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| Carbohydr Res, 1995 Jul 21, 272(1), 73 - 90 NMR reinvestigation of two N-acetylneuraminic acid-containing O-specific polysaccharides (O56 and O24) of Escherichia coli; Torgov VI et al.; Structures for the N-acetylneuraminic acid (Neu5Ac)-containing O56 and O24 polysaccharides of Escherichia coli have been reported previously . During these studies unusual chemical shifts had been observed for the NMR signals for H-3eq and C-3 of the Neu5Ac residues of both polysaccharides . In further pursuing this phenomenon, we have reinvestigated the O56 and O24 polysaccharides as well as derived oligosaccharides by one- and two-dimensional NMR spectroscopy . The results showed that structures of both polysaccharides (PSs) had to be modified and formulated as {formula: see text} 2D ROESY spectra revealed a strong NOE between H-3eq of Neu5Ac and the protons of the side-chain sugar (H-3 and H-5 of alpha-D-Gal p in the O56 PS and H-3 of alpha-D-Glc p in the O24 PS) and also between H-3ax of Neu5Ac and H-3 of beta-D-Glc p in the main chain . This indicated a close spatial association of the seven-linked alpha-Neu5Ac and the side-chain residues alpha-D-Gal p (O56 PS) and alpha-D-Glc p (O25 PS), respectively . The strong long-range spatial contacts caused the unusual chemical shifts of H-3eq and C-3 of Neu5Ac. J Mol Biol, 1995 Jul 21, 250(4), 407 - 19 Structural changes in base-paired region 28 in 16 S rRNA close to the decoding region of the 30 S ribosomal subunit are correlated to changes in tRNA binding; Ericson G et al.; Escherichia coli 30 S ribosomal subunits undergo a reversible change under low monovalent or divalent cation concentration and become inactive in tRNA binding and 50 S subunit association . In the inactive form, 16 S rRNA base-pairs (921-922).(1395-1396) and (923-925).(1391-1393), which are part of region 28, are unstable and an alternate arrangement, (921-923).(1532-1534), is detected by psoralen photochemical crosslinking . Site-directed mutagenesis has been used to investigate whether changes in base-paired region 28 or the alternate secondary structure is responsible for the inactivity of the subunit . 30 S subunits with the substitution C1533A or with deletion of nucleotides 1534 to 1542 can still be inactivated like the wild-type 30 S subunit . On the other hand, 30 S subunits that contain sequence changes in the 920 to 926 region show moderate to severe decreases in tRNA binding even under activating conditions . When 30 S subunits containing these mutations were subjected to chemical probing, they failed to show the normal hyper-reactivity of nucleotide G926 and, instead, reactivity was shifted to G925 or to G928, and G929 . Two mutations in the 920 region result in structures in which A1394 is base-paired rather than being unpaired as normal; deletion but not substitution of A1394 resulted in loss of tRNA binding activity and depression of the reactivity of G926 . Mutations were made to insert or delete a nucleotide at position 920 . The deletion mutant but not the insertion mutant has decreased tRNA binding activity and also low reactivity of G926 . We conclude that structural changes in region 28 account for the active/inactive difference in tRNA binding . Molecular models of region 28 were made using the program MC-SYM . Models that include a hydrogen bond interaction between A1394 and G1392 account for the G926 reactivity in the wild-type sequence and account for the effects of most of the mutations in changing the G926 reactivity. J Biol Chem, 1995 Jul 21, 270(29), 17394 - 9 A stable carbocyclic analog of 5-phosphoribosyl-1-pyrophosphate to probe the mechanism of catalysis and regulation of glutamine phosphoribosylpyrophosphate amidotransferase; Kim JH et al.; Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-alpha-pyrophosphoryl-2-alpha, 3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5-phosphate (cPRPP) . Although cPRPP competes with PRPP for binding to the catalytic C site of the Escherichia coli enzyme, two lines of evidence demonstrate that cPRPP, unlike PRPP, does not promote an active enzyme conformation . First, cPRPP was not able to "activate" Cys1 for reaction with glutamine or a glutamine affinity analog . The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys1 for catalysis . Second, binding of cPRPP to the C site blocks binding of AMP and GMP, nucleotide end product inhibitors, to this site . However, the binding of nucleotide to the allosteric site was essentially unaffected by cPRPP in the C site . Since it is expected that nucleotide inhibitors would bind with low affinity to the active enzyme conformation, the nucleotide binding data support the conclusion that cPRPP does not activate the enzyme. Anal Biochem, 1995 Jul 20, 229(1), 99 - 105 Selection of high-affinity binding sites for sequence-specific, DNA binding proteins from random sequence oligonucleotides; Pierrou S et al.; We describe a rapid and sensitive method to isolate sets of high-affinity binding sites for sequence-specific DNA binding proteins . The DNA binding domain of the protein is expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) . Binding reactions are set up with total soluble extract from induced bacteria and a double-stranded oligonucleotide for which the central 32 bp have been randomized . To ensure stringent conditions, binding is done in the presence of high levels of poly(dI:C) . The GST fusion protein is recovered by the addition of glutathione-Sepharose . Following extensive washing of the Sepharose beads, the bound oligonucleotides are rescued by polymerase chain reaction amplification . The amplified material is used in the next cycle of selection and amplification . Approximately five cycles are needed to obtain a pure population of high-affinity sites, which are then cloned and sequenced . This procedure should be applicable to any sequence-specific DNA binding protein for which the cDNA is available and which can be expressed in bacteria in a functional form. Biochim Biophys Acta, 1995 Jul 20, 1268(1), 81 - 7 Rotational asymmetry of Escherichia coli flagellar motor in the presence of arsenate; Welch M et al.; The flagellar motor of Escherichia coli (E . coli) is driven by a proton-motive force (PMF), hence it was of interest to determine whether the motor is symmetrical in the sense that it can be rotated by any polarity of PMF . For this purpose the cells had to be deenergized first . Conventional deenergization procedures caused irreversible loss of motility, presumably due to ATP-dependent degradative processes . However, E . coli cells deenergized by incubation with arsenate manifested a slow, reversible depletion of PMF . In this procedure there was a sufficiently long time window, during which a considerable proportion of the cells lost their motility and could be made to rotate again by an artificially-imposed PMF . The motors of these cells rotated in response to any PMF polarity, but positive and negative polarities rotated different sub-populations of cells and the direction was almost exclusively counterclockwise . The reason for the unidirectionality of the rotation was not the intervention of the chemotaxis system . A number of potential reasons are suggested . One is the arsenate effect on the motor function found previously {Margolin, Y., Barak, R . and Eisenbach, M . (1994) J . Bacteriol . 176, 5547-5549} . A possible interaction between arsenate and the motor is discussed. Nature, 1995 Jul 20, 376(6537), 274 - 7 Three-dimensional structure of the kinesin head-microtubule complex; Kikkawa M et al.; Kinesin is a microtubule (MT)-associated 'motor' molecule fundamental to organelle transport . Recently, various kinesin superfamily members (KIFs) have also been identified and suggested as being responsible for the transport of specific organelles . Kinesin is a heterotetramer composed of two heavy chains and two light chains . The heavy chains form two globular heads, a rod and a fan-like tail completed by the light chains . The globular head, which is composed of approximately 340 amino-terminal residues of the heavy chain, includes both ATP-binding and MT-binding domains, and its recombinant protein also has these properties . To improve the understanding of the mechanism of force generation by an MT-based molecular motor, kinesin, we report here the three-dimensional structure of the complex of a recombinant kinesin head and MTs, as revealed by helical reconstruction from cryo-electron micrographs . A kinesin head is a globular teardrop-like structure binding to the ridge of one protofilament of MTs . We have determined the polarity of the structure of the complex of MTs and the kinesin head in relation to MT polarity. Nature, 1995 Jul 20, 376(6537), 260 - 3 A novel receptor involved in T-cell activation; Cocks BG et al.; Optimal T-cell activation and T-cell expansion require triggering by T-cell antigen receptors and co-stimulatory signals provided by accessory cells . A major co-stimulatory pathway involves crosslinking the CD28 molecule on T cells by its ligands CD80 or CD86 expressed on antigen-presenting cells . But recent studies on CD28-deficient mice have indicated that CD28 is not required for all T-cell responses and that additional T-cell co-stimulatory pathways exist . Here we describe a novel glycoprotein, of relative molecular mass 70,000 (M(r) 70K), designated SLAM, that belongs to the immunoglobulin gene superfamily, which is involved in T-cell stimulation . SLAM is constitutively expressed on peripheral-blood CD45ROhigh memory T cells, T-cell clones, immature thymocytes, and a proportion of B cells, and is rapidly induced on naive T cells after activation . Engagement of SLAM enhances antigen-specific proliferation and cytokine production by T cells carrying the CD4 antigen (CD4+) . Particularly, the production of interferon-gamma (IFN-gamma) is strongly upregulated, even in T helper type 2 (Th2) CD4+ T-cell clones, whereas no induction of interleukin (IL)-4 or IL-5 production was observed in Th1 clones . In addition, the engagement of SLAM induces directly the proliferation of CD4+ T-cell clones and preactivated T cells, in the absence of any other stimuli, and without CD28 involvement . Thus SLAM is a novel receptor on T cells that, when engaged, potentiates T-cell expansion in a CD28-independent manner and induces a Th0/Th1 cytokine production profile. Nature, 1995 Jul 20, 376(6537), 230 - 5 Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor; Walter MR et al.; The crystal structure of interferon-gamma bound to the extracellular fragment of its high-affinity cell-surface receptor reveals the first view of a class-2 cytokine receptor-ligand complex . In the complex, one interferon-gamma homodimer binds two receptor molecules . Unlike the class-1 growth hormone receptor complex, the two interferon-gamma receptors do not interact with one another and are separated by 27 A . Upon receptor binding, the flexible AB loop of interferon-gamma undergoes a conformational change that includes the formation of a 3(10) helix. Mol Cell Biochem, 1995 Jul 19, 148(2), 105 - 13 Partial characterization of the RNA from LPS-stimulated macrophages that induces the release of chemotactic cytokines by resident macrophages; Ribeiro RA et al.; It is well established that exogenous RNA is incorporated into eukaryotic cells and is able to exert various biological responses . Little, however, is known about the effects of such RNA on macrophages . In this study, we demonstrate that RNA extracted from macrophages stimulated with Escherichia coli lipopolysaccharide (LPS), referred to as L-RNA, in contrast to RNA from non-stimulated macrophages (N-RNA), induces the release of a macrophage-derived neutrophil chemotactic factor (MNCF) and interleukin-8 (IL-8) from macrophage monolayers . The effect of L-RNA was dependent of the integrity of the polynucleotide chain and was not due to LPS contamination since its ability to induce MNCF and IL-8 release was strongly reduced by RNase but was not affected by DNase or polymyxin B . The poly A(+)L-RNA and poly A(-)L-RNA fractions were able to induce the release of MNCF and IL-8, indicating that the L-RNA could be acting at transcriptional and translational levels . The demonstration that actinomycin-D and cycloheximide inhibited the release of MNCF and IL-8 by L-RNA-stimulated macrophages confirms this assumption . Fractionation of the total L-RNA by centrifugation on a 5-20% sucrose gradient showed that the L-RNA which sediments in the 4-5S region of the gradient is the only fraction capable of inducing the release of MNCF from naive macrophages . We have previously shown that macrophage monolayers stimulated with interleukin-1 beta or LPS release a low molecular RNA which also sediments in the same 4-5S region . Taken together, these results support our proposal that resident macrophages, when activated by injurious stimuli, in addition to secreting cytokines, also release a low molecular weight (4-5S) RNA which may act on the surrounding macrophages to further stimulate the release of cytokines . This process would amplify the inflammatory response and would increase the mechanisms involved in the defense response or tissue injury. Biochim Biophys Acta, 1995 Jul 19, 1250(2), 197 - 203 Identification of two specific lysines responsible for the inhibition of phospholipase A2 by manoalide; Bianco ID et al.; Manoalide, a natural product of sponge, irreversibly inhibits phospholipase A2 (PLA2) by reacting with lysine residues . Cobra venom PLA2 mutants were constructed in which four of the six lysine residues were independently replaced by arginine or methionine, which cannot react with manoalide . The mutants were overexpressed in Escherichia coli, renatured, and purified . The enzyme mutants lacking Lys-6 (K6R and K6M) or Lys-79 (K79R) were inhibited only 40% by manoalide while the native cobra venom PLA2 was inhibited 80% under the same conditions . This means that the manoalide modification of either Lys-6 or Lys-79 accounted for only half of the manoalide inhibition . The double mutant (K6R79R) was not inhibited by manoalide at all . Lys-56 (K56R) and Lys-65 (K65R) mutants were inhibited to the same extent as the native enzyme which indicates that these residues are not responsible for any of the inhibitory effects produced by manoalide . These results demonstrate that the reaction of manoalide with both Lys-6 and Lys-79 can account for all of its inhibition of cobra venom PLA2 . The inhibition of PLA2 and its mutants with manoalide did not affect the activity of the enzyme toward monomeric substrate, which suggests that manoalide does not modify the catalytic site residues, that it does not block access to this site, and that its inhibition requires an interface . Furthermore, as with native PLA2, the activation of phosphatidylethanolamine hydrolysis by phosphorylcholine-containing compounds was exhibited by all of the mutants suggesting that none of the lysines examined are essential for this activation. Proc Natl Acad Sci U S A, 1995 Jul 18, 92(15), 7021 - 5 A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity; Mack M et al.; Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker . One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer . The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only . The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments . By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines . The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column . As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells. Biochemistry, 1995 Jul 18, 34(28), 9227 - 34 Role of Asp274 in lac repressor: diminished sugar binding and altered conformational effects in mutants; Chang WI et al.; The role of Asp274 in inducer binding of lac repressor has been explored by spectroscopic measurements, fluorescence quenching, in vitro induction assays, and chemical modification of mutants with conservative substitutions at this site . Although no fluorescence emission shift or characteristic UV difference spectrum was observed at high inducer concentration, fluorescence quenching, effects on operator binding, and chemical modification results indicate indirectly that the mutants Asp274-->Asn and Asp274-->Glu bind sugar, albeit with very low affinity (> 0.1 M) . Consistent with very weak inducer binding indicated by protection from fluorescence quenching by iodide, operator binding activity of these two mutant proteins is altered at very high IPTG concentration, although in opposite directions . The distinct effects of inducer on operator binding in these two mutant proteins as well as substantial differences in the effect of sugar ligand on chemical modification of Cys107 and Cys140 by 2-(bromoacetamido)-4-nitrophenol suggest that the conformation of the protein before and after association with sugar may differ in these mutant proteins . Fluorescence quenching assays of lac mutant proteins at Asp274 indicate the proximity of Trp220 to the side chain at position 274, consistent with the location of this residue in the structural model of lac repressor and in the crystallographic structure of the homologous purine repressor . From these results, we conclude that Asp274 is in the inducer binding site, that the character of this residue is crucial to inducer binding, and that interaction of sugar with the side chain at this position may be associated with the conformational change necessary for generating high affinity ligand binding. Biochemistry, 1995 Jul 18, 34(28), 9172 - 6 The binding site of the nicotinic acetylcholine receptor in animal species resistant to alpha-bungarotoxin; Barchan D et al.; The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193 . We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX) . Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX . In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human . Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli . The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder . The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment . The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 18, 34(28), 9166 - 71 Tryptophan-scanning mutagenesis of MotB, an integral membrane protein essential for flagellar rotation in Escherichia coli; Sharp LL et al.; The MotB protein of Escherichia coli is an essential component of the flagella that functions together with the MotA protein in transmembrane proton conduction . MotB has a single hydrophobic segment that spans the membrane . In order to determine which parts of the membrane-spanning segment can tolerate the introduction of a large, hydrophobic side chain, single Trp residues were substituted into many consecutive positions in the segment and the effects on function were measured . Trp residues were tolerated at positions near the periplasmic end of the MotB segment but not at positions near the cytoplasmic end . These results are different from what was seen in a similar mutational study of MotA, in that protein Trp residues were tolerated at positions that would be clustered together on one face of each hydrophobic segment if they are alpha-helices {Sharp, L . L., Zhou, J., & Blair, D . F . (1995) Proc . Natl . Acad . Sci . U.S.A . (in press)} . Those results suggested that the membrane-spanning segments of MotA are alpha-helices arranged in a bundle so that each has a face directed toward the lipid . The contrasting results seen with MotB indicate that its relationship to neighboring protein segments is different . Double-Trp substitutions, one each in MotA and MotB, also were studied . Many double substitutions had strongly synergistic effects which imply that the membrane segments of these proteins interact . (ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 18, 34(28), 9103 - 10 Cloning, Zn2+ binding, and structural characterization of the guanine nucleotide exchange factor human Mss4; Yu H et al.; The Sec4/Ypt1/Rab family of small GTP-binding proteins are involved in the regulation of intracellular vesicular transport . A rat gene, mss4, that encodes a guanine nucleotide exchange factor (GEF) for Sec4 was recently cloned by its ability to rescue defects in protein transport of a yeast temperature-sensitive (ts) mutant, sec4-8 . We describe herein the cloning, bacterial expression, and biochemical characterization of human Mss4 . As expected, both the cDNA and its encoded amino acid sequences are highly conserved between the human and rat mss4 . Soluble and functional Mss4 protein was obtained by expressing the gene as a glutathione-S-transferase fusion protein in Escherichia coli . Subsequent biochemical analysis revealed that Mss4 binds 1 equiv of Zn2+, and zinc is essential for the stability of the protein . Utilizing multidimensional heteronuclear NMR techniques, we assigned most of the 1H, 15N, and 13C resonances of this 14-kDa protein . Its secondary structure was also deduced from slowly exchanging amide protons, characteristic NOEs, and 3JNH-C alpha H coupling constants . The protein contains a central seven-stranded antiparallel beta sheet, flanked by two small beta sheets . Many resonances pertaining to a loop region of the molecule cannot be identified, suggesting that it might be involved in local movements . These studies provide the first structural insights into a protein possessing GEF activity. Biochemistry, 1995 Jul 18, 34(28), 9095 - 102 Rat guanylyl cyclase C expressed in COS-7 cells exhibits multiple affinities for Escherichia coli heat-stable enterotoxin; Deshmane SP et al.; Intestinal cells exhibit binding sites with different affinities for Escherichia coli heat-stable enterotoxin (ST) and guanylin, suggesting the existence of different receptors for these peptides . Guanylyl cyclase C from intestinal cells has been identified as one receptor for these peptides . Equilibrium and kinetic binding characteristics of rat guanylyl cyclase C expressed in COS-7 cells were examined, employing ST, to determine if this receptor exhibited multiple affinities . Scatchard analysis of equilibrium binding yielded curvilinear isotherms consistent with the presence of high (pM) and low (nM) affinity sites . Kinetic analysis of binding demonstrated that these sites exhibited similar dissociation but different association kinetics . In addition, two distinct affinity states of low affinity sites were identified with dissociation constants of 0.15 and 5.85 nM . Association of ST and low affinity sites was biphasic, while dissociation from these sites was unimodal . Close agreement of equilibrium and kinetic dissociation constants suggested that low affinity sites were in the lowest affinity state at equilibrium . Comparison of the ligand dependence of guanylyl cyclase activity (EC50 = 110 nM) with receptor occupancy revealed that binding of ST to the lowest affinity state of low affinity sites (EC50 = 80 nM) is directly coupled to catalytic activation . These studies suggest that binding sites with different affinities for ST exhibited by intestinal cells reflect the expression of a single gene product, guanylyl cyclase C, rather than different receptors for the ligand . The shift in affinity state of low affinity sites and its correlation with catalytic activation suggest a central role for this phenomenon in mechanisms mediating receptor-effector coupling of membrane guanylyl cyclases. Biochemistry, 1995 Jul 18, 34(28), 9000 - 8 Amino acid differences at positions 10, 11, and 104 explain the profound catalytic differences between two murine pi-class glutathione S-transferases; Bammler TK et al.; The glutathione S-transferases play a pivotal role in the detoxification of toxic and carcinogenic electrophiles . We have previously reported the isolation of two actively transcribed murine pi-class glutathione S-transferase genes . In this study the two proteins encoded by these genes, Gst p-1 and Gst p-2, were expressed in Escherichia coli and found to exhibit profoundly different catalytic activities, the activity of Gst p-2 toward a panel of electrophilic substrates being 1-3 orders of magnitude lower than that of Gst p-1 . In order to establish the basis for the difference between these highly homologous proteins, mutants were generated where specific amino acids had been exchanged . Kinetic analysis of the wild-type and mutant enzymes revealed that the amino acid differences occurring at positions 10 (Val/Ser), 11 (Arg/Pro), and 104 (Val/Gly) are responsible for the reduced enzymatic activity of Gst p-2 . This analysis together with computer graphics modeling for Gst p-2 indicated that these changes affected both substrate and glutathione binding to the enzyme. Biochemistry, 1995 Jul 18, 34(28), 8931 - 9 Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa > 9; Wilson NA et al.; Apparent pKa values of active site residues Asp26, Cys32, and Cys35 in reduced thioredoxin have been characterized . Both wild-type thioredoxin and mutant D26A thioredoxin were selectively 13C-enriched on cysteine beta-carbons . In both proteins, the variation with pH of 1HB1, 1HB2, and 13CB NMR chemical shifts has been measured . In wild-type reduced thioredoxin, for both cysteines, the pH versus chemical shift plots of HB1 protons can be fit to one titration with pKa values of 7.0-7.1 . In contrast, the HB2 protons and beta-carbons give pH--chemical shift plots that clearly reflect more than one titration; fits to the data give apparent pKa values of 7.0-7.3 and 9.5 for HB2 protons and 7.5-7.9 and 9.2-10.2 for CB carbons . In reduced D26A, all three probe chemical shifts have a pH dependence that is fit by one titration with pKa of 7.4-7.9 . The absence of a titration with pKa > 9 in D26A, taken together with cysteine thiol pKa values of 7.1 and 7.9 determined by Raman spectroscopy {Li et al . (1993) Biochemistry 32, 5800-5808}, indicates that the pKa > 9 in reduced thioredoxin is that of Asp26 . This is highly significant in view of the previous observation that, in oxidized thioredoxin, Asp26 pKa is 7.5 {Langsetmo et al . (1991) Biochemistry 30, 7603-7609} . The very high pKa values of these carboxyls is consistent with their local environment in the three-dimensional structure; the Asp26 side chain in oxidized thioredoxin is almost but not completely buried, and in reduced thioredoxin it may be even more buried.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1995 Jul 18, 92(15), 6883 - 6 The tRNA processing enzyme RNase T is essential for maturation of 5S RNA; Li Z et al.; The maturation of 5S RNA in Escherichia coli is poorly understood . Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated . We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis . Our results indicate that no mature 5S RNA is made in RNase T-deficient strains . Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate . Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type . Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction . In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation . Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action . These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival. Biochem Pharmacol, 1995 Jul 17, 50(2), 163 - 8 Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase; Johansson M et al.; Human and mouse deoxycytidine kinase (dCK) (EC 2.7.1.74) were cloned and expressed in Escherichia coli . Michaelis-Menten kinetics were determined for the purified enzymes with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), 2-chloro-2'-deoxyadenosine (CdA), 2',3'-dideoxycytidine (ddCyd) and 9-beta-D-arabinofuranosylguanine (araG) as substrates and ATP and UTP as phosphate donors . Both human and mouse dCK showed highest affinity to dCyd with Km values of 0.05-0.2 microM . The anti-leukaemic compound CdA was the superior substrate of the nucleoside analogues tested . Both enzymes were able to efficiently utilize ATP and UTP as phosphate donors . However, the use of UTP instead of ATP as phosphate donor decreased Km values for all substrates investigated . The kinetic properties of mouse and human dCK differed in that the human enzyme showed higher affinity for the substrates dAdo, CdA, ddCyd and araG . The human enzyme also showed higher affinity for ATP and UTP . The ability to phosphorylate dCyd was, however, similar for both human and mouse dCK . At physiological concentration of the feedback inhibitor dCTP, mouse dCK showed lower activity than human dCK for all substrates investigated. FEBS Lett, 1995 Jul 17, 368(2), 315 - 20 Recognition of 2'-hydroxyl groups by Escherichia coli ribonuclease HI; Iwai S et al.; In order to investigate the hydrogen-bonding interactions between Escherichia coli ribonuclease HI and the 2'-hydroxyl functions of the substrate, oligonucleotide duplexes containing 2'-amino-2'-deoxyuridine or 2'-fluoro-2'-deoxyuridine at a specific site were used, and their affinities for the enzyme were determined by kinetic analyses . The results indicate that the hydroxyl groups of the nucleoside 3'-adjacent to the cleaved phosphodiester linkage and the second nucleoside 5' to the cleaved phosphodiester act as both a proton donor and an acceptor and as a proton acceptor, respectively, in the enzyme-substrate complex . A molecular model was constructed using the interactions derived from the results. FEBS Lett, 1995 Jul 17, 368(2), 263 - 6 Evidence for SecA- and delta pH-independent insertion of D1 into thylakoids; van Wijk KJ et al.; Many nuclear-encoded proteins are targeted into chloroplast thylakoids by an azide sensitive Sec-related mechanism or by a delta pH-driven mechanism . In this report, the requirements for the integration of chloroplast-encoded thylakoid proteins have been analysed in pulse-labeled intact chloroplasts . We show that the integration of the photosystem II reaction centre protein, D1, continues in the absence of a delta pH and in the presence of azide . A range of other proteins are similarly targeted to thylakoids in the presence of azide, suggesting that the SecA-related mechanism is not widely used for the targeting of chloroplast-encoded proteins. FEBS Lett, 1995 Jul 17, 368(2), 235 - 8 The gamma subunit in the Escherichia coli ATP synthase complex (ECF1F0) extends through the stalk and contacts the c subunits of the F0 part; Watts SD et al.; A mutant, in which a cysteine has been site-directed into the polar loop region of the c subunit at residue 44, has been studied . Cross-linking of the c subunit to both the gamma and epsilon subunits was observed with cupric 1,10-phenanthrolinate treatment . The linkage between the c and gamma subunits was localized to that part of the gamma subunit between residues 202-286, based on peptide analysis . Reference to the high resolution structure of F1 {Abrahams et al . (1994) Nature 370, 621-628} appears to limit this contact site to the region including residues 202-230 . This segment contains 4 tyrosines and 1 tryptophan as possible reactive residues for cross-linking with the c subunit cysteine. FEBS Lett, 1995 Jul 17, 368(2), 220 - 4 Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties; Aliverti A et al.; Mutations of the conserved residue Glu-92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves . The purified ferredoxin mutants were found twice as active with respect to wild-type protein in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase in the presence of ferredoxin . Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the {2Fe-2S} cluster geometry, whose redox potential becomes approximately 80 mV less negative . These data point to a role of the Glu-92 side-chain in determining the low redox potential typical of the {2Fe-2S} cluster of chloroplast and cyanobacterial ferredoxins . Also a ferredoxin/ferredoxin-NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified . Fusion of the ferredoxin with its reductase causes only minor effects to the iron-sulfur cluster, as judged by cyclic voltammetry and EPR measurements. EMBO J, 1995 Jul 17, 14(14), 3415 - 24 Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli; Missiakas D et al.; Previous studies have established that DsbA and DsbC, periplasmic proteins of Escherichia coli, are two key players involved in disulfide bond formation . A search for extragenic mutations able to compensate for the lack of dsbA function in vivo led us to the identification of a new gene, designated dsbD . Lack of DsbD protein leads to some, but not all, of the phenotypic defects observed with other dsb mutations, such as hypersensitivity to dithiothreitol and to benzylpenicillin . In addition, unlike the rest of the dsb genes, dsbD is essential for bacterial growth at temperatures above 42 degrees C . Cloning of the wild-type gene and sequencing and overexpression of the protein show that dsbD is part of an operon and encodes an inner membrane protein . A 138 amino acid subdomain of the protein was purified and shown to possess an oxido-reductase activity in vitro . Expressing this subdomain in the periplasmic space helped restore the phenotypic defects associated with a dsbD null mutation . Interestingly, this domain shares 45% identity with the portion of the eukaryotic protein disulfide isomerase carrying the active site . We further show that in dsbD mutant bacteria the dithiol active sites of DsbA and DsbC proteins are mostly oxidized, as compared with wild-type bacteria . Our results argue that DsbD generates a reducing source in the periplasm, which is required for maintaining proper redox conditions . The finding that overexpression of DsbD leads to a Dsb- phenotype, very similar to that exhibited by dsbA null mutants, is in good agreement with such a model. EMBO J, 1995 Jul 17, 14(14), 3365 - 72 A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane; Matsuyama S et al.; Lipoproteins are localized in the outer or inner membrane of Escherichia coli, depending on the species of amino acid located next to the N-terminal fatty acylated Cys . The major outer membrane lipoprotein (Lpp) expressed in spheroplasts was, however, retained in the inner membrane as a mature form . A novel protein that is essential for the release of Lpp from the inner membrane was discovered in the periplasm and purified . The partial amino acid sequence of this 20 kDa protein (p20) was determined and used to clone a gene for p20 . Sequencing of the gene revealed that p20 is synthesized as a precursor with a signal sequence . p20 formed a soluble complex only with outer membrane-directed lipoproteins such as Lpp, indicating that p20 plays a critical role in the sorting of lipoproteins . Lpp released from the inner membrane in the presence of p20 was specifically assembled into the outer membrane in vitro . These results indicate that p20 is a periplasmic carrier protein involved in the translocation of lipoproteins from the inner to the outer membrane. EMBO J, 1995 Jul 17, 14(14), 3302 - 10 Branch migration of three-strand recombination intermediates by RecG, a possible pathway for securing exchanges initiated by 3'-tailed duplex DNA; Whitby MC et al.; RecG protein is required for normal levels of recombination and DNA repair in Escherichia coli . This 76 kDa polypeptide is a junction-specific DNA helicase that acts post-synaptically to drive branch migration of Holliday junction intermediates made by RecA during the strand exchange stage of recombination . To gain further insight into the role of RecG, we studied its activity on three-strand intermediates formed by RecA between circular single-stranded and linear duplex DNAs . Once RecA is removed, RecG drives branch migration of these intermediates by a junction-targeted activity that depends on hydrolysis of ATP . RuvAB has a similar activity . However, when RecG is added to a RecA strand exchange reaction it severely reduces the accumulation of joint molecule intermediates by driving branch migration of junctions in the reverse direction to that catalysed by RecA strand exchange . In comparison, RuvAB has little effect on the reaction . We discuss how reverse branch migration by RecG, which acts counter of the 5'-->3' polarity of RecA binding and strand exchange, could serve to promote or abort the early stages of recombination, depending on the orientation of the single DNA strand initiating the exchange relative to the adjacent duplex region. Biochem Biophys Res Commun, 1995 Jul 17, 212(2), 317 - 25 Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase; Miranda EA et al.; In order to examine the structure-function relationship of the poly (ADP-ribose) polymerase (PARP) catalytic domain, potential active-site residues in the catalytic domain have previously been described . Here, we have used mutagenesis with hydroxylamine to generate a random library of PARP mutants . The identification, overproduction in insect cells, purification and characterization of a gain-of-function mutant (L713F) is described . We show that the kcat of this mutant is increased over nine times compared to the wild-type enzyme; the Km for NAD+ is unchanged . The size and the branching structure of the ADP-ribose polymers are similar in both the wild-type and the mutant enzyme . This mutation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproduction in vivo on cell survival following DNA damage. J Immunol Methods, 1995 Jul 17, 184(1), 101 - 12 Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression . Comparison of different methods; De Jong MO et al.; The main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that binding of the biotinylated ligand to the receptor does not inhibit the subsequent interaction of biotin with fluorescently tagged avidin or streptavidin . Using interleukin-2 (IL-2), we compared the usefulness of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS-LC-biotin, DBB and photobiotin, and developed optimal biotinylation conditions for the preparation of biologically active biotin-labeled IL-2 and the detection of IL-2 receptor expressing cells by flow cytometry . As determined by spot blot analysis, biotinylation of IL-2 was most efficient at the highest biotin-to-protein (B:P) ratio used . At a B:P ratio of 100, most of the biological activity of IL-2 was retained when S-NHS-LC-biotin was used . In contrast, most of the biological activity of IL-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions . Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cells by flow cytometry after sequential staining with the biotinylated IL-2 and fluorescence tagged streptavidin . A high B:P ratio generally resulted in a high specific fluorescence intensity of the cells, particularly when S-NHS-LC-biotin was used as the biotinylation reagent . Biotin-IL-2 could also be used to detect IL-2 receptors expressed by lymphocytes in peripheral blood and bone marrow . Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 receptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated IL-2 expressed high numbers of the IL-2 receptor alpha chain . This is in agreement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain . After stimulation with PHA, virtually all lymphocytes expressed the alpha chain, whereas only part of these cells showed a strong fluorescence signal after staining with biotin-IL-2, while the rest of the cells had very low numbers of IL-2 binding sites . Our results demonstrate that, in addition to staining individual receptor subunits with antibodies, staining with biotinylated IL-2 is a useful indicator of functional IL-2 receptor expression. Biochem Biophys Res Commun, 1995 Jul 17, 212(2), 705 - 11 Homology cloning of GTP-cyclohydrolase I from various unrelated eukaryotes by reverse-transcription polymerase chain reaction using a general set of degenerate primers; Maier J et al.; GTP-cyclohydrolase I is the primary enzyme of tetrahydrobiopterin and folic acid biosynthesis . cDNA fragments of GTP-cyclohydrolase I were obtained from rainbow trout, chicken, the fungi Neurospora crassa, Phycomyces blakesleeanus and Saccharomyces cerevisiae, the cellular slime mold Dictyostelium discoideum, the phytoflagellate Euglena gracilis and the higher plant Mucuna hassjo using primers specific for conserved regions of the open reading frame and the reverse transcription polymerase chain reaction (RT-PCR) technique . A number of regions were found to be strictly conserved between unrelated eukaryotes . These regions may be essential for the function of GTP-cyclohydrolase I and are discussed with respect to the recently resolved crystal structure of the Escherichia coli enzyme. Anal Chem, 1995 Jul 15, 67(14), 2493 - 7 Ion/molecule reactions for improved effective mass resolution in electrospray mass spectrometry; McLuckey SA et al.; Tandem mass spectrometry is shown to improve the effective mass resolution in electrospray mass spectrometry . The technique involves selecting a population of ions within a narrow range of mass-to-charge values and allowing the ions to undergo proton transfer reactions . The shifts in mass-to-charge ratios associated with product ions formed by proton transfer allow for mass and charge assignment . The success of the technique relies on the relative enrichment of ions of a particular charge state that occurs in the mass-to-charge selection step . This approach can be used to extend the polymer mass range amenable to measurement, analyze mixtures that might otherwise be too complex for reliable mass measurements, and improve mass measurement precision when a mixture of cations is present within a given charge state . The technique is illustrated with a quadrupole ion trap using multiply-charged ions of cytochrome c, transfer ribonucleic acid from E . coli, strain W, and a synthetic deoxyribonucleic acid 30-mer. Structure, 1995 Jul 15, 3(7), 729 - 41 Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition; Mattevi A et al.; BACKGROUND: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis . Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effectors . The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known . We have determined the three-dimensional structure of the allosteric type I PK from Escherichia coli, in order to study the mechanism of allosteric regulation . RESULTS: The 2.5 A resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)8-barrel domain, a flexible beta-barrel domain and a C-terminal domain . The allosteric and active sites are located at the domain interfaces . Comparison of the T-state E . coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively . Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes . Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites . The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme . CONCLUSIONS: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations . On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations . These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site. Structure, 1995 Jul 15, 3(7), 669 - 79 Three new crystal structures of point mutation variants of monoTIM: conformational flexibility of loop-1, loop-4 and loop-8; Borchert TV et al.; BACKGROUND: Wild-type triosephosphate isomerase (TIM) is a very stable dimeric enzyme . This dimer can be converted into a stable monomeric protein (monoTIM) by replacing the 15-residue interface loop (loop-3) by a shorter, 8-residue, loop . The crystal structure of monoTIM shows that two active-site loops (loop-1 and loop-4), which are at the dimer interface in wild-type TIM, have acquired rather different structural properties . Nevertheless, monoTIM has residual catalytic activity . RESULTS: Three new structures of variants of monoTIM are presented, a double-point mutant crystallized in the presence and absence of bound inhibitor, and a single-point mutant in the presence of a different inhibitor . These new structures show large structural variability for the active-site loops, loop-1, loop-4 and loop-8 . In the structures with inhibitor bound, the catalytic lysine (Lys13 in loop-1) and the catalytic histidine (His95 in loop-4) adopt conformations similar to those observed in wild-type TIM, but very different from the monoTIM structure . CONCLUSIONS: The residual catalytic activity of monoTIM can now be rationalized . In the presence of substrate analogues the active-site loops, loop-1, loop-4 and loop-8, as well as the catalytic residues, adopt conformations similar to those seen in the wild-type protein . These loops lack conformational flexibility in wild-type TIM . The data suggest that the rigidity of these loops in wild-type TIM, resulting from subunit-subunit contacts at the dimer interface, is important for optimal catalysis. Structure, 1995 Jul 15, 3(7), 635 - 9 Counting the calories to stay in the groove; Ladbury JE; High-sensitivity microcalorimetry is beginning to make an impact on the determination of thermodynamic parameters associated with protein-DNA interactions and the understanding of the relationship of these data to structural details of complex formation. Eur J Biochem, 1995 Jul 15, 231(2), 491 - 7 Human topoisomerase II alpha is phosphorylated in a cell-cycle phase-dependent manner by a proline-directed kinase; Wells NJ et al.; Topoisomerase II is essential for chromosome condensation and segregation at mitosis in eukaryotic cells, but the mechanism of its regulation is not clearly understood . We have investigated whether or not the alpha isozyme of human topoisomerase II is phosphorylated in a cell-cycle phase-dependent manner . Two-dimensional tryptic phosphopeptide mapping revealed that several sites on HeLa topoisomerase II alpha protein were phosphorylated predominantly or exclusively during the G2 and M phases . To identify the protein kinases involved in this cell-cycle phase-specific phosphorylation, oligohistidine-tagged recombinant domains of the topoisomerase II alpha protein were expressed in Escherichia coli, purified by affinity chromatography and phosphorylated in vitro by different protein kinases . Phosphorylation of the C-terminal domain of the topoisomerase II alpha protein by the universal mitotic controller, p34cdc2, generated multiple tryptic phosphopeptides, many of which corresponded to the G2/M-phase-specific phosphorylation sites observed in vivo . The same phosphopeptides were obtained following phosphorylation of the C-terminal domain in vitro by the mitogen-activated protein kinase . Site-directed mutagenesis studies identified five of these sites of phosphorylation, each of which comprised a serine-proline motif . Our data implicate one or more proline-directed kinases in the cell-cycle-dependent regulation of topoisomerase II alpha enzyme activity in human cells. Eur J Biochem, 1995 Jul 15, 231(2), 396 - 404 Characterization of a 2{4Fe-4S} ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus; Armengaud J et al.; The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one {3Fe-4S} cluster and one {4Fe-4S} cluster . This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin . The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions . A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms . The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH . The reconstituted ferredoxin was purified and shown to be distinct from the native {7Fe-8S} ferredoxin, based on several biochemical and spectroscopic criteria . In the oxidized state, EPR revealed the quasi-absence of {3Fe-4S} cluster . 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2{4Fe-4S} ferredoxin . This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur . Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of {7Fe-8S} form . Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity . The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins. Eur J Biochem, 1995 Jul 15, 231(2), 306 - 11 Recombinant production and biological properties of rat cytokine-induced neutrophil chemoattractants, GRO/CINC-2 alpha, CINC-2 beta and CINC-3; Shibata F et al.; Recently we found four cytokine-induced neutrophil chemoattractants, CINC-1, CINC-2 alpha, CINC-2 beta and CINC-3/macrophage inflammatory protein 2 (MIP-2), in conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in rats {Nakagawa, H., Komorita, N., Shibata, F., Ikesue, A., Konishi, K., Fujioka, M . & Kato, H . (1994) Biochem . J . 301, 545-550} . In the present report, we describe recombinant production of CINC-2 alpha, CINC-2 beta and CINC-3 in Escherichia coli, and biological properties of these chemokines . Neutrophil chemotactic activities of CINC-2 alpha and 2 beta in vitro were the same as the activity of CINC-1 . CINC-3 had an activity comparable to other CINCs, but showed a decrease at high concentrations . Stimulation of neutrophils with CINCs induced an increase in intracellular {Ca2+} dose-dependently . CINC-3 was more potent than the other CINCs and still induced an increase in intracellular {Ca2+} in rat neutrophils stimulated first with other CINCs . CINC-2 alpha, CINC-2 beta and CINC-3 induced a comparable response to CINC-1 in the release of cathepsin G from rat neutrophils . Injection of CINC-2 alpha, 2 beta and 3 into preformed air-pouch on the back of rat induced infiltration of neutrophils to an extent similar to that caused by the injection of CINC-1 . These data indicate CINC-2 alpha, 2 beta and 3 as well as CINC-1 are chemoattractants specific for neutrophil in vivo. Biochem J, 1995 Jul 15, 309 ( Pt 2), 689 - 93 Over-expression and characterization of active recombinant rat liver carnitine palmitoyltransferase II using baculovirus; Johnson TM et al.; The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system . Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II) . CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates . Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus . The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated . Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively. Biochem J, 1995 Jul 15, 309 ( Pt 2), 601 - 5 Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli; Balme A et al.; Flavocytochrome b2 consists of two distinct domains . The N-terminal domain contains protohaem IX and the larger, C-terminal domain contains flavin mononucleotide (FMN) . We describe here the isolation of the flavin-binding domain expressed in Escherichia coli independent of the cytochrome domain . The isolated domain is an efficient lactate dehydrogenase with ferricyanide as electron acceptor but reduces cytochrome c, the physiological oxidant for flavocytochrome b2, extremely poorly; electron transfer from the flavin-binding domain to the separately expressed cytochrome domain is undetectable . FMN reduction by lactate occurs as a single exponential process in the isolated flavin-binding domain, in contrast to the biphasic kinetics observed with native flavocytochrome b2. Biochem J, 1995 Jul 15, 309 ( Pt 2), 465 - 72 Structural and functional properties of heparin analogues obtained by chemical sulphation of Escherichia coli K5 capsular polysaccharide; Razi N et al.; Capsular polysaccharide from Escherichia coli K5, with the basic structure (GlcA beta 1-4GlcNAc alpha 1-4)n, was chemically modified through N-deacetylation, N-sulphation and O-sulphation {Casu, Grazioli, Razi, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr . Res . 263, 271-284} . Depending on the reaction conditions, the products showed different proportions of components with high affinity for antithrombin (AT) . A high-affinity subfraction, M(r) approx . 36,000, was shown by near-UV CD, UV-absorption difference spectroscopy and fluorescence to cause conformational changes in the AT molecule very similar to those induced by high-affinity heparin . Fluorescence titrations demonstrated about two AT-binding sites per polysaccharide chain, each with a Kd of approx . 200 nM . The anti-(Factor Xa) activity was 170 units/mg, similar to that of the IIId international heparin standard and markedly higher than activities of previously described heparin analogues . Another preparation, M(r) approx . 13,000, of higher overall O-sulphate content, exhibited a single binding site per chain, with Kd approx . 1 microM, and an anti-(Factor Xa) activity of 70 units/mg . Compositional analysis of polysaccharide fractions revealed a correlation between the contents of -GlcA-GlcNSO3(3,6-di-OSO3)- disaccharide units and affinity for AT; the 3-O-sulphated GlcN unit has previously been identified as a marker component of the AT-binding pentasaccharide sequence in heparin . The abundance of the implicated disaccharide unit approximately equalled that of AT-binding sites in the 36,000-M(r) polysaccharide fraction, and approached one per high-affinity oligosaccharide (predominantly 10-12 monosaccharide units) isolated after partial depolymerization of AT-binding polysaccharide . These findings suggest that the modified bacterial polysaccharide interacts with AT and promotes its anticoagulant action in a manner similar to that of heparin. Biochem J, 1995 Jul 15, 309 ( Pt 2), 425 - 30 Processing of iduronate 2-sulphatase in human fibroblasts; Froissart R et al.; Iduronate 2-sulphatase (IDS) is a lysosomal enzyme involved in degradation of dermatan sulphate and heparan sulphate . Antigenic material was obtained either by purification of placental IDS (A and B forms) or by expression of three different fusion peptides in Escherichia coli allowing the production of five specific antibodies . Pulse-chase-labelling experiments in over-expressing fibroblasts showed poor IDS processing but large amounts of precursors were secreted into the medium . The endocytosis of the 35S- or 33P-labelled precursors by deleted fibroblasts together with glycosylation studies and proteolysis inhibition by leupeptin allowed better elucidation of IDS maturation . The initial 73-78 kDa form is converted into a phosphorylated 90 kDa precursor after modification of its oligosaccharide chains in the Golgi apparatus . This precursor is processed by proteolytic cleavage through various intermediates to a major 55 kDa intermediate, with the release of an 18 kDa polypeptide . Further proteolytic cleavage by a thiol protease gives the 45 kDa mature form containing hybrid and complex-type oligosaccharide chains. Biochem J, 1995 Jul 15, 309 ( Pt 2), 411 - 7 Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli; Lu KV et al.; The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids . In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis . Expression of this DNA via a plasmid vector in E . coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating . Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids . Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161 . Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E . coli should be avoided when possible. FEMS Microbiol Lett, 1995 Jul 15, 130(1), 13 - 7 Dr fimbriae coding region associated hemolytic activity of Escherichia coli; Goluszko P et al.; We investigated the hemolytic activity of Escherichia coli strain EC901 carrying plasmid pBJN406 containing genes draA-E involved in expression of the mannose-resistant Dr hemagglutinin, and in its isogenic insertion mutants devised with Tn5, Tn3, and TnphoA . While E . coli BN406 displayed rapid hemolytic activity against equine erythrocytes, insertion mutations in draD and draE, but not in draA, draB, and draC, abolished all hemolytic activity . These data suggest a role for draD and draE in the expression of hemolysis. FEMS Microbiol Lett, 1995 Jul 15, 130(1), 1 - 6 Tyrosine protein phosphorylation in murine B lymphocytes by stimulation with lipopolysaccharide from Porphyromonas gingivalis; Kimura S et al.; The molecular effect of lipopolysaccharides (LPS) from porphyromonas gingivalis as well as Escherichia coli on the tyrosine protein phosphorylation in the splenic B lymphocytes from LPS-responsive C3H/HeN and LPS-hyporesponsive C3H/HeJ mice was examined . P . gingivalis LPS induced tyrosine phosphorylation of selected membrane proteins that included the phosphoproteins with apparent molecular masses of 24.8 kDa and 26.0 kDa (p24.8 and p26.) in the B lymphocytes from both strains of mice, while E . coli LPS induced p24.8 and p26.0 in C3H/HeN B Lymphocytes only . These findings suggest that through the same tyrosine phosphorylation pathway as observed in C3H/HeN B lymphocytes, P . gingivalis LPS induced the activation of C3H/HeJ B lymphocytes in which a trigger signal by E . coli LPS could not be transduced to initiate tyrosine protein phosphorylation. Eur J Pharmacol, 1995 Jul 14, 280(3), 339 - 42 Nitric oxide and sensory afferent neurones modulate the protective effects of low-dose endotoxin on rat gastric mucosal damage; Barrachina D et al.; Pretreatment (1 h) with low doses (5-40 micrograms/kg i.p.) of Escherichia coli endotoxin dose dependently reduced the gastric mucosal damage induced by a 10 min challenge with 1 ml ethanol (50% and 100%) in conscious rats . Treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 5 and 10 mg/kg i.p.), significantly inhibited the protective effects of endotoxin (40 micrograms/kg i.p.) . The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.p.) . The protective effects of endotoxin were not influenced by pretreatment with dexamethasone (5 mg/kg s.c . twice) or indomethacin (5 mg/kg s.c.) . However, ablation of sensory afferent neurons by capsaicin pretreatment (20, 30 and 50 mg/kg s.c.) abolished the mucosa protective effects of endotoxin (40 micrograms/kg) . These findings suggest that the protection elicited by low doses of endotoxin against ethanol-induced mucosal damage involves synthesis of nitric oxide and activation of sensory neurones. J Biol Chem, 1995 Jul 14, 270(28), 16903 - 10 Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication; Buchberger A et al.; Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP . Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence . Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected . Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP . N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain . They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions . Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant . Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction . Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site. J Biol Chem, 1995 Jul 14, 270(28), 16653 - 9 Proton-translocating nicotinamide nucleotide transhydrogenase of Escherichia coli . Involvement of aspartate 213 in the membrane-intercalating domain of the beta subunit in energy transduction; Yamaguchi M et al.; Mutations in the beta subunit of Escherichia coli proton-translocating nicotinamide nucleotide transhydrogenase of the conserved residue beta Asp-213 to Asn (beta D213N) and Ile (beta D213I) resulted in the loss, respectively, of about 70% and 90% NADPH-->3-acetylpyridine adenine dinucleotide (AcPyAD) transhydrogenation and coupled proton translocation activities . However, the cyclic NADP(H)-dependent NADH-->AcPyAD transhydrogenase activities of the mutants were only approximately 35% inhibited . The latter transhydrogenation, which is not coupled to proton translocation, occurs apparently via NADP under conditions that enzyme-NADP(H) complex is stabilized . Mutations beta D213N and beta D213I also resulted in decreases in apparent KmNADPH for the NADPH-->AcPyAD and S0.5NADPH (NADPH concentration needed for half-maximal activity) for the cyclic NADH-->AcPyAD transhydrogenation reactions, and in KdNADPH, as determined by equilibrium binding studies on the purified wild-type and the beta D213I mutant enzymes . These results point to a structural role of beta Asp-213 in energy transduction and are discussed in relation to our previous suggestion that proton translocation coupled to NADPH-->NAD (or AcPyAD) transhydrogenation is driven mainly by the difference in the binding energies of NADPH and NADP. J Biol Chem, 1995 Jul 14, 270(28), 16610 - 4 Factor for inversion stimulation-dependent growth rate regulation of serine and threonine tRNA species; Emilsson V et al.; We have previously shown that the accumulation of 20 tRNA species in Escherichia coli is individually regulated as a function of cellular growth rate . We have also reported that the growth rate regulation of some but not all tRNA species is dependent on the activity of the factor for inversion stimulation (FIS) . In present work, we studied the growth rate regulation of the serine- and threonine-accepting tRNA families . We show that the levels of tRNA(3Thr), tRNA(3Ser), tRNA(2Thr), tRNA(3Thr), and tRNA(4Thr) are reduced in fis cells as the growth rate increases . The accumulation of these tRNA species is reduced 2-5-fold at the fastest bacterial growth rate . The strongest effect is observed for the two minor tRNA species; tRNA(2Ser) and tRNA(2Thr) . In contrast, we find that the accumulation of tRNA(1Ser), tRNA(5Ser), and tRNA(1Thr) is similar in wild type and fis bacteria . The data presented provide further evidence for the suggestion that FIS is a stimulating factor that is involved, directly or indirectly, in the high expression level of some tRNA genes at fast bacterial growth rates. J Biol Chem, 1995 Jul 14, 270(28), 16499 - 502 Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes; Enenkel C et al.; Targeting of import substrate to nuclear pore complexes of permeabilized vertebrate cells was previously shown to require a protein complex composed of two subunits, termed karyopherin . Yeast contain a homologue of karyopherin alpha named Srp1p, which was initially identified as a genetic suppressor of mutations in a subunit of RNA polymerase I . To determine whether yeast contain a karyopherin complex that includes Srp1p as the karyopherin alpha homologue, we genetically replaced Srp1p with a Srp1-Protein A chimera . Cytosol from this strain contained a complex, composed of the chimera and a protein of 95 kDa, that was purified using affinity chromatography on IgG Sepharose . Microsequence analysis showed that the 95-kDa protein was identical with a yeast protein encoded by gene L8300.15 on chromosome XII . Sequence comparison revealed that the L8300.15 gene product is the closest structural homologue of vertebrate karyopherin beta . The yeast alpha and beta karyopherin subunits were expressed in Escherichia coli and were purified . When combined, they formed a heterodimeric complex and were active in targeting import substrate to nuclear envelopes of mammalian cells . We propose that all karyopherins function as alpha/beta heterodimers. J Mol Biol, 1995 Jul 14, 250(3), 309 - 14 Monomer-dimer equilibrium of the pSC101 RepA protein; Ingmer H et al.; The pSC101 RepA protein, which is required for plasmid DNA replication, but is inhibitory to replication at high concentration, has been found in both monomeric and dimeric forms . While RepA monomers bind to direct repeat iterons near the pSC101 replication origin, dimers bind to sequences that autoregulate RepA synthesis . We investigated the solution properties of purified RepA protein by analytical ultracentrifugation analysis, and found that RepA exists in Escherichia coli cells in a monomer-dimer equilibrium (Kd = 4 microM), and, moreover, that RepA is primarily in the monomeric form at the concentration (500 molecules per cell; 2 microM) we found by Western blot analysis to occur in cells carrying replicating wild-type pSC101 plasmids . However, at concentrations inhibitory to pSC101 DNA replication, the majority of RepA molecules exist as dimers . Our findings provide experimental support for the proposal that the equilibrium between monomer and dimer forms of RepA has a key role in determining its effect on the replication of pSC101. Nature, 1995 Jul 13, 376(6536), 191 - 4 Crystal structure of the zeta isoform of the 14-3-3 protein; Liu D et al.; The 14-3-3 family of proteins have recently been identified as regulatory elements in intracellular signalling pathways: 14-3-3 proteins bind to oncogene and proto-oncogene products, including c-Raf-1 (refs 2-5), c-Bcr (ref . 6) and polyomavirus middle-T antigen; overexpression of 14-3-3 activates Raf kinase in yeast and induces meiotic maturation in Xenopus oocytes . Here we report the crystal structure of the major isoform of mammalian 14-3-3 proteins at 2.9 A resolution . Each subunit of the dimeric protein consists of a bundle of nine antiparallel helices that form a palisade around an amphipathic groove . The groove is large enough to accommodate a tenth helix, and we propose that binding to an amphipathic helix represents a general mechanism for the interaction of 14-3-3 with diverse cellular proteins . The residues in the dimer interface and the putative ligand-binding surface are invariant among vertebrates, yeast and plants, suggesting a conservation of structure and function throughout the 14-3-3 family. Nature, 1995 Jul 13, 376(6536), 188 - 91 Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways; Xiao B et al.; A broad range of organisms and tissues contain 14-3-3 proteins, which have been associated with many diverse functions including critical roles in signal transduction pathways, exocytosis and cell cycle regulation . We report here the crystal structure of the human T-cell 14-3-3 isoform (tau) dimer at 2.6 A resolution . Each monomer (Mr 28K) is composed of an unusual arrangement of nine antiparallel alpha-helices organized as two structural domains . The dimer creates a large, negatively charged channel approximately 35 A broad, 35 A wide and 20 A deep . Overall, invariant residues line the interior of this channel whereas the more variable residues are distributed on the outer surface . At the base of this channel is a 16-residue segment of 14-3-3 which has been implicated in the binding of 14-3-3 to protein kinase C. Nature, 1995 Jul 13, 376(6536), 184 - 8 A giant nucleopore protein that binds Ran/TC4; Yokoyama N et al.; Ran/TC4 is a small nuclear G protein that forms a complex with the chromatin-bound guanine nucleotide release factor RCC1 (ref . 2) . Loss of RCC1 causes defects in cell cycle progression, RNA export and nuclear protein import . Some of these can be suppressed by overexpression of Ran/TC4 (ref . 1), suggesting that Ran/TC4 functions downstream of RCC1 . We have searched for proteins that bind Ran/TC4 by using a two-hybrid screen, and here we report the identification of RanBP2, a novel protein of 3,224 residues . This giant protein comprises an amino-terminal 700-residue leucine-rich region, four RanBP1-homologous (refs 9, 10) domains, eight zinc-finger motifs similar to those of NUP153 (refs 11, 12), and a carboxy terminus with high homology to cyclophilin . The molecule contains the XFXFG pentapeptide motif characteristic of nuclear pore complex (NPC) proteins, and immunolocalization suggests that RanBP2 is a constituent of the NPC . The fact that NLS-mediated nuclear import can be inhibited by an antibody directed against RanBP2 supports a functional role in protein import through the NPC. Nucleic Acids Res, 1995 Jul 11, 23(13), 2519 - 25 The site-specific DNA endonuclease encoded by a group I intron in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene introduces a single-strand break at low concentrations of Mg2+; Turmel M et al.; Two group I introns (CpSSU.1 and CpSSU.2) that each potentially encode a protein with two copies of the LAGLI-DADG motif were identified in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene . They both belong to subgroup IA3 and represent novel insertion positions in this gene (sites 508 and 793 in the Escherichia coli 16S rRNA) . The proteins encoded by the two introns were synthesized in vitro and tested for their ability to cleave the homing site of their respective introns . Only the CpSSU.1-encoded protein (I-CpaII) was found to display specific DNA endonuclease activity . At 0.1 mM MgCl2, I-CpaII nicks only the bottom (transcribed) DNA strand, but at concentrations ranging from 0.5 to 5.0 mM, it cleaves both DNA strands (leaving a 4 nucleotide single-stranded extension with 3'-OH overhangs) while preferentially nicking the bottom strand . The rate of cleavage of the top strand increases with increasing concentration of MgCl2 . The preliminary data derived from these endonuclease assays suggest that the mode of DNA cleavage by I-CpaII is directed by the availability of Mg2+ and the affinity of different binding sites for this cation. Nucleic Acids Res, 1995 Jul 11, 23(13), 2506 - 11 Phosphorylation of a chloroplast RNA-binding protein changes its affinity to RNA; Lisitsky I et al.; An RNA-binding protein of 28 kDa (28RNP) was previously isolated from spinach chloroplasts and found to be required for 3' end-processing of chloroplast mRNAs . The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic- and glycine-rich amino terminal domain . Upon analysis of the RNA-binding properties of the 'native' 28RNP in comparison to the recombinant bacterial expressed protein, differences were detected in the affinity to some chloroplastic 3' end RNAs . It was suggested that post-translational modification can modulate the affinity of the 28RNP in the chloroplast to different RNAs . In order to determine if phosphorylation accounts for this post-translational modification, we examined if the 28RNP is a phosphoprotein and if it can serve as a substrate for protein kinases . It was found that the 28RNP was phosphorylated when intact chloroplasts were metabolically labeled with {32P} orthophosphate, and that recombinant 28RNP served as an excellent substrate in vitro for protein kinase isolated from spinach chloroplasts or recombinant alpha subunit of maize casein kinase II . The 28RNP was apparently phosphorylated at one site located in the acidic domain at the N-terminus of the protein . Site-directed mutagenesis of the serines in that region revealed that the phosphorylation of the protein was eliminated when serine number 22 from the N-terminus was changed to tryptophan . RNA-binding analysis of the phosphorylated 28RNP revealed that the affinity of the phosphorylated protein was reduced approximately 3-4-fold in comparison to the non-phosphorylated protein . Therefore, phosphorylation of the 28RNP modulates its affinity to RNA and may play a significant role in its biological function in the chloroplast. Nucleic Acids Res, 1995 Jul 11, 23(13), 2472 - 8 Metalloregulation of the cyanobacterial smt locus: identification of SmtB binding sites and direct interaction with metals; Erbe JL et al.; The smtB gene of Synechococcus PCC 7942 encodes a trans-acting repressor of the metal-regulated smtA gene that encodes a class II metallothionein . Recombinant SmtB has been expressed in Escherichia coli and purified . Electrophoretic mobility shift assays using recombinant SmtB or a protein extract from Synechococcus PCC 6301 reveal the concentration-dependent formation of three specific complexes with the smt operator/promoter . SmtB is also capable of direct interaction with metals as evidenced by 65Zn binding to the SmtB protein as well as the inhibition of repressor-DNA complex formation in the presence of various metal ions . Methylation interference analysis of such complexes identifies four protein contact points within the smt operator/promoter DNA . The points of contact appear to represent two pairs of binding sites, one pair in each of two inverted repeats (nt 548-563, 589-602) . The contact points within each pair lie on opposing DNA strands and are separated by 10 bp, placing the repressor binding sites on opposite sides of the DNA helix . Based on electrophoretic mobility shift assays, methylation interference and molecular size calculations we propose that recombinant SmtB binds to the smt operator/promoter in multimeric fashion. Nucleic Acids Res, 1995 Jul 11, 23(13), 2396 - 403 Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs; Rosendahl G et al.; The ribosomal protein L11 binds to the region of 23S rRNA associated with the GTPase-dependent steps of protein synthesis . Nucleotides 1054-1107 within this region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite . Twenty point mutations (G-->A and C-->T transitions) and numerous multiple mutations were generated . Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability . Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions . These effects are relieved by point mutations at other sites, indicating functional interconnections within the higher order structure of this 23S rRNA region . Several mutations prevent direct binding of r-protein L11 to 23S rRNA in vitro . These mutations are mainly in a short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where the protein probably makes nucleotide contacts . Some of these mutations also interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site on the rRNA . When added together to rRNA, proteins L10.(L12)4 and L11 bind cooperatively to overcome the effects of mutations at 1091 and 1099 . The proteins also stimulate each others binding to rRNA mutated at 1087 or 1092, although in these cases binding remains clearly substoichiometric . Surprisingly, none of the mutations prevents incorporation of L11 into ribosomes in vivo, indicating that other, as yet unidentified, factors are involved in the cooperative assembly process. Nucleic Acids Res, 1995 Jul 11, 23(13), 2389 - 95 Cloning and characterisation of a nuclear, site specific ssDNA binding protein; Smidt MP et al.; Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins . In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D) . Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF) . Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins . Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription . A protein with similar binding characteristics is present in liver nuclear extract . The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. Nucleic Acids Res, 1995 Jul 11, 23(13), 2371 - 80 Mapping the path of the nascent peptide chain through the 23S RNA in the 50S ribosomal subunit; Stade K et al.; Peptides of different lengths encoded by suitable mRNA fragments were biosynthesized in situ on Escherichia coli ribosomes . The peptides carried a diazirine derivative bound to their N-terminal methionine residue, which was photoactivated whilst the peptides were still attached to the ribosome . Subsequently, the sites of photo-cross-linking to 23S RNA were analyzed by our standard procedures . The N-termini of peptides of increasing length became progressively cross-linked to nucleotide 750 (peptides of 6, 9 or 13-15 amino acids), to nucleotide 1614 and concomitantly to a second site between nucleotides 1305 and 1350 (a peptide of 25-26 amino acids), and to nucleotide 91 (a peptide of 29-33 amino acids) . Previously we had shown that peptides of 1 or 2 amino acids were cross-linked to nucleotides 2062, 2506 and 2585 within the peptidyl transferase ring, whereas tri-and tetrapeptides were additionally cross-linked to nucleotides 2609 and 1781 . Taken together, the data demonstrate that the path of the nascent peptide chain moves from the peptidyl transferase ring in domain V of the 23S RNA to domain IV, then to domain II, then to domain III, and finally to domain I . These cross-linking results are correlated with other types of topographical data relating to the 50S subunit. Biochemistry, 1995 Jul 11, 34(27), 8924 - 30 Mutagenesis in Escherichia coli by three O6-substituted guanines in double-stranded or gapped plasmids; Pauly GT et al.; Plasmids were constructed with guanine (G) or O6-methyl- (m6G), O6-ethyl-(e6G), or O6-benzyl- (b6G) guanine in the initiation codon (ATG) of the lacZ' gene . Four deoxyuridine residues were incorporated near the modified guanine in the complementary strand . The deoxyuridine-containing plasmids exhibited similarly high transformation efficiencies in ung- Escherichia coli, although the frequency of mutations induced by m6G, e6G, and b6G residues was relatively low . Treatment of the plasmids with uracil-DNA glycosylase (UDG), to remove the uracil residues, or UDG and exonuclease III, to create a gap in the deoxyuridine-containing strand, reduced transformation efficiency for adduct-containing plasmids but did not affect transformation efficiency for control plasmids . However, the same treatments dramatically enhanced mutagenesis by m6G, e6G, and b6G . These results were consistent with blockage of replication by the modified guanines in double-stranded plasmids resulting in preferential replication of the complementary strand . Replication past the modified guanines was forced in the gapped plasmids . The frequency of modified guanine-induced mutations in gapped vectors was similar in strains of E . coli that were proficient in DNA polymerase III but deficient in either DNA polymerase I or II or both polymerase I and II suggesting either that polymerase III was primarily responsible for adduct bypass in all strains or that the probability of base misinsertion during bypass by either polymerase I or II was similar to that for polymerase III . Repair studies with gapped plasmids indicated that m6G was subject to repair by Ada methyltransferase and to postreplication processing by methylation-directed mismatch repair . Neither e6G nor b6G were similarly repaired.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 11, 34(27), 8914 - 23 Cytosine methyltransferase from Escherichia coli in which active site cysteine is replaced with serine is partially active; Gabbara S et al.; EcoRII methyltransferase (M.EcoRII) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine (SAM) to C-5 position of second cytosine in the DNA sequence 5'-CCWGG (W = A or T) . The reaction is initiated by a nucleophilic attack of the C-6 of target cytosine by a cysteine that is conserved among all cytosine methyltransferases . We have replaced this cysteine in M.EcoRII with serine or alanine and purified the proteins to homogeneity . The catalytic efficiency (kcat/Km) of the mutant enzyme with serine (C186S) for methyl transfer is about 10,000 times less than that of WT but is substantially higher than the efficiency of the C186A mutant . We show that the WT enzyme and C186S mutant are proficient in exchange of proton at C-5 and that this activity is reduced in the mutant to the same extent as the methyl transfer activity . The C186S mutant is insensitive to a cysteine-specific inhibitor, and it transfers methyl groups to the same position of cytosine as the WT enzyme . The ability of serine to act as a nucleophile in the enzyme reaction suggests that it--and probably the cysteine in the WT enzyme--is activated by a nearby base . Like the WT enzyme, C186S forms stable SDS-resistant complexes with DNA containing 5-azacytosine; but unlike the WT enzyme, the mutant reacts faster with 5-azacytosine than with normal cytosine . Apparently, greater reactivity of 5-azacytosine assists the C186S mutant in catalysis. Biochemistry, 1995 Jul 11, 34(27), 8896 - 903 Isolated RNA binding domain of a class I tRNA synthetase; Gale AJ et al.; Class I tRNA synthetases typically have two major domains consisting of a class-defining N-terminal nucleotide binding fold, which contains the active site, and an idiosyncratic C-terminal domain, which in many instances provides for interactions with the tRNA anticodon . Whether the C-terminal domain can function in specific RNA binding when disconnected from the catalytic core is not known . We fused the anticodon binding domain of Escherichia coli methionyl tRNA synthetase to maltose binding protein . This fusion protein and the released, isolated domain are stable and have native-like structural characteristics, as shown by circular dichroism and thermal denaturation studies . Both the fusion protein and the isolated domain bind specifically to a small RNA hairpin oligonucleotide that recapitulates the anticodon stem-loop of tRNAfMet . Neither protein binds to an RNA hairpin with a point mutation in the anticodon trinucleotide . The binding specificity and affinity of these proteins duplicate those of the interaction between methionyl tRNA synthetase and the anticodon stem-loop oligonucleotide . Thus, the anticodon binding domain is functionally independent of the class-defining catalytic core and can be joined to another protein with little change in RNA binding characteristics. Biochemistry, 1995 Jul 11, 34(27), 8752 - 62 Self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme; Harris SJ et al.; The extent of self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme has been investigated by velocity sedimentation as a function of both NaCl and protein concentrations . The core enzyme, existing as essentially monomeric species having a sedimentation coefficient of 13.1 S at NaCl concentrations greater than 0.2 M, undergoes reversible self-association at lower salt concentrations . Estimates for the stoichiometry of association and equilibrium constants of reaction were determined from the effect of protein concentration on the weight-average sedimentation coefficient measured at different NaCl concentrations . Data analysis by a nonlinear curve-fitting procedure indicated that protein self-association is best described by a sequential model characterized by weaker association constants for each additional step of oligomerization, and any model that involves cooperative formation of oligomeric species can be excluded . These findings are at variance with the conclusion of a previous study {Shaner, S . L., Platt, D . M., Wensley, C . G., Yu, H., Burgess, R . R., & Record, M . T . (1982) Biochemistry 26, 5539-5551} which suggested that core RNA polymerase exists in equilibrium between monomeric and tetrameric forms of the enzyme and excluded the existence of intermediate species . Simulation of sedimentation velocity boundary and gradient profiles are used to assess the validity of both models of association of core protein . It was clear that had the core enzyme undergone a cooperative monomer<-->tetramer mode of association, then bimodality would have been observed in the derivative tracings of the sedimentation boundary under these experimental conditions . Nevertheless, no such observation was reported by Shaner et al . and this study . The sequential model favored by the results of this study is consistent with the proposed model resulted from a small-angle X-ray study {Heumann, H, Meisenberger O., & Pilz, I . (1982) FEBS Lett . 138, 273-276} . Further analysis of the data by the Wyman linked-function relationship {Wyman, J . (1964) Adv . Protein Chem . 19, 223-286} implies that core enzyme monomer loses approximately three counterions per contact upon association to higher oligomeric species. Biochemistry, 1995 Jul 11, 34(27), 8513 - 9 Interactions of Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA . The nucleic acid does not wrap around the protein hexamer; Bujalowski W et al.; The interactions of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA have been studied using the thermodynamically rigorous fluorescence titration technique, which allowed us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal change and the degree of binding . Binding of the DnaB protein to the ssDNA fluorescent derivative poly(d epsilon A) is accompanied by a strong increase of the nucleic acid fluorescence . We show that, in the presence of the ATP nonhydrolyzable analog AMP-PNP, the DnaB helicase binds polymer ssDNA with the site-size of 20 +/- 3 nucleotides per protein hexamer . This stoichiometry has been fully confirmed in the binding experiments with ssDNA oligomers of 40 and 20 residues in length . Two DnaB hexamers bind to 40-mer, and one DnaB hexamer binds to 20-mer . Thermodynamic studies of the 20-mer binding to the DnaB hexamer show that the hexamer has a single, strong binding site for ssDNA . Moreover, photo-cross-linking experiments indicate that only a single subunit is primarily in contact with ssDNA . This surprisingly very low site-size of the large hexameric helicase--ssDNA complex, the existence of only a single, strong ssDNA binding site on the hexamer, and the results of photo-cross-linking experiments preclude the possibility of extensive wrapping of the ssDNA around the hexamer and formation of the complex in which all six protomers are simultaneously bound to ss nucleic acid.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Biochem Biophys, 1995 Jul 10, 320(2), 224 - 35 Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin-binding peptide derived from Bordetella pertussis adenylate cyclase; Munier H et al.; This paper reports the solution conformation of a peptide (P196-267) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase . P196-267 corresponding to the protein fragment situated between amino acid residues 196-267 was overproduced by a recombinant Escherichia coli strain . Its affinity for calmodulin is only one order of magnitude lower (Kd = 2.4 nM) than that of the whole bacterial enzyme (Kd = 0.2 nM) . The proton resonances of the NMR spectra of P196-267 were assigned using homonuclear two-dimensional techniques (double-quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser enhancement spectroscopy) and a standard assignment procedure . Analysis of the nuclear Overhauser effect connectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular secondary structure . The peptide is flexible in solution, being in equilibrium between random coil and helical structures . Two segments of 11 amino acids (situated between V215 and A225) and 15 amino acids (situated between L233 and A247) populate in a significant proportion the helix conformational state . The two helices can be considerably stabilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a similar secondary conformation . No elements of tertiary structure organization were detected by the present experiments . The conformational properties of the isolated calmodulin target fragment are discussed in relation with the available NMR and X-ray data on various peptides complexed to calmodulin. FEBS Lett, 1995 Jul 10, 368(1), 77 - 80 ERK2/p42 MAP kinase stimulates both autonomous and SRF-dependent DNA binding by Elk-1; Sharrocks AD; A ternary complex comprised of SRF, ternary complex factor (TCF) and the c-fos SRE is the target of several extracellular signal regulated pathways . Phosphorylation of the TCF Elk-1 is a key event in the activation of this complex . We demonstrate that ERK2/p42 phosphorylation of Elk-1 stimulates its recruitment into ternary complexes with SRF . Moreover, phosphorylation of Elk-1 also stimulates its autonomous SRF-independent binding to high affinity binding sites . Thus part of the effect of ERK2/p42 phosphorylation is to stimulate DNA-binding by the ETS DNA-binding domain of Elk-1. FEBS Lett, 1995 Jul 10, 368(1), 55 - 8 Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins; Jones DH et al.; 14-3-3 proteins play a role in many cellular functions: they bind to and regulate several proteins which are critical for cell proliferation and differentiation . 14-3-3 proteins exist as dimers, and in this study we have shown that diverse 14-3-3 proteins can form both homo- and heterodimers in vitro (by cross-linking studies) and in vivo (by coimmunoprecipitation and Western blot analysis); this interaction is mediated solely through the N-terminal domain of the proteins . The composition of 14-3-3 dimers within a cell may play a key part in the role of this family of proteins as modulators or adapters which facilitate the interaction of distinct components of signalling pathways. FEBS Lett, 1995 Jul 10, 368(1), 49 - 54 Analysis and crystallization of a 25 kDa C-terminal fragment of cloned elongation factor Ts from Escherichia coli; Bogestrand S et al.; A 25 kDa C-terminal tryptic fragment of elongation factor Ts has been purified to homogeneity . Experimental evidence suggests that the 25 kDa C-terminal and the 5.3 kDa N-terminal fragments are structurally independent domains . The N-terminal fragment is shown to be essential for the nucleotide exchange activity . Crystals of the C-terminal fragment belong to space group P2 or P2(1) . The diffraction pattern shows a pronounced pseudo-C2 symmetry at low resolution . This pseudo symmetry increases when the crystals are irradiated with X-rays for a few hours. FEBS Lett, 1995 Jul 10, 368(1), 39 - 44 Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance; Posas F et al.; The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis . The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein . The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase . It was also active towards p-nitrophenylphosphate . The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis . The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2 . The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2 . Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase. FEBS Lett, 1995 Jul 10, 368(1), 15 - 8 Interaction between cardiolipin and the mitochondrial presequence of cytochrome c oxidase subunit IV favours lipid mixing without destabilizing the bilayer structure; Mandieau V et al.; We demonstrate the ability of a peptide corresponding to the presequence of the cytochrome c oxidase subunit IV to induce lipid mixing between large unilamellar liposomes . This lipid mixing requires the presence of CL or PE, lipids able to form non-bilayer structures, and is not observed with other negatively charged lipids . However, the fact that this mixing occurs without mixing of the liposome aqueous phases and without destabilizing the lipid organisation is unusual and has not been observed for other amphiphilic peptides . This observation supports the idea that the presequence could play a role in the formation of translocation contact sites between the two mitochondrial membranes and facilitate the structural rearrangements of the outer and inner membrane proteins involved in the two import machineries in a way to permit the formation of a continuous import channel through the two mitochondrial membranes without mixing the cytoplasmic and mitochondrial aqueous contents. FEBS Lett, 1995 Jul 10, 368(1), 105 - 9 Identification of additional homologues of subunits VII and VIII of the ubiquinol-cytochrome c oxidoreductase enables definition of consensus sequences; Boumans H et al.; The Candida utilis QCR7 gene encoding subunit VII of the ubiquinol-cytochrome c oxidoreductase was isolated by functional complementation of the Saccharomyces cerevisiae subunit VII-null mutant . Several other subunit VII homologues as well as homologues for subunit VIII were identified by screening the GenBank database . Some of these homologues for subunit VII could only be identified as such using a consensus sequence that was derived from the multiple sequence alignment . Definition of the consensus should facilitate further analysis of structure/function relationships in this protein. Virology, 1995 Jul 10, 210(2), 391 - 9 Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus; Martinez-Torrecuadrada JL et al.; Overlapping fragments of the outermost capsid protein VP2 of African horse sickness virus serotype 4 (AHSV-4) have been expressed in Escherichia coli . Horse sera from infected and vaccinated animals, rabbit sera, and mice monoclonal antibodies specific for AHSV were used to screen these fragments for antigenic regions . The screening revealed that the major antigenic domain of the AHSV-4 VP2 is localized in a central region (amino acids 200 to 413) and that both the N-terminal region (aa 1-159) and the half C-terminal region (aa 414-1060) are not immunogenic . All the fragments containing a region between amino acids 253 and 413 (fragment H) were able to elicit consistently high titers of neutralizing antibodies . The ability of several subfragments of this region to evoke neutralizing antibodies indicates the presence of several sites inside this domain . However, neutralizing antibodies in sera of horse infected or vaccinated with attenuated viruses were not absorbed by fragment H, indicating that this domain is not immunodominant in AHSV . This information might be useful in designing a subunit vaccine against AHSV infection. J Biol Chem, 1995 Jul 7, 270(27), 16476 - 81 Disruption of base-paired U4.U6 small nuclear RNAs induced by mammalian heterogeneous nuclear ribonucleoprotein C protein; Forne T et al.; Due to 3' end modifications, mammalian U6 small nuclear RNA (snRNA) is heterogeneous in size . The major form terminates with five U residues and a 2',3'-cyclic phosphate, but multiple RNAs containing up to 12 U residues have a 3'-OH end . They are labeled in the presence of {alpha-32P}UTP by the terminal uridylyl transferase activity present in HeLa cell nuclear extracts . That these forms all enter the U6 snRNA-containing particles, U4.U6, U4.U5.U6, and the spliceosome, has been demonstrated previously . Here, we report an interaction between the heterogeneous nuclear ribonucleoprotein (hnRNP) C protein, an abundant nuclear pre-mRNA binding protein, and the U6 snRNAs that have the longest uridylate stretches . This U6 snRNA subset is free of any one of the other snRNPs, since anti-Sm antibodies failed to immunoprecipitate hnRNP C protein . Furthermore, isolated U4.U6 snRNPs containing U6 snRNAs with long oligouridylate stretches are disrupted upon binding of hnRNP C protein either purified from HeLa cells or produced as recombinant protein from Escherichia coli . In view of these data and our previous proposal that the U6 snRNA active in splicing has 3'-OH end, we discuss a model where the hnRNP C protein has a decisive function in the catalytic activation of the spliceosome by allowing the release of U4 snRNP. J Biol Chem, 1995 Jul 7, 270(27), 16360 - 70 Role of the Escherichia coli recombination hotspot, chi, in RecABCD-dependent homologous pairing; Dixon DA et al.; Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as chi sites, 5'-GCTGGTGG-3' . In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a chi sequence in the donor linear double-stranded DNA . We show that this stimulation is due to two factors: 1) the enhanced production of chi-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step . Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and chi-specific single-stranded DNA fragment production are observed . Also, under these conditions, chi-specific fragments derived from both the upstream and downstream regions of the DNA strand containing chi and from cleavage of the non-chi-containing DNA strand are detected . Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD not equal to) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to chi stimulation of recombination . Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, chi itself may be a preferred site for initiation of homologous pairing in this concerted process. J Biol Chem, 1995 Jul 7, 270(27), 16321 - 6 Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon; Saito K et al.; Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the reaction connecting serine and cysteine/methionine metabolism, plays a regulatory role in cysteine biosynthesis in plants . We have isolated a cDNA clone encoding SATase by direct genetic complementation of a Cys- mutation in Escherichia coli using an expression library of Citrullus vulgaris (watermelon) cDNA . The cDNA encodes a polypeptide of 294 amino acids (31,536 Da) exhibiting 51% homology with that of E . coli SATase . DNA-blot analysis indicated the presence of a single copy of the SATase gene (sat) in watermelon . RNA hybridization analysis suggested the relatively ubiquitous and preferential expression in the hypocotyls of etiolated seedlings . Immunoblot analysis indicated the accumulation of SATase predominantly in etiolated plants . L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the SATase in an allosteric manner, indicating the regulatory function of SATase in this metabolic pathway, whereas beta-(pyrazole-1-yl)-L-alanine, a secondary metabolite formed partly through the cysteine biosynthetic pathway, showed no inhibitory effect . A multi-enzyme complex was formed from recombinant proteins of SATase and cysteine synthase (O-acetylserine(thiol)-lyase) from watermelon, suggesting efficient metabolic channeling from serine to cysteine, preventing the diffusion of intermediary O-acetyl-L-serine. J Biol Chem, 1995 Jul 7, 270(27), 16251 - 7 The conserved motif, GXXX(D/E)(R/K)XG{X}(R/K)(R/K), in hydrophilic loop 2/3 of the lactose permease; Jessen-Marshall AE et al.; A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified among a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane . To determine the importance of this motif within the lactose permease of Escherichia coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed . A dramatic inhibition of activity was observed with all bulky mutations at the first-position glycine . Based on these results, together with sequence comparisons within the superfamily, it seems likely that small side chain volume (and possibly high beta-turn propensity) may be structurally important at this position . The acidic residue at the fifth position was also found to be very important for transport activity and even a conservative glutamate at this location exhibited marginal transport activity . In contrast, many substitutions at the eighth-position glycine, even those with a high side chain volume and/or low beta-turn propensity, still retained high levels of transport activity . Similarly, none of the basic residues within the motif were essential for transport activity when replaced individually by nonbasic residues . However, certain substitutions at the basic residue sites as well as the eighth-position glycine were observed to have moderately reduced levels of active transport of lactose . Taken together, the results of this study confirm the importance of the conserved loop 2/3 motif in transport function . It is suggested that this motif may be important in promoting global conformational changes within the permease. J Biol Chem, 1995 Jul 7, 270(27), 16230 - 5 Cytosolic mercaptopyruvate sulfurtransferase is evolutionarily related to mitochondrial rhodanese . Striking similarity in active site amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-directed mutagenesis; Nagahara N et al.; Rat liver mercaptopyruvate sulfurtransferase (MST) was purified to homogeneity . MST is very similar to rhodanese in physicochemical properties . Further, rhodanese cross-reacts with anti-MST antibody . Both purified authentic MST and expressed rhodanese possess MST and rhodanese activities, although the ratio of rhodanese to MST activity is low in MST and high in rhodanese . In order to compare the active site regions of MST and rhodanese, the primary structure of a possible active site region of MST was determined . The sequence showed 66% homology with that of rat liver rhodanese . An active site cysteine residue (Cys246; site of formation of persulfide in catalysis) and an arginine residue (Arg185; substrate binding site) in rhodanese were also conserved in MST . On the other hand, two other active site residues (Arg247 and Lys248) were replaced by Gly and Ser, respectively . Conversion of rhodanese to MST was tried by site-directed mutagenesis . After cloning of rat liver rhodanese, recombinant wild type and three mutants (Arg247-->Gly and/or Lys248-->Ser) were constructed . The enzymes were expressed in Escherichia coli strain BL21 (DE3) with a T7 promoter system . The mutation of these residues decreases rhodanese activity and increases MST activity. J Biol Chem, 1995 Jul 7, 270(27), 16213 - 20 Cytochrome bd oxidase from Azotobacter vinelandii . Purification and quantitation of ligand binding to the oxygen reduction site; Junemann S et al.; Cytochrome bd has been purified from Azotobacter vinelandii by a new simplified procedure . The heme and total iron content has been measured, as has the number of high affinity CO and NO binding sites . Spectral changes indicate high affinity binding of CO and NO to heme d only, with a stoichiometry of 1 molecule of gas per 2 molecules of heme b or per 3 atoms of iron . The results clearly define a stoichiometry of one heme d per complex . Low affinity binding of CO and NO to heme b595 also occurs at higher ligand concentrations . EPR heme-nitrosyl signals are seen with NO bound to both hemes b595 and d but with no indication of spin exchange coupling . Exposure of the air-oxidized complex to alkaline pH results in removal of molecular oxygen from heme d and a change in line shape of the high spin region of the EPR spectrum . Cyanide binds to both heme d and heme b595 in the air-oxidized complex, displacing molecular oxygen from heme d . The rate of cyanide binding to heme d as assessed by spectral changes at 650 nm does not correlate with the rate of binding to heme b595 as assessed by the loss of the high spin EPR signal . In addition, the cyanide binding rate in the presence of reductant is only 3 times that of the rate of binding to the air-oxidized enzyme, in contrast to the copper-containing oxidases where strong redox cooperativity makes these two rates differ by a factor of at least 10(6) . The results do not support the idea of the presence of two strongly interacting hemes in a binuclear center. J Biol Chem, 1995 Jul 7, 270(27), 16128 - 33 The isolation and characterization of cDNA encoding human and rat brain inositol polyphosphate 4-phosphatase; Norris FA et al.; Inositol polyphosphate 4-phosphatase, an enzyme of the inositol phosphate signaling pathway, catalyzes the hydrolysis of the 4-position phosphate of inositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and phosphatidylinositol 3,4-bisphosphate . The amino acid sequences of tryptic and CNBr peptides of the enzyme isolated from rat brain were determined . Degenerate oligonucleotide primers based on this sequence were used to amplify a 74-base pair polymerase chain reaction product . This product was used to isolate a 5607-base pair composite cDNA, which had an open reading frame encoding a protein with 939 amino acids with a predicted molecular mass of 105,588 Da . The rat brain polymerase chain reaction product was used as a probe to isolate a human brain cDNA that predicts a protein with 938 amino acids and a molecular mass of 105,710 Da . Remarkably, the human and rat proteins were 97% identical . Recombinant rat protein expressed in Escherichia coli catalyzed the hydrolysis of all three substrates of the 4-phosphatase . Northern blot hybridization indicates that the 4-phosphatase is widely expressed in rat tissues with the highest levels of expression occurring in brain, heart, and skeletal muscle . Polyclonal antiserum directed against the carboxyl terminus of the 4-phosphatase immunoprecipitated > 95% of the 4-phosphatase activity in crude homogenates of rat brain, heart, skeletal muscle, and spleen, suggesting that this enzyme accounts for the 4-phosphate activity present in rat tissues . This antiserum also immunoprecipitated the 4-phosphatase from human platelet sonicates. Biochem Biophys Res Commun, 1995 Jul 6, 212(1), 84 - 9 Phenotypes of dnaA mutants of Escherichia coli sensitive to detergents and organic solvents; Shinpuku T et al.; We examined growth sensitivities of dnaA mutants of Escherichia coli to detergents (deoxycholic acid, cholic acid) and organic solvents (ethanol, glycerol) . The dnaA602, dnaA604, dnaA5, and dnaA46 mutants with mutations in the putative ATP binding site of DnaA protein showed higher sensitivities to the detergents than did the wild-type strain . The dnaA508 and dnaA167 mutants with mutations in the N-terminal region of DnaA protein, however, showed higher sensitivities to organic solvents . The dnaA204 and dnaA205 mutants with a mutation in the C-terminal region of DnaA protein showed similar sensitivity as did the wild-type strain to reagents . Complementation analysis with a plasmid carrying the wild-type dnaA gene confirmed that dnaA mutations are responsible for these phenotypes that are sensitive to the reagents. Biochem Biophys Res Commun, 1995 Jul 6, 212(1), 159 - 64 A test of the role of electrostatic interactions in determining the CO stretch frequency in carbonmonoxymyoglobin; Decatur SM et al.; The vibrational frequency of CO bound to myoglobin can be varied by up to 60 cm-1 by making site-specific mutations in the distal pocket . These changes may result from specific chemical interactions between distal amino acids and the CO or from changes in the electrostatic field of the distal pocket . In this paper, we separate the relative contributions of these two effects by comparing the IR spectra of the carbonmonoxy complexes of human myoglobin mutants V68N, V68D, and V68E . The effect of replacing valine with these polar amino acids on the electrostatic environment of the distal heme pocket has been independently determined earlier by measurements of the heme reduction potential and electronic absorption spectral band shifts . While all three mutations result in a negative dipole pointing towards the CO ligand, the CO stretch frequency shifts differently in each case . These differences are attributed to specific chemical interactions between the amino acids and the CO ligand. Biochem Biophys Res Commun, 1995 Jul 6, 212(1), 104 - 9 Enhancement of beta-galactosidase gene expression in rat pheochromocytoma cells by exposure to extremely low frequency magnetic fields; Ohtsu S et al.; Exposure of PC12-VG cells to an extremely low frequency magnetic field (ELFMF) enhanced the beta-galactosidase gene expression stimulated by treatment of the cells with forskolin . The enhancing effect of the ELFMF was inhibited by treatment of the cells with a specific inhibitor of PKC, calphostin C, as well as with the Ca2+ entry blockers nifedipin and dantrolen . Enhancement appeared within the first hour of a 4h forskolin treatment when the ELFMF was given at different times during culture . We speculate that exposure of PC12-VG cells to an ELFMF during the early response to forskolin treatment affects cell signal transduction, resulting in enhanced gene expression. Mol Cell Biochem, 1995 Jul 5, 148(1), 79 - 88 The role of tau phosphorylation in transfected COS-1 cells; Medina M et al.; Tau cDNAs from each of the six human isoforms were transfected into COS-1 cells and, in every case, more than one peptide was observed . The diversity of expressed isoforms was due to different levels of tau phosphorylation . Tau phosphorylation results in a decrease of the protein electrophoretic mobility . The major contribution to this mobility shift is due to the phosphorylation at the at the C-terminus of the molecule, as inferred from the expression of tau fragments . Phosphorylation takes place in some of the sites modified in neural cells and in the basis of AD patients . Copolymerization studies indicate that the level of phosphorylation, as well as the localization of the modified residues, may affect the binding of the protein to microtubules . These results indicate that phosphorylation regulates tau function inside the cell. Gene, 1995 Jul 4, 160(1), 95 - 100 Identification and characterization of IS1296 in Mycoplasma mycoides subsp . mycoides SC and presence in related mycoplasmas; Frey J et al.; IS1296, a new insertion sequence belonging to the IS3 family of insertion elements has been identified in Mycoplasma mycoides subsp . mycoides (Mmm) biotype small colony (SC), the agent of contagious bovine pleuropneumonia (CBPP) . IS1296 is 1485-bp long and has 30-bp inverted repeats . It contains two open reading frames, ORFA and ORFB, which show significant similarities to the ORFs which encode the transposase function of IS elements of the IS3 family, in particular IS150 of Escherichia coli . IS1296 is present in 19 copies in Mmm SC-type strain PG1 and in 18 copies in a recently isolated field strain L2 . It seems to transpose at low frequency in Mmm SC . IS1296 is also present in 5 copies in Mmm biotype large colony (LC)-type strain Y-goat, and in two copies in Mycoplasma sp . 'bovine group 7' reference strain PG50 . It is, however, not present in other species of the 'mycoides cluster' or other closely related Mycoplasma sp . of ruminants. Gene, 1995 Jul 4, 160(1), 87 - 8 Construction of an Actinobacillus pleuropneumoniae-Escherichia coli cosmid cloning vector {Gene 160 (1995) 81-86}; West SE et al.; We constructed a cosmid vector, pSW206, which should be useful for the construction of genomic libraries of Actinobacillus pleuropneumoniae (Apl) DNA . pSW206 is based on the broad-host-range plasmid RK2 and can be introduced into Apl by conjugation. Gene, 1995 Jul 4, 160(1), 75 - 9 A second putative mRNA binding site on the Escherichia coli ribosome; Ivanov IG et al.; Translation in bacteria is initiated by a base-pairing interaction between the extreme 3'-end of the small-subunit rRNA and a purine-rich domain (Shine-Dalgarno (SD) sequence) preceding the initiation codon at the 5'-end of most bacterial mRNAs . Here, we describe the identification of a second functional and alternative site on the Escherichia coli ribosome which is capable of interacting with mRNA devoid of SD sequences and initiate the translation . This site is localized between nt 1340 and 1360 of the 16S rRNA in E . coli and is complementary to the untranslated region at the 5'-end of tobacco mosaic virus RNA (omega sequence). Gene, 1995 Jul 4, 160(1), 69 - 74 The partition region of plasmid QpH1 is a member of a family of two trans-acting factors as implied by sequence analysis; Lin Z et al.; Sequencing analysis revealed that the partition region of the Coxiella burnetii plasmid QpH1 contains a putative operon, designated qsopAB . The two open reading frames (ORFs), qsopA and qsopB, specify the QsopA and QsopB proteins, with deduced molecular masses of 45.7 and 37.6 kDa, respectively . Maxicell analysis demonstrated that although qsopB was located downstream from qsopA, it had its own promoter that was active in Escherichia coli . Several direct or inverted repeats were found around this operon . The most distinct was a 20-bp long imperfect palindrome in the promoter region of qsopA, with homology to a palindrome in the promoter region of P1 parA . Structurally qsopAB was similar to parAB of the P1 plasmid . However, at the amino acid (aa) sequence level, QsopA and QsopB were closest to the F plasmid SopA and SopB proteins, respectively . QsopA shared 58.0% homology and 32.7% identity with SopA, but only 45-50% homology and 22-26% identity with other members of the protein A partition family . QsoB had even lower (41-45%) homology to other members of the protein B partition family, with the highest homology and identity to SopB . Despite lower homologies, both QsopA and QsopB did share conserved aa sequence regions and invariant residues with other members within each family. Gene, 1995 Jul 4, 160(1), 25 - 31 Integrative vectors for heterologous gene expression in Streptomyces spp; Motamedi H et al.; Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed . The vectors are comprised of a strong constitutive promoter, PE, a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene . The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site . HyR transformants are stable in the absence of drug selection . Conjugative derivatives were also constructed by incorporating oriT, the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E . coli donor to Steptomyces lividans (Sl) . Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described . Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems . The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl. Gene, 1995 Jul 4, 160(1), 133 - 4 Cloning of the pfaP gene of Leptospira borgpetersenii; Trueba GA et al.; A lambda gt11 library constructed with Leptospira borgpetersenii DNA was screened with monoclonal antibodies (mAb) recognizing a periplasmic flagella-associated protein . A plaque expressing a fusion protein (lambda F15) which reacted with the mAb was isolated and the nucleotide sequence analyzed . The deduced amino-acid (aa) sequence indicates that the pfaP gene belongs to a group of bacterial genes whose products share aa sequence and possibly functional homologies with sppA, an Escherichia coli signal peptidase-encoding gene. Gene, 1995 Jul 4, 160(1), 101 - 3 Cloning and sequencing of a gene from the archaeon Pyrococcus furiosus with high homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli; Jones CE et al.; A gene from the hyperthermophilic archaeon Pyrococcus furiosus, strain Vc1 (DSM 3638), contains an 817-amino-acid open reading frame which shows 42% identity to the phosphoenolpyruvate (PEP) synthetase of Escherichia coli . This putative P . furiosus PEP synthetase is slightly larger than the E . coli enzyme, the region between residues 58 and 89 being absent from the latter. Gene, 1995 Jul 4, 159(2), 255 - 60 Cloning, sequencing and functional expression of a cDNA encoding a NADP-dependent malic enzyme from human liver; Gonzalez-Manchon C et al.; This work reports the structure of a cDNA (ME) encoding a human malic enzyme (ME) (malate NADP oxidoreductase, EC 1.1.1.40) elucidated by joining several overlapping fragments amplified by PCR from human hepatic cDNA or from cDNA libraries . The full-length cDNA has an open reading frame (ORF) of 1719 bp that encodes a 572-amino-acid protein of 64 113 Da, similar to the native monomeric, cytosolic, NADP-dependent ME isolated from human liver . The comparison of the structure of this cDNA with that of the human mitochondrial NAD(P)-dependent ME (EC 1.1.1.39) shows a homology of 63%, suggesting that these two forms originated from the same gene . The expression of the cDNA in Escherichia coli as a translational fusion (glutathione S-transferase::ME) protein yielded a product of the predicted mass . The recombinant protein shows NADP-dependent malate oxidoreductase activity and is virtually inactive with NAD . It also shows other distinct features of the native cytosolic NADP-dependent ME, like Mn2+ dependence, similar substrate (Km = 117 microM) and cofactor affinity (Km = 2 microM) constants, and a lack of allosteric regulation . In human proliferative cells, the NADP-dependent ME activity is poorly expressed and barely inducible by thyroid hormones. Gene, 1995 Jul 4, 159(2), 203 - 7 Functional antibody single-chain fragments from the cytoplasm of Escherichia coli: influence of thioredoxin reductase (TrxB); Proba K et al.; The cytoplasmic expression of a functional antibody (Ab) fragment, containing the correct intradomain disulfide bonds, was investigated in E . coli . We used a single-chain Fv (scFv) fragment of the levan-binding Ab ABPC48, which was shown to be functional only in the presence of the disulfide bonds . Significant amounts of functional, disulfide-containing scFv could be produced in the cytoplasm of E . coli in the absence of thioredoxin reductase (TrxB) activity . The amount of soluble protein remained largely unchanged by this null mutation . A stronger promoter did not result in further improved yields of functional Ab fragment, despite much higher protein production, suggesting that inefficient disulfide formation was still limiting the yield of active scFv . This method of expressing functional Ab fragments in the cytoplasm of E . coli may be important for screening and selection systems. Biochemistry, 1995 Jul 4, 34(26), 8380 - 9 Roles of divalent metal ions in oxidations catalyzed by recombinant cytochrome P450 3A4 and replacement of NADPH--cytochrome P450 reductase with other flavoproteins, ferredoxin, and oxygen surrogates; Yamazaki H et al.; Recombinant cytochrome P450 (P450) 3A4 was most active in nifedipine and testosterone oxidation in a system including NADPH-P450 reductase, cytochrome b5 (b5), a semisynthetic phospholipid mixture plus cholate, glutathione, and MgCl2 . The MgCl2 effect could be seen with high concentrations of Ca2+ or Sr2+ but not readily when these cations were replaced with monovalent cations . The divalent cation effect was also seen in liver microsomes . Part of the basis of this effect appears to be enhanced rates of b5 reduction, as judged from studies on deletions of reconstitution components and analysis of steady-state spectral studies . Rapid reduction of ferric P450 3A4 to ferrous was dependent upon the presence of substrate, either testosterone or ethylmorphine . When testosterone was present, reduction was also highly dependent upon the presence of b5 and Mg2+ . In the case of the substrate ethylmorphine, the need to add b5 and Mg2+ to obtain optimal reduction rates was less pronounced . These patterns are consistent with the dramatic dependence of testosterone 6 beta-hydroxylation on b5 and the lack of dependence of ethylmorphine N-demethylation on b5 . Our interpretation is that divalent cations stimulate electron transfer from NADPH-P450 reductase to several acceptors and that substrates and b5 can bind to P450 3A4 to influence its rate of reduction by the reductase . P450 3A4 catalyzed testosterone 6 beta-hydroxylation within Escherichia coli cells . The reactions could be supported by E . coli cytosol or by purified E . coli flavodoxin and NADPH-flavodoxin reductase . Spinach ferredoxin and NADPH-ferredoxin reductase also supported catalytic activities.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 4, 34(26), 8348 - 56 Calcineurin subunit interactions: mapping the calcineurin B binding domain on calcineurin A; Sikkink R et al.; Recombinant forms of the A and B subunits of the protein phosphatase calcineurin were produced in Escherichia coli, reconstituted into a heterodimer and purified to homogeneity . The reconstituted heterodimer exhibited properties like that of bovine brain calcineurin . This included calmodulin-stimulated activity and a subunit stoichiometry and Stokes radius consistent with native-like structure . In order to map the region on the A subunit where calcineurin B binds, a series of overlapping 20-residue peptides corresponding to this putative domain were synthesized . Using isolated calcineurin A and B subunits, an assay that relied upon peptide inhibition of calcineurin B stimulation of calcineurin A activity was developed . All five peptides, but not a control peptide, inhibited calcineurin B-dependent stimulation of calcineurin A although with different potencies . The three most effective inhibitory peptides spanned calcineurin A residues 338-377 . These three peptides also altered the electrophoretic mobility of the isolated calcineurin B subunit during native polyacrylamide gel electrophoresis indicating a direct interaction between these peptides and calcineurin B . The peptide corresponding to residues 348-367 was also able to block binding of calcineurin B to the catalytic subunit. Biochemistry, 1995 Jul 4, 34(26), 8330 - 40 Calcium binding to the regulatory N-domain of skeletal muscle troponin C occurs in a stepwise manner; Li MX et al.; Ca2+ binding to a recombinant regulatory N-domain (residues 1-90) of chicken troponin C (NTnC) has been investigated with the use of heteronuclear multidimensional NMR spectroscopy . The protein has been cloned in pET3a vector and expressed in minimal media in Escherichia coli to allow uniform 15N and 13C labeling . The NMR spectra have been resolved and completely assigned {Gagne et al . (1994) Protein Sci . 3, 1961-1974} . Ca2+ titration monitored by 2D (1H, 15N)-HMQC NMR spectral changes revealed that Ca2+ binding to sites I and II of NTnC is a stepwise process and that chemical shift changes occur throughout the N-domain upon the binding of each Ca2+ . The Ca2+ dissociation constants for the binding of the first and second Ca2+ were determined to be 0.8 microM < or = Kd1 < or = 3 microM and 5 microM < or = Kd2 < or = 23 microM, respectively . This mechanism is believed to represent that of the N-domain in intact TnC since we have shown earlier that the properties of the N-domain (1-90) were identical to those of the N-domain in intact TnC {Li et al . (1994) Biochemistry 33, 917-925} . In contrast, however, our previous Ca2+ fluorescence and far-UV CD studies on F29W NTnC and F29W TnC indicated cooperative Ca2+ binding to sites I/II and no detectable differences in their affinities . To rationalize these observations, a direct comparison was made of the Ca2+ titration of NTnC and F29W NTnC as monitored by far-UV CD spectroscopy . Unlike F29W NTnC, NTnC gave a biphasic curve with binding constants in reasonable agreement with the NMR data . Although the far-UV CD spectra of NTnC and the F29W NTnC domain were the same in the absence of Ca2+, the Ca(2+)-induced negative ellipticity increase for NTnC is significantly smaller than for F29W NTnC . These observations indicate that the F29W mutation has perturbed the Ca2+ binding properties of the N-domain and its CD spectroscopic properties in the Ca(2+)-saturated state. Biochemistry, 1995 Jul 4, 34(26), 8257 - 63 Dynamics of lactose permease of Escherichia coli determined by site-directed chemical labeling and fluorescence spectroscopy; Wu J et al.; Mutants with a single Cys residue in place of Phe27, Pro28, Phe29, Phe30, or Pro31 at the periplasmic end of putative transmembrane helix I were used to study the interaction of lactose permease with ligand by site-directed chemical modification or fluorescence spectroscopy . With permease embedded in the native membrane, mutant Phe27-->Cys or Phe28-->Cys is readily labeled with {14C}-N-ethylmaleimide (NEM), while mutant Phe29-->Cys, Phe30-->Cys, or Phe31-->Cys reacts less effectively . beta,D-Galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) has little or no effect on the reactivity of Phe27-->Cys, Phe29-->Cys, or Phe30-->Cys permease . Remarkably, however, Pro31-->Cys permease which is essentially unreactive in the absence of ligand becomes highly reactive in the presence of TDG . Ligand also enhances the NEM reactivity of the mutant with Cys in place of Pro28 which is presumably on the same face of helix I as position 31 . The five single-Cys mutants which also contain a biotin acceptor domain in the middle cytoplasmic loop were purified by monomeric avidin-affinity chromatography in dodecyl beta,D-maltoside and subjected to site-directed fluorescence spectroscopy . Mutants Phe27-->Cys, Phe29-->Cys, and Phe30-->Cys react rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), and reactivity is not altered in the presence of TDG . In striking contrast, mutants Pro28-->Cys and Pro31-->Cys react extremely slowly with MIANS in the absent of ligand, and TDG dramatically enhances reactivity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochim Biophys Acta, 1995 Jul 3, 1250(1), 69 - 75 The binding of propionyl-CoA and carboxymethyl-CoA to Escherichia coli citrate synthase; Man WJ et al.; The interaction of propionyl-CoA and acetyl-CoA with E . coli citrate synthase has been studied in order to gain insight into the structural requirements for substrate binding by this enzyme . In contrast to the enzyme from pig heart, the E . coli enzyme was unable to catalyse significant exchange of the methylene protons of propionyl-CoA while overall activity was very low with this enzyme . Carboxymethyl-CoA is a presumptive transition state analogue of acetyl-CoA using pig heart citrate synthase . The effect of carboxymethyl-CoA on both the native enzyme from E . coli and a catalytically active aspartate mutant (D362E) was investigated . Whereas the native enzyme was inhibited by carboxymethyl-CoA, the mutant enzyme (D362E) shows either no inhibition or minimal inhibition depending on the assay conditions . The binding of acetyl-CoA is not inhibited as a result of the mutation . The results with propionyl-CoA and carboxymethyl-CoA suggest that the active site of the E . coli enzyme is more restricted as compared with the enzyme from pig heart and, in the case of propionyl-CoA, this restriction prevents the formation of a catalytically productive enzyme-substrate complex. Biochim Biophys Acta, 1995 Jul 3, 1250(1), 29 - 34 Single step purification of biologically active recombinant rat basic fibroblast growth factor by immobilized metal affinity chromatography; Kroiher M et al.; The construction and use of a plasmid which allows the expression and single step purification of recombinant rat basic fibroblast growth factor (bFGF) is described . A cDNA encoding rat bFGF was subcloned into the expression plasmid pQE-9 (Qiagen) in such a way that the bFGF which is produced from the resulting construct contains 6 histidine residues near the amino terminus . The resulting plasmid, pQE-9-bFGF, was expressed in the E . coli strain M15{pREP4} and the 6 x His-bFGF was purified to homogeneity from the soluble fraction of the bacterial cell lysate in a single step by affinity chromatography on a nickel chelate resin . About 5 mg of 6 x His-bFGF was obtained from the soluble fraction from one liter of bacterial cell culture . Testing of the 6 x His-bFGF in a PC12 cell differentiation assay showed that its activity was comparable to the activities for native bFGF and recombinant bFGF purified by multistep methods. FEBS Lett, 1995 Jul 3, 367(3), 280 - 2 The first 37 residues are sufficient for dimerization of ribosomal L7/L12 protein; Gudkov AT et al.; The ribosomal protein L7/L12 with the substitution of Cys38 for the Val38 residue was obtained and studied to test the orientation of polypeptide chains in the N-terminal region of the dimer . The results show that the L7/L12 dimer has a parallel (head-to-head) orientation of subunits and that its first 37 N-terminal residues are sufficient for dimerization. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6640 - 4 The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression; Xu YL et al.; The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs . Previously, a cDNA clone encoding a GA 20-oxidase {gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-} was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase . Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe . This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns . The three exons are 1131-bp long and encode 377 amino acid residues . A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20 . The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level . Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases . Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis . The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase . Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6630 - 4 Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis; Yuan Y et al.; Mycolic acids represent a major constituent of the mycobacterial cell wall complex, which provides the first line of defense against potentially lethal environmental conditions . Slow-growing pathogenic mycobacteria such as Mycobacterium tuberculosis modify their mycolic acids by cyclopropanation, whereas fast-growing saprophytic species such as Mycobacterium smegmatis do not, suggesting that this modification may be associated with an increase in oxidative stress experienced by the slow-growing species . We have demonstrated the transformation of the distal cis double bond in the major mycolic acid of M . smegmatis to a cis-cyclopropane ring upon introduction of cosmid DNA from M . tuberculosis . This activity was localized to a single gene (cma1) encoding a protein that was 34% identical to the cyclopropane fatty acid synthase from Escherichia coli . Adjacent regions of the DNA sequence encode open reading frames that display homology to other fatty acid biosynthetic enzymes, indicating that some of the genes required for mycolic acid biosynthesis may be clustered in this region . M . smegmatis overexpressing the cma1 gene product significantly resist killing by hydrogen peroxide, suggesting that this modification may be an important adaptation of slow-growing mycobacteria to oxidative stress. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6552 - 6 Alternate protein frameworks for molecular recognition; Ku J et al.; In an effort to determine whether proteins with structures other than the immunoglobulin fold can be used to mimic the ligand binding properties of antibodies, we generated a library from the four-helix bundle protein cytochrome b562 in which the two loops were randomized . Panning of this library against the bovine serum albumin (BSA) conjugate of N-methyl-p-nitrobenzylamine derivative 1 by phage display methods yielded cytochromes in which residues Trp-20, Arg-21, and Ser-22 in loop A and Arg-83 and Trp-84 in loop B were conserved . The individual mutants, which fold into native-like structure, bind selectively to the BSA-1 conjugate with micromolar dissociation constants (Kd), in comparison to a monoclonal antibody that binds selectively to 1 with a Kd of 290 nM . These and other antibody-like receptors may prove useful as therapeutic agents or as reagents for both intra- and extracellular studies. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6518 - 21 Direct evidence for secondary loss of mitochondria in Entamoeba histolytica; Clark CG et al.; Archezoan protists are though to represent lineages that diverged from other eukaryotes before acquisition of the mitochondrion and other organelles . The parasite Entamoeba histolytica was originally included in this group . Ribosomal RNA based phylogenies, however, place E . histolytica on a comparatively recent branch of the eukaryotic tree, implying that its ancestors had these structures . In this study, direct evidence for secondary loss of mitochondrial function was obtained by isolating two E . histolytica genes encoding proteins that in other eukaryotes are localized in the mitochondrion: the enzyme pyridine nucleotide transhydrogenase and the chaperonin cpn60 . Phylogenetic analysis of the E . histolytica homolog of cpn60 confirmed that it is specifically related to the mitochondrial lineage . The data suggest that a mitochondrial relic may persist in this organism . Similar studies are needed in archezoan protists to ascertain which, if any, eukaryotic lineages primitively lack mitochondria. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6499 - 503 The GroES homolog of Helicobacter pylori confers protective immunity against mucosal infection in mice; Ferrero RL et al.; Helicobacter pylori is an important etiologic agent of gastroduodenal disease . In common with other organisms, H . pylori bacteria express heat shock proteins that share homologies with the GroES-GroEL class of proteins from Escherichia coli . We have assessed the heat shock proteins of H . pylori as potential protective antigens in a murine model of gastric Helicobacter infection . Orogastric immunization of mice with recombinant H . pylori GroES- and GroEL-like proteins protected 80% (n = 20) and 70% (n = 10) of animals, respectively, from a challenge dose of 10(4) Helicobacter felis bacteria (compared to control mice, P = 0.0042 and P = 0.0904, respectively) . All mice (n = 19) that were immunized with a dual antigen preparation, consisting of H . pylori GroES-like protein and the B subunit of H . pylori urease, were protected against infection . This represented a level of protection equivalent to that provided by a sonicated Helicobacter extract (P = 0.955) . Antibodies directed against the recombinant H . pylori antigens were predominantly of the IgG1 class, suggesting that a type 2 T-helper cell response was involved in protection . This work reports a protein belonging to the GroES class of heat shock proteins that was shown to induce protective immunity . In conclusion, GroES-like and urease B-subunit proteins have been identified as potential components of a future H . pylori subunit vaccine. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6334 - 8 Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor; Daly NL et al.; The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma . Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis . The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins . Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement . The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions . The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns . Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module . To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins . Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6244 - 8 The recombination hot spot chi activates RecBCD recombination by converting Escherichia coli to a recD mutant phenocopy; Myers RS et al.; The products of the recB and recC genes are necessary for conjugal recombination and for repair of chromosomal double-chain breaks in Escherichia coli . The recD gene product combines with the RecB and RecC proteins to comprise RecBCD enzyme but is required for neither recombination nor repair . On the contrary, RecBCD enzyme is an exonuclease that inhibits recombination by destroying linear DNA . The RecD ejection model proposes that RecBCD enzyme enters a DNA duplex at a double-chain end and travels destructively until it encounters the recombination hot spot sequence chi . Chi then alters the RecBCD enzyme by weakening the affinity of the RecD subunit for the RecBC heterodimer . With the loss of the RecD subunit, the resulting protein, RecBC(D-), becomes deficient for exonuclease activity and proficient as a recombinagenic helicase . To test the model, genetic crosses between lambda phage were conducted in cells containing chi on a nonhomologous plasmid . Upon delivering a double-chain break to the plasmid, lambda recombined as if the cells had become recD mutants . The ability of chi to alter lambda recombination in trans was reversed by overproducing the RecD subunit . These results indicate that chi can influence a recombination act without directly participating in it. EMBO J, 1995 Jul 3, 14(13), 3252 - 61 The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation; Iost I et al.; We have used either Escherichia coli or T7 RNA polymerase to transcribe in E . coli a series of lacZ genes that differ in the nature of their ribosome binding sites (RBS) . Each T7 RNA polymerase transcript yields from 15- to 450-fold less beta-galactosidase than its E . coli polymerase counterpart, the ratio being larger when weaker RBS are used . The low beta-galactosidase yield from T7 transcripts reflects their low stability: the ams-1/rne-50 mutation, which inactivates RNase E, nearly equalizes the beta-galactosidase yields from T7 and E . coli RNA polymerase transcripts . T7 RNA polymerase transcribes the lacZ gene approximately 8-fold faster than the E . coli enzyme . We propose that this higher speed unmasks an RNase E cleavage site which is normally shielded by ribosomes soon after its synthesis when the slower E . coli enzyme is used . This leads to degradation of the T7 transcript, unless the leading ribosome comes in time to shield the cleavage site: the weaker the RBS, the lower this probability and the more severe the inability of T7 RNA polymerase transcripts for beta-galactosidase synthesis. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6329 - 33 Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns; Horlacher J et al.; The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N1-methyloxoformycin B (pi) has been investigated . The kappa-X and kappa-pi base pairs are jointed by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs . Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity . With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template . With d pi in the template, no incorporation of d kappa TP was observed with HIV reverse transcriptase . The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa . Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi . DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base . These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6249 - 53 Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit; Koppen A et al.; The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities . The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity . Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E . coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h . It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD . After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion . Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination . The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants . It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination. J Gen Virol, 1995 Jul, 76 ( Pt 7), 1651 - 63 Comparative studies of bacterially expressed integrase proteins of caprine arthritis-encephalitis virus, maedi-visna virus and human immunodeficiency virus type 1; Stormann KD et al.; Integrase (IN) proteins mediate an essential step in retroviral life cycles, the integration of reverse-transcribed viral DNA into the host genome . To create tools for direct comparative investigations, hexahistidine-tagged IN proteins of the phylogenetically related lentiviruses caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1) were expressed in Escherichia coli . After purification by affinity chromatography, the active enzymes were compared in vitro for their site-specific cleavage, integration and disintegration activities on cognate and non-cognate oligonucleotide substrates . It was found that CAEV IN and MVV IN catalyse both site-specific cleavage and disintegration with high efficiencies, reduced substrate specificities and similar reaction patterns . Comparisons with the respective activities of HIV-1 IN revealed basic functional similarities as well as considerable differences such as more restricted substrate requirements for site-specific cleavage . On the other hand, all three enzymes catalyse disintegration almost independent of the substrate origin . Furthermore, MVV IN was shown to join oligonucleotides as efficiently as HIV-1 IN, albeit with reduced substrate specificity . In contrast, no detectable strand transfer activities occurred with CAEV IN. Rev Latinoam Microbiol, 1995 Jul-Sep, 37(3), 291 - 304 {DNA supercoiling and topoisomerases in Escherichia coli}; Gomez-Eichelmann MC et al.; The chromosomal DNA of all cells is under helical tension or supercoiling . There are two classes of DNA supercoiling: plectonemic and toroidal . Plectonemic supercoiling is generated by the action of DNA topoisomerases, while toroidal supercoiling is generated by DNA-protein interactions and by topoisomerase activitities . DNA supercoiling plays an important role in replication, repair, recombination, transposition and transcription . DNA topoisomerases type I are ATP-independent enzymes that cut one DNA strand and relax supercoiled molecules . DNA topoisomerases type II requiere ATP, cut both DNA strands and supercoil relaxed molecules . All organisms have more than one topoisomerase of each, type I and type II . Escherichia coli has two topoisomerases type I: topoisomerase I and topoisomerase III and two topoisomerases type II: topoisomerase II or gyrase and topoisomerase IV . In this review we discuss the concept of DNA supercoiling and present current knowledge on E . coli DNA topoisomerases. Int J Microcirc Clin Exp, 1995 Jul-Aug, 15(4), 170 - 80 Effects of iloprost, a stable prostacyclin analog, and its combination with NW-nitro-L-arginine on early events following lipopolysaccharide injection: observations in the hamster cheek pouch microcirculation; Bouskela E et al.; The effects of iloprost, a stable prostacyclin analog, and of its combination with NW-nitro-L-arginine (L-NAG) on the microcirculatory changes observed in early stages of endotoxemia were investigated in male hamsters treated with Escherichia coli lipopolysaccharide (LPS) . The cheek pouch was studied in vivo by means of intravital microscopy and mean arterial and venous pressures, mean arteriolar internal diameter, spontaneous arteriolar vasomotion, microvascular blood flow, macromolecular permeability, leukocyte adhesion and mean survival time were evaluated in animals treated with LPS alone or with the combination of LPS+iloprost and LPS+iloprost+L-NAG . Intravenous injection of LPS (100 mg/kg) per se elicited a significant reduction in mean arterial blood pressure (MABP) and arteriolar blood flow . The observed arterioles dilated and spontaneous vasomotion ceased . Iloprost (40 ng/kg/min) prevented LPS-induced reduction of MABP, ameliorated the decrease in arteriolar blood flow and reduced the vasodilation . Arteriolar vasomotion was reduced but it did not cease . The combination of L-NAG (0.5 mg/kg) + iloprost (40 ng/kg/min) with LPS (100 mg/kg) did not improve the results obtained with iloprost alone, except that it prevented the vasodilation and the vasomotion ceased 1 h after the bolus injection of LPS+L-NAG . The mean survival time compared to LPS alone (57 +/- 7 h) was significantly increased by the combination of LPS+iloprost (102 +/- 6 h) and it did not change significantly with the combination L-NAG+iloprost (64 +/- 9 h) . Topical addition of iloprost (10 ng/kg/min) evoked an early increase in macromolecular permeability and a significant decrease in leukocyte adhesion compared with animals treated with topical LPS (0.7 microgram/ml/min) alone . The combination of L-NAG+iloprost (1.3 + 10 ng/ml/min) enhanced the early increase in macromolecular permeability even further, tended to increase the macromolecular permeability and evoked a smaller decrease in leukocyte adhesion than the one observed with iloprost combined with LPS . Our results, in the hamster cheek pouch microcirculation, suggest that the use of prostacyclin could be beneficial in the treatment of early changes in endotoxic shock, but L-NAG showed no benefit in this preparation. Rev Assoc Med Bras, 1995 Jul-Aug, 41(4), 259 - 65 {Clinical and epidemiological characteristics of acute diarrhea by classical enteropathogenic Escherichia coli}; Fagundes Neto U et al.; Acute diarrhea is usually considered as a self limited disease, but under certain circumstances, mainly owing to the age of the patient, the nutritional status and the enteropathogenic agent the illness can evolve for a protracted evolution . PURPOSE: In the present study we report the clinical and epidemiological features of a group of infants under two years of age with acute diarrhea caused by serogroups of enteropathogenic Escherichia coli (EPEC) . PATIENTS AND METHODS: During a two year period 200 infants under two years of age, mean 8.2 months, with acute diarrhea less than 5 days duration, were consecutively studied . A control group of 40 healthy infants matched for age was also made up . The nutritional status was determined and the occurrence of food intolerance was also monitored . The patients were followed up for 4 weeks after been discharged from the hospital . Stool samples were obtained for research of bacterial, viral and protozoan enteropathogenic agents . RESULTS: EPEC was isolated in the stools of 84 (42.0%) infants, as a sole enteropathogenic agent in 55 (27.5%) and in the remaining 29 (14.5%) infants associated with some other agent . EPEC was isolated in the stools of 9 (22.5%) infants of the control group (p < 0.05) . Food intolerance was the main digestive complication and also the most important factor that caused perpetuation of diarrhea . The mean duration of the disease was 11.2 days, varying from 2 to 40 days . In 53 (71.6%) infants the disease lasted less than 14 days, while in the remaining 21 (28.4%) it lasted more than 14 days, and all these infants presented with food intolerance . CONCLUSIONS: Enteric infections caused by EPEC serogroups, particularly O111 and O119, are more prevalent in weaned infants under 1 year of age belonging to families of low income rates . These infants present heavy fluid and electrolyte losses in the stools associated to high rates of food intolerance. Arq Gastroenterol, 1995 Jul-Sep, 32(3), 152 - 7 Ultrastructural study of enteropathogenic Escherichia coli O111ab:H2 infection in an infant with acute diarrhea; Fagundes-Neto U et al.; Enteropathogenic Escherichia coli is the most important cause of acute diarrhea in developing countries, specially in infants under one year of age . Enteropathogenic Escherichia coli strains are able to induce profound cytoskeletal alterations in the enterocyte known as attaching and effacing lesions, associated with the formation of cuplike pedestals . We report an Enteropathogenic Escherichia coli O11ab:H2 strain isolated from an infant with acute diarrhea, on the eleventh day of disease, that caused attaching and effacing lesion and penetrated the enterocyte, as well as invaded the HeLa cell tissue culture in vitro and the rabbit ileal loop assay in vivo, in the ultrastructural study . This observation indicates that the severe lesions of the small bowel caused by an enteropathogenic Escherichia coli O111ab:H2 strain can occur even in the early stages of the infection. Res Immunol, 1995 Jul-Aug, 146(6), 373 - 82 Similar binding properties for a neutralizing anti-tetanus toxoid human monoclonal antibody and its bacterially expressed Fab; Lafaye P et al.; A high-affinity anti-tenanus toxoid (TT) human monoclonal antibody showing neutralizing activity was isolated from a fusion between mouse myeloma and human splenic cells . Fab fragments from this antibody were obtained using a recombinant phage surface-display expression system . The parental antibody and the corresponding Fab had identical immunological activities, including specificity and affinity . These results confirm the feasibility of developing Escherichia coli expression of monoclonal human Fab from hybridoma cells. J Protein Chem, 1995 Jul, 14(5), 341 - 7 Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified from Escherichia coli; Violand BN et al.; The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified from Escherichia coli have been determined . This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds . The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest . However, thermolysin digestion did not cleave any peptide bonds within domain 2 . The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain . These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified from Escherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor. J Protein Chem, 1995 Jul, 14(5), 299 - 308 Fluorescence study of Escherichia coli cyclic AMP receptor protein; Wasylewski M et al.; Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP) . The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein . Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76-83% to the total fluorescence emission . The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm . The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time . The Trp-85 exhibits a shorter fluorescence decay time . In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers . The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone . It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains . This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix. Dev Dyn, 1995 Jul, 203(3), 337 - 51 Gsh-1: a novel murine homeobox gene expressed in the central nervous system; Valerius MT et al.; We report the characterization of Gsh-1, a novel murine homeobox gene . Northern blot analysis revealed a transcript of approximately 2 kb in size present at embryonic days 10.5, 11.5, and 12.5 of development . The cDNA sequence encoded a proline rich motif, a polyalanine tract, and a homeodomain with strong homology to those encoded by the clustered Hox genes . The Gsh-1 expression pattern was determined for days E8.5 to E13.5 by whole mount and serial section in situ hybridizations . Gsh-1 transcription was restricted to the central nervous system . Expression is present in the neural tube and hindbrain as two continuous, bilaterally symmetrical stripes within neural epithelial tissue . In the mesencephalon, expression is seen as a band across the most anterior portion . There is also diencephalon expression in the anlagen of the thalamus and the hypothalamus as well as in the optic stalk, optic recess, and the ganglionic eminence . Moreover, through the use of fusion proteins containing the Gsh-1 homeodomain, we have determined the consensus DNA binding site of the Gsh-1 homeoprotein to be GCT/CA/CATTAG/A. Dev Dyn, 1995 Jul, 203(3), 324 - 36 Migration of myogenic cells from the somites to the fore-limb buds of developing mouse embryos; Sze LY et al.; In this study, we have isolated newly formed somites from the caudal regions of 8.5 day mouse embryos and transplanted them orthotopically into correspondingly staged hosts at the level of the prospective limb-forming region . The experimental embryos were then cultured intact for 32-36 hr . The donor somites used were pre-labelled with DiI, a fluorescent lipophilic dye, or were obtained from transgenic embryos that carried a 1 kb 5' regulatory sequence of the desmin gene linked to the gene encoding Escherichia coli beta-galactosidase . The transgene is specifically expressed in skeletal muscles (Li et al . {1993} Development 117:947-959) . The aim of these experiments was to show definitively that the musculature of the mammalian limb is derived from the somites . The results demonstrated that DiI-labelled cells from the implanted somites were able to invade the proximal region of the fore-limb bud during the course of development . The use of transgenic somites as grafts confirmed that some of the somitic cells found in the limbs were myogenic cells . To determine whether the displacement of somitic cells is an active or passive process, somatopleure obtained from the prospective limb-forming regions of day 8.5 day embryos was implanted into 8.5 day hosts . We did not detect the presence of DiI-labelled somatopleural cells in the fore-limb after 32-36 hr of culture . This suggests that somitic cells reached the limb bud via active locomotion rather than as a result of being passively dragged there, as the limb elongates during development . In addition, we injected latex beads into the somites, as probes, to determine whether extracellular matrix-driven translocation plays a role in driving the somitic cells to the limb bud . In a majority of the specimens examined, we could not detect the presence of these beads in the limb bud . However, in the trunk of these embryos, the beads were found dispersed throughout the ventral neural crest pathway. Int J Immunopharmacol, 1995 Jul, 17(7), 571 - 80 Reduction in endotoxin-induced organ dysfunction and cytokine secretion by a cyclic nitrone antioxidant; Downs TR et al.; Multiple organ dysfunction (MOD) is the leading cause of mortality in septic patients with circulatory shock . Recent evidence suggests that the overproduction of the cytokine, tumor necrosis factor-alpha(TNF), and oxygen free radical molecules may mediate the progression of sepsis to MOD and death . In this study, we have examined the ability of MDL 101,002, a free radical scavenger, to reduce organ dysfunction and cytokine secretion induced by lipopolysaccharide (LPS) administration in rats . Treatment with MDL 101,002(10-60 ng/kg, i.p.) 30 min prior to an LPS challenge resulted in a dose-dependent reduction in several markers indicative of organ dysfunction and mortality . MDL 101,002 markedly decreased LPS-induced liver and kidney damage as indicated by serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) or urea and creatinine, respectively . MDL 101,002 also prevented LPS-induced pulmonary edema, but did not prevent leukopenia and only partially reduced thrombocytopenia . Associated with these improvements in organ dysfunction and survival was a modest decrease in LPS-stimulated interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) secretion and a marked ( > 90%) inhibition of TNF secretion by MDL 101,002 . The data are consistent with a role for oxygen free radicals in the development of endotoxin-induced organ dysfunction and shock and suggest that free radical scavengers could reduce the mortality consequent to sepsis by decreasing organ dysfunction, at least in part, through a reduction in free radical stimulated cytokine secretion. Protein Eng, 1995 Jul, 8(7), 725 - 31 Properties of a single-chain antibody containing different linker peptides; Alfthan K et al.; Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody . Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues . The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues . Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains . The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM . The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases . Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity . The fragments with linkers longer than 11 residues remained monomeric. Protein Eng, 1995 Jul, 8(7), 711 - 6 Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the gamma-L-glutamyl-L-cysteine-binding site; Hara T et al.; Lys18, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathione synthetase from Escherichia coli B are presumed to be highly concerned with the substrate, gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), binding by X-ray crystallography and affinity labeling studies . Using site-directed mutagenesis, we investigated functional roles of those residues for gamma-Glu-Cys binding . The mutant enzymes of Arg86 and Asn283 altered their kinetic parameters, especially the Michaelis constants of gamma-Glu-Cys . In the case of Asn283, the residue is not likely to have an essential role in gamma-Glu-Cys binding but its side chain would extend to make a van der Waals contact with bound gamma-Glu-Cys . Chemical modification of a cysteine residue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86 would not only be much responsible for gamma-Glu-Cys binding but would also have a role in maintaining the structural integrity of the enzyme . The other mutant enzymes showed little defect in their kinetic parameters of gamma-Glu-Cys. Mol Biochem Parasitol, 1995 Jul, 73(1-2), 7 - 18 Sequence, characterization and localization of a cysteine proteinase cathepsin L in Schistosoma mansoni; Michel A et al.; A cDNA encoding Schistosoma mansoni cathepsin L was isolated from a cDNA library and sequenced . Alignment of the proposed amino-acid sequence with known members of cathepsin L shows highest homologies with sequences from mouse and rat . An expression plasmid was constructed in Escherichia coli to produce recombinant schistosome cathepsin L with an extension of six histidines at its N terminus . Using antibodies raised against the purified fusion protein, two polypeptide bands with approx . molecular masses of 38 and 31 kDa were identified in a schistosome extract . By use of specific radioiodinated inhibitors, a radioactively labeled protein could be detected at 31 kDa, suggesting that this is the active mature enzyme . The larger protein of 38 kDa did not react with the inhibitor, indicating that it represents the inactive precursor molecule . Immunohistological experiments revealed that the proteinase is localized in structures associated with the reproductive system of females and with the subtegumental region of the gynecophoric canal of males . However, Northern blot hybridization demonstrates that more transcripts are present in female parasites than in males . Genomic Southern blotting suggests that schistosome cathepsin L is expressed from a single-copy gene. Mol Biochem Parasitol, 1995 Jul, 73(1-2), 189 - 98 Molecular analysis of two hexokinase isoenzymes from Entamoeba histolytica; Ortner S et al.; The zymodemes, electrophoretic patterns of hexokinase, phosphoglucomutase and glucose phosphate isomerase isoenzymes, have been widely used to determine the pathogenicity of Entamoeba histolytica isolates . Although pathogenic and nonpathogenic forms of E . histolytica differ clearly in sequences of many homologous genes, a conversion between pathogenic and nonpathogenic zymodemes has been reported by several laboratories . To approach the question what might be the basis for the observed conversion, we examined the molecular biology of the hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzymes in pathogenic E . histolytica . We isolated two different cDNAs pHXK1 and pHXK2 coding for polypeptides with significant sequence similarity to hexokinases and deduced molecular masses of 49.8 kDa and 49.4 kDa . The two hexokinase sequences differed by 11% on the amino acid and by 8% on the nucleotide level . Expression of the cDNAs in Escherichia coli as nonfusion proteins gave two polypeptides with hexokinase activity . The recombinant Hxk1 and Hxk2 polypeptides comigrated with the more basic and more acidic isoforms of pathogenic amoebae in starch gel electrophoresis, as well as in low and high resolution isoelectric focussing gels . This identified the observed hexokinase isoenzymes of pathogenic E . histolytica as the products of two genes, hxk1 and hxk2. Mol Biochem Parasitol, 1995 Jul, 73(1-2), 133 - 43 Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani; Allen TE et al.; The gene encoding the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani has been cloned and sequenced . The hgprt open reading frame encoded a polypeptide of 211 amino acids that exhibited 3 regions of significant homology with other eukaryotic HGPRTs and a C-terminal tripeptide compatible with a glycosomal targeting signal . Northern blot analysis of L . donovani RNA revealed two hgprt transcripts, a 1.9-kb mRNA and a 1.7-kb transcript . The expression of the 1.7-kb hgprt mRNA and the activity of HGPRT enzyme were both augmented approx . 5-fold in parasites incubated in the absence of purines . Southern blots of genomic DNA indicated only a single hgprt locus within the L . donovani genome . Overexpression of L . donovani hgprt in E . coli complemented genetic deficiencies in hypoxanthine and guanine phosphoribosylating activities and yielded abundant quantities of enzymatically active HGPRT . The recombinant HGPRT was purified to homogeneity and recognized hypoxanthine, guanine and allopurinol, but not adenine or xanthine, as substrates . The hgprt clone and pure HGPRT protein provide essential reagents for validating HGPRT as a therapeutic target for the treatment of leishmaniasis and other diseases of parasitic origin. Mol Biochem Parasitol, 1995 Jul, 73(1-2), 111 - 21 Cloning and characterization of a Plasmodium falciparum cyclophilin gene that is stage-specifically expressed; Reddy GR; An immunosuppressive agent, cyclosporin A (CsA), has antimalarial activity in several Plasmodium species . Cyclophilins of several species including Plasmodium falciparum exhibit peptidyl-prolyl cis-trans isomerase activity which is inhibited by CsA . A gene encoding P . falciparum cyclophilin (PFCyP) was cloned and characterized . This gene has the entire coding sequence for the mature protein plus a 39-amino-acid-long N-terminal extension . Most of the amino acids predicted to be involved in the peptidyl-prolyl cis-trans isomerase activity and CsA binding are present in the cloned gene . The PFCyP also has the single highly conserved tryptophan residue that is a major determinant in the inhibition of PPIase activity by CsA . The PFCyP coding sequence with or without the N-terminal amino-acid extension was used to construct recombinant expression vectors which were transformed into E . coli . Both vectors produced enzymatically active mature PFCyP proteins that were sensitive to CsA . Northern blot analysis of RNA isolated from the synchronized parasite cultures verified the expression of PFCyP in all erythrocytic stages of the parasite, but at variable levels . The highest level of expression was observed in ring-stage parasites, a stage shown to be more susceptible to CsA . Inhibition of P . falciparum growth in vitro by CsA was re-evaluated for chloroquine-sensitive and chloroquine-resistant strains of the parasite . Essentially, there was no difference between the two strains for the concentration of CsA required to yield 50% inhibition in 48 h of exposure (0.25-0.4 microM). Anal Biochem, 1995 Jul 1, 228(2), 208 - 20 Quantitative fluorescence method for continuous measurement of DNA hybridization kinetics using a fluorescent intercalator; Yguerabide J et al.; We present a quantitative fluorescence method for continuous measurement of DNA or RNA hybridization (including renaturation) kinetics using a fluorescent DNA intercalator . The method has high sensitivity and can be used with reaction volumes as small as 1 microliter and amounts of DNA around 1 ng . The method is based on the observations that (i) for the usual hybridization conditions, intercalators such as ethidium bromide bind (intercalate) to double-stranded DNA (dsDNA) but not single-stranded DNA or RNA and (ii) there is a large increase in fluorescence intensity when intercalators such as ethidium bromide bind to dsDNA . In this application, the intercalator can be considered as a quantitative indicator of dsDNA concentration . When a small amount of intercalator is added to a hybridizing solution, the fluorescence intensity of the intercalators increases with increase in dsDNA . The hybridization reaction can thus be monitored by continuously recording fluorescence intensity vs time . Because the amount of intercalator bound to dsDNA is not necessarily proportional to dsDNA concentration, the time-dependent fluorescence intensity graph is not identical to the kinetic graph {dsDNA} vs t . However, the fluorescence intensity vs time graph can easily be converted to the true {dsDNA} vs t graph by means of an experimental calibration graph of fluorescence intensity vs {dsDNA} . This calibration graph is obtained in a separate experiment using samples containing known amounts of dsDNA in the ethidium bromide buffer used in the kinetic measurement . We present results of experimental tests of the intercalator technique using ethidium bromide as an intercalator and DNA from Escherichia coli and lambda-phage and Poly(I)-Poly(C) RNA hybrids . These DNA and RNA samples have Cot1/2 values that cover a range of 10(6) . Our experimental results show that (i) the kinetics of hybridization are not significantly perturbed by the intercalator at concentrations where no more than 10% of the binding sites on DNA or RNA hybrids are occupied, (ii) the kinetic graphs obtained by the intercalator fluorescence method and corrected with the calibration graph agree with kinetic graphs obtained by optical absorbance measurements at 260 nm, and (iii) the intercalator technique can be used in the different salt environments often used to increase the velocity of the hybridization reaction and at the hybridization temperatures (35-75 degrees C) normally used to minimize nonspecific hybridization. Inflamm Res, 1995 Jul, 44(7), 275 - 80 Modulation of the endothelial procoagulant response to lipopolysaccharide and tumour necrosis factor-alpha in-vitro: the effects of dexamethasone, pentoxifylline, iloprost and a polyclonal anti-human IL-1 alpha antibody; Heyderman RS et al.; Endothelial expression of tissue factor (TF), a potent procoagulant molecule, is increased in response to inflammatory mediators such as lipopolysaccharide (LPS), tumour necrosis factor (TNF) and interleukin-1 (IL-1) . We have examined the effects of three antiinflammatory agents and a polyclonal anti-human IL-1 alpha antibody on the human endothelial TF response to E . coli 0111:B4 LPS and recombinant TNF alpha (rTNF alpha) in vitro . In contrast to the expected inhibitory effect, dexamethasone, pentoxyfilline and iloprost failed to block TF expression when administered simultaneously or 30 minutes prior to stimulation with either LPS or rTNF alpha . Inhibition of procoagulant activity was demonstrated with the anti-IL-1 alpha antibody, suggesting that endothelial derived IL-1 alpha is partially responsible for the TF response to the agonists employed . The failure of the antiinflammatory agents to inhibit endothelial TF expression highlights the possibility that therapeutic agents that modulate the circulating monocyte response to LPS and TNF alpha may not ameliorate the endothelial dysfunction that is also induced by these inflammatory mediators. Avian Dis, 1995 Jul-Sep, 39(3), 636 - 7 Relationship between body-weight gain after movement of chickens to an unfamiliar cage and response to Escherichia coli challenge infection; Gross WB; One day after chickens were moved from a brooder to unfamiliar cages, there was a high negative correlation (p < 0.001) between an individual's body-weight gain and the severity of response to Escherichia coli challenge infection. Avian Dis, 1995 Jul-Sep, 39(3), 480 - 8 Dose titration study of enrofloxacin (Baytril) against respiratory colibacillosis in Muscovy ducks; Kempf I et al.; Four-week-old specific-pathogen-free Muscovy ducks were inoculated with reovirus . One week later, they were inoculated intratracheally with a O78:K80 strain of Escherichia coli . The next day, they were given enrofloxacin at different doses in the drinking water . Comparison of mortality rates, weight gain, macroscopic lesions, and E . coli re-isolations among treated and untreated birds showed that a 5-day treatment course with 12.5 or 25 ppm enrofloxacin in water for 4 hours in the morning provided good therapeutic efficacy against respiratory colibacillosis. Microb Pathog, 1995 Jul, 19(1), 19 - 29 Ex vivo induction of TNF-alpha and IL-6 mRNA in bovine whole blood by Mycobacterium paratuberculosis and mycobacterial cell wall components; Adams JL et al.; Johne's disease is a chronic enteritis of cattle and other ruminant species that is of worldwide economic importance . The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes . The potential role of these cytokines in the development and progression of Johne's disease has not been investigated . Using reverse transcriptase polymerase chain reaction (RT-PCR) and specific bovine oligonucleotide cytokine primers and probes for bovine TNF-alpha and IL-6, we examined the ex vivo expression of mRNA for these inflammatory cytokines in whole blood from healthy cattle . Cytokine mRNA levels increased after a brief incubation of bovine whole blood with Mycobacterium paratuberculosis or its lipoarabinomannan (LAM) . Muramyl dipeptide (MDP) and Escherichia coli LPS also stimulated TNF-alpha and IL-6 mRNA expression . Several strains of M . paratuberculosis were tested and found to have similar abilities to stimulate TNF-alpha and IL-6 mRNA expression . Several strains of the closely related Mycobacterium avium, and the unrelated saprophyte, Mycobacterium phlei, had somewhat less ability to stimulate TNF-alpha and IL-6 mRNA expression. J Biochem (Tokyo), 1995 Jul, 118(1), 75 - 80 Effect of site-directed mutations on processing and activity of gamma-glutamyltranspeptidase of Escherichia coli K-12; Hashimoto W et al.; gamma-Glutamyltranspeptidase {EC 2.3.2.2} of Escherichia coli K-12 is thought to be synthesized from a single precursor polypeptide into a heterodimeric form through post-translational processing . Cells of a gamma-glutamyltranspeptidase-overproducing transformant of E . coli K-12 were fractionated and the localization of the enzyme was examined by Western blot analysis . The periplasmic fraction only contained the mature form of gamma-glutamyltranspeptidase, membrane fraction only contained the precursor of gamma-glutamyltranspeptidase, and no precursor of gamma-glutamyltranspeptidase was detected in the cytoplasmic fraction . Amino acid residues at the cleavage site for processing into the large and small subunits were substituted by site-directed mutagenesis . The processing phenotypes of six mutants were examined by Western blot analysis, and their gamma-glutamyltranspeptidase activities were measured . Mutations at the N-terminal amino acid residues of the small subunit (Thr-391, Thr-392, and His-393) prevented the maturation of the enzyme and the immature mutants exhibited no enzymatic activity . A mutation at the C-terminal residue of the large subunit (Gln-390) had less effect on the processing and enzymatic activity . These results suggest that the sequence of threonyl-threonyl-histidinyl residues at the N-terminal of the small subunit is very important for the processing of E . coli K-12 gamma-glutamyltranspeptidase and this processing is essential to the expression of gamma-glutamyltranspeptidase activity of E . coli K-12. J Biochem (Tokyo), 1995 Jul, 118(1), 67 - 74 Production of hetero-dimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme; Iwakura M et al.; As a result of the expression of a single open reading frame composed of the coding sequence for a cysteine-free mutant (Cys85-->Ala, Cys152-->Ser) of Escherichia coli dihydrofolate reductase (DHFR; 18K monomeric protein), that for the E . coli thymidylate synthase (TS; dimeric protein with a 30K promoter), and a spacer sequence (coding 7 amino acids) with a Shine-Dargarno sequence, an active hetero-dimeric bifunctional enzyme with 50K DHFR-TS and 30K TS polypeptides was stably produced in the transformed E . coli cell in addition to an overproduction of the TS dimeric enzyme . The highly purified hetero-dimeric enzyme has similar Vmax and Km values in both DHFR and TS activities to those of the natural counterparts, monomeric DHFR and dimeric TS . Although the hetero-dimeric enzyme did not show an apparent channeling transfer of dihydrofolate (the intermediate substrate) between the spatially discrete DHFR and TS active sites, the coupling efficiency of the TS and DHFR reactions in the artificial enzyme was better than that in the separated enzymes, as shown by a decrease in the intermediate concentration at the steady state in the coupled reaction. J Biochem (Tokyo), 1995 Jul, 118(1), 23 - 7 Iron-regulated expression and membrane localization of the magA protein in Magnetospirillum sp . strain AMB-1; Nakamura C et al.; The magA gene from Magnetospirillum sp . strain AMB-1 is required for the synthesis of bacterial magnetic particles (BMPs) . This gene has been cloned, sequenced and found to encode a protein which is homologous to the Escherichia coli potassium efflux membrane-binding protein, KefC . By using the firefly luciferase gene (luc) cloned downstream of the magA promoter, the effect of iron on regulation of magA expression was investigated, and transcription of magA was found to be enhanced by low concentrations of iron . Intracellular localization of the MagA protein was studied using magA-luc fusion proteins . The luc gene was cloned downstream of the magA hydrophilic C-terminal domain . Detection of luciferase activity in the cytoplasm, cell membrane, and magnetic particle membrane subcellular fractions confirmed that the MagA fusion protein was localized in the cell membrane . The fusion protein was also detected on the surface of the lipid bilayer covering the magnetic particles . These results suggest that MagA is a membrane-bound protein, the expression of which is enhanced at low iron concentrations. J Biochem (Tokyo), 1995 Jul, 118(1), 183 - 8 Drosophila melanogaster aldolase: characterization of the isozymes alpha, beta, and gamma generated from a single gene; Zhang R et al.; Three isozymic forms, alpha, beta, and gamma, of Drosophila melanogaster aldolase are produced from a single gene by alternative usage of the triple exons 4 (4 alpha, 4 beta, and 4 gamma) {Shaw-Lee et al . (1992) J . Biol . Chem . 267, 3959-3967; Kim et al . (1992) Mol . Cell . Biol . 12, 773-783; Kai et al . (1992) J . Biochem . 112,677-688} . The expression plasmids for the respective isozymes were transfected into Escherichia coli cells, and the isozymes alpha and beta were purified to homogeneity by a simple procedure, though isozyme gamma was only partially purified . These isozymes are active towards two substrates, fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P), with a preference for Fru-1,6-P2 over Fru-1-P, but they have different kcat/Km values towards these two substrates; isozyme alpha shows the highest value for Fru-1,6-P2 . These isozymes show similarity in optimal pHs, thermal stability, and Km values for both Fru-1,6-P2 and Fru-1-P . They are composed of four identical subunits of 40 kDa, forming a tetramer with a molecular weight of approximately 160 kDa . The three isozymes are different in primary structure only at the carboxyl-terminal region encoded by the respective exon 4 . Therefore, this region should be primarily responsible for the distinct characteristics of these isozymes. J Hepatol, 1995 Jul, 23(1), 8 - 13 Prospective study of plasma fibronectin in fulminant hepatitis: association with infection and mortality; Acharya SK et al.; BACKGROUND/AIMS: Plasma fibronectin is an opsonic glycoprotein, normally synthesized by the liver, which decreases subsequent to severe liver damage and low levels of which may contribute to reticuloendothelial system dysfunction by compromising opsonic activity . This may result in an increased frequency of infection and death . The present study was conducted to evaluate the association of plasma fibronectin activity with infection and mortality in patients with fulminant hepatic failure . METHODS: Plasma fibronectin was estimated serially in 69 consecutive patients with fulminant hepatic failure, nine patients with uncomplicated acute viral hepatitis and 32 normal volunteers . RESULTS: Plasma fibronectin levels in patients with fulminant hepatic failure (85.6 +/- 75.8 micrograms/ml) were significantly lower than in patients with uncomplicated acute viral hepatitis (295.5 +/- 88.5 micrograms/ml) and healthy volunteers (362.6 +/- 69.2 micrograms/ml) . Forty-nine (72%) patients with fulminant hepatic failure died . The initial values of fibronectin in fulminant hepatic failure did not correlate with mortality . Patients with fulminant hepatic failure who survived showed a progressive rise in the fibronectin levels compared to the absence of an increase in fibronectin levels in the non-survivors . The mortality in patients with fulminant hepatic failure with infection (24/27) was significantly higher (p < 0.05) compared to those without infection (25/42) . Initial fibronectin levels in patients with infection (70.3 +/- 54.2 micrograms/ml) were significantly lower (p < 0.05) than in those without infection (92.3 +/- 64.4 micrograms/ml) . We conclude that plasma fibronectin levels in patients with fulminant hepatic failure are decreased compared to healthy subjects and the absence of an increase in levels indicates a poor prognosis . Low levels of fibronectin are associated with an increased incidence of infection, which increases the mortality in these patients. Hum Mol Genet, 1995 Jul, 4(7), 1209 - 12 Recessively inherited L-DOPA-responsive dystonia caused by a point mutation (Q381K) in the tyrosine hydroxylase gene; Knappskog PM et al.; Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in the biosynthesis of dopamine . Recently, we described a point mutation in hTH (Q381K) in a family of two siblings suffering from progressive L-DOPA-responsive dystonia (DRD), representing the first reported mutation in this gene . We here describe the cloning, expression and steady-state kinetic properties of the recombinant mutant enzyme . When expressed by a coupled in vitro transcription-translation system and in E . coli, the mutant enzyme represents a kinetic variant form, with a reduced affinity for L-tyrosine . The 'residual activity' of about 15% of the corresponding wild-type hTH (isoform hTH1), at substrate concentrations prevailing in vivo, is compatible with the clinical phenotype of the two Q381K homozygote patients carrying this recessively inherited mutation. Res Vet Sci, 1995 Jul, 59(1), 50 - 5 Effects of concanavalin A and pokeweed lectins on microvillar membrane proteins during the organ culture of rabbit intestinal mucosa; Embaye H et al.; The effects of pokeweed lectin (PWL) and concanavalin A (Con A) on microvillar membrane (MVM) proteins during the organ culture of rabbit ileal explants for 24 hours were compared with the known effects of enteropathogenic Escherichia coli (EPEC) . PWL resulted in the accelerated release of brush border enzymes into the culture medium, accompanied by decreased tissue activities and an increase in the total activity present in the tissue and culture medium . Con A had less effect on the release and tissue activities of brush border enzymes and the total activity was not increased . Sucrose density gradient centrifugation of the culture medium showed that MVM enzymes were predominantly particulate, consistent with their release as vesicles . Centrifugation of ileal explants showed that PWL, but not Con A, resulted in a decrease in the modal density of the brush border which was consistent with a lower glycoprotein-to-lipid ratio . These findings suggest that PWL, in common with EPEC, may cause the disruption and vesiculation of microvilli and the compensatory stimulation of MVM protein synthesis. Res Microbiol, 1995 Jul-Aug, 146(6), 445 - 55 Analysis of genes encoding the cell division protein FtsZ and a glutathione synthetase homologue in the cyanobacterium Anabaena sp . PCC 7120; Zhang CC et al.; Heterocysts, cells specialized in nitrogen fixation in Anabaena sp . PCC 7120, lose the potential for cell division once fully differentiated . This suggests that cell division activity is differentially regulated in heterocysts and vegetative cells . FtsZ has been shown to play a crucial role in bacterial cell division . Two degenerate oligonucleotide primers were designed to detect, by polymerase chain reaction (PCR), an ftsZ homologue from the heterocystous cyanobacterium Anabaena sp . PCC 7120 . A PCR-amplified DNA fragment was cloned and used as a probe to isolate the entire ftsZ gene of Anabaena sp . PCC 7120 . The deduced amino acid sequence shares strong similarities with other FtsZ proteins, suggesting remarkable conservation of the FtsZ protein during evolution . An ORF downstream of ftsZ, which would be transcribed in the opposite direction compared to ftsZ, could encode a polypeptide with significant sequence similarity to the glutathione synthetase from Escherichia coli . Inactivation experiments in vivo for both ftsZ and the glutathione synthetase gene did not yield any double recombinants either in the presence or in the absence of combined nitrogen, suggesting that both genes are essential for cell growth under these conditions. J Cereb Blood Flow Metab, 1995 Jul, 15(4), 547 - 51 Attenuation of stroke size in rats using an adenoviral vector to induce overexpression of interleukin-1 receptor antagonist in brain; Betz AL et al.; Adenoviruses have been proposed as potential vectors for gene therapy in the central nervous system, but there are no reports of their use in the treatment of a brain disease . Because central administration of interleukin-1 receptor antagonist protein (IL-1ra) reduces ischemic brain damage, we determined whether a recombinant adenovirus vector carrying the human IL-1ra cDNA (Ad.RSVIL-1ra) could be used to ameliorate brain injury in permanent focal ischemia . Groups of six rats received intraventricular injections of Ad.RSVIL-1ra or a control adenovirus containing the Escherichia coli beta-galactosidase gene (Ad.RSVlacZ) . Histochemical staining for beta-galactosidase 5 days after virus injection indicated that transgene expression was confined primarily to the cells lining the ventricle . The concentrations of IL-1ra injected animals, achieving levels of 9.1 +/- 3.3 ng/g in brain and 23.7 +/- 22.5 ng/ml in CSF . In these animals, cerebral infarct volume resulting from 24 h of permanent middle cerebral artery occlusion was reduced 64% . These studies demonstrate that adenoviral vectors can be used to deliver genes that attenuate brain injury. Infect Immun, 1995 Jul, 63(7), 2797 - 800 Cloning and characterization of a protective outer membrane lipoprotein of Actinobacillus pleuropneumoniae serotype 5; Bunka S et al.; The gene encoding an outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae serotype 5 was cloned, and the protein was expressed in Escherichia coli . One open reading frame of 1,104 bp was detected that encoded a protein (OmlA) with a predicted molecular mass of 40 kDa . A comparison with the omlA gene and the corresponding protein of A . pleuropneumoniae serotype 1 (G.-F . Gerlach, C . Anderson, S . Klashinsky, A . Rossi-Kampos, A.A . Potter, and P.J . Wilson, Infect . Immun . 61:565-572, 1993) revealed that the nucleic acid sequences had an overall sequence identity of 62.9% and the deduced amino acid sequences showed a sequence agreement of 57.3% . Both proteins were antigenically distinct . In a Western blot (immunoblot) analysis using a specific antiserum against A . pleuropneumoniae serotype 5 OmlA, a homologous protein was detected in the reference strains of A . pleuropneumoniae serotypes 5A, 5B, and 10 . Pigs immunized with this recombinant protein were protected from death in an aerosol challenge experiment with an A . pleuropneumoniae serotype 5 isolate. Infect Immun, 1995 Jul, 63(7), 2652 - 7 A Mycobacterium leprae gene encoding a fibronectin binding protein is used for efficient invasion of epithelial cells and Schwann cells; Schorey JS et al.; Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen . M . leprae can infect a variety of cells in vivo, including epithelial cells, muscle cells, and Schwann cells, in addition to macrophages . The ligand-receptor interactions important in the attachment and ingestion of M . leprae by these nonmacrophage cells remains unknown . Fibronectin (FN) significantly enhances both attachment and ingestion of M . leprae by epithelial and Schwann cell lines . We cloned an M . leprae FN binding protein (FN attachment protein {FAP}) distinct from the 85ABC complex which has been shown previously to bind FN . The FAP open reading frame predicts a protein of 29.5 kDa with a 39-amino-acid signal peptide and was previously described as an antigen in leprosy patients . M . leprae FAP has homologies in M . vaccae, M . avium, and M . tuberculosis, as determined by Southern blotting and direct peptide analysis . Both anti-FAP antibodies and an Escherichia coli-expressed recombinant protein significantly blocked M . leprae attachment and internalization by T-24, an epithelial cell line, and JS1, a Schwann cell line . These data suggest that FN can be a bridging opsonic ligand for attachment of mycobacteria to nonphagocytes and that FAP plays an important role in this process . This may be an important step in the initiation of M . leprae infection in vivo. Infect Immun, 1995 Jul, 63(7), 2587 - 95 Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis; Gomez FJ et al.; HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum . It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H . capsulatum yeast cells . In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen . The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation . Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR . One 680-bp segment that corresponded to the known peptide sequence was amplified from H . capsulatum DNA . This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced . The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively . A cDNA was synthesized by reverse transcription PCR and was expressed in E . coli . Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells . Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells . Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice. Infect Immun, 1995 Jul, 63(7), 2581 - 6 Characterization of a novel Mycobacterium bovis secreted antigen containing PGLTS repeats; Bigi F et al.; Serum from naturally infected cattle was used to identify a novel Mycobacterium bovis antigen from an expression library . The first recombinant product identified was a fusion protein with lacZ (55 kDa) . A clone containing the whole gene was also obtained . This clone expressed a 38-kDa protein . A rabbit serum against the recombinant antigen reacts in M . bovis supernatants with two proteins of 36 and 34 kDa . The new protein was called P36/P34 . The gene cloned has a deduced amino acid sequence with a predicted molecular mass of 28 kDa, showing a characteristic signal sequence for exportation . The protein bears partial homology to a 28-kDa protein from M . leprae . An interesting feature of the P36/P34 sequence is that it contains several PGLTS repeats, which are not present in the M . leprae protein . Antigenic determinants seem also to be conserved between the two proteins because sera from leprosy patients recognized the recombinant M . bovis protein . The discrepancy among the molecular mass deduced from the sequence (28 kDa), that of the recombinant protein in Escherichia coli (38 kDa), and that of the native protein in M . bovis (36 and 34 kDa) could be attributed to posttranslational modifications or to the high proline content that may alter the migration properties of the protein . This antigen seems to be immunodominant during bovine tuberculosis, because 8 of 9 serum specimens from diseased cattle are reactive . The homology among the M . leprae 28-kDa protein, the protein described in this article, and a recently described M . tuberculosis protein suggests the existence of a new protein family in mycobacteria. Infect Immun, 1995 Jul, 63(7), 2499 - 507 Cloning and genetic characterization of the flagellum subunit gene (flaA) of Legionella pneumophila serogroup 1; Heuner K et al.; The gene flaA, encoding the flagellum subunit protein of Legionella pneumophila serogroup 1, has been isolated from an expression library of L . pneumophila isolate Corby in Escherichia coli K-12 by using an antiflagellin specific polyclonal antiserum . DNA sequence analysis of the flaA gene revealed the presence of a 1,428-bp open reading frame encoding a protein of 475 amino acids with an apparent molecular mass of 48 kDa that is expressed independently of an E . coli vector promoter . Peptide sequencing of the N terminus of the isolated flagellum subunit protein confirmed that this open reading frame encodes the flagellin . By comparing the FlaA amino acid sequence with those of flagellins of various other bacteria, high degrees of homology in the N-terminal and C-terminal amino acids could be observed . The flaA-specific mRNA was determined to be 1.6 kb in size, the expected size of a monocistronic mRNA . Temperature-dependent expression of flagellin was found to be regulated at the transcriptional level . Sequence analysis and primer extension experiments indicated that the transcription of the gene flaA is directed by a sigma 28-like RpoF-FliA factor . By using fliA and fliA+ E . coli K-12 mutants, it was shown that flaA expression in E . coli required the sigma 28 factor . A flaA-specific DNA probe hybridizes with genomic DNA isolated from L . pneumophila and with most of the genomic DNAs from non-L . pneumophila Legionella strains . Two L . pneumophila strains and isolates of Legionella bozemanii and Legionella feeleii (serogroup 1) carry flaA-specific sequences but were not able to produce flagella. Infect Immun, 1995 Jul, 63(7), 2409 - 17 Clonal relationships among bloodstream isolates of Escherichia coli; Maslow JN et al.; The clonal relationships among 187 bloodstream isolates of Escherichia coli from 179 patients at Boston, Mass., Long Beach, Calif., and Nairobi, Kenya, were determined by multilocus enzyme electrophoresis (MLEE), analysis of polymorphisms associated with the ribosomal operon (ribotyping), and serotyping . MLEE based on 20 enzymes resolved 101 electrophoretic types (ETs), forming five clusters; ribotyping resolved 56 distinct patterns concordant with the analysis by MLEE . The isolates at each study site formed a genetically diverse group and demonstrated similar clonal structures, with the same small subset of lineages accounting for the majority of isolates at each site . Moreover, two ribotypes accounted for approximately 30% of the isolates at each study site . One cluster contained the majority (65%) of isolates and, by direct comparison of the ETs and ribotypes of individual isolates, was genetically indistinguishable from the largest cluster for each of two other collections of E . coli causing pyelonephritis and neonatal meningitis (R . K . Selander, T . K . Korhonen, V . Vaisanen-Rhen, P . H . Williams, P . E . Pattison, and D . A . Caugent, Infect . Immun . 52:213-222, 1986; M . Arthur, C . E . Johnson, R . H . Rubin, R . D . Arbeit, C . Campanelli, C . Kim, S . Steinbach, M . Agarwal, R . Wilkinson, and R . Goldstein, Infect . Immun . 57:303-313, 1989), thus defining a virulent set of lineages . The isolates within these virulent lineages typically carried DNA homologous to the adhesin operon pap or sfa and the hemolysin operon hly and expressed O1, O2, O4, O6, O18, O25, or O75 antigens . DNA homologous to pap was distributed among isolates of each major cluster, whereas hly was restricted to isolates of two clusters, typically detected in pap-positive strains, and sfa was restricted to isolates of one cluster, typically detected in pap- and hly-positive strains . The occurrence of pap-positive isolates in the same geographically and genetically divergent lineages suggests that this operon was acquired early in the radiation of E . coli, while hly and sfa were acquired subsequently, most likely by pap-positive and pap- and hly-positive precursors, respectively. Endocrinology, 1995 Jul, 136(7), 3037 - 45 Tissue-specific expression of novel messenger ribonucleic acids cloned from a renin-expressing kidney tumor cell line (As4.1); Thompson HA et al.; As4.1 cells are derived from a renin-expressing kidney tumor induced by tissue-specific oncogene-mediated tumorigenesis in transgenic mice . These cells express high levels of renin messenger RNA (mRNA) and synthesize prorenin and renin; they were therefore used as a model to further investigate the molecular biology of renin-producing kidney cells by cloning and characterizing novel mRNAs expressed in these cells . One clone, designated 1.5, was randomly selected from an As4.1 complementary DNA (cDNA) library, and two other cDNA clones, designated 4.9 and 6.9, were obtained by screening the cDNA library using a strategy to identify As4.1 cell-specific mRNAs . Each clone exhibited a highly restricted tissue-specific expression profile, including high level expression in As4.1 cells and low level expression in kidney . No homology was found between the sequence of the partial 1.5 and 4.9 cDNAs and sequences in Genbank . Southern blot analysis revealed that clone 4.9 is encoded by a single copy gene containing at least two separate exons . A homology search of the sequence of clone 6.9 revealed it to encode a cDNA to serum amyloid A protein; consistent with this identification, expression of 6.9 mRNA was highly induced in both kidney and liver after treatment of mice with Escherichia coli lipopolysaccharide. Endocrinology, 1995 Jul, 136(7), 2896 - 903 Retinoid X receptor alpha binds with the highest affinity to an imperfect direct repeat response element; Yang YZ et al.; The regulation of gene expression by retinoids is mediated by two classes of receptors, retinoic acid receptors and retinoid X receptors (RXR) . RXR can bind to specific target genes as homodimers, and these homodimers can activate gene expression in the presence of the ligand 9-cis-retinoic acid . A direct repeat of AGGTCA with a 1 base pair spacer (DR1) acts as a RXR homodimer response element in the presence of 9-cis-retinoic acid . However, it is not known if this represents the highest affinity binding site for the RXR homodimer . To investigate this question, we used a nonbiased strategy to isolate from a pool of random DNA those sequences that have the highest affinity for RXR alpha homodimers . The imperfect DR1 sequence 5'-GGGGTCAAAGGTCA displayed the highest in vitro binding affinity for RXR alpha homodimers . Transient transfection studies confirmed that this sequence is a more potent response element than is a perfect DR1 of either AGGTCA or GGGGTCA . The results also indicate that for RXR alpha homodimers, the receptor bound to the 5' half-site dislays different DNA binding specificity than that bound to the 3' half-site . Thus, DNA binding specificity is determined not only by the amino acid sequence of the protein but also by its protein-protein interactions and its position on the response element (5' vs . 3'). J Virol, 1995 Jul, 69(7), 4331 - 8 Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity; Ziebuhr J et al.; The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses . In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity . The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity . Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain . Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene . Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells . This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts . These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E. J Virol, 1995 Jul, 69(7), 4255 - 60 The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A; Satoh S et al.; Hepatitis C virus proteins are produced by proteolytic processing of the viral precursor polyprotein that is encoded in the largest open reading frame of the viral genome . Processing of the nonstructural viral polyprotein requires the viral serine-type proteinase present in nonstructural protein 3 (NS3) . The cleavage of the junction between NS4B and NS5A is mediated by NS3 only when NS4A is present . NS4A is thought to be a cofactor that enhances the cleavage efficiency of NS3 in hepatitis C virus protein-producing cells . Stable NS3-NS4A complex formation required the N-terminal 22 amino acid residues of NS3 . This interaction contributed to stabilization of the NS3 product as well as increased the efficiency of cleavage at the NS4B/5A site . The N-terminal 22 amino acid residues fused to Escherichia coli dihydrofolate reductase also formed a stable complex with NS4A . NS3 derivatives which lacked the N-terminal 22 amino acid residues showed drastically reduced cleavage activity at the NS4B/5A site even in the presence of NS4A . These data suggested that the interaction with NS4A through the 22 amino acid residues of NS3 is primarily important for the NS4A-dependent processing of the NS4B/5A site by NS3. J Virol, 1995 Jul, 69(7), 4095 - 102 Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41; Arroyo J et al.; The transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1) has been implicated in the cytopathology observed during HIV infection . The first amino acids located at the amino terminus are involved in membrane fusion and syncytium formation, while sequences located at the carboxy terminus have been predicted to interact with membranes and modify membrane permeability . The HIV-1 gp41 gene has been cloned and expressed in Escherichia coli cells by using pET vectors to analyze changes in membrane permeability produced by this protein . This system is well suited for expressing toxic genes in an inducible manner and for analyzing the function of proteins that modify membrane permeability . gp41 enhances the permeability of the bacterial membrane to hygromycin B despite the low level of expression of this protein . To localize the regions of gp41 responsible for these effects, a number of fragments spanning different portions of gp41 were inducibly expressed in E . coli . Two regions of gp41 were shown to increase membrane permeability: one located at the carboxy terminus, where two highly amphipathic helices have been predicted, and another one corresponding to the membrane-spanning domain . Expression of the central region of gp41 comprising this domain was highly lytic for E . coli cells and increased membrane permeability to a number of compounds . These findings are discussed in the light of HIV-induced cytopathology and gp41 structure. Rinsho Byori, 1995 Jul, 43(7), 679 - 85 {Mutation detection by PCR-TGGE}; Shirakawa T et al.; The variants in enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) gene were detected by temperature gradient gel electrophoresis (TGGE) . 15 clinical strains isolated from patients and a wild type strain (B2C) were analyzed after the conventional PCR . Although all PCR products (707bp) corresponded to A-subunit of LT, three strains were the different electrophoretic patterns after silver staining as compared to the wild type . For further electrophoretic analyses, the 707bp region was divided into 4 parts . The different ones were localized in the downstream part (183bp), but each those DNA bands was not so clear than DNA bands in 707bp . The clearer patterns were obtained by using a primer attached GC-clamp . The hetero-duplex assays in TGGE were proceeded by a series of procedures in mixing with the equal quantity of PCR products derived from a variant and a wild type, heat-denaturation and then annealing . TGGE of those mixed samples had 4 bands that were 2 front bands as homo-duplex and 2 slower migration bands as hetero-duplex . To Confirm the site of the mutations, the nucleotide sequences in each 183bp PCR products were decided by dideoxynucleotide-fluorescent dye method . Indeed, two variants were recognized four one-base substitutions without deletions and the one was five . Thus, the difference of migration in TGGE depended on the number and the localization in mutation sites. Genetics, 1995 Jul, 140(3), 897 - 907 Genetic instability within monotonous runs of CpG sequences in Escherichia coli; Bichara M et al.; Genetic information can be altered by base substitutions, frameshift mutations, and addition or deletion of nucleotides . Deletions represent an important class of genetic aberration occurring at DNA sequences where it is often possible to predict the existence of intermediates of mutation . Instability within tracts of repetitive sequence have recently been associated with several genetic disorders, including the so-called triplet repeat diseases and certain forms of colorectal cancers . In Escherichia coli, (GpC)n repetitive sequences have been shown to be deletion prone, but the precise mechanism of this mutagenic pathway is still unknown . We show here that interrupting the monotony of the (GpC)n run with an ApT or a GpT dinucleotide decreases the rate of deletions within these sequences . On the other hand, introducing purine-pyrimidine alternating sequences beside the GpC insert results in an increased rate of deletion . Two pathways can be envisioned: (1) (GpC)n tracts can be seen as potential Z-forming DNA sequences, and this unusual DNA structure can be processed by an unknown cellular mechanism to give rise to the observed deletions and (2) (GpC)n monotonous runs can be considered as a succession of direct or palindromic repeats, allowing formation of DNA structures that are known to participate to frameshift mutagenesis . The results presented in this article are discussed in the light of these two alternative pathways. Genetics, 1995 Jul, 140(3), 889 - 96 Molecular analysis of lambda bio transducing phage produced by oxolinic acid-induced illegitimate recombination in vivo; Shimizu H et al.; To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we examined the formation of lambda Spi- phage during prophage induction . The frequency of Spi- phage was two to three orders of magnitude higher in the presence of oxolinic acid, an inhibitor of DNA gyrase A subunit, than in the absence of the drug, while it was very low in nalAr bacteria with the drug . RecA function is not required for the formation of these phages, indicating that this enhancement is not caused by the expression of SOS-controlled genes . Analyses of att region and recombination junctions of Spi- phages revealed that they have essentially the same structures as lambda bio transducing phages but are classified into two groups with respect to recombination sites . In the majority class of the transducing phages, there were not more than 3-bp homologies between the parental E . coli bio and lambda recombination sites . In the minority class of the transducing phages, on the other hand, 9-10-bp homologies were found between the parental recombination sites . These results suggested that oxolinic acid-induced illegitimate recombination takes place by two variants of a DNA gyrase-dependent mechanism. Genetics, 1995 Jul, 140(3), 861 - 74 Intermolecular transposition of IS10 causes coupled homologous recombination at the transposition site; Eichenbaum Z et al.; Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid . This was detected by the activity of the tet gene expressed from the phage 434 PR promoter . Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site . Cointegrate formation was abolished in delta recA strains, although simple insertions of IS10 were observed . This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor . Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event . Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome . This was accompanied by the formation of an additional copy of IS10 in the chromosome . Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10. Gene Ther, 1995 Jul, 2(5), 351 - 4 Adenovirus-mediated arterial gene transfer does not require prior injury for submaximal gene expression; Li JJ et al.; We investigated whether gene expression in arterial wall following adenovirus-mediated gene transfer would be enhanced by prior injury . We introduced Escherichia coli lacZ into the balloon-injured canine femoral arteries through a double-balloon catheter by either a replication-defective adenovirus or liposome-mediated transfection (lipofection) using an expression plasmid, and quantified beta-galactosidase activity in arterial homogenates harvested 5 days after gene transfer . Gene expression by lipofection was enhanced six-fold when gene transfer was performed at 3 days, instead of 0 days after injury . However, gene expression achieved by adenovirus infection was not significantly changed irrespective of when gene transfer was performed between 0 and 12 days after injury, and beta-galactosidase activity was 25-fold higher than the enhanced value obtained by lipofection performed 3 days after injury . Our study indicates that submaximal gene expression in arterial wall can be achieved when adenovirus-mediated gene transfer is performed at the same time as angioplasty. Biosci Biotechnol Biochem, 1995 Jul, 59(7), 1323 - 5 soxRS gene increased the level of organic solvent tolerance in Escherichia coli; Nakajima H et al.; Escherichia coli strain JA300 grows in the presence of n-hexane, but not in the presence of cyclohexane . We isolated a 10.5-kb DNA fragment that provided cyclohexane tolerance on a multi-copy plasmid, from the chromosomal DNA of JA300 . In this fragment, there were found C-terminal 10-amino acids truncated soxR (soxR') and soxS which control the superoxide response regulon genes . Characterization of subclones found that both the soxR' and overexpression of the soxS increased the levels of organic solvent tolerance in several E . coli strains. Biosci Biotechnol Biochem, 1995 Jul, 59(7), 1304 - 8 Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens; Alvarez AM et al.; Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E . coli . The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates . Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa . Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies . These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments. Biophys J, 1995 Jul, 69(1), 250 - 63 Fluctuations in rotation rate of the flagellar motor of Escherichia coli; Kara-Ivanov M et al.; The purpose of this work was to study the changes in rotation rate of the bacterial motor and to try to discriminate between various sources of these changes with the aim of understanding the mechanism of force generation better . To this end Escherichia coli cells were tethered and videotaped with brief stroboscopic light flashes . The records were scanned by means of a computerized motion analysis system, yielding cell size, radius of rotation, and accumulated angle of rotation as functions of time for each cell selected . In conformity with previous studies, fluctuations in the rotation rate of the flagellar motor were invariably found . Employing an exclusively counterclockwise rotating mutant ("gutted" RP1091 strain) and using power spectral density, autocorrelation and residual mean square angle analysis, we found that a simple superposition of rotational diffusion on a steady rotary motion is insufficient to describe the observed rotation . We observed two additional rotational components, one fluctuating (0.04-0.6 s) and one oscillating (0.8-7 s) . However, the effective rotational diffusion coefficient obtained after taking these two components into account generally exceeded that calculated from external friction by two orders of magnitude . This is consistent with a model incorporating association and dissociation of force-generating units. Trends Biochem Sci, 1995 Jul, 20(7), 285 - 6 Purification of His-Tag fusion proteins from Escherichia coli; Hengen P; Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet . This month's column discusses a contaminating protein from Escherichia coli found when histidine-tagged fusion proteins are purified by metal chelate affinity chromatography . For details on how to partake in the newsgroup, see the accompanying box. J Clin Microbiol, 1995 Jul, 33(7), 1961 - 2 Retrospective case-control study of diffusely adhering Escherichia coli and clinical features in children with diarrhea; Poitrineau P et al.; A retrospective case-control study with a small population group revealed that, among clinical signs, vomiting but not diarrhea was significantly associated with the presence of diffusely adhering Escherichia coli (DAEC) in children suffering from gastroenteritidis (P < 0.05) . Of the children carrying DAEC strains, those who were F1845 DNA probe positive had a significantly longer hospital stay than those who were F1845 DNA probe negative . We believe that the heterogeneity of DAEC strains is responsible for the discrepant results concerning their involvement in disease and that only some of these strains are really pathogenic for children. J Clin Microbiol, 1995 Jul, 33(7), 1704 - 11 Isolation and characterization of a new strain of Ehrlichia chaffeensis from a patient with nearly fatal monocytic ehrlichiosis; Dumler JS et al.; Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis, a disease that ranges in severity from asymptomatic infection to death . Only one isolate of E . chaffeensis has been made, the Arkansas strain, upon which all characterizations of the agent of human monocytic ehrlichiosis have been based . We report the isolation and characterization of a new strain of E . chaffeensis, the 91HE17 strain, which was cultivated from a patient with a nearly fatal illness . The new isolate grows best in culture with careful control of pH . The two isolates are nearly identical as determined by light and electron microscopy and have significant antigenic identity in fluorescent-antibody and immunoblot assays using polyclonal antisera and the E . chaffeensis-specific monoclonal antibody 1A9 . Isolate 91HE17 had 99.9% nucleotide sequence identity with the Arkansas strain in the 16S rRNA gene . Parts of the Escherichia coli GroE operon homologs had identical restriction enzyme digestion patterns, and a 425-bp region of the GroEL gene had at least 99.8% sequence identity between the E . chaffeensis Arkansas and 91HE17 strains . Isolate 91HE17 lacked an epitope identified in E . chaffeensis Arkansas by the monoclonal antibody 6A1 . This new E . chaffeensis isolate is very similar to the Arkansas strain and provides the opportunity to substantiate the existence of diversity among ehrlichiae which infect humans . Specific factors which differ among strains may then be compared to assess their potential contributions toward cellular pathogenicity and ultimately toward the development of disease in humans. Nat Struct Biol, 1995 Jul, 2(7), 561 - 8 Identification of critical active-site residues in the multifunctional human DNA repair enzyme HAP1; Barzilay G et al.; All organisms express dedicated repair enzymes for counteracting the cytotoxic and mutagenic potential of apurinic/apyrimidinic (AP) lesions, which would otherwise pose a serious threat to genome integrity . We present the predicted three-dimensional structure of the major human AP site-specific DNA repair endonuclease, HAP1, and show that an aspartate/histidine pair, in conjunction with a metal ion-coordinating glutamate residue, are critical for catalyzing the multiple repair activities of HAP1 . We suggest that this catalytic mechanism is conserved in certain reverse transcriptases, but is distinct from the two metal ion-mediated mechanism defined for other hydrolytic nucleases. Nat Struct Biol, 1995 Jul, 2(7), 548 - 53 Alternating arginine-modulated substrate specificity in an engineered tyrosine aminotransferase; Malashkevich VN et al.; Mutation of six residues of Escherichia coli aspartate aminotransferase results in substantial acquisition of the transamination properties of tyrosine amino-transferase without loss of aspartate transaminase activity . X-ray crystallographic analysis of key inhibitor complexes of the hexamutant reveals the structural basis for this substrate selectivity . It appears that tyrosine aminotransferase achieves nearly equal affinities for a wide range of amino acids by an unusual conformational switch . An active-site arginine residue either shifts its position to electrostatically interact with charged substrates or moves aside to allow access of aromatic ligands. J Biomol NMR, 1995 Jul, 6(1), 23 - 32 Solution structure of the Oct-1 POU homeodomain determined by NMR and restrained molecular dynamics; Cox M et al.; The POU homeodomain (POUhd), a divergent member of the well-studied class of homeodomain proteins, is the C-terminal part of the bipartite POU domain, the conserved DNA-binding domain of the POU proteins . In this paper we present the solution structure of POUhd of the human Oct-1 transcription factor . This fragment was overexpressed in Escherichia coli and studied by two- and three-dimensional homo- and heteronuclear NMR techniques, resulting in virtually complete 1H and 15N resonance assignments for residues 2-60 . Using distance and dihedral constraints derived from the NMR data, 50 distance geometry structures were calculated, which were refined by means of restrained molecular dynamics . From this set a total of 31 refined structures were selected, having low constraint energy and few constraint violations . The ensemble of 31 structures displays a root-mean-square deviation of the coordinates of 0.59 A with respect to the average structure, calculated over the backbone atoms of residues 6 to 54 . The fold of POUhd is very similar to that of the canonical homeodomains . Interestingly, the recognition helix of the free POUhd ends at residue 53, while in the cocrystal structure of the intact POU domain with the DNA octamer motif {Klemm, J.D., Rould, M.A., Aurora, R., Herr, W . and Pabo, C.O . (1994) Cell, 77, 21-32} this helix in the POUhd subdomain is extended as far as residue 60. Mol Biol Evol, 1995 Jul, 12(4), 533 - 45 Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion); Turmel M et al.; We describe here a case of homologous introns containing homologous open reading frames (ORFs) that are inserted at the same site in the large subunit (LSU) rRNA gene of different organelles in distantly related organisms . We show that the chloroplast LSU rRNA gene of the green alga Chlamydomonas pallidostigmatica contains a group I intron (CpLSU.2) encoding a site-specific endonuclease (I-CpaI) . This intron is inserted at the identical site (corresponding to position 1931-1932 of the Escherichia coli 23S rRNA sequence) as a group I intron (AcLSU.m1) in the mitochondrial LSU rRNA gene of the amoeboid protozoon Acanthamoeba castellanii . The CpLSU.2 intron displays a remarkable degree of nucleotide similarity in both primary sequence and secondary structure to the AcLSU.m1 intron; moreover, the Acanthamoeba intron contains an ORF in the same location within its secondary structure as the CpLSU.2 ORF and shares with it a strikingly high level of amino acid similarity (65%; 42% identity) . A comprehensive survey of intron distribution at site 1931 of the chloroplast LSU rRNA gene reveals a rather restricted occurrence within the polyphyletic genus Chlamydomonas, with no evidence of this intron among a number of non-Chlamydomonad green algae surveyed, nor in land plants . A parallel survey of homologues of a previously described and similar intron/ORF pair (C . reinhardtii chloroplast CrLSU/A . castellanii mitochondrial AcLSU.m3) also shows a restricted occurrence of this intron (site 2593) among chloroplasts, although the intron distribution is somewhat broader than that observed at site 1931, with site-2593 introns appearing in several green algal branches outside of the Chlamydomonas lineage . The available data, while not definitive, are most consistent with a relatively recent horizontal transfer of both site-1931 and site-2593 introns (and their contained ORFs) between the chloroplast of a Chlamydomonas-type organism and the mitochondrion of an Acanthamoeba-like organism, probably in the direction chloroplast to mitochondrion . The data also suggest that both introns could have been acquired in a single event. Transgenic Res, 1995 Jul, 4(4), 288 - 90 pBINPLUS: an improved plant transformation vector based on pBIN19; van Engelen FA et al.; We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector . Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number in E . coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes. Plant J, 1995 Jul, 8(1), 133 - 8 Thioredoxin activity in the C terminus of Phalaris S protein; Li X et al.; Self-incompatibility in the grass Phalaris coerulescens is controlled by two genes S and Z . Isolation and sequencing of two S alleles showed that they encode proteins that are highly conserved at the C terminus with significant homology to thioredoxin H proteins . In particular, the residues in and around the active site of thioredoxin, Trp-Cys-Gly-Pro-Cys, are perfectly conserved . The C terminus of the S protein has been expressed in Eschericia coli and purified to homogeneity on Ni-NTA resin . Functional assays showed that the protein has thioredoxin activity; it can act as a substrate for E . coli thioredoxin reductase and also catalyse the reduction of insulin by dithiothreitol . The possible role of thioredoxin-like activity of the S protein in mediating the incompatibility reaction in Phalaris is discussed. Plant J, 1995 Jul, 8(1), 121 - 31 Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida; Urwin PE et al.; The cysteine proteinase inhibitor, oryzacystatin-I (Oc-I), and several engineered Oc-I variants have been tested for efficacy in inhibiting growth and development of both the free-living nematode, Caenorhabditis elegans, and the plant parasitic nematode Globodera pallida . To assist in the design of protein engineering experiments to improve the efficacy of Oc-I, an alignment of 28 cystatins and a molecular model of Oc-I were generated . Inhibitory activities (Ki) of wild-type and variant forms of Oc-I against both papain and the C . elegans cysteine proteinase, gcp-1, were measured . For one variant, in which residue Asp86 was deleted (Oc-I deltaD86), the Ki was reduced by 13- to 14-fold . LD50 studies to test the effect of Oc-I and Oc-I delta D86 against C . elegans showed the relative median potency of Oc-I delta D86 to be 0.76 that of wild-type Oc-I . When expressed in tomato hairy roots both Oc-I and Oc-I delta D86 had a detrimental effect on growth and development of G . pallida . This effect was significantly greater on Oc-I deltaD86-expressing roots leading to a reduction in size of G . pallida females to a level at which fecundity is profoundly affected. Biometals, 1995 Jul, 8(3), 223 - 30 The mobile ferrous iron pool in Escherichia coli is bound to a phosphorylated sugar derivative; Bohnke R et al.; Based on in vivo Mossbauer spectroscopy it has previously been demonstrated that the intracellular iron pool of Escherichia coli, grown in iron deficient media supplemented with siderophores as the sole iron source, is dominated by a single Fe2+ and a single Fe3+ species . We have isolated the ferrous ion species and have purified it employing native column PAGE, chromatography and ultrafiltration . The purified compound displays an Mapp of 2.2 kDa and an extremely low isoelectric point (pI) of 1.05 . It is shown that this ferrous ion binding compound is neither a protein nor a nucleotide, rather it is composed mainly of phosphorylated sugar derivatives . This compound binds approximately 40% of the cytoplasmic iron . Therefore it is proposed that this oligomeric ferrous carbohydrate phosphate represents the long sought after mobile, low molecular mass iron pool. J Chem Technol Biotechnol, 1995 Jul, 63(3), 279 - 89 Cloning, expression and characterization of a single-chain antibody fragment to the herbicide paraquat; Graham BM et al.; New cost effective methods for the detection and removal of pesticides from water samples are required to meet modern safety standards . The recent development of techniques to produce antibody fragments in bacteria has provided the opportunity to exploit antibodies as specialized chemicals for affinity detection/removal technologies . The variable heavy and light polypeptide chains of the anti-paraquat monoclonal antibody PQXB1/2 have been cloned into the single-chain antibody (ScAb) expression vector pBG1 . The construct was expressed in Escherichia coli and 0.4 mg functional antibody produced from 1 dm3 of induced culture . Characterization of ScAb by antigen binding profile and competition ELISA showed it to have a sensitivity one order of magnitude below that of the parent monoclonal . ScAb was purified as a monomer or dimer and analysed by HPLC size exclusion chromatography . When immobilized on polystyrene beads the ScAb could remove 85% of a paraquat-bovine serum albumin conjugate from solution in a single step. Arch Microbiol, 1995 Jul, 164(1), 24 - 8 Glutamate excretion in Escherichia coli: dependency on the relA and spoT genotype; Burkovski A et al.; Glutamate excretion due to amino acid starvation was investigated in "stringent" and "relaxed" strains of Escherichia coli . The observed excretion process is relA-dependent, carrier-mediated, and glutamate-specific . After induction, excretion was detected within less than 2 min and continued for more than 5 h with a rate of 7-10 nmol (mg dry weight)-1 min-1 . Using carbonyl cyanide m-chlorophenylhydrazone or polymyxin B nonapeptide, together with valinomycin, it was shown that glutamate excretion is driven by the membrane potential. Can J Microbiol, 1995 Jul, 41(7), 601 - 11 Effects of leader sequences upon the heterologous expression of restrictocin in Aspergillus nidulans and Aspergillus niger; Brandhorst T et al.; The effects of altered leader sequences on the secretion and localization of restrictiocin expression in Aspergillus nidulans and Aspergillus niger were investigated . The region encoding the leader sequence of the Aspergillus restrictus restrictocin (res) gene was altered and variants were expressed under the glucoamylase (glaA) promoter in A . nidulans and A . niger . The entire restrictocin leader sequence was replaced by the glaA leader sequence in one variant . In another, the signal sequence of restrictocin was replaced with the glaA signal, leaving a hybrid with the putative restrictocin pro region in place of the glaA pro region . The putative pro region was deleted from the restrictocin leader of a third variant . Toxic effects, such as reduced transcript levels and cellular lysis, were minimal when restrictocin was expressed with the native leader sequence, but became more pronounced as the leader sequence was varied . These toxic effects were inversely proportional to the level of restrictocin secreted . In all transformed strains, restrictocin secretion appeared at the periphery of colonies and was observed to occur at the tips of hyphae . Localization of restrictocin to differentiated structures (conidiophores), as occurs in A . restrictus, was observed only in transformed strains containing the complete restrictocin leader sequence. J Natl Med Assoc, 1995 Jul, 87(7), 508 - 9 Tumor necrosis factor-induced necrosis: a monocyte-mediated hypercoagulable effect; Spillert CR et al.; The mechanisms by which tumor necrosis factor (TNF) exerts its necrotic effects are somewhat obscure . We hypothesize that TNF, by monocyte activation, produces the procoagulant tissue factor, thus leading to a state of hypercoagulability with resultant thrombotic vascular occlusion and tissue necrosis . To test this hypothesis, modified recalcification time values (in minutes +/- standard deviation) were obtained on aliquots of blood with A) 20 microL of albumin, B) 20 microL of saline containing endotoxin, and C) 20 microL of albumin with 450 units of TNF . No differences were noted if the samples were not incubated . We conclude that TNF, can cause tumor (tissue) necrosis, and since incubation is required, TNF alone (without monocyte activation) has no procoagulant activity. Plant Physiol, 1995 Jul, 108(3), 1049 - 57 Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis; Phillips AL et al.; Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from arabidopsis thaliana genomic DNA . One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1-2 . A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags . The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product . The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12 . The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only . In the floral shoots of the ga1-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes. Genes Dev, 1995 Jul 1, 9(13), 1633 - 44 Ectopic production of guanosine penta- and tetraphosphate can initiate early developmental gene expression in Myxococcus xanthus; Singer M et al.; Amino acid or carbon limitation is sufficient to initiate fruiting body development in Myxococcus xanthus . In both Escherichia coli and M . xanthus the levels of guanosine 3'-di-5'-(tri)di-phosphate nucleotides {(p)ppGpp} rise transiently when cells are starved for amino acids or carbon . Ectopic increase in the intracellular concentration of (p)ppGpp was achieved in M . xanthus by introducing a copy of the E . coli relA gene, whose product catalyzes pyrophosphate transfer from ATP- to GTP-forming pppGpp . The E . coli RelA protein was detected in these M . xanthus strains, and a rise in (p)ppGpp was observed chromatographically . This increase in the intracellular (p)ppGpp levels was sufficient to activate developmentally specific gene expression . Although (p)ppGpp is made from GTP, the intracellular GTP pool from these strains was not significantly decreased . Moreover, when the GTP pool was lowered by either of two specific inhibitors of GTP synthesis, mycophenolic acid or decoyinine, development was not induced . These results suggest that M . xanthus cells can assess their nutritional status by monitoring the internal availability of amino acids through (p)ppGpp levels. Eur J Biochem, 1995 Jul 1, 231(1), 181 - 5 Molecular chaperones protect against glycation-induced inactivation of glucose-6-phosphate dehydrogenase; Ganea E et al.; Glucose-6-phosphate dehydrogenase is inactivated slowly by reaction with sugars (glycation), a process thought to be important in the development of diabetic complications . A major protein from the ocular lens, alpha-crystallin . which exhibits some chaperone-like properties, protects against this inactivation . The well-known molecular chaperone GroEL (chaperonin 60 from Escherichia coli) also protects . On a molar basis, alpha-crystallin is better than GroEL at protecting against glycation-induced inactivation of glucose-6-phosphate dehydrogenase . The relative amounts of enzyme/chaperone indicate that each molecule of alpha-crystallin binds two molecules of the damaged enzyme . This supports the view that alpha-crystallin has a chaperone-like structure as well as a chaperone-like function. Mutat Res, 1995 Jul, 343(4), 219 - 27 Determination of mutational spectrum of the pesticide, captan, with an improved set of Escherichia coli LacZ mutants; Lu C et al.; The mutational spectrum of the fungicide, captan, was determined using a set of improved Escherichia coli lacZ mutants . Captan created mutations mostly at dA-dT sites (83%) with only 17% occurring at dG-dC sites . The hydrolysis products of captan do not appear to be mutagenic because samples of captan at different hydrolysis stages showed basically the same mutational spectra: 31% at AT --> CG transversions, 8% of GC --> AT transitions, 2% of GC --> CG transversions, 8% of GC --> TA transversions, 19% of AT --> TA transversions, and 32% of AT --> GC transitions . Prepared solutions of captan lost their mutational activity gradually over time, indicating that the rate of decrease in mutagenicity agreed with the kinetics of captan hydrolysis reported in other studies . Using the change in mutagenicity to predict degradation, the hydrolysis of captan in pH 7.0 buffer was about three times faster than the hydrolysis carried out in pH 4.5 buffer . To our knowledge, this is the first presentation of mutational spectrum of captan. Mol Pharmacol, 1995 Jul, 48(1), 72 - 9 Biphasic binding of 5-fluoro-2'-deoxyuridylate to human thymidylate synthase; Reilly RT et al.; Thymidylate synthase (TS) is a homodimeric enzyme that catalyzes the reductive methylation of dUMP by N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, to form dTMP . Inhibition of TS by the dUMP analog 5-fluoro-dUMP (FdUMP) occurs through the formation of a covalent ternary complex containing the nucleotide analog, N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, and the enzyme; this complex is termed the inhibitory ternary complex (ITC) . In the present report, the kinetics of FdUMP binding into an ITC with purified preparations of human TS were examined . Rapid chemical-quench techniques, as well as steady state binding methods, showed that the enzyme contains two distinct FdUMP binding sites with different affinities for the nucleotide analog . Binding to the first, or high affinity, site was rapid and reached a maximum stoichiometry of 1.0 mol of FdUMP/mol of dimer; binding to the second, or low affinity, site was much slower and reached a stoichiometry of 1.7 mol of FdUMP/mol of dimer . Rate constants for FdUMP binding to and dissociation from the ITC (kon and koff, respectively) were determined, as were equilibrium dissociation constants (Kd) . A naturally occurring mutant form of TS, which contains a tyrosine to histidine substitution at residue 33 and renders cells relatively resistant to fluoropyrimidines, exhibited a lower affinity for FdUMP specifically at the second binding site, with little or no change at the first . Hill coefficients were < 1.0, with the His-33 enzyme having a significantly lower coefficient than the wild-type enzyme . The results, in total, indicate that the two FdUMP binding sites on the TS dimer are nonequivalent . We suggest that such nonequivalence may be due to negative cooperativity, where nucleotide binding to the first subunit elicits a conformational change that results in reduced affinity for ligand at the second subunit . This negative cooperativity may be stronger for the His-33 mutant . Thus, the relative fluoropyrimidine resistance conferred by the His-33 substitution may be due to enhanced negative cooperative effects on FdUMP binding into the ITC, thereby reducing the effectiveness of the pyrimidine analog as an inhibitor of thymidylate biosynthesis. Biochem J, 1995 Jul 1, 309 ( Pt 1), 77 - 83 Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase alpha-subunit; Savery NJ et al.; Transcription activation at two semi-synthetic Escherichia coli promoters, CC(-41.5) and CC(-72.5), is dependent on the cyclic AMP receptor protein (CRP) that binds to sites centred 41.5 and 72.5 bp upstream from the respective transcription startpoints . An UP-element that can bind the C-terminal domain of the RNA polymerase (RNAP) alpha-subunit was cloned upstream of the DNA site for CRP at CC(-41.5) and downstream of the DNA site for CRP at CC(-72.5) . In both cases CRP-dependent promoter activity was increased by the UP-element, but CRP-independent activity was not increased . DNase I footprinting was exploited to investigate the juxtaposition of bound CRP and RNAP alpha-subunits . In both cases, CRP and RNAP alpha-subunits occupy their cognate binding sites in ternary CRP-RNAP promoter complexes . RNAP alpha-subunits can occupy the UP-element in the absence of CRP, but this is not sufficient for open complex formation . The positive effects of binding RNAP alpha-subunits upstream of the DNA site for CRP at -41.5 are suppressed if the UP-element is incorrectly positioned. Biochem J, 1995 Jul 1, 309 ( Pt 1), 203 - 7 Rat thimet oligopeptidase: large-scale expression in Escherichia coli and characterization of the recombinant enzyme; McKie N et al.; The coding sequence for rat testis thimet oligopeptidase (TOP) (EC 3.4.24.15) was placed under the control of the T7 polymerase/promoter system . Cultures of Escherichia coli transfected with the resulting plasmid expressed the enzyme as a soluble cytoplasmic protein . Medium-scale cultures allowed isolation of the enzyme in quantities of tens of milligrams . The availability of the recombinant enzyme permitted the determination of such chemical properties as epsilon 280 (48,960), zinc content (2 atom/molecule) and available thiol content (8-10/molecule) for TOP . The recombinant enzyme showed the catalytic activities previously reported for the naturally occurring enzyme, so that we can now conclude with confidence that these are all due to TOP and there is no need to postulate the existence of separate 'Pz-peptidase' or 'endo-oligopeptidase A' enzymes. Biochem J, 1995 Jul 1, 309 ( Pt 1), 195 - 202 Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle; Dahlmann B et al.; The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells . In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner . Both proteolytic processes are paralleled by the hydrolysis of ATP . Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue . Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis . Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex . Although ATP cannot be substituted for by adenosine 5'-{beta,gamma-methylene}triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-{beta,gamma-imido}triphosphate, adenosine 5'-O-{gamma-thio}triphosphate, adenosine 5'-O-{beta-thio}-diphosphate and adenosine 5'-{alpha,beta-methylene}triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation . These effects are independent of the presence of Mg2+ . Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli. Appl Environ Microbiol, 1995 Jul, 61(7), 2482 - 6 A beta-glucuronidase reporter gene construct for monitoring aflatoxin biosynthesis in Aspergillus flavus; Flaherty JE et al.; Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A . parasiticus . Current research is directed at the elimination of these compounds in important food sources . The objective of this research was to develop a method to study the induction and regulation of aflatoxin biosynthesis by examining the expression of one aflatoxin pathway gene, ver1 . The promoter region of ver1 was fused to the beta-glucuronidase (GUS) gene (uidA) from Escherichia coli to form the reporter construct, GAP13 . A . flavus 656-2 was transformed with this construct . Aflatoxin production, GUS activity, and transcript accumulation were determined in transformants after shifting the cultures from a nonconducive medium to a medium conducive to aflatoxin biosynthesis . Transformants harboring GAP13 displayed GUS expression only when aflatoxin was detected in culture . Further, the transcription of the uidA gene driven by the ver1 promoter followed the same profile as for the ver1 genes . The results show that the GAP13 construct may be useful as a genetic tool to study the induction of aflatoxin in situ and to identify substances that affect the expression of genes involved in aflatoxin biosynthesis . The utility of this construct to detect inducers of aflatoxin biosynthesis in maize kernels was tested in a bioassay . A heat-stable inducer of aflatoxin with a molecular size of less than 10 kDa was detected in extracts from maize kernels colonized by A . flavus. J Pharmacol Exp Ther, 1995 Jul, 274(1), 427 - 36 Modulation of superoxide generation in in vivo lipopolysaccharide-primed rat alveolar macrophages by arachidonic acid and inhibitors of protein kinase C, phospholipase A2, protein serine-threonine phosphatase(s), protein tyrosine kinase(s) and phosphatase(s); Mayer AM et al.; Ninety minutes after i.v . injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) into rats, phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide anion (O2-) secretion was enhanced in suspensions of in vivo LPS-treated alveolar macrophages (AM phi) when compared with saline (SAL)-treated AM phi . The purpose of this investigation was to dissect the in vitro mechanism of PMA-stimulated O2- generation in both LPS and SAL-treated rat AM phi, with a panel of inhibitors of protein kinase C (PKC), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), phospholipase A2 (PLA2), cyclooxygenase (CO) and 5-lipoxygenase (5-LO) . The following agents blocked PMA-stimulated O2- generation in both LPS- and SAL-treated AM phi (expressed as percentage of control): 1) PKC inhibitors: staurosporine: 100 nM, 7.0% (LPS) and 5.6% (SAL); sphingosine: 10 microM, 21% (LPS) and 10.5% (SAL); 2) PTK inhibitor: genistein: 100 microM, 44% (LPS) and 31% (SAL); 3) PTP inhibitors: phenylarsine oxide, 10 microM, 12.1% (LPS) and 18% (SAL); diamide, 1000 microM, 10.1% (LPS) and 10.5% (SAL); and 4) PLA2 inhibitors: manoalide: 1 microM, 29.3% (LPS) and 5.2% (SAL); scalaradial: 1 microM, 7.7% (LPS) and 7.1% (SAL); and WAY 125,984: 10 microM, 17.1% (LPS) and 14.5% (SAL) . In addition, it was observed that exogenously added arachidonic acid (AA)-stimulated O2- generation in a time- and dose-dependent manner in both LPS and SAL-treated AM phi . The following inhibitors enhanced or did not affect PMA-stimulated O2- generation in LPS- and SAL-treated AM phi (expressed as percentage of of control): 1) PSP inhibitors: okadaic acid: 0.5 microM, 117% (LPS) and 153% (SAL); calyculin A: 1 microM, 112% (LPS) and 101% (SAL); 2) CO and 5-LO inhibitors: indomethacin: 10 microM, 107% (LPS) and 90% (SAL); WY 50, 295: 1 microM, 99% (LPS) and 103% (SAL); and 3) the PTP inhibitor orthovanadate upon prolonged preincubation . In both in vivo LPS- or SAL-primed AM phi, PMA-stimulated O2- generation appears to be modulated by PKC, PLA2, AA, PTK, PTP and PSP . No modulatory role was evident for either CO or 5-LO metabolites . These findings might bear on the design of therapeutic approaches for the modulation of O2- release by AM phi in the early stages of sepsis and adult respiratory distress syndrome. J Invest Dermatol, 1995 Jul, 105(1 Suppl), 58S - 61S Murine epidermal V gamma 5/V delta 1-T-cell receptor+ T cells respond to B-cell lines and lipopolysaccharides; Reardon CL et al.; The V gamma 5/V delta 1(+)-T-cell receptor (TCR)-bearing T-cell clone, 2CBET-3, was generated from C57BL/6 mice . Upon stimulation, 2CBET-3 cells produce interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha, but not IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, macrophage colony-stimulating factor, or interferon-gamma . These cells were evaluated for their ability to be stimulated by a variety of murine cell lines, including fibroblasts, trophoblasts, melanoma cells, embryonic carcinomas, B-cell lymphomas, mastocytoma cells, and keratinocytes . The human B-lymphoma cell line, Daudi, also was included in these studies . We found that 2CBET-3 cells produced cytokines up to several hundredfold above the control levels in response to the B-cell lines, Daudi, and A20/2J, but not to the B-cell line 439.4.2 . After fixation with glutaraldehyde, Daudi and A20/2J continued to stimulate this gamma delta T-cell line . 2CBET-3 cells also responded to the keratinocyte line PAM212, but not to another, XB-2 . When lipopolysaccharides (LPS) from Escherichia coli or S . typhimurium were added to 2CBET-3 cells in the presence of A20/2J cells, 2CBET-3 cells responded with increased cytokine production compared with the cytokine production in the presence of A20/2J cells alone . 2CBET-3 cells by themselves did not respond to LPS alone or to supernatants from A20/2J cells incubated with LPS . Unlike 2CBET-3, the epidermal T-cell hybridoma 70BET-49, expressing a V gamma 5/V delta 1-TCR identical to that of 2CBET-3, did not respond to A20/2J cells in the presence or absence of LPS, suggesting a requirement for molecules other than the TCR for V gamma 5/V delta 1-TCR+ T-cell stimulation by the B-cell lines and by LPS . This unique reactivity of gamma delta-TCR+ cells is different from that of alpha beta-TCR+ cells and may reflect a functional specialization of gamma delta-TCR+ cells in the response to bacterial infections. J Clin Invest, 1995 Jul, 96(1), 361 - 9 Regulation of taurine transport by Escherichia coli heat-stable enterotoxin and guanylin in human intestinal cell lines; Brandsch M et al.; The human colon carcinoma cell lines Caco-2 and HT-29 take up taurine actively . Treatment of Caco-2 cells with Escherichia coli heat-stable enterotoxin (STa) or with guanylin inhibited taurine uptake by approximately 40% . In contrast, neither STa nor guanylin changed the uptake of taurine in HT-29 cells . The inhibition in Caco-2 cells was associated with a decrease in the maximal velocity as well as in the affinity of the transporter . STa caused a 21-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels in Caco-2 cells with no change in cAMP levels . Neither cGMP nor cAMP levels were affected by STa treatment in HT-29 cells . Experiments with protein kinase inhibitors suggested that protein kinase A may mediate the observed effects of STa on taurine uptake . In accordance with this suggestion, treatment of Caco-2 cells with cholera toxin, which elevated intracellular cAMP levels, was found to inhibit taurine uptake . The steady state levels of the taurine transporter mRNA transcripts were not altered as a result of STa treatment . Studies with Caco-2 cells grown on permeable filters revealed that STa acts from the apical side . The taurine uptake from the apical side was inhibited by STa, but the taurine uptake from the basolateral side remained unaffected . It is suggested that the activity of the intestinal taurine transporter may be regulated by protein kinase A at a posttranslational level and that the intestinal absorption of taurine may be impaired during infection with enterotoxigenic strains of E . coli. Carcinogenesis, 1995 Jul, 16(7), 1637 - 42 The role of tyrosine-158 in O6-alkylguanine-DNA alkyltransferase activity; Edara S et al.; The tyrosine residue present at position 158 in the human O6-alkylguanine-DNA alkyltransferase is one of 22 amino acid residues that are conserved in all known alkyltransferase protein sequences . The importance of this amino acid in the reactions brought about by the alkyltransferase was studied by changing this residue to alanine or to phenylalanine . The control and mutant alkyltransferase proteins were expressed in an Escherichia coli strain lacking alkyltransferase activity and the proteins purified to near homogeneity and their activities measured using both methylated DNA and O6-benzylguanine (BG) as substrates . The alteration to alanine led to a very large decrease in activity towards both substrates but removal of O6-methylguanine from DNA and the conversion of BG to guanine could still be detected when large amounts of the protein were used . The activity of the Y158A mutant was at least 800 times less than that of the control alkyltransferase . The change of tyrosine-158 to phenylalanine reduced the rate of reaction with methylated DNA only slightly (to about one-third) . The conversion of BG to guanine by the Y158F mutant was also reduced to about one-third when assayed in the absence of DNA and by about one-half in the presence of DNA . These results suggest that the presence of tyrosine at position 158 plays an important but not absolutely essential role in the reactions brought about by the alkyltransferase . This role is likely to involve the stabilization of the bound substrate by interaction with the aromatic ring of the tyrosine . The hydrogen bond formed by the hydroxyl group from tyrosine-158 may also facilitate the reaction but the contribution from this interaction is relatively small. Carcinogenesis, 1995 Jul, 16(7), 1595 - 602 Mouse methyltransferase for repair of O6-methylguanine and O4-methylthymine in DNA; Kawate H et al.; cDNA for mouse O6-methylguanine-DNA methyltransferase was expressed in methyltransferase-deficient Escherichia coli mutant cells, and the overproduced mouse enzyme was purified to a homogeneous state . Using this purified product, polyclonal antibodies were prepared and used to estimate amounts of the methyltransferase protein in cells . A single cell of NIH3T3 contained 1.8 x 10(4) molecules of the methyltransferase protein . When mouse fibroblasts were immunostained, it was shown that most of the methyltransferase protein exists in the cytoplasm rather than in the nucleus . Using double-stranded oligomers containing a single O6-methylguanine or O4-methylthymine at predetermined sites, the mouse enzyme repaired O6-methylguanine and O4-methylthymine, at an almost equal efficiency . In the LacZ reversion assay, MNNG-induced A:T to G:C as well as G:C to A:T transition mutations were efficiently suppressed by the function of mouse methyltransferase, in vivo. J Med Microbiol, 1995 Jul, 43(1), 33 - 6 An ascending non-obstructive model for chronic pyelonephritis in BALB/c mice; Gupta R et al.; Chronic pyelonephritis was successfully produced in female BALB/c mice with Escherichia coli after introduction of the bacterial inoculum by the ascending route . E . coli strain 31-B, a nalidixic acid-resistant derivative of strain TN675 expressing type-1 pili, and E . coli strain BH-5, a mutant of 31-B, not expressing type-1 pili, were used in the model . Both the strains were able to induce chronic renal inflammation in the experimental animals . Whereas the initial colonisation was greater with strain 31-B, its non-pilate mutant BH-5 caused a significantly greater inflammatory response and also caused renal scarring observable 5 months after the infection. J Bacteriol, 1995 Jul, 177(14), 4194 - 7 MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli; Brown ED et al.; The Escherichia coli gene murZ was recently shown to encode UDP-N-acetylglucosamine enolpyruvyl transferase, which catalyzes the first committed step of peptidoglycan biosynthesis (J . L . Marquardt, D . A . Siegele, R . Kolter, and C . T . Walsh, J . Bacteriol . 174:5748-5752, 1992) . The map position of murZ (69.3 min) differed from that determined for murA (90 min), a gene which had been previously proposed to encode the same activity (P.S . Venkateswaran and H . C . Wu, J . Bacteriol . 110:935-944, 1972) . Here we describe the construction of a chromosomal deletion of murZ and a plasmid containing murZ under arabinose control . Growth of cells containing the murZ deletion was dependent on the expression of murZ from the plasmid . We conclude that murZ is an essential gene and encodes the sole UDP-N-acetylglucosamine enolpyruvyl transferase of E . coli . To simplify the nomenclature, we recommend that murA be used to designate the gene at 69.3 min that encodes this activity and that the designation murZ be abandoned. J Bacteriol, 1995 Jul, 177(14), 4179 - 82 Two recA genes in Myxococcus xanthus; Norioka N et al.; Two recA genes, recA1 and recA2, in Myxococcus xanthus were cloned by using the recA gene of Escherichia coli, and their DNA sequences were determined . On the basis of deduced amino acid sequences, RecA1 and RecA2 have 67.0% identity to each other and 60.5 and 60.9% identities to E . coli RecA, respectively . Expression of recA2 was detected in both vegetative and developmental cells by Northern blot (RNA) analysis, and a threefold induction was observed when cells were treated with nalidixic acid . Repeated attempts to isolate a recA2 disruption mutant have failed, while a recA1 disruption mutant was readily isolated . Both the recA1 and recA2 genes expressed in E . coli complement the UV sensitivity of an E . coli recA strain. J Bacteriol, 1995 Jul, 177(14), 4152 - 6 rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation; Condon C et al.; Here we present evidence that only five of the seven rRNA operons present in Escherichia coli are necessary to support near-optimal growth on complex media . Seven rrn operons are necessary, however, for rapid adaptation to nutrient and temperature changes, suggesting it is the ability to adapt quickly to changing environmental conditions that has provided the selective pressure for the persistence of seven rrn operons in E . coli . We have also found that one consequence of rrn operon inactivation is a miscoordination of the concentrations of initiation factor IF3 and ribosomes. J Bacteriol, 1995 Jul, 177(14), 4137 - 9 Oligoribonuclease is distinct from the other known exoribonucleases of Escherichia coli; Yu D et al.; Oligoribonuclease, an exoribonuclease specific for small oligoribonucleotides, was initially characterized 20 years ago (S . K . Niyogi and A . K . Datta, J . Biol . Chem . 250:7307-7312, 1975) and shown to be different from RNase II and polynucleotide phosphorylase . Here we demonstrate, using mutant strains and purified enzymes, that oligoribonuclease is not a manifestation of RNases D, BN, T, PH, and R, exoribonucleases discovered subsequently . Thus, oligoribonuclease is the eighth distinct exoribonuclease discovered in Escherichia coli . We also show that oligoribonuclease copurifies with polynucleotide phosphorylase. J Bacteriol, 1995 Jul, 177(14), 4134 - 6 Sulfate and thiosulfate transport in Escherichia coli K-12: evidence for a functional overlapping of sulfate- and thiosulfate-binding proteins; Sirko A et al.; In Escherichia coli, sulfate and thiosulfate ions are transported by an ABC-type transporter consisting of both the membrane components (the products of cysT, cysW, and cysA genes) and the periplasmic binders (the products of cysP and sbp genes) . The single cysP and sbp mutants are able to utilize both sulfate and thiosulfate as a sole sulfur source, while the inactivation of both genes leads to cysteine auxotrophy resulting from the block in the transport of both ions. J Bacteriol, 1995 Jul, 177(14), 4121 - 30 Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter; Guzman LM et al.; We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC . Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors . phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression . Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain . Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature . We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system. J Bacteriol, 1995 Jul, 177(14), 4113 - 20 Characterization of the MetR binding sites for the glyA gene of Escherichia coli; Lorenz E et al.; Sequence analysis of the glyA control region of Escherichia coli identified two regions with homology to the consensus binding sequence for MetR, a lysR family regulatory protein . Gel shift assays and DNase I protection assays verified that both sites bind MetR . Homocysteine, a coregulator for MetR, increased MetR binding to the glyA control region . The MetR binding sites were cloned into the pBend2 vector . Although the DNA did not show any significant intrinsic bend, MetR binding resulted in a bending angle of about 33 degrees . MetR-induced bending was independent of homocysteine . To verify that the MetR binding sites play a functional role in glyA expression, site-directed mutagenesis was used to alter the two binding sites in a lambda glyA-lacZ gene fusion phage . Changing the binding sites toward the consensus MetR binding sequence caused an increase in glyA-lacZ expression . Changing either binding site away from the consensus sequence caused a decrease in expression, suggesting that both sites are required for normal glyA regulation. J Bacteriol, 1995 Jul, 177(14), 4053 - 8 Guanosine 3',5'-bispyrophosphate (ppGpp) synthesis in cells of Escherichia coli starved for Pi; Spira B et al.; Cells of Escherichia coli which enter a phase of starvation for Pi induce the synthesis of the nucleotide guanosine 3',5'-bispyrophosphate (ppGpp) . This induction is relA independent but depends on the spoT gene product . A mutant unable to produce ppGpp is impaired in the expression of two genes which belong to the pho regulon, a defect which is dependent on the product of spoT . We suggest that ppGpp is essential for the proper induction of the genes which belong to the pho regulon. J Bacteriol, 1995 Jul, 177(14), 4043 - 52 Molecular analysis of treB encoding the Escherichia coli enzyme II specific for trehalose; Klein W et al.; A gene bank of partially Sau3A-digested Escherichia coli DNA ligated in plasmid pBR322 was screened for the ability to complement a mutant unable to metabolize trehalose at low osmolarity . The resulting plasmid was shown to contain the genes encoding transport (treB) and metabolic (treC) functions . The complementing DNA region was sequenced and shown to contain an operon of two genes, with treB as the promoter proximal gene and with treC as the promoter distal gene . The transcriptional start point was determined, and one major transcript was detected . The control region of the operon was found to contain consensus binding motifs for the cyclic AMP-catabolite activator protein complex and for a specific repressor protein whose gene, treR, is located immediately upstream of treB, being transcribed in the same direction as treB treC . The products of both genes could be expressed in minicells in which TreB revealed itself as a protein with an apparent molecular weight of 42,000 . The gene product of treB consists of 485 amino acids with a calculated molecular weight of 52,308 . It showed high homology to enzymes IIScr of enteric bacteria specific for the uptake of sucrose and encoded by plasmid pUR400 of enteric bacteria . Like enzyme IIScr, enzyme IITre belongs to the EIIBC domain type and lacks a covalently bound EIIA domain . Instead, enzyme IITre-mediated phosphorylation of trehalose requires the activity of enzyme IIAGlc, a component of the major glucose transport system. J Bacteriol, 1995 Jul, 177(14), 4028 - 35 Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA; Tabuchi A et al.; The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication . To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA . Six hybrid proteins were examined for function . The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro . For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required . None of the hybrid initiator proteins activated the P1 origin . Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation . A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication. J Bacteriol, 1995 Jul, 177(14), 3992 - 7 Cloning and analysis of gene clusters for production of the Escherichia coli K10 and K54 antigens: identification of a new group of serA-linked capsule gene clusters; Pearce R et al.; The polysaccharide capsules of Escherichia coli have been classified into three groups: I, II, and I/II . The third group, I/II, has been poorly studied and possesses characteristics of both group I and group II capsules . In this report, we describe the cloning of the K10 and K54 capsule gene clusters, two representatives of group I/II capsules . Probes taken from DNA flanking regions 1 and 3 of the group II capsule clusters hybridized to these group I/II clones, confirming that the group I/II capsule genes are flanked by the same DNA and are therefore located in the same serA-linked region of the chromosome as group II capsule gene clusters . Southern blotting showed that homologous sequences were present in both the K10 and K54 capsule gene clusters and in other group I/II strains . No homology was detected between these sequences and the chromosomal DNA of either a group I or a group II strain . Likewise, no homology was detected to the chromosomal DNA of either a K11 or K19 strain, both of which had previously been classified as group I/II strains . In the K10 and K54 capsule gene clusters, these conserved sequences flanked a serotype-specific region in a manner analogous to group II capsule gene organization . Complementation of mutations in the kpsE, kpsD, and kpsC genes in region 1 of the K5 capsule gene cluster by subclones of the K10 and K54 capsule gene clusters indicated that certain stages in the export of group II and I/II capsules may be conserved . In the light of the findings presented here, we suggest that the group I/II capsule gene clusters are sufficiently different from group II capsule gene clusters to justify their renaming as group III. J Bacteriol, 1995 Jul, 177(14), 3972 - 8 Characterization of FNR* mutant proteins indicates two distinct mechanisms for altering oxygen regulation of the Escherichia coli transcription factor FNR; Bates DM et al.; In order to gain insight into the mechanism by which the Escherichia coli transcription factor FNR* is activated in response to anaerobiosis, we have analyzed FNR mutant proteins which, unlike the wild-type protein, stimulate gene expression in the presence of oxygen in vivo . Cell extracts containing seven different FNR* mutant proteins were tested in vitro for the ability to bind to the FNR consensus DNA site in a gel retardation assay under aerobic conditions . At the concentration of protein tested, only extracts which contained FNR* mutant proteins with amino acid substitutions at position 154 showed significant DNA binding . The three position-154 FNR* mutant proteins could be further distinguished from the other mutant proteins by analysis of the in vivo phenotypes of FNR* proteins containing amino acid substitutions at either of two essential cysteine residues . In the presence of oxygen, FNR* mutant proteins with amino acid substitutions at position 154 were the least affected when either Cys-23 or Cys-122 was substituted for Ser . On the basis of these in vivo and in vitro analyses, FNR* mutant proteins appear to segregate into at least two classes . Thus, it appears that each class of FNR* substitutions alters the normal pathway of FNR activation in response to oxygen deprivation by a different mechanism. J Bacteriol, 1995 Jul, 177(14), 3917 - 22 Degradation of Escherichia coli uncB mRNA by multiple endonucleolytic cleavages; Patel AM et al.; The mechanism of segmental decay of the uncB sequence near the 5' end of the 7-kb Escherichia coli unc operon mRNA was investigated . Northern (RNA) blots of mRNA expressed from a plasmid carrying the uncBE portion of the operon revealed that the uncB message was rapidly degraded by multiple internal cleavages which resulted in the formation of at least five discrete species having a common 3' end . Turnover studies indicated that processing rapidly converted all species to the smallest . Identification of the 5' ends by primer extension analysis revealed that the cleavages were made either in the uncB coding region or in the intercistronic region between uncB and uncE, the latter being the most 3' cleavage . An rne mutant strain contained much higher levels of the uncBE message, implying that RNase E, the product of the rne gene, is essential for the normal degradation of uncB, and a number of the 5' ends were not detected in the rne mutant . The cleavage sites in chromosomally encoded unc mRNA were also identified by primer extension . These studies reveal that the segmental decay of the uncB region of unc mRNA occurs rapidly through a series of endonucleolytic cleavages . The rapid decay of uncB is expected to play a role in limiting expression of this gene relative to that of the other genes of the operon. Clin Immunol Immunopathol, 1995 Jul, 76(1 Pt 1), 90 - 5 Prevention of autoimmune diabetes in nonobese diabetic female mice by treatment with recombinant glutamic acid decarboxylase (GAD 65); Pleau JM et al.; The nonobese diabetic (NOD) mouse spontaneously develops insulin-dependent diabetes (IDDM or type I diabetes), resulting from T-lymphocyte-mediated destruction of pancreatic beta cells . This autoimmune phenomenon includes mononuclear cell infiltration of the islets of Langerhans (insulitis) and the presence of circulating autoantibodies . The specificity of the autoantibodies and of the autoreactive T cells was investigated and several autoantigens were proposed, in particular glutamic acid decarboxylase (GAD) . This enzyme exists in two forms (GAD 65 and GAD 67) encoded by two independent genes . To explain the role of GAD in type I diabetes, we prepared recombinant rat GAD 65 as fusion protein, produced in an Escherichia coli expression system, and we treated NOD female mice from 4 to 7 weeks of age by repeated intraperitoneal injections of 5 micrograms fusion protein (3 injections per week); control groups received the fusion partner, maltose binding protein (MBP) or dissolving agent (NaCl 0.9%) . We investigated two parameters, the degree of insulitis 5 weeks after the last injection and the overall incidence of the disease . Histological examination of the pancreata from GAD-treated mice revealed a significant reduction in the severity of insulitis compared with the two control groups . Furthermore, we observed that the time of onset and the frequency of diabetes in NOD females injected with GAD fusion protein differed significantly from the control groups receiving MBP or NaCl (P < 0.0001) . These results show that a 3-week treatment of NOD female mice starting at 4 weeks of age protects them from diabetes, again emphasizing the crucial role of GAD as autoantigen in type I diabetes. Anesthesiology, 1995 Jul, 83(1), 169 - 77 Does early posttreatment with lidocaine attenuate endotoxin-induced acute injury in rabbits? Nishina K, Mikawa K, Maekawa N, Takao Y, Obara H. BACKGROUND: It is well known that endotoxin causes acute lung injury, resulting in adult respiratory distress syndrome . Lidocaine pretreatment has recently been shown to attenuate endotoxin-induced lung injury in rabbits . The aim of the current study was to determine whether early postinjury treatment with intravenous lidocaine could attenuate acute lung injury induced by endotoxin in rabbits . METHODS: Thirty-two male anesthetized rabbits were randomly assigned to receive one of four treatments (n = 8 for each group): infusion of saline (group S-S), infusion of saline with lidocaine treatment (group S-L), infusion of Escherichia coli endotoxin (100 micrograms.kg-1 over a 60-min period) without lidocaine treatment (group E-S), or infusion of endotoxin with lidocaine treatment (group E-L) . Ten minutes after the end of infusion of endotoxin (groups E-L and E-S) or saline (groups S-S and S-L), the animals received a bolus injection followed by continuous infusion of lidocaine (2 mg.kg-1 + 2 mg.kg-1.h-1 in groups S-L and E-L) or saline (groups S-S and E-S) . The rabbits' lungs were ventilated with 40% O2 . Hemodynamics, peripheral leukocyte and platelet counts, and arterial O2 tension (PaO2) were recorded during the ventilation period (6 h) . After the observation, lung mechanics; the cell fraction of bronchoalveolar lavage fluid (BALF); and concentrations of activated complement components C3a and C5a, cytokines, and arachidonic acid metabolites in BALF were measured and analyzed . The ratio of lung wet weight to dry weight (W/D weight ratio) and albumin concentrations in BALF were analyzed as indexes of pulmonary edema . The Cypridina luciferin analogue-dependent chemiluminescence (representing O2 production) by neutrophils isolated from the pulmonary artery and light-microscopic findings of the lung were compared among the four groups . RESULTS: Endotoxin caused decreases in peripheral leukocyte and platelet counts, lung compliance, and PaO2 . It caused increases in lung W/D weight ratio; polymorphonuclear cell counts in BALF; and albumin, C3a, C5a, tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, IL-8, and thromboxane B2 concentrations in BALF . Lidocaine attenuated the changes in W/D weight ratio and morphologic lung damage . The change in compliance, decrease in PaO2, and albumin concentrations in BALF were slightly but significantly less in rabbits receiving lidocaine after injury . The Cypridina luciferin analogue-dependent chemiluminescence by neutrophils was greater in rabbits receiving endotoxin without lidocaine than in those receiving endotoxin with lidocaine . CONCLUSIONS: These results indicate that early treatment with lidocaine attenuates endotoxin-induced lung edema in rabbits without affecting chemical mediators in BALF . However, the improvement is slight and likely to be of little clinical significance. Am J Pathol, 1995 Jul, 147(1), 176 - 82 Mesangial cell-derived interleukin-10 modulates mesangial cell response to lipopolysaccharide; Fouqueray B et al.; Interleukin (IL)-10 is a novel cytokine produced by a variety of cells, including monocytes/macrophages, upon exposure to lipopolysaccharide (LPS) . Recent observations indicate that, in turn, IL-10 exerts suppressive effects on macrophage response to LPS . Because mesangial cells are also a target for LPS, we have examined the potential role of IL-10 in the regulation of mesangial cell response to LPS . To this aim, we have studied the synthesis and the autocrine/paracrine function of IL-10 in cultured mouse mesangial cells . IL-10 mRNA expression and IL-10 protein secretion were determined by a reverse transcription polymerase chain reaction technique and a specific enzyme-linked immunosorbent assay, respectively . No IL-10 mRNA expression was detectable in unactivated cells . LPS induced IL-10 mRNA expression in a dose-dependent fashion (1 to 100 micrograms/ml) . In addition, LPS induced IL-10 protein release that was both dose dependent (1 to 100 micrograms/ml) and time dependent (24 to 72 hours) . We have also studied the effect of IL-10 on the production of inflammatory mediators by LPS-activated mouse mesangial cells . Whereas recombinant IL-10 inhibited the generation of tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta by 90 and 60%, respectively, it did not affect the formation of nitric oxide-derived nitrite (NO2-) and nitrate (NO3-) . As shown by the use of anti-IL-10 monoclonal antibody, endogenously produced IL-10 affected the generation of TNF-alpha but neither that of IL-1 beta nor that of NO2- and NO3- . Finally, we have examined whether conditions known to also reduce the generation of TNF-alpha modified the expression of IL-10 . Of all the conditions tested, only the addition of desferrioxamine and transforming growth factor-beta were found to increase IL-10 release . Together, these data demonstrate that mesangial cell-derived IL-10 has important regulatory effects on the inflammatory response of these cells to LPS because of its capacity to blunt TNF-alpha generation. Surgery, 1995 Jul, 118(1), 63 - 72 Reduced tumor necrosis factor production in endotoxin-spiked whole blood after trauma: experimental results and clinical correlation; Fabian TC et al.; BACKGROUND . The overproduction of tumor necrosis factor-alpha (TNF) plays a key role in virtually every experimental model of septic shock, which has led to the development of several therapies that target TNF and other cytokines in clinical sepsis . However, our previous work showed that plasma TNF was reduced, rather than increased, when a septic challenge was administered 3 days after hemorrhagic shock . In this study we compared whole-blood TNF production ex vivo in human beings and animals after trauma . METHODS . TNF was measured before and after a 4-hour incubation of whole blood with 0 or 5 micrograms/ml Escherichia coli endotoxin (LPS) at 37 degrees C ex vivo . Samples were obtained from trauma patients with (n = 8) and without (n = 14) sepsis and compared with those obtained in healthy volunteers (n = 11) . In parallel experiments in a pig model TNF was measured before and after fluid resuscitation from trauma after an ex vivo (0 or 5 micrograms/ml LPS) or an in vivo (5 micrograms/kg LPS, 30 minutes intravenously) challenge . RESULTS . With either an immunoassay or a bioassay in either human beings or pigs before or after trauma, TNF was at or below the threshold of detection, unless the blood sample was spiked with LPS . After spiking, TNF was markedly elevated, but the increment was reduced after trauma . In pigs an LPS challenge in vivo delayed 3 days after trauma evoked an increment in plasma TNF that was blunted compared with that in an uninjured control . This trauma-induced reduction in blood TNF could not be attributed to a simple reduction in the number of monocytes nor to changes in cortisol, nor to increased numbers of neutrophils, whose proteolytic enzymes can impair production or increase the degradation of TNF . Although the plasma concentration of soluble TNF-binding proteins (60 kd) was elevated in nonsepsis (p = 0.0358) and sepsis trauma patients (p = 0.0148), the correlation with TNF production was relatively weak (R2 = 0.260) . CONCLUSIONS . There was no evidence of TNF overproduction in whole blood after trauma . If these results could be generalized to other tissues, it would be difficult to justify therapeutic targeting of TNF in exaggerated inflammatory response (or septic complications) after trauma. J Immunol, 1995 Jul 1, 155(1), 236 - 43 Humoral response to a carboxyl-terminal region of the merozoite surface protein-1 plays a predominant role in controlling blood-stage infection in rodent malaria; Daly TM et al.; The developmental stages of malaria parasites that infect E are responsible for the morbidity and mortality associated with this disease . One of the leading candidates for a blood-stage vaccine against malaria is a surface protein of merozoites, the infectious stages for E, designated merozoite surface protein-1 (MSP-1) . The rodent malarial parasite Plasmodium yoelii yoelii (Py) has provided a model system for the study of this Ag, and previous studies from our laboratory had demonstrated that the carboxyl-terminal, cysteine-rich region of MSP-1, when expressed in a native configuration, could immunize mice against a normally lethal challenge infection with Py . We have now prepared a new fusion construct with the glutathione-S-transferase gene of Schistosoma japonicum joined to the carboxyl-terminal 11 kDa of Py MSP-1 . This includes only the two epidermal growth factor-like domains of the MSP-1 protein . When expressed in recombinant Escherichia coli, the fusion protein induces a strong protective response in BALB/c mice as judged by the resistance of immunized animals to a virulent challenge infection . Moreover, we demonstrate that this resistance can be transferred passively by immune serum or by purified Ig, establishing a significant role for humoral immunity in protection . No role for CD4+ or CD8+ T cells could be identified in the first 12 days after challenge infection in immune mice selectively depleted of these cells; however, after this time, parasitemias gradually increased in mice depleted of CD4+ T cells, suggesting an active host response is necessary to completely eliminate the infection. J Bacteriol, 1995 Jul, 177(13), 3894 - 6 A study of the double mutation of dnaJ and cbpA, whose gene products function as molecular chaperones in Escherichia coli; Ueguchi C et al.; The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli . To gain insight into the function of CbpA, we examined the nature of a cbpA null mutation with special reference to those of dnaK and dnaJ null mutations . In particular, the cbpA dnaJ double-null mutant was found to exhibit severe defects in cell growth, namely, a very narrow temperature range for growth, a defect in cell division, and susceptibility to killing by carbon starvation . These phenotypes are very similar to those reported for dnaK null mutants but not to those of dnaJ null mutants . Our results are best interpreted by assuming that CbpA is capable of compensating for DnaJ for cell growth and thus that the function(s) of CbpA is closely related to that of DnaJ. J Bacteriol, 1995 Jul, 177(13), 3890 - 3 Cellular localization of the Escherichia coli SpoT protein; Gentry DR et al.; The SpoT protein of Escherichia coli serves as a source of degradation as well as an apparent source of synthesis of (p)ppGpp . Since the subcellular localization of SpoT might be a clue to its function, we have used SpoT-specific antisera to analyze cell extracts fractionated on sucrose gradients . We find that the SpoT protein is not bound to ribosomes or to either inner or outer membrane fractions . Although the SpoT protein is found in large aggregates, its localization is probably cytosolic. J Bacteriol, 1995 Jul, 177(13), 3873 - 8 Molecular cloning of the cyanobacterial adenylate cyclase gene from the filamentous cyanobacterium Anabaena cylindrica; Katayama M et al.; Molecular cloning of the structural gene for adenylate cyclase (cya) of the cyanobacterium Anabaena cylindrica was carried out by complementation of an Escherichia coli strain defective in the cya gene . The cya-defective strain produced significant amounts of cyclic AMP when it was transformed with the cya gene isolated from A . cylindrica . This gene encodes a polypeptide consisting of 502 amino acid residues (molecular weight, 55,300) . The deduced primary protein structure showed that the carboxyl-terminal region of the adenylate cyclase of A . cylindrica shows strong structural similarity to the conserved regions of the adenylate cyclases of various eukaryotes . No similarity was found between the amino acid sequences of the cya gene of A . cylindrica and that of E . coli . A hydropathy plot suggests that this protein has two hydrophobic regions, a transmembrane span and a signal peptide . An antiserum specific to this adenylate cyclase was prepared by immunizing a rabbit with a glutathione S-transferase-adenylate cyclase fusion protein expressed in E . coli . This antiserum recognized a 55-kDa protein in Anabaena cell lysates . Subcellular fractionation analysis showed that A . cylindrica adenylate cyclase localized in the thylakoid membrane. J Bacteriol, 1995 Jul, 177(13), 3865 - 9 Expression of the narX, narL, narP, and narQ genes of Escherichia coli K-12: regulation of the regulators; Darwin AJ et al.; The products of four Escherichia coli genes (narX, narL, narQ, and narP) regulate anaerobic respiratory gene expression in response to nitrate and nitrite . We used lacZ gene and operon fusions to monitor the expression of these nar regulatory genes in response to different growth conditions . Maximal expression of the narXL operon required molybdate, nitrate, and integration host factor . Expression of the narP and narQ genes was weakly repressed by nitrate . The NarL and NarP proteins were required for full nitrate induction of narXL operon expression, whereas the nitrate repression of narP and narQ expression was mediated solely by the NarL protein . narXL operon expression was unaffected by anaerobiosis, whereas expression of narP and narQ was induced approximately fourfold . The Fnr and ArcA proteins were not required for this anaerobic induction. J Bacteriol, 1995 Jul, 177(13), 3801 - 7 Identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli; DeShazer D et al.; Transcription of the pertussis toxin operon (ptx) is positively regulated in Bordetella pertussis by the bvgAS locus . However, a ptx-lacZ transcriptional fusion in Escherichia coli cannot be activated by bvgAS in trans . This suggests that an additional factor(s) is required for transcription of ptx . A gene encoding a Bvg accessory factor (Baf) was identified by its ability to activate an E . coli ptx-lacZ fusion in the presence of bvgAS . The expression of ptx-lacZ was decreased by the addition of 40 mM MgSO4, a compound that also modulates ptx expression in B . pertussis . Baf alone did not activate expression of an E . coli fhaB-lacZ fusion, nor did it increase expression of fhaB-lacZ in trans with bvgAS . The gene encoding Baf was localized, sequenced, and found to produce a novel 28-kDa protein . Sequences homologous to B . pertussis baf were identified in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium . When an additional copy of baf was integrated into the chromosome of BC75, a B . pertussis mutant that produces a low level of pertussis toxin, pertussis toxin production was partially complemented in the cointegrate strain. J Bacteriol, 1995 Jul, 177(13), 3793 - 800 Point mutations in the leader boxA of a plasmid-encoded Escherichia coli rrnB operon cause defective antitermination in vivo; Heinrich T et al.; We have introduced point mutations into the leader boxA of a plasmid-encoded Escherichia coli rrnB operon to study the in vivo role of this regulatory element in the natural context of rRNA synthesis . The same mutations were previously shown to cause severe antitermination defects in vitro and in the context of a reporter gene assay . The plasmid-encoded rrnB mutant constructs studied here also contained point mutations in the 16S and 23S rRNA genes, which were used to distinguish rRNAs derived from plasmid and chromosomal rrn operons by primer extension analysis . Point mutations in boxA reduced the fraction of plasmid-derived rRNA in the cell from 75% to about 50% . The reduction was similar for both 30S and 50S subunits as well as 70S ribosomes, suggesting that no transcriptional polarity occurred between the expression of the 16S and 23S rRNA genes in plasmid rrnB operons carrying a mutant boxA . The boxA mutations do not affect the amount of transcription initiation, suggesting that a suboptimal leader boxA causes premature transcription termination at an early stage of transcription . Our results are consistent with a role for antitermination in the completion of full-length rrn transcripts but give no indications of posttranscriptional boxA functions. J Bacteriol, 1995 Jul, 177(13), 3764 - 70 Escherichia coli alkaline phosphatase localized to the cytoplasm slowly acquires enzymatic activity in cells whose growth has been suspended: a caution for gene fusion studies; Derman AI et al.; Alkaline phosphatase is normally localized to the periplasm of Escherichia coli and is unable to fold into its native conformation if retained in the cytoplasm of growing cells . The alkaline phosphatase activity of E . coli expressing a version of the protein without a signal sequence was nonetheless found to increase gradually when the growth of cells was suspended . At least 30% of the protein was activated over the course of several hours when freshly grown exponential-phase cells were held on ice . Similar behavior was observed with cells expressing certain other mutant versions of alkaline phosphatase that are retained in the cytoplasm . The activation resulted not from the passage of the alkaline phosphatase into the periplasm but from the slow folding of alkaline phosphatase into its native conformation in the cytoplasm . These findings indicate that the mechanism by which proteins are normally kept reduced in the cytoplasm fails to function if cells are not growing . It was found that the addition of the sulfhydryl-alkylating agent iodoacetamide to cells after growth blocks this activation completely . This treatment can therefore diminish the likelihood of spurious enzyme activity measurements in studies that make use of alkaline phosphatase fusion proteins. J Bacteriol, 1995 Jul, 177(13), 3728 - 35 Isolation and identification of menaquinone-9 from purified nitrate reductase of Escherichia coli; Brito F et al.; On the basis of the observation that nitrate reductase from Escherichia coli is sensitive to UV irradiation with an action spectrum indicative of a naphthoquinone (F . Brito and M . Dubourdieu, Biochem . Int . 15:1079-1088, 1987), we extracted and characterized quinone components from two different preparations of purified nitrate reductase . A soluble form of nitrate reductase, composed of alpha and beta subunits, was purified after release from the membrane fraction by heat treatment, and a detergent-solubilized form, containing alpha, beta, and gamma (cytochrome bNR) subunits, was purified in the presence of Triton X-100 . Extraction of soluble alpha beta form with chloroform-methanol yielded several UV-absorbing components, which were characterized as menaquinone-9 with an oxidized side chain and further photodestruction products of the menaquinone . The total amount of menaquinone extracted into the organic phase was estimated to be 0.97 mol/mol of alpha beta dimer . Extraction of the detergent-solubilized alpha beta gamma form by a similar procedure yielded two naphthoquinone-like components which were characterized by mass spectrometry as the oxidized forms of menaquinone-9 and demethylmenaquinone-9 . In this case, the molar ratio of total naphthoquinone to the alpha beta dimer was estimated to be greater than 6:1 . When cytochrome bNR and detergent were eliminated from the detergent-solubilized enzyme by heat treatment and ion-exchange chromatography, only menaquinone-9 could be identified in the organic extract of the active alpha beta product . These results suggest that menaquinone-9 is specifically bound to the alpha beta dimer and may be the UV-sensitive component in the pathway of electron transfer catalyzed by nitrate reductase. J Bacteriol, 1995 Jul, 177(13), 3704 - 13 Ordered translocation of 987P fimbrial subunits through the outer membrane of Escherichia coli; Cao J et al.; The 987P fimbria of enterotoxigenic Escherichia coli is a heteropolymeric structure which consists essentially of a major FasA subunit and a minor subunit, the FasG adhesin . The latter harbors the binding moiety for receptor molecules on piglet intestinal epithelial cells . In this study, anti-FasF antibody probes were developed and used to demonstrate that the FasF protein represents a new minor fimbrial component . FasF was identified in highly purified fimbriae, and its sequence demonstrated significant levels of similarity with that of FasA . Immune electron microscopy localized both the FasG and FasF proteins at the fimbrial tip as well as at broken ends and at various intervals along the fimbrial length . The presence of these minor proteins in purified 987P fimbriae was corroborated by enzyme-linked immunosorbent assay inhibitions . Finally, the use of nonfimbriated fasG, fasF, and fasA mutants indicated that subunit translocation through the outer membrane follows a specific order, FasG being the first, FasF being the second, and FasA being the third type of exported subunit . Since fimbriae are thought to grow from the base, FasG is proposed to be a tip adhesin and FasF is proposed to be a linker molecule between the adhesin and the fimbrial shaft . Moreover, export of FasG (or FasF) in the absence of FasF (or FasA) indicates that during the process of fimbrial biogenesis in the outer membrane, translocating events precede the initiation of subunit heteropolymerization. J Bacteriol, 1995 Jul, 177(13), 3695 - 703 An essential role for the Escherichia coli DnaK protein in starvation-induced thermotolerance, H2O2 resistance, and reductive division; Rockabrand D et al.; During a 3-day period, glucose starvation of wild-type Escherichia coli produced thermotolerant, H2O2-resistant, small cells with a round morphology . These cells contained elevated levels of the DnaK protein, adjusted either for total protein or on a per-cell basis . Immunoprecipitation of {35S}methionine-labeled protein produced by such starving cells demonstrated that DnaK underwent continuous synthesis but at decreasing rates throughout this time . Glucose resupplementation of starving cells resulted in rapid loss of thermotolerance, H2O2 resistance, and the elevated DnaK levels . A dnaK deletion mutant, but not an otherwise isogenic wild-type strain, failed to develop starvation-induced thermotolerance or H2O2 resistance . The filamentous phenotype associated with DnaK deficiency was suppressed by cultivation in a defined glucose medium . When starved for glucose, the nonfilamentous and rod-shaped dnaK mutant strain failed to convert into the small spherical form typical of starving wild-type cells . The dnaK mutant retained the ability to develop adaptive H2O2 resistance during growth but not adaptive resistance to heat . Complementation of DnaK deficiency by using Ptac-regulated dnaK+ and dnaK+J+ expression plasmids confirmed a specific role for the DnaK molecular chaperone in these starvation-induced phenotypes. J Bacteriol, 1995 Jul, 177(13), 3623 - 30 Expression of the sodium ion pump methylmalonyl-coenzyme A-decarboxylase from Veillonella parvula and of mutated enzyme specimens in Escherichia coli; Huder JB et al.; The structural genes of the sodium ion pump methylmalonyl-coenzyme A (CoA)-decarboxylase from Veillonella parvula have recently been cloned on three overlapping plasmids (pJH1, pJH20, and pJH40) and sequenced . To synthesize the complete decarboxylase in Escherichia coli, the genes were fused in the correct order (mmdADECB) on a single plasmid (pJH70) . A DNA region upstream of mmdA apparently served as promoter in E . coli because expression of the mmd genes was not dependent on the correct orientation of the lac promoter present on the pBluescript KS(+)-derived expression plasmid . To allow controlled induction of the mmd genes, the upstream region was deleted and the mmd genes were cloned behind a T7 promoter . The derived plasmid, pT7mmd, was transformed into E . coli BL21(DE3) expressing T7 RNA polymerase under the control of the lac promoter . The synthesized proteins showed the typical properties of methylmalonyl-CoA-decarboxylase, i.e., the same migration behavior during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stimulation of the decarboxylation activity by sodium ions, and inhibition with avidin . In methylmalonyl-CoA-decarboxylase expressed in E . coli from pT7mmd, the gamma subunit was only partially biotinylated and the alpha subunit was present in substoichiometric amounts, resulting in a low catalytic activity . This activity could be considerably increased by coexpression of biotin ligase and by incubation with separately expressed alpha subunit . After these treatments methylmalonyl-CoA-decarboxylase with a specific activity of about 5 U/mg of protein was isolated by adsorption and elution from monomeric avidin-Sepharose . To analyze the function of the delta and epsilon subunits, the corresponding genes were deleted from plasmid pT7mmd . E . coli cells transformed with pJHdelta2, which lacks mmdE and the 3' -terminal part of mmdD, showed no methylmalonyl-CoA-decarboxylase activity . In addition, a contrast, catalytically active methylmalonyl-CoA-decarboxylase was expressed in E . coli from plasmid pJHdelta1, which contained a deletion of the mmdE gene only . The mutant enzyme could be isolated, reconstituted into proteolipsomes, and shown to function in the transport of Na+ ions coupled to methylmalonyl-CoA decarboxylation . The small epsilon subunit therefore has no catalytic function within the methylmalonyl-CoA-decarboxylase complex but appears to increase the stability of this complex. Crit Care Med, 1995 Jul, 23(7), 1217 - 26 Endotoxemia causes ileal mucosal acidosis in the absence of mucosal hypoxia in a normodynamic porcine model of septic shock; VanderMeer TJ et al.; OBJECTIVE: To evaluate the hypothesis that splanchnic ischemia and mucosal hypoxia are responsible for lipopolysaccharide-induced intramucosal acidosis in pigs . DESIGN: Prospective, randomized, unblinded study . SETTING: Surgical research laboratory at a large, university-affiliated medical center . SUBJECTS: Anesthetized, mechanically ventilated swine . INTERVENTIONS: Pigs were infused with lactated Ringer's solution (12 mL/kg/hr) and, starting at 30 mins, 25-mL boluses of dextran-70 (maximum 15 mL/kg/hr) to maintain cardiac output at 90% to 110% of the baseline value for each pig . Ileal mucosal hydrogen ion concentration was measured tonometrically . A segment of distal ileum was exteriorized, opened, and placed on a platform to permit measurement of mucosal PO2, using an array of Clark-type microelectrodes and a computerized data acquisition and analysis system . Mucosal perfusion was measured using laser-Doppler flowmetry . The control group (n = 4) received no further interventions . Pigs in the lipopolysaccharide group (n = 6) were infused with 150 micrograms/kg of Escherichia coli lipopolysaccharide over 60 mins . To assess the effect of mucosal acidosis on mucosal PO2 in nonendotoxemic animals, intramucosal hydrogen ion concentration, mucosal PO2, and mucosal perfusion were measured in pigs rendered hypercarbic through deliberate hypoventilation (hypercarbia group; n = 4) . MEASUREMENTS AND MAIN RESULTS: Infusion of lipopolysaccharide resulted in a significant increase in intramucosal hydrogen ion concentration . However, in the lipopolysaccharide group, mucosal perfusion did not change significantly and mucosal PO2 increased significantly . In the hypercarbia group, hypercarbia was associated with significant increases in both intramucosal hydrogen ion concentration and mucosal PO2 . CONCLUSIONS: Mucosal hypoxia is not responsible for lipopolysaccharide-induced mucosal acidosis in this normodynamic pig model of septic shock . A rightward shift of the oxyhemoglobin dissociation curve (the Bohr effect) can explain the increase in mucosal oxygenation observed in endotoxemic pigs. Am J Respir Crit Care Med, 1995 Jul, 152(1), 348 - 54 Endotoxin-induced hydrogen peroxide production in intact pulmonary circulation of rat; Minamiya Y et al.; Although the importance of free oxygen radical has been reported in acute lung injury, the direct evidence in vivo model was lacking . We report a new method, which for the first time allows direct detection of hydrogen peroxide in the intact rat pulmonary microcirculation . We used the computer image-analyzing system and 2',7'-dichlorofluorescin diacetate for the marker of hydrogen peroxide production in vivo . A rat sepsis model was produced by continuous infusion of endotoxin for 30, 60, and 120 min . Hydrogen peroxide production in the pulmonary microcirculation of the sepsis rat was higher than in the control rat at each time point (p < 0.01) and increased time-dependently (p < 0.01) . Catalase (5,000 U/kg) almost completely inhibited the hydrogen peroxide production in the sepsis rat (p < 0.01) . In high-power view, hydrogen peroxide was detected in granulocytes that adhered to the capillaries and endothelial cells that were adjoining adherent granulocytes . These observations suggest that hydrogen peroxide in the endothelium was diffused from granulocytes . In this study, we demonstrated direct evidence of hydrogen peroxide production from adherent granulocytes in intact rat lung treated with endotoxin . We conclude that endotoxin causes the granulocyte adhesion and oxidative stress to the endothelium due to adherent granulocytes within 30 min in the pulmonary microcirculation. Ann Thorac Surg, 1995 Jul, 60(1), 12 - 8 Cellular cardiomyoplasty: myocardial regeneration with satellite cell implantation; Chiu RC et al.; BACKGROUND . Damaged skeletal muscle is able to regenerate because of the presence of satellite cells, which are undifferentiated myoblasts . In contrast, destruction of cardiac myocytes is associated with an irreversible loss of myocardium and replacement with scar tissue, because it lacks stem cells . We tested the hypothesis that skeletal muscle satellite cells implanted into injured myocardium can differentiate into cardiac muscle fibers and thus repair damaged heart muscle . METHODS . Two series of canine studies were performed . In the first series (n = 26), satellite cells were isolated from skeletal muscle, cultured, and labeled with tritiated thymidine . The cells were implanted into acutely cryoinjured myocardium and the specimens harvested 4 to 18 weeks later . In the second series (n = 20), satellite cells in culture were labeled with lacZ reporter gene, which encodes production of Escherichia coli beta-galactosidase . Four to 6 weeks later, beta-galactosidase activity was studied using X-Gal stain . RESULTS . New striated muscles were found in the first series of experiments at the site of implantation, within a dense scar created by cryoinjury . These muscles showed histologic evidence of intercalated discs and centrally located nuclei, similar to those seen in cardiac muscle fibers . Tritiated thymidine radioactivity was not identified clearly, presumably due to dilutional effect as the stem cells replicated repeatedly . In the second series, histochemical studies of reporter gene-labeled and implanted satellite cells revealed the presence of beta-galactosidase within the cells at the implant site, which confirmed the survival of implanted cells . CONCLUSIONS . Our data are consistent with the hypothesis of milieu-influenced differentiation of satellite cells into cardiac-like muscle cells . Confirmation of these findings and its functional capabilities could have important clinical implications. J Lipid Res, 1995 Jul, 36(7), 1489 - 97 Purification and characterization of recombinant squalene epoxidase; Nagumo A et al.; Recombinant rat squalene epoxidase (rSE) was expressed in E . coli and purified to an apparent homogeneity . This expression system was constructed using squalene epoxidase (SE) cDNA in which nucleotides coding 99 amino acids in the N-terminal were deleted and nucleotides coding hexa-histidine in the C-terminal were added . Purification was carried out using Ni-chelate affinity agarose and Cibacron Blue Sepharose column chromatography . Purification was achieved 100-fold over the crude E . coli extract with a yield of about 50% . The purified enzyme demonstrated a single band on SDS-polyacrylamide gel electrophoresis . The enzyme showed no distinct absorption spectrum in the visible regions . The properties of rSE were compared with those of rat liver microsomal SE . The requirement of the co-factors, the S105 fraction or Triton X-100, and NADPH-cytochrome c reductase, the pH dependency for enzyme activity, and the sensitivity to NB-598 seen with both enzymes suggest that rSE has properties very similar to rat microsomal SE . 2,3-Oxi-dosqualene (OSQ) and 2,3;22,23-dioxidosqualene (DOSQ) were formed by rSE in a completely reconstituted system . It is suggested that recombinant squalene epoxidase catalyzes the conversion of squalene to 2,3-oxidosqualene and of 2,3-oxidosqualene to 2,3;22,23-dioxidosqualene. Mol Cell Endocrinol, 1995 Jul, 112(1), 15 - 9 Desethylamiodarone is a competitive inhibitor of the binding of thyroid hormone to the thyroid hormone alpha 1-receptor protein; van Beeren HC et al.; Desethylamiodarone (DEA), the major metabolite of the potent antiarrythmic drug amiodarone, is a non-competitive inhibitor of the binding of thyroid hormone (T3) to the beta 1-thyroid hormone receptor (T3R) . In the present study, we investigated whether DEA acts in a similar way with respect to the alpha 1-T3R . The chicken alpha 1-T3R, expressed in an E . coli system, was incubated in the presence or absence of DEA with {125I}T3 in buffer containing 0.05% Triton X-100, 0.05% BSA and 1% ethanol (v/v) in order to solubilise DEA . DEA, but not amiodarone, inhibited T3 binding in a dose-dependent manner; the IC50 value was 3.5 x 10(-5) M . Scatchard analyses in the presence of DEA demonstrated a dose-dependent decrease in Ka values, but no change in MBC . Lineweaver-Burk plots clearly indicated competitive inhibition by DEA . Pre-incubation of the alpha 1-receptor with DEA decreased maximal {125I}T3 binding, which was independent of the duration of pre-incubation . In conclusion, in contrast to the beta 1-T3R, where DEA acts as a non-competitive inhibitor, we now report as a new finding the competitive action of DEA to the alpha 1-T3R. Drug Metab Dispos, 1995 Jul, 23(7), 702 - 7 Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis; Born SL et al.; Canine hepatic cytochrome P450 PBD-2 metabolizes 2,2',4,4',5,5'-hexachlorobiphenyl and catalyzes the 21-hydroxylation of progesterone, thereby distinguishing PBD-2 as unique among 2B P450s . Heterologous expression of the PBD-2 cDNA, P450 2B11, in COS and yeast systems produced a protein capable of androstenedione metabolism; however, this P450 did not metabolize progesterone in a manner consistent with PBD-2 . Modification of PBD-2 reconstitution parameters resulted in significantly increased catalytic activities and further emphasized differences between PBD-2 and the heterologously expressed enzyme . Subsequent Escherichia coli expression of 2B11 generated a protein that possessed substrate specificities indistinguishable from those of PBD-2 and provided a system in which the determinants of 2B11 progesterone 21-hydroxylation could be examined via site-directed mutagenesis . Site-directed mutants of 2B11 expressed in E . coli revealed that substitution of Ile with Val at position 363 converted 2B11 into a highly active and specific progesterone 16 alpha-hydroxylase . Mutants Val-114 --> Ile, Asp-290 --> Ile, and Ile-365 --> Phe exhibited decreased progesterone 21- and 16 alpha-hydroxylase activities, in accordance with decreases in androstenedione hydroxylase activities . In contrast, replacement of Ile-365 with Val or Leu resulted in much greater changes in progesterone than androstenedione hydroxylation . Thus, the combination of P450 reconstitution techniques, heterologous expression, and site-directed mutagenesis has revealed PBD-2 to be an important progesterone 21-hydroxylase in canine liver and has identified several amino acid residues that alter progesterone metabolism by 2B11. Bioorg Med Chem, 1995 Jul, 3(7), 945 - 53 A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase; Garcia-Junceda E et al.; Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA . In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus . The three enzymes have been purified in only one step by chelation affinity chromatography . The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies. Nucl Med Biol, 1995 Jul, 22(5), 643 - 8 Comparison of the infection imaging properties of a 99mTc labeled chemotactic peptide with 111In IgG; Babich JW et al.; The biodistribution and infection imaging properties of a 99mTc labeled hydrazino nicotinamide (HYNIC) derivatized chemotactic peptide analog (For-Met-Leu-Phe-Lys-HYNIC) and 111In-DTPA-IgG were compared in rabbits with Escherichia coli infection . Six New Zealand white rabbits were injected in the left posterior thigh with a suspension of E . coli . Twenty four hours later, the animals were injected with: 1.0 mCi of 99mTc labeled peptide plus 0.1 mCi of 111In-DTPA-IgG . At 2-3 and 16-18 h, dual photon scintigrams were acquired and the images were corrected for crossover between the two windows . After recording the final images, the animals were sacrificed and biodistribution was determined . At both imaging times the biodistributions of the two reagents were markedly different . The highest concentrations of 111In-DTPA-IgG were detected in blood pool structures, liver and kidney . In contrast localization of 99mTc labeled peptide was greatest in spleen, lung and liver (consistent with binding to leukocytes) . In general, the sites of infection were better visualized with the radiolabeled peptide and T/B ratios increased with time (P < 0.01) . At both times, the T/Bs for 99mTc-peptide were higher (P < 0.01); 3.54 +/- 0.47 vs 2.52 +/- 0.38 at 2-3 h and 6.88 +/- 0.79 vs 3.78 +/- 0.36 at 16-18 h . These results indicate that although both radiopharmaceuticals localize at sites of infection, the radiolabeled peptide are superior reagents for the rapid detection of focal sites of infection . However, since the mechanisms of localization are different the combined use of both agents could have value in the general evaluation of infection/inflammation. Biokhimiia, 1995 Jul, 60(7), 995 - 1004 {Import of a modified form of a cytochrome P450scc precursor into mitochondria from various sources}; Novikova LA et al.; An Escherichia coli strain providing hypersynthesis of a recombinant cytochrome P450scc precursor supplemented with the extra MetArgGlySerHis6GlyIleArg sequence at the NH2-terminus (6His-pP450scc) has been constructed . A procedure for isolation and purification of 6His-pP450scc from the cell homogenate has been elaborated . It has been found that the recombinant precursor is imported into isolated rat liver and rat heart mitochondria as well as into yeast mitochondria . The import is coupled with proteolytic processing resulting in the mature size form of cytochrome P450scc . Modification of the targeting P450scc presequence resulting in its increased positive charge is supposed to relieve tissue-specific restrictions on the P450scc import into mitochondria. Bioconjug Chem, 1995 Jul-Aug, 6(4), 361 - 6 Galactose-containing amphiphiles prepared with a lipophilic radical initiator: association processes between liposomes triggered by enzymatic reaction; Ohno K et al.; A galactose-containing monomer (2-(methacryloyloxy)ethyl beta-D-galactopyranoside, MEGal) was polymerized by using a lipophilic radical initiator . The amphiphile obtained formed a liposome by mixing with bis(trans,trans-2,4-dioctadecadienoyl)phosphatidylcholine (DDPC), and the liposome obtained was physically stabilized by the polymerization of DDPC by UV irradiation . The enzymatic treatment of the galactose-containing liposomes with galactose oxidase resulted in the formation of aldehyde groups on the liposome surface . By the subsequent mixing of the liposome suspension with the amino group-containing liposome suspension, a rapid increase in turbidity was observed due to the formation of Schiff bases between the aldehyde groups and the amino groups at the interface of the liposomes . The rate of turbidity change strongly depended on the degree of polymerization of MEGal, the surface densities of galactose and amino groups on the liposome, the distance from the liposome surface to amino end groups, and the flexibility and deformability of the liposomes. Arch Oral Biol, 1995 Jul, 40(7), 639 - 44 Expression of a functional rat salivary cystatin S polypeptide in Escherichia coli; Sharma A et al.; Cystatin S is a cysteine proteinase inhibitor that is transiently expressed during rat submandibular gland development and can be induced by isoproterenol in the adult . A cDNA for rat cystatin S which included the entire coding sequence of the secreted cystatin was cloned . A coding region of the cystatin gene was amplified by polymerase chain reaction and cloned into the pGEX-2T expression vector . The chimeric plasmid was transformed into Escherichia coli, and protein expression was induced by isopropyl-beta-D-thiogalactopyranoside . The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and fast protein liquid chromatography on a MonoQ column . The purified recombinant cystatin reacted with antibodies to cystatin S purified from rat submandibular glands and showed an amino-terminal amino acid sequence identical to that of rat cystatin S . The recombinant protein exhibited papain inhibition activity comparable to natural cystatin . This was a successful expression and purification of a functionally and immunologically reactive recombinant cystatin from E . coli, an approach which will be used later towards generating recombinant variants to study the binding and functional domains of this cysteine protease inhibitor. Vet Microbiol, 1995 Jul, 45(2-3), 171 - 83 Selective humoral immune response of Balb/C mice to Brucella abortus proteins expressed by vaccinia virus recombinants; Toth TE et al.; Genes encoding Brucella abortus Cu/Zn superoxide dismutase (SOD) and a 54 kDa Escherichia coli HtrA homologue were cloned into shuttle plasmids pUV-1 and pSC11, and transfected into vaccinia virus to develop recombinants vUBSOD and vSB54 . Control vaccinia virus recombinants vUV-1 and vSC11, carrying only the beta-gal reporter gene but no B . abortus DNA were also developed . Recombinants were analyzed in Western blotting using a polyclonal B . abortus immune serum . vUBSOD expressed a protein of apparent molecular weight of 28 kDa, composed of the 20 kDa B . abortus Cu/Zn-SOD and a protein approximately 8 kDa encoded by a portion of the vaccinia virus TK gene . vSB54 expressed a 54 kDa protein corresponding to the 54 kDa HtrA homologue . Recombinants vUSV-1 and vSC11 did not express B . abortus proteins . Groups of mice were inoculated intraperitoneally with 10(7) TCID50 of 1 of the 4 different recombinant vaccinia viruses and 5 weeks later their sera were analyzed for antibodies against vaccinia virus and B . abortus proteins . Each group of mice responded with antibodies to vaccinia virus . Sera of vSB54-inoculated mice recognized the 54 kDa HtrA homologue . vUBSOD did not induce a humoral immune response . These results represent the first report on the expression of B . abortus proteins by vaccinia virus recombinants and the first demonstrated immune response against a B . abortus protein expressed by such a recombinant. Neuroendocrinology, 1995 Jul, 62(1), 55 - 61 Production of systemic and hypothalamic cytokines during the early phase of endotoxin fever; Jansky L et al.; Changes in concentrations of cytokines in plasma and in hypothalamic push-pull perfusates of guinea pigs were measured within the 1st hour after intramuscular injections of bacterial lipopolysaccharide (LPS; Escherichia coli, 20 micrograms/kg) or solvent (0.9% saline) . In control animals injected with solvent, interleukin (IL)-1 and tumor necrosis factor alpha (TNF-alpha) were not detectable in plasma . Only IL-6 was present in picogram quantities . Within 45 min after injection of LPS, the concentrations of IL-1, TNF-alpha, and IL-6 increased in the plasma: by several orders of magnitude for TNF-alpha and about tenfold for IL-G . Picogram amounts of biologically active IL-1 were detected in plasma after injection of LPS . No steady state levels of systemic cytokines were reached during the experimental period . In hypothalamic perfusates of animals injected with the solvent, no IL-1 was detectable . TNF-alpha could be detected at higher concentrations than IL-6 . IL-6 was detectable at tenfold lower concentrations than in the plasma . In animals injected with LPS, the hypothalamic concentration of IL-6 started to increase during the period 15-30 min and the concentrations of TNF-alpha during the period 30-45 min after LPS injection . The concentrations of IL-6 increased by 300-400% and did not exceed picogram values . No progressive increase of hypothalamic levels of these cytokines was observed during the time course of the experiment . The method used did not detect any changes in the amount of biologically active IL-1 in hypothalamic perfusates of LPS-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS) Kansenshogaku Zasshi, 1995 Jul, 69(7), 772 - 6 {Experimental infection of infant rabbits with verocytotoxin-producing Escherichia coli of bovine origin}; Nakazawa M et al.; Infant rabbits were used as a model to study the diarrhogenicity of verocytotoxin-producing Escherichia coli (VTEC) isolated from bovine . Infant rabbits aged 6 days were inoculated intragastrically with 10(9) viable bacteria of four VTEC or three non-VTEC strains . Of these strains, three strains (VT+, eaeA+), one strain (VT+, eaeA-) and one strain (VT-, eaeA+) caused diarrhea in rabbits 48 to 60 hr after inoculation . None of the two strains (VT-, eaeA-) caused symptoms . Based on these results, it is suggested that the 6-day-old infant rabbit is a suitable animal for studying diarrhea caused by VTEC or eaeA-positive E . coli strains of bovine origin. Eur J Pediatr, 1995 Jul, 154(7), 530 - 3 Fulminant hepatic failure in a child as a potential adverse effect of trimethoprim-sulphamethoxazole; Simma B et al.; Trimethoprim-sulphamethoxazole (TMP-SMZ) is considered a safe drug for treatment of infectious bacterial diseases in children . Side-effects are rare and generally take the form of a hypersensitivity reaction to the sulphamethoxazole component of the drug . Hepatic injury usually presents as a transient elevation of liver enzymes, which is of little clinical relevance . Fulminant liver failure due to TMP-SMZ has been reported in only six adults and never in children . We here report a 5-year-old girl who developed fulminant liver failure 3 weeks after her third exposure to TMP-SMZ . After a biphasic clinical course she underwent successful liver transplantation . CONCLUSION: Trimethoprim-Sulphamethoxazole may cause fulminant liver failure in children . The disease can run a biphasic clinical course and liver transplantation must be considered as the therapeutic option for these patients. Chin Med J (Engl), 1995 Jul, 108(7), 548 - 50 Endotoxin-induced liver injury and changes in the levels of plasma tumor necrosis factor-alpha and interleukin-6 in rabbits; Wu C et al.; ELISA was employed to detect changes in plasma TNF-alpha and IL-6 level in rabbits having endotoxin-induced generalized Shwartzman reaction . Liver injury was assessed by the increase of serum ALT and hepatic histopathologic changes . The results showed that plasma TNF-alpha and IL-6 levels increased remarkably 12h after intravenous injection of endotoxin twice at a 24h interval, and these changes corresponded with the degree of liver injury . Coinjection of dexamethasone and endotoxin partly prevented the elevation of TNF-alpha and IL-6 levels and reduced liver injury . These results indicated that TNF-alpha and IL-6 were involved in endotoxin-induced liver injury. Cent Afr J Med, 1995 Jul, 41(7), 216 - 9 Indomethacin and alpha-tocopheral enhanced survival in endotoxic rats; Ashorobi RB et al.; Induced endotoxaemia was studied in healthy male albino rats injected intravenously with lipopolysaccharide from Escherichia coli at 10, 20 and 30 mg/kg dose levels . The endotoxic rats were closely observed for mortality within 48 hours and subsequently for seven days . Within five hours post administration over 50 pc mortality was observed at dose levels of 20 to 30 mg/kg . Pre-treatment of rats with Indomethacin (250 mg/kg) alone and alpha-tocopheral (100 mg/kg) produced significant protective effects with the mortality rate reduced to 50 pc at the highest endotoxin dose level . Interestingly a combination of both drugs significantly improved survival with an observed mortality of 20 pc . Prednisolone (500 mg/kg) either alone or in combination with indomethacin or alpha-tocopherol did not offer any advantage . Instead the mortality rate was significantly high . A combination of Indomethacin and alpha-tocopheral significantly improved survival in endotoxic rats. Cardiovasc Res, 1995 Jul, 30(1), 97 - 105 Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus; Li JJ et al.; OBJECTIVE: The aim was to examine the feasibility, efficiency and safety of adenovirus-mediated in vivo gene transfer into the canine myocardium by a percutaneous transluminal method using a needle-catheter . METHODS: Either a replication-defective adenovirus (Adex1SRLacZL) or a plasmid (pSRLacZ), both expressing E . coli lacZ coding beta-galactosidase (beta-gal), was directly injected into the left ventricle of dogs through a needle-catheter inserted via a femoral artery . Expression of lacZ was examined by histochemical staining and quantified by measuring beta-gal activity . RESULTS: Injections with Adex1SRLacZL induced lacZ expression as a result of 40 out of 41 injections; the expression level was 10 times higher than that obtained with pSRLacZ . Induced beta-gal activity was detected within 24 h, peaked at 7 days and retained for 2 weeks after gene transfer . A repetitive administration of the same adenovirus at 14 days after the first injection also evoked a reduced but significant level of expression despite neutralizing antibodies to adenovirus in serum . Although injection induced an inflammatory response that peaked at 3 days after injection and gradually subsided without a second peak, the temporal change and the extent of inflammation induced by adenovirus injection was not significantly different from those induced by injection with either saline or plasmid . Neither leakage of enzymes such as CPK or LDH nor alteration in the ECG was detected in the 30 days following gene transfer . CONCLUSIONS: Our findings demonstrate that a catheter-mediated direct injection with an adenovirus can induce gene expression in the ventricle more efficiently without additional myocardial damage and inflammation compared with injection with a plasmid . A repeat dose of the same adenovirus elicited gene expression at an attenuated but significant level . This method may potentially have clinical applications: in modifying myocardial phenotype and/or improving general circulation under certain circumstances. Plant Cell Physiol, 1995 Jul, 36(5), 779 - 87 Genomic organization of 251 kDa acetyl-CoA carboxylase genes in Arabidopsis: tandem gene duplication has made two differentially expressed isozymes; Yanai Y et al.; Acetyl-CoA carboxylase (ACCase) catalyzes the carboxylation of acetyl-CoA, forming malonyl-CoA a key intermediate in the biosynthesis of fatty acids and a variety of secondary metabolites . Based upon amino acid sequences conserved among rat, chicken, and E . coli ACCases, PCR-primers were used to amplify a genomic fragment which codes for an ACCase of Arabidopsis . The resulting fragment was used for isolation of genomic and cDNA clones . We have determined the complete cDNA sequence coding for an Arabidopsis ACCase consists of 2,254 amino acids with the molecular mass of 251 kDa . This enzyme contains no recognizable plastid transit-peptide sequence . Therefore, this ACCase is presumably the cytosolic isozyme . Southern analysis indicates that there are two ACCase genes in the Arabidopsis genome . Surprisingly, the results of RFLP analysis and physical mapping of the isolated genomic clones demonstrate that these two genes, acc1 and acc2, are contiguously located within a 25-kbp genomic region near the middle of chromosome 1 . Both genes are transcriptionally active, as transcripts from each gene were detected by reverse transcription-PCR analysis using gene-specific primers . The acc1 and acc2 transcripts accumulate in leaves and seedlings but only the acc1 transcript accumulates in developing siliques, unexpectedly . The differences in the expression patterns may be indicative of the differential role of the two genes. Biochem Mol Biol Int, 1995 Jul, 36(3), 483 - 90 Unexpected nuclear localization of a chimeric beta-galactosidase lacZ reporter gene product in mammalian cells; McInnis R et al.; A lacZ cassette was designed to include a synthetic amino terminus optimized for translation in eukaryotic cells, as well as multiple restriction sites for the insertion of heterologous regulatory elements both 5' and 3' of the reporter . The cassette was placed under the control of the metallothionein promoter in combination with the SV40 enhancer and this plasmid was introduced into mammalian cells . High levels of beta-galactosidase were observed in several cell types, demonstrating efficient expression of the reporter . Unexpectedly, most of the chromogenic reaction product appeared to be intra- or peri-nuclear, indicating that the enzyme is similarly localized . The synthetic amino terminus does not resemble known nuclear localization signals and thus may constitute a novel signal. Genetics, 1995 Jul, 140(3), 909 - 15 Short-patch reverse transcription in Escherichia coli; Thaler DS et al.; Chimeras of RNA and DNA have distinctive physical and biological properties . Chimeric oligonucleotides that contained one, two or three ribonucleotides whose phosphodiester backbone was covalently continuous with DNA were synthesized . Site-directed mutagenesis was used to assess genetic information transfer from the ribonucleotide positions . Transfer was scored by the formation or reversion of an ochre site that also corresponded to a restriction cleavage site . This allowed physical as well as genetic assay of mutational events . Bases attached to the ribonucleotides were able to accurately direct the synthesis of progeny DNA . The results suggest that in vivo DNA polymerases utilize a "running start" on a DNA backbone to continue across a covalent backbone junction into a region of ribonucleotides and then back again onto a normal DNA backbone . The phenomenon is designated short-patch reverse transcription (SPRT) by analogy to short-patch mismatch correction and reverse transcription as the term is generally used . The possibility is considered that SPRT contributes to an unrecognized pathway of mutagenesis. J Clin Microbiol, 1995 Jul, 33(7), 1797 - 803 Differentiation of Bartonella-like isolates at the species level by PCR-restriction fragment length polymorphism in the citrate synthase gene; Norman AF et al.; The citrate synthase gene (gltA) of Bartonella henselae was cloned and sequenced to compare genetic divergence among alpha and gamma branches of the class Proteobacteria and to develop enhanced genotypic reagents for B . henselae identification . B . henselae gltA is 1,293 nucleotides in length and 63 to 66% homologous with corresponding gene sequences of Rickettsia prowazekii, Escherichia coli, and Coxiella burnetii . The observed genetic variability suggests that gltA sequences can provide a useful means for studying moderate divergence among related bacteria . Oligonucleotides specific for B . henselae gltA were evaluated for the ability to prime PCR amplification within the alpha and gamma branches of the proteobacteria . Under the conditions used, only B . henselae, Bartonella quintana, and R . prowazekii template DNAs yielded amplification products (approximately 380 bp) . DNAs from 28 Bartonella-like isolates of feline origin were amplified by B . henselae primers and analyzed for restriction fragment length polymorphism . The resulting patterns for all 28 isolates were similar or identical to that of the recognized B . henselae strain . Current studies are aimed at optimization of PCR conditions for specificity and sensitivity of amplification of Bartonella sequences from clinical isolates. Biotechnol Prog, 1995 Jul-Aug, 11(4), 397 - 402 Process characterization of a novel cross-regulation system for cloned protein production in Escherichia coli; Chen W et al.; A novel cross-regulation expression system has been shown previously to be very effective for regulated recombinant protein production . Earlier studies established that this system offers better control of basal expression and higher maximal induced expression than more traditional vectors . Using production of cloned chloramphenicol acetyltransferase (CAT) as a model system, several factors determining the performance of this system were examined . Specifically, the effects of varying induction times and inducer (IPTG) concentrations on cell growth and the rate of CAT production were examined . The CAT expression was maximally induced with at least 0.5 mM IPTG added at the midexponential growth phase . Specific CAT content (on a total protein basis) was correlated with the CAT mRNA level . CAT message levels were minimal preinduction and far above background postinduction, consistent with prior simulation results . Cessation of CAT accumulation as the culture entered the stationary phase coincided with a corresponding 10-fold decrease in the level of CAT mRNA which was likely caused by an increased mRNA degradation rate . Maintenance of significant CAT message levels with a concomitant 2-fold increase in CAT accumulation was achieved by extending cell growth in a fed-batch process. Arthritis Rheum, 1995 Jul, 38(7), 990 - 8 Autoepitopes of the 52-kd SS-A/Ro molecule; Kato T et al.; OBJECTIVE . This study was undertaken to clarify the mechanisms responsible for the generation of anti-52-kd SS-A/Ro autoantibodies and to elucidate why, as has recently been reported, anti-52-kd autoantibodies preferentially recognize the denatured form rather than the native 52-kd molecule . METHODS . Using a series of truncated 52-kd autoantigens, produced as beta-galactosidase fusion proteins in Escherichia coli, the B cell epitope distribution was probed with 18 anti-Ro-positive sera by immunoblotting and by enzyme-linked immunosorbent assay . RESULTS . Nearly all the antigenicity of the molecule was found to be linked to its leucine zipper region . In a further study using 9 of the 18 sera, the antigenicity of the molecule was found to be mainly formed by multiple conformational epitopes, and one of these epitopes appeared to be universally recognized by all the sera tested . CONCLUSION . The recognition of multiple epitopes indicates that the Ro 52-kd antigen itself drives the autoimmunity to this molecule . Further, the concentration of the antigenicity at the leucine zipper region may explain why anti-52-kd antibodies preferentially recognize the denatured protein rather than its native form. Am J Respir Cell Mol Biol, 1995 Jul, 13(1), 45 - 53 Regulation of nitric oxide release by macrophages after intratracheal lipopolysaccharide; Shellito JE et al.; We investigated the effect of intratracheal (i.t.) lipopolysaccharide (LPS) on alveolar macrophage release of nitric oxide . Mice received i.t . LPS at doses ranging from 1 to 100 micrograms/100 g body weight and were killed at serial intervals for bronchoalveolar lavage . Control mice received i.t . phosphate-buffered saline . We found that after i.t . LPS, there was an early (1 to 3 days) influx of neutrophils followed by a later (5 to 7 days) influx of macrophages into the lungs . Alveolar macrophages lavaged from mice given i.t . LPS did not spontaneously release nitric oxide (measured as nitrite), but the capacity of these cells to release nitric oxide in vitro in response to interferon-gamma (IFN-gamma) or LPS was markedly upregulated . Alveolar macrophages lavaged from mice given i.t . LPS but not i.t . phosphate-buffered saline also expressed mRNA for inducible nitric oxide synthase as measured by semiquantitative reverse-transcription polymerase chain reaction . To investigate possible mechanisms for cellular priming for increased nitric oxide release after i.t . LPS, mice were depleted of CD4+ lymphocytes with an anti-CD4 antibody . Alveolar macrophages from CD4-depleted mice given i.t . LPS released significantly less nitric oxide in vitro in comparison to macrophages from nondepleted mice . Additional mice were treated with neutralizing doses of anti-tumor necrosis factor or anti-IFN-gamma antibody before i.t . LPS . Pretreatment with each cytokine antibody decreased but did not eliminate macrophage priming for nitric oxide release after i.t . LPS . We conclude that intratracheal LPS induces mRNA for nitric oxide synthase in alveolar macrophages, priming the cells for increased release of nitric oxide in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) J Infect Dis, 1995 Jul, 172(1), 136 - 43 Increased adherence of Escherichia coli RDEC-1 to human tissue culture cells results in the activation of host signaling pathways; Philpott DJ et al.; Attaching and effacing (AE) adhesion is associated with the pathogenesis of enteropathogenic Escherichia coli (EPEC) and rabbit diarrheogenic E . coli (RDEC-1) . Although RDEC-1 provides an animal model for investigating pathophysiology of EPEC infection, RDEC-1 does not adhere to human cell lines, thereby limiting in vitro investigation . Therefore, transformed RDEC-1 strains expressing adhesins derived from human diarrheogenic E . coli were studied . These adhesins promoted AE adhesion of RDEC-1 and led to the accumulation of alpha-actinin aggregates in the cytoplasm of infected cells . Furthermore, these strains induced host signal transduction pathways, resulting in tyrosine phosphorylation of host proteins and an intracellular elevation of calcium . These results demonstrate that RDEC-1 and EPEC stimulate similar signal transduction pathways in infected epithelial cells, thus lending additional support for the use of RDEC-1 as a model for the study of human EPEC infection. Mol Cell Biol, 1995 Jul, 15(7), 3882 - 91 RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element; Luan DD et al.; R2 is a non-long terminal repeat-retrotransposable element that inserts specifically in the 28S rRNA gene of most insects . The single protein encoded by R2 has been shown to contain both site-specific endonuclease and reverse transcriptase activities . Integration of the element involves cleavage of one strand of the 28S target DNA and the utilization of the exposed 3' hydroxyl group to prime the reverse transcription of the R2 RNA transcript . We have characterized the RNA requirement of this target DNA-primed reverse transcription reaction and found that the 250 nucleotides corresponding to the 3' untranslated region of the R2 transcript were necessary and sufficient for the reaction . To investigate the sequence requirements at the site of reverse transcription initiation, a series of RNA templates that contained substitutions and deletions at the extreme 3' end of the RNA were tested . The R2 templates used most efficiently had 3' ends which corresponded to the precise boundary of the R2 element with the 28S gene found in vivo . Transcripts containing short polyadenylated tails (8 nucleotides) were not utilized efficiently . R2 RNAs that were truncated at their 3' ends by 3 to 6 nucleotides were used less efficiently as templates and then only after the R2 reverse transcriptase had added extra, apparently nontemplated, nucleotides to the target DNA . The ability of the reverse transcriptase to add additional nucleotides to the target DNA before engaging the RNA template might be a mechanism for the generation of poly(A) or simple repeat sequences found at the 3' end of most non-long terminal repeat-retrotransposable elements. J Virol, 1995 Jul, 69(7), 4440 - 52 Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase; Blain SW et al.; To determine the various roles of RNase H in reverse transcription, we generated a panel of mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase based on sequence alignments and the crystal structures of Escherichia coli and human immunodeficiency virus type 1 RNases H (S . W . Blain and S . P . Goff, J . Biol . Chem . 268:23585-23592, 1993) . These mutations were introduced into a full-length provirus, and the resulting genomes were tested for infectivity by transient transfection assays or after generation of stable producer lines . Several of the mutant viruses replicated normally, some showed significant delays in infectivity, and others were noninfectious . Virions were collected, and the products of the endogenous reverse transcription reaction were examined to determine which steps might be affected by these mutations . Some mutants left their minus-strand strong-stop DNA in RNA-DNA hybrid form, in a manner similar to that of RNase H null mutants . Some mutants showed increased polymerase pausing . Others were impaired in first-strand translocation, independently of their wild-type ability to degrade genomic RNA, suggesting a new role for RNase H in strand transfer . DNA products synthesized in vivo by the wild-type and mutant viruses were also examined . Whereas wild-type virus did not accumulate detectable levels of minus-strand strong-stop DNA, several mutants were blocked in translocation and did accumulate this intermediate . These results suggest that in vivo wild-type virus normally translocates minus-strand strong-stop DNA efficiently. Mol Microbiol, 1995 Jul, 17(2), 303 - 12 Leucine-responsive regulatory protein plays dual roles as both an activator and a repressor of the Escherichia coli pap fimbrial operon; van der Woude MW et al.; The expression of the pap pilus operon of Escherichia coli is under a phase-variation control mechanism in which cells undergo a reversible transition between transcriptionally active (phase ON) and inactive (phase OFF) states . In this study, we explore the roles of leucine-responsive regulatory protein (Lrp) and the histone-like protein H-NS in the regulation of pap phase variation . Our data indicate that the phase OFF state results from repression of the intrinsically active papBA promoter by Lrp and H-NS, each of which can act independently as transcriptional repressors . Lrp requires pap DNA sequences upstream of the papBA promoter for its repressor activity whereas H-NS does not . In contrast, in the ON state, Lrp, in conjunction with PapI, activates pap transcription . This activation is not merely a result of alleviating the H-NS mediated repression, but induces a level of transcription that is eightfold higher than the basal level of transcription from the papBA promoter measured in the absence of both H-NS and Lrp . Analysis of Lrp activation mutants indicates that binding of Lrp to pap DNA sequences is not sufficient for transcription activation, consistent with a model in which an additional domain of Lrp interacts with the transcriptional apparatus . Together, our results show that Lrp functions as a transcriptional activator in phase-ON cells and as a repressor of basal transcription in phase-OFF cells . Because pap phase variation occurs in the absence of H-NS, it is not clear what role this regulatory protein plays in pap gene regulation. Mol Microbiol, 1995 Jul, 17(2), 291 - 301 A transcription terminator signal necessary for plasmid ColIb-P9 replication; Mori A et al.; Replication of the IncI alpha plasmid ColIb-P9 requires the repZ gene, which encodes an essential, unstable initiator protein termed RepZ . Although many functional features of the ColIb-P9 replicon resemble those of structurally unrelated IncFII plasmids R1 and NR1, the role of transcription of repZ towards the replication origin is poorly understood . Using a series of deletion and substitution mutants of the ColIb-P9 replicon, we found that RepZ prefers to act in cis and that a spacer sequence between repZ and the origin is required for replication . This spacer element, referred to as CIS, retained strong transcription terminator activity . Efficient transcription terminators, whether Rho-dependent or -independent, were capable of replacing CIS function for in vivo replication; ColIb-P9 replicated better as transcription terminated more efficiently within CIS . When the CIS element was substituted for by a strong Rho-dependent terminator, such as lambda tR1 or E . coli trp t', in vivo replication of these recombinant replicons became dependent on the Rho factor, in contrast to the authentic ColIb-P9 replicon. Mol Microbiol, 1995 Jul, 17(2), 251 - 8 Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP; Tagami H et al.; CRP-cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene (crp*) . It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery . Here we address the question of how glucose inhibits the expression of beta-galactosidase in the absence of cAMP . We have isolated several mutations in the crp gene that confer a CRP* phenotype . The expression of beta-galactosidase is reduced by glucose in cells carrying these mutations . Using Western blotting and/or SDS-PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp* mRNA levels . The level of CRP* protein correlates with beta-galactosidase activity . When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp* expression is virtually abolished . These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya- crp* cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose. Mol Microbiol, 1995 Jul, 17(2), 231 - 40 Characterization of a new rho mutation that relieves polarity of Mu insertions; Peters JE et al.; We report the identification and characterization of a new rho mutation, rho614, that relieves polarity of Mu insertions in Escherichia coli . The mutation was identified by its ability to suppress the polarity of the Mu-mediated phi(lamB'-'lacZ)hyb61-4 fusion that is located at codon four of the lamB signal sequence . The rho614 mutation alters residue 80 in the proposed RNA-binding domain of Rho and is recessive to wild-type rho . We suggest that in the presence of the rho614 allele transcripts initiated at the Mu promoter PcM fail to terminate at presumptive Rho-dependent termination sites, namely rut1 and rut2, and continue into the 3' 'lamB gene allowing a LamB+ phenotype . This contention is supported by deletion analysis of the region and the observation that insertional inactivation of genes that reduce transcription from PcM, clpP (ATP protease), himA (IHF-alpha), and himD (IHF-beta), block the LamB+ phenotype . rho614, rho4 and rho201 alleles suppress the polarity of a malK::Mu insertion on the downstream lamB gene . However, the polarity of the phi(lamB'-'lacZ)hyb61-4 insertion is only suppressed by the rho614 mutation . We propose that the rho614 mutation allows suppression of transcriptional polarity without interfering with translation initiation signals of the truncated 'lamB gene . In addition to identifying a new rho mutation and Rho-dependent terminator sequence, this system provides a means of studying Rho protein/terminator relationships through the identification of new classes of rho mutations.
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