Escherichia coli

Biochemistry, 1993 Aug 10, 32(31), 7893 - 903
Preparation and characterization of a bifunctionally spin-labeled mutant of murine epidermal growth factor for saturation-transfer electron paramagnetic resonance studies of the growth factor/receptor complex; Rousseau DL Jr et al.; In this report we describe the production of a {Lys3,Tyr22} murine epidermal growth factor (mEGF) mutant for spin-labeling with bis(sulfo-N-succinimidyl)-{15N,2H16}doxyl-2-spiro-4'-pimelate ({15N,2H16}BSSDP) in order to study the rotational dynamics of the EGF/EGF receptor complex by saturation-transfer electron paramagnetic resonance (ST-EPR) . Previous results {Faulkner-O'Brien et al . (1991) Biochemistry 30,8976-8985} indicated that the reaction of {15N,2H16}BSSDP with wild-type mEGF did not yield a product useful for ST-EPR studies of the EGF/EGF receptor complex because the major product, in which {15N,2H16}BSSDP was attached only at the amino terminus of mEGF, lacked rigid motional coupling of the spin probe to the protein, and the more tightly coupled bidentate product was unstable . Using oligonucleotide-mediated site-directed mutagenesis of a synthetic gene for mEGF, we replaced Tyr3 with Lys and His22 with Tyr in wild-type mEGF to produce a mutant mEGF suitable for {15N,2H16}BSSDP labeling . The {Lys3,Tyr22} mEGF was expressed in Escherichia coli HB101 transformed with a pIN-III-ompA3-{Lys3,Tyr22} mEGF plasmid and was purified from the bacterial periplasm using a simple two step purification method . The {15N,2H16}BSSDP reacted with {Lys3,Tyr22}mEGF in high yield, and EPR analysis of the major product revealed tight motional coupling between the spin probe and the protein . Biological activity, as assessed by stimulation of EGF receptor autophosphorylation and dimerization, was not affected by either the mutations or the addition of the spin label . The {15N,2H16}BSSDP-modified {Lys3,Tyr22} mEGF was shown to be equipotent with mEGF in EGF receptor competition binding assays using A431 cells; in EPR studies, mEGF also was shown to specifically block {15N,2H16}BSSDP-modified {Lys3,Tyr22}mEGF binding to the EGF receptor in A431 membrane vesicles . Using the {15N,2H16}BSSDP-modified {Lys3,Tyr22}mEGF, we now report the first measurement of the rotational dynamics of the EGF/EGF receptor complex in A431 membrane vesicles by ST-EPR.

Biochemistry, 1993 Aug 10, 32(31), 7939 - 45
In vitro protein engineering using synthetic tRNA(Ala) with different anticodons; Ma C et al.; The use of synthetic tRNA for in vitro protein engineering was tested in a coupled transcription/translation system prepared from Escherichia coli . DNA sequences similar to the natural tRNA(Ala/UGC) gene from E . coli but with different anticodons were synthesized in vitro, cloned into a DNA plasmid, and then transcribed in vitro with T7 RNA polymerase . The UGC alanine anticodon was changed to CUA corresponding to the UAG stop codon, CCU corresponding to the rarely used AGG arginine codon, and two four-nucleotide anticodons used to suppress stop codons . Bacterial dihydrofolate reductase was the test protein . Its cloned coding sequence was mutagenized at the GUG codon for valine-75 to correspond to the anticodons of the tRNA constructs, and then the plasmids were used to direct the synthesis of dihydrofolate reductase in the coupled transcription/translation system containing the corresponding synthetic tRNA . The results indicate that all four synthetic tRNAs were functionally active in the synthesis of full-length, enzymatically active dihydrofolate reductase protein.

Biochemistry, 1993 Aug 10, 32(31), 7866 - 71
Mutational effects on the cooperativity of Ca2+ binding in calmodulin; Waltersson Y et al.; The importance of the aspartate ligand in the +Y Ca2+ coordinating position of two EF-hands of calmodulin has been investigated . Synthetic calmodulin genes were used to produce engineered proteins with the wild-type bovine sequence as well as with aspartate 58 in Ca(2+)-binding site II and/or aspartate 95 in site III changed to asparagine . The macroscopic Ca(2+)-binding constants of the intact calmodulins and of tryptic fragments comprising the N- and C-terminal domains were determined from titrations with Ca2+ in the presence of 5,5'-Br2BAPTA . Substitution of aspartate by asparagine in Ca(2+)-binding site II led to a slight increase in the total free energy change on Ca2+ binding, and the cooperativity of Ca2+ binding to the N-terminal sites was substantially increased . The change from aspartate to asparagine in site III decreased the Ca2+ affinity and also appeared to decrease the positive cooperativity between the sites in the C-terminal domain . Thus, identical mutations in sites II and III were found to result in opposite effects . The data imply that involvement of liganding side chains in interactions other than direct calcium attraction and calcium coordination is of considerable importance for the Ca(2+)-binding process, particularly for the cooperativity.

Biochemistry, 1993 Aug 10, 32(31), 7999 - 8003
A model for oxidative modification of glutamine synthetase, based on crystal structures of mutant H269N and the oxidized enzyme; Liaw SH et al.; Proteolytic degradation of glutamine synthetase (GS) in Escherichia coli is known to follow "marking" by oxidative modification . At an early stage of the degradative pathway, oxidation of His 269 and Arg 344 abolishes GS enzymatic activity . We propose a mechanism for the early stage of oxidative inactivation of GS on the basis of the crystal structure of H269N and tryptophan fluorescence spectra of H269N and H269NR344Q: (1) Oxidation of Arg 344, adjacent to the n2 metal ion site, decreases ATP binding . (2) Oxidation of His 269 to Asn destroys the n2 site, consistent with the function of His 269 as a ligand for the n2 metal . (3) Loss of Mn2+ at the n2 site destroys the integrity of the ATP binding site . (4) Destruction of the ATP site results in the observed low enzymatic activity of H269N and H269NR344Q . During later stages of oxidative modification, the n1 metal ion site is destroyed and the active site of the enzyme becomes flexible as suggested by X-ray data collected from an oxidized crystal of GS . Thus, studies of mutant and oxidized enzymes confirm that there are at least two stages of oxidative modification of GS . These studies suggest that the early modification occurs at the n2 metal ion site, eliminating enzyme activity, and the later modification occurs at the n1 metal ion site, relaxing the GS structure, perhaps enabling proteolytic degradation . These studies also illuminate the differing roles of the two bound metal ions: the tightly bound n1 ion enhances the stability of the catalytically active conformation, and the less tightly bound n2 ion participates in ATP binding.

FEBS Lett, 1993 Aug 9, 328(1-2), 35 - 40
Expression of heterologous phosphofructokinase genes in yeast; Heinisch JJ; Genes encoding phosphofructokinases (PFK) from Escherichia coli and from the human muscle were expressed in PFK-deficient strains of Saccharomyces cerevisiae under the control of an inducible GAL1 promoter . They restored PFK activity under inducing conditions and complemented the galactose-negative growth phenotype of the recipient strains . The PFK enzymes expressed appear to be stable in yeast . The human muscle enzyme crossreacts with specific antibodies and shows the expected subunit size . As expected, its activity can be activated by fructose-2,6- bisphosphate, in contrast to the bacterial enzyme.

FEBS Lett, 1993 Aug 9, 328(1-2), 130 - 8
Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of beta-carotene; Cunningham FX Jr et al.; Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms . These yellow pigments serve the vital role of protecting against potentially lethal photo-oxidative damage . Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified . We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule beta-carotene . The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria . Chemically-induced mutants of the cyanobacterium Synechococcus sp . PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells . A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli . The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase . The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA . The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.

FEBS Lett, 1993 Aug 9, 328(1-2), 111 - 4
Determination of a functional lysine residue of a plant cysteine synthase by site-directed mutagenesis, and the molecular evolutionary implications; Saito K et al.; Comparison of seven deduced amino acid sequences of cysteine synthase (O-acetyl-L-serine (thiol)-lyase, EC 4.2.99.8) from plants and bacteria disclosed the presence of 12 conserved Lys residues, which can be candidates for a functional binding site for pyridoxal phosphate cofactor . These 12 conserved Lys residues in a cDNA clone encoding spinach cysteine synthase A were replaced with Gly by oligonucleotide-directed in vitro mutagenesis . These Lys-->Gly mutated cDNAs were transferred into Escherichia coli NK3, a cysteine auxotroph lacking both cysteine synthase loci, cysK and cysM . One mutant replaced at Lys-49 could not complement the cysteine requirement of NK3, whereas other mutants and wild-type clone could . No enzymatic activity of cysteine synthase A was detected either in the cell-free extracts of E . coli NK3 transformed with the Lys-49 mutant . These results indicated that Lys-49 is a functional residue for the catalytic activity of cysteine synthase . This Lys residue is conserved in other evolutionarily related amino acid-metabolizing enzymes catalyzing reactions involving the beta-carbon of amino acids.

Biochim Biophys Acta, 1993 Aug 7, 1164(3), 299 - 304
Identity and some properties of the L-threonine aldolase activity manifested by pure 2-amino-3-ketobutyrate ligase of Escherichia coli; Marcus JP et al.; 2-Amino-3-ketobutyrate ligase catalyzes the reversible, pyridoxal 5'-phosphate-dependent condensation of glycine with acetyl CoA forming the unstable intermediate, 2-amino-3-ketobutyrate . Several independent lines of evidence indicate that the pure protein obtained in the purification of this ligase from Escherichia coli also has L-threonine aldolase activity . The evidence includes: (a), a constant ratio of specific activities (aldolase/ligase) at all stages of purifying 2-amino-3-ketobutyrate ligase to homogeneity; (b), the same rate of loss of aldolase and ligase activities during controlled heat inactivation of the pure protein at 60 degrees C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymatic activities that are not significantly different during slow inactivation by iodoacetamide, with and without L-threonine added; (d), coincident rates of loss and essentially identical rates of recovery of aldolase activity and ligase activity during resolution of the holoenzyme with hydroxylamine followed by reconstitution with pyridoxal 5'-phosphate . No aldolase activity is observed with D-threonine as substrate and L-allothreonine is about 25% as effective as L-threonine . Whereas ligase activity has a sharp pH optimum at 7.5, the aldolase activity of this pure protein is maximal at pH 9.0 . Comparative apparent Km values for glycine (ligase) and L-threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective Vmax values were found to be 2.5 mumol of CoA released/min per mg vs . 0.014 mumol of acetaldehyde formed (NADH oxidized)/min per mg.

J Med Chem, 1993 Aug 6, 36(16), 2279 - 91
Benzoquinazoline inhibitors of thymidylate synthase: enzyme inhibitory activity and cytotoxicity of some 3-amino- and 3-methylbenzo{f}quinazolin-1(2H)-ones; Pendergast W et al.; The synthesis and thymidylate synthase (TS) inhibitory activity of a series of simple benzo{f}-quinazolin-1(2H)-ones are described . Fully aromatic 3-amino compounds with compact lipophilic substituents in the 9-position were found to have I50 values as low as 20 nM on the isolated enzyme, and represent the first examples of potent, folate-based TS inhibitors that completely lack any structural feature corresponding to the (p-aminobenzoyl)glutamate moiety of the cofactor . A number of the compounds also showed moderate growth inhibitory activity against a human colon adenocarcinoma cell line (SW480), with IC50 values as low as 2 microM.

Science, 1993 Aug 6, 261(5122), 762 - 5
Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure; Nolan JM et al.; Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity . A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5' ends of circularly permuted tRNAs (cptRNAs) . The polymerase chain reaction was used for the production of cptRNA templates . After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA . Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension . These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site . The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions.

Nature, 1993 Aug 5, 364(6437), 548 - 9
CAP interacts with RNA polymerase in solution in the absence of promoter DNA; Heyduk T et al.; Protein-protein interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation . Interactions between catabolite gene activator protein (CAP) and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA . Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending) . All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP . Here we use the thermodynamically rigorous technique of fluorescence polarization to show that CAP interacts with RNA polymerase in solution in the absence of promoter DNA (KD,app = 2.8 x 10(-7) M), whereas {Ala158}CAP, a mutant of CAP specifically defective in transcription activation, does not.

J Biol Chem, 1993 Aug 5, 268(22), 16639 - 47
Effects of substitutions of closely related amino acids at the contact surface in an antigen-antibody complex on thermodynamic parameters; Ito W et al.; We constructed a library of 512 kinds of Fv fragment, derivatives of a monoclonal antibody, D1.3, specific for hen egg-white lysozyme, in which a total of nine of the original amino acids were replaced by closely related amino acids at positions in the complementarity-determining regions of the H chain . More than 80% of the clones in the library produced Fv fragments in Escherichia coli . Two wild-type and 13 mutant Fv fragments were prepared in large quantities and subjected to analysis by differential titration calorimetry . The association constants of the 15 Fv fragments with hen egg-white lysozyme were distributed between 0.12 x 10(7) and 1.59 x 10(8) M-1 . The changes in delta H0 and -T delta S0 caused by one-point mutation at each position did not have intrinsic values for each change . The same changes at one position had different effects on KA, delta H0, and -T delta S0 when differences had been introduced in other regions . The delta(delta G0) caused by a single-point mutation ranged from -0.56 to 1.56 kcal/mol . By contrast, the delta(delta H0) and delta(-T delta S0) caused by a single-point mutation ranged from -3.5 to 3.4 and from -3.8 to 3.4 kcal/mol, respectively . When antibodies gain the binding energy contributed by the effects of enthalpy, they lose the binding energy contributed by the effects of entropy and vice versa . In general, changes in entropy compensate for changes in enthalpy.

J Biol Chem, 1993 Aug 5, 268(22), 16519 - 27
HIV nucleocapsid protein . Expression in Escherichia coli, purification, and characterization; You JC et al.; The single-stranded nucleocapsid protein that coats the RNA genome of human immunodeficiency virus within the virion core has been produced in Escherichia coli and purified to homogeneity . The mature 55-amino acid protein, normally generated from the gag polyprotein precursor by HIV protease-catalyzed processing of both its amino and carboxyl termini, was produced in E . coli with authentic termini directly, without the need for processing . The protein was purified 30-fold to apparent homogeneity, as determined by both amino acid analysis and SDS-polyacrylamide gel electrophoresis . Sequencing of each terminus of the purified protein indicated that no proteolytic degradation occurred . A molar extinction coefficient (epsilon 280 = 8350 cm-1 M-1) was determined . The purified nucleocapsid protein binds tightly to single-stranded RNA as judged by a nitrocellulose filter binding assay . A binding constant (Kw) of 1 x 10(8) M-1 was calculated . Using fluorescence quenching of nucleocapsid protein upon RNA binding as an assay, a binding site size of seven nucleotides was determined . These results contrast to a larger 15-nucleotide site measured by others for a larger form of nucleocapsid protein-containing sequences from its immature precursor . The possible relevance of these findings are discussed.

J Biol Chem, 1993 Aug 5, 268(22), 16302 - 8
Energy transduction between membranes . TonB, a cytoplasmic membrane protein, can be chemically cross-linked in vivo to the outer membrane receptor FepA; Skare JT et al.; TonB, a cytoplasmic membrane protein, couples cytoplasmic membrane protonmotive force to active transport across the outer membrane of Escherichia coli . In vivo cross-linking studies were initiated to analyze TonB interactions with other cell envelope proteins . Four TonB-specific cross-linked complexes were detected with apparent molecular masses of 195, 77, 59, and 43.5 kDa . The 195-kDa complex was shown to contain both TonB and FepA, the outer membrane receptor for the siderophore enterochelin . The 195-kDa complex is absent in strains missing either TonB or FepA and can be detected by either TonB-specific or FepA-specific monoclonal antibodies . This is the first direct in vivo evidence that TonB can span the periplasmic space to interact physically with outer membrane receptors . Consistent with that observation, the outer membrane protease OmpT was shown to play a role in TonB turnover, both in the presence and absence of ExbB results in the rapid degradation of TonB . The absence of OmpT could be used to stabilize TonB in an exbB::Tn10 strain such that steady state levels of TonB protein are identical to a wild-type strain . Under those conditions, the absence of ExbB results in greatly reduced TonB activity, indicating that ExbB plays a direct role in energy transduction and probably secondarily protects TonB protein from proteolysis . The 59-kDa complex was absent in an exbB::Tn10 strain, suggesting either that ExbB is in the complex with TonB or that ExbB is required to form the 59-kDa complex . A tolQ nonsense mutation had no effect on the cross-linking profile observed, confirming that its participation in TonB-dependent phenomena is minor and most likely the result of evolutionary cross-talk.

J Biol Chem, 1993 Aug 5, 268(22), 16259 - 64
Identification of the metal ligands and characterization of a putative zinc finger in methionyl-tRNA synthetase; Xu B et al.; A truncated form of the methionyl-tRNA synthetase (delta MTS), which has been cloned, overproduced, and characterized, was used in an attempt to better understand the role of the enzyme-bound zinc in the amino-acylation process . Apo-, Zn(2+)-, Co(2+)-, and 113Cd(2+)-substituted delta MTS proteins were prepared in vivo and purified to homogeneity . Apo-delta MTS was devoid of enzymatic activity in the aminoacylation of tRNA(fMet) and in the methionine-dependent ATP-pyrophosphate exchange reactions . Kinetic constants in both the aminoacylation and ATP-pyrophosphate exchange reactions for the Co(2+)- and 113Cd(2+)-substituted delta MTS proteins were found to be identical with those of the native Zn2+ protein . The low energy absorption spectrum of Co(2+)-substituted delta MTS resembles the d-d transition bands characteristic of tetrahedrally coordinated Co(2+)-substituted proteins . A strong S-->Co2+ charge transfer absorption at 350 nm was clearly evident having a molar absorptivity consistent with four thiolate ligands . The environment of the metal center was further probed by measuring the 113Cd chemical shift of 113Cd(2+)-substituted delta MTS . A single resonance at 759.6 ppm was observed . This chemical shift is consistent with Cd2+ coordinated to four thiolate ligands . The Escherichia coli methionyl-tRNA synthetase contains a potential metal binding sequence Cys-X2-Cys-X9-Cys-X2-Cys in a connecting polypeptide within the nucleotide fold . Titration of a 21-amino acid peptide corresponding to this putative metal binding site, Cys145-Cys161, was shown to bind Co2+ with a Kd of 120 +/- 11 microM . These results demonstrate that the isolated zinc finger binding domain is capable of specifically forming a stoichiometric complex with the divalent cation . Taken together, our studies identify the 4 cysteine residues in the zinc finger-like domain as the metal binding ligands in the E . coli methionyl-tRNA synthetase . The role of the enzyme-bound metal appears to be structural and not directly involved in catalysis.

J Biol Chem, 1993 Aug 5, 268(22), 16241 - 7
Leucine/isoleucine/valine-binding protein contracts upon binding of ligand; Olah GA et al.; Small-angle x-ray scattering and computer modeling have been used to study the effects of ligand binding to the leucine/isoleucine/valine-binding protein, an initial component of the high-affinity active transport system for branched-chain aliphatic amino acids in Escherichia coli . Measurements were made with no ligand present and with either L-leucine or L-valine present . Upon binding of either leucine or valine, there is a decrease in the radius of gyration, from 23.2 +/- 0.2 to 22.2 +/- 0.2 A, and in the maximum particle dimension, from 82 +/- 3 to 73 +/- 3 A . The x-ray structure of the unbound form has been determined and gives a radius of gyration and a maximum dimension consistent with the values found for the solution structure in this study (Sack, J . S., Saper, M . A., and Quiocho, F . A . (1989) J . Mol . Biol . 206, 171-191) . The reduction in the radius of gyration and maximum dimension upon ligand binding can be accounted for by a substrate-induced cleft closure in a combined "hinge-twist" motion . Modeling of the substrate-bound state was done by comparison of this protein with another periplasmic binding protein (L-arabinose-binding protein), which possesses a similar two-lobe structure and for which the x-ray structure is known in its ligand-bound form.

J Biol Chem, 1993 Aug 5, 268(22), 16216 - 22
A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus; Friedrich T et al.; A thrombin-specific inhibitor with an apparent molecular mass of 11 kDa has been purified from the insect Rhodnius prolixus . Amino-terminal protein sequence analysis allowed the molecular cloning of the corresponding cDNA . The open reading frame codes for a protein of about 103 amino acid residues and displays an internal sequence homology of residues 6-48 with residues 57-101 indicating a two-domain structure . Based on the amino acid sequence the two domains exhibit high homology to protease inhibitors belonging to the Kazal-type family . Model building suggests that the first domain binds to the active site with residue His10 pointing into the specificity pocket . From gel filtration and tight-binding inhibition experiments the inhibitor appears to form 1:1 complexes with thrombin . Periplasma-directed heterologous expression of the rhodniin cDNA in Escherichia coli yields the intact thrombin inhibitor . Natural and recombinant rhodniin both display inhibition constants of about 2 x 10(-13) M.

J Biol Chem, 1993 Aug 5, 268(22), 16105 - 8
The DNA-binding subunit of human transcription factor IID can interact with the TATA box as a multimer; Jupp R et al.; Transcription initiation from eukaryotic protein-coding genes is a complex process that minimally requires RNA polymerase (pol) II (B) and at least seven general transcription factors . The 38-kDa subunit (TBP) of the human general transcription factor TFIID recognizes the TATA sequence element and initiates the assembly of the other general transcription factors and RNA pol II . It is believed, based on experiments with yeast recombinant protein, that TBP binds as a monomer to DNA . Using purified recombinant human TBP protein we find that TBP interacts with the TATA element as both a monomer and a dimer . The multimeric binding of TBP to DNA revealed by this study has important implications for the role of TBP in transcription initiation and suggests novel mechanisms whereby other transcription factors may interact with a RNA pol II preinitiation complex.

J Biol Chem, 1993 Aug 5, 268(22), 16528 - 36
Human immunodeficiency virus reverse transcriptase . Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase; Thimmig RL et al.; Human immunodeficiency virus (HIV) reverse transcriptase isolated from viral particles contains two subunits, p51 and p66 . We have produced both subunits in separate Escherichia coli strains using expression vectors . Stop codons were placed immediately after the codon for the carboxyl-terminal residue of the mature processed p51 and p66 subunits found in viral particles . Insertion of a methionine in front of the HIV protease cleavage site in the recombinant protein enabled synthesis of both subunits with the natural amino-terminal proline, since E . coli methionine aminopeptidase cleaves a Met-Pro amino-terminal linkage . That this occurred to an extent greater than 95% was confirmed by sequencing the purified subunits . Examination of the activities of the individual p51 and p66 subunits on a variety of templates and under solution conditions optimized for each subunit revealed a significant catalytic activity for the natural p51 subunit . This result contrasts to results reported earlier for many recombinant forms without the natural amino and/or carboxyl termini . As expected from earlier work, the optimal homopolymeric template for the p66 subunit was poly(rA) . For the p51 subunit, poly(dC) was found to be the optimal template; its activity is 2- to 4-fold greater than p66 on poly(dC) . The p51 subunit is 13- to 50-fold less active on poly(rC) . These findings are discussed in the context of our earlier hypothesis (McHenry, C . S . (1989) in Molecular Biology of Chromosome Function (Adolph, K., ed) Chap . 5, Springer-Verlag, New York) that the HIV reverse transcriptase might be functionally asymmetric with distinct plus- and minus-strand polymerases.

Biochemistry, 1993 Aug 3, 32(30), 7765 - 71
Kinetics of ATP hydrolysis during the DNA helicase II-promoted unwinding of duplex DNA; Yodh JG et al.; The ATP hydrolysis activity of DNA helicase II from Escherichia coli was examined in the presence of linear single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) . In the presence of ssDNA, the ATP hydrolysis reaction followed a linear time course until the ATP was depleted . In the presence of dsDNA, in contrast, there was a kinetic lag before a linear phase of ATP hydrolysis was achieved . The nonlinear kinetics of the dsDNA-dependent ATP hydrolysis reaction could be modeled by a kinetic scheme in which helicase II undergoes a time-dependent transition from an ATPase-inactive to an ATPase-active form . Order of addition experiments indicated that this transition was not due to a rate-limiting association event between helicase II and any other component of the reaction . Instead, agarose gel assays showed that progressive unwinding of the dsDNA occurs during the same time period as the lag phase of the ATP hydrolysis reaction . No significant ATP hydrolysis was observed when the linear dsDNA was replaced with closed circular dsDNA, suggesting that the ATP hydrolysis reaction requires a dsDNA substrate that can be unwound to the complementary single strands . These results are consistent with a model in which the lag phase of the dsDNA-dependent ATP hydrolysis reaction corresponds to progressive unwinding of the dsDNA, with the ATP hydrolysis reaction arising from helicase II molecules that are bound to the separated single strands.

Biochemistry, 1993 Aug 3, 32(30), 7692 - 7
Genetic fusion of subunits I, II, and III of the cytochrome bo ubiquinol oxidase from Escherichia coli results in a fully assembled and active enzyme; Ma J et al.; The cytochrome bo ubiquinol oxidase from Escherichia coli is a five-subunit enzyme which is a member of the superfamily of heme-copper respiratory oxidases . Three of the subunits (I, II, and III) are homologous to the three mitochondrial encoded subunits of the eukaryotic aa3-type cytochrome c oxidase . Subunits, I, II, and III of the eukaryotic oxidase contain 12, 2, and 7 putative transmembrane spans, respectively . The hydropathy profiles of the subunits of most other members of this oxidase superfamily are consistent with these structures . However, subunit I from the E . coli oxidase contains 15 transmembrane spans, with one additional span at the N-terminus and two additional spans at the C-terminus in comparison to the eukaryotic oxidase . The additional transmembrane helix at the N-terminus predicts that the amino terminal residue should be on the periplasmic side of the membrane . By deleting the intergenic region between the cyoA and cyoB genes, an in-frame fusion between subunit II (cyoA) and subunit I (cyoB) was generated . This links the C-terminus of subunit II, known to be on the periplasmic side of the membrane, to the N-terminus of subunit I . The resulting oxidase is fully active, and supports the toplogical folding pattern previously suggested for subunit I with the N-terminus in the periplasm . Whereas subunit I of the E . coli oxidase has two additional membrane-spanning helices at the C-terminus, subunit III has two fewer helices than does the corresponding subunit III of the eukaryotic oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Aug 3, 32(30), 7753 - 8
Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding; LeTilly V et al.; The study of interactions between proteins and nucleic acids is central to the understanding of the control of genetic expression . Fluorescence anisotropy has been used to measure, in solution, the equilibrium binding profiles of a bacterial repressor protein, the tryptophan repressor (TR), to a fluorescently labeled oligonucleotide containing one of its target operator sequences . Investigation of the effects of changing concentrations of corepressor, operator DNA, and protein implicate TR oligomers in the regulation of DNA binding . These studies also demonstrate that the relatively straightforward technique of fluorescence anisotropy can be applied to the study of the interactions between proteins and nucleic acids . The fluorescence technique exhibits sufficient sensitivity to replace radioactive methods of detection in most cases . In addition, since it is a solution-based methodology, it offers a true equilibrium measure of the protein-nucleic acid equilibria, and the effects of changes in solution conditions such as salt and ligand concentration, pH, and temperature can be readily evaluated . Data acquisition is relatively simple and rapid, and the data are of sufficient quality for detailed thermodynamic analyses of complex systems . Given these attributes, fluorescence anisotropy will find multiple applications in the area of genetic regulation.

Biochemistry, 1993 Aug 3, 32(30), 7720 - 6
Orientation of functional and nonfunctional PTS permease signal sequences in lipid bilayers . A polarized attenuated total reflection infrared study; Tamm LK et al.; Synthetic peptides corresponding to the N-terminal 23 and 22 residues, respectively, of two integral plasma membrane proteins of Escherichia coli, namely the mannitol- and glucitol-specific permeases of the bacterial sugar phosphotransferase system, were incorporated into single planar phospholipid bilayers supported on germanium plates . Polarized attenuated total reflection infrared spectra were recorded, and order parameters were derived from the measured dichroic ratios . The order parameters of the two wild-type peptides which form amphiphilic alpha-helices in membranes were -0.4 to -0.5, indicating a preferential alignment of the alpha-helix long axis parallel to the membrane surface . Nonfunctional mutant peptides of the mannitol permease sequence in which serine-3 or aspartate-4 were substituted with prolines (S3P and D4P) or lysine (D4K), but which were still largely alpha-helical, exhibited peptide order parameters close to zero, indicating a high degree of disorder of these peptides in the lipid bilayers . The lipid was well ordered at low concentrations of peptides in the membranes but became disordered at high peptide concentrations . This effect of lipid disordering was more pronounced for the D4K mutant than for the wild-type mannitol peptide.

Biochemistry, 1993 Aug 3, 32(30), 7650 - 7
Effects of mutations in the hinge region of serpins; Hopkins PC et al.; An expression system for alpha 1-antitrypsin in Escherichia coli was developed using a T7 RNA polymerase promoter . Addition of rifampicin to inhibit the E . coli RNA polymerase after induction of the T7 RNA polymerase gene resulted in about 30% of newly synthesized protein being alpha 1-antitrypsin . This expression system was then used to examine the effect of mutations in the hinge region of alpha 1-antitrypsin on its activity . The mutations were based on ones in antithrombin III that had previously been shown to have adverse effects on activity . Mutation of Ala347 to threonine in alpha 1-antitrypsin did not affect the kinetic behavior of the protein with trypsin or human leukocyte elastase . In contrast, mutation of Gly349 to proline converted the majority of the protein into a substrate for both proteinases . The small fraction of this mutant that was active, however, had kinetic parameters that were indistinguishable from wild-type alpha 1-antitrypsin . Cleavage within the reactive-site loop of wild-type alpha 1-antitrypsin causes a conformational change in the molecules (the S-to-R transition) and results in a marked increase in heat stability . This increase in heat stability was also seen upon cleavage within the reactive-site loops of both of the alpha 1-antitrypsin mutants . The results are discussed in terms of a kinetic mechanism for serpin-proteinase interactions, in which after the formation of an initial complex the serpin partitions between the formation of a stable complex and a cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Aug 3, 32(30), 7623 - 9
Expression of CheA fragments which define domains encoding kinase, phosphotransfer, and CheY binding activities; Swanson RV et al.; The histidine protein kinase CheA is a central component of the Escherichia coli chemotaxis system . The autophosphorylation activity of CheA is controlled by membrane-bound chemoreceptors and by the CheW coupling protein . CheA phosphorylates the CheY and CheB proteins which respectively control the direction of flagellar rotation and the level of receptor adaptation, thereby regulating the cells' chemotactic response . Genes encoding three polypeptide fragments of CheA were constructed and expressed in order to better define the functional organization of the wild-type protein . These fragments allowed the identification of regions of the protein responsible for CheY binding, phosphotransfer, and kinase activity . The kinase domain was expressed as a 30-kDa polypeptide corresponding to the central portion of the wild-type protein which contains sequences homologous to other histidine kinases . It was able to phosphorylate a 15-kDa amino-terminal phosphotransfer domain which was separately expressed and purified . This latter domain is capable of phosphotransfer to CheY despite the fact that it lacks the ability to stably bind CheY . CheY was immobilized to a dextran matrix through a single cysteine residue which was introduced into the protein at a position far removed from the active site . A stable binding site for CheY was mapped to a segment between the site of autophosphorylation and the kinase domain by using surface plasmon resonance to detect binding to the immobilized CheY . The region of the kinase which tightly binds the unphosphorylated substrate may play an important role in regulating the specificity of the signal transducing system.

Biochemistry, 1993 Aug 3, 32(30), 7703 - 11
Partially structured self-associating states of acidic fibroblast growth factor; Mach H et al.; A combination of near- and far-UV circular dichroism, Fourier-transform infrared spectroscopy, tryptophan fluorescence, size-exclusion chromatography, and a fluorescent extrinsic hydrophobic probe has been employed to characterize partially structured states of human recombinant acidic fibroblast growth factor (aFGF) . At low pH, the addition of specific polyanionic ligands or moderate amounts of salts induces states with high secondary but low tertiary structure content . At neutral pH, intermediate amounts of chaotropic agents impose similar partially structured conformational states which also display noncooperative unfolding transitions . Kinetic evidence indicates that similar forms of the protein exist in the first few hundred milliseconds in the refolding pathway of aFGF . The kinetics of their formation appear to be temperature-independent, implying lack of an energy barrier, which is characteristic for further slow folding into the native state . Unlike the native and fully unfolded states, these partially structured conformations exhibit very low solubility, resulting in irreversible aggregation . Potential physiological implications of the existence of such "molten globule" states with regard to the growth factor's transport and biological activity are considered.

Biochemistry, 1993 Aug 3, 32(30), 7617 - 22
In vitro analysis of translational rate and accuracy with an unmodified tRNA; Harrington KM et al.; Escherichia coli tRNA(Phe) transcript lacking all the modified nucleosides was investigated in an in vitro translation system . To estimate the affinity of tRNA toward EF-Tu, Kd and K-1 were measured by the nuclease protection assay, and it was shown that the absence of modifications decreases ternary complex stability less than 2-fold . The activity of unmodified Phe-tRNA(Phe) on E . coli ribosomes was compared to modified Phe-tRNA(Phe) using the framework of the kinetic proofreading mechanism (Thompson & Dix, 1982) with both cognate and noncognate codons . Values of the individual rate constants in the elongation process showed that the modifications increased the accuracy of translation by (1) decreasing the rate of dipeptide synthesis and (2) increasing the rate of rejection with noncognate codons.

Eur J Pharmacol, 1993 Aug 2, 248(2), 157 - 62
Inhibition of 5-hydroxytryptamine- and enterotoxin-induced fluid secretion by 5-HT receptor antagonists in the rat jejunum; Beubler E et al.; The effects of cholera toxin and heat stable Escherichia coli (E . coli) enterotoxin on intestinal fluid secretion are commonly considered to be mediated by cyclic nucleotides . It was demonstrated recently, by using the 5-hydroxytryptamine (5-HT)2 receptor antagonist ketanserin and the 5-HT3 receptor antagonist tropisetron, that 5-HT acts as an important mediator in cholera toxin- and heat stable E . coli enterotoxin-induced fluid secretion . In the present investigation ketanserin and tropisetron were compared with the newer 5-HT3 receptor antagonists ondansetron and granisetron versus 5-HT-, cholera toxin- and heat stable E . coli enterotoxin-induced fluid secretion in the rat jejunum in vivo . Both ondansetron and granisetron dose-dependently inhibited 5-HT- and enterotoxin-induced fluid secretion . Ketanserin blocked 5-HT-induced fluid secretion, but only diminished enterotoxin-induced effects even at higher doses . Tropisetron inhibited 5-HT- and cholera toxin-induced effects at high dose but only diminished heat stable E . coli enterotoxin-induced effects . We conclude that 5-HT3 receptors, located on enterochromaffin cells and nervous structures, are more important in mediating fluid secretion than 5-HT2 receptors, located on the epithelial cells.

Photochem Photobiol, 1993 Aug, 58(2), 219 - 25
In vivo repair of cytosine hydrates in DNA of cultured human lymphoblasts; Weiss RB et al.; Ultraviolet irradiation of DNA in vitro results in the production of a wide variety of pyrimidine base alterations, including cytosine hydrates . Enzymes that initiate the repair of monomeric pyrimidine damage have been identified in both bacterial and mammalian systems; however, the in vivo formation and repair of cytosine photohydrates has not been demonstrated in cellular DNA . Using Escherichia coli endonuclease III as a damage-specific probe, we have shown that ring-saturated pyrimidines are formed in cultured human cells by irradiation with broad-spectrum UV light . In addition, these types of base damage are removed from the DNA of human lymphoblasts within 5 h following the irradiation . Analysis of the action spectrum for the formation of cytosine hydrates in DNA reveals that these photoproducts are formed most efficiently by irradiation in the range of 255-265 nm light, coinciding with the wavelengths that are maximally absorbed by the DNA bases.

Mol Microbiol, 1993 Aug, 9(3), 557 - 68
Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA; Mudd EA et al.; Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo . Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180 kDa . However, proteolytic fragments as small as 70 kDa, which can arise during purification, also exhibit RNase E activity . In vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites . RNase E cleaves rne mRNA and autoregulates the expression of the rne gene . In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cleaved by RNase K . Our data are consistent with RNase K being a proteolytic fragment of RNase E.

Mol Microbiol, 1993 Aug, 9(3), 443 - 57
The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli; van Heeswijk WC et al.; Regulation of glutamine-synthetase (GS) activity in enteric bacteria involves a complex cascade of events . In response to nitrogen limitation, a transferase catalyses the uridylylation of the PII protein, which in turn stimulates deadenylylation of GS . Deadenylylated GS is the more active form of the enzyme . Here we characterize in detail the genes from Escherichia coli encoding uridylyl-transferase (glnD), the PII protein (glnB), and adenylyl-transferase (glnE) . glnD is transcribed from its own promoter, glnE is contranscribed with another gene, orfXE, whereas glnB is partly contranscribed with a gene encoding a homologue of the transcription activator NtrC . All three gln regulatory genes were constitutively expressed at a low level, i.e . their expression was independent of the nitrogen status and the RNA polymerase sigma factor sigma 54 . We conclude that the functioning of the GS adenylylation cascade is regulated by modulation of the activities of uridylyl-transferase and adenylyl-transferase, rather than by changes in the expression of their genes.

Mol Microbiol, 1993 Aug, 9(3), 425 - 34
Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli; Stewart V; Synthesis of most anaerobic respiratory pathways is subject to dual regulation by anaerobiosis and nitrate . Anaerobic induction is mediated by the FNR protein . Dual interacting two-component regulatory systems mediate nitrate induction and repression . The response regulator protein NARL binds DNA to control nitrate induction and repression of genes encoding nitrate respiration enzymes and alternate anaerobic respiratory enzymes, respectively . The homologous protein NARP controls nitrite induction of at least two operons . Nitrate and nitrite signalling are both mediated by the homologous sensor proteins NARX and NARQ . Recent mutational analyses have defined a heptamer sequence necessary for specific DNA binding by the NARL protein . These heptamers are located at different positions in the control regions of different operons . The NARL protein-binding sites in the narG (nitrate reductase) and narK (nitrate-nitrite antiporter) operon control regions are located approximately 200bp upstream of the transcription initiation site . The integration host factor (IHF) greatly stimulates nitrate induction of these operons, indicating that a specific DNA loop brings NARL protein, bound at the upstream region, into the proximity of the promoter for transcription activation . Other NARL protein-dependent opersons do not require IHF for nitrate induction, and the arrangement of NARL heptamer sequences in these control regions is quite different . This complexity of signal transduction pathways, coupled with the diversity of control region architecture, combine to provide many interesting areas for future investigation . An additional challenge is to determine how or if the FNR and NARL proteins interact to mediate dual positive control of transcription initiation.

Mol Microbiol, 1993 Aug, 9(3), 419 - 24
Assembly of the Escherichia coli F1F0 ATPase, a large multimeric membrane-bound enzyme; Brusilow WS; The F1F0 proton translocating ATPase of Escherichia coli is a large membrane-bound enzyme complex consisting of more than 20 polypeptides that are encoded by the unc operon . Besides being a system for analysing the enzymology of ATP synthesis and energy coupling, the ATPase is a model system for determining how large oligomeric membrane-bound proteins are synthesized and assembled . The assembly of the ATPase involves differential gene expression and assembly of the subunits within the membrane and with each other . This review discusses the influence of F1 subunits on the assembly and proton permeability of the F0 proton channel, and the possible advantages to assembly of the particular arrangement of genes in the unc operon.

J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1869 - 75
Molybdenum uptake in Escherichia coli K12; Lopez Corcuera G et al.; Molybdenum uptake was examined in Escherichia coli K12 using the radionuclide 99Mo . The molybdenum uptake system was characterized in an unusual chlD strain, which appeared to be normal in uptake of the MoO4(2-) ion but altered in subsequent molybdenum processing . As a consequence, molybdenum could be chased from cells in the chlD strain, while it was irreversibly assimilated in the wild-type strain . Molybdenum uptake showed a biphasic kinetic curve, with a very rapid binding followed by a slow uptake phase . The uptake appeared to involve an active transport system . Molybdenum, probably in the form of molybdate, accumulated by a factor of about 30 in the cells . An energy source was necessary and uptake was inhibited by arsenate, but not by CCCP (carbonyl cyanide m-chlorophenylhydrazone) . The uptake system saturated with a Km of 2.5-2.7 x 10(-8) M . Uptake seemed to depend on a periplasmic binding protein, since cold shock treatment and arsenate abolished uptake . A molybdate binding protein activity was detected in the periplasmic fluid with a KD of 9 nM . Sulphate inhibited uptake and the uptake activity was pH dependent, with an apparent pK of 6.7 . These results imply that molybdate transport belongs to the family of energy-dependent periplasmic binding protein systems . An explanation for the peculiar behaviour of the chlD strain used in this work is proposed.

J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1829 - 40
Identification of the formate dehydrogenases and genetic determinants of formate-dependent nitrite reduction by Escherichia coli K12; Darwin A et al.; The formate dehydrogenases of Escherichia coli involved in electron transfer from formate to nitrite (Nrf activity: nitrite reduction by formate) have been identified . No previously undescribed selenoprotein was detected in bacteria grown under conditions optimal for the expression of Nrf activity . The Nrf activities of single mutants defective in either FdhN or FdhH were between 50 and 60% that of the parental strain . A double mutant defective in both FdhN and FdhH retained less than 10% of the activity of the FdhN+ FdhH+ strain . No Nrf activity was detected in a triple mutant defective in FdhN, FdhH and FdhO or in the selC strain . It is concluded that all three of the known formate dehydrogenases of E . coli can contribute to the transfer of electrons from formate to the Nrf pathway . Mutants defective in Nrf activity and cytochrome c552 synthesis were isolated by insertion mutagenesis or identified amongst strains received from the E . coli Genetic Stock Center . The mutations were located in at least three regions of the chromosome, including the 92 to 94 minute region which includes fdhF, the gene encoding FdhH required for formate hydrogenlyase activity . Fine structure mapping by P1 transduction established that the nrf mutations in the fdhF region were due to defects in three separable loci, all of which were independent of but close to fdhF . Clones were isolated from a cosmid library that complemented a deletion extending from fdhF into a region essential for Nrf activity . From these clones, plasmids were isolated that complemented only some of the Nrf- mutations in the 92 to 94 minute region, confirming the presence of different operons essential for Nrf activity and cytochrome c552 synthesis in this region . Suggested reasons for this genetic complexity include the need for proteins involved in electron transfer from the various formate dehydrogenases to cytochrome c552, for the attachment of the haem group to the apocytochrome and for cytochrome c552 export into the periplasm.

J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1807 - 15
Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene; Cluness MJ et al.; The filamentous fungus Fusarium lateritium is cyanide tolerant, due, at least in part, to the induction by cyanide of the enzyme formamide hydrolyase (EC 4.2.1.66) . This enzyme, more commonly known as cyanide hydratase, catalyses the hydration of cyanide to formamide . The enzyme was purified from F . lateritium and showed a subunit molecular mass of 43 kDa (as judged by SDS-PAGE), while the native protein appeared to form aggregates of up to 1217 kDa (as judged by gel-filtration and non-denaturing PAGE) . mRNA samples from cultures grown with and without cyanide were in vitro translated and immunoprecipitated . This demonstrated that, in this species, the gene encoding the enzyme designated chy1, is cyanide inducible . Differential screening was used to isolate a cyanide hydratase cDNA clone which was subsequently used to obtain the corresponding genomic clone . A fragment of the cDNA clone encoding all but the first seven amino acids of the protein was expressed in E . coli using the expression vector pGEX-2T . Features of F . lateritium cyanide hydratase together with an analysis of the nucleotide sequence encoding this enzyme are presented.

J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1715 - 21
Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of legionella pneumophila; High AS et al.; A genomic library derived from a virulent isolate of Legionella pneumophila was constructed in Escherichia coli JM 83 using the cloning vector pUC19 . The clones were screened by filter immunoassay using L . pneumophila rabbit polyclonal antisera and in the absence of in situ bacterial lysis one such clone, LP 116, expressed L . pneumophila-specific antigens on the surface of E . coli . Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp . Southern hybridization confirmed that the fragment was L . pneumophila DNA . Sequencing data showed that the fragment was 810 bp in length with an open reading frame (ORF) of 678 bp . The outer-membrane profiles of the E . coli parent, the L . pneumophila DNA-contributing strain and clone LP 116 were compared by SDS-PAGE . A protein of 25 kDa was found in outer-membrane preparations of both the clone LP 116 and L . pneumophila but not in E . coli JM 83 . This was in agreement with the molecular mass of the deduced peptide of the mature protein . Immunoblots using L . pneumophila-specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was a L . pneumophila polypeptide . Both direct immunofluorescence assay and immunoblots using the commercially produced monoclonal antibody specific for the common antigen of the major outer-membrane protein (MOMP) confirmed that the 25 kDa protein produced by LP 116 was involved with the MOMP complex . The gene encoding this protein has been designated ompM.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1707 - 14
The Escherichia coli serA-linked capsule locus and its flanking sequences are polymorphic, genetic evidence for the existence of more than two groups of capsule gene clusters; Drake CR et al.; Two families of Escherichia coli capsules, termed groups I and II, have been defined previously on the basis of a number of biochemical and genetic criteria . Recently, a third group of capsules, termed I/II has been suggested on the basis of chemical structure and mode of expression . In this paper, we show that group I capsule-producing strains lack the serA-linked group II capsule genes . In addition, group I/II capsule-producing strains lack the group II capsule genes despite the former genes also mapping near to serA . Therefore, the genetic data presented in this paper support the existence of three groups of capsule gene clusters, two of which are linked to serA . Sequences flanking the K4 capsule genes were found in the chromosome of all E . coli strains examined and were sometimes present in multiple copies at different loci, indicating that this chromosomal region is highly polymorphic.

Kansenshogaku Zasshi, 1993 Aug, 67(8), 753 - 7
{Serotypes of enteroadherent Escherichia coli exhibiting diffuse pattern of adherence isolated from diarrheal patients and healthy controls in Brazil, Myanmar and Japan}; Tsukamoto T et al.; We tried to detect enteroadherent Escherichia coli exhibiting a diffuse pattern of adherence to HeLa cells from stock strains derived from patients with or without diarrhea in Brazil, Myanmar and Japan (Osaka) . Enteroadherent E . coli was found from 16 (23 strains) out of 126 (384 strains) in diarrheal infant cases (12.7%), 26 (29 strains) out of 126 (348 strains) in healthy control cases (20.6%) in Brazil, from 15 (18 strains) out of 221 (542 strains) in diarrheal infant cases (6.8%), 4 (4 strains) out of 87 (212 strains) in healthy control cases (4.6%) in Myanmar . In Japan (Osaka), enteroadherent E . coli was detected from 7 (7 strains) out of 123 (198 strains) in diarrheal cases (5.7%) . These results show that enteroadherent E . coli (diffuse) may not be associated with diarrhea . Forty-four (62.9%) out of 70 strains belonged to 19 O serogroups or 21 O:H serotypes . All strains were H antigen serotypable, and 26 strains were found to be nonmotile . The predominant O:H serotypes were O11:H15, O11:H-, O20:H34, O21:H5, O89:H-, O99:H33 and O154:H45 . Only one strain belonged to enteropathogenic E . coli serogroup O127.

FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 189 - 95
Transient repression of the synthesis of OmpF and aspartate transcarbamoylase in Escherichia coli K12 as a response to pollutant stress; Faber F et al.; The synthesis of total cellular proteins in Escherichia coli K12 was studied in batch culture following exposure of cells to low concentrations of monochlorophenol, pentachlorophenol and cadmium chloride . Changes in protein patterns were identified after pulse-chase labelling of proteins with {35S}methionine and subsequent two-dimensional gel electrophoresis (2D-PAGE) . We demonstrated that besides the induction of some stress proteins, also a transient decrease in the rate of synthesis of other proteins occurred . Two of these proteins were identified as OmpF and aspartate transcarbamoylase (ATCase) . Their transient repression appeared to be a general response to stress elicited by different pollutants and may therefore be used as a general and sensitive early warning system for pollutant stress.

Electrophoresis, 1993 Aug, 14(8), 753 - 8
Analysis of plasmid-DNA and cell protein of recombinant Escherichia coli using capillary gel electrophoresis; Hebenbrock K et al.; Plasmid DNA prepared from cultivation samples of recombinant Escherichia coli was analyzed by capillary gel electrophoresis . We used this method to control the genetic stability during a fed-batch culture . The plasmid DNA and a standard DNA mixture with molecular weights in the size range of 3000-22,000 base pairs (bp) were analyzed in capillaries that were filled with solutions of non-cross-linked polyacrylamide . With this method the plasmid DNA from recombinant E . coli cultivation samples was analyzed within 30 min . Separation parameters such as gel concentration, capillary length and current were optimized for that purpose . The method was modified to allow the analysis of proteins . Crude cell lysate samples could be screened for the protein pattern within 15 min in sodium dodecyl sulfate-polyacrylamide filled capillaries . The method was also used to verify product purity after the down-stream process.

Electrophoresis, 1993 Aug, 14(8), 713 - 9
Analysis of trp repressor-DNA interactions using gel electrophoresis; Lewis DE et al.; Quantitative analysis of the DNA-binding equilibria of E . coli trp repressor by gel electrophoresis led to reevaluation of our understanding of this complex system . In this review, the data leading to controversy about the trp system are discussed, and our current understanding is presented.

Electrophoresis, 1993 Aug, 14(8), 704 - 12
Effects of anomalous migration and DNA to protein ratios on resolution of equilibrium constants from gel mobility-shift assays; Senear DF et al.; Numerical resolution of binding constants from data generated by the gel mobility-shift assay is dependent on the experimental resolution of the different ligation states of the DNA . Previously we showed that the populations of the intermediate ligation states at partial saturation with the protein ligand are extremely sensitive to cooperativity (Senear, D . F . and Brenowitz, M . J . Biol . Chem . 1991, 266, 13661-13671) . This makes accurate gel mobility-shift data extremely useful to the demonstration of cooperativity . However, the accuracy with which the intermediate ligation state populations are resolved has been questioned . Thus, two additional and related questions are now considered . First, what information is available if the intermediate ligation state populations are not used in the analysis of binding constants . Second, is accurate information obtained from those states under conditions of high DNA concentration . These questions are addressed by using the interactions of the lambda cI repressor protein with the three site operator, OR, and the interaction of the E . coli GalR protein with the single site operator, OE . Both simulated and experimental data are analyzed . The results point to two conclusions . First, precise resolution of all macroscopic constants for binding of proteins to DNA is critically dependent on the intermediate ligation state populations; resolution is limited to at most two DNA sites if these states are not used in the analysis . Second, when the DNA and protein concentrations used in the titrations are comparable, the resolution of binding constants is extremely sensitive to experimental uncertainty in the macromolecule concentrations.

Electrophoresis, 1993 Aug, 14(8), 693 - 8
Electrophoretic analysis of protein-induced DNA bending and twist changes; Niederweis M et al.; DNA fragments with a varied phasing of two intrinsic bends at their ends and a tet operator in-between were constructed to determine Tet repressor-induced twist changes in the operator DNA . These distance variants show a sinusoidal dependence of their electrophoretic mobilities on the phasing of their bends in polyacrylamide gels . Complex formation with Tet repressor leads to a displacement of the first minimum, indicating an unwinding of the tet operator DNA . Model calculations were performed to assess the contribution of Tet repressor-induced DNA bending to this result . They revealed that the amplitude of the electrophoretic mobilities of the distance variants may be used as a parameter to separate the effects of Tet repressor-induced twist change and bending . The systematic analysis presented here may help to improve the quantitative interpretation of gel shifts and promote the use of this highly sensitive method to gain structural information about protein-DNA complexes.

Electrophoresis, 1993 Aug, 14(8), 669 - 79
Theoretical studies on the mobility-shift behavior of binary protein-DNA complexes; Cann JR; The theory of mass transport coupled to reversible interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility-shift behavior of binary protein-DNA complexes . Several systems have been modeled in terms of either (i) specific binding of a protein molecule to a single site on the DNA molecule; (ii) cooperative binding to two or three sites; (iii) noncooperative binding to two sites, both of which bind protein with equal affinity; (iv) statistical binding to multiple sites having identical intrinsic binding constants; or (v) protein-induced DNA loop formation . Both models (iii) and (v) embody the concept of reversible isomerization of protein-DNA complexes . The resulting simulations have provided fundamental information concerning (i) the factors governing the electrophoretic persistence and separation of protein-DNA complexes; (ii) the shape of experimental mobility-shift patterns; (iii) the generation of the protein-DNA ladder upon titration, for example, of the 203-base pair operator with lac repressor; and (iv) the theoretical bases for quantitative interpretation of the patterns in terms of thermodynamic and kinetic parameters . The practical implications of these findings are discussed.

Comput Appl Biosci, 1993 Aug, 9(4), 435 - 40
CURVATURE: software for the analysis of curved DNA; Shpigelman ES et al.; Software is presented to plot the sequence-dependent spatial trajectory of the DNA double helix and/or distribution of curvature along the DNA molecule . The nearest-neighbor wedge model is implemented to calculate overall DNA path using local helix parameters: helix twist angle, wedge (deflection) angle and direction (of deflection) angle . The procedures described proved to be very convenient as tools for investigation of a relationship between overall DNA curvature and its gel electrophoretic mobility . All parameters of the model had been estimated from experimental data . Using these wedge parameters the program takes, as input, any DNA sequence and calculates the likely degree of curvature at each point along the molecule . This information is displayed both graphically and in the form of simplified representations of curved double helices . The Software, CURVATURE, can thus be used to investigate possible roles of curvature in modulation of gene expression and for location of curved portions of DNA, which may play an important role in sequence-specific protein--DNA interactions.

Protein Sci, 1993 Aug, 2(8), 1320 - 30
Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers; Baudin V et al.; A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates . While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers . While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity . These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA . X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer . We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers . Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers . Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior . In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.

Protein Sci, 1993 Aug, 2(8), 1210 - 9
Synthetic chimeras of mouse growth factor-associated glandular kallikreins . I . Kinetic properties; Blaber M et al.; A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--were expressed in Escherichia coli and isolated, and their kinetic properties were characterized . The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes . The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors . Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins . Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J . Mol . Biol . 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor . Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF . Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates . The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.

J Pharmacol Toxicol Methods, 1993 Aug, 29(4), 185 - 93
The use of cyclic voltammetry for the evaluation of oxidative damage in biological samples; Kohen R; A method using cyclic voltammetry to evaluate oxidative damage in biological systems is presented . Three biological systems were tested: Escherichia coli cells, the rat jejunal mucosa, and the enzyme, lactate dehydrogenase . Exposure of E . coli cells to oxidative stress resulted in a rapid decrease in their survival and a decrease in their ability to accumulate 14C-leucine . This was accompanied by a significant increase in the oxidation potential of the cells . Similar results were obtained when the rat jejunal mucosa was exposed in a perfusion system to oxidative stress induced by the hydroxyl radical produced by either hydrogen peroxide and ferrous ions or the combination of ascorbic acid and copper ions . Loss of cellular potassium was taken as an indication of damage to the rat jejunum . Exposure of lactate dehydrogenase to oxidative stress induced by hydroxyl and peroxyl radicals also resulted in a significant loss of enzyme activity along with a pronounced change in the cyclic voltammogram of the enzyme . It was concluded that measurement of the oxidation potentials of these biological systems can give an indication of the occurrence of oxidative damage.

Biokhimiia, 1993 Aug, 58(8), 1240 - 6
{Thioredoxin-reductase: structure, properties, and function}; Labudova O et al.; Thioredoxin reductase (TR-RED) pertains to the family of pyridine nucleotide disulfide oxidoreductases distinguished by their remarkable structural homology . The enzyme is a constituent component of the thioredoxin complex which is present in all types of organisms and is universal in respect of the numerous physiological functions it performs . The ability of TR-RED to protect the skin from UV-generated free oxygen species has been found . Owing to its ability to control melanin biosynthesis, the enzyme "doses" the suntan.

J Vet Med Sci, 1993 Aug, 55(4), 607 - 11
Changes in reticuloendothelial function in dogs with endotoxin-induced shock; Okano S et al.; A shock model was experimentally produced by intravenous injection of a lethal dose (3 mg/kg) of endotoxin under general anesthesia induced by pentobarbital sodium using 7 beagles . The effect of this endotoxic shock on the reticuloendothelial function was investigated . The blood endotoxin concentration peaked immediately after administration and decreased subsequently . However, the value still remained on an increased level (1,051 pg/ml) even at 360 min after endotoxin treatment . The lipid emulsion test as an index of reticuloendothelial phagocytotic activity and the arterial ketone body ratio as an index of the energy charge in the liver decreased after endotoxin treatment and failed to recover during the experiment . Fibronectin, one of opsonic proteins, tended to decrease after injection of the endotoxin and was significantly (p < 0.01) low at 180 and 360 min compared with the value before injection of the endotoxin . These results suggested the depression of the reticuloendothelial function during endotoxin-induced shock.

Hum Gene Ther, 1993 Aug, 4(4), 403 - 9
Assessment of recombinant adenoviral vectors for hepatic gene therapy; Li Q et al.; Recombinant adenoviral vectors have recently been used to transfer genes into a number of different cell types in vitro and in vivo . A recombinant adenoviral vector bearing the Escherichia coli beta-galactosidase (beta-gal) gene was used to quantitate the frequency of hepatocyte transduction in the mouse after direct viral infusion into the portal vein . When 10(10) adenoviral particles were infused, over 95% of the hepatocytes were transduced in vivo as determined by x-gal staining . The transduction protocol is relatively safe in that there is no detectable helper virus production in transduced animals and that very few extrahepatic cells are transduced by this method . There is also no evidence of significant liver pathology unless substantially greater quantities of virus are used . However, the transduced hepatocytes do not appear to persist in vivo because the percentage of hepatocytes expressing beta-gal declined over time . Four months after the procedure, 0.5-10% of the hepatocytes contain detectable beta-gal activity in vivo . The change in beta-gal-positive cells correlates with decreasing amounts of adenoviral DNA . Thus, current recombinant adenoviral vectors may have clinical applications in gene therapy for acute hepatic disorders.

Am J Physiol, 1993 Aug, 265(2 Pt 1), G370 - 8
Na(+)-K(+)-2Cl- cotransport and Cl- secretion evoked by heat-stable enterotoxin is microfilament dependent in T84 cells; Matthews JB et al.; We previously reported that adenosine 3',5'-cyclic monophosphate-mediated stimulation of Cl- secretion in the human intestinal epithelial cell line T84 is accompanied by significant remodeling of F-actin and that both the secretory and cytoskeletal responses may be inhibited by phalloidin derivatives, agents that polymerize actin and prevent dynamic reorganization of microfilaments . In contrast, the carbachol-elicited Cl- secretory response (Ca2+ mediated) was not attenuated by phalloidin (J . Clin . Invest . 87: 1903-1909, 1991) . In the present study, we examine the effect of phalloidin on the Cl- secretory response elicited by the heat-stable enterotoxin of Escherichia coli (STa), which induces elevations in intracellular guanosine 3',5'-cyclic monophosphate . We find that apical administration of 1 microM STa results in a regionally restricted redistribution of F-actin confined to the basal pole of the cells . In monolayers pretreated with phalloidin, the Cl- secretory response to STa was inhibited by > 60% . Sequential treatment of phalloidin-loaded monolayers with STa followed by carbachol resulted in a synergistic secretory response that was not different from control (unloaded) monolayers . Examination of efflux/uptake through specific membrane transport pathways involved in STa-stimulated Cl- secretion indicated normal activation of apical Cl- and basolateral K+ channels under phalloidin-loaded conditions . The ability of STa-treated monolayers to pump Na+ in an absorptive direction was also unaffected by phalloidin . der phalloidin-loaded conditions, STa-stimulated Na(+)-K(+)-2Cl- cotransporter activity was reduced by approximately 60%, sufficient to account for the observed inhibition of net Cl- secretory response.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Biochem Biophys, 1993 Aug 1, 304(2), 367 - 70
A new approach to measurement of redox-cycling activity in Escherichia coli; McManus DC et al.; Redox-cycling agents catalyze the flow of reducing equivalents to oxygen; this process generates superoxide ion and other reduced oxygen species . Measurements of redox-cycling activity have been performed previously by studying cyanide-resistant oxygen consumption (respiration) of Escherichia coli cells . E . coli strain GK100, lacking both terminal oxidases, has almost no measurable respiration . We show that the use of this strain eliminates the requirement for cyanide in measurements of redox-cycling activity . The addition of either menadione sodium bisulfite or plumbagin, well-known redox-cycling agents, to GK100 cells resulted in high levels of oxygen consumption . The rate of menadione bisulfite-induced oxygen consumption in this respiration-deficient strain, in the absence of cyanide, was comparable to the cyanide-resistant respiration of isogenic respiration-proficient E . coli strains . In GK100 cells, cyanide increased menadione bisulfite-induced oxygen consumption but had no effect on plumbagin-induced oxygen consumption.

Arch Biochem Biophys, 1993 Aug 1, 304(2), 338 - 44
Cryogenic solvents inhibit the binding of the Escherichia coli heat-stable enterotoxin to intestinal brush border membranes; Katwa LC et al.; The cryogenic solvents ethylene glycol, glycerol, dimethyl sulfoxide (Me2SO) and dimethylformamide (Me2FM), with increasing potency, produced concentration-dependent inhibition of the binding of the Escherichia coli heat-stable enterotoxin (STa) to pig intestinal brush border membranes . Inhibition increased with time, and both Me2SO and Me2FM appeared to decrease both the affinity of the STa receptor and the effective receptor number . Both solvents stimulated the release of previously bound 125I-STa (Me2FM > Me2SO), 3 M Me2FM inducing 93% release by 120 min . These effects were reversible, and preincubation of membranes with up to 3 M Me2SO or Me2FM at 37 degrees C for 30 min, followed by washing, did not alter subsequent 125I-STa binding . Also, 125I-STa released from membranes by 3 M Me2FM was shown to rebind to the membranes after 10-fold dilution of Me2FM . Since pretreating membranes with the thiol reagent p-chloromercuribenzenesulfonate had no effect on the release of bound 125I-STa by Me2SO or Me2FM, and since neither of these can reduce disulfide bonds, the formation of mixed disulfides between STa and receptor is unlikely . Me2SO inhibition of 125I-STa binding was greater with membranes than with a partially purified receptor preparation, which may result from the substitution of detergent for the phospholipid normally associated with the receptor(s).

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7029 - 33
Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase; Keasling JD et al.; An exopolyphosphatase {exopoly(P)ase; EC 3.6.1.11} activity has recently been purified to homogeneity from a mutant strain of Escherichia coli which lacks the principal exopoly(P)ase . The second exopoly(P)ase has now been identified as guanosine pentaphosphate phosphohydrolase (GPP; EC 3.6.1.40) by three lines of evidence: (i) the sequences of five tryptic digestion fragments of the purified protein are found in the translated gppA gene, (ii) the size of the protein (100 kDa) agrees with published values for GPP, and (iii) the ratio of exopoly(P)ase activity to GPP activity remains constant throughout a 300-fold purification in the last steps of the procedure . The enzyme liberates orthophosphate by processive hydrolysis of the phosphoanyhydride bonds of polyphosphate {poly(P)} chains (1000 residues) or by hydrolysis of the 5'-gamma-phosphate of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) to guanosine 5'-diphosphate 3'-diphosphate (ppGpp or "magic spot") . The Km for long-chain poly(P) as a substrate (approximately 0.5 nM) is far lower than that for pppGpp (0.13 mM); the kcat for the poly(P)ase activity is 1.1 s-1 and that for pppGpp hydrolase is 0.023 s-1 . These and other findings direct attention to possible functions of poly(P) in the response of E . coli to stresses and deprivations.

J Gen Virol, 1993 Aug, 74 ( Pt 8), 1649 - 52
Inducible expression of a foreign gene inserted into the human cytomegalovirus genome; Takekoshi M et al.; We previously described the insertion of a foreign gene into a non-essential region of human cytomegalovirus (HCMV) by homologous recombination . Here we report insertion of the Escherichia coli lacZ gene downstream of the mouse metallothionein promoter into the HindIII-O region of HCMV by replacement-type recombination . Expression of the lacZ gene in the recombinant was independent of viral growth, but dependent on induction by heavy metals . Of several metals tested for beta-galactosidase induction and also for their toxicity to HEL cells, Zn was found to be the most suitable for use as an inducer . In HEL cells infected with the recombinant in the presence of 50 microsM-Zn, beta-galactosidase activity was maximal 3 days after infection, and reached levels 27 times higher than the value obtained in the absence of Zn.

EMBO J, 1993 Aug, 12(8), 3123 - 32
The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2; Poon RY et al.; Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE . We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15 . Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2 . In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form . Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP . We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.

Virology, 1993 Aug, 195(2), 638 - 48
Selection of Marek's disease virus recombinants expressing the Escherichia coli gpt gene; Marshall DR et al.; We developed a positive selection method for recovering Marek's disease virus (MDV) recombinants . The Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt), under the control of the major immediate-early promoter from cytomegalovirus, was inserted into the inverted repeats flanking the unique long (UL) region of a non-pathogenic serotype 2 MDV strain 281MI/1 . In a second demonstration of the usefulness of the positive selection system, the gpt gene was inserted into the inverted repeats flanking the unique short (US) region of the turkey herpesvirus (HVT) strain FC126 . The targeted insertion site in 281MI/1 was in a previously established nonessential site for virus replication . The targeted insertion site for FC126, at the junction of the UL and US regions, is a nonessential site for in vitro replication of herpes simplex virus . Recombinant viruses were easily selected by incubating the transfected cells in mycophenolic acid (MPA)-containing medium . Purification of recombinants resulted from a series of trypsinization and sonication steps combined with the culturing of virus in MPA-containing medium to inhibit wild-type virus replication . This simple technique for recovering MDV and HVT recombinants should increase the efficiency of identifying nonessential sites and gene function analysis by insertional mutagenesis.

J Bacteriol, 1993 Aug, 175(15), 4905 - 6
Delayed expression of in vivo restriction activity following conjugal transfer of Escherichia coli hsdK (restriction-modification) genes; Prakash-Cheng A et al.; Following conjugal transfer of the hsdK genes (hsdRK, hsdMK, and hsdSK) of Escherichia coli K-12, restriction activity was first detected only after approximately 15 generations, whereas modification activity was observed immediately . This sequential expression explains the establishment of hsdK genes in a nonmodified host and suggests regulation of restriction activity after conjugal transfer.

J Bacteriol, 1993 Aug, 175(15), 4843 - 50
Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences; Belogurov AA et al.; The IncN plasmid pKM101 (a derivative of R46) encodes the antirestriction protein ArdB (alleviation of restriction of DNA) in addition to another antirestriction protein, ArdA, described previously . The relevant gene, ardB, was located in the leading region of pKM101, about 7 kb from oriT . The nucleotide sequence of ardB was determined, and an appropriate polypeptide was identified in maxicells of Escherichia coli . Like ArdA, ArdB efficiently inhibits restriction by members of the three known families of type I systems of E . coli and only slightly affects the type II enzyme, EcoRI . However, in contrast to ArdA, ArdB is ineffective against the modification activity of the type I (EcoK) system . Comparison of deduced amino acid sequences of ArdA and ArdB revealed only one small region of similarity (nine residues), suggesting that this region may be somehow involved in the interaction with the type I restriction systems . We also found that the expression of both ardA and ardB genes is controlled jointly by two pKM101-encoded proteins, ArdK and ArdR, with molecular weights of about 15,000 and 20,000, respectively . The finding that the sequences immediately upstream of ardA and ardB share about 94% identity over 218 bp suggests that their expression may be controlled by ArdK and ArdR at the transcriptional level . Deletion studies and promoter probe analysis of these sequences revealed the regions responsible for the action of ArdK and ArdR as regulatory proteins . We propose that both types of antirestriction proteins may play a pivotal role in overcoming the host restriction barrier by self-transmissible broad-host-range plasmids . It seems likely that the ardKR-dependent regulatory system serves in this case as a genetic switch that controls the expression of plasmid-encoded antirestriction functions during mating.

J Bacteriol, 1993 Aug, 175(15), 4764 - 71
DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro; Scarlato V et al.; The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes . We show that in Escherichia coli, the PTOX promoter cloned in cis of the bvg locus is activated and environmentally regulated . Cotransformation of E . coli with the bvg locus cloned in a low-copy-number plasmid and with the PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results . If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent . If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished . Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases . In vitro, the transcription of the PTOX promoter is activated on E . coli RNA polymerase supplemented with cell extracts from wild-type B . pertussis . Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized . Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the PTOX promoter in E . coli and that DNA topology may play a role in the induction of transcription.

J Bacteriol, 1993 Aug, 175(15), 4744 - 55
Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties; Romeo T et al.; Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase . One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen . To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-lacZ fusion . The glycogen excess mutation TR1-5 was found to be pleiotropic . It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties . The mutated gene was designated csrA for carbon storage regulator . Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators of glgC expression . A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth . Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis . Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product.

J Bacteriol, 1993 Aug, 175(15), 4670 - 80
A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells; Donnenberg MS et al.; Enteropathogenic Escherichia coli (EPEC) is capable of attaching intimately to epithelial cells and effacing their microvilli . A chromosomal locus, eaeA (originally eae), is required for the intimate attachment aspect of this effect . We report the mapping of a region of the EPEC chromosome that is located immediately downstream of the eaeA gene and that is also necessary for intimate attachment . An isogenic in-frame deletion mutation in one of the open reading frames identified in this region was engineered . Because the resulting mutant, like an eaeA deletion mutant, is deficient in the ability to attach intimately to epithelial cells, the mutated gene is designated eaeB . Full activity is restored to the eaeB mutant when the cloned gene is reintroduced on a plasmid . The eaeB mutant remains capable of producing intimin, the product of the eaeA gene . No differences in the fractionation properties or electrophoretic mobility of intimin are apparent in the eaeB mutant . The product of the eaeB locus was identified by in vitro transcription-translation . The nucleotide sequence of the eaeB gene predicts a protein that contains a sequence motif common to several aminotransferase enzymes . These results indicate that the attaching and effacing effect is a complex phenotype dependent on a gene cluster present on the EPEC chromosome.

J Virol, 1993 Aug, 67(8), 4566 - 79
Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli; Luckow VA et al.; The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks . This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells . The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7 . Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E . coli when Tn7 transposition functions are provided in trans by a helper plasmid . The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.

J Virol, 1993 Aug, 67(8), 4464 - 73
Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI; Yao Z et al.; Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G . Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture . To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI . The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro . Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo . Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues . Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues . Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect . Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products . Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II . Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane.

J Urol, 1993 Aug, 150(2 Pt 2), 759 - 62
The diagnosis of acute pyelonephritis in the piglet using single photon emission computerized tomography dimercaptosuccinic acid scintigraphy: a pathological correlation; Giblin JG et al.; Single photon emission computerized tomography (SPECT) scintigraphy has proved to be an extremely sensitive renal imaging modality in children with genitourinary pathology, including pyelonephritis, particularly when compared to 2-dimensional planar imaging . This study was undertaken to corroborate SPECT dimercaptosuccinic acid (DMSA) scintigraphic findings with specific histopathology in acute pyelonephritis . Unilateral vesicoureteral reflux was produced in 19 Yorkshire piglets 3 to 4 weeks old . The bladders of 12 animals were inoculated with Escherichia coli 2 weeks later, after baseline SPECT DMSA scans had been obtained . The animals were then re-imaged at 3 (4), 7 (4) or 14 (4) days after infection and sacrificed for histological evaluation . Seven purposefully uninfected piglets with unilateral reflux served as controls and were followed for up to 6 weeks before imaging and sacrifice . SPECT proved to be 97% sensitive and 93% specific in providing the diagnosis of acute pyelonephritis . The SPECT findings were manifest by a spectrum of abnormal findings (mottling, striations, inner cortical scalloping and focal cortical defects), which correlated precisely with the extent and severity of cortical involvement in the acute pyelonephritic process . We propose a new classification scheme for SPECT DMSA renal scintigraphic imaging, and believe that this modality is exquisitely sensitive in providing the diagnosis as well as in evaluating the extent of renal parenchymal involvement when acute pyelonephritis is induced in the animal model.

Nippon Geka Gakkai Zasshi, 1993 Aug, 94(8), 809 - 15
{Experimental study on the effects of endotoxemia as a retardation-factor influencing on the decrease of serum bilirubin after the relief of obstructive jaundice}; Idei T; I examined the effects of endotoxemia influencing on obstructive jaundice and the decrease of serum bilirubin after the relief of it . Experimental obstructive jaundice and its alleviation by external biliary drainage was made in Donryu-rats . Serum bilirubin was higher (p < 0.01) in the group treated by continuous infusion of low-dose endotoxin (LPS E . coli 0111:B4, 6 micrograms/hr/100gBW) during 72 hours biliary obstruction (10.48 +/- 1.54mg/dl) than in the control group (6.76 +/- 0.71mg/dl) . After the relief of biliary obstruction, 4 kinds of experimental conditions were set up as follows: CONTROL; infusion of sterile saline, ET; continuous infusion of low-dose endotoxin, ET + UDCA; continuous infusion of endotoxin and ursodeoxycholic acid (UDCA, 10mg/hr/100gBW) through another intravenous tube simultaneously, ET + MP; continuous infusion of endotoxin after one-shot injection of methylprednisolone (MP, 3mg/100gBW) . Serum bilirubin 7.5 hours after the relief of biliary obstruction was as follows: CONTROL: 0.48 +/- 0.18mg/dl, ET: 3.41 +/- 1.13mg/dl, ET + UDCA: 2.80 +/- 1.28mg/dl, ET + MP: 1.18 +/- 0.50mg/dl . ET-group showed retardation of the decrease of serum bilirubin (p < 0.01) . MP showed improvement of the impaired decreasing rate of serum bilirubin by endotoxemia (p < 0.01) . Bile-output from the external biliary drainage after the relief of biliary obstruction was decreased significantly in the ET-group . ET + UDCA-group showed remarkable increase of the bile-output, but no increase of excretion of bilirubin in the bile compared with ET-group . While ET + MP-group showed improvement of the bile-output and increase of excretion of bilirubin in the bile compared with ET-group.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetics, 1993 Aug, 134(4), 1031 - 8
The mutational specificity of two Escherichia coli dnaE antimutator alleles as determined from lacI mutation spectra; Schaaper RM; In a companion study we have described the isolation of a series of mutants of Escherichia coli that replicate their DNA with increased fidelity . These mutants carry a mutation in the dnaE gene, encoding the alpha (polymerase) subunit of DNA polymerase III holoenzyme, which is responsible for the faithful replication of the bacterial chromosome . The mutants were detected as suppressors of the high mutability of a mutL strain (defective in postreplicative mismatch correction), in which mutations may be considered to arise predominantly from errors of DNA replication . To investigate the specificity of these antimutator effects, we have analyzed spectra of forward mutations in the N-terminal part of the lacI gene (i-d mutations) for two of the mutL dnaE derivatives (dnaE911 and dnaE915), as well as the control mutL strain . DNA sequencing of over 600 mutants revealed that in the mutL background both antimutator alleles reduce specifically transition mutations (A.T-->G.C and G.C-->A.T) . However, the two alleles behave differently in this respect . dnaE911 reduces A.T-->G.C more strongly than it does G.C-->A.T, whereas the reverse is true for dnaE915 . Second, dnaE911 does not appear to affect either transversion or frameshift mutations, whereas dnaE915 displays a distinct mutator effect for both . This mutator effect of dnaE915 for frameshift mutations was confirmed by the frequency of reversion of the trpE9777 frameshift mutation . The discovery that dnaE antimutator alleles possess distinct specificities supports the notion that DNA polymerases discriminate against errors along multiple pathways and that these pathways can be influenced independently.

Circ Shock, 1993 Aug, 40(4), 268 - 75
Repeated plasma therapy induces fatal shock in experimental septicemia; Busund R et al.; Plasma therapy in severe septicemia, either as part of plasma exchange or alone, was evaluated in a model of lethal septic shock induced with live Escherichia coli in pigs . The following groups were studied: group I, septic animals treated with repeated plasma exchanges, n = 8; group II, nontreated septic controls, n = 8; group III, septic animals treated with repeated plasma infusions, n = 14; and group IV, nonseptic animals treated with repeated plasma infusions, n = 7 . In the septic animals treated with plasma (groups I and III), a rapid fatal response was observed between 2 and 5 min after the start of plasma therapy, while the septic controls (group II) showed a progressively longer lasting septic shock . The nonseptic animals (group IV) were unaffected by the plasma infusions . Plasma levels of endotoxin above 2 ng/ml were associated with rapid death during plasma therapy . Ionized Ca2+ fell abruptly in this situation . This study indicates that commonly used plasma therapies (exchanges or transfusions) in septic animals may have acute deleterious effects . These effects may be explained by depletion of ionized calcium.

Circ Shock, 1993 Aug, 40(4), 235 - 42
Effect of tirilazad mesylate (U74006F) on eicosanoid and tumor necrosis factor generation in healthy and endotoxemic neonatal calves; Semrad SD et al.; The effects of a 21-aminosteroid, tirilazad mesylate (U74006F; The Upjohn Co., Kalamazoo, MI), developed for treatment of central nervous system trauma in human beings, on eicosanoid and tumor necrosis factor (TNF) generation were evaluated in both healthy and endotoxin-challenged neonatal calves . Endotoxemia was induced in 24 neonatal calves by intravenous infusion of Escherichia coli lipopolysaccharide (3.25 micrograms/kg) over 3 hr . Group I calves received the endotoxin infusion alone; group II calves received an infusion of 0.9% saline and were treated with tirilazad mesylate (1.5 mg/kg) 1 hr after the infusion was started, group III and IV calves were treated with tirilazad mesylate 1 hr after or before endotoxin infusion was started . Tirilazad mesylate effectively suppressed production of plasma prostacyclin (6-keto-PGF1 alpha) and TNF in endotoxin-challenged neonatal calves . In addition, production of thromboxane B2 was mitigated by treatment with tirilazad mesylate both 1 hr before and 1 hr after initiation of endotoxin infusion . It appears that tirilazad mesylate effectively suppresses eicosanoid and TNF generation induced by endotoxin, and thus may be beneficial in the treatment of endotoxemia and septicemia in neonatal calves.

Protein Expr Purif, 1993 Aug, 4(4), 337 - 44
Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase expressed in Escherichia coli: production of homogeneous protein; Frimpong K et al.; When overexpressed in Escherichia coli, the catalytic domain of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) is catalytically active, but exhibits major heterogeneity . This heterogeneity reflects deletion of about 60 aminoacyl residues from the C-terminus, presumably a result of proteolytic cleavage or premature termination of translation . With the intent of separating the intact and truncated proteins via immunoaffinity chromatography, we constructed the expression phagemid pKFT7-21 . This construct encodes the catalytic domain of Syrian hamster HMG-CoA reductase with the C-terminal extension Glu-Glu-Phe, an epitope recognized by a specific antibody . Following overexpression, the modified catalytic domain RcatEEF had high catalytic activity and exhibited no heterogeneity . It therefore was possible to purify RcatEEF to over 95% homogeneity without resorting to immunoaffinity chromatography . The yield of homogeneous protein averaged 20-25 mg per liter of cells with a final specific activity of up to 40 mumol NADPH oxidized per minute per milligram . The EEF modification thus should prove useful for the purification of the catalytic domains of other eukaryotic HMG-CoA reductases which exhibit heterogeneity.

Protein Expr Purif, 1993 Aug, 4(4), 320 - 7
Use of nonreducing SDS-PAGE for monitoring renaturation of recombinant protein synthesis initiation factor, eIF-4 alpha; Stern BD et al.; The purification of biologically active human protein synthesis initiation factor 4 alpha, eIF-4 alpha, overexpressed in Escherichia coli, is complicated by its localization in insoluble inclusion bodies, as well as its possession of four cysteines . Two of these cysteines have been reported to be reduced in the native molecule and two form a disulfide bond . A method is described, using nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, for monitoring renaturation of the polypeptide during staged dialyses in decreasing urea concentrations . The production of biologically active eIF-4 alpha only occurs when the polypeptide is totally reduced during solubilization of the inclusion bodies in 8 M urea . This requires a minimum dithiothreitol concentration of 50 mM . Conversely, reformation of the disulfide bonds only occurs when the staged dialyses are performed at lower concentrations of sulfhydryl reagents . Once renatured, as described, eIF-4 alpha can be purified by affinity chromatography on m7GTP-Sepharose . Approximately 20 micrograms of biologically active eIF-4 alpha per milliliter of bacterial culture can be obtained . The affinity-purified eIF-4 alpha has activity equivalent to that reported for purified native human eIF-4 alpha, as measured by its activity in a rabbit reticulocyte translation system . The method described is applicable to the purification of other cysteine-containing polypeptides that accumulate to high levels in inclusion bodies.

Protein Expr Purif, 1993 Aug, 4(4), 282 - 9
Expression, purification, and characterization of recombinant ornatin E, a potent glycoprotein IIb-IIIa antagonist; Mazur P et al.; A synthetic gene encoding ornatin E (OrnE), a 50-amino acid glycoprotein IIb-IIIa (GP IIb-IIIa) antagonist and platelet aggregation inhibitor isolated from the leech Placobdella ornata, was designed, constructed, and expressed in Escherichia coli . The OrnE gene was fused to the heat stable enterotoxin stII signal sequence and expressed under the transcriptional control of the E . coli alkaline phosphatase promoter . This construction directed secretion of recombinant ornatin E (rOrnE) into the extracellular medium at levels of 7-19 mg/liter . The protein was purified to apparent homogeneity in 18-38% yields by ammonium sulfate precipitation, Q-Sepharose and S-Sepharose ion exchange chromatography, and reverse-phase HPLC . Purified rOrnE was found to be indistinguishable from leech-derived OrnE as judged by amino acid composition, N-terminal sequencing, mass spectroscopic analysis, and HPLC coelution . In addition, rOrnE exhibits similar activity in fibrinogen/GP IIb-IIIa ELISA and platelet aggregation assays . Purified rOrnE possesses three disulfide bonds, the reduction and carboxymethylation of which results in a ca . 60-fold reduction in biological activity . A misfolded variant of rOrnE was characterized and shown to have a ca . 6-fold reduction in activity . These data demonstrate that the native disulfide bonds are required for the optimal GP IIb-IIIa antagonist activity of the ornatins.

Protein Expr Purif, 1993 Aug, 4(4), 275 - 81
High yield of active STb enterotoxin from a fusion protein (MBP-STb) expressed in Escherichia coli; Handl CE et al.; Maltose binding protein (MBP) fused to STb, a heatstable enterotoxin of Escherichia coli, was secreted into the periplasm . A factor Xa cleavage site is present between MBP and STb allowing MBP to be cleaved from STb . The gene fusion is under the control of the strong and inducible Ptac promoter . Three hours after induction with IPTG, cells were harvested . Following osmotic shock treatment of the cells, the MBP-STb fusion protein was released and affinity-purified using an amylose resin . The fusion protein purified in this way was biologically active in ligated intestinal segments of rats . Digestion of MBP-STb with factor Xa released native STb which was purified to homogeneity by reverse-phase chromatography using a PepRPC column . The toxin was eluted at approximately 38% acetonitrile . The 5000-Da toxin was shown to be pure by SDS-PAGE and immunoblotting . The recovered enterotoxin was active in the rat loop assay . Amino acid sequence analysis showed that the first eight residues were identical to those of native STb, confirming the identity of STb . The ultraviolet absorption spectra of purified STb revealed low absorption at 254 and 280 nm compared to 210-230 nm . Isoelectric focusing under nondenaturing conditions indicated a pI of 9.6 . Typically, 8 liters of bacterial culture resulted in 2.2 mg of pure STb . This genetic construction provides a readily obtainable source of biologically active STb toxin.

Protein Expr Purif, 1993 Aug, 4(4), 265 - 74
Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: expression of the Escherichia coli F1-ATPase epsilon subunit; Van Heeke G et al.; A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest . Carbonic anhydrase is soluble and stable in E . coli and serves as a highly specific purification tag in the recovery of the fusion protein by a single affinity chromatography step . The enterokinase cleavage site was engineered into the construct to allow accurate and efficient release of the target protein . To demonstrate the practical value of this vector, the E . coli F1-ATPase epsilon subunit was expressed as a fusion with hCAII . After a single purification step, biologically active recombinant E . coli F1-ATPase epsilon subunit was recovered following proteolytic removal of the hCAII moiety.

Biotechniques, 1993 Aug, 15(2), 264 - 6
In vitro transcription of transfer RNAs with 3'-end modifications; Liu M et al.; It is difficult to modify the 3'-end of RNA transcribed in vitro from recombinant plasmid or phagemid DNA because of the need to retain a restriction endonuclease recognition site at the downstream end of the template in order to linearize the DNA before transcription . To avoid limitations on the 3'-sequence of RNA transcripts, we have modified a template-containing phagemid by inserting a FokI site outside the RNA gene sequence, positioned to cleave the template DNA to yield the desired 3' terminus . We demonstrate that this phagemid is an efficient template for tRNA transcription and use it to prepare mutants of Escherichia coli tRNA(Val) with modifications at the 3'-CCA end . Phagemids containing a FokI site should be suitable for in vitro transcription of large quantities of RNA of any length and with an unlimited variety of 3'-terminal sequences.

J Clin Microbiol, 1993 Aug, 31(8), 2163 - 6
Evaluation of conventional media for detection of colonization factor antigens of enterotoxigenic Escherichia coli; Ghosh AR et al.; Strains of enterotoxigenic Escherichia coli producing either colonization factor antigen (CFA) I or II were tested for expression of CFA when grown on 16 different agar media by using agglutination and coagglutination with monoclonal antibodies, mannose-resistant hemagglutination, and a salt aggregation assay . CFA was detected from the CFA-positive strains when CFA agar was used, and it was also detected when other commercially available media were used, notably nutrient agar . CFA was not detected when other commercial media such as MacConkey agar were used . The use of nutrient agar with monoclonal antibody-based coagglutination reagents offers a potentially simple and rapid method for detecting E . coli which express CFA I or II.

J Clin Microbiol, 1993 Aug, 31(8), 2146 - 51
Characterization of a chromosomal gene and the antigen it expresses from the Lyme disease agent, Borrelia burgdorferi; LeFebvre RB et al.; The sequence and characterization of a chromosomal gene from the Lyme disease agent Borrelia burgdorferi and the antigen it encodes are described . The gene was cloned and expressed in transformed Escherichia coli cells . The gene is composed of 597 bases and expresses a predicted protein of 199 amino acids . Antibodies specific for the recombinant antigen reacted with a single B . burgdorferi protein with a molecular mass of approximately 22 kDa . The protein was not susceptible to proteinase digestion but was extracted by n-butanol phase partitioning, suggesting a periplasmic location of the antigen . Sera from humans and canines seropositive for B . burgdorferi reacted with the recombinant antigen . The antigen characterized in this report appears to be immunologically significant in naturally infected hosts.

J Clin Microbiol, 1993 Aug, 31(8), 2031 - 7
Escherichia coli strains involved in diarrhea in France: high prevalence and heterogeneity of diffusely adhering strains; Jallat C et al.; Two hundred sixty-two strains of Escherichia coli isolated from diarrheal stool specimens from infants, children, and adults hospitalized in Clermont-Ferrand, France, were studied to classify them in the previously described pathogenic groups of E . coli involved in diarrheal diseases . A total of 1.5% of them belonged to the enterotoxigenic E . coli pathotype, but none belonged to the enteroinvasive E . coli, enterohemorrhagic E . coli, or enteropathogenic E . coli pathotypes . Seventeen strains (6.5%) exhibited an aggregative pattern of adhesion to HEp-2 cells (EAggEC pathotype), but of these, three (17.6%) did not hybridize with the EAggEC DNA probe . Most of the strains involved in diarrhea belonged to the diffusely adhering E . coli group; 100 strains (38.2%) exhibited a diffuse adhesion (DA) to HEp-2 cells . Only eight strains (8.9%) from controls diffusely adhered to HEp-2 cells . The highly significant difference (P < 0.0001) between DA strains from patients and from controls suggests that the diffusely adhering E . coli strains should be considered pathogens . Only 33 of them (33%) hybridized with the previously described DA DNA probe, and only 2 (2%) hybridized with the AIDA DNA probe . Four different major proteins were observed in the bacterial surface extracts of the 33 strains positive with the DA DNA probe . In addition, 16 strains that diffusely adhered to HEp-2 cells induced a cytotoxic effect on HEp-2 cells that was characterized by pyknosis and lysis of the cytoplasmic membrane . This cytotoxic effect was correlated with the synthesis of a hemolysin . The genes involved in diffuse adhesion to HEp-2 cells were located on conjugative R plasmids in strains that did not hybridize with the DA or AIDA DNA probes.

Appl Environ Microbiol, 1993 Aug, 59(8), 2760 - 2
Starved cells of the fatty acid auxotroph Escherichia coli AK7 develop abnormal sensitivity to media with low osmolarity; Scaravaglio OR et al.; The unsaturated fatty acid auxotroph Escherichia coli AK7 supplied with linolenic acid, while appearing normal during logarithmic growth, showed a fast decline in CFU during starvation as a result of an osmotic downshift when transferred to standard agar plates unsupplemented with an osmolyte such as 300 mosM sucrose or salt (NaCl or KCl) . The starved cells could recover their osmoresistance when an energy source was added to the starvation medium.

Appl Environ Microbiol, 1993 Aug, 59(8), 2758 - 9
Evaluation of indoxyl-beta-D-glucuronide as a chromogen in media specific for Escherichia coli; Haines JR et al.; Indoxyl-beta-D-glucuronide (indoxyl) was evaluated as a specific chromogen for detection of Escherichia coli by the membrane filter method . In all, 413 colonies were tested from the indoxyl-supplemented media, yielding 93.3% confirmation, as E . coli . Compared with the indoxyl medium, other media gave either much lower recovery with high verification or equal recovery with poor verification.

Appl Environ Microbiol, 1993 Aug, 59(8), 2465 - 73
Stoichiometric interpretation of Escherichia coli glucose catabolism under various oxygenation rates; Varma A et al.; Metabolic by-product secretion is commonly observed in oxygen-limited cultures . Oxygen limitations occur because of limits in the capacity of the respiratory system or because of the oxygenation limits of the cultivation method used . The latter restriction is of considerable practical importance since it results in a critical cell concentration above which oxygenation is insufficient, leading to by-product secretion . In this study we used a flux balance approach to determine optimal metabolic performance of Escherichia coli under variable oxygen limitations . This method uses linear optimization to find optimal metabolic flux patterns with respect to cell growth . Cell growth was defined as precursor requirements on the basis of a composition analysis . A growth-associated maintenance requirement of 23 mmol of ATP per g of biomass and a non-growth-associated maintenance value of 5.87 mmol at ATP per g (dry weight)-h were incorporated on the basis of a comparison with experimental data . From computations of optimal growth increased oxygen limitations were found to result in the secretion of acetate, formate, and ethanol in that order . Consistent with the experimental data in the literature, by-product secretion rates increased linearly with the growth rate . The computed optimal growth under increasing oxygen limitation revealed four critical growth rates at which changes in the by-product secretion pattern were observed . Concomitant with by-product secretion under oxygen limitations were changes in metabolic pathway utilization . The shifts in metabolism were characterized by changes in the metabolic values (computed as shadow prices) of the various redox carriers . The redox potential was thus identified as a likely trigger that leads to metabolic shifts.2+ a

Appl Environ Microbiol, 1993 Aug, 59(8), 2359 - 63
Analysis of Tox5 gene expression in Gibberella pulicaris strains with different trichothecene production phenotypes; Hohn TM et al.; The Tox5 gene encodes trichodiene synthase, the first unique enzyme in the trichothecene biosynthetic pathway . In Gibberella pulicaris R-6380, the level of Tox5 mRNA was found to increase 47-fold in early stationary phase . Sequence analysis of the Tox5 promoter regions from geographically distinct strains of G . pulicaris revealed the existence of two Tox5 alleles (Tox5-1 and Tox5-2) . All G . pulicaris strains that produce high levels of trichothecenes in liquid culture carry a 42-nucleotide (nt) tandem repeat sequence (Tox5-1) in the Tox5 promoter region, whereas strains that produce low levels of trichothecenes carry a single copy of this sequence (Tox5-2) . A genetic cross between high- and low-level trichothecene producers resulted in the cosegregation of higher-level trichothecene production with the Tox5-1 allele . To determine the importance of the 42-nt repeat sequence in the regulation of Tox5 expression, reporter gene constructs carrying either the Tox5-1 or the Tox5-2 promoter region fused to the beta-galactosidase gene of Escherichia coli were introduced into the high-level-trichothecene-producing strain, R-6380 . Expression of reporter gene activity in transformants was found to be regulated in a manner similar to Tox5 expression but appeared to be independent of the 42-nt sequence copy number . These results indicate that transcriptional controls play an important role in the regulation of Tox5 expression and that genes involved in trichothecene biosynthesis in G . pulicaris may be linked to Tox5.

Appl Environ Microbiol, 1993 Aug, 59(8), 2347 - 51
Analysis of the Bradyrhizobium japonicum hemH gene and its expression in Escherichia coli; Frustaci JM et al.; Complementation analysis showed that the Bradyrhizobium japonicum hemH gene was both necessary and sufficient to rescue mutant strains I110ek4 and I110bk2 in trans with respect to hemin auxotrophy, protoporphyrin accumulation, and the deficiency in ferrochelatase activity . The B . japonicum hemH gene was expressed in an Escherichia coli T7 expression system and yielded a 39-kDa protein, which was consistent with the predicted size of the deduced product . The overexpressed protein was purified and shown to contain ferrochelatase activity, thereby demonstrating that the hemH gene encodes ferrochelatase . When expressed from the lac promoter, the B . japonicum hemH gene was able to complement the enzyme activity of a ferrochelatase-defective E . coli mutant, and it also conferred hemin prototrophy on those cells . These latter findings confirm the identity of the hemH gene product and demonstrate that B . japonicum ferrochelatase can interact with the E . coli heme synthesis enzymes for heme formation in complemented cells.

Am J Physiol, 1993 Aug, 265(2 Pt 2), H767 - 9
Calcitonin gene-related peptide mediates hypotension and tachycardia in endotoxic rats; Huttemeier PC et al.; Circulating calcitonin gene-related peptide (CGRP) concentrations are elevated in experimental and clinical sepsis . CGRP causes hypotension and tachycardia, suggesting that the peptide might mediate the acute circulatory changes in sepsis . To test this hypothesis we administered Escherichia coli endotoxin (8 mg/kg iv) to Nembutal- (pentobarbital sodium; 50 mg/kg) anesthetized rats . Endotoxin caused hypotension and tachycardia within 60 min that stabilized for 90 min . After 2 h more severe hypotension developed, and 80% of rats died spontaneously after 3 h . In other endotoxic rats we administered 20 nmol of the CGRP receptor antagonist hCGRP (8-37) intravenously at 60 min . hCGRP (8-37) transiently reversed tachycardia (from 469 +/- 11 to 407 +/- 7 beats/min, P < 0.05) and increased mean blood pressure (from 63 +/- 4 to 93 +/- 11 mmHg, P < 0.05) over 30 min, after which hemodynamics and survival rates were no different from untreated animals . The results suggest that CGRP plays an important role in the acute circulatory changes of endotoxemia . More detailed work is necessary to determine the effects of CGRP antagonism on cardiac function, regional blood flow, and overall survival rates in sepsis.

Radiat Res, 1993 Aug, 135(2), 229 - 33
Exonuclease I of Escherichia coli removes phosphoglycolate 3'-end groups from DNA; Sandigursky M et al.; Exonuclease I of E . coli is a 3'-->5' exonuclease acting on single-stranded DNA . We further demonstrate that the enzyme can remove phosphoglycolate groups at 3' termini in DNA . These types of lesions are introduced into DNA by agents that cause oxidative damage such as ionizing radiation . An oligonucleotide substrate pd(T)20{32P}dA was treated with acid to remove the adenine base to generate 3' termini containing 2-deoxyribose-5-phosphate end groups . This substrate was then treated with periodate to generate 3'-phosphoglycoaldehyde groups and was further oxidized with I2 to generate 3'-phosphoglycolate groups . The pd(T)20{32P}PGA substrate was annealed to pd(A)40-60 to produce a double-stranded substrate . Exonuclease I was effective in the removal of the 3'-phosphoglycolate groups from this substrate as determined by HPLC separation . With exonuclease III and endonuclease IV of E . coli, exonuclease I is the third activity found in E . coli that is able to excise deoxyribose-phosphate fragments at 3' termini in DNA . These sugar fragments are blocks to DNA polymerase, and their removal is necessary to complete the base excision repair process.

Mol Immunol, 1993 Aug, 30(12), 1077 - 87
Purification and immunochemical characterization of recombinant and native ragweed allergen Amb a II; Kuo MC et al.; The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described {Rogers et al . (1991) J . Immun . 147, 2547-2552; Griffith et al . (1991a), Int . Archs Allergy appl . Immun . 96, 296-304} . In this study, we present results generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both . The specificity of two anti-Amb a II antipeptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb aI.1 . These two sera, directed against separate regions of the Amb a II molecule, detected three individual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragweed pollen extract . These Amb a II isoforms have pI in the 5.5-5.85 range and can be easily distinguished from Amb a I isoforms with pI in the 4.5-5.2 range detected by an anti-Amb a I specific peptide antiserum . The Amb a II isoforms have also been individually purified from pollen, positively identified as Amb a II by amino acid sequencing, and visualized as separate bands on IEF gels . An analysis of Amb a II cDNA sequences generated by PCR led to the prediction of three Amb a II isoforms with pI of 5.74, 5.86 and 5.97 that are very similar to the pI deduced from 2D Western blot analysis . Recombinant Amb aI.1 and Amb a II have been expressed in E . coli, purified in their denatured form, and examined by ELISA for their capacity to bind pooled allergic human IgE . Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties . In contrast, Amb a II had a markedly reduced IgE-binding capacity as compared to Amb a I.1 . These data suggest that recombinant Amb a I.1 and Amb a II, isolated in a denatured form, differ significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.

Leukemia, 1993 Aug, 7 Suppl 2, S13 - 7
Identification of P-glycoprotein/multidrug resistance genes from model organisms; Allikmets R et al.; Using degenerate oligonucleotides from conserved portions of the ATP-binding domain of the active transporter genes, several new members of this gene superfamily have been cloned from Drosophila, Saccharomyces cerevisiae, and E . coli DNA . The Drosophila and E . coli genes contain two sets of transmembrane domains and two ATP-binding domains, whereas the yeast gene contains single transmembrane and ATP-binding domains . All three genes show a high degree of similarity to the mammalian P-glycoprotein/multidrug resistance (MDR) genes . The E . coli sequence is the only known transporter gene containing both ATP and transmembrane domains in a single open reading frame . While the function of these sequences has not been determined, they may prove to be useful for developing a model to study the function of P-glycoproteins.

J Leukoc Biol, 1993 Aug, 54(2), 133 - 7
Suppressive effect of interleukin-4 on the differentiation of M1 and HL60 myeloid leukemic cells; Yamashita U et al.; The effect of interleukin-4 (IL-4) on the proliferation and differentiation of myeloid leukemic cell lines was studied in vitro . A culture of murine myeloid cell line, M1, with lipopolysaccharide (LPS) from Escherichia coli, induced differentiation into macrophages that expressed Fc receptors and phagocytic activity . IL-4 did not induce the differentiation of M1 cells but inhibited the differentiation of M1 cells induced with LPS . On the other hand, LPS arrested the proliferation of M1 cells . IL-4 had no effect on the proliferation of M1 cells but restored the LPS-induced arrest of the proliferation of M1 cells . IL-1, IL-6 and tumor necrosis factor alpha (TNF-alpha) also induced the differentiation of M1 cells into macrophages and arrested proliferation . IL-4 suppressed the IL-1-, IL-6-, and TNF-induced differentiation of M1 cells and restored the arrested proliferation with IL-1, IL-6, and TNF . Similar results were obtained with human myeloid cell line HL60 . These results suggest that IL-4 has a suppressive effect on the differentiation of myeloid cells into macrophages.

J Dent Res, 1993 Aug, 72(8), 1171 - 79
Characterization of Actinomyces with genomic DNA fingerprints and rRNA gene probes; Bowden G et al.; Cellular DNA from 25 Actinomyces naeslundii and Actinomyces viscosus strains belonging to the 7 taxonomic clusters of Fillery et al . (1978) and several unclustered strains was obtained by enzymatic and N-lauroylsarcosine/guanidine isothiocyanate treatment of whole cells, followed by extraction of the nucleic acid . The DNA samples were digested with restriction endonucleases BamHI or PvuII, and agarose gel electrophoresis was used to obtain DNA fingerprints . The DNA fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA . The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for comparison between the taxonomic cluster strains and strains within clusters . Representative strains from each taxonomic cluster provided different BamHI DNA fingerprints and ribotype patterns with 3 to 9 distinct bands . Some strains within a cluster showed identical ribotype patterns with both endonucleases (A . naeslundii B120 and A . naeslundii B102 from cluster 3), while others showed the same pattern with BamHI but a different pattern with PvuII (A . naeslundii ATCC 12104 and 398A from cluster 5) . A viscosus ATCC 15987 (cluster 7) and its parent strain T6 yielded identical fingerprint and ribotype patterns . The genomic diversity revealed by DNA fingerprinting and ribotyping demonstrates that these techniques, which do not require phenotypic expression, are suited for study of the oral ecology of the Actinomyces, and for epidemiological tracking of specific Actinomyces strains associated with caries lesions and sites of periodontal destruction.

Mol Gen Genet, 1993 Aug, 240(2), 296 - 301
Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele; Cazaux C et al.; Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities . Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264-->Glu) and recA665 (Tyr264-->His) bearing mutations at this site . As expected both mutations affected all RecA activities in vivo . Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways . Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.

Mol Gen Genet, 1993 Aug, 240(2), 286 - 9
The gdhA1 point mutation in Escherichia coli K12 CLR207 alters a key lysine residue of glutamate dehydrogenase; Jones KM et al.; gdhA1 is a spontaneous mutant of Escherichia coli that causes complete loss of activity of the NADP-specific glutamate dehydrogenase (GDH) encoded by the gdhA gene . The gdhA1 mutational site has been identified by recombinational mapping, polymerase chain reaction (PCR) amplification and DNA sequencing, as an A to G transition at nucleotide 274 of the gdhA coding sequence, resulting in an amino acid change of lysine 92 to glutamic acid . The mutant enzyme forms hybrid hexamers with a wild-type GDH, providing a useful system for analysis of conformational integrity of mutational variants.

Eur J Biochem, 1993 Aug 1, 215(3), 653 - 61
Folding and assembly of phage P22 tailspike endorhamnosidase lacking the N-terminal, head-binding domain; Danner M et al.; Tryptic digestion of a thermal unfolding intermediate of the phage P22 tailspike endorhamnosidase produces an N-terminally shortened protein fragment comprising amino-acid residues 108-666 {Chen, B.-L . & King, J . (1991) Biochemistry 30, 6260-6269} . In the present work, the 60-kDa C-terminal fragment was purified to homogeneity from the tryptic digest by gel-fitration chromatography . As in the case for the whole tailspike protein (72 kDa), the purified fragment was found to remain stably folded as a highly soluble, SDS-resistant, enzymatically active trimer . However, its unfolding in the presence of guanidinium chloride was accelerated at least 10-fold compared to the complete, native tailspike protein . Shortened tailspike trimers reconstituted spontaneously and with high yield after diluting a solution containing acid-urea-unfolded fragment polypeptides with neutral buffer . Upon recombinant expression of the 60-kDa polypeptide in Escherichia coli, it also assembled efficiently and formed SDS-resistant trimers . The refolding and assembly pathway of the N-terminally shortened tailspike paralleled that of the complete protein with slightly, but significantly, accelerated folding reactions, at both the subunit and the trimer levels . As found for the complete tailspike protein, yields of refolding and assembly of the 60-kDa fragments into SDS-resistant trimers decreased with increasing temperature . The refolding yield of fragments derived from a temperature-sensitive mutant (Gly244-->Arg) tailspike protein was affected in similar fashion as shown for the whole protein . We conclude that the N-terminal domain (residues 1-107) is dispensable for folding and assembly of the P22 tailspike endorhamnosidase both in vitro and in vivo.

Eur J Biochem, 1993 Aug 1, 215(3), 573 - 85
1H- and 15N-NMR assignment and solution structure of the chemotactic Escherichia coli Che Y protein; Bruix M et al.; Che Y is a 129-residue parallel alpha/beta protein involved in bacterial chemotaxis . We have used this protein as a model to study the folding reaction of parallel alpha/beta proteins . As a first step we carried out the complete assignment of the 1H and 15N spectra from Escherichia coli Che Y protein on the basis of two-dimensional 1H homonuclear and 1H-15N heteronuclear experiments by using sequence-specific methods . Our assignments differ from the preliminary assignments made by Kar et al . {Kar, L., Matsumura, P . & Johnson, M.E . (1992) Biochem . J . 287, 521-531} of aromatic residues obtained by comparison of NOEs with short proton-proton distances in the crystal structure of Che Y . The analysis of the extension of the secondary elements, as well as a preliminary calculation of the three-dimensional structure, indicate that the solution structure is closely coincident with the single crystal structure determined by X-ray diffraction.

Carcinogenesis, 1993 Aug, 14(8), 1633 - 8
Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases; Hein DW et al.; A genetic polymorphism at the NAT2 gene locus, encoding for polymorphic N-acetyltransferase (NAT2), segregates individuals into rapid, intermediate or slow acetylator phenotypes . Both rapid and slow acetylator phenotypes have been associated with increased incidence of cancer in certain target organs related to arylamine exposure, suggesting a role for acetylation in both the activation and deactivation of arylamine carcinogens . A second gene (NAT1) encodes for a different acetyltransferase isozyme (NAT1) that is not subject to the classical acetylation polymorphism . In order to assess the relative ability of NAT1 and NAT2 to activate and deactivate arylamine carcinogens, we tested the capacity of recombinant human NAT1 and NAT2, expressed in Escherichia coli XA90 strains DMG100 and DMG200 respectively, to catalyze the N-acetylation (deactivation) and O-acetylation (activation) of a variety of carbocyclic and heterocyclic arylamine carcinogens . Both NAT1 and NAT2 catalyzed the N-acetylation of each of the 17 arylamines tested . Rates of N-acetylation by NAT1 and NAT2 were considerably lower for heterocyclic arylamines such as 2-amino-3-methyl-imidazo{4,5-f}quinoline (IQ), particularly those (e.g . IQ) with steric hindrance to the exocyclic amino group . For carbocyclic arylamines such as 4-aminobiphenyl and beta-naphthylamine, the apparent affinity was significantly (P < 0.05) higher for NAT2 than NAT1 . NAT1/NAT2 activity ratios and clearance calculations suggest a significant role for the polymorphic NAT2 in the N-acetylation of carbocyclic arylamine carcinogens . Both NAT1 and NAT2 catalyzed acetyl coenzyme A-dependent O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-4-aminobiphenyl to yield DNA adducts . NAT1 catalyzed paraoxon-resistant, intramolecular N,O-acetyltransferase-mediated activation of N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylaminobiphenyl at low rates; catalysis by NAT2 was not readily detectable in the presence of paraoxon . In summary these studies strongly suggest that the human acetylation polymorphism influences both the metabolic activation (O-acetylation) and deactivation (N-acetylation) of arylamine carcinogens via polymorphic expression of NAT2 . These findings lend mechanistic support for human epidemiological studies suggesting associations between both rapid and slow acetylator phenotype and cancers related to arylamine exposure.

Biochem J, 1993 Aug 1, 293 ( Pt 3), 751 - 6
Processing of chimeric mammalian cytochrome b5 precursors in Escherichia coli: reaction specificity of signal peptidase and identification of an aminopeptidase in post-translocational processing; Harding V et al.; A chimeric precursor interlinked by an arginine residue between the full-length signal sequence of alkaline phosphatase and the eukaryotic cytoplasmic cytochrome b5 was constructed . Expression of the chimeric precursor protein in Escherichia coli resulted in efficient export of spectrally authentic cytochrome b5 into the periplasm {Karim, Harding, Evans, Kaderbhai and Kaderbhai (1993) Bio/Technology 11, 612-618} . On sequencing, the apparent absence of arginine at the N-terminus of the secreted cytochrome b5 implied that the chimera was either miscleaved by signal peptidase or further processed following signal excision by an uncharacterized peptidase . The influence of the N-terminal region of cytochrome b5 on the unusual processing of the chimeric precursor was investigated by engineering a number of variant forms in which the region between Arg+1 and the mature portion of cytochrome b5 was extended and varied . Observations of the in vivo processed patterns of these variant cytochrome b5 forms exported into the periplasm revealed that the absence of arginine was due to neither miscleavage of the translocated precursor by the signal peptidase nor the nature of the early region of cytochrome b5 . In fact, the selective excision of the arginine residue occurred subsequent to signal sequence deletion by an aminopeptidase which was sensitive to the metal chelator o-phenanthroline . We show that this aminopeptidase also participates in the trimming of the N-terminal arginine residue of the bacterial alkaline phosphatase to generate the three isoenzymes in the periplasm.

Biochem J, 1993 Aug 1, 293 ( Pt 3), 735 - 8
Cloning of the gene for glutamate decarboxylase and its expression during conidiation in Neurospora crassa; Hao R et al.; Neurospora crassa glutamate decarboxylase (GAD) is produced during conidiation and stored in dormant conidia . Polyclonal antibody was generated to GAD that had been purified to homogeneity . The anti-GAD antibody was specific for N . crassa GAD and inhibited GAD activity . The level of GAD protein decreased during conidial germination, indicating that GAD was degraded during this phase of development . The anti-GAD antibody was used to isolate a cDNA clone of GAD from a lambda ZAP cDNA expression library . Escherichia coli containing a plasmid with the cDNA insert produced GAD activity . The cDNA clone contained a 2.6 kbp insert and hybridized to a 2.6 kb mRNA species from conidiating cultures of N . crassa . GAD mRNA was not present in vegetative hyphae . In conidiating cultures, GAD mRNA was first detected when conidia began to appear . The level of GAD mRNA increased as conidiation progressed . This is the first example of the cloning of an enzyme that is regulated at the level of mRNA during the asexual developmental cycle of N . crassa.

Neurology, 1993 Aug, 43(8), 1581 - 5
Titin antibodies in myasthenia gravis: identification of a major immunogenic region of titin; Gautel M et al.; Approximately 15% of patients with myasthenia gravis (MG) have thymus neoplasia . These MG with thymoma (MGT) patients show autoantibodies to striated muscle as well as autoantibodies to acetylcholine receptor . To characterize these thymoma-associated muscle antigens, we cloned a number of immunopositive cDNAs by immunoscreening muscle cDNA libraries with sera from MGT patients . Analysis of the isolated cDNAs show that all share a common sequence encoding a distinct region of the titin gene . We expressed this main immunogenic region (MIR) of titin in Escherichia coli, and determined autoantibody serum titers directed against the obtained recombinant antigen in a variety of patients . We could detect titin MIR autoantibodies in 97% of sera from MGT patients but not in control sera from healthy blood donors . Therefore, expressed titin from the MIR of the molecule is a sensitive marker antigen for evaluating the presence of thymoma in MG.

J Clin Invest, 1993 Aug, 92(2), 984 - 92
Immunochemical and molecular characterization of anti-RNA polymerase I autoantibodies produced by tight skin mouse; Shibata S et al.; Autoantibodies against nuclear proteins like RNA polymerase I (RNA pol I) are produced in a number of rheumatic autoimmune diseases . Production of antibodies specific for the 190-kD subunit of RNA pol I appears to be characteristic in the patients with systemic sclerosis . Previous investigations have shown that the tight skin (TSK) mouse is an experimental model for systemic sclerosis . In the present study we show that the TSK mice produce high titers of anti-RNA pol I antibodies, both of IgM and IgG classes . To characterize the immunochemical properties of these antibodies we obtained a large panel of hybridomas from these mice . Analysis of these hybridomas revealed that clonal frequency of autoreactive B cells specific for RNA pol I are higher in the TSK mice that in the controls . mAbs obtained from the TSK mice were specific for the 190-kD subunit and cross-reacted with Escherichia coli and phage T7 RNA polymerases (155-, 150-, and 107-kD polypeptides) . We have also demonstrated that these antibodies bind better to the phosphorylated enzymes . The anti-RNA pol I mAbs were divided into three groups in terms of their functional property . The first group of antibodies increased the catalytic activity of the enzyme whereas the antibodies of the second group inhibited the enzymatic activity . Competitive inhibition RIAs showed that these two groups of antibodies bound to distinct epitopes . The third group of antibodies was neutral and had no activity on the enzyme function . These results suggest that TSK mouse anti-RNA pol I antibodies recognize three or more conserved epitopes . To understand the molecular basis of the generation of such autoreactive antibodies we analyzed their V gene repertoire . Northern analysis of RNAs of 14 TSK hybridomas showed that the VH genes encoding these antibodies were mainly from VH J558 family . It is possible that these genes were derived from a single germline gene or from a set of related genes of a single subgroup.

J Clin Invest, 1993 Aug, 92(2), 786 - 90
A molecular map of G protein alpha chains in microdissected rat nephron segments; Senkfor SI et al.; Membrane-associated guanine nucleotide binding proteins regulate many receptor-mediated signals . Heterogeneity of biochemical and functional properties in nephron segments could be due to differences in G protein expression . To ascertain whether such heterogeneity of G proteins is present in various nephron segments, this study examines the distribution and relative abundance of G protein alpha chains in microdissected medullary thick ascending limb, cortical collecting tubules, outer medullary collecting tubules, proximal inner medullary tubules, and distal inner medullary tubules . Reverse transcription and polymerase chain reactions were employed using oligonucleotides encoding highly conserved regions of all known alpha chains . The cDNA was sequenced for alpha chain identification . The alpha i2 versus alpha s distribution was different in the outer medullary collecting tubules, when compared with the medullary thick ascending limb (P < 0.001) or the cortical collecting tubule, the proximal inner medullary tubules, and the distal inner medullary tubules (P < 0.05) . These latter four segments did not significantly differ from each other . A similar analysis was applied to the frequently used line of kidney cells, LLC-PK1, whose exact cellular origin remains unclear . Interestingly, we detected both alpha i2 and alpha i3, while only alpha i2 was detected in the rat distal nephron . No alpha o or alpha z reverse transcription PCR products were detected . In contrast alpha 11 and alpha 14 members of the more recently described alpha q family were detected in the outer medullary collecting tubules and the proximal inner medullary tubules, respectively . We conclude that the majority of nephron segments have a relatively constant distribution of G protein alpha chains.

J Clin Invest, 1993 Aug, 92(2), 1085 - 92
Adenoviral-mediated gene transfer to rabbit synovium in vivo; Roessler BJ et al.; Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium . Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes) . Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions . In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques . Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits . Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy . High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection.

J Bacteriol, 1993 Aug, 175(16), 5281 - 5
Identification of Mycoplasma pirum genes involved in the salvage pathways for nucleosides; Tham TN et al.; Genes encoding enzymes involved in the salvage pathway for nucleosides have been cloned and sequenced from the mollicute Mycoplasma pirum . One of them, encoding deoxyriboaldolase, was functionally identified by complementation of an Escherichia coli mutant . These genes are clustered, suggesting an operon organization, and they are immediately followed by the putative gene for the triose phosphate isomerase, an enzyme used during glycolysis.

J Bacteriol, 1993 Aug, 175(16), 5242 - 52
Characterization of twenty-six new heat shock genes of Escherichia coli; Chuang SE et al.; Most organisms respond to heat by substantial alteration of the pattern of gene expression . This has been particularly well studied with Escherichia coli although the response has by no means been completely characterized . Here we report the characterization of 26 new heat shock genes of E . coli, termed hsl, discovered by global transcription analysis with an overlapping lambda clone bank . We have measured the molecular weights of the corresponding heat shock proteins and mapped each of them to within a few kilobases on the E . coli genome . In vitro, 16 of them can be activated by the E sigma 32 RNA polymerase, which specifically transcribes heat shock genes . In vivo expression kinetics of seven of eight examined new proteins were found to be similar to those of the four most studied heat shock proteins, DnaK, DnaJ, GroEL (MopA), and GroES (MopB) . In the course of this work, we confirmed that the catalytic subunit of the ATP-dependent Clp protease (also known as Ti protease), ClpP, is derived from a larger precursor protein . Possible assignments of some of the hsl genes to known proteins are discussed.

J Bacteriol, 1993 Aug, 175(16), 5186 - 92
Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector; Das Gupta SK et al.; We have constructed a promoter selection vector for mycobacteria to analyze the sequences involved in mycobacterial transcriptional regulation . The vector pSD7 contains extrachromosomal origins of replication from Escherichia coli as well as from Mycobacterium fortuitum and a kanamycin resistance gene for positive selection in mycobacteria . The promoterless chloramphenicol acetyltransferase (CAT) reporter gene has been used to detect mycobacterial promoter elements in a homologous environment and to quantify their relative strengths . Using pSD7, we have isolated 125 promoter clones from the slowly growing pathogen Mycobacterium tuberculosis H37Rv and 350 clones from the fast-growing saprophyte Mycobacterium smegmatis . The promoters exhibited a wide range of strengths, as indicated by their corresponding CAT reporter activities (5 to 2,500 nmol/min/mg of protein) . However, while most of the M . smegmatis promoters supported relatively higher CAT activities ranging from 100 to 2,500 nmol/min/mg of protein, a majority of those from M . tuberculosis supported CAT activities ranging from 5 to only about 100 nmol/min/mg of protein . Our results indicate that stronger promoters occur less frequently in the case of M . tuberculosis compared with M . smegmatis . To assess the extent of divergence of mycobacterial promoters vis-a-vis those of E . coli, the CAT activities supported by the promoters in E . coli were measured and compared with their corresponding activities in mycobacteria . Most of the mycobacterial promoter elements functioned poorly in E . coli . The homologous selection system that we have developed has thus enabled the identification of mycobacterial promoters that apparently function optimally only in a native environment.

J Bacteriol, 1993 Aug, 175(16), 5176 - 85
Genetic analysis of double-strand break repair in Escherichia coli; Takahashi NK et al.; We had reported that a double-strand gap (ca . 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models . We have now examined various mutants for this repair capacity . (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background . This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break . (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background . (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair . In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction . (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background . (v) The RecBCD enzyme does not abolish the gap repair . We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active . We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant . We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds . We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.

J Bacteriol, 1993 Aug, 175(16), 5159 - 67
Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes; Cohen G et al.; The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined . Previously, we showed that S . clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics . It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant . In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins . The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E . coli thioredoxin reductase and is partially able to accept E . coli thioredoxin as a substrate . The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins . However, in vivo it is unable to donate electrons to E . coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E . coli thioredoxin reductase . The S . clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster . They are transcribed in the same direction and separated by 33 nucleotides . In contrast, the trxA and trxB genes of E . coli, the only other organism in which both genes have been characterized, are physically widely separated.

J Bacteriol, 1993 Aug, 175(16), 5129 - 34
Roles of the GcvA and PurR proteins in negative regulation of the Escherichia coli glycine cleavage enzyme system; Wilson RL et al.; When Escherichia coli was grown in medium containing both inosine and glycine, the PurR repressor protein was shown to be responsible for a twofold reduction from the fully induced glycine cleavage enzyme levels . This twofold repression was also seen by measuring beta-galactosidase levels in cells carrying a lambda gcvT-lacZ gene fusion . In this fusion, the synthesis of beta-galactosidase is under the control of the gcv regulatory region . A DNA fragment carrying the gcv control region was shown by gel mobility shift assay and DNase I footprinting to bind purified PurR protein, suggesting a direct involvement of the repressor in gcv regulation . A separate mechanism of purine-mediated regulation of gcv was shown to be independent of the purR gene product and resulted in an approximately 10-fold reduction of beta-galactosidase levels when cells were grown in medium containing inosine but lacking the inducer glycine . This additional repression was dependent upon a functional gcvA gene, a positive activator for the glycine cleavage enzyme system . A dual role for the GcvA protein as both an activator in the presence of glycine and a repressor in the presence of inosine is suggested.

J Bacteriol, 1993 Aug, 175(16), 5078 - 90
kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus; Kornacki JA et al.; The kil-kor regulon of IncP plasmid RK2 is a complex regulatory network that includes genes for replication and conjugal transfer, as well as for several potentially host-lethal proteins encoded by the kilA, kilB, and kilC loci . While kilB is known to be involved in conjugal transfer, the functions of kilA and kilC are unknown . The coregulation of kilA and kilC with replication and transfer genes indicates a possible role in the maintenance or broad host range of RK2 . In this work, we found that a fourth kil locus, designated kilE, is located in the kb 2.4 to 4.5 region of RK2 and is regulated as part of the kil-kor regulon . The cloned kilE locus cannot be maintained in Escherichia coli host cells, unless korA or korC is also present in trans to control its expression . The nucleotide sequence of the kilE region revealed two potential multicistronic operons . The kleA operon consists of two genes, kleA and kleB, predicted to encode polypeptide products with molecular masses of 8.7 and 7.6 kDa, respectively . The kleC operon contains four genes, kleC, kleD, kleE, and kleF, with predicted products of 9.2, 8.0, 12.2, and 11.3 kDa, respectively . To identify the polypeptide products, each gene was cloned downstream of the phage T7 phi 10 promoter and expressed in vivo in the presence of T7 RNA polymerase . A polypeptide product of the expected size was observed for all six kle genes . In addition, kleF expressed a second polypeptide of 6 kDa that most likely results from the use of a predicted internal translational start site . The kleA and kleC genes are each preceded by sequences resembling strong sigma 70 promoters . Primer extension analysis revealed that the putative kleA and kleC promoters are functional in E . coli and that transcription is initiated at the expected nucleotides . The abundance of transcripts initiated in vivo from both the kleA and kleC promoters was reduced in cells containing korA or korC . When korA and korC were present together, they appeared to act synergistically in reducing the level of transcripts from both promoters . The kleA and kleC promoter regions are highly homologous and contain two palindromic sequences (A and C) that are the predicted targets for KorA and KorC proteins . DNA binding studies showed that protein extracts from korA-containing E . coli cells specifically retarded the electrophoretic mobility of DNA fragments containing palindrome A . Extracts from korC-containing cells altered the mobility of DNA fragments containing palindrome C . These results show that KorA and KorC both act as repressors of the kleAand kleC promoters . In the absence of korA and korC, expression of the cloned kleA operon was lethal to E.coli cells, whereas the cloned kleC operon gave rise to slowly growing, unhealthy colonies . Both phenotypes depended on at least one structural gene in each operon, suggesting that the operons encode genes whose products interact with critical host functions required for normal growth and viability . Thus, the kilA, kilC, and kilE loci of RK2 constitute a cluster of at least 10 genes that are coregulated with the plasmid replication initiator and the conjugal transfer system . Their potential toxicity to the host cell indicates that RK2 is able to establish a variety of intimate plasmid-host interactions that may be important to its survival in nature.

J Bacteriol, 1993 Aug, 175(16), 5049 - 56
OmpF assembly mutants of Escherichia coli K-12: isolation, characterization, and suppressor analysis; Misra R; This paper describes a novel genetic method used to isolate mutations that alter proper assembly of OmpF in the outer membrane . The thermolabile nature of assembly intermediates allowed selection of temperature-sensitive mutations within the ompF gene . A variant allele of ompF (ompF-Dex) was used because it provided a convenient selectable phenotype (Dex+) . Assembly mutants were isolated in two steps . First, amber mutations were obtained that mapped in ompF-Dex . This resulted in a Dex- phenotype . Starting with these Dex- strains, Dex+ revertants were isolated . Mutants that displayed a temperature-sensitive Dex+ phenotype were further characterized . Three such mutants possessed a single substitution within ompF that reverted the nonsense codon to a sense codon which replaced W214 with either an E or Q and Y231 with a Q residue in the mature OmpF protein . All three mutant OmpF proteins showed an assembly defect . This defect led to a substantial reduction in the amount of stable OmpF trimers with the concomitant increase of a high-molecular-weight form of OmpF which migrated at the top of the gel . Suppressor mutations were sought that corrected the assembly defect of OmpF . These extragenic suppressor mutations were mapped at 45 min on the Escherichia coli chromosome . The suppressor mutations displayed no allele specificity and were recessive to the wild-type allele . In the presence of a suppressor, mutant stable trimers appeared in an almost normal manner . The appearance of stable trimers concurred with a substantial loss of the high-molecular-weight OmpF species . At this stage, it is not clear whether the high-molecular-weight species of OmpF is a normal assembly intermediate or a dead-end assembly product . The results presented in this study raise the intriguing possibility of a chaperone-like activity for the wild-type suppressor gene product.

J Bacteriol, 1993 Aug, 175(16), 5035 - 42
Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64; Kim SR et al.; A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized . Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region . The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene . The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface . Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS . Upstream of the traA gene, a promoter sequence for sigma 70 of E . coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.

J Bacteriol, 1993 Aug, 175(16), 5028 - 34
Interplay between the membrane-associated UhpB and UhpC regulatory proteins; Island MD et al.; Expression of the Escherichia coli uhpT gene, encoding the sugar phosphate transport protein, is induced by extracellular glucose-6-phosphate and requires the function of the uhpABC regulatory genes . The UhpA and UhpB proteins are related to the response-regulator and sensor-kinase proteins of two-component regulatory systems, whereas the UhpC protein is related to UhpT and homologous transport proteins . To investigate the role of segments of the membrane-associated UhpB and UhpC regulatory proteins, a series of mutations were constructed in vitro by insertion of a 12- or 24-bp oligonucleotide linker at 44 sites within the uhpABCT locus . The effect of these mutations on regulation of a uhpT-lacZ transcriptional reporter was assayed with the mutated uhp alleles in single copy on the chromosome . All but one of the insertions in uhpA or uhpT were inactive for transcription activation or transport, respectively . In contrast, about half of the insertions in uhpB and uhpC retained Uhp expression, and insertions at four sites in uhpB and at one site in uhpC conferred high-level constitutive expression . The constitutive mutants in UhpB resulted from insertions in the nonpolar amino-terminal half of the protein, and all insertions in that half of UhpB affected Uhp expression in some manner, which suggests that the transmembrane segments of UhpB might negatively regulate the kinase activity of the carboxyl portion . The constitutive behavior of all but one of these uhpB alleles was dependent on the presence of active forms of both UhpA and UhpC, which suggests that UhpB and UhpC act jointly as a complex in the signaling process.

J Bacteriol, 1993 Aug, 175(16), 4985 - 9
Isolation and characterization of an Escherichia coli strain with a high frequency of C-to-T mutations at 5-methylcytosines; Ruiz SM et al.; We used a genetic selection system to isolate a strain of Escherichia coli with a high frequency of C-to-T transition mutations at the second C of the sequence CCAGG . Cytosines in other sequences do not mutate to thymine at a high frequency in this strain, and the frequencies of other base substitution mutations are not increased to the same extent . The gene responsible for the mutator phenotype has been mapped to 43 min on the E . coli chromosome . Several lines of evidence indicate that this gene is distinct from the very short patch repair gene vsr.

J Bacteriol, 1993 Aug, 175(16), 4970 - 8
Minimum domain of the Shiga toxin A subunit required for enzymatic activity; Haddad JE et al.; The minimum sequence of the enzymatic (A) subunit of Shiga toxin (STX) required for activity was investigated by introducing N-terminal and C-terminal deletions in the molecule . Enzymatic activity was assessed by using an in vitro translation system . A 253-amino-acid STX A polypeptide, which is recognized as the enzymatically active portion of the 293-amino-acid A subunit, expressed less than wild-type levels of activity . In addition, alteration of the proposed nicking site between Ala-253 and Ser-254 by site-directed mutagenesis apparently prevented proteolytic processing but had no effect on the enzymatic activity of the molecule . Therefore, deletion analysis was used to identify amino acid residue 271 as the C terminus of the enzymatically active portion of the STX A subunit . STX A polypeptides with N-terminal and C-terminal deletions were released into the periplasmic space of Escherichia coli by fusion to the signal peptide and the first 22 amino acids of Shiga-like toxin type II, a member of the STX family . Although these fusion proteins expressed less than wild-type levels of enzymatic activity, they confirmed the previous finding that Tyr-77 is an active-site residue . Therefore, the minimum domain of the A polypeptide which was required for the expression of enzymatic activity was defined as StxA residues 75 to 268.

J Bacteriol, 1993 Aug, 175(16), 4951 - 6
Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12; Plumbridge JA et al.; The intracellular concentration of the enzyme glucosamine-6-phosphate synthase, encoded by the gene glmS in Escherichia coli, is repressed about threefold by growth on the amino sugars glucosamine and N-acetylglucosamine . This regulation occurs at the level of glmS transcription . It is not due just to the presence of intracellular amino sugar phosphates, because mutations which derepress the genes of the nag regulon (coding for proteins involved in the uptake and metabolism of N-acetylglucosamine) also repress the expression of glmS in the absence of exogenous amino sugars.

Arch Biochem Biophys, 1993 Aug 1, 304(2), 415 - 9
Overexpression in Escherichia coli of soluble aristolochene synthase from Penicillium roqueforti; Cane DE et al.; Aristolochene synthase, a fungal cyclase which has been isolated from Aspergillus terreus and Penicillium roqueforti, catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon aristolochene . The aristolochene synthase gene (Ari1) of P . roqueforti has previously been cloned and expressed at low levels as a protein A-aristolochene synthase fusion protein in Escherichia coli . We have now used the polymerase chain reaction to amplify the aristolochene synthase coding sequence using engineered primers which produced dsDNA carrying an EcoRI restriction site and the T7 gene 10 ribosome binding site and translational spacer element immediately upstream of the ATG start codon and a BamHI site adjacent to the TAA stop codon . The PCR product was digested with EcoRI and BamHI and inserted into the multiple cloning site of the expression vector pLM1 which carried the promoter and translational leader sequence from T7 gene 10 and the E . coli rrnBT1T2 tandem transcription terminator . Cloning of the resulting construct into E . coli XL1-Blue and subcloning into the expression host E . coli BL21 (DE3) gave transformants which expressed aristolochene synthase at levels up to 40% of soluble protein when induced with isopropyl beta-D-thiogalactoside . Purification of the recombinant protein by ammonium sulfate precipitation, ion-exchange chromatography on Q Sepharose, and affinity dye chromatography on Reactive Blue 4-agarose gave homogenous aristolochene synthase which had the expected N-terminal sequence, ATSTE, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and steady-state kinetic parameters when compared to native fungal protein.

Arch Biochem Biophys, 1993 Aug 1, 304(2), 345 - 51
Steady-state kinetic evaluation of the reverse reaction for Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase; Gruys KJ et al.; Recently it has been found that the kinetic mechanism for Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) in the forward direction is random with synergistic binding of substrates and inhibitors (K . J . Gruys, M . C . Walker, and J . A . Sikorski, 1992, Biochemistry 31, 5534) . This work, however, did not address the reverse reaction with 5-enolpyruvoylshikimate-3-phosphate (EPSP) and phosphate (Pi) as substrates where a similar question of random versus ordered addition of substrates remained . Previous transient-state kinetic results led to a proposal for an equilibrium-ordered mechanism, where binding of EPSP occurs first followed by Pi (K . S . Anderson, and K . A . Johnson, 1990, Chem . Rev . 90, 1131) . Steady-state kinetic results of the reverse reaction presented here suggest that, like the forward reaction, addition of substrates occurs randomly . Initial velocity studies with EPSP and Pi show a normal intersecting pattern in the reciprocal plots, consistent with a random or steady-state-ordered mechanism, but not with equilibrium-ordered addition of substrates . Inhibition of the EPSPS reverse reaction by 5-amino-S3P or the S3P-glyphosate hybrid molecule gave the expected competitive patterns versus EPSP, but mixed noncompetitive patterns versus Pi . These results also disfavor an equilibrium-ordered model, but again are consistent with a random or steady-state-ordered mechanism . A more quantitative mechanistic analysis of the inhibition data to determine the true rather than apparent Ki values provides evidence for a random over a steady-state-ordered addition of substrates . These results in combination with previous findings lead to the conclusion that the mechanism is random addition of EPSP and Pi since it is the only possible model for substrate addition that is consistent with the cumulative data from all kinetic (transient- as well as steady-state) and direct binding studies.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7356 - 60
Fractionated nuclear extracts from hamster cells catalyze cell-free recombination at selective sequences between adenovirus DNA and a hamster preinsertion site; Tatzelt J et al.; We have explored the mechanism of adenovirus type 12 (Ad12) DNA integration because of its importance for viral oncogenesis and as an example of insertional recombination . We have used a fractionated cell-free system from nuclear extracts of hamster cells and have partly purified nuclear proteins that could catalyze in vitro recombination . As recombination partners, the 20,880- to 24,049-nucleotide Pst I D fragment of Ad12 DNA and the hamster preinsertion sequence p7 from the Ad12-induced tumor CLAC1 have proven to recombine at higher frequencies than randomly selected adenoviral or cellular DNA sequences . A preinsertion sequence might carry elements essential in eliciting recombination . Patch homologies between the recombination partners seem to play a role in the selection of sites for recombination in vivo and in the cell-free system . Nuclear extracts from BHK21 cells were prepared by incubating the nuclei in 0.42 M (NH4)2SO4 and fractionated by Sephacryl S-300 gel filtration, followed by chromatography on Mono S and Mono Q columns . The purified products active in recombination contained a limited number of different protein bands, as determined by polyacrylamide gel electrophoresis and silver staining . The most highly purified fraction IV had helicase and topoisomerase I activities . We used two different methods to assess the in vitro generation of hamster DNA-Ad12 DNA recombinants upon incubation with the purified protein fractions: (i) transfection of the recombination products into recA- strains of Escherichia coli and (ii) the polymerase chain reaction by using amplification primers unique for each of the two recombination partners . In p7 hamster DNA, the nucleotide sequence 5'-CCTCTCCG-3' or similar sequences served repeatedly as a preferred recombination target for Ad12 DNA in the tumor CLAC1 and in five independent cell-free recombination experiments.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7186 - 90
Inhibition of RNA polymerase II transcription by oligonucleotide-RecA protein filaments targeted to promoter sequences; Golub EI et al.; In the presence of RecA protein, which plays a major role in genetic recombination in Escherichia coli, an oligodeoxyribonucleotide can find its homologous counterpart in double-stranded DNA and form triple-stranded structures . A triple-stranded structure formed by an oligonucleotide with a sequence overlapping essential regulatory elements of a viral promoter, such as TATA or GC boxes, inhibited in vitro transcription driven by RNA polymerase II . An oligonucleotide with eight nucleotides homologous to its target suppressed RNA polymerase II activity in HeLa cell extracts . This procedure offers a potential alternative to the usual mutational analysis of transcriptional promoters.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7149 - 52
The discriminator base influences tRNA structure at the end of the acceptor stem and possibly its interaction with proteins; Lee CP et al.; For many tRNAs, the discriminator base preceding the CCA sequence at the 3' end is important for aminoacylation . We show that the discriminator base influences the stability of the 1.72 base pair onto which it is stacked . Mutations of the discriminator base from adenosine to cytidine or uridine make the cytidine residue in the C1-G72 base pair of mutant Escherichia coli initiator tRNAs more reactive toward sodium bisulfite, the single-strand-specific reagent . The activity of the enzyme Met-tRNA transformylase toward these and other mutant initiator tRNAs is also consistent with destabilization of the 1.72 base pair in vitro and in vivo . By influencing the strength of the 1.72 base pair, the discriminator base could affect the energetic cost of opening the base pair and modulate the structure of the tRNA near the site of aminoacylation . For some aminoacyl-tRNA synthetases and other proteins that interact with tRNA, these factors could be important for specific recognition and/or formation of the transition state during catalysis.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7129 - 33
Identification, cloning, and nucleotide sequencing of the ornithine decarboxylase antizyme gene of Escherichia coli; Canellakis ES et al.; The ornithine decarboxylase antizyme gene of Escherichia coli was identified by immunological screening of an E . coli genomic library . A 6.4-kilobase fragment containing the antizyme gene was subcloned and sequenced . The open reading frame encoding the antizyme was identified on the basis of its ability to direct the synthesis of immunoreactive antizyme . Antizyme shares significant homology with bacterial transcriptional activators of the two-component regulatory system family; these systems consist of a "sensor" kinase and a transcriptional regulator . The open reading frame next to antizyme is homologous to sensor kinases . Antizyme overproduction inhibits the activities of both ornithine and arginine decarboxylases without affecting their protein levels . Extracts from E . coli bearing an antizyme gene-containing plasmid exhibit increased antizyme activity . These data strongly suggest that (i) the cloned gene encodes the ornithine decarboxylase antizyme and (ii) antizyme is a bifunctional protein serving as both an inhibitor of polyamine biosynthesis as well as a transcriptional regulator of an as yet unknown set of genes.

J Clin Endocrinol Metab, 1993 Aug, 77(2), 433 - 8
Tissue specificity and serologic reactivity of an autoantigen associated with autoimmune thyroid disease; Ross PV et al.; A recent report has identified a new autoantigen called D1 that appears to be associated with Graves' ophthalmopathy and is expressed in the thyroid and eye muscle . To better characterize the tissue specificity and disease relevance of this antigen, we evaluated the expression of D1 RNA in various human tissues using a reverse transcriptase polymerase chain reaction assay . These studies indicate a wide tissue distribution of the messenger RNA for this antigen, including the thyroid, eye muscle, parathyroid, spleen, skeletal muscle, and uterus . There were variations in the relative amounts of specific message for D1 in the different tissues, with the uterus, thyroid, and eye muscle having the greatest amount of product per microgram of total RNA . A maltose binding protein-D1 fusion protein was expressed in Escherichia coli, purified, and used to assess serologic reactivity to D1 by Western blot . Autoantibodies to this antigen were noted in 19 of 24 (78%) of Hashimoto's disease patients, 26 of 41 (63%) of Graves' disease patients, and in 9 of 17 (53%) of normal controls . Sixty percent of Graves' disease patients with clinical ophthalmopathy had antibodies to D1, as did 63% of Graves' patients without signs or symptoms of clinical ophthalmopathy . There was no correlation between reactivity to D1 and either clinical measures of hyperthyroidism or antibody titers to thyroid peroxidase or thyroglobulin . The presence of autoantibodies to this antigen in patients with Hashimoto's disease, in Graves' disease patients without ophthalmopathy and in normal controls indicate that serologic recognition of this antigen is not restricted to patients with ophthalmopathy . In addition, the expression of messenger RNA for this antigen in multiple types of cells questions the tissue specificity of this autoantigen.

J Cell Physiol, 1993 Aug, 156(2), 421 - 7
High-level expression of cytokine-induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: purification and production of blocking antibodies; Wittwer AJ et al.; Significant levels of cytokine-induced neutrophil chemoattractant (CINC) were found in serum-free medium conditioned by a highly metastatic rat cell line, RC20 . To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits . CINC was a potent chemoattractant for rat neutrophils (EC-50 0.5 nM) . A fusion protein of glutathione-S-transferase and CINC (GST-CINC) was produced in E . coli . Anti-CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST-CINC affinity chromatography . Both goat and rabbit anti-CINC antibody preparations at 4 micrograms/mL (an 11-fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay . These antibodies have been used to develop a sensitive immunoassay for CINC . The availability of large amounts of affinity-purified blocking anti-CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells.

Exp Parasitol, 1993 Aug, 77(1), 97 - 110
Babesia bovis: characterization of the T helper cell response against the 42-kDa merozoite surface antigen (MSA-1) in cattle; Brown WC et al.; The Babesia bovis major merozoite surface antigen (MSA-1) is a 42-kDa integral membrane glycoprotein previously shown to induce immunodominant antibody responses in cattle protectively immune to B . bovis and to induce neutralizing antibody . Recent studies have also shown that MSA-1 B cell epitopes common to New World strains of B . bovis are not present in either Israel or Australia strains . To understand the potential role of this protein in protective immunity, T helper cell responses specific for MSA-1 were characterized in Babesia-immune cattle . Peripheral blood mononuclear cells from immune cattle proliferated against affinity-purified recombinant MSA-1 protein expressed in Escherichia coli . MSA-1 preferentially stimulated the growth of CD4+ T cells in cell lines cultured with antigen for 4 weeks . MSA-1-reactive cell lines responded to a membrane fraction of B . bovis merozoites, suggesting recognition of the native protein . However, B . bovis-reactive T cell lines and T helper clones established by stimulation with crude parasite membrane antigen failed to respond to recombinant MSA-1, indicating that this antigen is not immunodominant for T cells . The majority of MSA-1-specific T helper clones reacted to unfractionated merozoite membrane antigen from New World B . bovis strains, but none of the clones responded to Australia B . bovis or to a Mexico strain of Babesia bigemina . Several T helper clones produced low levels of cytokines when stimulated with concanavalin A and interleukin-2 . Northern blot analysis revealed the expression of interleukin-2, interleukin-4, interferon-gamma, and tumor necrosis factor-alpha messenger RNA in mitogen-stimulated T helper clones, showing that the clones examined expressed an unrestricted T helper phenotype . We conclude that the MSA-1 protein, although serologically immunodominant and capable of inducing neutralizing antibodies as well as a T helper cell response, is not an immunodominant T cell antigen . Furthermore, the parasite strain specificity of the Th clones supports previous findings of extensive polymorphism in the MSA-1 glycoprotein and suggests that like B cell epitopes, T cell epitopes reside in a nonconserved portion of the protein.

EMBO J, 1993 Aug, 12(8), 3333 - 8
Identity determinants of human tRNA(Ser): sequence elements necessary for serylation and maturation of a tRNA with a long extra arm; Achsel T et al.; Recently, there has been much progress in understanding tRNA identity, i.e . in elucidating the sets of nucleotides that are responsible for the specific aminoacylation of a tRNA with its cognate amino acid . Interest focused, however, on tRNAs from Escherichia coli and yeast . Here we have identified the major and minor determinants of human tRNA(Ser) which were revealed by an identity switch from human tRNA(Val) to tRNA(Ser) . We used in vitro transcripts and subsequent aminoacylation by HeLa S100 extract to determine the kinetic parameter Vmax/Km . The two major identity elements which are absolutely required for aminoacylation by human seryl-tRNA synthetase are the discriminator base and the long extra arm . This is in contrast to E . coli tRNA(Ser) where the discriminator base is unimportant, whereas identity determinants in the acceptor stem are required . Other sequence elements have an influence not only on serylation, but also on tRNA maturation in vitro, i.e . on pre-tRNA processing and base modification . These nucleotides are located in the DHU and the T phi C arm and are probably necessary for the proper folding of tRNAs containing a long extra arm . A34 to inosine modification depends highly on the correct three-dimensional structure of the tRNA, whereas A58 to m1A methylation does not rely on the three-dimensional folding of the substrate . This is the first tRNA identity switch involving the exchange of a short versus a long extra arm.

EMBO J, 1993 Aug, 12(8), 3287 - 95
Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli; Asai T et al.; Escherichia coli cells induced for the SOS response express inducible stable DNA replication (iSDR) as an SOS function . Initiation of iSDR is independent of transcription, translation and DnaA protein, which are essential for initiation of DNA replication from oriC . We found that a recA mutant that is defective in recombination but proficient in SOS induction could not elicit iSDR . In contrast, iSDR was enhanced by recD and recJ mutations that inactivate the exonuclease V activity of the RecBCD enzyme and the RecJ exonuclease activity, respectively . A mutation in the ruvC gene that blocks the resolution of recombination intermediates (i.e . Holliday structures) also enhanced iSDR . Furthermore, inhibition of branch migration by recG or ruvAB mutations dramatically increased the iSDR activity . recBC mutants are defective in iSDR induction but the defect was suppressed by a mutation in the sbcA gene . The major product of minichromosomes replicated by iSDR was covalently closed circular monomers . We propose that recombination intermediates (i.e . D-loop structures) created by the action of RecA recombinase and RecBC(D) helicase play a central role in initiation of iSDR.

Endocrinology, 1993 Aug, 133(2), 815 - 21
Endotoxin-induced corticotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is mediated centrally by interleukin-1; Kakucska I et al.; In the acute phase of bacterial infection, a variety of cytokines, including interleukin-1 (IL-1), are elicited by bacterial endotoxin in both the periphery and the central nervous system . Bacterial endotoxin has been previously reported to profoundly activate the hypothalamic-pituitary-adrenal axis, resulting in elevated glucocorticoid secretion that may serve an important role as part of the inhibitory feedback mechanisms on the activated immune system . To determine whether IL-1 acts within the brain to mediate endotoxin-induced CRH gene expression in the hypothalamic paraventricular nucleus (PVN), we studied the effect of administering the human IL-1 receptor antagonist (IL-1ra) into the brain, a competitive inhibitor of IL-1, on CRH gene expression in the PVN after systemic lipopolysaccharide (LPS) treatment . Eight hours after the ip administration of LPS, the paraventricular CRH mRNA content was elevated 3-to 4-fold (P < 0.01) compared to the control value, and this elevation could be completely abolished by central IL-1ra pretreatment (P < 0.05 compared to LPS-treated group; P > 0.05 compared to controls) . In contrast, systemic IL-1ra administration did not inhibit endotoxin-induced CRH gene expression in the PVN . These studies demonstrate that LPS stimulates hypothalamic CRH by a mechanism that involves the action of IL-1 within the central nervous system and may proceed independently of peripheral actions of IL-1 circulating in the bloodstream.

Arch Surg, 1993 Aug, 128(8), 920 - 4
Endotoxin stimulates lymphocyte glutaminase expression; Sarantos P et al.; BACKGROUND/HYPOTHESIS: Glutamine is the principal fuel used by lymphocytes . It is hydrolyzed by the glutaminase enzyme, which regulates the rate of intracellular glutamine metabolism . Since lymphocyte glutamine utilization is increased during infection to support cellular proliferation, we hypothesized that endotoxin regulates lymphocyte glutaminase expression at the molecular level . METHODS: Adult rats received Escherichia coli endotoxin (one dose of 7.5 mg/kg) or saline . Total RNA from lymphocytes in the ileocolic lymph node chain was extracted for Northern hybridization and labeled with an alpha-phosphorus 32 rat glutaminase cDNA probe . The mRNA of the constitutively expressed gene beta-actin was the control for RNA loading . Quantitation of glutaminase transcripts was determined by densitometric scanning and values were normalized to actin . Glutaminase-specific activity (nanomoles per milligram of protein per hour) and glutaminase kinetic parameters were also determined . RESULTS: Treatment with a single dose of endotoxin resulted in a 53% increase in glutaminase activity at 4 hours . Kinetic analysis showed that the increase in glutaminase activity was due to an 84% increase in Vmax (maximal enzyme velocity) with no change in Km (enzyme affinity) . Endotoxin increased glutaminase mRNA twofold at 2 hours and more than fourfold at 4 hours . The increase in message preceded the increase in activity consistent with gene transcription prior to enzyme biosynthesis . CONCLUSION/CLINICAL RELEVANCE: The increase in glutaminase activity provides lymphocytes in the mesenteric lymph nodes with more glutamine for energy and cellular proliferation during times of infection when the gut mucosal barrier may become compromised.

Am Rev Respir Dis, 1993 Aug, 148(2), 281 - 7
Tumor necrosis factor and endotoxin do not directly affect in vitro diaphragm function; Diaz PT et al.; Ventilatory pump failure can occur in the setting of severe infection . Recent in vivo studies have shown a significant decrease in diaphragm force production in rats with pneumococcal sepsis and sepsis secondary to Escherichia coli endotoxin . We hypothesized that diaphragm impairment during sepsis may be mediated by a direct effect of tumor necrosis factor-alpha (TNF) or endotoxin . To test this hypothesis we studied the mechanical characteristics of isolated rat diaphragm strips in tissue baths containing rTNF-alpha or endotoxin and compared the results with control strips . The strips were stimulated to contract isometrically in the tissue baths that were aerated with 95% O2-5% CO2 . Baseline force-frequency determinations were made at 60 min . Following this, the strips were fatigued over a 4-min period (20 Hz, 0.33-s trains, 1 train/s) and force-frequency relationships determined 30 s, 10 min, and 60 min post-fatigue . There were no significant differences found between control and experimental strips in any aspect of contractile function tested, including force-frequency characteristics, fatiguability, and recovery from fatigue . Using an isolated cell line assay (L929), we found evidence of attenuated cytotoxicity of TNF at 26 degrees C compared with 37 degrees C . Therefore, we repeated the experiments studying the effects of TNF on in vitro muscle at 37 degrees C . We once again found no effect of TNF on contractile function . We conclude that the impairment of diaphragm function during sepsis is not mediated by a direct effect of TNF or endotoxin.

Surgery, 1993 Aug, 114(2), 199 - 204; discussion 204-5
Endotoxin stimulates arginine transport in pulmonary artery endothelial cells; Lind DS et al.; BACKGROUND . The pulmonary endothelium plays an important role in the metabolism of the amino acid arginine, the exclusive precursor molecule for nitric oxide (NO) . Despite decreased circulating arginine levels, endothelial NO production is elevated during endotoxemia . However, the regulation of pulmonary artery endothelial arginine transport has not been studied . We hypothesized that endotoxin stimulates carrier-mediated arginine transport by the pulmonary endothelium . METHODS . The relative contributions of the various transport systems to total arginine transport by porcine pulmonary artery endothelial cells (PAECs) was determined by assaying the uptake of 3H-L-arginine in the presence or absence of Na+ . PAECs were then incubated with various concentrations of Escherichia coli endotoxin, and y(+)-mediated arginine transport was measured at different time points thereafter . Kinetic studies were performed over a range of arginine concentrations to determine changes in transport affinity and maximum rate of metabolism . To address the role of RNA and protein synthesis in the increased transport, uptake was measured after exposure of cells to the transcriptional inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide . RESULTS . Most (75%) of arginine transport by PAECs was mediated by the high-affinity Na(+)-independent transport system y+ . Endotoxin stimulated y(+)-mediated arginine transport by PAECs twofold to fivefold, a response that was time and dose dependent . The accelerated transport was detectable within 2 hours and maximal at 12 hours . Kinetic studies revealed that the accelerated arginine transport was the result of a 68% increase in the maximal transport velocity (1519 +/- 65 pmol/mg protein/30 sec in endotoxin-treated cells vs 903 +/- 96 in control cells; p < 0.01) without a change in transport affinity . The endotoxin-mediated increase in arginine uptake was abrogated by actinomycin D and cycloheximide . CONCLUSIONS . Endotoxin stimulates Na(+)-independent arginine transport by PAECs through a process that requires de novo RNA and protein synthesis, possibly of the transporter itself . This response may be designed to support arginine-dependent biosynthetic pathways in the lung during septic states.

J Neurosci, 1993 Aug, 13(8), 3238 - 51
Targeted ablation of diverse cell classes in the nervous system in vivo; Nirenberg S et al.; The study of both the function and development of complex neural systems would be greatly facilitated by a means for systematically blocking intercell communication . One way of preventing cells from signaling each other is to remove them from the system by ablation . Here we present a general technique for visualizing and ablating selected cell classes in vivo . The cells of interest are genetically engineered so that they can be selectively labeled with a photoactivatable dye and visualized in living preparations; the dye-labeled cells can then be photoablated . This approach is applicable to a broad range of cell types, in organisms amenable to gene transfer, and permits ablations to be performed at different developmental stages or in the adult . We demonstrate the use of this technique on several cell types in the mouse retina and cerebral cortex, and in the zebrafish embryo.

Mol Cell Biol, 1993 Aug, 13(8), 4986 - 98
Alpha 2-chimerin, an SH2-containing GTPase-activating protein for the ras-related protein p21rac derived by alternate splicing of the human n-chimerin gene, is selectively expressed in brain regions and testes; Hall C et al.; n-Chimerin (alpha 1-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p21rac . We now report the occurrence of another form of chimerin, termed alpha 2-chimerin . This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains . alpha 1- and alpha 2-chimerin mRNAs were expressed differently . In the rat brain, only alpha 1-chimerin mRNA was expressed in cerebellar Purkinje cells, although both alpha 1- and alpha 2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus . Only alpha 2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes . A 45-kDa SH2-containing chimerin corresponding to the alpha 2 form was purified from rat brain . As with Escherichia coli 45-kDa recombinant alpha 2-chimerin, purified brain alpha 2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine . The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor . To examine the relationships of alpha 1- and alpha 2-chimerin transcripts, human genomic DNA clones were characterized . In alpha 2-chimerin mRNA, a 3' splice acceptor site within exon 1 of alpha 1-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence . The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32) . It contained several splice junctions conserved with the sequence-related protein kinase C and bcr genes . alpha 2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing.

Mol Cell Biol, 1993 Aug, 13(8), 4588 - 99
A bipartite nuclear localization signal in the retinoblastoma gene product and its importance for biological activity; Zacksenhaus E et al.; The retinoblastoma gene product, p110RB1, appears to regulate cell growth by modulating the activities of nuclear transcription factors . The elements that specify the transport of p110RB1 into the nucleus have not yet been explored . We now report the identification of a basic region, KRSAEGGNPPKPLKKLR, in the C terminus of p110RB1, which has sequence similarity to known bipartite nuclear localization signals (NLSs) . A two-amino-acid mutation introduced into this putative NLS {to give mutant NLS(NQ)} or deletion of the entire NLS (delta NLS) abrogated exclusive nuclear localization, yielding proteins which were distributed either equally throughout the cell or predominantly in the cytoplasm . A mutant protein {NLS(NQ)/delta 22} containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110RB1 with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm . When fused to the C terminus of Escherichia coli beta-galactosidase, the RB1 NLS directed this protein to the nucleus, indicating that the motif is not only necessary but also sufficient for nuclear transport . Neither NLS(NQ) nor delta NLS was hyperphosphorylated in vivo, but both retained their abilities to interact, in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F . When transfected at multiple copy number, the NLS mutant alleles displayed reduced biological activity, measured by inhibition of growth of the osteogenic sarcoma cell line Saos-2, which has no wild-type RB1 . Naturally occurring mutations and deletions in exon 25 of RB1 which disrupt the NLS may lead to partial or complete inactivation of p110RB1 and may be responsible for some retinoblastoma and other tumors.

J Infect Dis, 1993 Aug, 168(2), 476 - 9
Susceptibility to hemolytic-uremic syndrome relates to erythrocyte glycosphingolipid patterns; Newburg DS et al.; Hemolytic-uremic syndrome (HUS) is usually preceded by enteric infection by Shiga-like toxin-producing Escherichia coli (SLT-EC), but most children with SLT-EC diarrhea do not develop HUS . SLT toxicity depends on entry into the target cell via its host cell glycolipid receptor, globotriaosylceramide (Gb3) . The relationship between differential susceptibility to HUS and erythrocyte Gb3 levels, as measured by high-pressure liquid chromatography, was studied . Erythrocytes of children with histories of HUS had lower nonhydroxylated fatty acyl (NFA) Gb3 levels than did erythrocytes of controls (1.6 vs . 2.0 nmol/mL of packed cells); these erythrocytes had lower ratios of NFA-Gb3 to lactosylceramide (0.16) than did erythrocytes of SLT-EC diarrheal patients without subsequent HUS (0.30; P < .003) or of healthy controls (0.28; P < .001) . The lower erythrocyte Gb3 levels associated with HUS may reflect a genetic predisposition for differential outcomes of SLT-EC gastroenteritis.

J Bacteriol, 1993 Aug, 175(15), 4917 - 21
Menaquinone (vitamin K2) biosynthesis: cloning, nucleotide sequence, and expression of the menC gene from Escherichia coli; Sharma V et al.; The benzenoid aromatic compound o-succinylbenzoic acid is formed by dehydration of the prearomatic compound 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid by the enzyme o-succinylbenzoate synthase, encoded by the menC gene . A 1.3-kb PstI-PvuII fragment was found to complement the menC mutation . The complete nucleotide sequence of this fragment revealed a single open reading frame of 954 bp capable of encoding a 35-kDa protein . A consensus sequence for a ribosomal binding site but no promoter consensus sequences were found . However, the first base of the initiating codon of this open reading frame overlaps the upstream menB gene termination codon, suggesting an operon-like organization for these genes . Consistent with this suggestion, the menB promoter can initiate transcription of the menC gene.

J Bacteriol, 1993 Aug, 175(15), 4834 - 42
Recombinant expression of the pufQ gene of Rhodobacter capsulatus; Fidai S et al.; Genetic studies have shown that the expression of the pufQ gene is required for normal levels of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus . Yet, the exact function of the pufQ gene is unknown, and a pufQ gene product has never been isolated . We describe the recombinant overexpression of pufQ in Escherichia coli, as well as the purification and characterization of its gene product, the 74-amino-acid PufQ protein . Site-directed mutagenesis was used to facilitate the cloning of the pufQ gene into various expression vector systems of E . coli, including pKK223-3, pLcII-FX, and pMal-c . Although high levels of pufQ transcription were evident from constructs of all three vectors, high levels of protein expression were apparent only in the pMal-c system . In vector pMal-c, the recombinant PufQ protein is expressed as a fusion with an amino-terminal maltose-binding domain . After affinity purification on an amylose column, full-length PufQ protein was released from the fusion protein by limited proteolysis with the enzyme factor Xa . The PufQ protein demonstrated a strong tendency to associate with phospholipid vesicles, consistent with the view that it is an integral membrane protein . The PufQ protein was subsequently purified by high-performance liquid chromatography and identified by amino-terminal sequence analysis . A possible role for the PufQ protein in the transport of bacteriochlorophyll biosynthetic intermediates is discussed.

J Bacteriol, 1993 Aug, 175(15), 4699 - 711
The hmc operon of Desulfovibrio vulgaris subsp . vulgaris Hildenborough encodes a potential transmembrane redox protein complex; Rossi M et al.; The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp . vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2 . Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W . B . R . Pollock, M . Loutfi, M . Bruschi, B . J . Rapp-Giles, J . D . Wall, and G . Voordouw, J . Bacteriol . 173:220-228, 1991) . Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli . Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase . Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of {Fe} hydrogenase from Desulfovibrio species . Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence . The expression of individual genes in E . coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1 . Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D . desulfuricans G200, indicating this region to contain the hmc operon promoter . The expression of two truncated hmc genes in D . desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc . We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate . The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.

J Bacteriol, 1993 Aug, 175(15), 4625 - 30
Characterization of cytR mutations that influence oligomerization of mutant repressor subunits; Barbier CS et al.; In Escherichia coli, the transport and catabolism of nucleosides require expression of the genes composing the CytR regulon . The role of the CytR repressor in transcriptional regulation has been examined through a study of mutant CytR proteins . Two important and interrelated CytR mutants are encoded by cytR delta M149, a dominant negative allele, and cytRC289R . Studies with CytR delta M149 indicated that the native, repression-competent CytR protein is multimeric while the CytR amino acid substitution C-289-->R has been proposed to affect subunit oligomerization on the basis of its ability to suppress the transdominance of CytR delta M149 . The present study identifies other CytR amino acid residues proximal to Cys-289 that may also participate in normal subunit oligomerization . Mutations in these CytR residues, cytRA307P, cytRM308R, and cytRL309P, encoded inactive repressors in a CytR- background and, when combined with cytR delta M149, yielded hybrid repressors that were recessive in a CytR+ genetic background . Because the stability and solubility observed for the new, mutant CytR proteins and the wild-type CytR protein were indistinguishable, these residue replacements, like the C-289-->R substitution, are envisaged as being located at the subunit interface and thus suppress the CytR delta M149 transdominance by blocking efficient and stable assembly of wild-type and hybrid CytR subunits . The assignment of CytR amino acids to a protein region involved in subunit association is also consistent with the observations that these CytR amino acids are roughly colinear with regions of the LacI repressor that influence monomer-dimer association and would be surface located by alignment to the E . coli galactose-binding protein crystal structure.

Infect Immun, 1993 Aug, 61(8), 3544 - 7
Conjugal transfer of broad-host-range incompatibility group P and Q plasmids from Escherichia coli to Actinobacillus actinomycetemcomitans; Goncharoff P et al.; The first example of conjugal transfer of DNA from Escherichia coli to the periodontal pathogen Actinobacillus actinomycetemcomitans is presented . Derivatives of the incompatibility group P (IncP) plasmid RK2 successfully transferred from an E . coli donor to an A . actinomycetemcomitans recipient . The resulting A . actinomycetemcomitans transconjugants transferred the plasmids back to E . coli recipients . The IncP transfer functions were also used in trans to mobilize the IncQ plasmid pBK1 from E . coli to A . actinomycetemcomitans . The IncP and IncQ plasmids both transferred into A . actinomycetemcomitans at high frequencies (0.3 to 0.5 transconjugants per donor) and showed no gross deletions, insertions, or rearrangements . Determinations of MICs of various antibiotics for the A . actinomycetemcomitans transconjugant strains demonstrated the expression of ampicillin, chloramphenicol, and kanamycin resistance determinants.

Infect Immun, 1993 Aug, 61(8), 3490 - 5
Analysis of the human antibody response to thrombospondin-related anonymous protein of Plasmodium falciparum; Scarselli E et al.; Thrombospondin-related anonymous protein (TRAP) of the malaria parasite Plasmodium falciparum shares two sequence motifs with other proteins which possess adhesive properties . Recently, findings indicate that TRAP is an antigen which contributes to antisporozoite immunity . We have cloned and expressed the TRAP coding sequences in Escherichia coli to investigate the human humoral immune response against this protein in a region of malaria endemicity of West Africa characterized by a seasonal transmission . Our results show that antibodies against TRAP are present in infected individuals . The anti-TRAP antibodies were analyzed in both a longitudinal and a prospective study . The longitudinal analysis shows seasonal fluctuations of the levels of specific antibodies as well as age-dependent quantitative differences . The immune response is long-lived in most of the adults and some of the older children but short-lived in young children . More importantly, the prospective analysis suggests that the presence of anti-TRAP antibodies in older children before the beginning of malaria transmission correlates with the subsequent control of parasite densities.

J Virol, 1993 Aug, 67(8), 4905 - 13
Location and unusual membrane topology of the immunity protein of the Escherichia coli phage T4; Lu MJ et al.; The immunity protein (Imm) encoded by the Escherichia coli phage T4 effects exclusion of phage superinfecting cells already infected with T4 . The 83-residue polypeptide possesses two long lipophilic areas (from residues 3 to 32 and 37 to 65) interrupted by a hydrophilic stretch including two positively charged residues . The charge distribution of the protein very strongly suggested that it is a plasma membrane protein with the C terminus facing the periplasm . While it could be shown that the expected location was correct, fusions of Imm to alkaline phosphatase or beta-galactosidase showed that the C terminus was at the cytosolic side of the membrane . Also, concerning function, there was almost no structural specificity to this part of the protein . Even removal of the two positively charged residues did not completely abolish function . Evidence suggesting that Imm is associated with the membrane at specific sites is presented . It is proposed that Imm is localized to the membrane with the help of a receptor and that, therefore, it does not follow the established rules for the topology of other membrane proteins . The results also suggest that Imm acts indirectly, possibly by altering the conformation of a component of a phage DNA injection site.

J Virol, 1993 Aug, 67(8), 4549 - 56
African swine fever virus encodes a serine protein kinase which is packaged into virions; Baylis SA et al.; Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases . This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase . The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved . The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro . An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions.

J Acquir Immune Defic Syndr, 1993 Aug, 6(8), 898 - 903
Prevalence and persistence of antibody titers to recombinant HIV-1 core and matrix proteins in HIV-1 infection; Janvier B et al.; Numerous studies have established the correlation between antibodies to the core protein p24 of HIV-1 and the progression of the acquired immunodeficiency syndrome . In this study, we analyzed the immune response to two recombinant gag proteins, p24 and p17, in order to evaluate their diagnostic or prognostic significance . Immune response to the immunodominant domain of the transmembrane glycoprotein gp41 was used as a reference . Sera collected from individuals from France and Burundi (Central Africa) at various CDC stages of HIV-1 infection were tested using three sandwich enzyme-linked immunoassays developed with a synthetic peptide corresponding to the immunodominant domain of gp41, SP gp41, or recombinant p24 and p17 cloned and expressed in Escherichia coli . These assays allowed detection of titer antibodies to the three cited antigens . Antibodies to SP gp41 were detected in every HIV-1-positive patient from France and Burundi, generally at a high and stable level . Results obtained with p24 confirmed the value of antibodies to p24 as a prognostic marker only in European and North American populations, since the African population had very high levels of these antibodies even at an advanced stage of the disease . They also confirmed that initial antibody response to p24 is more predictive of outcome than antibody titer change over time . Although antibodies to p17 decline during progression to AIDS, they are frequently absent in French patients at early, asymptomatic stages and therefore could not be used as a prognostic marker . In contrast, antibodies to p17 are significantly less common in African patients with AIDS when compared with symptomless HIV-1-infected African individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

Gen Physiol Biophys, 1993 Aug, 12(4), 317 - 24
Tris buffer protects DNA backbone against breakage upon irradiation with ultraviolet light; Kejnovsky E et al.; We show that Tris molecules protect DNA against nicking upon irradiation with ultraviolet light . However, the protective effect only concerns DNA backbone but not bases and it is observed in aqueous solution but not in formamide . Changes of pH or ionic strength due to Tris have no effect on the protection . The present observation has a practical importance for photofootprinting studies of DNA and its complexes with proteins but it can also serve as a basis for a development of a novel method reflecting DNA hydration and conformation.

Sheng Li Xue Bao, 1993 Aug, 45(4), 368 - 74
{The effect of trilostane and dexamethasone on serum amino acids and TNF in endotoxemic rats}; Fu CG et al.; By the use of specific endogenous GC biosynthesis inhibitor Trilostane and exogenous GC Dexamethasone (Dex), we established three rat models with similar severity of endotoxemia, but different plasma GC level . With these models we studied the effect of elevated plasma GC on serum TNF, pathological changes of vital organs and the changes in serum amino acids and glucose concentration in comparable endotoxemic condition . It was observed that plasma GC level was negatively correlated with the maximum TNF concentration, but positively correlated with the liver tyrosine transaminase activity, serum amino acids and glucose concentrations and as the serum GC level was elevated, the pathological changes of intestine, stomach, liver and lungs became less pronounced . From these results, it was supposed that in severe endotoxemia hypersecration of endogenous GC and maintenance of high level plasma GC are essential for animals to alleviate tissue injuries by reducing the overproduction of the host-derived mediators such as TNF and promoting the metabolism of amino acids and glucose.

Plant Physiol, 1993 Aug, 102(4), 1129 - 37
Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions; Paul K et al.; The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S . Knight, I . Andersson, and C.-I . Branden {1990} J Mol Biol 215: 113-160) . The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli . Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme . Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold . Removal of an additional single residue (i.e . removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold . Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme . None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2 . Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme . However, this truncated small subunit was still synthesized by the E . coli cells . These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme.

Indian J Biochem Biophys, 1993 Aug, 30(4), 218 - 23
Evidence for beta-galactosidase catalyzed hydrolysis of paranitrophenyl-beta-D-galactopyranoside anchored in cyclodextrins; Jyothirmayi N et al.; The absorption maximum of p-nitrophenol upon mixing with molar equivalents of alpha-cyclodextrin (alpha-CD) or hydroxypropyl-beta-cyclodextrin (HPB) showed nearly 5 nm shift to the longer wavelength region, indicative of complex formation, while beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD) and hydroxyethyl-beta-cyclodextrin (HEB) produced only a marginal shift of about 1-2 nm, suggestive of a weaker interaction . It has been shown by circular dichroism spectral studies that the aglycon part of p-nitrophenyl-beta-D-glycoside (PNPG) is also encapsulated by alpha-cyclodextrin . The encapsulated form of PNPG could be hydrolyzed by beta-galactosidase, the temperature and pH-optima for hydrolysis of anchored substrates being essentially identical to that of free substrate . However, small but consistent increase in Km values were obtained for alpha-cyclodextrin-, HEB- and HPB-anchored substrates . The kcat values also registered an increase for the HEB- and HPB-anchored substrates . However, there was no increase in kinetic efficiency (kcat/Km) of enzyme . The inhibition noted at higher concentrations of HEB- and gamma-cyclodextrin-anchored PNPG but not with o-nitrophenyl-beta-D-galactoside (ONPG)-cyclodextrin mixture suggests that PNPG-cyclodextrin complexes were responsible for the inhibition . Taken together, these results suggest that the enzyme catalyses the hydrolysis of anchored substrates.

J Biochem (Tokyo), 1993 Aug, 114(2), 255 - 62
The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor derived from mammalian cells; Yamanishi K et al.; The structure of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor expressed in CHO cells was investigated . The bioactive protein ({-32-153}M-CSF), expressed from a nucleotide sequence that encoded a signal peptide of 32 amino acids and N-terminal amino acids numbers 1-153, was heterogeneous in terms of molecular mass, as analyzed by SDS-PAGE, because of the presence of N-linked sugar moieties . The primary structure of the polypeptide was determined by sequence analysis and amino acid analysis of the fragments obtained from lysylendopeptidase digests of reduced and alkylated M-CSF, and from pepsin digests of the intact molecule . A sugar chain was located only at Asn-122 of the two putative sites of N-glycosylation that were present per subunit . The homodimeric structure appeared to have seven disulfide bonds, formed by inter- or intra-molecular linkages, since there were no free thiol groups in the molecule . The assignment of disulfide bonds by sequence analysis using peptide fragments indicated the combinations of Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146 . Gel-filtration analysis of Ser31{-32-153}M-CSF, in which the remaining Cys31 was replaced by Ser and which was expressed in COS cells, suggested that the mutein existed as a monomer . Our study shows that the disulfide-bond pairings of {-32-153}M-CSF that is expressed and post-translationally modified in mammalian cells are identical to those of Escherichia coli-derived {3-153}M-CSF with only one intermolecular disulfide bond, namely, Cys31-Cys31.

J Biochem (Tokyo), 1993 Aug, 114(2), 171 - 6
The alpha subunit of ATP synthase (F0F1): the Lys-175 and Thr-176 residues in the conserved sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) are located in the domain required for stable subunit-subunit interaction; Jounouchi M et al.; The sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) is conserved in nucleotide binding proteins including the alpha and beta subunits of the ATP synthase . Various mutations were introduced in the alpha Lys-175 and alpha Thr-176 residues in the sequence (Gly-Asp-Arg-Gln-Thr-Gly-Lys-Thr, residues 169-176) of the Escherichia coli ATP synthase alpha subunit . Surprisingly, single amino acid substitutions drastically affected the subunit assembly of the enzyme . The entire enzyme assembly was lost by alpha Lys-175-->Phe (or Trp) or alpha Thr-176-->Phe (or Tyr) mutation . Other mutants had similar (alpha His-175, alpha Ser-175, alpha Gly-175, alpha Ser-176, and alpha His-176 mutants) or lower (alpha Ala-176, alpha Cys-176, alpha Leu-176, and alpha Val-176 mutants) effects on assembly of the active enzyme compared with that of the wild-type . However, all these mutant enzymes except the alpha Ser-176 enzyme showed enhanced cold sensitivities and reduced stabilities at high temperature . Mutant enzymes such as alpha Gly-175 and alpha His-176 showed low multi-site (steady state) catalysis, possibly due to loss of proper subunit-subunit interactions . These results suggest that the alpha Lys-175 and alpha Thr-176 residues are not absolutely essential for catalysis, but that they, or possibly the entire conserved sequence, are located in the key domain for the subunit-subunit interactions essential for enzyme stability and steady state activity.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1993 Aug, 114(2), 167 - 70
Requirement of brain extract for the activity of brain calmodulin-dependent protein kinase IV expressed in Escherichia coli; Okuno S et al.; Calmodulin-dependent protein kinase IV (CaM-kinase IV) is a Ca(2+)-responsive multifunctional protein kinase which occurs abundantly in the brain . When cDNA for rat brain CaM-kinase IV was expressed in Escherichia coli, the enzyme was produced in a good yield, but it did not show significant activity . The inactive recombinant CaM-kinase IV was phosphorylated and became highly active on incubation with a rat brain extract in the presence of both Ca2+/calmodulin and ATP/Mg2+ . The recombinant CaM-kinase IV-activating activity in brain was one to two orders of magnitude higher than that in the other tissues examined . These observations suggest that CaM-kinase IV may undergo a posttranslational modification, probably Ca2+/calmodulin-dependent phosphorylation by CaM-kinase IV kinase, before exhibiting activity in the central nervous system.

J Toxicol Sci, 1993 Aug, 18 Suppl 3, 11 - 9
Mutagenic and clastogenic properties of (3-{3-(6-benzoyloxy-3-cyano-2- pyridyloxycarbonyl) benzoyl}-1-ethoxymethyl-5-fluorouracil) (BOF-A2), a new 5-fluorouracil derivative with anti-tumour activity; Shibahara T et al.; As part of the safety assessment of (3-{3-(6-benzoyloxy-3-cyano-2- pyridyloxycarbonyl)benzoyl}-1-ethoxymethyl-5-fluorouracil) (BOF-A2), a new 5-fluorouracil (5-FU) derivative with anti-tumour activity, its potential genotoxicity was studied in 3 different tests . BOF-A2 was negative in a reverse mutation (Ames) test in strains of S . typhimurium and E . coli . BOF-A2 induced chromosomal aberrations in Chinese hamster cells in vitro especially in the presence of exogenous metabolic activation, and was clastogenic in vivo, inducing micronuclei in mouse bone marrow . The clastogenic activity of BOF-A2 was similar to that of 5-FU.

Fiziol Zh Im I M Sechenova, 1993 Aug, 79(8), 41 - 9
{The effect of captopril on the arterial pressure, structural resistance and vascular reactivity of the kidney in rats with nephrogenic hypertension}; Rodionov IM et al.; The angiotensin-converting enzyme inhibiting agent captopryl suppressed a moderate hypertension in rats with pyelonephritis and with ureteral obstruction, but not in rats with ureteral obstruction combined with the renal artery constriction . The suppression of the hypertension was accompanied by a reversion of structural alterations in the blood vessels.

Trends Biochem Sci, 1993 Aug, 18(8), 294 - 6
Translational introns: an additional regulatory element in gene expression; Engelberg-Kulka H et al.; The linear expression of a gene can be interrupted by the well-known RNA introns and the recently discovered protein introns . In both cases, splicing mechanisms physically excise the unexpressed segments . In this article we describe a third category of introns that we call 'translational introns' . These functional introns are not excised through a splicing mechanism; instead, the translational machinery bypasses a segment of the coding sequence of an mRNA . We suggest that 'translational introns' are part of a regulatory mechanism that may sense changes in the rate of translation and thereby control the ratio of alternative gene products.

Pediatr Res, 1993 Aug, 34(2), 217 - 21
The effect of caloric supplementation on selected milk protective factors in undernourished Guatemalan mothers; Herias MV et al.; The level and avidity indices of specific antibodies against tetanus toxoid, Escherichia coli O6 and a pool of 10 common E . coli O antigens, as well as the concentration and daily output of lactoferrin and total secretory IgA (SIgA), were evaluated in the milk of moderately undernourished mothers who were in a random blind design divided into two groups and given different caloric supplementations . Group A received a high caloric supplement (500 kcal/d), and group B received a low caloric supplement (140 kcal/d) . Determinations were done using ELISA in various modifications, except for lactoferrin, which was quantified by single radial immunodiffusion . The avidity indices were investigated as an evaluation of the antibody quality . In all the parameters evaluated, the only difference found between the two groups at the end of the supplementation period was in the content of total SIgA, which was lower in group B, both in concentration and daily output . However, the SIgA remained within the normal range . Increases as well as decreases in the levels of specific IgA antibodies occurred within both groups . Avidity was decreased in group B only against one of the antigens tested . We conclude that moderate undernutrition does not impair the levels of milk antibodies, and supplementation does not enhance them but prevents the decrease in the content of total milk SIgA . There is a suggestion that the avidity of certain antibody specificities could be hampered.

Pediatr Res, 1993 Aug, 34(2), 182 - 6
Escherichia coli 0111 B4 lipopolysaccharide given intracisternally induces blood-brain barrier opening during experimental neonatal meningitis in piglets; Temesvari P et al.; Neonatal bacterial meningitis remains a life-threatening infection, and severe neurologic sequelae may be left in survivors as well . The goal of the study was to develop and characterize a porcine model of the disease with intravital observation of the permeability changes in cerebral microvessels . Eighteen newborn piglets were given doses of 0 ng (group 1), 20 ng (group 2), and 200 ng (group 3) of Escherichia coli 0111 B4 endotoxin (LPS) intracisternally (n = 6 in each group) . Cardiovascular parameters were without changes, but a compensated metabolic acidosis occurred in group 3 4 h after LPS injection . Using the open cranial window technique combined with fluorescence excitation, there was no blood-brain barrier leakage in pial-arachnoid microvessels for sodium fluorescein during the 4 h of experiments in group 1 piglets, whereas spotty extravasations occurred in group 2 and in group 3 after the LPS injections (70.5 +/- 10.5 and 55.2 +/- 4.1 min, respectively, mean +/- SEM) . A dose-dependent increase in sodium fluorescein uptake in brain regions examined (parietal and occipital cortex, cerebellum, and periventricular white matter) was also found by fluorescence spectrophotometry . LPS-treated piglets had developed pleocytosis . Four h after the challenge, the white blood cell counts in cerebrospinal fluid were (mean +/- SD): group 1, 8.2 +/- 7.6 microL-1; group 2, 453 +/- 703 microL-1; and group 3, 1 027 +/- 620 microL-1, respectively, whereas there was no change in white blood cell count of peripheral blood samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropathol Appl Neurobiol, 1993 Aug, 19(4), 324 - 8
Ependymal cells of the choroid plexus express tumour necrosis factor-alpha; Tarlow MJ et al.; Tumour necrosis factor-alpha (TNF alpha) is a major proinflammatory cytokine which appears in the cerebrospinal fluid very early after endotoxin challenge, and is likely to be produced locally . Following in vivo and in vitro challenge with endotoxin, we have demonstrated immunocytochemically and by in situ hybridization that pig and guinea-pig choroid plexus ependymal cells can produce TNF alpha . Immuno-electron microscopy shows that this protein is localized within ependymal cells to the cytoplasm and microvilli . We suggest that this TNF alpha may be important in the initiation of the inflammatory response in bacterial meningitis.

Mol Biochem Parasitol, 1993 Aug, 60(2), 265 - 72
Characterization of a divergent glycosomal microbody phosphoglycerate kinase from Trypanosoma brucei; Alexander K et al.; There are 3 loci in the phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei . The PGK-A gene product, which we term 56PGK, is targeted to glycosomal microbodies and is highly homologous to the parasite's 2 known PGKs (one cytoplasmic and one glycosomal) . However, 56PGK contains an 80 amino acid insertion as well as numerous substitutions compared to the other PGKs . The complementation and kinetic analyses described here demonstrate that 56PGK is an authentic phosphoglycerate kinase--the largest yet described . When expressed in Escherichia coli, 56PGK complements the pgk- phenotype . 56PGK was expressed as a fusion protein and purified to near homogeneity . The Michaelis constants are similar to those of other PGKs, being 0.12 and 2.4 mM for Mg-ATP and 3-phosphoglycerate, respectively . As with other T . brucei PGKs, ATP but not GTP or ITP can serve as a phosphate donor during catalysis . No evidence was obtained for phosphate transfer to atypical substrates . 56PGK shows sulfate inhibition at all concentrations tested, rather than the sulfate activation observed with yeast PGK.

Mol Biochem Parasitol, 1993 Aug, 60(2), 171 - 85
Reduced purine accumulation is encoded on an amplified DNA in Leishmania mexicana amazonensis resistant to toxic nucleosides; Kerby BR et al.; Nucleoside analogs are potential anti-Leishmania agents . To better understand how these compounds might lose their effectiveness, Leishmania were independently selected for resistance to inosine dialdehyde or tubercidin . Each of the resistant cells exhibited resistance to inosine dialdehyde and tubercidin as well as to formycin B and allopurinol ribonucleoside . Resistant cells had a greatly reduced capability of accumulating exogenous adenosine, guanosine, thymidine and guanine . This decreased ability to accumulate nucleosides and at least one nucleobase appeared to be due to reduced activity of a number of distinct purine transporters, as the differences between purine metabolizing enzymes were not sufficiently different to account for the decreased accumulation capability . The resistance to toxic nucleosides and the decreased ability to accumulate purines were due to the presence in the resistant cells of an extrachromosomal DNA approximately 55 kb in size . The extrachromosomal DNA was not detected in wild-type cells or revertants which have lost resistance to toxic nucleosides . Except for a 1.2-kb difference, the extrachromosomal DNA from both independently selected resistant cells appeared to be identical . The resistant cells contained 2-4 times as much DNA homologous to the extrachromosomal DNA as compared to wild type cells . When cloned into an E . coli/Leishmania shuttle vector, a portion of the amplified DNA had the ability to confer upon wild-type cells resistance to the toxic purine nucleoside analogs tubercidin and inosine dialdehyde . These transformed cells also exhibited a decreased ability to accumulate non-toxic purine nucleosides.

Mol Microbiol, 1993 Aug, 9(4), 695 - 702
The partition (par) locus of pSC101 is an enhancer of plasmid incompatibility; Miller CA et al.; The incompatibility that pSC101-derived plasmids express toward each other is mediated by directly repeated sequences (iterons) located near the plasmid's replication origin . We report here that the pSC101 par locus, which stabilizes plasmid inheritance in dividing cell populations and alters DNA superhelicity, can function as a cis-acting enhancer of incompatibility, which we show is determined jointly by the copy number of the plasmid and the number of iterons per copy . A single synthetic 32 bp iteron sequence carried by the pUC19 plasmid confers strong pSC101-specific incompatibility in the absence of any other pSC101 sites but requires the par locus to express strong incompatibility when carried by a lower-copy-number plasmid . We propose a model by which the par locus can enhance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.

Mol Microbiol, 1993 Aug, 9(4), 681 - 6
The interaction between coumarin drugs and DNA gyrase; Maxwell A; The coumarin group of antibiotics have as their target the bacterial enzyme DNA gyrase . The drugs bind to the B subunit of gyrase and inhibit DNA supercoiling by blocking the ATPase activity . Recent data show that the binding site for the drugs lies within the N-terminal part of the B protein, and individual amino acids involved in coumarin interaction are being identified . The mode of inhibition of the gyrase ATPase reaction by coumarins is unlikely to be simple competitive inhibition, and the drugs may act by stabilizing a conformation of the enzyme with low affinity for ATP.

Mol Microbiol, 1993 Aug, 9(4), 653 - 60
The filamentous haemagglutinin, a multifaceted adhesion produced by virulent Bordetella spp; Locht C et al.; Filamentous haemagglutinin (FHA) is the major attachment factor produced by virulent Bordetella spp . Similar to the other virulence factors, its production is tightly regulated by a two-component system in response to environmental changes . Although of impressive size (c . 220 kDa), it is very efficiently released into the culture supernatant of Bordetella pertussis . Its biogenesis involves complex processing of a larger precursor with a calculated molecular mass of 370 kDa . Export of FHA into the culture medium depends on an outer membrane protein homologous to haemolysin accessory proteins . Purified extracellular FHA is able to increase the adherence of other pathogens to the host, which may contribute to super-infection in whooping cough . Although FHA- mutants colonize lungs as efficiently as the wild-type parent strains, immune responses against FHA appear to protect against colonization . Unlike many other adhesins, FHA expresses at least three different attachment activities, one specific for the CR3 integrins of macrophages, one involving a carbohydrate-binding site, specific for interactions with cilia, and a heparin-binding activity that may be important for interaction of B . pertussis with epithelial cells or extracellular matrices.

J Nat Prod, 1993 Aug, 56(8), 1246 - 54
The identification of 5-hydroxy-L-norvaline in cultures of pyridoxine auxotrophs of Escherichia coli B; Hill RE et al.; Gc-ms and hplc have been employed to identify 5-hydroxy-L-norvaline (L-2-amino-5-hydroxypentanoic acid) in the culture fluids of pyridoxine-starved cultures of two pyridoxine auxotrophs of Escherichia coli B . The production of 5-hydroxynorvaline is shown to be secondary to pyridoxine starvation of the cultures, and the quantities of 5-hydroxynorvaline are similar to those of the common protein amino acids present in these cultures . This is the first time that 5-hydroxynorvaline has been identified as a product of bacterial metabolism.

J Muscle Res Cell Motil, 1993 Aug, 14(4), 385 - 91
Identification of functioning regulatory sites and a new myosin binding site in the C-terminal 288 amino acids of caldesmon expressed from a human clone; Huber PA et al.; A partial clone of caldesmon, coding for the C-terminal 288 amino acids, was isolated from a human fetal liver cDNA library and sequenced . Expression of the clone in Escherichia coli produced a peptide called H1 (M(r) 32,549), which inhibited tropomyosin-enhanced actomyosin Mg(2+)-ATPase activity by 90% with half maximal inhibition at 0.03-0.04 mol H1 per mol actin . The inhibition could be reversed by Ca(2+)-calmodulin . H1 bound actin, Ca(2+)-calmodulin and tropomyosin and smooth muscle myosin with high affinities . This latter finding shows the presence of a second myosin-binding site in caldesmon . This was confirmed in thrombic digests of native sheep aorta and chicken gizzard caldesmon.

J Cell Sci, 1993 Aug, 105 ( Pt 4), 903 - 11
The highly divergent alpha- and beta-tubulins from Dictyostelium discoideum are encoded by single genes; Trivinos-Lagos L et al.; As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium alpha- and beta-tubulins, as well as the Dictyostelium alpha-tubulin gene . Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms . The complete alpha-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids . The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids . Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described . Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development . The highest levels of message accumulate late in germinating spores and vegetative amoebae . Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development . We have expressed the carboxy-terminal two-thirds of the alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins . These antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.

J Bioenerg Biomembr, 1993 Aug, 25(4), 331 - 7
Attempts to define distinct parts of NADH:ubiquinone oxidoreductase (complex I); Friedrich T et al.; The NADH:ubiquinone oxidoreductase (complex I) is made up of a peripheral part and a membrane part . The two parts are arranged perpendicular to each other and give the complex an unusual L-shaped structure . The peripheral part protrudes into the matrix space and constitutes the proximal segment of the electron pathway with the NADH-binding site, the FMN and at least three iron-sulfur clusters . The membrane part constitutes the distal segment of the electron pathway with at least one iron-sulfur cluster and the ubiquinone-binding site . Both parts are assembled separately and relationships of the major structural modules of the two parts with different bacterial enzymes suggest, that both parts also emerged independently in evolution . This assumption is further supported by the conserved order of bacterial complex I genes, which correlates with the topological arrangement of the corresponding subunits in the two parts of complex I.

Biochem Soc Trans, 1993 Aug, 21 ( Pt 3)(3), 727 - 30
Ribonucleotide reductases: radical enzymes with suicidal tendencies; Booker S et al.; Ribonucleotide reductases isolated from E . coli and from L . leichmannii differ considerably in their primary and quaternary structures, as well as in their cofactor requirements . Despite these differences, studies with the wt enzymes and the normal substrate, and with the wt enzymes and a variety of mechanism-based inhibitors, demonstrate amazing mechanistic similarities between the two reductases . Recent studies with five cysteine mutants of both reductases reveal strikingly similar phenotypes, indicating that, despite the differences in the primary structures, the groups involved in catalysis in both enzymes appear to be similar.

Eur J Clin Chem Clin Biochem, 1993 Aug, 31(8), 531 - 5
Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results; Victor T et al.; Despite the widespread use of polymerase chain reaction (PCR) for diagnosis of infectious diseases, the technology has not been generally introduced into routine diagnostic laboratories . One of the most serious problems which has influenced the acceptance of this technology is the occurrence of false positive PCR results . This study describes the experience, in a hospital laboratory setting, of using PCR for the diagnosis of heat-labile enterotoxin-producing E . coli, M . tuberculosis, M . paratuberculosis and human papillomavirus . Results indicate that a build-up of amplicons, generated during the amplification process in the laboratory, is the main source of PCR-contamination . Protocols are described that include both physical and chemical procedures to prevent contamination . The use of photo-induced psoralen is recommended for those laboratories already involved in PCR work where amplicons are likely to be present . An enzymatic system (uracil-N-glycosylase) was evaluated and is recommended for workers intending to start diagnostic PCR . Attention was given to simple control measures which are easily implemented in a routine diagnostic laboratory . Protocols such as these are likely to have a major impact on the introduction of PCR-based methods into routine laboratories.

Antimicrob Agents Chemother, 1993 Aug, 37(8), 1580 - 6
Treatment of hospitalized patients with complicated skin and skin structure infections: double-blind, randomized, multicenter study of piperacillin-tazobactam versus ticarcillin-clavulanate . The Piperacillin/Tazobactam Skin and Skin Structure Study Group; Tan JS et al.; We compared the efficacy and safety of two beta-lactam-beta-lactamase inhibitor combinations, namely, piperacillin-tazobactam and ticarcillin-clavulanate, in the treatment of complicated bacterial infections of skin that required hospitalization . The study was a randomized, double-blind, comparative trial involving 20 centers . The infections were classified as (i) cellulitis with drainage, (ii) cutaneous abscess, (iii) diabetic or ischemic foot infection, and (iv) infected wounds and ulcers with drainage . The clinical response rates were comparable for the two treatment regimens (61% of the patients were cured with piperacillin-tazobactam and ticarcillin-clavulanate and improvement was seen in 15 and 16% of patients treated with piperacillin-tazobactam and ticarcillin-clavulanate, respectively) . Both regimens were found to be safe and well tolerated . These data support the use of piperacillin-tazobactam for initial empiric therapy of hospitalized patients with complicated skin and skin structure infections.

Am J Vet Res, 1993 Aug, 54(8), 1230 - 4
Hemostasis in cows with endotoxin-induced mastitis; Welles EG et al.; Hemostasis was evaluated in cows with experimentally induced endotoxemia and mastitis, caused by intramammary infusion of endotoxin (1 mg) derived from Escherichia coli . Hemostatic tests included prothrombin time; activated partial thromboplastin time; thrombin time; fibrinogen, fibrin(ogen) degradation products, and platelet concentrations; and antithrombin-III and plasminogen activities . Significant alterations were observed in the mean values of most analytes (prothrombin time was increased; thrombin time was increased with subsequent decrease; activated partial thromboplastin time, fibrinogen concentration, plasminogen activity, and platelet concentration were decreased; and antithrombin-III activity and fibrin(ogen) degradation products concentration were unchanged) at 1 or more postchallenge sample collection times (3, 12, or 24 hours) after endotoxin administration, compared with mean values obtained from samples prior to endotoxin administration . These data indicated activation of hemostatic mechanisms, initiated either directly by endotoxin or by inflammatory mediators released or produced in response to endotoxin infusion.

Anal Biochem, 1993 Aug 1, 212(2), 394 - 401
A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli; Feliciello I et al.; We have developed a very efficient and rapid method for the preparation on a small or large scale of highly purified plasmid DNA from Escherichia coli . The procedure consists of five steps: (1) cell lysis by NaOH-SDS, (2) precipitation of cell lysate with 2 M potassium acetate-1 M acetic acid, (3) precipitation of the resulting supernatant with isopropanol, (4) treatment of the precipitate with RNase, and (5) a second isopropanol precipitation . The new procedure yields a plasmid DNA that is more than 90% in the supercoiled form and virtually free from proteins, RNA, and chromosomal DNA . We have thoroughly tested the method in the preparation of several thousand samples of different plasmids from various E . coli strains . We found that it consistently produced samples of plasmid DNA suitable for all routine uses such as restriction analysis, sequencing, and preparation of DNA probes for cloning and hybridization experiments . Moreover, plasmids purified by this procedure could fully replace plasmids purified on CsCl gradients for more demanding tasks such as the in vitro synthesis of RNA probes by phage RNA polymerases, the generation of deletion mutants with exonuclease III, and the transfection of mammalian cells by the calcium phosphate coprecipitation method, as tested on human fibroblasts and on CV-1 cells.

Anal Biochem, 1993 Aug 1, 212(2), 388 - 93
Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin; Mecklenburg M et al.; A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described . The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection . The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated . The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml . This corresponds to a 10-fold increase in sensitivity over the unamplified system . A recombinant human insulin-proinsulin conjugate was also tested . The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors . The implications of these results upon on-line analysis are discussed.

Alcohol Clin Exp Res, 1993 Aug, 17(4), 828 - 31
Cloning and expression of the full-length cDNAS encoding human liver class 1 and class 2 aldehyde dehydrogenase; Zheng CF et al.; The amino acid sequences of both human class 1 and 2 aldehyde dehydrogenase (ALDH) and the sequences of the genes coding for them are known . Based on this sequence data, we designed primers and isolated the full-length cDNAs encoding both isozymes from a human liver mRNA pool . cDNAs were subcloned in the plasmid pT7-7 and expressed in Escherichia coli with a yield of approximately 3 mg ALDH protein/liter of cell culture, although only one-third of the enzyme was soluble . The soluble recombinantly expressed ALDHs were purified to homogeneity using a hydroxyacetophenone-Sepharose affinity column . The mitochondrial isozyme had a subunit molecular weight of 55 kDa, an isoelectric point of 4.9, and a specific activity of 1.10 units/mg, which were in good agreement with that from the native enzyme . The expressed cytosolic isozyme had the same subunit molecular weight (55 kDa) and pI (5.4) as that reported for the native enzyme and had a specific activity of 0.26 units/mg . The expressed mitochondrial isozyme could be recognized by antibodies raised against rat mitochondrial ALDH, whereas the cytosolic isozyme could be recognized by antibody raised against horse cytosolic ALDH.

Acta Anaesthesiol Scand, 1993 Aug, 37(6), 602 - 6
Hemodynamic changes associated with thermodilution cardiac output determination in canine acute blood loss or endotoxemia; Nishikawa T; Since the technique of thermodilution (TD) cardiac output measurement, per se, causes hemodynamic alterations, the author examined whether the alterations elicited by iced injectate are augmented in the presence of acute blood loss or endotoxemia, compromised conditions frequently associated with critically ill patients . Acute blood loss (N = 8) and endotoxemia (N = 8) were induced by withdrawing arterial blood approximately 20-30 ml.kg-1 over 30 min and by a slow intravenous infusion of E . coli endotoxin 2.5-3.0 mg.kg-1 over 10 min, respectively, in anesthetized dogs . The magnitudes of decreases in mean arterial and pulmonary artery pressures during slowing of heart rate (HR) following injection of iced injectate 3 ml were slightly less in acute blood loss than in normovolemia, whereas in endotoxemia the degree of mean arterial pressure decrease during slowing of HR following iced injectate 3 ml was slightly less as compared with that before endotoxemia . However, the alterations in other hemodynamic variables following injection of iced injectate 3 ml were similar between dogs with and without acute blood loss or endotoxemia . No profound hemodynamic changes were observed during any TD cardiac output measurements under both conditions . Cardiac output estimated by TD correlated closely with pulmonary blood flow measured by electromagnetic flowmeter in endotoxemia (r > 0.9) but not during acute blood loss . These results indicate that TD cardiac output determination does not cause serious hemodynamic alterations in endotoxemia or acute blood loss, and can estimate right ventricular output accurately in endotoxemia but not in acute blood loss.

Virus Res, 1993 Aug, 29(2), 125 - 40
Identification of an infectious laryngotracheitis virus gene encoding an immunogenic protein with a predicted M(r) of 32 kilodaltons; Kongsuwan K et al.; The nucleotide sequence of an infectious laryngotracheitis virus (ILTV) gene which maps immediately upstream from the glycoprotein 60 (gp60) gene was determined . The gene, designated p32, encodes a predicted polypeptide of 298 amino acids with an estimated M(r) of 32,000 daltons . The predicted protein sequence has four potential N-glycosylation sites and a signal sequence at the N-terminal region . Amino acid residues in the NH2-terminal region of the p32 protein exhibit similarity to glycoprotein X (gX) of pseudorabies virus (PRV) and its homolog in equine herpesvirus type 1 (EHV-1) . Within the conserved (N-terminus) region, one putative N-linked glycosylation site and four cysteine residues are aligned in these proteins . These common structural features of the gX-like proteins were also found in glycoprotein G (gG) of human herpes simplex virus type 2 (HSV-2) and equine herpesvirus type 4 (EHV-4) . High level bacterial production of the p32 protein was achieved by cloning the p32 open reading frame into a pGEX-2T expression vector . Western blot analysis of the fusion protein produced in E . coli using immune chicken sera confirms that p32 protein is of viral origin and is an immunogen in birds with infectious laryngotracheitis (ILT) . An antiserum from chicken immunized with the fusion protein detected a substantial amount of p32 protein in the medium of ILTV-infected cells in Western blotting . Moreover tunicamycin treatment of cells infected with the virus indicated that p32 was glycosylated . This allows us to conclude that p32 is a glycoprotein and like gX of PRV accumulates in the medium of infected cells.

Respir Physiol, 1993 Aug, 93(2), 189 - 201
Transient ventilatory responses to endotoxin infusion in the cat are mediated by thromboxane A2; Orr JA et al.; We tested the hypothesis that ventilatory responses to endotoxin infusion in the anesthetized cat are mediated by thromboxane A2 (TxA2) . Intravenous infusion of endotoxin (1.6 mg/kg of E . coli, strain 05:B55, delivered over 1 min) in six cats elicited increases in right ventricular blood pressure (Prv) and a transient systemic hypotension . These hemodynamic changes were accompanied by an abrupt apnea, followed by a transient period of rapid, shallow breathing, Cardiorespiratory changes coincided with large increases (> 10-fold) in the plasma concentration of TxB2, the stable metabolite of TxA2 . These effects and the release of TxA2 did not occur if endotoxin was infused a second time into the same animal . In addition, animals that were pretreated with either indomethacin (n = 3; 3.0 mg/kg) or the TxA2 receptor antagonist, daltroban, (n = 4; 7.5 mg/kg) exhibited no change in Prv, arterial blood pressure, or respiration when given equivalent doses of endotoxin . We conclude that the release of TxA2 is responsible for the early pulmonary hypertension and rapid, shallow breathing observed during endotoxin infusion in the anesthetized cat . These TxA2-mediated responses are severe but transient in nature.

J Neurooncol, 1993 Aug, 17(2), 99 - 109
Partial characterization of glioma-derived growth factor 2: a novel mitogenic activity from human cell line D-54 MG; Lyon E et al.; We have shown that several human malignant glioma cell lines are stimulated by bacterial lipopolysaccharide (E . coli 0111:B4, 1 microgram/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T3, clone A31 cells . This glioma-derived growth factor (GDGF-2) acts like a 'competence' factor . Malignant glioma cell line D-54 MG constitutively produced GDGF-2, which we have partially characterized from serum-free conditioned culture medium . GDGF-2 is resistant to heat (100 degrees C, 5 min), acidic (pH 2, 2 hr) or reducing (0.5 M 2 ME, 30 min) conditions as well as exposure to RNases; however, it is sensitive to > 4 freeze-thaw cycles, alkaline (pH 11, 2 hr) conditions or pre-treatment with proteolytic enzymes . GDGF-2 had a pl of 6.8 determined by preparative isoelectric focusing, bound to DEAE, with elution at 35 and 185 mM NaCl and at 43% acetonitrile from a C4 reversed phase column . GDGF-2 activity was not neutralized by antibodies to TGF alpha, TGF beta, PDGF, VEGF or TNF alpha indicating that it is not immunochemically related to these growth factors . However GDGF-2 co-chromatographed on Superose 12 HPLC (250 x 9 mm; 5% isopropanol, 6 mM CHAPS in PBS) with a substance that suppressed growth of mink lung epithelial cells (Mv1Lu), but not BALB 3T3 cells, and could be neutralized by anti-TGF beta antibodies . GDGF-2 activity eluted from heparin columns in 0.6 M NaCl; thus, it is not a heparin binding growth factor . D-54 MG cell line produced alpha 2-macroglobulin (alpha 2M), which is known to bind TGF beta; however, immunoprecipitation of alpha 2M did not deplete TGF beta or GDGF-2 activity . Further, neither GDGF-2 or TGF beta can be dissociated into lower molecular weight active components by chromatography in high salt (2 M NaCl) or 2-ME (0.5 M) . GDGF-2 may be a novel autocrine or paracrine mitogen, stimulating mitotic division or interfering with normal cell growth regulation.

Plant J, 1993 Aug, 4(2), 385 - 98
Nucleotide sequences recognized by the AGAMOUS MADS domain of Arabidopsis thaliana in vitro; Shiraishi H et al.; The AGAMOUS gene of Arabidopsis thaliana is a homeotic gene involved in the development of stamens and carpels . This gene encodes a putative DNA-binding protein sharing a homologous region with the DNA-binding domains, MADS boxes, of yeast MCM1 and mammalian SRF . To examine the DNA-binding activity of the AGAMOUS protein, double-stranded oligonucleotides with random sequences of 40 bp in the central region were synthesized and mixed with the AGAMOUS MADS domain overproduced in Escherichia coli . Oligonucleotides which bound to the MADS domain were recovered by repeated immunoprecipitation with an antibody which recognizes the overproduced protein . From a comparison of the recovered DNA sequences, the consensus sequence of the high-affinity binding-sites for the AGAMOUS MADS domain was determined to be 5'-TT(A/T/G) CC(A/T)6GG(A/T/C)AA-3' . DNase I footprinting and methylation interference experiments showed that the MADS domain binds to this motif . Comparisons with the binding-site sequences of other MADS-box proteins revealed that the MCM1 binding-sites in a-mating type-specific promoters of Saccharomyces cerevisiae show similarities with the binding-site sequence of the AGAMOUS MADS domain . A synthetic MCM1 binding-site in the upstream region of the STE2 gene is recognized by the AGAMOUS MADS domain.

Am J Physiol, 1993 Aug, 265(2 Pt 1), G394 - 402
Peptide-regulated guanylate cyclase pathways in rat colon: in situ localization of GCA, GCC, and guanylin mRNA; Li Z et al.; Guanylate cyclases play a role in both physiological and pathological secretion in the mammalian intestine . Agents that raise guanosine 3',5'-cyclic monophosphate (cGMP) levels, such as atrial natriuretic peptide (ANP), guanylin (an endogenous intestinal peptide), or Escherichia coli heat-stable enterotoxin type a (STa; a bacterial toxin), enhance electrolyte secretion and the accumulation of luminal fluid . Although secretion in all parts of intestine is sensitive to changes in cGMP metabolism, an increasing body of evidence suggests that these responses are particularly important in proximal colon . To date, three peptide-sensitive membrane-bound guanylate cyclases {types A, B, and C (GCA, GCB, and GCC, respectively)} have been cloned from mammalian tissues . GCA responds to ANP, GCB to C-type natriuretic peptide, and GCC to guanylin and STa . Expression of these receptor/cyclase genes has not previously been investigated at the cellular level in the colon . Nucleotide probes specific for GCA, GCB, GCC, and guanylin were generated by polymerase chain reaction . These probes were used to evaluate colonic cyclase and guanylin mRNA expression in the rat . GCB mRNA is not detectable in this tissue either by in situ hybridization or by Northern blot analysis . In contrast, GCA, GCC, and guanylin mRNAs are all conspicuously expressed . With the in situ hybridization technique, GCA mRNA expression is seen in cells in the lamina propria . GCC mRNA expression is seen in epithelial cells throughout colonic crypts, and also, although at a slightly lower level, in cells of the surface epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

J Leukoc Biol, 1993 Aug, 54(2), 111 - 3
The leukocyte surface antigens CD11b and CD18 mediate the oxidative burst activation of human peritoneal macrophages induced by type 1 fimbriated Escherichia coli; Gbarah A et al.; Analysis by immunofluorescence-activated cell sorting of human peritoneal macrophages from patients undergoing intermittent peritoneal dialysis revealed that they express the CD11/CD18 surface antigens, with CD11b and CD18 as the predominant ones . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from lysates of 125I-labeled macrophages with rabbit polyclonal antibodies against the CD11a-c/CD18 complex or against CD18, revealed four radioactive bands corresponding to CD11a, CD11b, CD11c, and CD18 . Monoclonal antibodies against CD11b and CD18 inhibited by 80 and 90%, respectively, the oxidative burst activation of the macrophages by type 1 fimbriated Escherichia coli, whereas monoclonal antibodies against CD11a, CD11c, and CD43 were without effect . Our results suggest that CD11b and CD18 (receptors for C3bi) serve also as receptors for mannose-specific E . coli on human peritoneal macrophages and may be involved in the lectinophagocytosis of the bacteria by these cells.

Clin Chem, 1993 Aug, 39(8), 1573 - 89
Application of gene transfer technologies to the production of enzyme reference materials: example of gamma-glutamyltransferase; Siest G et al.; Protein reference materials are traditionally prepared by purification from mammalian or human tissues . The supply of these tissues is limited; consequently, there is a growing need for applied molecular and cellular biology technologies for the production of human recombinant proteins . This is especially true when only small amounts of the proteins are available in the tissues . We review the current knowledge necessary for high-level production of such proteins in different heterologous expression systems, using our data on gamma-glutamyltransferase (EC 2.3.2.2) as an example . We describe the steps required to achieve the expression of enzymes and other proteins in Escherichia coli, yeast, or mammalian cells . We list many of the problems investigators may face in preparing recombinant proteins, and provide information on selecting the most appropriate system as well as the most favorable experimental conditions . Depending on the expression system, recombinant proteins can potentially be obtained for most, if not all, enzymes of interest in clinical chemistry, and such proteins should possess characteristics very similar to those of the corresponding human native proteins . Studies suggest that these products can be used as reference materials in clinical chemistry laboratories.

J Bacteriol, 1993 Aug, 175(16), 5268 - 72
Genes for two subunits of acetyl coenzyme A carboxylase of Anabaena sp . strain PCC 7120: biotin carboxylase and biotin carboxyl carrier protein; Gornicki P et al.; Genes for two subunits of acetyl-coenzyme A carboxylase, biotin carboxylase and biotin carboxyl carrier protein, have been cloned from Anabaena sp . strain PCC 7120 . The two proteins are 181 and 447 amino acids long and show 40 and 57% identity to the corresponding Escherichia coli proteins, respectively . The sequence of the biotinylation site in Anabaena sp . strain PCC 7120 is MetLysLeu, not the MetLysMet found in other sequences of biotin-dependent carboxylases . The amino acid sequence of biotin carboxylase is also very similar (32 to 47% identity) to the sequence of the biotin carboxylase domain of other biotin-dependent carboxylases . Genes for these two subunits of acetyl-coenzyme A carboxylase are not linked in Anabaena sp . strain PCC 7120, contrary to the situation in E . coli, in which they are in one operon.

J Bacteriol, 1993 Aug, 175(16), 5009 - 21
Identification and transcriptional analysis of the Escherichia coli htrE operon which is homologous to pap and related pilin operons; Raina S et al.; We have characterized a new Escherichia coli operon consisting of two genes, ecpD and htrE . The ecpD gene encodes a 27-kDa protein which is 40% identical at the amino acid level to the pilin chaperone PapD family of proteins . Immediately downstream of the ecpD gene is the htrE gene . The htrE gene encodes a polypeptide of 95 kDa which is processed to a 92-kDa mature species . The HtrE protein is 38% identical to the type II pilin porin protein PapC . The ecpD htrE operon is located at 3.3 min on the genetic map, corresponding to the region from kbp 153 to 157 of the E . coli physical map . The htrE gene was identified on the basis of a Tn5 insertion mutation which resulted in a temperature-sensitive growth phenotype above 43.5 degrees C . The transcription of this operon is induced with a temperature shift from 22 to 37 or 42 degrees C but not to higher temperatures, e.g., 50 degrees C . Consistent with this result, the temperature-induced transcription was shown to be independent of the rpoH gene product (sigma 32) . The transcription of this operon was further shown to require functional integration host factor protein, since himA or himD mutant bacteria possessed lower levels of ecpD htrE transcripts . Among the three transcriptional start sites discovered, one, defined by the P2 promoter, was found to be under the positive regulation of the katF (rpoS) gene, which encodes a putative sigma factor required for the transcription of many growth phase-regulated genes.

Infect Immun, 1993 Aug, 61(8), 3536 - 9
Ehrlichia chaffeensis expresses an immunoreactive protein homologous to the Escherichia coli GroEL protein; Sumner JW et al.; A clone expressing a 58-kDa protein reactive with human convalescent-phase serum was isolated from a recombinant library of Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis . Sequencing identified two open reading frames, one encoding a 10.3-kDa polypeptide consisting of 94 amino acids and another encoding a 58-kDa polypeptide consisting of 550 amino acids . The sequences of the 10.3- and 58-kDa polypeptides were homologous to those of the Escherichia coli GroES and GroEL heat shock proteins, respectively.

C R Acad Sci III, 1993 Aug, 316(8), 793 - 8
Identification of barley oxalate oxidase as a germin-like protein; Dumas B et al.; A barley oxalate oxidase was purified to homogeneity and the N-terminal sequences of the protein and of two peptides generated by CNBr cleavage of this protein were determined . Searches for similarities in data bank revealed that the sequences are highly homologous to the amino-acid sequence of a wheat protein, germin, which is synthesized de novo during germination . The similarity of the two proteins was confirmed by showing that anti-oxalate oxidase antibodies strongly recognize germin produced in Escherichia coli . We show that like germin, oxalate oxidase is glycosylated, resistant to SDS denaturation, heat stable, and protease resistant . Moreover, oxalate oxidase activity is strongly induced during germination of barley embryos resulting from an accumulation of the protein . Thus, we conclude that barley oxalate oxidase is a germin-like protein.

Q Rev Biophys, 1993 Aug, 26(3), 225 - 331
Multi-stage proofreading in DNA replication; Beckman RA et al.; The mechanisms by which DNA polymerases achieve their remarkable fidelity, including base selection and proofreading, are briefly reviewed . Nine proofreading models from the current literature are evaluated in the light of steady-state and transient kinetic studies of E . coli DNA polymerase I, the best-studied DNA polymerase . One model is demonstrated to predict quantitatively the response of DNA polymerase I to three mutagenic probes of proofreading: exogenous pyrophosphate, deoxynucleoside monophosphates, and the next correct deoxynucleoside triphosphate substrate, as well as the response to combinations of these probes . The theoretical analysis allows elimination of many possible proofreading mechanisms based on the kinetic data . A structural hypothesis links the kinetic analysis with crystallographic, NMR and genetic studies . It would appear that DNA polymerase I proofreads each potential error twice, at the same time undergoing two conformational changes within a catalytic cycle . Multi-stage proofreading is more efficient, and may be utilized in other biological systems as well . In fact, recent evidence suggests that fidelity of transfer RNA charging may be ensured by a similar mechanism.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1993 Aug, 15(4), 250 - 4
{Study on infectious mechanism of type 1 fimbriated Escherichia coli in experimental cystitis of mice}; Jie L; The course of experimental cystitis in mice with Escherichia coli was studied by electron microscopy . The inoculated E . coli did not adhere to the intact bladder epithelium . However, when the mouse bladder was treated with trypsin, superficial cells were exfoliated, and the inoculated type 1 fimbriated E . coli adhered to the exposed intermediate cells and caused cystitis . The nonfimbriated E . coli showing no adherence to the superficial cells or intermediate cells and failed to cause cystitis . The present study demonstrated that the type 1 fimbriated E . coli play a role in causing cystitis, and the bladder superficial cells play a role in resisting the adherence of E . coli . The removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E . coli onto the bladder epithelium in vivo.

Plant Physiol, 1993 Aug, 102(4), 1337 - 40
Isolation and characterization of a cDNA clone for a harvest-induced asparagine synthetase from Asparagus officinalis L; Davies KM et al.; A full-length cDNA clone (pTIP27) encoding asparagine synthetase (AS; EC 6.3.5.4) was isolated from a cDNA library prepared from the tip section (apex to 30 mm) of Asparagus officinalis L . spears . The cDNA clone encodes an mRNA of 1978 bp, giving a derived protein of 66.5 kD molecular mass . The derived amino acid sequence is 81% homologous to AS from Pisum sativum . Only low levels of transcript for AS could be detected in growing spears, roots, or ferns . However, AS mRNA levels began to increase in the tips of harvested spears after 2 h at 20 degrees C, and in the other sections of the spear after 4 h, suggesting that all sections of the spear were responding to the same postharvest signal . The results are discussed in relation to metabolic changes occurring in harvested spears.

Pediatr Res, 1993 Aug, 34(2), 187 - 91
Effect of experimental Escherichia coli meningitis on concentrations of excitatory and inhibitory amino acids in the rabbit brain: in vivo microdialysis study; Perry VL et al.; Excessive extracellular fluid concentrations of the amino acids glutamate and aspartate play an important role in the pathogenesis of neuronal cell damage during hypoxia, hypoglycemia, and seizure . The purpose of these investigations was to test the hypothesis that bacterial meningitis causes progressive increase in excessive extracellular fluid concentrations of excitatory and inhibitory neurotransmitters . To test this hypothesis, Escherichia coli was injected intracisternally in juvenile rabbits after which neurotransmitter concentrations were measured with in vivo microdialysis . The data showed significant elevation of the excitatory amino acids aspartate and glutamate, as well as of the inhibitory neurotransmitters gamma-amino butyric acid and taurine in the excessive extracellular fluid of animals injected with E . coli compared with control animals injected with saline . However, concentrations of these excitatory and inhibitory amino acids rose late in the course of meningitis, at a time when the animals were hypotensive (mean blood pressure < or = 40 mm Hg) . These data show that the major increase in excitatory neurotransmitters during experimental meningitis occurs in association with the cerebral ischemia produced by septic shock rather than being produced by the meningitis itself.

Mol Microbiol, 1993 Aug, 9(4), 671 - 80
Regulation of the Escherichia coli heat-shock response; Bukau B; Steady-state- and stress-induced expression of Escherichia coli heat-shock genes is regulated at the transcriptional level through controls of concentration and activity of the positive regulator, the heat-shock promoter-specific subunit of RNA polymerase, sigma 32 . Central to these controls are functions of the DnaK, DnaJ, GrpE heat-shock proteins as negative modulators that mediate degradation as well as repression of activity and, in some conditions, of synthesis of sigma 32 . DnaJ has a key role in modulation since it binds sigma 32 and, jointly with DnaK and GrpE, represses its activity . Furthermore, DnaJ is capable of binding heat-damaged proteins, targeting DnaK and GrpE to these substrates, and thereby mediating DnaK-, DnaJ-, GrpE-dependent repair . It is proposed that one important signal transduction pathway that converts stress to a heat-shock response relies on the sequestering of DnaJ through binding to damaged proteins which derepresses and stabilizes sigma 32 . Damage repair ameliorates the inducing signal and frees DnaJ, DnaK, GrpE to shut off the heat-shock response.

Appl Microbiol Biotechnol, 1993 Aug, 39(6), 732 - 7
Improved leader and putative terminator sequences for high-level production of Streptomyces subtilisin inhibitor in Escherichia coli; Taguchi S et al.; A high-level production system in Escherichia coli for an alkaline serine protease inhibitor, termed Streptomyces subtilisin inhibitor (SSI), from S . albogriseolus S-3253 was established by replacing the SSI signal sequence with the OmpA signal sequence using the inducible pIN-III-ompA vector . Significant amounts of recombinant SSI, resulting from accurate cleavage of the OmpA signal peptide, were accumulated in the periplasmic space or excreted into the culture medium . The inhibitory activity of the processed protein against subtilisin BPN' was identical with that of authentic SSI . Furthermore, deletion of one of the putative dual terminators (terminator 1) resulted in a 1.9-fold increase in production . This effect on SSI gene expression efficiency was found to be governed mainly at the transcription level.

Biosci Biotechnol Biochem, 1993 Aug, 57(8), 1243 - 8
Molecular cloning and expression of proteinaceous alpha-amylase inhibitor gene from Streptomyces nitrosporeus in Escherichia coli; Sumitani J et al.; The proteinaceous alpha-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor . The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons . The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor . Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space . The amino-terminal sequence of the inhibitor produced by an E . coli transformant was identical to that of the T-76 inhibitor secreted by S . nitrosporeus . The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous alpha-amylase inhibitors.

Srp Arh Celok Lek, 1993 Aug-Dec, 121(8-12), 152 - 4
{Biologic effects and possible therapeutic use of the granulocyte-macrophage colony-stimulating factor . Modern treatment of leukopenia}; Jovanovic-Micic D et al.; This review is a brief overview of recent advances in biology as well as in potential clinical application of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) . Biologically active rhGM-CSF is a recombinant human protein expressed in Escherichia coli . GM-CSF is produced by nontransformed (T-lymphocytes, trophoblasts, keratinocytes, osteoblasts, tracheal epithelial cells, renal mesangial cells, endothelial cells, macrophages, fibroblasts, smooth muscle cells) and transformed (murine plasmocytoma, bladder carcinoma HIBY cell line, anaplastic carcinoma of the gall bladder, Yoshida sarcoma cell line, HC3T3-osteoblast cell line) cells . RhGM-CSF increases the number of circulating neutrophils, monocytes and eosinophils and increases chemotactic, microcidal killing and cytotoxic activity of monocytes and granulocytes . The present clinically relevant uses of rhGM-CSF two general areas: restoration of haematopoietic dysfunction by raising cell counts from suppressed to normal levels, and augmentation of host defence against infection . Thus, rhGM-CSF reduces risk of infections . In addition, rhGM-CSF may increase tumour cell destruction in some malignant diseases . RhGM-CSF produces dose-dependent toxicity consisting of myalgic fever, fluid retention and serosal effusions.

Mol Biochem Parasitol, 1993 Aug, 60(2), 303 - 11
Monoclonal antibodies that inhibit Plasmodium falciparum invasion in vitro recognise the first growth factor-like domain of merozoite surface protein-1; Chappel JA et al.; A major protein found on the surface of the invasive stage of the malaria parasite Plasmodium falciparum, merozoite surface protein-1 (MSP1), has been proposed as a vaccine candidate . Antibodies which recognise a single fragment of this molecule (MSP1(19)), composed of 2 regions related to epidermal growth factor (EGF), also inhibit parasite growth in vitro . It is shown by direct expression of the individual EGF-like domains in Escherichia coli, that the first domain is the target of growth-inhibitory antibodies . A single amino acid difference influences the binding of some antibodies to this domain.

Mol Microbiol, 1993 Aug, 9(4), 823 - 33
Mechanism of expression of DNA repair gene vsr, an Escherichia coli gene that overlaps the DNA cytosine methylase gene, dcm; Dar ME et al.; The DNA cytosine methylase gene of Escherichia coli, dcm, overlaps an open reading frame (ORF) that continues in +1 register past the end of dcm . This ORF codes for a gene, vsr, that is required for a T:G to C:G base mismatch correction process . In this study, mutants that affect the level of expression of the two genes were constructed and characterized . Further, a previously isolated mutant, dcm-6, was cloned and mutations within it were identified . Northern blots were used to identify dcm-specific RNA species in wild type and dcm-6 cells . Based on these studies we conclude that there is a six-codon overlap between vsr and dcm . The two proteins appear to be made from a single RNA transcript and translation of dcm is required for the efficient synthesis of Vsr . Further, Vsr is active by itself and may not be produced as a fusion with Dcm . This is the first example of chromosomal genes that overlap in their coding regions and produce proteins with distinct functions.

Mol Microbiol, 1993 Aug, 9(4), 717 - 27
Expression of gene 19 of the conjugative plasmid R1 is controlled by RNase III; Koraimann G et al.; Specific cleavage of mRNAs by RNase III has been shown to control the expression of several Escherichia coli genes . We show here that the expression of gene 19 of the conjugative resistance plasmid R1 is controlled in its expression by the same endoribonuclease . In vivo studies revealed that a DNA fragment of 150 nucleotides including a perfect 22 nucleotide inverted repeat in the gene 19 coding region is responsible for the low expression of the gene both at the protein and the RNA levels . By using a translational gene 19-lacZ fusion in isogenic RNase III+ and RNase III- strains we could identify RNase III as the key element in the down-regulation of gene 19 expression . The sequencing of in vitro generated and RNase III-digested transcripts confirmed the in vivo studies and revealed the exact positions of the RNase III cleavage sites within the coding part of the gene 19 transcript . The in vitro determined RNase III cleavage of gene 19 mRNA was confirmed by in vivo primer extension analysis . Finally, we could show that an exchange of three nucleotides within the RNase III recognition site abolished RNase III cleavage in vitro.

J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1681 - 9
Characterization of Leptospiraceae by 16S DNA restriction fragment length polymorphisms; Hookey JV; Chromosomal DNA from 37 Leptospiracae representing genetic species, groups and reference strains together with five leptospire isolates and Escherichia coli were digested with the restriction endonucleases BamHI, ClaI and EcoRI . The Southern blots were hybridized with a biotinylated E . coli 1.5 kb 16S rDNA probe and gave 36 reproducible and unique patterns . With the exception of the type strain (Leptospira interrogans serovar icterohaemorrhagiae RGA) and neotype strain (serovar icterohaemorrhagiae Ictero no . 1) all the species and taxa examined could be differentiated from each other on the basis of their BamHI, ClaI and EcoRI restriction fragment length polymorphisms (RFLP) . L . interrogans and L . borgpetersenii reference strains were heterogeneous, whereas Leptonema illini and L . parva incertae sedis were distinct and separate . Strains representing L . biflexa sensu lato presented divergent RFLP patterns . A porcine isolate was identified to be L . interrogans pomona Pomona.

Protein Expr Purif, 1993 Aug, 4(4), 298 - 303
Expression and purification of the HIV-1 reverse transcriptase using the baculovirus expression vector system; Kawa S et al.; We have successfully expressed and purified the human immunodeficiency virus type-1 reverse transcriptase (RT) using the baculovirus expression vector system . This expression system provides a eukaryotic environment in which post-translational modifications of foreign gene products can occur . After infection with recombinant virus, Western blot analysis confirmed the presence of an immunoreactive polypeptide of approximately 66 kDa from insect Sf9 cell lysates . RT was then purified from crude extracts of baculovirus-infected Sf9 cells; SDS-PAGE analysis of fractions obtain from partial purification showed that in contrast to the Escherichia coli-expressed RT, the baculovirus-expressed RT corresponded to a doublet of peptides at approximately 66 kDa . Further purification of the protein resulted in a p66 protein, judged to be more than 90% pure by SDS-PAGE and Coomassie blue stain . Following purification, the baculovirus derived RT had specific activity for DNA polymerase similar to that of the E . coli-derived RT . Therefore, RT purified from Sf9 cells appears to be suitable for structure-function studies of this enzyme.

Am J Physiol, 1993 Aug, 265(2 Pt 1), G214 - 8
Nitric oxide synthase induction and intestinal epithelial cell viability in rats; Tepperman BL et al.; The present study determined the presence of constitutive and inducible nitric oxide (NO) synthase activities in intestinal isolated epithelial cells and the effects of NO induction on intestinal epithelial cell viability . Epithelial cells were isolated from rat proximal small intestine by dispersion using citrate and EDTA . Constitutive NO synthase activity, determined by {14C}arginine conversion to citrulline that was inhibited by in vitro incubation with the arginine analogue NG-monomethyl-L-arginine (L-NMMA; 300 microM) or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 1 mM), was observed in these cells . Administration of Escherichia coli lipopolysaccharide (LPS; 3 mg/kg iv) significantly augmented NO synthase activity (determined 4 h later), which was inhibited in vitro by incubation with L-NMMA but not by EGTA . The highest level of constitutive and inducible NO synthase activity occurred in intestinal villus cells, and the lowest was in crypt cells . Induction of NO synthase activity was associated with a decrease in cellular viability as assessed by a decrease in trypan blue exclusion . Dexamethasone pretreatment (1 mg/kg iv 2 h before LPS administration) significantly reduced both induction of NO synthase activity and the reduction in cellular viability . Likewise concurrent administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg sc) ameliorated the reduction in cell viability induced by LPS administration, an effect abolished by pretreatment with the NO substrate L-arginine (350 mg/kg sc) . Whereas constitutively formed NO may have a physiological role in these cells, the results in this study suggest that induction of NO synthase in epithelial cells may represent a mechanism of local intestinal damage.

Eur J Biochem, 1993 Aug 1, 215(3), 787 - 92
Minimal 16S rRNA binding site and role of conserved nucleotides in Escherichia coli ribosomal protein S8 recognition; Mougel M et al.; Escherichia coli ribosomal protein S8 was previously shown to bind a 16S rRNA fragment (nucleotides 584-756) with the same affinity as the complete 16S rRNA, and to shield an irregular helical region (region C) {Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert-Bezancon, Ebel, J . P., Ehresmann, B . & Ehresmann, C . (1987) . J . Mol . Biol . 198, 91-107} . Region C was postulated to display characteristic features: three bulged adenines (A595, A640 and A642), a non-canonical U598-U641 pair surrounded by two G.C pairs . In order to delineate the minimal RNA binding site, deletions were introduced by site-directed mutagenesis and short RNA fragments were synthesized . Their ability to bind S8 was assayed by filter binding . Our results show that the RNA binding site can be restricted to a short helical stem (588-605/633-651) containing region C . The second part of the work focused on region C and on the role of conserved nucleotides as potential determinants of S8 recognition . Single and double mutations were introduced by site-directed mutagenesis in fragment 584-756, and their effect on S8 binding was measured . It was found that the three bulged positions are essential and that adenines are required at positions 640 and 642 . U598 is also crucial and the highly conserved G597.C643 pair cannot be inverted . These conserved nucleotides are either directly involved in the recognition process as direct contacts or required to maintain a specific conformation . The strong evolutionary pressure and the small number of positive mutants stress the high stringency of the recognition process.

Biochem J, 1993 Aug 1, 293 ( Pt 3), 713 - 9
Insulin-like growth factor (IGF)-II binding to IGF-binding proteins and IGF receptors is modified by deletion of the N-terminal hexapeptide or substitution of arginine for glutamate-6 in IGF-II; Francis GL et al.; Recombinant insulin-like growth factor-II (IGF-II) and two structural analogues, des(1-6)IGF-II and {Arg6}-IGF-II, were produced to investigate the role of N-terminal residues in binding to IGF-binding proteins (IGFBPs) and hence the biological properties of the modified peptides . The growth factors were modelled on two previously characterized variants of IGF-I, des(1-3)IGF-I and {Arg3}-IGF-I, which both show substantially decreased binding to IGFBPs and were expressed as fusion proteins in Escherichia coli . The biological activities of the corresponding analogues of IGF-I and IGF-II were compared in rat L6 myoblasts and H35B hepatoma cells . In the L6-myoblast protein-synthesis assay, the IGF-II analogues, des(1-6)IGF-II and {Arg6}-IGF-II, were slightly more potent than IGF-II but about 10-fold less potent than IGF-I and 100-fold less potent than the respective IGF-I analogues, des(1-3)IGF-I and {Arg3}IGF-I . In H35 hepatoma cells the anabolic response measured was the inhibition of protein breakdown, and the potency order was insulin >>> {Arg3}-IGF-I > des(1-3)IGF-I > {Arg6}-IGF-II > des(1-6)IGF-II > IGF-I > IGF-II . Binding of the IGFs and their analogues to the type 1 IGF receptor in L6 myoblasts and to the insulin receptor in H35 hepatoma cells did not fully explain the observed anabolic potency differences . Moreover, binding of all four analogues to the IGFBPs secreted by L6 myoblasts and H35B hepatoma cells was greatly decreased compared with the parent IGF . We conclude that the observed anabolic response to each IGF was determined by their relative binding to the competing cell receptor and IGFBP binding sites present.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7240 - 4
Nitric oxide activates cyclooxygenase enzymes; Salvemini D et al.; We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-1 and COX-2, respectively) . Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (1 microgram/ml, 18 h) caused an increase in the release of nitrite (NO2-) and prostaglandin E2 (PGE2), products of NOS and COX, respectively . Production of both NO2- and PGE2 was blocked by the NOS inhibitors NG-monomethyl-L-arginine or aminoguanidine . The effects of NG-monomethyl-L-arginine or aminoguanidine were reversed by coincubation with L-Arg, the precursor for NO synthesis, but not by D-Arg . RAW264.7 cells stimulated for 18 h with lipopolysaccharide in L-Arg-free medium (to reduce NO generation by the endogenous NOS pathway) failed to release NO2- and accumulated at least 4-fold less PGE2 when compared to cells in the presence of L-Arg . PGE2 production elicited by a 15-min arachidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the release obtained with cells induced in medium containing L-Arg . To examine the NO activation of the induced form of COX in the absence of an endogenous L-Arg, human fetal fibroblasts were first stimulated for 18 h with interleukin 1 beta . These cells released PGE2 but not NO2-, consistent with the induction of COX but not NOS in the fibroblast . Exogenous NO either as a gaseous solution or released by a NO donor, sodium nitroprusside or glyceryl trinitrate, increased COX activity in the interleukin 1 beta-stimulated fibroblasts by 5-fold; these effects were abolished by coincubation with hemoglobin (10 microM), which binds and inactivates NO, but not by methylene blue, an inhibitor of the soluble guanylate cyclase . Furthermore, sodium nitroprusside (0.25-1 mM) increased arachidonic acid-stimulated PGE2 production by murine recombinant COX-1 and COX-2 . These results demonstrate that NO enhances COX activity through a mechanism independent of cGMP and suggest that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response . The data suggest that NO directly interacts with COX to cause an increase in the enzymatic activity.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 6952 - 6
Human immunodeficiency virus type 1 (HIV-1) strains selected for resistance against the HIV-1-specific {2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)}-beta-D-pentofurano syl (TSAO) nucleoside analogues retain sensitivity to HIV-1-specific nonnucleoside inhibitors; Balzarini J et al.; We recently reported that a newly discovered class of nucleoside analogues--{2',5'-bis-O-(tert-butyldimethylsilyl)- 3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)}-beta-D - pentofuranosyl derivatives of pyrimidines and purines (designated TSAO)--are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) and targeted at the nonsubstrate binding site of HIV-1 reverse transcriptase (RT) . We now find that HIV-1 strains selected for resistance against three different TSAO nucleoside derivatives retain sensitivity to the other HIV-1-specific nonnucleoside derivatives (tetrahydroimidazo{4,5,1-jk}{1,4}benzodiazepin-2(1H)-one and -thione (TIBO), 1-{(2-hydroxyethoxy)methyl}-6-phenylthiothymine, nevirapine, and pyridinone L697,661, as well as to the nucleoside analogues 3'-azido-3'-deoxythymidine, ddI, ddC, and 9-(2-phosphonylmethoxyethyl)adenine . Pol gene nucleotide sequence analysis of the TSAO-resistant and -sensitive HIV-1 strains revealed a single amino acid substitution at position 138 (Glu-->Lys) in the RT of all TSAO-resistant HIV-1 strains . HIV-1 RT in which the Glu-138-->Lys substitution was introduced by site-directed mutagenesis and expressed in Escherichia coli could not be purified because of rapid degradation . However, HIV-1 RT containing the Glu-138-->Arg substitution was stable . It lost its sensitivity to the TSAO nucleosides but not to the other HIV-1-specific RT inhibitors (i.e., TIBO and pyridinone) . Our findings point to a specific interaction of the 4''-amino group on the 3'-spiro-substituted ribose moiety of the TSAO nucleosides with the carboxylic acid group of glutamic acid at position 138 of HIV-1 RT.

EMBO J, 1993 Aug, 12(8), 3007 - 16
Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli; Killmann H et al.; The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli . Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane . By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel . The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance . The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E . coli outer membrane . It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels . Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel . The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane . The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM . This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.

Mutat Res, 1993 Aug, 288(2), 249 - 55
Implication of uracil in spontaneous mutagenesis on a single-stranded shuttle vector replicated in mammalian cells; Cabral-Neto JB et al.; Almost all spontaneous point mutations found on a single-stranded shuttle vector after its transfection and replication in monkey cells were located at cytosine residues . In order to understand this very specific type of targeting we have studied the possible implication of uracil residues in the induction of these spontaneous mutations . The single-stranded shuttle vector pCF3A carrying the supF tRNA gene as a mutagenesis target has been allowed to replicate in mammalian COS7 cells, mutations being screened in bacteria using the beta-galactosidase assay . Progenies from untreated DNA and DNA treated with the uracil-DNA glycosylase prior to transfection were analyzed to determine the amount and classes of mutations . While spontaneous mutation frequency was 9.7 x 10(-4) for control DNA, single-stranded vector treated with the E . coli uracil-DNA glycosylase exhibited a reduced mutation frequency of about 30% . The abolished mutations were mainly confined to the cytosine to thymine transitions for which a decrease by a factor of 5 was indeed observed . This finding fits well with the fact that it is usually admitted that uracil pairs with adenine, indicating therefore that approximately 30% of spontaneous mutations observed in our experimental conditions and 80% of C to T transitions may be due to the presence of uracil instead of cytosine.

Oncogene, 1993 Aug, 8(8), 2283 - 92
Regulation of the human c-fes protein tyrosine kinase (p93c-fes) by its src homology 2 domain and major autophosphorylation site (Tyr-713); Hjermstad SJ et al.; The c-fes proto-oncogene product is expressed predominantly in hematopoietic cells of the myeloid lineage and has been implicated in the regulation of myeloid differentiation . The c-fes locus encodes a 93-kDa protein tyrosine kinase (p93c-fes) that possesses several structural features characteristic of the cytoplasmic class of protein tyrosine kinases, including a consensus sequence for autophosphorylation surrounding Tyr-713 and a src homology 2 (SH2) domain . To assess the effect of each of these potential regulatory sites on p93c-fes protein tyrosine kinase activity, we specifically deleted the c-fes SH2 domain using the polymerase chain reaction and replaced Tyr-713 with phenylalanine by oligonucleotide-directed mutagenesis (Y713F mutant) . The resulting mutants were expressed in Escherichia coli and assayed for changes in protein tyrosine kinase activity using an immune complex kinase assay . Both mutations produced a marked decrease in the rate and extent of autophosphorylation and phosphorylation of the model substrate, enolase . To test whether the c-fes SH2 domain could interact with the autophosphorylated kinase domain, the SH2 domain was expressed as a fusion protein with glutathione S-transferase and immobilized on glutathione-agarose . The recombinant c-fes SH2 domain precipitated p93c-fes as readily as a monoclonal antibody . Binding of the SH2 domain to p93c-fes was completely dependent upon autophosphorylation, as a kinase-defective mutant of p93c-fes was not precipitated by the SH2 domain . High-affinity binding was also observed with recombinant SH2 domains from v-src and v-fps, raising the possibility of protein-protein interactions between various members of the cytoplasmic PTK family . These results indicate that the c-fes SH2 domain and consensus autophosphorylation site (Tyr-713) play major roles in the positive regulation of p93c-fes tyrosine kinase activity, possibly through intramolecular interaction.

Mol Cell Biol, 1993 Aug, 13(8), 4679 - 90
Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1; Charest DL et al.; p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control . A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library . The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis . Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo . It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues . Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium . Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A . The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site . A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets . Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1 . This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.

J Immunol, 1993 Aug 1, 151(3), 1637 - 45
Intravenous endotoxin suppresses the cytokine response of peripheral blood mononuclear cells of healthy humans; Granowitz EV et al.; When administered parenterally, endotoxin stimulates the synthesis of IL-1, TNF-alpha, and IL-6 . However, this initial injection induces tolerance; a second injection of endotoxin results in lower levels of circulating cytokines . In our study, five healthy male volunteers between the ages of 18 and 30 were injected with Escherichia coli endotoxin . Four subjects received only saline . Immediately before the injection and 3, 6, and 24 h afterward, PBMC were isolated and stimulated in vitro with endotoxin, IL-1, or toxic shock syndrome toxin-1 . Inasmuch as CD14+ monocytes are the primary source of the cytokines induced by these stimuli, results are expressed as cytokine production per 10(6) CD14+ cells . Six h after endotoxin injection, endotoxin-stimulated CD14+ cells synthesized 66% less IL-1 beta (p < 0.01), 47% less TNF-alpha (p < 0.001), 56% less IL-6 (p < 0.01), and 49% less IL-8 (p < 0.01) than cells obtained before the injection . This suppression was not specific for endotoxin; IL-1 beta-induced IL-1 alpha and TNF-alpha were reduced by 84% (p = 0.01) and 68% (p < 0.001), respectively . A decrease in cytokine synthesis was also observed using toxic shock syndrome toxin-1 as a stimulus: 57% for IL-1 beta (p = 0.06), 70% for TNF-alpha (p < 0.01), 56% for IL-6 (p < 0.05), and 71% for IL-8 (p = 0.001) . When data were expressed as cytokine production per 10(6) PBMC, cells isolated 3 h after endotoxin injection synthesized significantly less stimulus-induced IL-1, TNF-alpha, IL-6, and IL-8 than did PBMC from saline-injected controls . We conclude that endotoxin tolerance is due, in part, to changes in the stimulus-induced cytokine response of circulating CD14+ cells.

J Virol, 1993 Aug, 67(8), 4886 - 95
Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation; Fouillot N et al.; The hepatitis B virus (HBV) P gene which encodes the reverse transcriptase and other proteins required for replication is expressed on the bicistronic mRNA pregenome which also encodes the capsid protein in its first cistron . Recent results have suggested that the hepadnaviral P gene is translated by internal entry of ribosomes upstream from the P gene, in the overlapping C gene . Using a reporter gene fused to the HBV C or P gene, we demonstrate that the C sequence does not allow internal initiation of translation . Alternatively, our results support a model in which the HBV P gene is translated by ribosomes which scan from the capped extremity of the bicistronic mRNA pregenome . The mechanism by which the ribosomes scan past four AUGs before they initiate translation at the P AUG was analyzed . Our results show that these AUGs are skipped via two mechanisms: leaky scanning on AUGs in a weak or suboptimal initiation context and translation of an out-of-C-frame minicistron followed by reinitiation at P AUG . The minicistron translation allows ribosomes to bypass an AUG in a favorable context that would otherwise be used as a start codon for translation of a truncated capsid protein . Our results suggest that this elaborated scanning mechanism permits the coordinate expression of the HBV C and P genes on the viral bicistronic mRNA pregenome.

Mutat Res, 1993 Aug, 294(2), 91 - 100
Recovery of DNA replication in UV-damaged Escherichia coli; Altshuler M; Following exposure to UV light DNA replication stops and then resumes . The SOS response is required for the restoration of replication . Replication recovery occurs in lexA(Ind) cells carrying a high constitutive level of RecA protein . Replication is also affected by UmuCD proteins, photoreactivation, and excision repair . In addition, there is a constitutive and recA independent way to replicate over UV photoproducts associated with the production of gaps in daughter DNA strands . There are two ways to account for the replication in UV-irradiated cells . A stalled replication fork can be reactivated . Alternatively, a replication fork could be destroyed irreparably, with no available way to complete the round of replication . In that case, postirradiation replication could be due exclusively to replication forks assembled de novo at the origin(s) . Changes in replication initiation are observed following UV irradiation . Initiations are first inhibited and then stimulated . They become independent of de novo protein synthesis and sometimes do not stop in dnaA(ts) mutants shifted to 42 degrees C . Although the inducible functions are involved in the recovery of replication at different levels of UV damage, some modifications of the replication initiation mechanism appear to be specific to severely damaged cells . Such modifications seem to include the dnaA(ts) independence for initiations and the transient initiation inhibition . RecA protein can be directly involved both in the modification of initiation and in reactivation of the stalled replication forks . Although the restoration of replication depends on the SOS response a synthesis of some protein(s) that do not belong to the LexA regulon seems to be required as well . These proteins can be under RecA control and one of their functions may be to inhibit the rnhA gene . Certain recA mutations may selectively affect different mechanisms of the replication recovery (namely, recA430, recA727, recA718, recA1730) . Overproduction of the photoreactivating enzyme in the dark could influence UmuCD activity in replication . The UmuCD function appears to be blocked in strains carrying the dnaE1026 mutation or overproducing the dnaQ protein . For some unknown reason the UmuCD-associated replication mechanism is the only one available for phage with damaged DNA.

Mutat Res, 1993 Aug, 294(2), 157 - 66
Cosuppression of recF, recR and recO mutations by mutant recA alleles in Escherichia coli cells; Wang TC et al.; The ability of recA718, recA720, recA730, recA750 and two known recA(Srf) alleles (recA2020 and recA441) to act as suppressors of recF, recR and recO mutations was examined by studying their UV radiation sensitivity in uvrA cells of Escherichia coli . With the exception of recA718, all the mutant recA alleles examined were able to suppress the UV radiation sensitivity caused by recF, recR or recO mutations, but not by the recB mutation . The suppression by recA750 was minimal . The suppression of recF, recR and recO mutations by other recA alleles was more pronounced, but none of them could exert full suppression . Heterozygotes containing a mutant recA allele (recA720, recA730 or recA441) and recA2020 (or recA803, another known recA(Srf) allele) failed to produce any additive or synergistic suppression of recF, indicating that these suppressors did not use different mechanisms for their suppression . Similar to a requirement of recJ+ in the suppression of recF, the suppression of recR and recO mutations by mutant recA alleles also required recJ+ . The similar phenotypes conferred by recF, recR, and recO mutations and the observations that a number of mutant recA alleles can cosuppress these three mutations suggest that the recF, recR and recO gene products may function together as a complex.

Mutat Res, 1993 Aug, 294(2), 127 - 38
N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli: multiple roles for UmuC protein; Fix D; Backmutations of an ochre (UAA) nonsense defect in the tyrA gene of Escherichia coli were induced by N-ethyl-N-nitrosourea (ENU) in both UmuC+ and UmuC- strains . This site is particularly flexible to base substitution mutation and all but one (TAA-->TGA) substitution can be recovered using a reversion assay . Employing direct sequencing of polymerase chain reaction-amplified genomic DNA and/or colony-hybridization methods, the changes induced by several separate doses of ENU in a total of 587 independent backmutations were investigated . In the UmuC+ strain, all possible single-base substitutions were recovered . Different frequencies for individual single-base substitutions were obtained and correlations with the surrounding base sequence could be seen . Transitions occurred most frequently at thymine residues having a purine on the 5'-side while transversions occurred more frequently at thymine residues having a cytosine on the 5'-side . In the UmuC- strain, ENU failed to induce A:T-->T:A and A:T-->C:G transversions and the frequency of A:T-->G:C transitions was reduced . However, an unidentified class of extragenic mutations were induced to a greater extent . These results suggest distinct pathways for ENU-induced mutagenesis at ethylated thymine residues and delineate several separate functions for the UmuC protein.

Mutat Res, 1993 Aug, 294(2), 109 - 16
Processivity of uracil DNA glycosylase; Higley M et al.; The purpose of this study was to determine the mechanism by which uracil DNA glycosylase locates uracil residues within double-stranded DNA . Using reaction conditions that contained low salt concentrations, the addition of uracil DNA glycosylase to plasmid DNAs containing multiple, randomly incorporated uracils resulted in the accumulation of form III DNA while unreacted form I DNA was still present . These data suggested that the enzyme utilizes a one-dimensional DNA-scanning mechanism such that this linear DNA arose by the accumulation of many single-strand breaks within the plasmid prior to enzyme dissociation . Reactions containing higher concentrations of uracil DNA glycosylase revealed a further accumulation of form III DNA after all form I DNA had been lost . These results suggested a partial (1.5-2 kb) enzyme processivity since the enzyme does not incise at all uracil bases on the DNA molecule prior to dissociation from that DNA . Since DNA scanning is regulated by electrostatic interactions, the processivity of the enzyme was evaluated through kinetic analyses of incision at various salt concentrations . At NaCl concentrations (< 50 mM), a significant amount of form III DNA accumulated while there were still unreacted form I DNAs present . In contrast, the accumulation of form III DNA was delayed at higher salt concentrations and the overall accumulation of form III DNA was less than that monitored at lower salt concentrations . DNAs were also analyzed by denaturing agarose gel electrophoresis in order to measure the average distance between strand breaks . Southern hybridizations showed a greater accumulation of breaks in DNAs that were reacted with the uracil DNA glycosylase at the lower salt concentrations, confirming a partial processivity for the enzyme.

Oral Microbiol Immunol, 1993 Aug, 8(4), 225 - 9
Ribotyping shows intrafamilial similarity in Actinobacillus actinomycetemcomitans isolates; Alaluusua S et al.; This study reports ribosomal RNA gene restriction patterns of 54 Actinobacillus actinomycetemcomitans isolates obtained from 9 families (12 children and 11 parents) . The isolates represented serotypes a, b, c and d . The chromosomal DNA extracted from A . actinomycetemcomitans isolates was digested with the restriction endonucleases EcoRI, BamHI, HindIII, ClaI and Bg/I . The DNA fragments were hybridized to the rrnB ribosomal RNA operon of the Escherichia coli chromosome . In 5 families, isolates belonging to the same family (mother and/or father and the children) had identical hybridization patterns when analyzed with all 5 enzymes . In 3 families, each family member harbored only one ribotype of A . actinomycetemcomitans, but at least one member harbored isolates that were of a different ribotype than the other members of the family . In one family, the mother harbored 2 ribotypes (and serotypes), one common with the daughter and one different . In conclusion, the study confirms the previous results that A . actinomycetemcomitans is transmitted intrafamilially.

Int J Cancer, 1993 Jul 30, 54(6), 1017 - 21
Adhesion of human lymphoid cell lines to immobilized carbohydrates and to bone-marrow stromal cell layers by surface sugar receptors; Gabius S et al.; Cell-surface sugar receptors may participate in interactions of lymphoid cells that influence their adhesive properties and proliferation . Their expression on cells of the pre-B line BLIN-I, the B-lymphoblastoid line Croco II, the myeloma line RPMI 8226 and the T-lymphoblastoid line CCRF-CEM was monitored with a panel of 14 types of chemically glycosylated E . coli beta-galactosidase at a non-saturating ligand concentration . Quantitative differences were determined for the capacity of the different cell types to bind constituents of the carbohydrate part of glycoconjugates . They were corroborated by analyses of binding for lactose-, beta-N-acetylgalactosamine-, beta-N-acetylglucosamine- and fucose-exposing neoglycoenzymes up to saturation levels . Values of dissociation constants of the tetrameric enzyme were in the range of 3-300 nM . Several types of sugar receptor led to carbohydrate-inhibitable adhesion of cells to 6 types of nitrocellulose-immobilized neoglycoprotein, their effectiveness being most obvious for the myeloma cells . Analyses of the carbohydrate-ligand-mediated adhesion of the other cell types revealed a comparatively decreased response . Only a few carbohydrates among the 7 types tested were effective in reducing cell adhesion to a far more complex ligand-bearing matrix than immobilized neoglycoproteins, namely bone-marrow stromal cell layers: sialic acid and N-acetylgalactosamine for B-lymphoblastoid cells and rhamnose for pre-B cells . These cellular interactions may encompass sugar receptors on the stromal cells and other types of molecular recognition in addition to the detected activities on the lymphoid cells.

Gene, 1993 Jul 30, 129(2), 239 - 47
The cDNA of a human lymphocyte cyclic-AMP phosphodiesterase (PDE IV) reveals a multigene family; Obernolte R et al.; Five protein families are needed to encompass the diversity of cyclic-AMP (cAMP) phosphodiesterases (PDE) . Family IV PDEs (PDE IV) specifically hydrolyze cAMP with a low Km, and are selectively inhibited by rolipram (Rp) and related drugs . Cloned cDNAs from rat (r) suggest that the PDE IV family comprises four distinct members, designated A, B, C and D . Using RN from a human lymphocytic B-cell line (43D-Cl2), we have isolated a 3.8-kb cDNA by low-stringency screening using a rat PDE IV member B (r-PDE IVB) probe . Expression of the human (h) cDNA in Escherichia coli results in cAMP-specific PDE activity that is Rp sensitive . A single large open reading frame (ORF) predicts a 564-amino-acid protein with 92.9% identity to r-PDE IVB; at the nucleotide level the identity is 86.3% . This h-PDE IVB clone, HPB106, differs from a related cDNA clone isolated by others from h-monocytes {Livi et al., Mol . Cell . Biol . 10 (1990) 2678-2686} . Our analysis identifies the monocyte clone with r-PDE IVA . Southern blots using a 1.2-kb h-PDE IVB probe at low stringency suggest the presence of additional uncloned human PDE IV family members . Analysis of genomic Southern blots using short specific probes from the h-PDE IVA and h-PDE IVB cDNAs indicates that distinct genes encode these two PDE IV family members . RNA from fractionated normal human leukocytes shows major specific messages of 3.0 and 4.6 kb for h-PDE IVA and 3.7 kb for h-PDE IVB.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1993 Jul 30, 194(2), 978 - 85
Implication of cysteine residues in the activity of single-chain urokinase-plasminogen activator; Hamelin J et al.; Single-chain urokinase-plasminogen activator contains 24 cysteine residues involved in 12 disulfide bonds and distributed all along the three domains of the protein . In order to investigate the role of these disulfide bridges in the catalytic activities of scu-PA, we used site-specific mutagenesis to construct 10 mutants in which some cysteine residues were changed to serine residues . Each mutated DNA fragment was cloned into a procaryotic expression vector and the protein expressed in E . coli . Mutant proteins of the expected size were produced and analyzed for amidolytic and fibrinolytic activities . From this, it is shown that: i) the disulfide bonds in the epidermal growth factor (EGF)-like and in the kringle domains are not necessary . Moreover, disulfide bond deletion in the kringle domain improves those catalytic activities; ii) on the contrary, the disulfide bridges in the catalytic domain are essential for maintaining both activities.

Biochem Biophys Res Commun, 1993 Jul 30, 194(2), 951 - 9
Analysis of cleavage-site patterns in protein precursor sequences with a perceptron-type neural network; Schneider G et al.; A method for feature extraction from protein sequences has been developed which is based on an artificial neural filter system . Amino acid sequences are analyzed with regard to physicochemical residue properties . This alternative representation of a sequence allows an interpretation of the networks' weight values in a comprehensive and biochemically meaningful way by displaying the optimized network weights in Hinton diagrams . Signal peptidase cleavage sites of E.coli periplasmic proteins, human mitochondrial precursors and chloroplast precursors from spinach have been investigated . The network for E.coli periplasmic protein precursors classified both training and test data with 100% accuracy . The interpretation of its network weights clearly confirms the "-3,-1 rule" and the existence of a hydrophobic core region starting at position -6 . Further striking features and dominant positions can be found for all three types of cleavage sites.

Science, 1993 Jul 30, 261(5121), 598 - 600
Greater susceptibility to mutations in lagging strand of DNA replication in Escherichia coli than in leading strand; Veaute X et al.; Models of DNA replication in Escherichia coli involve an asymmetric DNA polymerase complex that replicates concurrently the leading and the lagging strands of double-stranded DNA . The effect of asymmetry on mutagenesis was tested with pairs of plasmids containing the unidirectional ColE1 origin of replication and a single lesion located in the leading or lagging strand . The lesion used was the covalent adduct that the chemical carcinogen N-2-acetylaminofluorene (AAF) forms with the C-8 position of guanine . Whether SOS was induced or not, mutations arose at about a 20-fold higher frequency when the AAF adduct was located in the lagging strand than when in the leading strand.

Int J Cancer, 1993 Jul 30, 54(6), 1002 - 9
Modulating the metastatic potential of murine RAW117 large-cell lymphoma cells by selection for resistance to interferon-gamma; LaBiche RA et al.; Highly metastatic, in vivo-selected cells of RAW117-H10 large-cell lymphoma have been shown to be more resistant than poorly metastatic parental RAW117-P cells to the cytolytic and cytostatic activities of activated macrophages in co-culture experiments . Activated macrophages are known to produce soluble, cytostatic respiration-inhibiting factors, and such activities can be duplicated by interferon-gamma (IFN-gamma) or by combinations of IFN-gamma and Escherichia coli lipopolysaccharide (LPS) . Highly metastatic RAW117-H10 cells are more resistant to the cytostatic effects of IFN-gamma and LPS than poorly metastatic RAW117-P cells, and short-term (up to 72 hr) treatment with IFN-gamma and LPS does not change the metastatic potentials of RAW117 cells . We have studied the effects of sequential selection of RAW117-P cells for increased resistance to IFN-gamma and LPS . After 7 to 13 sequential selections, the resulting variant lines were completely refractory to the growth-inhibitory effects of IFN-gamma and LPS and cross-tolerant to macrophage-conditioned medium . The selected variants also completely lost their experimental metastatic potentials and their tumorigenicities after s.c . or i.m . injection . Cytofluorographic analysis indicated reduced cell-surface expression of H-2Kd antigen and fibronectin receptor on the variant cells but no change in surface mu heavy-chain immunoglobulin . The IFN-gamma-selected lines were less adhesive to liver microvascular endothelial cells than the unselected RAW117 cell lines, but were equally sensitive to NK cytolysis by spleen cells . Our results suggest that exposure to certain cytokines can affect the growth and metastatic potential of RAW117 cells and result in the selection of resistant variants with altered biologic properties.

Biochemistry, 1993 Jul 27, 32(29), 7526 - 30
Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position; Ladbury JE et al.; The effects of pH in the range 6.0-8.0 on the thermodynamics of the reversible thermal unfolding of Escherichia coli thioredoxin in the oxidized state have been determined over a range of concentrations using differential scanning calorimetry . The thermal denaturation indicated an inverse temperature dependence on concentration . The data were shown to fit a model based on dimerization of both the native and denatured states of the protein . The degree of dimerization of both states was found to be pH dependent . The previously described importance of protonation of the anomalously titrating aspartic acid 26 residue {Langsetmo, K., Fuchs, J., & Woodward, C . (1991) Biochemistry 30 ,7603-7609} was apparently verified by the agreement between the experimentally determined delta delta Gzerod and the calculated delta delta GzeroH in the pH range 7.0-8.0.

Biochemistry, 1993 Jul 27, 32(29), 7581 - 8
Purification of two picornaviral 2A proteinases: interaction with eIF-4 gamma and influence on in vitro translation; Liebig HD et al.; A mammalian cell infected with a human rhinovirus or enterovirus has a much reduced capability to translate capped mRNAs (the host cell shutoff), while still allowing translation of uncapped viral RNA . Biochemical and genetic evidence suggests that the viral proteinase 2A induces cleavage of the eukaryotic initiation factor (eIF) 4 gamma (also known as p220) component of eIF-4 (formerly called eIF-4F) . However, neither the mechanism underlying the specific proteolysis of eIF-4 gamma nor the influence of this cleavage on the translation of capped mRNAs has been clarified . Such studies have been hampered by a lack of large quantities of a purified 2A proteinase . Therefore, the mature proteinases 2A of human rhinovirus 2 and coxsackievirus B4 were expressed in soluble form in Escherichia coli . A four-step purification protocol was developed; 1 mg of highly purified 2A proteinase per gram wet weight of E . coli was obtained . Both enzymes cleaved directly eIF-4 gamma as part of the purified eIF-4 complex . Addition of HRV2 2A proteinase to HeLa cell cytoplasmic translation extracts resulted in eIF-4 gamma cleavage and drastically reduced the translation of capped mRNA; addition of purified eIF-4 restored translation to the initial level . However, translation of a reporter gene driven by the 5'-untranslated region of human rhinovirus 2 was translated 2-3-fold more efficiently in the presence of HRV2 2A proteinase.

Biochemistry, 1993 Jul 27, 32(29), 7531 - 41
Translesional synthesis on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene); Shibutani S et al.; Oligodeoxynucleotides modified site-specifically with dG-(+)-trans- and dG-(+)-cis-anti-BPDE (7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene) or dG-(-)-trans- and dG-(-)-cis-anti-BPDE were used as templates in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I . The primer could be extended past the dG-(-)-trans-BPDE adduct with small amounts of dAMP incorporated opposite the lesion . A small amount of base deletions was also observed while, with the dG-(-)-cis-BPDE adduct, one- and two-base deletions predominated . When templates containing dG-(+)-trans-BPDE were used, small amounts of products containing one-base deletions were observed; with dG-(+)-cis-BPDE, substitution of dAMP opposite the lesion was also detected . The frequency of nucleotide insertion for dAMP opposite dG-(-)-trans-BPDE and the frequency of extension from the primer terminus containing the dA:dG-(-)-trans-BPDE pair were much higher than those observed with the other, stereochemically different BPDE adducts . Kinetic studies were in agreement with the results of the primer extension study . When the base flanking the 5' side of dG-BPDE was changed from dC to dT, the frequency of one-base deletions increased . We conclude that the trans- or cis-addition product of dG-(-)-anti-BPDE has a higher miscoding potential than dG-(+)-anti-BPDE in our model system and that G-->T transversions and deletions predominate . These observations are consistent with the types of mutations observed in vivo.

Biochemistry, 1993 Jul 27, 32(29), 7460 - 5
Structure of the hormone binding domain of human beta 1 thyroid hormone nuclear receptor: is it an alpha/beta barrel?
McPhie P, Parkison C, Lee BK, Cheng SY.
To understand the structure of the hormone binding domain (HBD) of human beta 1 thyroid hormone nuclear receptor (h-TR beta 1), truncated h-TR beta 1 fragments, MD32 (M169-D456), KD29 (K201-D456), DD28 (D211-D456), KD25 (K235-D456), and KP28 (K201-P448), were analyzed by circular dichorism (CD) . MD32 and KD29 show intense CD spectra with double minima at 222 and 208-210 nm, indicating the presence of extensive regions of alpha-helix . DD28 and KD25 have spectra which are reduced in intensity with minima around 215 nm, characteristic of a beta-sheet . The observed spectra are compatible with sequence analysis which predicts that HBD contains alternating stretches of alpha-helix and beta-strand . These extensive decreases in secondary structure in DD28 and KP28 in which the predicted first beta-strand or last alpha-helix was deleted, respectively, were accompanied by the loss of hormone binding activity . On the basis of these results, we suggest a new model for h-TR beta 1 consisting of the known DNA binding domain linked by an alpha-helical hinge to the HBD, with the tertiary structure of an alpha/beta barrel . The model is compatible with previous chemical and genetic studies on the structure of this protein.

Biochemistry, 1993 Jul 27, 32(29), 7451 - 9
Mechanism of GTP hydrolysis by p21N-ras catalyzed by GAP: studies with a fluorescent GTP analogue; Moore KJ et al.; The mechanism of the hydrolysis of GTP by p21N-ras and its activation by the catalytic domain of p120 GTPase activating protein (GAP) have been studied using a combination of chemical and fluorescence measurements with the fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl)GTP (mantGTP) . Since the concentration of active p21 is important in these measurements, various assays for both total protein and active p21 were investigated . All assays gave good agreement except the filter binding assay of {3H}-GDP bound to p21, which gave values of 35-40% compared to the other methods . Concentrations of p21 were thus based on the absorbance of the mant-chromophore of the p21-mant-nucleotide complexes . The rate constants of the elementary steps of the p21 intrinsic GTPase activity and the GAP activated activity were similar between GTP and mantGTP . Incubation of a stoichiometric complex of p21.mantGTP results in a biphasic decrease in fluorescence . The second phase occurs with the same rate constant as the cleavage step and is accelerated by GAP . No other steps of the mechanism are affected by GAP . Incubation of a stoichiometric complex of p21.mantGpp{NH}p also results in a biphasic decrease in fluorescence even though cleavage does not occur . This is interpreted that the cleavage step of p21.GTP is preceded by and controlled by an isomerization of the p21.GTP complex . GAP accelerates the rate constant of the second fluorescence phase occurring with p21.mantGpp{NH}p . This result shows that GAP accelerates the proposed isomerization which limits GTP cleavage rather than the cleavage step itself.

Biochemistry, 1993 Jul 27, 32(29), 7435 - 44
Recognition of tRNA(Cys) by Escherichia coli cysteinyl-tRNA synthetase; Komatsoulis GA et al.; A study of the recognition of tRNA(Cys) by Escherichia coli cysteinyl-tRNA synthetase using in vivo and in vitro methods was performed . All three anticodon nucleotides, the discriminator nucleotide (73), and some elements within the tertiary domain (the D stem/loop, the T psi C stem/loop, and the variable loop) are important for recognition; the anticodon stem and acceptor stem appear to contain no essential elements . A T7 RNA polymerase transcript corresponding to tRNA(Cys) is only a 5.5-fold worse substrate than native tRNA(Cys) (in terms of the specificity constant, kcat/Km), mainly due to an increase in the value of Km for the transcript . The greatest loss of specificity caused by mutation of a single nucleotide occurs when the discriminator U73 is changed; kcat/Km declines 3-4 orders of magnitude depending on the substitution . Mutations in the wobble nucleotide of the anticodon also cause reductions in the specificity constant of 3 orders of magnitude, while mutations in the other anticodon nucleotides caused lesser effects . Interestingly, a C35A mutation (with the phenylalanine anticodon GAA) had no effect on aminoacylation by the cysteinyl-tRNA synthetase . Several amber suppressor tRNAs were constructed whose in vivo identity did not correlate with their in vitro specificity, indicating the need for both types of experiments to understand the factors which maintain tRNA specificity.

Biochemistry, 1993 Jul 27, 32(29), 7428 - 34
Inversion of receptor binding preferences by mutagenesis: free energy thermodynamic integration studies on sugar binding to L-arabinose binding proteins; Zacharias M et al.; The Escherichia coli L-arabinose-binding protein (ABP) participates as a specific receptor in the transport of L-arabinose, D-fucose, and D-galactose through the periplasmic space . The wild-type protein binds L-arabinose about 40 times more strongly than D-fucose . A mutation of the protein at position 108 (Met-->Leu) causes a specificity change . The Met108Leu ABP slightly prefers binding of D-fucose over L-arabinose . Molecular dynamics (MD) and thermodynamic integration (TI) computer simulations were performed to study the mechanism of sugar discrimination and specificity change based on the known high-resolution X-ray structures . The specificity change was evaluated by calculating the difference in free energy of L-arabinose versus D-fucose bound to wild-type and Met108Leu ABP . The calculated free energy differences are consistent with the experimentally observed specificity of wild-type and Met108Leu ABP . The simulations indicate that the specificity change of Met108Leu ABP is accomplished mainly by reduced Lennard-Jones interactions of residue 108 with L-arabinose and improved interactions with D-fucose . In addition to MD/TI calculations on sugar binding, finite difference Poisson-Boltzmann calculations were performed to identify the most stable ionization state of buried ionizable residues in ABP.

FEBS Lett, 1993 Jul 26, 327(2), 131 - 6
Comparison of ubiquinol and cytochrome c terminal oxidases . An alternative view; Musser SM et al.; There have been numerous instances in the recent literature where the properties of ubiquinol and cytochrome c terminal oxidases are compared . Here we specifically examine the cytochrome bo3-type ubiquinol oxidase from Escherichia coli and the cytochrome aa3-type cytochrome c oxidases . A second redox-active copper site (CuA) is present only in the cytochrome c oxidases and the physiological electron donors for the two enzymes are different (ubiquinol-8 vs . ferrocytochrome c) . In our opinion, these differences are significant and most likely indicate that distinct turnover mechanisms are operative in the two enzymes.

J Biol Chem, 1993 Jul 25, 268(21), 15887 - 99
Biochemical analysis of mutant alleles of the vaccinia virus topoisomerase I carrying targeted substitutions in a highly conserved domain; Klemperer N et al.; The 32-kDa topoisomerase I encoded by vaccinia virus relaxes supercoiled DNA in a manner which is mechanistically equivalent to that utilized by eucaryotic enzymes . Its amino acid sequence contains significant homology to the enzymes encoded by Saccharomyces cerevisiae, Saccharomyces pombe, human cells, and other poxviruses . The small size of the viral enzyme, and its essentiality in the viral life cycle, make it ideally suited for structural and functional analysis . In this report we present the construction and analysis of 15 mutant alleles of the topoisomerase containing amino acid substitutions in a highly conserved region . The enzymes encoded by these alleles were expressed in Escherichia coli and various parameters of their activity were examined . All of the alleles which show diminished (seven alleles) or abrogated (three alleles) DNA relaxation activity are deficient in DNA cleavage and the concomitant formation of the covalent enzyme/DNA intermediate . None are deficient in the prior step of noncovalent interaction with substrate DNA . Five of the mutant enzymes show significant temperature sensitivity in vitro . The extent of in vitro activity of the enzymes shows a good but incomplete correlation with the enzymes' abilities to lethally induce the resident lambda prophage within E . coli BL21(DE3) (via illegitimate recombination) . Mutations in 1 amino acid, in particular, impair prophage induction in vivo more significantly than DNA relaxation in vitro . In sum, these studies suggest that this region of the topoisomerase (amino acids 216-225) plays a proximal role in mediating DNA cleavage and the covalent interaction between the 3'-phosphoryl of the nicked DNA and tyrosine 274 of the vaccinia topoisomerase I . The studies also provide useful reagents for the molecular genetic analysis of the role of the topoisomerase within the context of vaccinia virus infection.

Nucleic Acids Res, 1993 Jul 25, 21(15), 3507 - 11
Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis; Woerner AM et al.; The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity . We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain . In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting . Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding . This defines a DNA binding domain contained within amino acids 180-248 . This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates . A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.

Nucleic Acids Res, 1993 Jul 25, 21(15), 3385 - 90
Genome sequencing on both strands: the Janus strategy; Burland V et al.; The design of large scale DNA sequencing projects such as genome analysis demands a new approach to sequencing strategy, since neither a purely random nor a purely directed method is satisfactory . We have developed a strategy that combines these two methods in a way that preserves the advantages of both while avoiding their particular limitations . Computer simulations showed that a specific balance of random and directed sequencing was required for the most efficient strategy, termed the Janus strategy, which has been used in the Escherichia coli genome sequencing project . This approach depended on obtaining sequence easily from either strand of a cloned insert, and was facilitated by inversion of the insert in the engineered M13 vector Janus, by site-specific recombination . The inversion was accomplished simply by growth on the appropriate host strain, when the DNA strand incorporated into the new single stranded phage was complementary to that in the original phage, and was sequenced by the same simple protocol as the first strand.

J Biol Chem, 1993 Jul 25, 268(21), 16002 - 8
Isolation and functional expression in Escherichia coli of a gene encoding phosphatidylethanolamine methyltransferase (EC 2.1.1.17) from Rhodobacter sphaeroides; Arondel V et al.; Phosphatidylcholine is a major component of membranes in most eukaryotes, but it is found only in a small number of bacteria, where it is synthesized by N-methylation of phosphatidylethanolamine . In yeast and other fungi the methylation of phosphatidylethanolamine to phosphatidylcholine proceeds in two steps: the methylation of phosphatidylethanolamine by phosphatidylethanolamine methyltransferase followed by the methylation of monomethylphosphatidylethanolamine by phospholipid methyltransferase . Here we describe the isolation of two allelic phosphatidylcholine-deficient mutants of Rhodobacter sphaeroides which are unable to methylate phosphatidylethanolamine, monomethylphosphatidylethanolamine, or dimethylphosphatidylethanolamine . A DNA fragment containing a gene designated pmtA, which encodes a 22.9-kDa protein, was found to complement both mutants . Expression of this gene in Escherichia coli, which normally lacks phosphatidylcholine or methylated derivatives of phosphatidylethanolamine, resulted in the formation of phosphatidylcholine . A protein extract derived from the E . coli strain expressing the pmtA gene was able to convert phosphatidylethanolamine, mono- and dimethylphosphatidylethanolamine into phosphatidylcholine . Based on these data we conclude that the product of the pmtA gene catalyzes a sequence of three chemically distinct, methylation reactions beginning with phosphatidylethanolamine and leading to the formation of phosphatidylcholine in R . sphaeroides.

J Biol Chem, 1993 Jul 25, 268(21), 15751 - 7
Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro; Hamel CP et al.; Studies reported previously from this laboratory have shown that microsomal membranes of the vertebrate retinal pigment epithelium (RPE) contain an RPE-specific 65-kDa protein, RPE65, which bears the determinant recognized by the strictly tissue-specific monoclonal antibody RPE9, and which is developmentally regulated (Hamel, C . P., Tsilou, E., Harris, E., Pfeffer, B . A., Hooks, J . J., Detrick, B., and Redmond, T . M . (1993) J . Neurosci . Res . 34, 414-425) . Microsequencing of 17 tryptic and chymotryptic peptides obtained from the in situ digestion of the RPE65 blotted on nitrocellulose yielded primary sequences that were used to generate oligonucleotide probes . An 84-nucleotide guessmer was used to isolate two clones from a bovine RPE lambda Zap II cDNA library . Rapid amplification of cDNA ends was used to complete the 5' and 3' ends, resulting in a 3,115-base pair composite cDNA . The open reading frame encodes a novel protein of 533 amino acid residues with a computed molecular weight of 60,940 . This protein does not match any other sequence in the data bases . The 231 amino acids obtained from peptide sequencing match 43% of the amino acid sequence deduced from the cDNA . The protein has a calculated pI of 6.41 and is not predicted to have any transmembrane segments . The open reading frame expressed in Escherichia coli has an apparent molecular weight identical to that of the native protein and is recognized by the monoclonal antibody RPE9, further corroborating its validity . Northern blot analysis detected a major mRNA species of 3.15 kilobases for RPE65, as well as shorter species, only in RPE and not in other tissues (including other ocular tissues) . Cultured RPE cells (7 weeks in primary culture) contained RPE65 mRNA in amounts equivalent to fresh RPE . Such cells, however, contained no immunodetectable RPE65 . The possible structure of this RPE-specific protein and hypotheses for the absence of translation in vitro are discussed.

J Biol Chem, 1993 Jul 25, 268(21), 15745 - 50
Discrete carboxyl-terminal segments of apolipoprotein E mediate lipoprotein association and protein oligomerization; Westerlund JA et al.; The carboxyl terminus of apolipoprotein (apo) E is required for lipoprotein association and for tetramer formation . To correlate these roles with specific regions within the carboxyl terminus, a series of apoE3 variants with carboxyl-terminal truncations at residues 266, 244, 223, and 191 were expressed in Escherichia coli . As determined by gel permeation and sedimentation equilibrium centrifugation, the four truncated variants were monomeric in solution . Compared to native apoE3 (299 residues), all had reduced affinity for lipoproteins, as assessed by incubation of 125I-labeled proteins with plasma followed by fractionation of lipoprotein classes by gel filtration . The 266-residue variant associated with very low density lipoproteins and high density lipoproteins, but was partly non-lipoprotein-bound (25% of total) . Shorter variants, with 244 or fewer residues, did not associate with very low density lipoproteins and only associated slightly (approximately 20%) with high density lipoproteins, with the major portion non-lipoprotein-bound (65-73%) . After these proteins were injected into rabbits, the clearance rate was proportional to the plasma level of non-lipoprotein-bound protein . These results indicate lipoprotein association modulates the clearance of apoE, residues within the segment 267-299 are critical for apoE tetramerization and facilitate lipoprotein association, and residues within the segment 245-266 also contribute to lipoprotein association.

J Biol Chem, 1993 Jul 25, 268(21), 15737 - 44
Human cystatin D . cDNA cloning, characterization of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva; Freije JP et al.; A cDNA coding for cystatin D, a human member of the cystatin protein family, has been cloned after specific amplification of reverse-transcribed parotid gland RNA . After replacing the segment encoding the putative 20-residue signal peptide with one encoding the Escherichia coli OmpA leader sequence, the cDNA was expressed in E . coli . The isolated recombinant protein exhibited Ki values of 1.2 nM and > 1 microM for papain and cathepsin B, respectively . An antiserum raised against recombinant cystatin D recognized a protein in human saliva with electrophoretical mobility identical to that of the recombinant protein . Immunoenzymatic analysis revealed that this cysteine proteinase inhibitor is present in human saliva and tears at concentrations of 3.8 and 0.5 mg/liter, respectively, while it was not detected in seminal plasma, blood plasma, milk, or cerebrospinal fluid . Cystatin D purified from human saliva by immunosorption displayed a heterogeneous N-terminal end, with sequences starting at residues 5, 7, 9, and 11 of the predicted N-terminal portion of the mature protein . On the basis of structural and functional properties, cystatin D represents a novel cysteine proteinase inhibitor possibly playing a protective role against proteinases present in the oral cavity.

J Biol Chem, 1993 Jul 25, 268(21), 15712 - 20
Laser cross-linking of proteins to nucleic acids . I . Examining physical parameters of protein-nucleic acid complexes; Hockensmith JW et al.; Pulsed laser cross-linking results in efficient and rapid formation of covalent bonds between proteins and the nucleic acids to which they are bound, creating a "snapshot" of the protein-nucleic acid equilibrium existing at the moment of irradiation . The "frozen" equilibrium allows the determination of protein-nucleic acid binding constants, confirming both theoretical predictions and experimental determinations by standard physical chemical methods . Laser cross-linking results accurately reflect the alteration of protein-nucleic acid interactions induced by traditional methods such as increasing the salt concentration or by the addition of a nucleic acid that competes for binding of the protein . Thus this technique is very useful for the study of the association of proteins and protein complexes with nucleic acids under environmental conditions at which the reactions are not amenable to study by traditional physical chemical methods . In this paper we continue our calibration of the method, focusing primarily on interactions with single-stranded DNA-binding proteins and describe techniques for measuring quantitative interactions between nucleic acid constructs and single-protein or multiprotein complexes . Laser cross-linking can also provide direct evidence that binding correlates with functional activity.

J Biol Chem, 1993 Jul 25, 268(21), 15655 - 8
Evidence against a relationship between fatty acid ethyl ester synthase and the Pi class glutathione S-transferase in humans; Board P et al.; Recently, Bora et al . (Bora, P . S., Bora, N . S., Wu, X., and Lange, L . G . (1991) J . Biol . Chem . 266, 16774-16777) reported the cloning and expression of a human fatty acid ethyl ester synthase III (FAEES-III) cDNA that has only four amino acid substitutions compared with human glutathione S-transferase (GST) GSTP1-1, and, when expressed in MCF-7 cells, the protein has both FAEES and GST activities . By site-directed mutagenesis of a GSTP1 cDNA, we have constructed a clone that encodes the FAEES-III protein described by Bora et al . (1991) . The recombinant FAEES-III protein was expressed in Escherichia coli and has been shown to be devoid of FAEES and GST activities . The recombinant FAEES-III protein does not bind to a glutathione agarose affinity matrix, presumably because two of the substituted amino acids, Trp-39-->Cys and Gln-52-->Glu, are thought to contribute to the GST glutathione binding site . One of the base substitutions in the FAEES-III cDNA encodes an extra SacI site not found in the GSTPI cDNA . Polymerase chain reaction amplification of human genomic DNA has identified the GSTPI gene, but no DNA from the proposed FAEES gene with a diagnostic SacI site has been detected . Evaluation of the hybridization pattern of HindIII genomic restriction fragments has identified fragments that contain the GSTPI gene and a pseudogene (Board et al . 1992), and there do not appear to be any hybridizing fragments that could contain the FAEES-III gene . Our results do not provide any evidence in support of a relationship between FAEES-III and GST, and the cDNA reported by Bora et al . (1991) may have resulted from a cloning artifact.

J Biol Chem, 1993 Jul 25, 268(21), 15435 - 41
The 28-kDa N-terminal domain of mouse stromelysin-3 has the general properties of a weak metalloproteinase; Murphy G et al.; The putative matrix metalloproteinase mouse stromelysin-3 was expressed from Escherichia coli and from a mouse myeloma cell line . In the former case a single major protein of 58-kDa was detectable by immunoblotting, but no proteolytic activity could be elicited by zymography or trypsin or organomercurial treatment as would be expected for a typical matrix metalloproteinase . In the latter case immunodetectable proteins of 55-58 and 27-28-kDa were produced . The effect of trypsin or organomercurial treatment of the 55-58-kDa forms was to generate a 51-kDa form and lower molecular mass fragments . Upon zymographic analysis only the 27-28-kDa forms showed caseinolytic activity . N-terminal sequencing and immunoblotting analysis with antibodies specific to distinct domains of stromelysin-3 indicated that the 27-28-Da stromelysin-3 forms had lost the predicted propeptide and the majority of the C-terminal domain . The purified 28-kDa form of stromelysin-3 could weakly degrade a number of extracellular matrix proteins and was inhibited by TIMP . However, the evidence that mature full-length stromelysin-3 is a metalloproteinase could not be substantiated and the precise role of this protein in vivo remains to be elucidated . By partial analogy with interstitial collagenase, one hypothesis is that stromelysin-3 with an intact C-terminal domain has specific properties for an as yet undefined substrate.

J Biol Chem, 1993 Jul 25, 268(21), 15510 - 6
Identification of the specific oligosaccharide sites recognized by type 1 fimbriae from Escherichia coli on nonspecific cross-reacting antigen, a CD66 cluster granulocyte glycoprotein; Sauter SL et al.; Nonspecific cross-reacting antigen (NCA), a CD66 cluster antigen, is a well characterized glycoprotein on granulocytes, macrophages, and lung epithelium . Structural studies at the protein and genomic levels have revealed that NCA is a member of the immunoglobulin (Ig) supergene family and contains a domain structure similar to Ig with an amino-terminal variable-like domain followed by disulfide loop-containing constant-like domains . Previous work by this laboratory and others has demonstrated that NCA is a receptor for binding of bacteria expressing type 1 fimbriae (pili) . This binding is mediated by interaction between lectins on the bacteria fimbriae and carbohydrate chains on NCA . In the present work we further characterize the specificity for bacterial binding by NCA using endoglycosidases and site-directed mutagenesis . Results of these studies demonstrate that Escherichia coli expressing type 1 fimbriae binds to high mannose oligosaccharide structures on NCA and that the functionally relevant sites are located in the variable-like domain of NCA.

Nature, 1993 Jul 22, 364(6435), 355 - 8
The role of turns in the structure of an alpha-helical protein; Brunet AP et al.; The turns joining segments of secondary structure have been proposed to be key elements in dictating the folded structures of native proteins . An alternative view assumes that turns play a passive role and are merely default structures that occur as a consequence of interactions between antiparallel segments of secondary structure, with chain reversal being dictated by the context surrounding the turn and not by the sequence of the turn itself . The solvent-exposure of turns and their tolerance to evolutionary variance suggests that they may have little or no effect on the formation of native structures . Previous investigations have focused on various types of beta-turns that connect antiparallel beta-strands with comparatively little reported on the structural role of interhelical turns . Here we probe the structural importance of such a turn in an antiparallel 4-helix bundle by randomly substituting an interhelical tripeptide in cytochrome b-562 with many different amino-acid sequences . Thirty-one of the resulting substituted proteins were characterized and all of them were shown to fold into stable, native-like structures . These results suggest that this interhelical turn does not does not play a dominant role in determining the folded structure of this antiparallel 4-helix bundle.

Biochim Biophys Acta, 1993 Jul 21, 1169(1), 1 - 11
Cloning of the cDNA coding for 14 kDa group II phospholipase A2 from rat liver; Van Schaik RH et al.; The amino acid sequence of rat liver phospholipase A2 was partially elucidated using peptide fragments generated by enzymatic or chemical cleavage . Based on this sequence information, two oligonucleotide probes were constructed which were applied in a polymerase chain reaction on cDNA generated from rat liver total RNA . This resulted in cloning of the cDNA corresponding to the coding region of the mature phospholipase A2 . The deduced amino acid sequence showed the enzyme belongs to the group II phospholipases, and is almost completely identical to rat platelet and spleen membrane-associated phospholipase A2 . However, in the cDNA isolated one codon was different as compared to the platelet and spleen enzymes, resulting in the substitution of Ala94 by Arg94 in the liver enzyme . In Northern blot analyses the mRNA for rat group II phospholipase A2 could not be detected in rat liver, neither in total RNA nor in poly(A)+ RNA . However, a polymerase chain reaction using total RNA originating from freshly isolated hepatocytes resulted in the amplification of the described phospholipase A2 cDNA . This indicates that group II PLA2 mRNA is present in these cells, but presumably at very low abundance . The observed increase in rat group II phospholipase A2 secretion in rat mesangial cells upon stimulation with interleukin-1 beta (Pfeilschifter et al . (1989), Biochem . Biophys . Res . Commun . 159, 385-394) was shown to be accompanied by an increased transcription of the rat group II phospholipase A2 gene, indicating interleukin exerts its effect via increased phospholipase A2 mRNA synthesis . Based on Northern blot analyses of stimulated rat mesangial cells, the size of the mRNA for rat group II phospholipase A2 was determined to be 0.9 kb.

J Mol Biol, 1993 Jul 20, 232(2), 493 - 8
Ligand binding induces an asymmetrical transmembrane signal through a receptor dimer; Yang Y et al.; Two ligand (aspartate)-binding pockets are formed at the interface between the subunits of the Tar homodimer, a bacterial chemoreceptor . Using mutant heterodimers of a hybrid receptor, Taz1, which consists of the external domain of Tar and the cytoplasmic domain of EnvZ, we disrupted either one or the other of the two ligand-binding pockets . We found that occupation of only one of the ligand-binding pockets was sufficient for induction of a transmembrane signal, and that the subunit responsible for the binding of the amino group of the ligand transduces the signal.

J Mol Biol, 1993 Jul 20, 232(2), 484 - 92
Ligand binding to the receptor domain regulates the ratio of kinase to phosphatase activities of the signaling domain of the hybrid Escherichia coli transmembrane receptor, Taz1; Jin T et al.; Taz1 is a hybrid receptor, in which the periplasmic receptor domain of Tar, an aspartate chemoreceptor, is fused with the cytoplasmic signaling domain of EnvZ, an osmosensor . Taz1 is able to induce ompC-lacZ expression in response to aspartate added to the medium . We introduced amino acid substitution mutations in the highly conserved region of the signaling domain of Tar near the Tar-EnvZ junction . The same mutations in Tsr, a serine chemoreceptor, are known to lock the flagella rotation in either a clockwise (CW) or in a counter-clockwise (CCW) mode . It was found that a CW-biased mutation in Taz1 resulted in ompC-lacZ expression in the "off mode", or low ompC-lacZ expression in both the absence and presence of aspartate, while CCW-biased mutations caused ompC-lacZ expression in the "on mode", or constitutive expression regardless of aspartate . The OmpR kinase and phospho-OmpR phosphatase activities of the wild-type and mutant Taz proteins were also examined in response to aspartate . The phosphatase activity of the wild-type Taz1 was found to decrease in the presence of aspartate, while the OmpR kinase activity remained constant . This indicated that aspartate binding to the Taz1 receptor domain modulates the ratio of kinase to phosphatase activity of the signaling domain . An increased kinase to phosphatase ratio in the presence of aspartate resulted in higher levels of phospho-OmpR in the cell and therefore induced ompC-lacZ expression . In contrast to the wild-type Taz1 protein, the enzymatic activities of CW as well as CCW mutants did not change in response to aspartate, indicating that mutant Taz proteins are incapable of transducing the signal across the membrane as a result of a locked conformation of the signaling domain in either the on or off mode.

J Mol Biol, 1993 Jul 20, 232(2), 419 - 45
XbaI and BlnI genomic cleavage maps of Escherichia coli K-12 strain MG1655 and comparative analysis of other strains; Perkins JD et al.; Complete XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 are presented, along with a comparison of the physical map of MG1655 with that of five other K-12 strains . We have mapped 35 XbaI cleavage sites generating 35 fragments ranging in size from 8 kb to 432 kb using methods similar to those used previously for the NotI and SfiI maps of MG1655 . The applicability of the MG1655 map to other strains of E . coli K-12 was assessed by comparing the NotI, SfiI and XbaI digestion patterns of EMG2, W1485, W3110, AB1157 and MC4100 with those of MG1655 . The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis . A model is presented to account for the differences between the strains on the basis of simple insertions, deletions and, in one case, an inversion . Insertions and deletions ranging in size from 1 kb to 86 kb are suggested by this model . Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains . The various features localized in these strains were used to place 9 of the 17 BlnI fragments on the E . coli physical map . The remaining fragments were placed by hybridization experiments similar to those used for the NotI, SfiI and XbaI maps . In this way, the complete BlnI map was constructed . The cleavage sites for XbaI and BlnI were assigned coordinates based on EcoMap6 developed by Rudd et al . The XbaI and BlnI maps of MG1655 presented here, when combined with the NotI (22 sites) and SfiI (31 sites) maps of MG1655 previously published, bring the total number of mapped rare restriction sites in MG1655 to 105 . The strain comparison analysis shows that this map is readily adaptable for use with other K-12 strains.

J Mol Biol, 1993 Jul 20, 232(2), 397 - 405
Formation of a RuvAB-Holliday junction complex in vitro; Parsons CA et al.; The ruvA and ruvB genes of Escherichia coli encode a novel DNA helicase that interacts with Holliday junctions and promotes branch migration . In this work, we have investigated the protein-DNA complexes formed between RuvA, RuvB and Holliday junctions . As shown previously, RuvA protein binds a synthetic Holliday junction in vitro, to form a specific protein-DNA complex that can be detected by a band-shift assay . We now show that the combined presence of RuvA and RuvB results in a super-shift of this complex indicative of the formation of a RuvAB-Holliday junction complex . In the absence of RuvA, the RuvB protein fails to bind Holliday junctions . The RuvAB-Holliday junction complex was detected by the band-shift assay only under conditions that favoured its stability, e.g . complex formation in the presence of a nucleoside triphosphate that can not be hydrolysed by RuvB (adenosine 5'-{gamma-thio}triphosphate) . In contrast, nucleoside triphosphates that can be hydrolysed (ATP, dATP, dCTP or TTP), lead to RuvAB-mediated branch migration of the junction . These results indicate that the formation of a (RuvAB-ATP)-Holliday junction complex represents the first step in the process of branch migration, and that branch migration is dependent upon ATP hydrolysis . In addition, we show that Holliday junction DNA stimulates the ATPase activity of RuvAB to a greater extent than either single-stranded or linear duplex DNA.

Biochemistry, 1993 Jul 20, 32(28), 7136 - 42
Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution; Ishikawa K et al.; The insertion of a Gly residue (designated as Gly-80b) between the C-cap of the alpha II-helix (Gln-80) and the N-cap of the alpha III-helix (Trp-81) in Escherichia coli ribonuclease HI enhances the protein stability by 0.4 kcal/mol in delta G (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., & Kanaya, S . (1992) J . Biol . Chem . 267, 21535-21542) . Another mutation within the alpha II-helix, Gly-77-->Ala, reduces the stability by 0.9 kcal/mol . Simultaneous introduction of these mutations enhances the stability by 0.8 kcal/mol, indicating that the effects of these mutations are cooperative and not simply independent . We determined the crystal structures of these three mutant proteins (G80b-, A77-, and A77/G80b-RNase H) to investigate this cooperative mechanism of the protein stabilization . The structures revealed that the inserted Gly-80b assumes a left-handed helical conformation in both the G80b- and the A77/G80b-RNase H . This inserted glycine residue allows the formation of a "paperclip", which is a common motif at the C-termini of alpha-helices . Accompanying the formation of the paperclip motif, two intrahelical hydrogen bonds are formed between the backbone atoms (O78-N80b and O80b-N84) . The stabilization caused by the insertion of Gly-80b can be ascribed to the formation of these hydrogen bonds . The Gly-77-->Ala substitution destabilizes the protein due to the deformed packing interactions in the hydrophobic core around Ala-77 and the stress in the wedged indole ring of Trp-81 . These effects are alleviated by the insertion of Gly-80b, which relaxes the backbone structure.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1993 Jul 20, 232(2), 406 - 18
The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an "extended minus 10" promoter; Kumar A et al.; It is known that Escherichia coli promoters having the major minus 10 consensus sequence TATAAT, but lacking significant resemblance to consensus in the minus 35 region, allow transcriptional initiation in vivo and in vitro if they have an additional TGn motif immediately upstream of minus 10 . To determine whether region 4.2 of sigma 70, whose normal role is sequence recognition at minus 35, is unnecessary for initiation of transcription at such "extended minus 10" promoters, we modified the sigma 70 gene so as to generate a carboxy-truncated polypeptide lacking the last 84 amino acids and therefore missing region 4.2 . Our results show that both the intact and truncated sigma 70 allow purified RNA polymerase to initiate efficiently and specifically at an extended minus 10 promoter, whereas only the intact sigma 70 permits efficient initiation at normal promoters (defined by minus 35 and minus 10 hexamer sequences).

J Mol Biol, 1993 Jul 20, 232(2), 362 - 74
Dynamics of in vitro assembly of 16 S rRNA into 30 S ribosomal subunits; Powers T et al.; One of the important unsolved problems in the ribosome field is the molecular basis for the sequential and co-operative nature of ribosome assembly . As an approach to this problem, we have taken advantage of the temperature dependence of in vitro reconstitution and have used chemical probing methods to examine the conformation and reactivity of 16 S rRNA at successive stages during subunit assembly . One class of nucleotides displays reactivities similar to those observed in native 30 S particles when the RNA and protein are incubated in the absence of any heat step (0 degrees C effects) . At 30 degrees C, where the assembly process takes 2 hours, other bases can be assigned to one of several additional kinetic classes, determined by the rate at which their chemical reactivities transit from levels observed in naked RNA to levels observed in fully assembled subunits: (1) fast (t1/2 = < 5 min at 30 degrees C); (2) slow (t1/2 = 15 to 30 min at 30 degrees C); (3) delayed slow (t1/2 = 30 to 60 min at 30 degrees C) . Finally, several nucleotides display transient kinetics in their reactivities, showing increasing reactivity at early time points and becoming protected later in assembly; most of these effects correspond to residues that were previously shown to display reciprocal enhancement and protection patterns during step-wise in vitro assembly . These findings, together with our previous studies using purified individual proteins lead to the following conclusions: (1) there is a predominant 5' to 3' polarity to in vitro assembly, even though it is uncoupled from transcription; (2) portions of the central and 3' major domains fold into an active conformation only at a very late stage of assembly; (3) bases footprinted by late-assembling proteins, according to the 30 S subunit assembly map, show generally slower kinetics than residues footprinted by proteins that bind early in the assembly map, providing direct evidence for the sequential nature of the in vitro assembly process; (4) most proteins are associated with nucleotides that fall into more than one kinetic class, suggesting that assembly proceeds through multiple pathways, or that individual proteins interact sequentially with different regions of the RNA.

Biochemistry, 1993 Jul 20, 32(28), 7302 - 9
Thermodynamics of ligand binding to trp repressor; Jin L et al.; The thermodynamics of L-tryptophan and operator DNA binding to the tryptophan repressor of Escherichia coli were analyzed by titration microcalorimetry and van't Hoff analysis of footprinting titrations, respectively . At 25 degrees C in 10 mM sodium phosphate, pH 7.6, and 0.1 M NaCl, the binding of L-tryptophan to the repressor is characterized by values of delta G degrees = -6.04, delta H degree = -14.7, and T delta S degree = -8.67 kcal/mol . The temperature dependence of delta H degree yields delta Cp degree = -0.46 +/- 0.08 kcal/(mol.K) per dimer . The binding is noncooperative at all temperatures studied . At 23 degrees C in 2.5 mM sodium phosphate, pH 7.6, and 25 mM NaCl, the binding of operator DNA to the repressor is characterized by values of delta G degree = -13.3 kcal/mol, delta H degree = -1.55 kcal/mol, T delta S degree = 11.8 kcal/mol, and delta Cp degree = -0.54 +/- 0.10 kcal/(mol.K) . Changes in water-accessible surface areas upon binding of L-tryptophan or DNA were calculated from X-ray crystal structures . The experimentally observed delta Cp degree values were compared with delta Cp degree values calculated according to several methods based on various proposed relationships between surface area changes and heat capacity changes . Regardless of which method is used, we find poor agreement between the calorimetric results for L-tryptophan binding and the surface areas calculated from X-ray data; the direction of the discrepancy is that the X-ray data underestimate the value of delta Cp degree.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Jul 20, 32(28), 7133 - 5
An elongated form of T4 glutaredoxin with four extra residues; Nikkola M et al.; Two different forms of T4 glutaredoxin (thioredoxin) arising from the same gene on a multicopy plasmid in an Escherichia coli expression system have been isolated and characterized . Up to one-fourth of purified T4 glutaredoxin has an extension of four amino acids in the carboxy terminus, with the sequence aspartate, arginine, isoleucine, lysine . This four-residue extension may be caused by a translational +1 frameshift at the UGA terminator codon.

Biochemistry, 1993 Jul 20, 32(28), 7069 - 78
Comparison of sequence preference of tomaymycin- and anthramycin-DNA bonding by exonuclease III and lambda exonuclease digestion and UvrABC nuclease incision analysis; Pierce JR et al.; The DNA bonding sites of two pyrrolo{1,4}benzodiazepine derivatives--tomaymycin (Tma) and anthramycin (Atm)--were identified by exonuclease III (exo III) digestion, lambda exonuclease (lambda exo) digestion, and UvrABC nuclease incision analysis . exo III digestion stalls 4-5 bases 3' to a drug-DNA adduct . While this method can recognize most of the Atm-and Tma-DNA modification sites, it is complicated in that exo III digestion is also stalled by certain unmodified sequences and by drug bound to the opposite strand . lambda exo digestion stalls 1-2 bases 5' to a drug-DNA adduct . The lambda exo method also recognizes most of the drug-DNA bonding sites and renders a cleaner background; however, it is also affected by opposite-strand drug bonding . Due to their intrinsic digestion polarities, these two exonucleases tend to be stalled by the drug-DNA adduct at one end of the DNA molecule . Purified UvrA, UvrB, and UvrC proteins acting together make dual incisions 6-8 bases 5' and 4 bases 3' to a Atm- or Tma-DNA adduct . This nuclease complex recognizes all the Tma- and Atm-DNA bonding sites identified by exonuclease digestion methods, and all the UvrABC incisions can be attributed to drug modifications in the incised DNA strand . The degree of UvrABC nuclease incision increases with increasing drug concentrations for DNA modification . Using the UvrABC incision method, we have identified the sequence preference of Tma- and Atm-DNA adduct formation in three DNA fragments, and we have found that these two drugs have different preferred sites for adduction . Both Tma- and Atm-DNA bonding is strongly influenced by the 5' and 3' neighboring bases; the orders of preferred 5' and 3' bases for Tma are A > G, T > C, and A, C > G, T, and for Atm the orders are A > G > T > C and A > G > T, C . The preferred triplets for Tma bonding are -AGA- > -GGC-, -TGC-, and AGC- and for Atm are -AGA-, -AGG- > -GGA-, -GGG-.

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