH synthase) . Rabbit alveolar macrophages were cultured with or without added LPS derived from Escherichia coli 0111:B4 for 4 h and then stimulated with opsonized zymosan (OPZ) . LPS priming of alveolar macrophages resulted in enhanced release of thromboxane (TX) upon stimulation with OPZ, when compared to stimulated non-LPS controls . Addition of exogenous arachidonic acid to LPS-primed alveolar macrophages also resulted in increased production of TX . The LPS-induced increase in TX formation, in response to OPZ or arachidonic acid, was abolished by the addition of actinomycin D or cycloheximide during the priming period . Gas chromatography/mass spectrometry analysis indicated that levels of prostaglandins D2, E2, and F2 alpha, along with TX, were augmented in stimulated LPS-primed alveolar macrophages, implicating PGH synthase in the priming process . PGH synthase enzymatic activity, as determined by addition of arachidonic acid to macrophage sonicates, was markedly enhanced in LPS-primed alveolar macrophages . This correlated with increased PGH synthase levels detected by immunoprecipitation of 35S-labeled proteins and by Western blot analysis . Finally, Northern blot analysis using a cDNA probe to the recently described mitogen-inducible mouse PGH synthase revealed strong induction of approximately 4.3-kilobase mRNA in LPS-primed alveolar macrophages . Taken together, these results reveal that induction of a novel PGH synthase, probably the rabbit homologue of PGH synthase-2, plays a role in the enhanced synthesis of prostanoids by LPS-primed alveolar macrophages. Biochemistry, 1992 Aug 4, 31(30), 7003 - 8 Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase; Thompson NE et al.; A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt . This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened . One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt . Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure . Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column . The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column . This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay . The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins. Biochemistry, 1992 Aug 4, 31(30), 6980 - 9 Interactions between DNA-bound transcriptional regulators of the Escherichia coli gal operon; Dalma-Weiszhausz DD et al.; Regulation of the initiation of gene transcription from the gal operon of Escherichia coli is activated by the binding of CAP (catabolite activator protein) to a site centered at base pair -41.5 relative to the S1 start site of transcription . This operon is repressed by the specific binding of Gal repressor (GalR) to two operators, OE and OI, centered at -60.5 and +53.5, respectively . It has been proposed that this negative regulation results from the interaction of GalR dimers bound to OE and OI to form a protein-mediated "looped complex" {cf . Adhya, S . (1989) Annu . Rev . Genet . 23, 207-230} . In order to test whether DNA-bound CAP would facilitate or inhibit the binding of GalR, the simultaneous binding of these proteins was studied by quantitative DNase I footprint titration analysis . These studies demonstrate that GalR binding is noncooperative in the presence and in the absence of CAP and that GalR and CAP bind to the gal operon independently . No evidence was found that CAP stabilizes a putative Gal repressor-mediated protein-DNA looped complex . It has been shown that the gal operon can be negatively regulated by the binding of Lac repressor (LacI) to a gal operon in which OE and OI were both modified to be recognized by LacI {Haber, R., & Adhya, S . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 9683-9687} . In contrast to GalR, LacI binds to the chimeric gal operon with moderate cooperativity via the formation of a stable protein-DNA looped complex.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Aug 4, 31(30), 6904 - 10 p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA; Roux B et al.; p-Aminobenzoic acid (PABA) is an important precursor in the bacterial biosynthetic pathway for folate enzymes . This biosynthesis requires three separate proteins: PabA, PabB, and PabC . Together PabA and PabB convert glutamine and chorismate to glutamate and 4-amino-4-deoxychorismate . This aminochorismate is subsequently transformed to PABA by PabC . In this study, PabA from Escherichia coli has been purified to homogeneity from an overproducing construct and found to have no detectable glutaminase activity until addition of the E . coli PabB subunit . PabB forms a 1:1 complex with PabA to yield a glutaminase k(cat) of 17 min-1 . The addition of chorismate, the substrate of PabB, induces a 2-fold increase of k(cat) as well as a 3-fold increase of Km for glutamine . The PabA/PabB complex has Kd less than 10(-8) M but does not form a stable complex isolable by gel filtration . Studies with the glutamine affinity label diazooxonorleucine (DON) reveal it is an inactivator of the glutaminase activity of the PabA/PabB complex, but DON does not alkylate and inactivate PabA alone . Similarly, while isolated PabA shows no tendency to form a glutamyl-enzyme intermediate, the PabA/PabB complex forms a covalent intermediate with {14C}glutamine on PabA that accumulates to 0.56 mol/mol in hydrolytic turnover . PabA is thus a conditional glutaminase, activated by 1:1 complexation with PabB. FEBS Lett, 1992 Aug 3, 307(3), 347 - 50 Ubiquinone biosynthesis . Cloning of the genes coding for chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyl transferase from Escherichia coli; Siebert M et al.; Chorismate pyruvate-lyase activity was detected in extracts of Escherichia coli . 4-Hydroxybenzoate was identified as the product of the enzymatic reaction by chemical derivatization and GC-MS analysis . The ubiC gene, coding for the chorismate pyruvate-lyase, was cloned and sequenced . The molecular weight of the gene product was calculated as 18,776 Da and confirmed by expression of the protein in E . coli minicells . The ubiA gene, coding for the 4-hydroxybenzoate octaprenyl transferase, was identified by sequence homology and complementation of a ubiA- strain . It is located directly downstream of ubiC in a typical operon structure. FEBS Lett, 1992 Aug 3, 307(3), 257 - 62 The active site structure of methane monooxygenase is closely related to the binuclear iron center of ribonucleotide reductase; Nordlund P et al.; Methane monooxygenase (MMO) catalyses the biological transformation of methane to methanol at a binuclear iron site . Guided by the three-dimensional structure of the R2 protein of E . coli ribonucleotide reductase (RNR), we have aligned the sequences of two different MMOs with the sequences of the iron coordinating four helix bundle in R2 . The model suggests that the central four helix bundle of R2 is present also in MMO . The iron coordination is similar in MMO and R2 with two histidine ligands and four carboxyl ligands in both cases . The residues lining the proposed oxygen binding site in MMO are significantly smaller in MMO than in R2 allowing binding of both molecular oxygen and methane at this site . This binding site is lined by residues Cys151, Thr213, Ile217 and Ile(Val)239. Carbohydr Res, 1992 Aug 3, 232(2), 227 - 33 Investigation of the active site of Escherichia coli beta-D-galactosidase by photoaffinity labelling; Kuhn CS et al.; 3,7-Anhydro-2-azi-1,2-dideoxy-D-glycero-L-manno-(8-3H)octitol++ + (1a) and 3-azibutyl 1-thio-beta-D-(6-3H)galactopyranoside (2a) were synthesised from the unlabelled compounds by reaction with galactose oxidase, then reduction with sodium borotritide . Whereas 1a was an efficient photoaffinity reagent for the beta-D-galactosidase from E . coli, 2a was ineffective . Three 3H-labelled peptides were isolated after digestion of the labelled enzyme with trypsin, one of which was an octapeptide (Trp 158 to Ser 165), which is remote from the segments detected as part of the active site of the enzyme. J Gen Virol, 1992 Aug, 73 ( Pt 8), 2093 - 8 Immunodetection of the proteins encoded by grapevine chrome mosaic nepovirus RNA2; Hibrand L et al.; Fragments of the putative non-structural proteins (44K and 46K) encoded by RNA2 of grapevine chrome mosaic nepovirus (GCMV) were expressed as fusion proteins in Escherichia coli and used to raise specific antisera . All three proteins encoded by GCMV RNA2 (viral coat protein, and the 44K and 46K proteins) were detected by immunoblotting in subcellular fractions prepared from the leaves of infected Chenopodium quinoa plants, confirming a previously proposed model of the GCMV RNA2-encoded polyprotein . In addition to the 44K protein, one of the antisera detected a 90K protein presumably representing a precursor of the 44K and 46K proteins . Whereas the 44K and coat proteins could be detected in both soluble and membrane fractions, the 46K protein was found to be specific to the membrane fraction . Analysis of the kinetics of accumulation of the proteins showed that the 44K and 46K proteins were very transient whereas the coat protein was more stable and could be detected up to 21 days after inoculation . These results provide the first direct in vivo data supporting the maturation map of the GCMV RNA2 polyprotein deduced from in vitro experiments. J Bacteriol, 1992 Aug, 174(16), 5454 - 6 The 55-kilodalton protein in an oriC complex fraction is glycogen synthase; Kaidow A et al.; Three proteins with molecular masses of 35, 55, and 75 kDa were found in an oriC complex fraction after purification through CsCl density gradient centrifugation (W . G . Hendrickson, T . Kusano, H . Yamaki, R . Balakrishnan, M . King, J . Murchie, and M . Schaechter, Cell 30:915-923, 1982) . Of these three proteins, the 55-kDa protein was determined to be glycogen synthase on the basis of the N-terminal amino acid sequence and the molecular weight . The oriC complex was formed in glgA mutant cells, which produce no detectable glycogen, as well as in wild-type cells . None of the 35-, 55-, and 75-kDa proteins were detected in the fraction from this mutant . The results indicate that these proteins were not constituents of the oriC complex. J Bacteriol, 1992 Aug, 174(16), 5436 - 41 Amino acid substitution in the lactose carrier protein with the use of amber suppressors; Huang AM et al.; Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains . The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside . Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity . In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found . The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity . Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine . It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition. J Bacteriol, 1992 Aug, 174(16), 5414 - 23 Isolation and characterization of ftsZ alleles that affect septal morphology; Bi E et al.; The ftsZ gene encodes an essential cell division protein that specifically localizes to the septum of dividing cells . In this study we characterized the effects of the ftsZ2(Rsa) mutation on cell physiology . We found that this mutation caused an altered cell morphology that included minicell formation and an increased average cell length . In addition, this mutation caused a temperature-dependent effect on cell lysis . During this investigation we fortuitously isolated a novel temperature-sensitive ftsZ mutation that consisted of a 6-codon insertion near the 5' end of the gene . This mutation, designated ftsZ26(Ts), caused an altered polar morphology at the permissive temperature and blocked cell division at the nonpermissive temperature . The altered polar morphology resulted from cell division and correlated with an altered geometry of the FtsZ ring . An intragenic cold-sensitive suppressor of ftsZ26(Ts) that caused cell lysis at the nonpermissive temperature was isolated . These results support the hypothesis that the FtsZ ring determines the division site and interacts with the septal biosynthetic machinery. J Bacteriol, 1992 Aug, 174(16), 5382 - 90 Decay of ompA mRNA and processing of 9S RNA are immediately affected by shifts in growth rate, but in opposite manners; Georgellis D et al.; By growing Escherichia coli in continuous cultures at various growth rates, we provide definitive evidence that the stability of the ompA mRNA is growth rate dependent . Shifting fast-growing cells into physiological salt buffer led to an immediately increased rate of ompA mRNA decay and to an instantly decreased rate of 9S RNA conversion into 5S rRNA . Shifting slowly growing cells into fresh medium had the opposite effect for each of the two RNA species . The observed regulatory patterns underline the need of cells to adjust the output of ompA and 9S RNAs in response to growth rate changes . At all growth rates and throughout all shift experiments, the half-life of bla mRNA was constant . A stabilization of the ompA transcript was even observed when slowly growing cells were shifted into fresh medium already containing the transcriptional inhibitor rifampicin . A hybrid bla transcript with the 5' untranslated region from the ompA gene behaved similarly to the wild-type ompA messenger in response to a shift in growth rate . In agreement with this result, we found that the same type of 5' cleavages as have been previously shown to initiate the decay of the ompA transcript seem to be involved in stability regulation . In E . coli the degradation of mRNA has been shown to depend on the ams/rne gene . This gene controls the stability-related cleavages in the ompA transcript, catabolic processes, and the cleavages which process the 9S rRNA into 5S RNA, an anabolic process . We discuss these results with respect to the ams/rne gene and the related nuclease activities that control the ompA and 9S RNA cleavages. J Bacteriol, 1992 Aug, 174(16), 5362 - 70 C-shaped cells caused by expression of an ftsA mutation in Escherichia coli; Gayda RC et al.; A plasmid, pDLL4, was isolated from a Tn5tac1 mutagenesis experiment with plasmid pZAQ . When pDLL4 was transformed into wild-type rod-shaped cells, it caused cells in the population to become curved (C-shaped or convoluted) . The Tn5tac1 transposon was integrated within the carboxyl end of the ftsA gene in pDLL4 . This mutation was designated ftsAc . Subcloning ftsAc DNA into another plasmid vector verified that the curved-cell phenotype was caused by the expression of this altered gene . DNA sequence analysis of the ftsAc mutation revealed that the transposition event changed the DNA so that the last 28 amino acids of the FtsA protein were lost and 5 new amino acids were added . A radioactive peptide band corresponding to this truncated FtsAc protein was identified by a T7 promoter-T7 polymerase protein labeling system . Observations of thin sections of these curved cells with an electron microscope revealed aggregates of striated cylindrical structures traversing the cytoplasm . The ends of these aggregates appear to be at or near the cell membrane . The linear periodicity of the cylinders was approximately 11 nm, and the diameter of a cylinder was about 15 nm . Aggregates of as many as five cylinders were arrayed diagonally to the long axis of the curved cells, a finding that suggests that some type of internal organization may be causing the curved cell shape. J Bacteriol, 1992 Aug, 174(16), 5309 - 16 Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase; Nichols BP et al.; In Escherichia coli, chorismate lyase catalyzes the first step in ubiquinone biosynthesis, the conversion of chorismate to 4-hydroxybenzoate . 4-Hydroxybenzoate is converted to 3-octaprenyl-4-hydroxybenzoate by 4-hydroxybenzoate octaprenyltransferase . These two enzymes are encoded by ubiC and ubiA, respectively, and have been reported to map near one another at 92 min on the E . coli chromosome . We have cloned the ubiCA gene cluster and determined the nucleotide sequence of ubiC and a portion of ubiA . The nucleotide sequence abuts with a previously determined sequence that encodes a large portion of ubiA . ubiC was localized by subcloning, and overproducing plasmids were constructed . Overexpression of ubiC allowed the purification of chorismate lyase to homogeneity, and N-terminal sequence analysis of chorismate lyase unambiguously defined the beginning of the ubiC coding region . Although chorismate lyase showed no significant amino acid sequence similarity to 4-amino-4-deoxychorismate lyase (4-amino-4-deoxychroismate----4-aminobenzoate), the product of E . coli pabC, chorismate lyase overproduction could complement the growth requirement for 4-aminobenzoate of a pabC mutant strain . Of the several enzymes that convert chorismate to intermediates of E . coli biosynthetic pathways, chorismate lyase is the last to be isolated and characterized. J Bacteriol, 1992 Aug, 174(16), 5219 - 27 Involvement of SecB, a chaperone, in the export of ribose-binding protein; Kim J et al.; Ribose-binding protein (RBP) is an exported protein of Escherichia coli that functions in the periplasm . The export of RBP involves the secretion machinery of the cell, consisting of a cytoplasmic protein, SecA, and the integral membrane translocation complex, including SecE and SecY . SecB protein, a chaperone known to mediate the export of some periplasmic and outer membrane proteins, was previously reported not to be involved in RBP translocation even though small amounts of in vitro complexes between SecB and RBP have been detected . In our investigation, it was shown that a dependence on SecB could be demonstrated under conditions in which export was compromised . Species of RBP which carry two mutations, one in the leader that blocks export and a second in the mature protein which partially suppresses the export defect, were shown to be affected by SecB for efficient translocation . Five different changes which suppress the effect of the signal sequence mutation -17LP are all located in the N domain of the tertiary structure of RBP . All species of RBP show similar interaction with SecB . Furthermore, a leaky mutation, -14AE, generated by site-specific mutagenesis causes reduced export in the absence of SecB . These results indicate that SecB can interact with RBP during secretion, although it is not absolutely required under normal circumstances. Gene, 1992 Aug 1, 117(1), 7 - 14 Expression of synthetic genes encoding fused proteins under tight control of modified regulatory regions of the colicin operon; Waleh NS et al.; A versatile expression vector system for construction of gene and protein fusions, specific radiolabeling of gene products and high-level protein production is described . Expression cassettes were constructed containing structural genes encoding native and analog forms of connective tissue-activating peptide-III (CTAP-III), beta-thromboglobulin, neutrophil-activating protein and modified regulatory sequences derived from the colicin E1 operon . Gene expression was enhanced by changes in the colicin promoter that increased the transcription initiation rate both in vivo and in vitro, and by deletion of a sequence affecting catabolite repression . High-level expression, producing recombinant protein up to 30% of the total cellular protein, was induced rapidly after stimulation of the SOS response by using either mitomycin C or nalidixic acid, by temperature shift using temperature-sensitive mutations in the LexA or RecA proteins, or by UV light . The presence of radiolabeled amino acids during induction resulted in greater than 95% preferential labeling of recombinant proteins . CTAP-III remained stable for more than 6 h following decay of the inducing signal . The use of CTAP-III in protein fusions improved stability of several therapeutically useful proteins including the thrombin-specific inhibitor, hirudin and a cell receptor-binding domain of laminin, when they were produced in Escherichia coli. Gene, 1992 Aug 1, 117(1), 23 - 9 Intragenic complementation between Escherichia coli trp repressors with different defects in the tryptophan-binding pocket; Storbakk N et al.; Site-directed mutagenesis of the trpR gene (encoding the trp repressor, TrpR) was used to replace Gly85 with tryptophan (Trp or W), in order to place Trp near its normal location in the L-tryptophan(L-W)-binding pocket . The resulting mutant protein (G85W) exhibits weak, but significant repressor activity in vivo that is independent of the presence of L-W in the media . This mutant negatively complements the chromosomal wild type (wt), but does not negatively complement either the wt or the super-repressor, E49K, when any of these alleles is expressed on a multicopy plasmid . Activity of the mutant repressor, G85W, when produced in vivo together with T44M, approaches that of the wt repressor . This result presumably reflects complementation between the two mutant polypeptides . Similar results are obtained when G85R or G85K are combined with T44M in vivo, but not when G85W is replaced by G85E . The level of repression is dependent on the presence of L-W in the media . The TrpR with two mutations altering both Gly85 (G85W, G85R, G85E or G85K) and Thr44 (T44M) has no repressor activity . These results suggest a type of site-specific intragenic complementation where only certain alterations at Gly85 complement T44M . In this study, a positive charge or an indole ring appears to be required for the observed intragenic complementation. Gene, 1992 Aug 1, 117(1), 15 - 22 Stepwise cloning and genetic organization of the seemingly unclonable HgiCII restriction-modification system from Herpetosiphon giganteus strain Hpg9, using PCR technique; Erdmann D et al.; The genes, hgiCIIR and hgiCIIM, that encode the HgiCII restriction and modification (R-M) system from Herpetosiphon giganteus strain Hpg9, an AvaII isoschizomer recognizing the sequence, GGATCC, were cloned in Escherichia coli . Cloning the respective hgiCIIM gene was achieved via in vitro selection both from a Sau3AI- and an NheI-generated plasmid gene library using AvaII, a commercially available isoschizomer of HgiCII . However, all attempts to clone the closely linked hgiCIIR and M genes in a single step resulted in deletions spanning parts of the coding region of hgiCIIR . Therefore, cloning of the missing 3'-terminal part of this gene was achieved by applying the inverse polymerase-chain-reaction technique . All attempts to construct an enzymatically active R.HgiCII failed; only the inactivated hgiCIIR gene could be cloned . Sequencing of the hgiCIIRM region (carrying predesigned small mutations in the R gene) disclosed three open reading frames (ORFs): one small ORF preceding the methltransferase (MTase)-encoding gene, plus those encoding M.HgiCII (49,620 Da) and R.HgiCII (30,891 Da) . M.HgiCII exhibits the common motif of ten conserved amino-acid blocks typically found within the group of m5C-MTases . The R-M system of HgiCII reveals strong homologies to the isoschizomeric R-M system of HgiBI from H . giganteus strain Hpg5, which, in contrast, could be cloned in one step. Gene, 1992 Aug 1, 117(1), 1 - 5 A signature element distinguishes sibling and independent mutations in a shuttle vector plasmid; Parris CN et al.; We have developed a new shuttle vector plasmid for studying mutagenesis in mammalian cells that permits proof of independence of identical mutations . Mutations occur more frequently at some sites in a gene than in others, and in a collection of mutant plasmids from a single transfection of mammalian cells the same mutation may appear several times . However, those arising from independent events cannot be distinguished from siblings of an initial event . The new vector system (pSP189) is a population of plasmids, each of which contains an 8-bp 'signature sequence' . This sequence confers a unique identification tag to each plasmid and allows individual members to be identified by a distinctive signature . The plasmid also carries the Escherichia coli bacterial supF gene as a marker for mutagenesis, as well as sequences which support replication in primate (including human) cells and E . coli . We have used the pSP189 system to generate a UV-induced spectrum of mutations in supF following replication in a single plate of human DNA-repair-deficient cells (xeroderma pigmentosum, complementation group A) . With the signature sequence, we were able to determine whether identical mutations derived from the transfection were of independent or sibling origin . There were eight identical mutations at the strongest hotspot, all of which had different signature sequences . Only one of these events would have been reported in previous experiments . This plasmid reduces the effort required to generate a spectrum of mutations caused by a DNA-damaging agent and allows a more accurate assessment of mutational hotspot intensity. Genes Dev, 1992 Aug, 6(8), 1553 - 61 The basis for the mechanistic bias for deletional over inversional V(D)J recombination; Gauss GH et al.; V(D)J recombination between recognition sites in the genome is characterized by certain biases . At some loci, proximal sites undergo recombination substantially more frequently than distal ones . The joining of DH/JH is an example of this . Because the DH element bears signal sequences on each side, inversion would be expected as often as deletion in DH/JH recombination . However, the markedly favored outcome is deletion, entailing utilization of the closer recombination site . One model proposed to explain these biases is the tracking model in which the recombinase tracks from one site to the other . Here, we have directly tested for various types of tracking in V(D)J recombination and have found no indication that it occurs . In addition, we have created DH-JH minilocus substrates for analysis of the basis for the preference for deletion . We find that we can reproduce the deletional bias for the system . Moreover, by flipping the orientation of the D segment, we can reverse the bias such that the frequency of inversions can exceed the number of deletions . These results indicate (1) that there is no intrinsic topological preference in this reaction, and (2) that the sequence of the signal and coding ends determines the bias. Genes Dev, 1992 Aug, 6(8), 1542 - 52 Functional analysis of Drosophila transcription factor IIB; Wampler SL et al.; We have isolated a cDNA encoding Drosophila transcription factor IIB (dTFIIB) and characterized the properties of recombinant dTFIIB with a reconstituted in vitro transcription system derived from Drosophila embryos . Purified, recombinant dTFIIB is fully active at a concentration of one molecule per template DNA . With different promoters, the transcriptional activity of dTFIIB was similar but not identical to that of human TFIIB, which suggests that there may be variations in the mechanisms by which TFIIB functions in transcription . We have also found that recombinant dTFIIB suppressed nonspecific initiation of transcription by RNA polymerase II by a mechanism that appears to involve direct interaction between TFIIB and the polymerase . Addition of excess dTFIIB to transcription reactions resulted in promoter-specific repression of transcription . These experiments have led to the hypothesis that TFIIB interacts with a basal transcription factor that is required for transcription of some, but not all, genes and that the presence of excess dTFIIB results in sequestration of the promoter-specific basal factor to prevent its assembly into a productive transcription complex . Excess dTFIIB did not, however, affect the ability of either GAL4-VP16 or Sp1 to stimulate transcription . These data indicate that in contrast to current models, GAL4 derivatives do not activate transcription by increasing the rate of assembly of TFIIB into the transcription complex. Int J Biochem, 1992 Aug, 24(8), 1341 - 5 Kinetic studies on activation of peptide bond formation by hyaluronic acid; Theocharis DA et al.; 1 . In this study, a cell-free system derived from Escherichia coli has been used in order to examine in detail the effect of hyaluronic acid on peptide bond formation with the aid of puromycin reaction . 2 . This reaction is activated by hyaluronic acid . 3 . The degree of activation of peptide bond formation depends on the molecular size of hyaluronic acid . 4 . The kinetic analysis revealed that the hyaluronic acid acts as a mixed-type nonessential activator . 5 . The presence of hyaluronic acid improves about 9-fold the activity status of ternary complex as it can be calculated by k3/k5 ratio. Crit Care Med, 1992 Aug, 20(8), 1146 - 51 Addition of alinidine, a specific bradycardic agent, to dobutamine in a canine model of endotoxic shock; Preiser JC et al.; BACKGROUND AND METHODS: Alinidine is a recently developed antiarrhythmic medication that acts directly on the cardiac pacemaker cells to reduce heart rate (HR) . At effective doses, alinidine might have cardiodepressant actions that could be hazardous in the presence of hemodynamic instability . On the other hand, one limitation of the use of catecholamines is tachycardia, and alinidine could be beneficial in situations such as septic shock, where adrenergic agents are commonly required . The present study explored the hemodynamic and gasometric effects of alinidine during dobutamine administration in a canine model of septic shock induced by endotoxin administration . In ten pentobarbital-anesthetized, mechanically ventilated dogs (weight 28 +/- 4 kg), Escherichia coli endotoxin (3 mg/kg) injection was followed 30 mins later by saline infusion to restore and maintain pulmonary artery occlusion pressure at the baseline value . Sixty minutes after the endotoxin administration, a dobutamine infusion was started at a rate of 10 micrograms/kg/min . Thirty minutes later, alinidine was administered as a bolus dosage of 1 mg/kg in five dogs; the other five dogs served as a control group . RESULTS: Alinidine administration resulted in a decrease in HR from 157 +/- 20 to 138 +/- 27 beats/min (p less than .01) and a nonsignificant increase in cardiac output from 5.2 +/- 3.0 to 6.8 +/- 2.8 L/min, as a consequence of increases in stroke volume from 31.9 +/- 15.3 to 49.2 +/- 13.9 mL (p less than .01) and in left ventricular stroke work from 32.1 +/- 20.0 to 57.4 +/- 32.1 g.m (p less than .05) . CONCLUSIONS: During experimental septic shock, alinidine administration can reverse dobutamine-induced tachycardia and simultaneously improve ventricular function. Plant Mol Biol, 1992 Aug, 19(5), 877 - 80 Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942; Scanlan DJ et al.; The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment . Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli. Plant Mol Biol, 1992 Aug, 19(5), 745 - 57 Molecular analysis of the plant gene encoding cytosolic phosphoglucose isomerase; Thomas BR et al.; The gene encoding a cytosolic isozyme of phosphoglucose isomerase (PGI, EC 5.3.1.9) was isolated from Clarkia lewisii, a wild flower native to California, and the structure and sequence of the entire coding region determined . PGI catalyzes an essential step in glycolysis and carbohydrate biosynthesis in plants . Spanning about 6 kb, the gene has 23 exons and 22 introns, the highest number yet reported in plants . The exons range in size from 43 to 156 nt and encode a protein of 569 amino acids . The protein is about 44-46% identical to the inferred protein sequences of pig, Escherichia coli and Saccharomyces cerevisiae . All of the introns are bordered with the consensus 5'-GT...AG-3' dinucleotides . The longest intron includes a large stem-loop structure bounded by a perfect 9 nt direct repeat . We cloned the PGI gene from a genomic library prepared from a single plant of known PGI genotype . The locus and allele of the clone were identified by matching restriction fragments to fragments from genetically defined genomic DNAs by Southern hybridization. Surgery, 1992 Aug, 112(2), 333 - 9; discussion 339-40 Retroviral-mediated gene transfer in human hepatocytes; Raper SE et al.; BACKGROUND . The ability to modify human hepatocytes genetically is an essential first step in the development of liver-directed ex vivo gene therapy for inherited metabolic disease . The purpose of these studies was to prove that the genome of human hepatocytes can be altered successfully to express foreign genetic material . METHODS . Human hepatocytes were plated at 2 or 4 x 10(6) cells/10 cm Primaria (Falcon, Oxnard, Calif.) plates . Fresh virus from the amphotropic viral producer cell line BAG, containing the Escherichia coli beta-galactosidase gene lacZ, was placed directly onto hepatocyte cultures and quantitative analysis of cells staining positive for the lacZ gene was undertaken . In a different human liver, a variety of viruses from producer cell lines containing clones of the human low-density lipoprotein (LDL) receptor were plated directly on cultures of human hepatocytes, and gene transfer was demonstrated by increased uptake of fluorescent-labeled LDL . RESULTS . Beta-galactosidase production in hepatocytes was assayed histochemically with the chromogenic substrate X-gal . The highest percentage of cells staining positive for expression of enzyme was seen at 4 x 10(6) cells/plate (43.66% +/- 1.02% vs 27.99% +/- 2.31%) . Gene transfer was also documented by the uptake of fluorescent-labeled LDL with a variety of different vectors containing the human LDL receptor . CONCLUSIONS . (1) Human hepatocytes can be cultured in vitro and are susceptible to retroviral infection, (2) functional gene transfer is demonstrated by intracellular function of foreign genes, and (3) the level of expression appears dependent on plating density . We conclude that human hepatocytes are suitable targets for genetic manipulation and may play an important role in human gene therapy trials. J Cell Biol, 1992 Aug, 118(3), 573 - 84 Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro; Wille H et al.; Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood . Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli . All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking . The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding . One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores . Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue . A similar self-assembly occurs with the chemically cross-linked dimers . In both cases there is no need for phosphorylation of the protein. Infect Immun, 1992 Aug, 60(8), 3464 - 7 Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation; Karch H et al.; Forty-five consecutive patients with various gastrointestinal disorders were identified as having Shiga-like toxin (SLT)-producing Escherichia coli infections . This was shown by the cytotoxic effect of stool extracts in Vero cell cultures which was neutralizable by antibodies to SLTs and by isolation of E . coli that hybridized with DNA probes complementary to SLT-I and SLT-II sequences . When we tested the same strains for SLT genes after subcultivation, the isolates from 15 patients became negative by colony hybridization and polymerase chain reaction and failed to produce SLTs . The instability of SLT genes warrants direct screening methods for clinical material and the development of new culture methods to prevent the loss of SLT genes. Infect Immun, 1992 Aug, 60(8), 3376 - 80 Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle; Mainil JG et al.; Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle . The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins . The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY) . The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate . The sizes of most of these virulence plasmids were from 65 to 95 MDa . The F41 probe failed to hybridize with any plasmid band . The virulence plasmids had multireplicon types typical of plasmids of the IncF groups . The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association. Infect Immun, 1992 Aug, 60(8), 3309 - 14 Biological activity of synthetic phosphonooxyethyl analogs of lipid A and lipid A partial structures; Ulmer AJ et al.; We investigated the biological activity of four new synthetic analogs of lipid A, termed PE-1, PE-2, PE-3, and PE-4 . All compounds contain an alpha-oxyethyl-linked (-O-CH2-CH2-) phosphoryl group in position 1 of the reducing glucosaminyl residue (GlcN I) of lipid A . PE-1 is a hexaacylated analog of Escherichia coli lipid A (compound 506) . PE-2 differs from PE-1 in carrying two myristic acid residues at GlcN I . PE-3 has the same acylation pattern as PE-2, but GlcN I is present in the beta anomeric form . Finally, PE-4 represents an analog of tetraacyl precursor Ia (compound 406) . Structure-activity relationships of these compounds were determined by measuring their capacity to induce tumor necrosis factor alpha, interleukin 1, and interleukin 6 release by human mononuclear cells and to cause mitogenicity of murine spleen cells . The results show that replacement of the glycosidic phosphoryl residue by a phosphonooxyethyl group had no substantial effect on the biological activity of compounds . However, the anomeric configuration of GlcN I was found to be of great biological relevance, as, in general, the alpha anomer (PE-2) expressed high activity, and the beta anomer (PE-3) expressed low mediator-inducing and mitogenic activity . The absence of the 3-hydroxyl groups within the acyl residues at GlcN I in PE-2 was found to only slightly affect the induction of monokines in human mononuclear cells compared with that of PE-1 or lipid A (506) . These stable 1-phosphonooxyethyl analogs of lipid A may be candidates in the development of immunomodulators for the treatment of systemic endotoxicosis. Infect Immun, 1992 Aug, 60(8), 3253 - 61 Characterization of two genes encoding distinct transferrin-binding proteins in different Actinobacillus pleuropneumoniae isolates; Gerlach GF et al.; The gene encoding the Actinobacillus pleuropneumoniae serotype 1 transferrin-binding protein (tfbA) was cloned, and the carboxy-terminal 70% of the protein was expressed as an aggregate protein in Escherichia coli . The nucleotide sequences of the tfbA genes from A . pleuropneumoniae serotypes 7 (G.-F . Gerlach, C . Anderson, A . A . Potter, S . Klashinsky, and P . J . Willson, Infect . Immun . 60:892-898, 1992) and 1 were determined, and a comparison revealed that they had 65% sequence identity . The deduced amino acid sequences showed a sequence agreement of 55%, and both proteins possessed a lipoprotein-like signal sequence . The serotype 1 TfbA protein had a predicted molecular mass of 65 kDa, compared with 60 kDa for the serotype 7 TfbA protein, and both proteins were immunologically distinct as assessed in a competitive enzyme-linked immunosorbent assay . Southern hybridization and Western blot (immunoblot) analysis of the 13 A . pleuropneumoniae type strains revealed that serotypes 2, 3, 4, 8, 9, 10, and 11 encode and express a TfbA protein highly homologous to that of A . pleuropneumoniae serotype 7 whereas the TfbA proteins and the encoding genes of serotypes 6 and 12 were highly homologous to that found in A . pleuropneumoniae serotype 1 . The tfbA genes of A . pleuropneumoniae serotypes 5A and 5B were recognized, under medium-stringency hybridization conditions, by the A . pleuropneumoniae serotype 1-derived tfbA probe, and the respective proteins were weakly reactive with the antibody raised against the A . pleuropneumoniae serotype 7 TfbA protein. Infect Immun, 1992 Aug, 60(8), 3143 - 9 Antibody responses to the chlamydial heat shock proteins hsp60 and hsp70 are H-2 linked; Zhong G et al.; The effects of both H-2 and non-H-2 genes on antibody responses to two Chlamydia trachomatis heat shock proteins (hsp60 and hsp70) were investigated . These chlamydial proteins are homologs of Escherichia coli GroEL (hsp60) and DnaK (hsp70) and are highly sequence conserved between bacterial and mammalian sources . Antibody responses among 17 different strains of mice immunized with C . trachomatis serovar B and serovar C elementary bodies were evaluated by immunoblot, radioimmunoprecipitation and enzyme-linked immunosorbent assay . Antibody responses to the two proteins displayed host genetic restriction . Of six distinctive H-2 haplotypes, only H-2d generated high antibody responses to hsp70 . Five of the six H-2 haplotypes, i.e., H-2a, H-2d, H-2k, H-2q, and H-2s, produced high antibody responses to hsp60 . Only the H-2b-bearing strain had low antibody responses to hsp60 . By using congenic and H-2 recombinant strains, the genes responsible for regulating antibody responses to hsp70 and hsp60 were mapped to the K-IA region of the H-2 locus . In F1 hybrid crosses between high and low responders, high responses to hsp60 and hsp70 were dominant traits . Other genes outside the H-2 locus also influenced antibody responses to hsp60 and hsp70, since inbred strains of identical H-2 but different background genes displayed variable antibody responses to the proteins . The genetic control of murine immune responses to C . trachomatis hsp60, a putative chlamydial immunopathologic antigen, suggests that a similar genetic mechanism may also exist in humans, and this observation may help to explain the observed variability in the spectrum of chlamydial diseases seen in humans. Infect Immun, 1992 Aug, 60(8), 3136 - 42 Monoclonal antibodies specific for elongation factor Tu and complete nucleotide sequence of the tuf gene in Mycobacterium tuberculosis; Carlin NI et al.; Monoclonal antibodies against mycobacterial antigens were produced by immunizing LOU/C rats with live Mycobacterium bovis BCG . The antibodies were characterized by an enzyme-linked immunosorbent assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting (immunoblotting) . One antibody, MAMB 2, reactive with a 47-kDa protein was used to screen a lambda gt11 M . tuberculosis gene library (R . A . Young, B . R . Bloom, C . M . Grosskinsky, J . Ivanji, D . Thomas, and R . W . Davis, Proc . Natl . Acad . Sci . USA 82:2583-2587, 1985) . Three recombinant phages reactive with MAMB 2 in plaque lysates were isolated, and part of the insert was sequenced . The mycobacterial inserts were all expressed as proteins fused with beta-galactosidase when the phages were induced as lysogens in Escherichia coli . The entire M . tuberculosis tuf gene was obtained by screening the lambda gt11 library with a DNA probe specific for the primary clones . A phage isolated from this screening was able to express the native protein in E . coli when introduced as a lysogen . A comparison of the entire gene sequence and the deduced protein sequence with the EMBL DNA and Swiss-Prot protein data libraries revealed strong homologies with elongation factors of bacteria, yeast mitochondria, and a plant chloroplast. Infect Immun, 1992 Aug, 60(8), 3117 - 21 Comparison of susceptibility of inbred and outbred infant mice to Escherichia coli heat-stable enterotoxin STa; Bertin A; Comparison of the susceptibility of outbred OF1 and inbred BALB/c, C57BL/6, DBA/2, and CBA mice to heat-stable toxin (STa) of enterotoxigenic Escherichia coli was made at different levels of induced secretion . STa was able to elicit fluid accumulation into the intestine of each strain of mice; however, quantitatively different results were obtained . Results were as usual expressed by gut weight/remaining body weight ratios . Fluid accumulation weight and fluid accumulation weight/remaining body weight ratios were also estimated . Values obtained for BALB/c and OF1 mice were never significantly different, but values for OF1 mice were significantly higher than those for DBA and C57BL/6 mice at the highest concentrations of toxin (toxin dilutions of 1/2, 1/4, and 1/5) . At the highest toxin concentration, gut weight/remaining body weight ratio in C57BL/6 mice was significantly lower than that for every other strain, but the fluid accumulation value obtained for DBA mice did not differ from that for C57BL/6 mice . Fluid accumulation values for DBA mice were also significantly lower at toxin dilutions of 1/5 and 1/8 than those for every other strain, and this was also the case when estimating the fluid accumulation weight/remaining body weight ratio at a dilution of 1/8 . Although the intestine of each strain of mice was able to respond to STa by fluid accumulation, differences in susceptibility of the STa receptor could exist and make DBA mice more resistant to enterotoxigenic E . coli diarrhea. Infect Immun, 1992 Aug, 60(8), 3059 - 64 Functional analysis of the Ca(2+)-regulated hemolysin I operon of Actinobacillus pleuropneumoniae serotype 1; Gygi D et al.; The genetic determinant encoding the synthesis and secretion of hemolysin I (HlyI; gene designation, hlyI) by Actinobacillus pleuropneumoniae serotype 1 4074T was cloned in the lambda vector EMBL4 . A 10.2-kb fragment that encoded hemolytic activity in the phage lysate was aligned by Southern blot hybridization to genes hlyC, hlyA, hlyB, and hlyD of the Escherichia coli hemolysin operon, and expression of the A . pleuropneumoniae genes in E . coli revealed that they have the same functions as their E . coli analogs: hlyIC encodes a protein that activates inactive 105-kDa prohemolysin I (encoded by hlyIA) to active hemolysin I, while hlyIB and hlyID are necessary for HlyIA secretion . Northern (RNA) hybridization of A . pleuropneumoniae RNA revealed that the gene cluster is transcribed as two RNA species, a major one of 3.5 kb, corresponding to hlyICA, and a second, minor one of 7.5 kb, corresponding to the whole operon, hlyICABD . The level of hlyI mRNA was substantially higher in A . pleuropneumoniae 4074T cells grown in the presence of Ca2+, supporting the view that the expression of the hlyI determinant is Ca2+ regulated . Parallel RNA hybridization with random gene probes suggested that this Ca2+ regulation is specific for the hlyI determinant. EMBO J, 1992 Aug, 11(8), 3039 - 44 Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix; van de Wetering M et al.; The high mobility group I (HMG) box is proposed to mediate DNA binding in a novel group of transcription-regulating proteins . Two of these, the proteins encoded by the T cell-specific TCF-1 and the mammalian sex-determining gene SRY, carry a single HMG box with specificity for the heptamer motif A/T A/T C A A A G . We have now analysed the mode of interaction of the HMG boxes of TCF-1 and SRY with this motif . Methylation interference footprinting revealed that both HMG boxes contacted adenines on both strands in the minor groove, whereas no major groove guanine contacts were discerned . Diethylpyrocarbonate (DEPC) carbethoxylation interference footprinting of TCF-1 indicated the absence of major groove contacts on positions 5, 6 and 7 of the motif . Carbethoxylation interference was observed, however, on positions 2, 3 and 4 and to a lesser extent on position 1 in the major groove . Combined T----C and A----I substitution, which changes the surface of the major groove but leaves the minor groove intact, did not interfere with sequence-specific binding by TCF-1 and SRY . These observations indicate that recognition of the heptamer motif by the HMG boxes of the distantly related TCF-1 and SRY proteins predominantly occurs through nucleotide contacts in the minor groove. EMBO J, 1992 Aug, 11(8), 3021 - 30 The DNA target of the trp repressor; Haran TE et al.; Unexpected features seen by high resolution X-ray crystallography at the interface of the trp repressor and the 'traditional' trp operator provoked the claim that the DNA fragment used in the crystal structure is not the true operator, and therefore that the crystal structure of the trp repressor-operator complex does not portray a specific interaction . An alternative sequence was proposed mainly on the basis of mutational studies and gel retardation analysis of short target duplexes (Staacke et al., 1990a,b) . We have reexamined the sequence consensus in trpR-repressible promoters and analyzed the mutagenesis experiments of others including Staacke et al . (1990a) and found them fully consistent with the interactions of the traditional operator sequence seen in the crystal structure, and stereochemically inconsistent with the above referenced alternative model . Moreover, an in vitro trp repressor-DNA binding analysis, employing both novel DNA constructs devised to avoid previously encountered artifacts as well as full-length promoter sequences, indicates that the traditional operator used in the crystal structure is the preferred target of the trp repressor. EMBO J, 1992 Aug, 11(8), 2925 - 33 Activation of mammalian DNA ligase I through phosphorylation by casein kinase II; Prigent C et al.; Mammalian DNA ligase I has been shown to be a phosphoprotein . Dephosphorylation of purified DNA ligase I causes inactivation, an effect dependent on the presence of the N-terminal region of the protein . Expression of full-length human DNA ligase I in Escherichia coli yielded soluble but catalytically inactive enzyme whereas an N-terminally truncated form expressed activity . Incubation of the full-length preparation from E . coli with purified casein kinase II (CKII) resulted in phosphorylation of the N-terminal region and was accompanied by activation of the DNA ligase . Of a variety of purified protein kinases tested, only CKII stimulated the activity of calf thymus DNA ligase I . Tryptic phosphopeptide analysis of DNA ligase I revealed that CKII specifically phosphorylated a major peptide also apparently phosphorylated in cells, implying that CKII is a protein kinase acting on DNA ligase I in the cell nucleus . These data suggest that DNA ligase I is negatively regulated by its N-terminal region and that this inhibition can be relieved by post-translational modification. Cancer Res, 1992 Aug 1, 52(15), 4183 - 9 Gene- and strand-specific damage and repair in Chinese hamster ovary cells treated with 4-nitroquinoline 1-oxide; Snyderwine EG et al.; 4-Nitroquinoline 1-oxide (4NQO) is a model chemical carcinogen that has often been referred to as a UV mimetic agent . Previous studies have indicated that UV-induced pyrimidine dimers are repaired preferentially and strand-specifically in actively transcribing genes . In the current study we have examined the gene-specific and strand-specific repair of 4NQO in Chinese hamster ovary B-11 cells treated with 2.5 microM 4NQO . The methodology used for detecting adducts involved the treatment of DNA from 4NQO-exposed cells with uvrABC excinuclease, which incises DNA at adduct sites, followed by denaturing gel electrophoresis of DNA, Southern hybridization, and probing for the sequence of interest . We examined the active and inactive coding regions of the DHFR gene, the active adenine phosphoribosyltransferase gene, relatively inactive c-fos oncogene, and the mitochondrial genome for 4NQO adducts . Initial 4NQO adduct levels found in these genes varied from 1.10 to 1.52 adducts/10 kilobases . Little difference in repair was found between active coding and inactive regions of the DHFR gene, or between DHFR, adenine phosphoribosyltransferase, and c-fos genes, which are transcribed at different levels . Approximately 71% of 4NQO adducts were repaired within 24 h in all gene sequences examined . During this same time period, approximately 51% of adducts were repaired from the genome overall, as determined by comparing the removal of bound radiolabeled 4NQO to total DNA . The results indicate that 4NQO adducts, unlike UV light-induced cyclobutane pyrimidine dimers (UV dimers), are not preferentially repaired in transcriptionally active genes . However, there may be regions of the genome that are not repaired with the same efficiency as the specific genes examined here . In addition, little to no difference was observed in the repair of 4NQO adducts in the transcribed and nontranscribed strands of the DHFR gene, a finding which is also in contrast to results with UV dimers . Interestingly, 4NQO adducts, unlike UV dimers, were removed from the mitochondrial genome, suggesting that repair of select lesions occurs in this organelle . Thus, there appear to be some differences in the repair pathways operating for 4NQO adducts and UV dimers, particularly with respect to gene- and strand-specific DNA repair. Arch Biochem Biophys, 1992 Aug 1, 296(2), 489 - 96 Indole protects tryptophan indole-lyase, but not tryptophan synthase, from inactivation by trifluoroalanine; Phillips RS et al.; Trifluoroalanine is a mechanism-based inactivator of Escherichia coli tryptophan indole-lyase (tryptophanase) and E . coli tryptophan synthase (R . B . Silverman and R . H . Abeles, 1976, Biochemistry 15, 4718-4723) . We have found that indole is able to prevent inactivation of tryptophan indole-lyase by trifluoroalanine . The protection of tryptophan indole-lyase by indole exhibits saturation kinetics, with a KD of 0.03 mM, which is comparable to the KI for inhibition of pyruvate ion formation (0.01 mM) and the Km for L-tryptophan synthesis . Fluoride electrode measurements indicate the formation of 28 mol of fluoride ion per mole of enzyme during inactivation of tryptophan indole-lyase, and 121 mol of fluoride ion are formed per mole of enzyme in the presence of 2 mM indole during the same incubation period . 19F NMR spectra of reaction mixtures of tryptophan indole-lyase and trifluoroalanine showed evidence only for fluoride ion formation, in either the absence or the presence of indole, and difluoropyruvic acid was not detected . The partition ratio, kcat/kinact, is estimated to be 9 . Tryptophan indole-lyase in the presence of trifluoroalanine exhibits visible absorption peaks at 446 and 478 nm, which decay at the same rate as inactivation . However, in the presence of 1 mM indole and trifluoralanine, tryptophan indole-lyase exhibits a peak only at 420 nm, and the spectra show a gradual increase at 300-310 nm with incubation . In contrast, tryptophan synthase is not protected by indole from inactivation by trifluoroalanine, and the absorption peak at 408 nm for the tryptophan synthase-trifluoroalanine complex is unaffected by indole . These results demonstrate that inactivation of tryptophan indole-lyase occurs via a catalytically competent species, probably the beta,beta-difluoro-alpha-aminoacrylate intermediate, which can be partitioned from inactivation to products by a reactive aromatic nucleophile, indole. Mol Cell Biol, 1992 Aug, 12(8), 3337 - 45 RCC1, a regulator of mitosis, is essential for DNA replication; Dasso M et al.; Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase . We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants . By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA . Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery . By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication. J Virol, 1992 Aug, 66(8), 4874 - 83 Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro; Ehrlich LS et al.; The capsid protein (CA) (p24) of human immunodeficiency virus (HIV) type 1 expressed in Escherichia coli and purified to greater than 90% homogeneity was used to examine assembly in vitro and to probe the nature of interactions involved in the formation of capsid structures . The protein was detected in dimeric and oligomeric forms as indicated by molecular size measurements by gel filtration column chromatography, sedimentation through sucrose, and nondenaturing gel electrophoresis . Chemical cross-linking of CA molecules was observed with several homobifunctional reagents . Oligomer size was dependent on cross-linker concentration and exhibited a nonrandom pattern in which dimers and tetramers were more abundant than trimers and pentamers . Oligomers as large as dodecamers were detected in native polyacrylamide gels . These were stable in solutions of high ionic strength or in the presence of nonionic detergent, indicating that strong interactions were involved in oligomer stabilization . Limited tryptic digestion converted the putative dodecamers to octamers, suggesting that a region involved in CA protein multimerization was exposed in the structure . This region was mapped to the central portion of the protein . The recombinant CA proteins assembled in vitro into long rodlike structures and were disassembled into small irregular spheres by alterations in ionic strength and pH . The observation that assembly and disassembly of purified HIV type 1 CA protein can be induced in vitro suggests an approach for identifying possible control mechanisms involved in HIV viral core assembly. J Med Microbiol, 1992 Aug, 37(2), 141 - 7 Plasmid content and localisation of the STaI (STaP) gene in enterotoxigenic Escherichia coli with a non-radioactive polynucleotide gene probe; Bertin A; Enterotoxigenic Escherichia coli (ETEC) strain P2200 of porcine origin possessed eight possibly plasmid-determined characters (K88+ Raf+ Hly+ Col+ Smr Tcr Su(r) STa+) and six plasmid DNA bands of 4.2-93 kb . Analysis of the spontaneous loss of characters and the results of matings with other E . coli strains revealed that the K88, Raf, Hly, Smr, Tcr and Su(r) characters could be transferred, and that the presence of the K88 and Raf characters was associated with an 83-kb plasmid . The presence and location of the STaI gene was investigated in several ETEC strains of bovine or porcine origin . Hybridisation with a non-radioactive polynucleotide probe associated the STaI gene with a plasmid in each strain; these plasmids were of 32-142 kb . In contrast, plasmids from a P2200 STa- variant and plasmids from two STa- variants of the bovine ETEC strain B41* (strain B41 obtained from a different source) did not hybridise with the probe . One of the B41*STa- variants had lost the STa plasmid, whereas the second variant retained a plasmid of the same size which did not hybridise . In contrast, a third B41*STa- variant retained a plasmid of the same size that still hybridised with the STaI probe . Plasmid DNA restriction fragment analysis, followed by hybridisation with the STaI probe, showed that the STaI gene was associated with 8.3-, 6.8- and 3.5-kb plasmid fragments in strain B41, and with 4.9-, 6.8- and 3.5-kb plasmid fragments in strain B41*, following digestion with EcoRI, BamHI, or EcoRI + BamHI, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1992 Aug, 174(15), 5165 - 7 Construction and characterization of Streptomyces coelicolor A3(2) mutants that are multiply deficient in the nonessential hrd-encoded RNA polymerase sigma factors; Buttner MJ et al.; Previous studies showed that Streptomyces coelicolor A3(2) has four genes (hrdA, hrdB, hrdC, and hrdD) that appear to encode RNA polymerase sigma factors very similar to the sigma 70 subunit of Escherichia coli and that hrdC and hrdD could be individually disrupted without causing obvious phenotypic defects . Here, hrdA was cloned and stable null hrdA and hrdD mutants were constructed by gene replacement . These two mutants and a previously constructed hrdC null mutant were used in crosses to generate hrdAC, hrdAD, hrdCD, and hrdACD strains . The inability to synthesize one, two, or all three of the nonessential hrd-encoded sigma factors had no obvious phenotypic consequences. J Bacteriol, 1992 Aug, 174(15), 5127 - 31 Homology among Escherichia coli K1 and K92 polysialytransferases; Vimr ER et al.; The neuS-encoded polysialytransferase (polyST) in Escherichia coli K1 catalyzes synthesis of polysialic acid homopolymers composed of unbranched sialyl alpha 2,8 linkages . Subcloning and complementation experiments showed that the K1 neuS was functionally interchangeable with the neuS from E . coli K92 (S . M . Steenbergen, T . J . Wrona, and E . R . Vimr, J . Bacteriol . 174:1099-1108, 1992), which synthesizes polysialic acid capsules with alternating sialyl alpha 2,8-2,9 linkages . To better understand the relationship between these polySTs, the complete K92 neuS sequence was determined . The results demonstrated that K1 and K92 neuS genes are homologous and indicated that the K92 copy may have evolved from its K1 homolog . Both K1 and K92 structural genes comprised 1,227 bp . There were 156 (12.7%) differences between the two sequences; among these mutations, 55 did not affect the derived primary structure of K92 polyST and hence were synonymous with the K1 sequence . Assuming maximum parsimony, another estimated 17 synonymous mutations plus 84 nonsynonymous mutations could account for the 70 amino acid replacements in K92 polyST; 36 of these replacements were judged to be conservative when compared with those of K1 polyST . There were no changes detected in the first 146 5' or last 129 3' bp of either gene, suggesting, in addition to the observed mutational differences, the possibility of a past recombination event between neuS loci of two different kps clusters . The results indicate that relatively few amino acid changes can account for the evolution of a glycosyltransferase with novel linkage specificity. J Bacteriol, 1992 Aug, 174(15), 5110 - 6 Interaction of LexA repressor with the asymmetric dinG operator and complete nucleotide sequence of the gene; Lewis LK et al.; The dinG gene was originally isolated during a search for Escherichia coli promoters which are components of the SOS regulon . The regulatory region of this gene contains a potential binding site for LexA repressor which is quite different from other known sites . All previously described chromosomal LexA operators are imperfect palindromes containing the sequence CTG(N10)CAG . The noncanonical dinG sequence breaks the symmetry and takes the form TTG(N10)CAG . In the present study, a search for mutations within dinGop::galK fusion plasmids which render transcription independent of intracellular levels of LexA has yielded mutations only within this 16-bp sequence . Electrophoretic mobility shift assays performed with purified mutant and wild-type operator fragments revealed that the affinity of LexA for each of the mutant sites is greatly reduced compared with that of the wild type . One of the mutants contained an alteration in the putative promoter of dinG which increased the similarity of the -35 region to the consensus sequence (TTGGCT----TTGACT); the apparent promoter activity of this construct was subsequently found to be approximately eight times higher than that of the wild type in vivo . Additional experiments have established the complete nucleotide sequence of the dinG gene . A long open reading frame located immediately downstream of the asymmetric operator segment which could potentially encode a 72.9-kDa DinG protein was identified. J Bacteriol, 1992 Aug, 174(15), 5057 - 62 Menaquinone (vitamin K2) biosynthesis: nucleotide sequence and expression of the menB gene from Escherichia coli; Sharma V et al.; In Escherichia coli, the biosynthesis of the electron carrier menaquinone (vitamin K2) involves at least seven identified enzymes . One of these, naphthoate synthase, forms the bicyclic ring system by catalyzing the conversion of o-succinylbenzoyl-coenzyme A to 1,4-dihydroxy-2-naphthoic acid . The gene for this enzyme has been previously identified as menB . By genetic and biochemical tests, a 1.349-kb DNA fragment from the E . coli men locus complements menB mutants . This fragment contains a single 285-codon open reading frame (ORF) . Recombinant plasmids containing deletions of either the amino or the carboxy region of the ORF fail to complement the mutants . The ORF is preceded by consensus sequences for a ribosomal binding site and a sigma 70 promoter . menB transcription sufficient to complement the menB mutant in vivo and in vitro can be initiated from the identified putative promoter, and that in the constructs, menB expression, can be made independent of read-through transcription from the lac promoter . However, multicopy plasmids containing menB fail to generate the expected levels of enzymatic activity. J Bacteriol, 1992 Aug, 174(15), 5005 - 12 A mutant sigma 32 with a small deletion in conserved region 3 of sigma has reduced affinity for core RNA polymerase; Zhou YN et al.; sigma 70, encoded by rpoD, is the major sigma factor in Escherichia coli . rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant sigma 70 is rapidly degraded at high temperature . Extragenic mutations which reduce the rate of degradation of RpoD285 sigma 70 permit growth at high temperature . One class of such suppressors is located in rpoH, the gene encoding sigma 32, an alternative sigma factor required for transcription of the heat shock genes . One of these, rpoH113, is incompatible with rpoD+ . We determined the mechanism of incompatibility . Although RpoH113 sigma 32 continues to be made when wild-type sigma 70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters . Glycerol gradient fractionation of sigma 32 into the holoenzyme and free sigma suggests that RpoH113 sigma 32 has a lower binding affinity for core RNA polymerase than does wild-type sigma 32 . The presence of wild-type sigma 70 exacerbates this defect . We suggest that the reduced ability of RpoH113 sigma 32 to compete with wild-type sigma 70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+ . The rpoH113 cells would have reduced amounts of sigma 32 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability. J Bacteriol, 1992 Aug, 174(15), 4935 - 42 Localization of upstream sequence elements required for nitrate and anaerobic induction of fdn (formate dehydrogenase-N) operon expression in Escherichia coli K-12; Li J et al.; Two transcriptional activators, the FNR and NARL proteins, are required for induction of the fdnGHI operon, encoding Escherichia coli formate dehydrogenase-N . The FNR protein is required for anaerobic expression, while the NARL protein mediates nitrate induction . We used primer extension to locate the transcription initiation site 29 nucleotides upstream of the fdnG translation initiation codon . Expression assays with single-copy phi (fdnG-lacZ) gene fusions containing various deletions in the fdn 5'-regulatory region delimited three distinct cis-acting elements . One site, which is located at approximately -110, was required for nitrate induction . Two other sites share sequence similarity with the FNR protein binding site core consensus . The first site, centered at -42.5, was required for anaerobic induction . We used site-specific mutagenesis to change this putative FNR protein binding site into the CRP protein binding site core consensus . This change caused the fdn operon to be expressed aerobically, subject to CRP protein control . On the other hand, converting this putative FNR protein binding site into the FNR protein binding site core consensus resulted in elevated anaerobic induction of the fdn operon and also caused weak aerobic expression . The other putative FNR protein binding site, centered at -97.5, was not involved in anaerobic induction . It might play a negative role in fdn operon expression during anaerobic growth in the absence of nitrate. J Bacteriol, 1992 Aug, 174(15), 4871 - 7 Multiple transcripts encoded by the ilvGMEDA gene cluster of Escherichia coli K-12; Huang F et al.; We report here that, using Northern (RNA) blots, we identified two relatively stable transcripts of 4.6 and 1.1 kb that correspond to the products of the ilvEDA and ilvE genes and two relatively unstable transcripts of 6.7 and 3.6 kb that correspond to the products of the ilvGMEDA and ilvDA genes . The transcripts were identified by the use of eight probes derived from segments of the ilvGMEDA cluster . In addition, we used two strains with deletions of ilvG or ilvDA and observed the expected decrease in transcript size in Northern blots . Primer extension with reverse transcriptase generated a 169-nucleotide product corresponding to a 5' end within the ilvED intercistronic region, 37 nucleotides from the AUG codon of the ilvD gene . This primer extension product presumably indicates the 5' end of the ilvDA transcript that we detected in Northern blots . The stability of the transcripts was monitored, and RNase E was found to play a major role in ilv transcript degradation . Transcript levels varied in response to growth in the presence of the end product amino acids and in response to the presence of the polar frameshift site in ilvG . Although there have been speculations about the identities and numbers of transcripts derived from the ilvGMEDA cluster on the basis of the identification of some of the sites of transcription initiation and termination, this is the first report of the use of Northern blots to determine the actual sizes and distribution of mRNAs present in vivo. J Gen Microbiol, 1992 Aug, 138 ( Pt 8), 1751 - 7 Peptidoglycan biosynthesis in Escherichia coli: variations in the metabolism of alanine and D-alanyl-D-alanine; de Roubin MR et al.; The in vivo functioning of the alanine/D-alanyl-D-alanine pathway of Escherichia coli was investigated by determining precursor pool levels and specific enzyme activities under various growth conditions . Cells grown on D- or L-alanine showed several remarkable features compared with cells grown on other carbon sources: 10-fold higher values of the D-alanyl-D-alanine and the UDP-MurNAc-pentapeptide pools, a 240-fold increase of the alanine racemase activity, and the absence of bacteriolysis after treatment with D-cycloserine at high concentrations (50 micrograms ml-1) . In cells grown on glucose, D-cycloserine (1 micrograms ml-1) led to depletion of the D-alanyl-D-alanine pool and to lysis, which was efficiently antagonized by chloramphenicol . A threefold increase of the dipeptide pool was observed when cells were treated with chloramphenicol alone . The alanine racemase activity was lowest in glucose-grown cells and the D-alanine:D-alanine ligase and D-alanyl-D-alanine-adding activities were the same whatever the carbon source . Molecular masses of 53-56 kDa and 56-60 kDa were estimated for the partially purified inducible alanine racemase and D-alanine:D-alanine ligase respectively. J Gen Microbiol, 1992 Aug, 138 ( Pt 8), 1697 - 705 Cloning, nucleotide sequence and heterologous expression of the protective outer-membrane protein P.68 pertactin from Bordetella bronchiseptica; Li J et al.; The prn gene encoding the 68 kDa protective outer-membrane protein of Bordetella bronchiseptica (P.68 pertactin) was cloned, sequenced and expressed in Escherichia coli . The gene was isolated by DNA:DNA hybridization experiments using a radioactively-labelled fragment of the homologous prn gene from Bordetella parapertussis . DNA sequence analysis reveals that the gene is capable of encoding a protein with a molecular mass of 93996 Da (P.94); this precursor molecule is processed to form the P.68 antigen on the surface of B . bronchiseptica . Heterologous expression of the full-length gene encoding P.94 in Escherichia coli results in similar processing, with the P.68 antigen targeted to the bacterial outer membrane . Comparison of P.94 with the P.93 and P.95 precursors, encoding homologous proteins from Bordetella pertussis and B . parapertussis, shows a high degree (greater than 90%) of homology . The major differences between all three proteins occur in the number of repeats of the two families (Gly-Gly-Xaa-Xaa-Pro)n and (Pro-Gln-Pro)n of reiterated sequence motifs. J Gen Microbiol, 1992 Aug, 138 ( Pt 8), 1593 - 8 Oxidation of L-thiazolidine-4-carboxylate by L-proline dehydrogenase in Escherichia coli; Deutch CE; L-Thiazolidine-4-carboxylate (T4C, gamma-thioproline) is a toxic analogue of L-proline . T4C can be oxidized by Escherichia coli to form N-formylcysteine, which is hydrolysed to yield formate and cysteine . To determine if L-proline dehydrogenase (EC 1.5.99.8) catalyses T4C degradation, membrane fractions from E . coli were tested for T4C and proline oxidation activity . The specific activity for T4C oxidation in membranes from bacteria grown with 10 mM-proline was similar to the specific activity for proline oxidation and about 100 times that in membranes from bacteria grown without proline . Both oxidation activities were inactivated at 45 degrees C at the same rate . Membranes from a strain with a deletion of the putA gene encoding L-proline dehydrogenase or a strain with a putA::Tn5 insertion mutation had no detectable activity with either substrate . Although T4C was a simple competitive inhibitor of proline oxidation, proline inhibited T4C oxidation in a way that gave competitive but sigmoidal kinetics . At low concentrations, T4C induced proline dehydrogenase synthesis . Cysteine auxotrophs containing the putA::Tn5 mutation could still use T4C as a cysteine source, and bacteria with this mutation consumed oxygen in the presence of T4C at half the control rate . These results indicate that T4C is a substrate and an inducer of L-proline dehydrogenase but suggest that E . coli also contains a second enzyme catalysing T4C degradation. FEMS Microbiol Immunol, 1992 Aug, 4(6), 317 - 22 Interleukin-6 is a better marker of lethality than tumor necrosis factor in endotoxin treated mice; Kelly NM et al.; We established a mouse model to differentiate between a lethal and non-lethal presentation of endotoxic shock . The model involved injecting different amounts of Escherichia coli LPS into C3H/HeN mice which had been 'primed' with BCG . We found that the mice receiving non-lethal and lethal doses of LPS could not be differentiated in terms of their physical symptoms for the first 8 h post-injection . Tumour necrosis factor (TNF) was detected at concentrations 2-9-fold greater in mice receiving lethal doses of LPS when compared with non-lethally injected mice . However, given that (i) the successful detection of this differential was dependent on the time of sampling and (ii) that TNF was only detected in the first 3-4 h post LPS challenge, we suggest that TNF may not be very useful as a prognostic marker in endotoxic shock . In contrast, circulating IL-6 appeared to mirror the symptoms of the endotoxic mice . The relative disappearance of IL-6 after 10 h in the non-lethally injected mice corresponded with their symptomatic recovery, while IL-6 continued to circulate up to the time of death in the lethally injected mice . Furthermore, there appeared to be a good correlation between the levels of injected LPS and the levels of IL-6 induced into the circulation . Our results suggest that IL-6, rather than TNF, may serve as a prognostic marker for endotoxic shock. Thymus, 1992 Aug, 20(1), 63 - 7 The influence of thymus extracts on the chemotaxis of polymorphonuclear neutrophils (PMN) from patients with insulin-dependent diabetes mellitus (IDD); Wysocki J et al.; The main goal of this study was to evaluate the possible influence of thymus extracts on the chemotaxis of PMN isolated from diabetic patients . The analyzed group consisted of fifty patients with insulin-dependent diabetes mellitus (IDD), and twenty healthy adults were taken as controls . The results showed, that PMN isolated from patients with IDD manifested the impaired chemotaxis toward zymosan-activated plasma (ZAP), plasma incubated with cellophane and supernate from E . coli culture . Both thymus extracts did not change the directed migration toward control plasma and toward ZAP . THYMEX-L did improve the migration of diabetic PMN toward plasma incubated with cellophane, whereas TFX-THYMOMODULIN ameliorated the impaired chemotaxis toward supernate from E . coli culture . Both thymus extracts can attract PMN obtained from healthy donors, but not the cells isolated from diabetic patients. J Am Acad Dermatol, 1992 Aug, 27(2 Pt 2), 340 - 4 Toxic epidermal necrolysis in early infancy; Scully MC et al.; Toxic epidermal necrolysis (TEN) is a life-threatening bullous dermatosis characterized by the sudden onset of full-thickness epidermal necrosis . TEN is a disease of both children and adults, but TEN in early infancy is a rare event; only two well-documented cases in infants less than 6 months of age have been reported . We report a third case of a 6-week-old infant with Escherichia coli sepsis who received ampicillin and other antibiotics and subsequently developed TEN . Despite the withdrawal of ampicillin and aggressive systemic and wound care, the infant died . The infants in the other two reported cases also died, which suggests that TEN in early infancy has an extremely poor prognosis. Genetics, 1992 Aug, 131(4), 783 - 9 Mechanisms of directed mutation; Foster PL et al.; Spontaneous mutants arise among nondividing populations of Escherichia coli in apparent response to selective conditions . In this report we investigate several hypotheses to account for the role of selection in the production of these "directed" or "adaptive" mutations . We found that the Lac+ phenotypes of some mutants that arise late after lactose selection are due to suppressor mutations that are unlinked to the mutant lacZ allele; thus the production of these Lac+ mutants does not require an information flow from successful proteins back to the DNA that encodes them . Transcriptional induction of the lac operon, even in the presence of another, utilizable carbon source, did not stimulate the occurrence of Lac+ mutants in the absence of lactose, indicating that the role of the selective agent is not merely to induce transcription . The absence of two DNA repair pathways-methyl-directed mismatch repair and alkylation repair-also did not result in an accumulation of Lac+ mutants in the absence of lactose, suggesting that these repair pathways are not normally responsible for correcting transient variants that might arise in the absence of selection . However, in one case the Lac+ mutation is likely to be due to a miscoding lesion occurring on the nontranscribed DNA strand, indicating that, at least in this instance, DNA replication is required before directed mutations can arise. FEMS Microbiol Lett, 1992 Aug 1, 74(1), 105 - 8 OmpF channel permeability of quinolones and their comparison with beta-lactams; Sawai T et al.; The diffusion rates of nalidixic acid, ofloxacin and ofloxacin's two optically active isomers through OmpF channels were measured in proteoliposomes and compared with the rates of beta-lactams . The four quinolones showed high diffusion rates, exceeding that of cephaloridine and being comparable to imipenem . There was no significant difference in diffusion rate between nalidixic acid and ofloxacin, or between the two optically active isomers . The diffusion rates of enoxacin and norfloxacin were also estimated to be higher than many beta-lactams. J Vasc Interv Radiol, 1992 Aug, 3(3), 557 - 8 Combined retrograde-antegrade ureteral stent passage: salvage procedure for a ureteral leak; Gray RJ et al.; The authors describe a stent placement procedure for treatment of an infected ureteral leak after failure of traditional antegrade and retrograde approaches . In this procedure, a guide wire was placed across the distal ureteral segment into a urinoma with use of cystoscopic guidance . Thereafter, an antegrade approach was used to pass a wire loop snare, capture the guide wire, and withdraw it through the proximal ureter for subsequent stent passage . This approach allowed percutaneous stabilization of a ureteral leak in a patient who would have otherwise required immediate surgical repair. J Mol Endocrinol, 1992 Aug, 9(1), 83 - 92 Production and characterization of recombinant chicken insulin-like growth factor-I from Escherichia coli; Upton FZ et al.; Recombinant chicken insulin-like growth factor-I (cIGF-I) has been produced in Escherichia coli after first modifying a plasmid that coded for a human IGF-I (hIGF-I) fusion protein, in order to introduce codons for the eight amino acid substitutions . The cIGF-I fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography to remove proteinases, refolded and partially purified by reverse-phase high-performance liquid chromatography . The fusion protein was cleaved with hydroxylamine after which cIGF-I was purified to homogeneity by three additional chromatographic steps . Recombinant cIGF-I was equipotent with hIGF-I in cell culture bioassays of protein synthesis and breakdown using rat L6 myoblasts and chick embryo fibroblasts . Binding of radiolabelled cIGF-I and hIGF-I was also equivalent in the two cell lines, as was their binding in ligand blots of chicken, sheep and human plasma . The cross-reactivity of cIGF-I in a polyclonal hIGF-I radioimmunoassay was 60% of that observed with hIGF-I . The availability of recombinant cIGF-I will facilitate investigations into the role of IGF-I in chicken growth and development. Appl Environ Microbiol, 1992 Aug, 58(8), 2592 - 8 Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase; Nelms J et al.; The bifunctional enzyme chorismate mutase/prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which is encoded by the pheA gene of Escherichia coli K-12, is subject to strong feedback inhibition by L-phenylalanine . Inhibition of the prephenate dehydratase activity is almost complete at concentrations of L-phenylalanine greater than 1 mM . The pheA gene was cloned, and the promoter region was modified to enable constitutive expression of the gene on plasmid pJN302 . As a preliminary to sequence analysis, a small DNA insertion at codon 338 of the pheA gene unexpectedly resulted in a partial loss of prephenate dehydratase feedback inhibition . Four other mutations in the pheA gene were identified following nitrous acid treatment of pJN302 and selection of E . coli transformants that were resistant to the toxic phenylalanine analog beta-2-thienylalanine . Each of the four mutations was located within codons 304 to 310 of the pheA gene and generated either a substitution or an in-frame deletion . The mutations led to activation of both enzymatic activities at low phenylalanine concentrations, and three of the resulting enzyme variants displayed almost complete resistance to feedback inhibition of prephenate dehydratase by phenylalanine concentrations up to 200 mM . In all four cases the mutations mapped in a region of the enzyme that has not been implicated previously in feedback inhibition sensitivity of the enzyme. Am J Physiol, 1992 Aug, 263(2 Pt 1), E301 - 9 Differential effects of interleukin-1 and tumor necrosis factor on ketogenesis; Memon RA et al.; To determine the role of cytokines in mediating the decrease in ketones associated with infection, we studied the effect of endotoxin (LPS), interleukin-1 (IL-1), and tumor necrosis factor (TNF) on serum and hepatic ketone body levels (KB), serum free fatty acids (FFA), and hepatic malonyl-CoA levels . LPS decreased serum and hepatic KB in C57Bl/6 (LPS sensitive) mice, whereas it had little effect in C3H/HeJ (LPS resistant) mice, whose macrophages lack the ability to produce IL-1 and TNF in response to LPS, suggesting that IL-1 and TNF may mediate this effect . IL-1 and TNF decreased serum KB in both strains of mice . As seen with LPS, IL-1 decreased hepatic KB, whereas TNF had no such effect . LPS, IL-1, and TNF increased hepatic malonyl-CoA levels . TNF acutely raised serum FFA, whereas LPS and IL-1 did not . Postulating that the TNF-induced increase in FFA overrides the inhibitory effect of malonyl-CoA on fatty acid oxidation and ketogenesis, we used R-2-phenylisopropyladenosine to block TNF-induced lipolysis and demonstrated that in the absence of increased fatty acid flux, TNF inhibits KB formation . As seen with LPS, IL-1, but not TNF, decreased KB in the fasting state . These data suggest that IL-1 and TNF may mediate the antiketogenic effect of infection and that IL-1 has properties closest to that of LPS. Am J Vet Res, 1992 Aug, 53(8), 1298 - 301 Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro; Morris DD et al.; A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (IL-6) in vitro in response to endotoxin . Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages . Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5) . Culture medium supernatants were collected after 3, 6, 12, and 24 hours' incubation and were frozen at -70 C until assayed for IL-6 activity . Supernatant IL-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on IL-6 for survival . Results indicated that equine peritoneal macrophages produce IL-6 in vitro and that supernatant medium IL-6 activity was significantly (P less than 0.05) increased by exposure to endotoxin . Significant (P less than 0.05) time and treatment effects on macrophage IL-6 production were apparent . The IL-6 activity peaked at 6 or 12 hours' incubation, then remained high through 24 hours' incubation, regardless of endotoxin exposure . Medium IL-6 activity during 3 and 6 hours' incubation was significantly (P less than 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak IL-6 activity was similar among all endotoxin concentrations . Endotoxin concentration did not have an effect on medium IL-6 activity from macrophages exposed to endotoxin for 12 or 24 hours. Am J Vet Res, 1992 Aug, 53(8), 1285 - 9 Induction of the acute-phase cytokine, hepatocyte-stimulating factor/interleukin 6, in the circulation of horses treated with endotoxin; MacKay RJ et al.; Because hepatocyte-stimulating factor/interleukin 6 (IL-6) is the principal inducer of acute-phase protein synthesis in the liver, quantification of its activity in blood provides an early and sensitive assessment of the acute-phase response . Circulating IL-6 activity was monitored in 4 adult horses for 72 hours after IV administration of endotoxin . In 4 experiments performed at weekly intervals and in randomized order, each horse was given endotoxin--1,000 30, 1, and 0 ng/kg of body weight . Plasma IL-6 activity was quantified as the ability to promote growth of the IL-6-dependent B-cell hybridoma, B13.29 clone B9 . Interleukin-6 activity (171 +/- 10.2 U/ml) was found in all pretreatment plasma samples and was significantly (P less than 0.05) increased above baseline from 2 to 12 hours after 1,000 ng of endotoxin/kg was given and at 3 hours after 30 ng of endotoxin/kg was given . After 1,000- or 30-ng/kgt dosage of endotoxin, peak plasma IL-6 activity (10,128 +/- 4,096 and 1,555 +/- 1,326 U/ml, respectively) was observed for 3 hours . The IL-6 response of endotoxin-treated horses began about 1 hour after tumor necrosis factor appeared in the circulation, and its course closely approximated the endotoxin-induced febrile reaction . Significant increase in plasma IL-6 activity was not detected in horses given 1 ng of endotoxin/kg or control buffer. Am J Physiol, 1992 Aug, 263(2 Pt 2), R423 - 7 Endotoxin-induced fever is modulated by endogenous glucocorticoids in rats; Coelho MM et al.; This study was designed to determine if the febrile response, similar to the inflammatory response, can be modulated by endogenous glucocorticoids . A single intraperitoneal injection of lipopolysaccharide (LPS, 200 micrograms/kg) induced a significant increase in rectal temperature of unrestrained rats maintained within the thermoneutral zone . The febrile response was fully abolished following treatment with a glucocorticoid, dexamethasone (DEX), but was not affected by a mineralocorticoid, deoxycorticosterone acetate (DOCA) . Adrenalectomized (ADX) rats were markedly more sensitive to the lethal effects of LPS (200 micrograms/kg), such that all the animals died during the course of the experiment . In addition, ADX rats showed an enhanced febrile response to LPS (10 micrograms/kg ip) in comparison with sham-operated or adrenal-demedullated rats . This enhanced response was reduced by chronic or acute treatment with DEX, but not DOCA . LPS (10 micrograms/kg) also induced a marked rise in plasma corticosterone levels in control rats . In contrast, ADX rats displayed very low plasma corticosterone levels, which were not changed by LPS . In conclusion, the present results reveal that endogenous glucocorticoids are important modulators of LPS-induced fever. Genomics, 1992 Aug, 13(4), 1056 - 64 Base compositional structure of genomes; Fickett JW et al.; We model the base compositional structure of the human and Escherichia coli genomes . Three particular properties are first quantified: (1) There is a significant tendency for any region of either genome to have a strand-symmetric base composition . (2) The variation in base composition from region to region, within each genome, is very much larger than expected from common homogeneous stochastic models . (3) A given local base composition tends to persist over a scale of at least kilobases (E . coli) or tens of kilobases (human) . Multidomain stochastic models from the literature are reviewed and sharpened . In particular, quantitative measurements of the third property lead us to suggest a significant shift in the style of domain models, in which the variation of A+T content with position is modeled by a random walk with frequent small steps rather than with large quantum jumps . As an application, we suggest a way to reduce the amount of computation in the assembly of large sequences from sequences of randomly chosen fragments. J Lab Clin Med, 1992 Aug, 120(2), 205 - 11 Lipopolysaccharide, tumor necrosis factor, and interleukin-1 interact to cause hypotension; Weinberg JR et al.; Lipopolysaccharide (LPS) causes the syndrome of septic shock by initiating the release of endogenous mediators such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) from macrophages . Hypotension is one of the important clinical features of septic shock; however, TNF is only hypotensive in high doses . Therefore we have investigated the interactions of low, nonhypotensive doses of LPS, IL-1, and TNF in the restrained unanesthetized rabbit . Combinations of nonhypotensive doses of TNF, IL-1, and LPS produced significant (p less than 0.05) decreases in blood pressure as compared with doses of each of the substances alone . TNF bioactivity in animals that were made hypotensive with combinations of TNF, IL-1, and LPS was lower than in animals that were made hypotensive with TNF alone . This suggests that TNF release that is stimulated by LPS is not the sole cause of the hypotension that is seen in this model of endotoxic shock . In this model, interactions of LPS, IL-1, and TNF occur and may explain hypotension during some episodes of sepsis. Eur J Biochem, 1992 Aug 1, 207(3), 903 - 13 Purification and characterization of a recombinant murine interleukin-6 . Isolation of N- and C-terminally truncated forms; Zhang JG et al.; Murine interleukin-6 (IL-6), when expressed in Escherichia coli using the pUC9 vector, accumulated as insoluble aggregates or 'inclusion bodies' . After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL-6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by gel-permeation chromatography followed by reversed-phase HPLC . It was demonstrated that complete disulfide bond formation in murine IL-6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required . When fully reduced murine IL-6 was dissolved in 8 M guanidine hydrochloride and allowed to air-oxidize, complete disulfide bond formation, monitored by analytical reversed-phase HPLC, was shown to occur within 13 h at 6 degrees C . About 25 mg pure protein was obtained from 37 g wet cells . This recombinant murine IL-6 had a specific activity in the hybridoma growth factor assay of 2 x 10(8) U/mg, which is equivalent to that of native murine IL-6 . During the purification procedure, a number of variant forms of murine IL-6 were isolated and partially characterized . Two of these forms, T1 and T3, were C-terminal deletants of murine IL-6 lacking about 60 and 20 amino acids from the C-terminus, respectively, while the other form, T2, was an N-terminal deletant lacking 37 amino acids from the N-terminus . None of these variant forms of murine IL-6 bound to the murine IL-6 receptor and, consequently, all were inactive in the hybridoma growth factor assay. Carcinogenesis, 1992 Aug, 13(8), 1455 - 9 Sequence specificity of DNA repair by Escherichia coli Fpg protein; Graves R et al.; The sequence specificity of guanine methylation in DNA by N-methyl-N-nitrosourea and the subsequent repair of ring-opened N7-methylguanine was studied using oligonucleotides of defined sequence . It was found that the methylation of TAGGGGCCCCTA was less than 2-fold greater than that occurring in TAGAGATCTCTA or TATGTGCACATA and 6-fold greater than in TACGCGCGCGTA . This is consistent with the concept that guanine methylation is least when the 5' preceding base is a pyrimidine and greatest when the 5' base is another guanine . These dodecamers were used to study repair by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) after the 7-methyl-guanine present in them was converted to the ring-opened form by alkaline treatment . The repair of ring-opened 7-methylguanine was much faster in self-complementary double-stranded 12mer substrates and was twice as rapid at 37 degrees C in TAGGGGCCCCTA compared with TACGCGCGCGTA . However, at 15 degrees C, the relative rates were reversed since TACGCGCGCGTA was repaired at the same rate as at 37 degrees C, whereas the repair of TAGGGGCCCCTA was much slower at 15 degrees C . The repair of TAGGGGCCCCTA at 37 degrees C was also much faster than the repair of TAGAGATCTCTA and was slightly more rapid than repair of TATGTGCACATA . Ligation of the dodecamer substrates to form 24mers or 36mers slightly reduced the initial rates of repair but did not abolish these differences . These results indicate that under physiological conditions, the Fpg protein is more active against adducts in guanine-rich regions and such regions may be the most likely sites of adduct formation at the N7-position of guanine which can then give rise to derivatives attacked by this enzyme. Carcinogenesis, 1992 Aug, 13(8), 1415 - 25 Mutagenesis by (+)-anti-B{a}P-N2-Gua, the major adduct of activated benzo{a}pyrene, when studied in an Escherichia coli plasmid using site-directed methods; Mackay W et al.; The suspected major mutagenic adduct of benzo{a}pyrene, (+)-anti-B{a}P-N2-Gua, is built into the unique PstI recognition site of the Escherichia coli plasmid, pUC19, in order to study its mutagenic potential . The adduct can either be at G437, which is replicated during leading strand DNA synthesis, or at G438, which is replicated during lagging strand DNA synthesis . The DNA strand complementary to the strand containing the (+)-anti-B{a}P-N2-Gua adduct is saturated with UV lesions to minimize its potential to generate progeny . Although all in-frame mutations could have been detected, a G437----T transversion mutation is virtually exclusively obtained at a frequency of approximately 0.04% per adduct following transformation into Uvr+ E . coli when SOS is not induced, and approximately 0.18% when SOS is induced . The mutation frequency of the adduct in a Uvr- background is estimated to be approximately 0.2% when SOS is not induced, and approximately 0.9% when SOS is induced . The absence of G438----T mutations is rationalized . G----T mutations from (+)-anti-B{a}P-N2-Gua are compared to the mutational specificity of the ultimate mutagenic form of activated benzo{a}pyrene. Biochem J, 1992 Aug 1, 285 ( Pt 3), 871 - 9 Solubilization of growth hormone and other recombinant proteins from Escherichia coli inclusion bodies by using a cationic surfactant; Puri NK et al.; Recombinant pig growth hormone (rPGH) was solubilized from inclusion bodies by using the cationic surfactant cetyltrimethylammonium chloride (CTAC) . The solubilizing action of CTAC appeared to be dependent on the presence of a positively charged head group, as a non-charged variant was inactive . Relatively low concentrations of CTAC were required for rapid solubilization, and protein-bound CTAC was easily removed by ion-exchange chromatography . Compared with solubilization and recovery of rPGH from inclusion bodies with 7.5 M-urea and 6 M-guanidinium chloride, the relative efficiency of solubilization was lower with CTAC . However, superior refolding efficiency resulted in final yields of purified rPGH being in the order of CTAC greater than urea greater than or equal to guanidinium chloride . Detailed comparison of the different rPGH preparations as well as pituitary-derived growth hormone by h.p.l.c., native PAGE, c.d . spectral analysis and radioreceptor-binding assay showed that the CTAC-derived rPGH was essentially indistinguishable from the urea and guanidinium chloride preparations . The CTAC-derived rPGH was of greater biopotency than pituitary-derived growth hormone . The advantages of CTAC over urea and guanidinium chloride for increasing recovery of monomeric rPGH by minimizing aggregation during refolding in vitro were also found with recombinant sheep interleukin-I beta and a sheep insulin-like growth factor II fusion protein . In addition, the bioactivity of the CTAC-derived recombinant interleukin-1 beta was approximately ten-fold greater than that of an equivalent amount obtained from urea and guanidinium chloride preparations . It is concluded that CTAC represents, in general, an excellent additional approach or a superior alternative to urea and in particular guanidinium chloride for solubilization and recovery of bioactive recombinant proteins from inclusion bodies. Biochem J, 1992 Aug 1, 285 ( Pt 3), 707 - 9 C-terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity; Elder RH et al.; A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; methylated-DNA: protein-cysteine methyltransferase) has been manipulated to generate a C-terminally deleted protein which retains full methyl-transfer activity . The elimination of 22 amino-acid residues from the C-terminus was achieved by endonuclease-SacI digestion of the 623 bp cDNA coding sequence and ligation of a SacI/HindIII linker containing an in-frame stop codon . The truncated protein was characterized by its reduced molecular mass in immunoblots probed with an antiserum against the full-length protein and by fluorography after incubation with {3H}methylated calf thymus DNA . The rate of methyl transfer was virtually identical for the full-length and truncated ATases . The construction of such a truncated, yet still functional, ATase, with a molecular mass of 19.7 kDa should facilitate a detailed n.m.r . structural study and help to determine the functional significance of the C-terminal domain of mammalian ATases. Am J Pathol, 1992 Aug, 141(2), 451 - 6 Effect of serum amyloid P component level on transthyretin-derived amyloid deposition in a transgenic mouse model of familial amyloidotic polyneuropathy; Murakami T et al.; To elucidate the pathogenesis of amyloid deposition associated with familial amyloidotic polyneuropathy (FAP), we developed several transgenic mouse lines carrying the human mutant transthyretin (TTR) gene . We found that human TTR and mouse serum amyloid P component (SAP) are deposited as amyloid in tissues of these mouse lines . Because SAP is a major acute-phase reactant in mice, we asked whether repeated injections of Escherichia coli lipopolysaccharide (LPS) would enhance the amyloid deposition in one of these transgenic mouse lines . During the course of repeated LPS injections, serum levels of SAP in the transgenic mice remained between severalfold to about 50-fold higher than seen in the absence of stimulation . As no significant difference was detected in the onset, progression, and tissue distribution of TTR-derived amyloid (ATTR) deposition between the LPS-stimulated and unstimulated transgenic mice, the induction of SAP synthesis by acute inflammation probably does not affect the onset and extent of ATTR deposition. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7022 - 5 Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA; Michaels ML et al.; It has been previously shown both in vivo and in vitro that DNA synthesis past an oxidatively damaged form of guanine, 7,8-dihydro-8-oxoguanine (8-oxoG), can result in the misincorporation of adenine (A) opposite the 8-oxodG . In this study we show that MutY glycosylase is active on a site-specific, oxidatively damaged A/8-oxoG mispair and that it removes the undamaged adenine from this mispair . Strains that lack active MutY protein have elevated rates of G.C----T.A transversions . We find that the mutator phenotype of a mutY strain can be fully complemented by overexpressing MutM protein (Fpg protein) from a plasmid clone . The MutM protein removes 8-oxoG lesions from DNA . In addition, we have isolated a strain with a chromosomal mutation that suppresses the mutY phenotype and found that this suppressor also overexpresses MutM . Finally, a mutY mutM double mutant has a 25- to 75-fold higher mutation rate than either mutator alone . The data strongly suggest that MutY is part of an intricate repair system directed against 8-oxoG lesions in nucleic acids and that the primary function of MutY in vivo is the removal of adenines that are misincorporated opposite 8-oxoG lesions during DNA synthesis. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7017 - 21 Phycocyanin alpha-subunit phycocyanobilin lyase; Fairchild CD et al.; Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores . The bilin chromophores are attached through thioether bonds to cysteine residues . The cyanobacterium Synechococcus sp . PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin . When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separately, the surprising result is elimination of correct bilin attachment at only one site, that on the alpha subunit of phycocyanin . We have overproduced CpcE and CpcF in Escherichia coli . In vitro, these proteins catalyze the attachment of phycocyanobilin to the alpha subunit of apophycocyanin at the appropriate site, alpha-Cys-84, to form the correct adduct . CpcE and CpcF also efficiently catalyze the reverse reaction, in which the bilin from holo-alpha subunit is transferred either to the apo-alpha subunit of the same C-phycocyanin or to the apo-alpha subunit of a heterologous C-phycocyanin . The forward and reverse reactions each require both CpcE and CpcF and are specific for the alpha-Cys-84 position . Phycocyanobilin is the immediate precursor of the protein-bound bilin. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7008 - 12 The budding yeast HRR25 gene product is a casein kinase I isoform; DeMaggio AJ et al.; The Saccharomyces cerevisiae HRR25 gene was identified as a regulator of DNA strand-break repair . HRR25 encodes a protein kinase that is closely related to bovine casein kinase I (CKI) . CKI is a ubiquitous multipotential protein kinase . Rabbit polyclonal antibodies that recognize and immunoprecipitate Hrr25p have been generated and an immune complex protein kinase assay has been developed . The reaction depends upon HRR25 and shows that Hrr25p uses casein as a substrate . The identity between Hrr25p and bovine CKI suggests that Hrr25p is a yeast isoform of the CKI family and that CKIs may play a role in regulating DNA metabolism. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6929 - 33 Identification and isolation of the polyferredoxin from Methanobacterium thermoautotrophicum strain delta H; Steigerwald VJ et al.; Sequencing the genes encoding the methyl viologen-reducing hydrogenase, cloned from Methanobacterium thermoautotrophicum strain delta H and Methanothermus fervidus, revealed the presence of tightly linked genes, designated mvhB, which were predicted to encode proteins containing six tandemly arranged bacterial ferrodoxin-like domains . A lacZ-mvhB gene fusion has been constructed and expressed in Escherichia coli . Rabbit antibodies raised against the fusion polypeptide purified from E . coli have been used to identify and isolate the polyferrodoxin from Mb . thermoautotrophicum strain delta H . The polyferredoxin accumulates in cells of the methanogen during exponential growth but decreases rapidly on entry into stationary phase . It is not processed into monoferredoxins and is located primarily in the soluble fraction of cell lysates of Mb . thermoautotrophicum . Metronidazole reduction by crude extracts of Mb . thermoautotrophicum strain delta H cells, dependent on the presence of hydrogen and the heterodisulfide CoM-S-S-HTP {formed from the two coenzymes 2-mercaptoethanesulfonic acid (coenzyme M, HS-CoM) and N-(7-mercaptoheptanoyl)threonine O3-phosphate (HS-HTP)}, was not inhibited by the antibodies raised against the LacZ-MvhB fusion polypeptide. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6756 - 60 Attenuation of sensory receptor signaling by covalent modification; Borkovich KA et al.; The Tar receptor is a transmembrane protein that regulates bacterial chemotaxis in response to changes in the level of aspartic acid in the medium . The extracellular portion of the protein can bind aspartate, and the cytoplasmic portion modulates CheA kinase activity . The receptor can either activate or inhibit the kinase . The cytoplasmic portion of the receptor can be modified by carboxymethylation of specific glutamic acid residues . To test the effects of differential methylation on receptor function, we prepared membranes from cells that have specifically modified forms of the receptor and tested the relative ability of each of these forms to activate or inhibit CheA kinase . Completely demethylated receptor was a potent inhibitor and poor activator of the kinase, while the fully modified receptor was an excellent activator but an inefficient inhibitor . Partially modified receptor could act both as an effective inhibitor and as an activator . Reversible modification provides a mechanism that allows the cell to accumulate a population of receptor molecules capable of generating a wide range of signaling intensities. Cell Mol Biol (Noisy-le-grand), 1992 Aug-Sep, 38(5-6), 643 - 51 Partially purified bacteriocin kills malignant cells by apoptosis: programmed cell death; Farkas-Himsley H et al.; Bacterial proteins, partially purified bacteriocin (PPB), were investigated for their selective killing of malignant cells . It is shown here that upon PPB-cell interaction DNA fragmentation starts within one hour and peaks at 6 hrs . This process requires an on-going cellular metabolism . It is prevented by both actinomycin D, a DNA dependent RNA synthesis inhibitor, and cycloheximide, a protein synthesis inhibitor . We show here that the DNA fragmentation is triggered by PPB-cell membrane-receptor interaction which signals the activation of endogenous cellular endonucleases rather than actually penetrating the cell and interacting directly with the DNA, or serving as a nuclease itself . Thus, it is suggested that the cell death initiated by the lethal bacterial proteins, PPB, is a programmed, step-wise cell death involving apoptosis. Cell Mol Biol, 1992 Aug, 38(5), 529 - 42 High level expression and purification of peptide methionine sulfoxide reductase in Escherichia coli; Rahman MA et al.; The enzyme peptide methionine sulfoxide reductase catalyzes the conversion of methionine sulfoxide residues in proteins to methionine . The 636 nucleotide coding region of the peptide methionine sulfoxide reductase gene has been amplified from a genomic clone using the polymerase chain reaction and the product was subcloned into plasmid pGEX-2T downstream of the glutathione S-transferase gene under control of the tac promoter . Escherichia coli XL1-Blue cells transformed with this plasmid and induced with isopropylthio-beta-galactoside expressed high levels of the fusion protein . The protein was soluble and was purified to homogeneity by affinity binding to a glutathione-agarose resin followed by cleavage of the fusion protein with thrombin . Both the fusion protein and the purified peptide methionine sulfoxide reductase protein showed high peptide methionine sulfoxide reductase activity. Indian J Exp Biol, 1992 Aug, 30(8), 756 - 8 Presence of RNA from yeast inhibits the photoreactivation of UV-irradiated DNA by Phr A photolyase from Escherichia coli; Hejmadi VS et al.; Photoreactivation of UV-irradiated DSNA with phr A photolyase from Escherichia coli was studied in the presence of yeast RNA . Mixing of RNA with UV-irradiated DNA before its treatment with photolyase inhibited the photoreactivation of DNA . Denatured (by sonication) RNA was found to be more effective in blocking photolyase action . Agarose gel electrophoresis experiments suggest that this inhibition of photoreactivation is due to interference in the binding of photolyase with UV-irradiated DNA by yeast RNA. Indian J Exp Biol, 1992 Aug, 30(8), 659 - 63 Cloning and nucleotide sequencing of sheep growth hormone cDNA; Guron C et al.; cDNA was prepared from the mRNA isolated from sheep anterior pituitary glands . On cloning cDNA in E . coli, a clone coding full sequence of sheep pre-growth hormone was determined . The sequence for the sheep growth hormone (GH) is in agreement with the amino acid sequence of the protein determined previously except for the asparagine residue at position 99 rather than aspartic acid and the arginine residue at position 146 in place of threonine . The cDNA sequence presented is also in accordance with the genomic sequence for the sheep GH gene that has been reported. Microb Pathog, 1992 Aug, 13(2), 157 - 60 Genetic control of resistance to enterotoxigenic Escherichia coli in infant mice; Duchet-Suchaux M et al.; DBA/2 and CBA infant mice orally challenged with bovine enterotoxigenic Escherichia coli (ETEC) strain B80 presented resistance and susceptibility respectively, as measured by mortality rates 6 days after inoculation . Serum antibodies agglutinating ETEC strain B80 had very low titers in both mouse strains . Mendelian analysis of resistance on F1 and on segregating back-crosses showed that resistance is genetic and dominant . Dominance may be explained either by a mixed control with an overdominant major gene or by a polygenic control with a large heterosis effect. Electrophoresis, 1992 Aug, 13(8), 547 - 51 Automated magnetic preparation of DNA templates for solid phase sequencing; Wahlberg J et al.; An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments . The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA . The DNA template is obtained by the polymerase chain reaction (PCR) . Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively . The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions . Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers. Electrophoresis, 1992 Aug, 13(8), 506 - 11 cDNA sequence of three cysteine-rich clusters in the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer; Kita K et al.; Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase) . Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains . In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR . Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used . The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu) . The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%) . As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme. Circ Shock, 1992 Aug, 37(4), 323 - 32 Cardiovascular response in canine endotoxic shock: effect of ibuprofen pretreatment; Pinsky MR; Ibuprofen pretreatment increases arterial pressure and reduces mortality in endotoxic dogs . The increase in arterial pressure may be caused by increases in arterial resistance, arterial sphincter tone, or both . Thus it is not clear if ibuprofen pretreatment prevents the hemodynamic effects of endotoxemia or merely masks such effects by producing concomitant increases in arterial resistance . Accordingly, this study was performed to determine the effects of ibuprofen pretreatment on arterial pressure-flow relations and other measures of cardiovascular function in a canine model of endotoxic shock . In 19 pentobarbital-anesthetized and splenectomized, closed-chest dogs, biventricular stroke volumes were measured with electromagnetic flow probes, and intrathoracic vascular and pleural pressures were measured with catheters . Instantaneous venous return curves (see Pinsky MR, J Appl Physiol 56:765, 1984) were generated during positive-pressure ventilation, and steady-state arterial pressure-flow relations, left ventricular function, peripheral vascular compliance, oxygen consumption/oxygen delivery ratio, and arterial blood lactate levels were measured during two sequential volume loading and removal (20 ml/kg) sequences . All but two dogs received a bolus infusion of Escherichia coli endotoxin (2 mg/kg) between the two fluid challenge runs . Eleven of the 17 endotoxic dogs also received ibuprofen (15 mg/kg) immediately before the initial fluid challenge . Ibuprofen pretreatment abolished all hemodynamic effects of endotoxin, whereas in the untreated group endotoxin caused decreases in calculated arterial outflow pressure and in peripheral vascular capacitance . Oxygen consumption remained constant despite changes in oxygen delivery in the nonendotoxic dogs and in the ibuprofen-pretreated dogs, whereas oxygen consumption covaried with oxygen delivery in the endotoxic dogs not pretreated with ibuprofen . Arterial lactate levels were higher after endotoxin infusion (2.1 +/- 0.5 to 3.1 +/- 0.6 mmol/liter; P less than 0.05 pre- to postvolume) but were not different between treatment groups . These data suggest that ibuprofen alters many, but not all, of the hemodynamic effects of endotoxin infusion in the dog. J Cell Sci, 1992 Aug, 102 ( Pt 4), 821 - 32 Alteration in glycosaminoglycan metabolism and surface charge on human umbilical vein endothelial cells induced by cytokines, endotoxin and neutrophils; Klein NJ et al.; There is increasing evidence that the glycosaminoglycan (GAG) component of the vascular endothelium is important in regulating vascular permeability, thromboresistance and cellular interactions . We have investigated the GAG metabolism of cultured human umbilical vein endothelial cells (HUVEC) in response to a range of inflammatory stimuli . Using both chemical measurement of cellular and supernatant GAGS and 35S labelling to identify newly synthesised GAGS, interleukin 1 (IL1), tumour necrosis factor (TNF) and interferon gamma (IFN gamma) were shown to influence sulphated GAG metabolism significantly . IL1 and TNF caused a marked increase in culture supernatant GAGS and a concomitant reduction in cell-associated GAGS . This was shown histochemically to be associated with a marked reduction and redistribution of endothelial surface anionic sites . The addition of neutrophils to HUVEC pretreated with Escherichia coli endotoxin, IL1 or TNF resulted in a further reduction in both cellular GAGS and surface anionic sites . These results suggest that changes in endothelial cell GAG metabolism during inflammation may contribute to the disturbance of vascular endothelial homeostasis associated with infectious and inflammatory states. Diagn Microbiol Infect Dis, 1992 Aug, 15(6), 505 - 10 Evaluation of three different STb assays and comparison of enterotoxin pattern over a five-year period in Swedish porcine Escherichia coli; Handl CE et al.; The pig intestinal loop (PIL) assay, inhibition enzyme-linked immunosorbent assay (ELISA), and DNA hybridization assay were compared for analysis of Escherichia coli heat-stable enterotoxin b (STb) on 201 porcine E . coli strains . The DNA hybridization had a 95% correlation with the STb ELISA and was therefore chosen as the method for subsequent screening of enterotoxin genes: heat labile (LT), heat-stable a (STa), and/or STb . In contrast to the PIL assay, both the STb ELISA and DNA hybridization assays were more sensitive, reliable, reproducible, and showed good correlation with each other . Consequently, the STb ELISA is preferable for analysis of toxin preparations and screening of E . coli, whereas the DNA hybridization is better for large-scale epidemiologic screening . Escherichia coli strains (n = 437) associated with porcine diarrhea isolated in Sweden during 1989 were investigated . Of the strains, 135 (31%) were positive for at least one of these toxins and, therefore, designated enterotoxigenic E . coli (ETEC) . Our results were compared with the enterotoxin pattern found in earlier studies of Swedish porcine strains . The only change in occurrence of toxins was found in strains isolated from piglets less than 1 week of age . LT- and STb-producing ETEC had decreased, and STa-producing ETEC had increased in prevalence . The occurrence of STb among ETEC of weaned pigs was 93% . This toxin was also found to be more common than STa when strains from all age groups were considered. Protein Expr Purif, 1992 Aug, 3(4), 332 - 6 Construction of a high-copy "ATG vector" for expression in Escherichia coli; Beernink PT et al.; We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli . This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19 . Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2 . Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C . 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number . An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number . Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E . coli JM83 . Aldolase A can compose up to 40% of the total protein in E . coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture. Protein Expr Purif, 1992 Aug, 3(4), 322 - 31 Expression and purification of recombinant and native calreticulin; Baksh S et al.; Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems . The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum . In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin . Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography . A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin . The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions . The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies. Protein Expr Purif, 1992 Aug, 3(4), 308 - 12 Expression and purification of the cytoplasmic tail of an endocytic receptor by fusion to a carbohydrate-recognition domain; Taylor ME et al.; Gene fusion has been used to produce the cytoplasmic domain of an endocytic receptor . DNA sequences coding for the 52 COOH-terminal amino acids of the mannose receptor from human macrophages, including the 41-amino acid cytoplasmic tail, were fused to the codons specifying the carbohydrate-recognition domain (CRD) of rat mannose-binding protein . The fusion protein was expressed in Escherichia coli and purified in one step on mannose-Sepharose, making use of the carbohydrate-binding activity of the CRD . The tail peptide was released from the fusion protein using endoproteinase Arg-C . This method provides an alternative to chemical synthesis for the production of midlength peptides. Protein Expr Purif, 1992 Aug, 3(4), 290 - 4 High-level expression and production of recombinant human interleukin-6 analogs; Dagan S et al.; We have constructed and analyzed different mutant forms of interleukin-6 (IL-6) expressed in Escherichia coli that can be divided into two groups . The first group contains four full-length IL-6 molecules that differ in the presence of cysteine residues involved in disulfide bridges . The second group contains 22 N-terminal amino acid deletions in addition to the differences in the cysteine residues . The different IL-6 muteins were extracted and their expression levels and solubility were compared . We found that the production levels of IL-6 can be dramatically improved by deleting the first 22 N-terminal amino acids of the molecule . We have also found that the production of IL-6 containing the four cysteine residues is lower than the production of the mutant molecules that lack one or both pairs of cysteines . The yield of soluble and properly refolded IL-6 was the highest when the disulfide bond between the cysteines at positions 74 and 84 was present in the mutein form, which also lacked the 22 N-terminal amino acids. Protein Expr Purif, 1992 Aug, 3(4), 282 - 9 Purification of an active species of recombinant kappa-bungarotoxin; Fiordalisi JJ et al.; The expression of kappa-bungarotoxin in Escherichia coli from a synthetic gene results in the production of multiple species of polypeptide . These include not only biologically active kappa-bungarotoxin but also a variety of inactive species, which include inactive monomers as well as disulfide-linked polymeric species . Identification of these species and their separation from the biologically active recombinant toxin is necessary for the use of the toxin in physiological and biochemical studies . This has been accomplished by a combination of ion-exchange and reverse-phase chromatography which results in a homogeneous toxin preparation . The active material produced is sufficient for many types of biological studies and for mutagenesis experiments directed at determining the structure function relationships of toxin interactions with the neuronal nicotinic acetylcholine receptor . In addition, the kappa-bungarotoxin produced in this manner has the distinct advantage over venom-purified kappa-bungarotoxin of not being contaminated with other venom components which could potentially affect experimental observations. Glycobiology, 1992 Aug, 2(4), 383 - 93 Cloning and expression of the murine gene and chromosomal location of the human gene encoding N-acetylglucosaminyltransferase I; Kumar R et al.; A mouse cDNA clone previously isolated from an F9 teratocarcinoma cell library and shown to confer N-acetylglucosaminyltransferase I (GlcNAc-TI) activity on Lec1 Chinese hamster ovary (CHO) cell transfectants {Kumar, R., Yang,J., Larsen,R.D . and Stanley,P . (1990) Proc . Natl . Acad Sci . USA, 87, 9948-9952} has been sequenced . The nucleotide and deduced amino acid sequences are highly homologous to previously described human and rabbit GlcNAc-TI cDNAs . A 1250 bp portion of the mouse cDNA encoding all but the first 34 amino acids of the deduced protein sequence was inducibly expressed in Escherichia coli and gave rise to a prominent fusion protein of mol . wt approximately 45 kDa whose presence correlated with high levels of GlcNAc-TI activity in cell lysates . Probes generated from the cDNA were used to show that the GlcNAc-TI gene is present in a single copy in mammals and that a homologous gene was not detectable (under low-stringency hybridization conditions) in DNA from yeast, sea urchin, Drosophila or Chaenorhaditis elegans . Genomic DNA clones that hybridized to probes generated from the GlcNAc-TI cDNA were isolated from a mouse liver library . Restriction analyses, Southern hybridization and DNA sequence analyses of subcloned genomic DNA fragments and a polymerase chain reaction (PCR) product provided evidence that the coding and 3' untranslated regions of the cDNA reside in a single exon . However, the mouse GlcNAc-TI gene (Mgat-1) includes at least one additional exon 5' of the coding region . Southern analyses of DNA from mouse-human somatic cell hybrids and in situ hybridization were used to locate the human GlcNAc-TI gene (MGAT-1) between positions q31.2 and q31.3 on chromosome 5, a region of chromosome 5 that is syntenic with a region of mouse chromosome 11 . Northern analyses of adult mouse tissues revealed two GlcNAc-TI gene transcripts that are differentially expressed in different tissues. Biophys Chem, 1992 Aug, 44(1), 61 - 70 On the base sequences of the promoters in transcription initiation; Shinoda T; The base sequence of a specific DNA region identified as the promoter is investigated by means of the quantity S(r) corresponding to "superdelocalizability" of oxygen ion of each phosphate for the ten DNA dimer units ({XY/Y'X'}2-) and ({XY/Y'X'}2- + H+)-complexes . A mechanism is proposed of how RNA polymerase can recognize its transcription site (phosphate), and is applied to the Escherichia coli promoters, lacUV5, recAp, rrnEpl, and rrnEp2 . The result explains fairly well the character of the promoters experimentally found. J Biomol Struct Dyn, 1992 Aug, 10(1), 73 - 82 Restriction enzyme mapping of carcinogen binding regions on pBR322; Mallamaci MA et al.; Previous equilibrium binding experiments (S.A . Winkle and T.R . Krugh, Nucleic Acids Res . 9, 3175-3186 (1981)) suggested that the carcinogen N-hydroxy-N-acetyl-2-aminofluorene might exhibit preferential binding to a small number of sites on phiX174 DNA . To examine whether the covalently binding analogue N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) also possesses high affinity sites, the plasmid pBR322 was reacted with 3H labeled acetoxyAAF to give one to sixteen adducts per DNA molecule . Thus only higher affinity sites would be affected . The DNA was subsequently cleaved with either Alu I, Hae III, Hha I, Hinf I or Hpa II restriction endonuclease and the restriction fragments isolated by gel electrophoresis . Examination of the distribution of 3H acetoxyAAF among the fragments was not random but, rather, with each enzyme, the acetoxyAAF was found predominantly in a few fragments . The locations of the bands containing the acetoxyAAF for each enzyme overlap--suggesting that there are regions on pBR 322 which contain high affinity sites for acetoxyAAF binding. J Biomol Struct Dyn, 1992 Aug, 10(1), 63 - 72 Using lambda exonuclease inhibition assays to map carcinogen binding sites; Combates NJ et al.; Previous restriction mapping studies (M.A . Mallamaci, D.P . Reed and S.A . Winkle, J . Biomolecular Structure and Dynamics, in press (1992)) have indicated that a small number of locations on the plasmid pBR322 may be high affinity binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) . PBR322 was reacted with acetoxyAAF to produce DNA with one, three or seven acetoxyAAF moieties per DNA molecule . Thus only the higher affinity binding sites are affected . Subsequent digestion with the restriction enzyme Hinf I produced fragments containing previously indicated locations of potential acetoxyAAF binding sites . Fragments thought not to contain binding sites were also examined as controls . The isolated fragments, singly 32P end-labeled, were digested with lambda exonuclease . The three fragments suspected of containing acetoxyAAF binding sites possess new lambda exonuclease inhibition sites when the fragments are obtained from acetoxyAAF reacted DNA . No such inhibition sites are found with the two fragments suggested previously not to contain acetoxyAAF binding sites . These carcinogen produced inhibition sites occur in sequences which are similar, suggesting that acetoxyAAF preferentially may target a small number of sequences. Arzneimittelforschung, 1992 Aug, 42(8), 1001 - 4 Platelet-activating factor antagonists in experimental shock; Muacevic G et al.; Platelet-activating factor (PAF) at 10 micrograms/kg i.v . induced profound hyperkalemia, changes of hematological parameters, patterns of ECG, and acid-base balance in rats . In separate experiments infusions of PAF at 30 ng/kg/min or injection of endotoxin from E . coli (15 mg/kg i.v.) induced a marked drop in blood pressure . All these changes were antagonized by pretreatment or post-treatment (in case of hypotension) by the selective hetrazepinoic PAF antagonists apafant (WEB 2086, CAS 105219-56-5; 0.1-5 mg/kg i.v.), bepafant (WEB 2170, CAS 114776-28-2; 0.05-1.0 mg/kg i.v.), or STY 2108 (0.01-0.1 mg/kg), respectively . The results support the view that PAF can mimic features of endotoxin shock and that the hetrazepines (like apafant, bepafant) are useful tools to clarify the role of PAF in such conditions. Acta Ophthalmol (Copenh), 1992 Aug, 70(4), 506 - 14 Polyamine and histopathological changes after unilateral endotoxin-induced uveitis and its contralateral effects; Wickstrom K; The polyamines putrescine, spermidine and spermine are essential for normal cell function and proposed to be involved in inflammatory reactions . The polyamines were measured bilaterally in rabbit aqueous humor after unilateral endotoxin-induced uveitis . The contralateral eyes were injected with saline or not touched . Aqueous protein levels and leukocytes were determined and a histological evaluation was performed . Protein, leukocytes, putrescine and spermine increased in the treated eyes, but not in the untouched eyes . When saline was injected in the contralateral eye, a small increase in spermine was seen . Spermidine decreased first, but increased later, in both the endotoxin-injected and the other eye . Histopathologically, the treated eyes showed an infiltration of leukocytes, vasodilatation and in some cases optic nerve involvements . A mild reaction was also seen in the unchallenged contralateral eyes . The results show that polyamines might serve as a marker for acute inflammation in the eye and that the mechanism of putrescine and spermine induction is different from the one of spermidine . Polyamines are suggested to play a role in the cellular immune response. Mol Endocrinol, 1992 Aug, 6(8), 1249 - 58 Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I; Lala DS et al.; We proposed that a cell-selective regulatory protein coordinately regulates the expression of three enzymes that are required for the biosynthesis of corticosteroids: cholesterol side chain cleavage enzyme, steroid 21-hydroxylase, and the aldosterone synthase isozyme of steroid 11 beta-hydroxylase . In this report, we identify a 53-kilodalton protein, termed steroidogenic factor 1 (SF-1), that interacts with the related promoter elements from these steroidogenic enzymes, and we isolate and characterize a cDNA that very likely encodes this protein . We first showed that nuclear extracts from bovine adrenal glands interact with the mouse steroidogenic regulatory elements, forming complexes indistinguishable from those produced by nuclear extracts from mouse Y1 adrenocortical cells . These bovine adrenal extracts were subjected to sequential ion exchange and affinity chromatography to yield a highly enriched preparation of SF-1 . The predominant protein in the affinity-purified preparation comigrated with shift activity and had a mol wt of 53,000; UV cross-linking experiments demonstrated directly that this 53-kilodalton protein interacted with the steroidogenic regulatory element . Even with this marked enrichment, affinity-purified SF-1 bound six steroidogenic regulatory elements . These results support strongly the model that a steroidogenic cell-selective protein interacts with related promoter elements from three steroidogenic enzymes to regulate their coordinate expression . The recognition sequence of SF-1 closely resembles those of nuclear hormone receptor family members, suggesting that SF-1 may belong to this supergene family . By screening a Y1 cell cDNA library with the DNA-binding region of the H-2RIIBP nuclear hormone receptor cDNA, we isolated a cDNA that is selectively expressed in steroidogenic cells . When expressed as a glutathione S-transferase fusion protein in Escherichia . coli, the protein encoded by this cDNA interacts with all six related steroidogenic regulatory elements with a binding specificity indistinguishable from that of SF-1 . Surprisingly, the sequence of the putative DNA-binding domain of this cDNA matches exactly the corresponding sequence of the mouse homolog of the Drosophila transcription factor fushi tarazu-factor I . The demonstration that a member of the nuclear hormone receptor family interacts with the steroidogenic regulatory elements provides intriguing insights into possible mechanisms by which these essential genes are regulated. Mol Microbiol, 1992 Aug, 6(16), 2397 - 406 Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity; Dreyfus LA et al.; The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis . The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E . coli inner membrane . Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59 . Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis . In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity. Mol Microbiol, 1992 Aug, 6(16), 2309 - 18 The stable BRP signal peptide causes lethality but is unable to provoke the translocation of cloacin DF13 across the cytoplasmic membrane of Escherichia coli; van der Wal FJ et al.; The bacteriocin release protein (BRP) mediates the secretion of cloacin DF13 . The BRP precursor is slowly processed to yield the mature BRP and its stable signal peptide which is also involved in cloacin DF13 secretion . The function of the stable BRP signal peptide was analysed by constructing two plasmids . First, the stable BRP signal peptide was fused to the murein lipoprotein and, second, a stop codon was introduced after the BRP signal sequence . Exchange of the unstable murein lipoprotein signal peptide for the stable BRP signal peptide resulted in an accumulation of precursors of the hybrid murein lipoprotein . This indicated that the BRP signal peptide, as part of this hybrid precursor, is responsible for the slow processing . The stable BRP signal peptide itself was not able to direct the transfer of cloacin DF13 into the periplasmic space or into the culture medium . Over-expression of the BRP signal peptide was lethal and caused 'lysis' . Subcellular fractionation experiments revealed that the BRP signal peptide is located exclusively in the cytoplasmic membrane whereas the mature BRP, targeted by either the stable BRP signal peptide or the unstable Lpp signal peptide, is located in both the cytoplasmic and outer membrane . These results are in agreement with the hypothesis that the stable signal peptide and the mature BRP together are required for the passage of cloacin DF13 across the cell envelope. Mol Microbiol, 1992 Aug, 6(16), 2243 - 51 Interrelated effects of DNA supercoiling, ppGpp, and low salt on melting within the Escherichia coli ribosomal RNA rrnB P1 promoter; Ohlsen KL et al.; The formation of complexes containing high levels of DNA melting at the ribosomal RNA rrnB P1 promoter in vitro is shown to be facilitated by DNA supercoiling or low salt . The effector nucleotide ppGpp is ineffective under these conditions . The loss of supercoils or addition of salt increases the effectiveness of ppGpp in inhibiting formation of these complexes . In vivo plasmid DNA supercoiling is shown to decrease during starvation protocols that also increase levels of ppGpp . The results suggest that ppGpp regulation may be affected by the state of DNA supercoiling in vivo. Mol Microbiol, 1992 Aug, 6(16), 2219 - 24 (A)BC excinuclease: the Escherichia coli nucleotide excision repair enzyme; Lin JJ et al.; Nucleotide excision repair is the major pathway for removing damage from DNA . (A)BC excinuclease is the nuclease activity which initiates nucleotide excision repair in Escherichia coli . In this review, we focus on current understanding of the structure-function of the enzyme and the reaction mechanism of the repair pathway . In addition, recent biochemical studies on preferential repair of actively transcribed genes in E . coli are summarized. Mol Microbiol, 1992 Aug, 6(15), 2073 - 83 Cell division in Escherichia coli minB mutants; Akerlund T et al.; In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non-polarly in the cell . We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minB deletion mutant, and compared them to the corresponding wild-type strain and an intR1 strain in which the chromosome is over-replicated . The main findings were as follows . In the minB mutants, polar and non-polar divisions appeared to occur independently of each other . Furthermore, the timing of cell division in the cell cycle was found to be severely affected . In addition, nucleoid conformation and distribution were considerably disturbed . The results obtained call for a re-evaluation of the role of the MinB system in the E . coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle. Mol Microbiol, 1992 Aug, 6(15), 2033 - 40 Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli; Dean DA et al.; Active transport of maltose in Escherichia coli requires the presence of both maltose-binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane . Earlier, mutants in malF or malG were isolated that are able to grow on maltose in the complete absence of MBP . When the wild-type malE+ allele, coding for MBP, was introduced into these MBP-independent mutants, they frequently lost their ability to grow on maltose . Furthermore, starting from these Mal- strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated . In this study, we examined the interaction of wild-type and mutant MBPs with wild-type and mutant FGK2 complexes by using right-side-out membrane vesicles . The vesicles from a MBP-independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells . However, addition of wild-type MBP to these mutant vesicles produced unexpected responses . Although malE+ malG511 cells could not utilize maltose, wild-type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles . At higher concentrations of the wild-type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited . This behaviour of the vesicles was also reflected in the phenotype of malE+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 microM), but apparently not from millimolar concentrations present in maltose minimal medium . We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild-type MBP than did the wild-type FGK2 complex.(ABSTRACT TRUNCATED AT 250 WORDS) J Surg Res, 1992 Aug, 53(2), 170 - 4 Lipopolysaccharide-induced stimulation of alveolar macrophage opsonin-independent phagocytosis; Cardozo C et al.; Alveolar macrophage (AM) opsonin-independent phagocytosis may be an important process by which bacteria are cleared from the airspaces . Although it has been shown that clearance of bacteria from the lung is altered in association with endotoxemia, the effect of endotoxemia on opsonin-independent phagocytosis is unknown . Because alterations in AM opsonin-independent phagocytosis could potentially influence bacterial clearance in the lung, we studied the effects of the intravenous injection of Escherichia coli lipopolysaccharide (LPS) into rats on AM opsonin-independent phagocytosis of latex particles . AM obtained by bronchoalveolar lavage (BAL) 2 or 4 hr after injection of LPS demonstrated phagocytosis comparable to AM from control animals . In contrast, AM obtained 12 hr after injection of LPS demonstrated a nearly threefold increase in phagocytosis . Depletion of serum complement with Naja naja cobra venom factor (CVF) did not alter LPS-induced augmentation of phagocytosis . Furthermore, phagocytosis of AM obtained by BAL 2 or 12 hr after activation of complement by injection of CVF was not significantly different from that of AM from control animals, indicating that complement activation alone was not sufficient to stimulate opsonin-independent phagocytosis . These observations suggest that opsonin-independent phagocytosis may represent an important route of bacterial uptake by AM following endotoxemia, and that LPS-induced stimulation of AM opsonin-independent phagocytosis may occur independently of activation of complement by LPS. J Reprod Fertil, 1992 Aug, 95(3), 649 - 56 Release of two-cell block by reduction of protein disulfide with thioredoxin from Escherichia coli in mice; Natsuyama S et al.; The development of mouse pronuclear-stage embryos in media containing various concentrations of thioredoxin was monitored and the influence of antithioredoxin immunoglobulin G (IgG) and heat-treated thioredoxin on the thioredoxin-induced effects was evaluated . A significant increase in the number of four-cell embryos (76.3%) and blastocysts (37.3%) was observed when embryos were cultured in the medium containing 50 micrograms thioredoxin ml-1 compared with the rates (55.8 and 3.8%, respectively) in the basic medium . The number of blastocysts increased significantly to a maximum of 70.2% at 500 micrograms ml-1 . The biological activity of thioredoxin was evident after dialysis, but was markedly impaired by the addition of anti-thioredoxin IgG to the culture medium . Treatment at 60 degrees C for 5 min did not affect the enzymatic and biological activity of thioredoxin . More severe heat treatment (121 degrees C for 30 min) attenuated the enzymatic activity to 40% of its initial value and reduced the biological activity (number of blastocysts, from 77.8 to 51.6%) . These results indicate that the effect of thioredoxin on the two-cell block is due to the thioredoxin molecule itself, and suggest that disulfide formation within or between proteins resulting from oxidative stress is one of the major causes of the two-cell block. Int J Oral Maxillofac Surg, 1992 Aug, 21(4), 236 - 8 Necrotizing fasciitis of the neck and chest . Report of a case; Muto T et al.; A case is presented of necrotizing fasciitis of the neck and chest characterized by rapid progressive necrosis of subcutaneous tissue, fascia and skin . The diagnosis and management is discussed. J Dairy Sci, 1992 Aug, 75(8), 2119 - 25 Effect of interferon-gamma on the production of tumor necrosis factor during acute Escherichia coli mastitis; Sordillo LM et al.; The effects of recombinant interferon-gamma on the production of tumor necrosis factor in 10 dairy cows with Escherichia coli mastitis were determined . Prophylactic administration of recombinant interferon-gamma prior to the experimental E . coli challenge was effective in modifying the production of endogenous tumor necrosis factor during acute stages of disease . Elevated tumor necrosis factor concentrations were especially evident in cows that developed severe clinical symptoms and eventually died from endotoxemia . These results indicate that both milk and sera tumor necrosis factor concentrations are associated closely with the manifestation of peracute signs of coliform mastitis and are important factors contributing to morbidity and mortality of endotoxic shock . Pretreatment of cows with recombinant interferon-gamma possibly may down-regulate the generation of this potent endogenous inflammatory mediator within infected quarters . Controlling severe inflammation with recombinant interferon-gamma may prevent the tremendous loss in milk production and death that often accompany acute coliform mastitis during the periparturient period. J Biochem (Tokyo), 1992 Aug, 112(2), 175 - 82 Tissue-specific regulation of renin-binding protein gene expression in rats; Tada M et al.; Rat gene for renin-binding protein (RnBP) was shown to be expressed in the kidney, adrenal gland, brain, lung, spleen, ovary, testis, and heart . On sodium depletion and captopril administration, the rat showed a marked increase in the adrenal RnBP mRNA level and a slight decrease in the kidney RnBP mRNA level . In two-kidney, one clip hypertensive rats, the RnBP mRNA levels of the clipped and contralateral kidneys were unchanged and also its adrenal mRNA level was maintained at the control level . The recombinant rat RnBP was synthesized in Escherichia coli cells and purified to apparent homogeneity . The RnBP existed as a homodimer and formed a heterodimer with rat renin to inhibit renin activity extensively . Intravenous injection of the RnBP into rats resulted in a rapid and strong inhibition of plasma renin activity, which persisted at least for 2 h . These results suggest that the expression of RnBP gene in the kidney and adrenal gland is regulated independently, and the function of RnBP is related to electrolyte homeostasis, probably through the interaction with renin. Eur J Haematol, 1992 Aug, 49(2), 63 - 6 Incidence of thrombotic complications in adult patients with acute lymphoblastic leukaemia receiving L-asparaginase during induction therapy: a retrospective study . The GIMEMA Group; Gugliotta L et al.; The incidence of thrombotic complications chronologically related to L-asparaginase administration is retrospectively analyzed in 238 adult ALL patients treated according to the GIMEMA protocol ALL 0288 . The patients (126 males and 112 females, aged 12-68 years, median 29) received E . coli L-asparaginase (L-ase) in the induction phase at a dosage of 6000 U/m2/day x 7 d starting on d 15, as well as vincristine, prednisone, daunorubicin and cyclophosphamide, the last-named by random 1:1 . Ten patients (4.2%) developed thrombotic complications 5-15 d (median 11 d) after the start of L-ase treatment . The thrombotic events, which were lethal in 5 patients, involved the cerebral sinus (5 cases), the cerebral arteria (2 cases), the portal vein (1 case), the pulmonary district (1 case), and a deep vein in the lower extremity (1 case) . The occurrence of these complications was not related to the general thrombotic risk factors, nor to the main clinical and laboratory data registered at diagnosis and immediately before the start of L-asparaginase treatment . The present study documents for the first time in a sufficiently large series of adult ALL patients that the incidence and the severity of thrombotic events related to L-ase administration are relevant and need further consideration. Zentralbl Hyg Umweltmed, 1992 Aug, 193(2), 99 - 105 An enzymatic procedure for the confirmation of total coliforms and Escherichia coli enumeration from water; Giammanco G et al.; A new procedure for the confirmation of total coliforms and Escherichia coli from presumptive most probable number (MPN) test is described . The procedure utilizes an enzymatic test apparatus composed of a couple of devices, one charged with ONPG-medium for the detection of beta-galactosidase, the other with PNPG-medium for the detection of beta-glucuronidase . The results obtained demonstrate that the procedure is sensitive, specific and accurate . Further, it presents some advantages from the practical point of view: the cost of the devices is relatively low, their use is extremely simple and short time consuming, a single thermostatic apparatus adjusted at 36 degrees C for the incubation of the devices is only required, the results can be obtained after 18 h without any apparatus for the lecture (e.g . UV apparatus), they are not submitted to subjective interpretation. Eur J Biochem, 1992 Aug 1, 207(3), 1109 - 14 Interaction between the carboxyl groups of Asp127 and Asp129 in the active site of Escherichia coli phosphofructokinase; Laine R et al.; The pH dependence of the enzymic properties of the phosphofructokinase from Escherichia coli was compared to those of two mutants in which one carboxyl group of the active site has been removed from either Asp127 or Asp129 . All measurements of activity were made in the presence of allosteric activator ADP or GDP to eliminate any cooperative process . Asp129 is a crucial residue for the activity of phosphofructokinase since its conversion to Ser decreases the catalytic activity by 2-3 orders of magnitude in both the forward and reverse reactions, but the ionization of Asp129 is not directly related the pH dependence of phosphofructokinase activity . This pH dependence is however modified by the Asp129----Ser mutation, which decreases the pK of another residue, Asp127, by as much as pH of 1.5 . The side chain of Asp127 has the catalytic role proposed earlier: its deprotonated form acts as a base in the forward reaction, and its protonated form acts as an acid in the reverse reaction . The protonated form of Asp127 is also required for the binding of fructose 1,6-bisphosphate . The electrostatic interaction between the carboxyl groups of Asp127 and Asp129 seems different in free phosphofructokinase to that in enzyme/substrate complexes, suggesting that a conformational change occurs upon substrate binding . The pH dependence of phosphofructokinase activity involves one other ionizable group with a pK of approximately 6 which does not belong to the side chains of Asp127 or Asp129. Biochem J, 1992 Aug 1, 285 ( Pt 3), 881 - 8 Nucleotide sequence, organization and characterization of the atp genes and the encoded subunits of Mycoplasma gallisepticum ATPase; Rasmussen OF et al.; The nucleotide sequence of a 7.8 kbp DNA fragment from the genome of Mycoplasma gallisepticum has been determined . The fragment contains a cluster of nine tightly linked genes coding for the subunits of the M . gallisepticum ATPase . The gene order is I (I-subunit), B (a-subunit), E (c-subunit), F (b-subunit), H (delta-subunit), A (alpha-subunit), G (gamma-subunit), D (beta-subunit) and C (epsilon-subunit) . Two open reading frames were identified in the flanking regions; one (ORFU), preceding the I gene, encodes at least 110 amino acids and the other (ORFS), following the C gene, encodes at least 90 amino acids . The deduced amino acid sequences of the various subunits are presented and discussed with regard to the structure, function and differing sensitivity of the M . gallisepticum enzyme to dicyclohexylcarbodiimide and aurovertin . The alpha- and beta-subunits of the F1 portion are well conserved (51% and 65% identity with those of Escherichia coli), whereas the gamma-, delta- and epsilon-subunits, as well as the F0-subunits, show a low percentage identity . Nonetheless, the secondary structure of the F0-subunits show a high degree of similarity to the corresponding subunits of E . coli . Two very strong potential amphipathic alpha-helices are predicted in the delta-subunit and the N-terminus of the b-subunit contains two hydrophobic helical stretches . The possible roles of these structural properties in the close association of the F1 and F0 multisubunit complexes among mycoplasmas are discussed. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7070 - 4 Human T-cell lymphotropic virus type I (HTLV-I) transcriptional activator, Tax, enhances CREB binding to HTLV-I 21-base-pair repeats by protein-protein interaction; Zhao LJ et al.; HTLV-I Tax protein activates transcription from three 21-base-pair (bp) repeat sequences in the viral enhancer . The HTLV-I 21-bp repeat contains a TGACGT motif that is homologous to the cAMP-responsive element (CRE) and crucial for tax transactivation . Tax exhibits marginal affinity for DNA but rather interacts with cellular CRE-binding proteins to enhance their affinity for the HTLV-I 21-bp repeats . Using the HTLV-I 21-bp repeat and Jurkat T-lymphocyte nuclear extract in a gel electrophoretic mobility-shift assay, we previously detected three protein-DNA complexes that are specific for the CRE in the 21-bp repeat (complexes I, II, and IV) . Complexes I and II but not IV interacted with Tax . We now show that complexes I, II, and IV are composed of CREB (CRE binding protein) homodimer, CREB/ATF-1 (activating transcription factor 1) heterodimer, and ATF-1 homodimer, respectively . Tax stabilizes complexes I and II via a direct interaction with the CREB moiety . In the absence of DNA, CREB and Tax continue to form a complex that can be immunoprecipitated by a Tax-specific antibody . These results suggest that one mechanism by which Tax activates transcription may be mediated through the direct interaction with CREB homodimer and/or CREB/ATF-1 heterodimer to stabilize their assembly on the Tax-responsive CRE motifs in the HTLV-I enhancer. EMBO J, 1992 Aug, 11(8), 3031 - 8 Lac repressor with the helix-turn-helix motif of lambda cro binds to lac operator; Kolkhof P et al.; Lac repressor, lambda cro protein and their operator complexes are structurally, biochemically and genetically well analysed . Both proteins contain a helix-turn-helix (HTH) motif which they use to bind specifically to their operators . The DNA sequences 5'-GTGA-3' and 5'-TCAC-3' recognized in palindromic lac operator are the same as in lambda operator but their order is inverted form head to head to tail to tail . Different modes of aggregation of the monomers of the two proteins determine the different arrangements of the HTH motifs . Here we show that the HTH motif of lambda cro protein can replace the HTH motif of Lac repressor without changing its specificity . Such hybrid Lac repressor is unstable . It binds in vitro more weakly than Lac repressor but with the same specificity to ideal lac operator . It does not bind to consensus lambda operator. Protein Expr Purif, 1992 Aug, 3(4), 301 - 7 Comparative analysis of native and cysteine-deficient HIV-1 reverse transcriptase; Fischer M et al.; To study the subunit structure and the active site of human immunodeficiency virus reverse transcriptase (RT), the enzyme was expressed in E . coli and purified to homogeneity in large quantities . The recombinant enzyme consists of two major polypeptides of 66,000 and 53,000 Da in equimolar amounts and a minor species of 51,000 Da . Amino acid sequence analysis of the recombinant proteins revealed that the amino termini of the two major subunits are identical to that of the virion-derived enzyme . The two cysteinyl residues at positions 38 and 280 in the RT amino acid sequence were replaced by alanine in an attempt to elucidate the role of the sulfhydryl groups in RT enzyme activities, heterodimer formation, and intrasubunit linkage . The results reported here show that the two cysteinyls are dispensable and their absence in the amino acid sequence of the reverse transcriptase does not affect DNA polymerase or ribonuclease H enzyme activities or the formation of heterodimer structures . Furthermore, inhibitors of polymerase activity such as 3-azidothymidine triphosphate, dideoxythymidine triphosphate, and tetrahydroimidazo{4,5,1-JK}{1,4}benzodiazepens (1H)-one are equally effective on the mutant containing no cysteinyl residues and the wild-type enzyme. Hybridoma, 1992 Aug, 11(4), 391 - 407 Biochemical characterization of 25 distinct carcinoembryonic antigen (CEA) epitopes recognized by 57 monoclonal antibodies and categorized into seven groups in terms of domain structure of the CEA molecule; Kuroki M et al.; The chemical nature of 25 distinct carcinoembryonic antigen (CEA) epitopes, which were recognized by 57 different monoclonal antibodies and categorized into 7 groups (Groups A to G) in terms of domain structure of the CEA molecule, was analyzed and the findings obtained were compared with the results of our previous studies using recombinant CEA proteins . All 21 epitopes of Groups A to F defined by 48 MAbs were resistant to periodate oxidation and were to a greater or lesser extent retained after deglycosylation of CEA, indicating that they are all protein in nature . The 21 epitopes were detected in recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells . Seven of the 21 epitopes of protein nature were partially or completely sensitive to reduction and alkylation of CEA and not detected or only slightly revealed in the recombinant CEA proteins expressed in E . coli, indicating that those epitopes are dependent on the tertiary structure of the peptide chain, which is formed by disulfide bonds . All 4 epitopes of Group G defined by 9 MAbs were sensitive to mild periodate oxidation and deglycosylation, but resistant to reduction and alkylation and to digestion with pepsin or pronase, indicating that those 4 epitopes are carbohydrate in nature . Although none of the 4 epitopes of Group G were detected in the recombinant CEA proteins expressed in E . coli, two were detected in those expressed in CHO cells . The biochemical studies reported here thus provide information as to the nature of the epitopes on the CEA molecule and help form the basis for selecting the anti-CEA MAbs for use in biological study and potential clinical applications of CEA. Neuron, 1992 Aug, 9(2), 307 - 13 Interaction of charybdotoxin with permeant ions inside the pore of a K+ channel; Park CS et al.; Charybdotoxin (CTX) blocks high conductance Ca(2+)-activated K+ channels by binding to a receptor site in the externally facing "mouth." Toxin bound to the channel can be destabilized from its site by K+ entering the channel from the opposite, internal, solution . By analyzing point mutants of CTX expressed in E . coli, assayed with single Ca(2+)-activated K+ channels reconstituted into planar lipid bilayers, we show that a single positively charged residue of the peptide, Lys-27, wholly mediates this interaction of K+ with CTX . If position 27 carries a positively charged residue, internal K+ accelerates the dissociation rate of CTX in a voltage-dependent manner; however, if a neutral Asn or Gln is substituted at this position, the dissociation rate is completely insensitive to either internal K+ or applied voltage . Position 27 is unique in this respect; charge-neutral substitutions made at other positions fail to eliminate the K+ destabilization phenomenon . The results argue that CTX bound to the channel positions Lys-27 physically close to a K(+)-specific binding site on the external end of the conduction pathway and that a K+ ion occupying this site destabilizes CTX via direct electrostatic repulsion with the epsilon-amino group of Lys-27. Gene, 1992 Aug 1, 117(1), 61 - 6 Sequences encoding the protein and RNA components of ribonuclease P from Streptomyces bikiniensis var . zorbonensis; Morse DP et al.; The genes encoding the RNA (rnpB) and protein (rnpA) subunits of ribonuclease P (RNase P) of Streptomyces bikiniensis var . zorbonensis have been cloned by complementing the temperature-sensitive growth phenotype of Escherichia coli strains that carry mutations in these genes . The rnpB sequence of S . bikiniensis includes new covariations that lead to refinement of the previous secondary structure models for RNase P RNAs . The deduced amino acid sequence of S . bikiniensis RNase P is conserved with that of other known RNase P proteins only to a limited extent . Immediately upstream from rnpA is an open reading frame that codes for the highly conserved ribosomal protein, L34 . This same gene arrangement occurs in all bacteria studied to date. Surgery, 1992 Aug, 112(2), 467 - 74 Influence of hypercortisolemia on the acute-phase protein response to endotoxin in humans; Rock CS et al.; BACKGROUND . The response to systemic infection includes the coordinated appearance of hepatic acute-phase proteins, the production of which may be influenced by a counterregulatory hormonal background . This study sought to assess the potential for hypercortisolemic conditions to influence fibrinogen kinetics and other acute-phase protein responses in humans with endotoxemia . METHODS . Eleven hospitalized healthy male volunteers underwent two separate determinations of fibrinogen kinetics, one baseline and one after administration of endotoxin (2 ng/kg intravenously; lot EC-5) . Seven volunteers were studied without hormonal manipulation and four in the presence of a hypercortisolemic background (hydrocortisone infusion, 3 micrograms/kg/min) . Fibrinogen fractional synthetic rates were estimated from the incorporation of orally administered 15N-glycine, and fibrinogen, C-reactive protein, cortisol, tumor necrosis factor-alpha, and interleukin-6 levels were also determined . RESULTS . The presence of an antecedent hypercortisolemic background resulted in an attenuated interleukin-6 response, as well as decreased fibrinogen synthesis and C-reactive protein appearance . CONCLUSIONS . The current data suggest that glucocorticoid hormonal influences are of importance in the regulation of endotoxin-induced cytokine and acute-phase protein responses. Eur J Immunol, 1992 Aug, 22(8), 1983 - 7 A method for rapid screening of recombinant proteins for recognition by T lymphocytes; Hickling JK et al.; A simple, cost-effective method is described that allows rapid screening of recombinant protein sequences for their ability to stimulate T cells . Individual microcultures of E . coli each expressing a gene product or peptide sequence fused to protein A are grown in 96-well plates . Following lysis of the bacteria, the fusion peptide is readily captured with immobilized immunoglobulin in tissue culture wells . No further purification is required . T lymphocytes plus appropriate antigen-presenting cells are added directly to the wells and assayed for proliferation . The DNA in bacteria from wells stimulating T cell proliferation is then sequenced . The technique allows rapid mapping of T cell epitopes by facilitating screening of truncation mutants without extensive purification . Described here is a further application of the technique to study monosubstituted analogues of a known T cell epitope. EMBO J, 1992 Aug, 11(8), 3105 - 16 Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues; Dontsova O et al.; Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio-uridine residues at selected positions . After binding to the ribosome in the presence of cognate tRNA, the thio-U residues were activated by UV irradiation and the resulting sites of cross-linking to 16S RNA analysed . Three distinct cross-links were consistently observed: (i) from position '+6' of the mRNA (the 3'-base of the A-site codon) to base 1052 of 16S RNA; (ii) from position '+7' of the mRNA to base 1395; and (iii) from '+11' to base 532 . Individual yields of the cross-links were strongly dependent on the particular mRNA sequence in each case . The '+11/532' and '+6/1052' cross-links were always entirely tRNA-dependent, whereas the '+7/1395' cross-link was observed at lower intensity in the absence of tRNA . In the presence of a second (A-site bound) tRNA the +6/1052 cross-link was markedly reduced . A cross-link to the 1050 region was again observed when a message carrying a thio-U at position '+9' was translocated on the ribosome so as to bring the thio-U to position +6 . Taken together, the data are incompatible with some current models both for the three-dimensional arrangement of 16S RNA and for the orientation of the tRNA-mRNA complex in the ribosome. Arch Biochem Biophys, 1992 Aug 1, 296(2), 685 - 90 Epitope mapping of monoclonal antibodies to the Escherichia coli F1 ATPase alpha subunit in relation to activity effects and location in the enzyme complex based on cryoelectron microscopy; Aggeler R et al.; The interaction of Escherichia coli F1 ATPase (ECF1) with several different monoclonal antibodies (mAbs) specific for the alpha subunit has been examined . The epitopes for each of the mAbs have been localized by using molecular biological approaches to generate fragments of the alpha subunit . The binding of several of the mAbs has also been examined by cryoelectron microscopy of ECF1 Fab complexes . One of the mAbs, alpha II, bound in the region Asn 109-Val 153 without affecting ATPase activity . Most of the mAbs bound in the C-terminal third of the alpha subunit . MAb alpha 1 bound between residues Gln 443 and Trp 513 . This mAb activated ATPase activity and was visualized in cryoelectron microscopy, superimposed on the alpha subunit, indicating that the epitope was on the top or bottom of ECF1 in the hexagonal projection . Other mAbs to the C-terminus, including alpha D which also activated the enzyme, reacted between Gly 371 and Trp 513 but failed to bind to small overlapping fragments within this sequence . The epitopes for these mAbs are probably formed by the folded polypeptide which occurs only in Western analysis when long stretches of the alpha subunit are present, suggesting that the C-terminus of alpha is a self-folding domain . In cryoelectron microscopy, Fab fragments for alpha D were seen extending from the sides of the ECF1 complex in hexagonal projection. Appl Microbiol Biotechnol, 1992 Aug, 37(5), 609 - 14 Protein compositional analysis of inclusion bodies produced in recombinant Escherichia coli; Rinas U et al.; Culture conditions favouring the simultaneous formation of soluble protein and inclusion bodies (IBs) were chosen for producing the cytoplasmic protein beta-galactosidase or the periplasmic protein TEM-beta-lactamase . Soluble and insoluble cell fractions of Escherichia coli producing either beta-galactosidase or TEM-beta-lactamase were analyzed by one- and two-dimensional gel electrophoresis and subsequent silver staining or immunodetection of the recombinant protein . The results show that truncated fragments of the recombinant protein were not present in the soluble cell fraction but accumulate in the IB fraction . The presence of other cellular, non-plasmid-encoded proteins in IB preparations such as the outer membrane proteins OmpF, OmpC, and OmpA or the ribosomal subunit proteins L7/L12 was attributed to co-precipitation of cell-debris-associated components . Protein-folding enzymes were not detected in IB preparations . The specificity of in-vivo protein association in the formation of IBs and its implication on protein purification is discussed. Biotechnology (N Y), 1992 Aug, 10(8), 900 - 4 A universal expression-purification system based on the coiled-coil interaction of myosin heavy chain; Wolber V et al.; We have constructed a series of Escherichia coli expression vectors that produce high yields of fusion proteins containing the C-terminal fragment of light meromyosin (LMM) from rabbit fast skeletal muscle . The fusion proteins retain the ability of LMM to form polymers in low salt and to be soluble in high salt . Thus they can be easily purified from bacterial extracts with a high salt-low salt extraction procedure and still retain their biochemical properties . We demonstrate the utility of this system for the heterologous production and simple purification of LMM fusions of p21H-ras, the neurofibromatosis type I protein and the Tat and protease proteins of HIV-1. Microb Pathog, 1992 Aug, 13(2), 161 - 6 Nucleotide sequences of the major subunits of F9 and F12 fimbriae of uropathogenic Escherichia coli; Garcia E et al.; An Eco RV-Cla I fragment containing the gene encoding the F9 fimbrial subunit of the human uropathogenic Escherichia coli strain C1018 and a PstI-PstI fragment containing the F12 fimbrial subunit gene of the dog uropathogenic strain 1442 have been cloned and the nucleotide sequence of the fragments determined . The structural gene of the F9 fimbriae (FniA) codes for a protein of 165 amino acid residues with a signal peptide of 25 amino acids . The F12 fimbrial gene (FtwA) codes for a protein of 155 amino acids which is preceded by a single peptide of 21 amino acids . The amino acid sequences of the FniA and FtwA proteins deduced from the nucleotide sequence were compared with sequences of other known P-fimbrial subunit proteins . As expected, most differences between the various proteins were found in the hypervariable regions defined by van Die et al . (1987) . The N-terminal sequence of the FtwA protein differs from the one published by Klemm et al . (1983) . FtwA contains two deletions found in comparison to the other fimbrial subunits. Mol Microbiol, 1992 Aug, 6(16), 2225 - 42 Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue-specific adhesive properties; Marklund BI et al.; Escherichia coli strains bind to Gal alpha 1-4Gal-containing glycolipids via P pili-associated G-adhesins . Three functional classes of adhesins with different binding specificities are encoded by conserved G-alleles . We suggest that the Class I papG-allele of strain J96 is a novel acquisition possibly introduced via horizontal gene transfer into one of the two P pili gene clusters carried by this strain . Closely related strains in the ECOR collection of natural E . coli isolates carry either a Class II or a Class III G-adhesin . Data indicate that genetic exchanges involving either entire pap or prs gene clusters or individual pap/prs genes have occurred . We propose that the retention and spread of pap/prs DNA among E . coli is the result of selection pressure exerted by mammalian intestinal isoreceptors. Gut, 1992 Aug, 33(8), 1050 - 6 Intestinal hypersecretion of the refed starved rat: a model for alimentary diarrhoea; Young A et al.; Fluid transport was gravimetrically measured in vivo in the duodenum, jejunum, and ileum of anaesthetised fed, 72 hour starved and 72 hour starved rats refed for up to five days after starvation . Basal unstimulated fluid transport was monitored by instilling 0.9% NaCl into the lumen and measuring the gain or loss in weight of the closed intestinal loop . Fluid was absorbed in all the areas of the intestine in the fed rats . Increasing basal fluid absorption was observed in the duodenum over the three days of starvation but in the jejunum there was no significant change . In the ileum, the pattern was very different, on day 1 the fluid was absorbed but on days 2 and 3 there was an increasing secretion of fluid . Refeeding the rats with their normal diet restored the basal absorption of fluid in the duodenum within 24 hours, had no effect in the jejunum but in the case of the ileum the hypersecretion of fluid observed in the day 3 starved rat was maintained on day 1 of refeeding, increased further on day 2, decreased on day 3 but returned to absorption on day 4 . The normal absorption was restored to the ileum on day 5 of refeeding . Fluid secretion was induced in all the rat groups by bethanechol (ip 60 micrograms/kg bw) a stable cholinergic agonist, PGE2 (ip 10 micrograms/kg (bw) and E coli STa (luminally instilled, 500 ng/ml) a secretory enterotoxin . All the secretagogues gave enhanced secretion compared with the fed by day 2 of starvation which increased considerably on day 3 . Refeeding returned their secretion back to the fed level in the duodenum within 24 hours, in the jejunum within 48 hours but in the ileum their induced secretion on day 2 of refeeding was greater than that of the day 2 of refeeding was greater than that of day 3 starved and took until day 4 to return to the fed levels for behanechol and PGE2 and until day 5 for E . coli STa . This behaviour of rat small intestine showing even greater hypersecretion in the refed state than the starved mimics the human condition of alimentary induced diarrhoea where incautious feeding of starved humans induces severe, often lethal diarrhoea . The refed starved rat appears to be a possible model for this condition. Mol Gen Genet, 1992 Aug, 234(2), 211 - 6 The acu-1 gene of Coprinus cinereus is a regulatory gene required for induction of acetate utilisation enzymes; Maconochie MK et al.; We have isolated a gene from Coprinus cinereus which cross-hybridises to the facA and acu-5 genes of Aspergillus nidulans and Neurospora crassa, respectively . These genes encode acetyl-CoA synthetase, an enzyme which is inducible by acetate and required for growth on acetate as sole carbon source . We have designated the C . cinereus gene acs-1 and have used transformation to demonstrate its functional homology to the ascomycete genes by complementation of an N . crassa acu-5 mutation . The acs-1 gene has never been identified by mutation; mutations leading to loss of acetyl-CoA synthetase function map to another gene, acu-1 . Using Northern analyses we have shown that acu-1 has a regulatory function that is required for acetate-induced transcription of acs-1 and of another acetate utilisation gene, acu-7, the isocitrate lyase structural gene. Am J Hum Genet, 1992 Aug, 51(2), 386 - 95 Molecular abnormalities of a human glucose-6-phosphate dehydrogenase variant associated with undetectable enzyme activity and immunologically cross-reacting material; Maeda M et al.; Among a large number of glucose-6-phosphate dehydrogenase (G6PD) variants associated with different severity of clinical manifestations, enzyme deficiency, and kinetic abnormalities found in humans, only one variant exhibits no measurable activity and lacks an immunologically cross-reacting material in blood cells and other tissues . The mRNA content of the patient's lymphoblastoid cells was found to be normal, and the size of mRNA was also normal (i.e., approximately 2.4 kb) . Western blot hybridization indicated that the patient's cells did not produce cross-reacting material . The variant mRNA was reverse transcribed and amplified by PCR . Nucleotide sequencing of the variant cDNA showed the existence of three nucleotide base changes, i.e., a C----G at nucleotide 317 (counting from adenine of the initiation codon), which should cause Ser----Cys substitution at the 106th position (counting from the initiation Met); a C----T at nucleotide 544, which induces the Arg----Trp at the 182d position; and a C----T at nucleotide 592, which induces Arg----Cys at the 198th position of the protein . The existence of three mutation sites was confirmed by sequencing of selected regions of the variant gene . No base deletion or frameshift mutation was found in the variant cDNA . No nucleotide change was detected in the extended 5' region, which included the most distal cap site . When the variant cDNA was expressed in Escherichia coli, the G6PD activity was approximately 2% of that expressed by the normal cDNA, and cross-reacting material was undetectable . However, when the variant mRNA was expressed in the in vitro translation system of rabbit reticulocytes, the variant protein was produced . These results suggest that extremely rapid in vivo degradation or precipitation of the variant enzyme induced by the three amino acid substitutions could be the major cause of the molecular deficiency. J Bacteriol, 1992 Aug, 174(15), 5145 - 8 Glycerol-induced unraveling of the tight helical conformation of Escherichia coli type 1 fimbriae; Abraham SN et al.; Glycerol was found to unravel the helical conformation of Escherichia coli type 1 fimbriae without appreciable depolymerization . The linearized fimbrial polymers have a diameter of 2 nm, react strongly with a monoclonal antibody directed at an inaccessible epitope on native fimbriae, and display greater mannose-binding activity and trypsin sensitivity than native fimbriae . Removal of glycerol by dialysis results in spontaneous reassembly of the linear polymers into structures morphologically, antigenically, and functionally indistinguishable from native fimbriae. Gene, 1992 Aug 1, 117(1), 91 - 7 Cloning and expression of the Acanthamoeba castellanii gene encoding transcription factor TFIID; Wong JM et al.; We have cloned and characterized the cDNA encoding transcription factor TFIID from the eukaryote, Acanthamoeba castellanii . The gene occurs as a single species, encodes one mRNA and, presumably, a single protein . A . castellanii TFIID contains two recognizable domains, a nonconserved N-terminal domain and a highly conserved C-terminal domain . Similarities between the amino acid (aa) sequences of TFIID from several organisms are also found within the N-terminal 78 aa, suggesting a potential role in TFIID function . Full-length or truncated A . castellanii TFIID produced in Escherichia coli binds to a TATA box and is able to activate transcription in a TFIID-depleted HeLa cell extract, but the C-terminal 180-aa domain was found to be less efficient in these reactions. Curr Genet, 1992 Aug, 22(2), 85 - 91 Functional elements of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase; Hata Y et al.; Analysis was made of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase . Northern blots using a glucoamylase cDNA as a probe indicated that the amount of mRNA corresponding to the glaA gene increased when expression was induced by starch or maltose . The promoter region of the glaA gene was fused to the Escherichia coli uidA gene, encoding beta-glucuronidase (GUS), and the resultant plasmid was introduced into A . oryzae . Expression of GUS protein in the A . oryzae transformants was induced by maltose, indicating that the glaA-GUS gene was regulated at the level of transcription in the presence of maltose . The nucleotide sequence 1.1 kb upstream of the glaA coding region was determined . A comparison of the nucleotide sequence of the A . oryzae glaA promoter with those of A . oryzae amyB, encoding alpha-amylase, and A . niger glaA showed two regions with similar sequences . Deletion and site-specific mutation analysis of these homologous regions indicated that both are essential for direct high-level expression when grown on maltose. Protein Sci, 1992 Aug, 1(8), 1032 - 49 Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins; Park K et al.; The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data . Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins . The convex constraint analysis algorithm (Perczel, A., Hollosi, M., Tusnady, G., & Fasman, G . D., 1991, Protein Eng . 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights . The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy . For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation . The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively. Zhonghua Yi Xue Za Zhi, 1992 Aug, 72(8), 468 - 70, 509 {Dynamic changes of pulmonary glucocorticoid receptor in endotoxin-induced lung injury}; Liu L; The changes of maximal binding capacity (Bmax) of glucocorticoid receptor (GCR) in the endotoxin-induced lung injury were observed by using radioligand binding assay . The content of glucocorticoid (GC) and the activity of phospholipase A2 (PLA2) were also measured . The results showed that the level of GCR in lung cytoplasma decreased continuously after endotoxin infusion, and its affinity decreased in the late phase while PLA2 activity markedly increased . There was a negative correlation between the Bmax of GCR and PLA2 activity (r = -0.87, P < 0.01) . These findings indicated that there is a secondary alteration in GCR during endo toxin-induced lung injury, and that the GC hypofunction caused by reduced GCR binding capacity may be of importance in the development of ARDS. Protein Expr Purif, 1992 Aug, 3(4), 295 - 300 Purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase, from a recombinant strain of Escherichia coli; Roessner CA et al.; The gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of Catharanthus roseus has been cloned into an inducible Escherichia coli expression vector using an expression cassette polymerase chain reaction technique . Induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies . Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell culture . The recombinant enzyme has good activity (approximately 30 nkat/mg). J Biomol Struct Dyn, 1992 Aug, 10(1), 83 - 96 Locating binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene using restriction enzyme inhibition assays; Mallamaci MA et al.; Restriction enzyme inhibition studies have been employed to map the locations of high affinity binding sites of the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) on pBR322, phiX174 and SV40 DNAs . Bound carcinogen levels were kept low (less than 20 bound AAF moieties per DNA molecule) in order to observe only the binding to the high affinity sites . Inhibition of certain restriction enzymes was observed in a limited number of locations on these DNAs . Inhibition increased as bound AAF increased and the particular restriction enzymes inhibited varied with location . On all three DNAs, activities of these enzymes was not affected in other locations . Comparison of the sequences at the sites of inhibition on the three DNAs indicates that all sites have common sequence elements: the presence of either the sequence T(C/G)TT(G/C) or the sequence T(G/C)CTT(G/C). Virus Genes, 1992 Aug, 6(3), 301 - 6 Overexpression and purification of enzymatically active recombinant integrase protein of Rous sarcoma virus; Marczinovits I et al.; The carboxy-terminal domain of polymerase gene of Rous sarcoma virus was cloned into an expression vector under the control of lac regulatory elements, resulting in the plasmid pMF1413 . Upon isopropyl-beta-D-thiogalactopyranoside induction, viral integration (IN) protein was expressed in large quantity in Escherichia coli . The expressed recombinant protein was prepurified by successive washing of the bacterial pellet with 0.1 M NaCl and detergents . Further purification was performed in high yield by standard chromatography methods . The purified enzyme revealed selective DNA cleaving activity on supercoiled plasmid with the LTR-LTR junction fragment . The reaction was metal ion dependent, with a preference for Mn2+ over Mg2+, and showed substrate specificity at 1 mM MnCl2. Oral Microbiol Immunol, 1992 Aug, 7(4), 218 - 24 Direct and indirect effects of Porphyromonas gingivalis lipopolysaccharide on interleukin-6 production by human gingival fibroblasts; Yamazaki K et al.; We examined the production of interleukin-6 (IL-6) by human gingival fibroblasts (ATCC CRL 1292) stimulated with lipopolysaccharide (LPS) from Porphyromonas gingivalis and Escherichia coli, or supernatant of human peripheral blood adherent cell culture medium incubated in the presence of IL-1 and the same two LPS . Confluent monolayers of gingival fibroblasts were incubated with stimulants for 6 h at 37 degrees C in 5% CO2 and air . After removal of stimulants, the cell cultures were incubated for an additional 2 or 24 h in the same environment . At the end of the culture period, supernatants were collected and assayed for IL-6 activity by stimulatory IgG production with the human B-lymphoblastoid cell line CESS . The direct effect of LPS on IL-6 production by gingival fibroblasts was much weaker than the indirect one via IL-1 production by adherent cells . The stimulating effect of culture supernatants of adherent cells stimulated with LPS on IL-6 production by gingival fibroblasts was as effective as that of recombinant IL-1, when this latter was added at a concentration equivalent to that contained in the culture supernatant of adherent cells . These results suggest that, although gingival fibroblasts may be involved in the pathogenesis of chronic periodontal disease by the production of cytokines, such a role may not result from a direct stimulation by periodontopathic bacteria . The phenomenon is more likely to be mediated indirectly by IL-1 produced by infiltrating inflammatory cells. J Appl Bacteriol, 1992 Aug, 73(2), 136 - 43 Intergeneric conjugation in Thiobacillus versutus; Read DL et al.; In plate matings with Escherichia coli HB101/pUW965::Tn5 (KmR) Thiobacillus versutus reacted as an efficient recipient, producing 10(-2) to 10(-3) kanamycin resistant (KmR) T . versutus exconjugants per donor cell . Analysis of agarose gels of plasmid DNA extracted from the exconjugants confirmed that the suicide vector pUW964 did not persist in the recipient, implying that the kanamycin resistance of the exconjugants is based on effective transposition of Tn5 in T . versutus as well as function of the E . coli kanamycin gene . Transfer was equally efficient when a nalidixate-resistant T . versutus mutant was used as recipient . Hybridization evidence for the presence of Tn5 was consistently negative . The significance of this anomalous result is discussed. Alcohol Clin Exp Res, 1992 Aug, 16(4), 788 - 94 Modulation of f-met-leu-phe induced chemotactic activity and superoxide production by neutrophils during chronic ethanol intoxication; Bautista AP et al.; Chronic alcohol consumption has been associated with increased migration of neutrophils into liver that could contribute to the development of alcoholic liver disease . Mild endotoxemia may be at least partially responsible for this condition since endotoxemia was shown to be present in virtually all chronic alcoholics . This study examines the release of superoxide anion and chemotactic activity by Kupffer cells and sequestered hepatic as well as blood neutrophils during chronic alcohol intoxication (16 weeks) alone, and following an intravenous injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) 3 hr before cell isolation . Chronic ethanol consumption increased the total neutrophil yield per liver, but did not change the f-met-leu-phe induced chemotactic activity by both hepatic and blood neutrophils . However, the combined insults of ethanol and LPS increased the chemotactic activity and superoxide anion generation by these cells . Plasma from ethanol-fed rats was highly chemotactic to syngeneic normal rat neutrophils . This activity was increased 1.75-fold in the plasma obtained from chronic ethanol plus endotoxin-injected rats . The chemotactic activity of Kupffer cells was not significantly modulated during ethanol intoxication plus endotoxin treatment . The f-met-leu-phe-induced superoxide anion release by Kupffer cells was enhanced after LPS treatment . Chronic ethanol consumption did not induce any effect on this parameter . These observations suggest that functional alterations in neutrophils during chronic ethanol intoxication may contribute to hepatic injury. Mol Cell Probes, 1992 Aug, 6(4), 319 - 25 Detection of antibodies to the E4 or E7 proteins of human papillomaviruses (HPV) in human sera by western blot analysis: type-specific reaction of anti-HPV 16 antibodies; Jochmus I et al.; To determine the cross-reactivity between early (E) proteins of different human papillomavirus (HPV) types, 346 serum samples were tested with E4 and E7 of HPV 16 . Two hundred and sixteen of them were also tested with HPV 1 E4, 21 with HPV 11 E4 and E7, and 109 with HPV 18 E4 and E7 . Viral fusion proteins were expressed in Escherichia coli and used as antigens in Western blot experiments . The sera were obtained from patients with HPV-associated genital lesions or cervical cancer, from renal transplant recipients and from patients hospitalized for reasons unrelated to HPV infections (the controls) . In contrast to findings relating to HPV 16 E4 specific antibodies, the prevalence of anti-HPV 1 E4 antibodies was not greater in renal transplant recipients than in the controls . In each age group of the control population more sera reacted with HPV 1 E4 than with HPV 16 E4 . Sera of patients with HPV-associated cervical diseases and cervical cancer reacted less frequently with HPV 11 E4 or E7 and HPV 18 E4 or E7, respectively, than with the corresponding HPV 16 proteins . Thirty of 117 HPV 16 E4 or E7 positive sera showed reactivity to the corresponding protein of either HPV 1, 11 or 18 . As demonstrated by cross-absorption experiments performed with 26 of the double-reacting sera, 24 contained two populations of antibodies reacting with proteins of different HPV types whereas only two contained cross-reacting antibodies . We concluded that in the majority of sera antibodies to the HPV 16 E4 and E7 proteins are type-specific. J Gen Microbiol, 1992 Aug, 138 ( Pt 8), 1729 - 36 p43, the protein product of the atypical insertion sequence IS900, is expressed in Mycobacterium paratuberculosis; Tizard ML et al.; The novel mycobacterial insertion sequence IS900 was analysed by coupled transcription-translation, of both strands independently, in a cell-free E . coli extract using an exogenous promoter . This revealed only one protein product, p43, as predicted from the nucleotide sequence . The protein was readily translated in recombinant E . coli, using the tac promoter, though it did not appear as a major product by SDS-PAGE analysis . A synthetic peptide was used to generate and affinity-purify a specific anti-p43 antibody, which clearly identified the protein in recombinant E . coli . p43 was relatively stable in exponential phase and stationery phase bacteria, though a 28 kDa processed form was seen to accumulate over a period of hours . Both forms appeared in the soluble fraction of the bacterial lysate . The anti-p43 antibody also identified p43, as a 28 kDa processed product, in Western blots of protein extracts from Mycobacterium paratuberculosis, indicating a level of expression which would be unusually high for a classical transposase . These data have important implications for the relationship between IS900 and its host. Pediatr Res, 1992 Aug, 32(2), 150 - 4 Presence of non-maternal antibodies in newborns of mothers with antibody deficiencies; Hahn-Zoric M et al.; To explain the mechanism for induction and production of specific antibodies found in the newborn already at birth, without previous known exposure to the antigen, we chose a model that presumably excluded the possibility of specific antibodies being transferred from the mother to the fetus . Specific IgG, IgA, and IgM antibodies against Escherichia coli and poliovirus antigens were determined with ELISA in serum, saliva, and amniotic fluid from hypogammaglobulinemic and IgA-deficient mothers as well as in cord serum, saliva, and meconium from their offspring . All the mothers lacked IgA and some also lacked IgM antibodies, which were found in their healthy newborns . The amniotic fluid from a hypogammaglobulinemic mother lacking IgA contained small amounts of IgA antibodies, which were also found in the neonate, suggesting a fetal origin . There was evidence for the presence of antiidiotypic antibodies to poliovirus in the cord sera . We propose that idiotypic and/or antiidiotypic IgG antibodies transferred via the placenta from the mother to the fetus can initiate specific immune responses seen in the newborn . Thus, it may be that transplacental IgG not only passively protects the newborn, but also actively primes the fetus during fetal life via its content of idiotypic and/or antiidiotypic antibodies. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7129 - 33 A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects; Apfel C et al.; Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs) . Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma . The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes . We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist . This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation . Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7056 - 9 Evidence for two promoters upstream of the pts operon: regulation by the cAMP receptor protein regulatory complex; Fox DK et al.; Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon . We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo . Promoter-reporter gene fusion studies identified two CRP.cAMP-dependent promoters (the previously identified P1 and another promoter, P0) upstream of ptsH . The crr promoters (P2) within ptsI may be negatively regulated by CRP.cAMP. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6741 - 5 Requirement for a conserved serine in both processing and joining activities of retroviral integrase; Katz RA et al.; Retroviruses encode a protein, the integrase (IN), that is required for insertion of the viral DNA into the host cell chromosome . IN alone can carry out the integration reaction in vitro . The reaction involves endonucleolytic cleavage near the 3' ends of both viral DNA strands (the processing step), followed by joining of these new viral DNA ends to host DNA (the joining step) . Based on their evolutionary conservation, we have previously identified at least 11 amino acid residues of IN that may be essential for the reaction . Here we report that even conservative replacements of one of these residues, an invariant serine, produce severe reductions in both the processing and joining activities of Rous sarcoma virus IN in vitro . Replacement of the analogous serine of the type 1 human immunodeficiency virus IN had similar effects on processing activity . These results suggest that this single conserved serine is a component of the active site and that one active site is used for both processing and joining . Replacement of this serine with certain amino acids resulted in a loss or reduction in DNA binding activities, while other replacements at this position appeared to affect later steps in catalysis . All of the defective Rous sarcoma virus INs were able to compete with the wild-type protein, which supports a model in which IN functions in a multimeric complex. J Gen Virol, 1992 Aug, 73 ( Pt 8), 1977 - 86 Analysis of the functional significance of amino acid residues in the putative NTP-binding pattern of the poliovirus 2C protein; Teterina NL et al.; The amino acid sequence of the poliovirus 2C protein contains two highly conserved stretches, GSPGTGKS136 and MDD177, which correspond to the consensus 'A' and 'B' motifs (GXXXXGKS/T and DD/E, respectively) found in nucleoside triphosphate-binding proteins . To assess the functional importance of these amino acid sequences, we changed conserved and non-conserved amino acids . The replacement of the non-conserved Thr133 residue with Ser or Ala did not markedly change the virus phenotype . Similarly, replacement of the non-conserved Pro131 residue by Ala did not abolish virus viability, but changes of this residue to Thr or Asn were not tolerated . No viable mutant could be isolated after transfection of cultured cells with transcripts mutated at the conserved Lys135, Ser136 or Asp177 residues . However, true revertants were selected from Arg135 and Ser135 mutants, from Glu177 and Gly177 mutants, and from Ala136 mutants . Thr136 mutants not only gave rise to true revertants, but also to two independent isolates of a suppressor mutant, Asn140----Tyr . All the lethal mutations resulted in severe inhibition of viral RNA synthesis in vivo, although no translational deficiency was detected in a cell-free system . This is the first direct evidence for the functional significance of the nucleoside triphosphate-binding pattern in the poliovirus 2C protein. J Clin Invest, 1992 Aug, 90(2), 533 - 6 Release of soluble receptors for tumor necrosis factor (TNF) in relation to circulating TNF during experimental endotoxinemia; Spinas GA et al.; Serial plasma samples from human volunteers obtained after intravenous administration of Escherichia coli endotoxin were analyzed for the presence of circulating soluble tumor necrosis factor receptors (sTNFR) . A four- to fivefold increase of type A (p75) and type B (p55) sTNFR was observed 3 h after endotoxin challenge . Pretreatment of the volunteers with ibuprofen before the injection of endotoxin resulted in a slight increase (3.87 +/- 0.2 vs . 3.27 +/- 0.3 ng/ml) and temporal shift of sTNFR-A release concurrent to a marked augmentation of TNF levels (603 +/- 118 vs . 338 +/- 56 pg/ml) as compared to the group without ibuprofen pretreatment . There was a significant correlation between peak sTNFR-A levels and peak TNF levels in the individual probands (r = 0.52, P = 0.04) . On the contrary, release kinetics and plasma concentrations of sTNFR-B were identical in both groups (7.38 +/- 0.69 vs . 7.44 +/- 0.33 ng/ml) and no correlation with individual TNF levels was observed . The amount of sTNFR liberated upon endotoxin challenge was not sufficient to block TNF-mediated cytotoxic effects . Our data indicate that the release in vivo of type A and type B sTNFR upon a short exposure to endotoxin is regulated differently. J Bacteriol, 1992 Aug, 174(16), 5479 - 81 Mutations causing aminotriazole resistance and temperature sensitivity reside in gyrB, which encodes the B subunit of DNA gyrase; Toone WM et al.; Certain mutations in gyrA and gyrB, the genes encoding the two subunits of DNA gyrase, are known to influence expression of the his operon (K . E . Rudd and R . Menzel, Proc . Natl . Acad . Sci . USA 84:517-521, 1987) . Such mutations lead to a decrease in tRNA(His) levels and consequently to an attenuator-dependent increase in his operon expression . This effect presumably is due to the dependence of the hisR promoter (hisR encodes tRNA(His) on supercoiling for maximal activity . We used a relaxed (Rel-) strain of Escherichia coli to isolate gyrB mutants by selecting for resistance to the histidine antimetabolite 3-amino-1,2,4-triazole and then screening for temperature-sensitive growth on rich medium . Rel- mutants, which generally have lower basal levels of ppGpp (a positive regulator of his operon transcription), are more sensitive than wild-type E . coli to aminotriazole . The chance of isolating spoT mutants, which can be selected with a similar procedure, was decreased by selecting in the presence of a multicopy plasmid that carries the wild-type spoT gene . Under these conditions, gyrB mutants were isolated preferentially . This scheme selects for loss of function of DNA gyrase, rather than for its alteration due to resistance to specific gyrase inhibitors, and thus a greater variety of gyrase mutations might be obtainable.
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