Escherichia coli

Biochem J, 1993 Sep 1, 294 ( Pt 2), 589 - 93
cDNA cloning and characterization of the protein encoded by RD, a gene located in the class III region of the human major histocompatibility complex; Cheng J et al.; The RD gene, initially defined in the mouse, has been mapped between the Bf and C4A genes in the human major histocompatibility complex class III region . Using the mouse cDNA as a probe, we isolated and sequenced human RD cDNA clones . The composite nucleotide sequence consisted of 1301 nucleotides, excluding a poly(A) tail at the 3' end . It contained a single open reading frame encoding a polypeptide of 380 amino acid residues with a calculated molecular mass of 42274 Da . The most striking structural feature of the deduced amino acid sequence is a region consisting entirely of 24 tandem repeats of an Arg-Asp (or Glu) dipeptide . The human RD cDNA was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and used to produce antisera in rabbits . Western blot analysis and immunoprecipitation of lysates of biosynthetically labelled HeLa cells indicated that RD is a 44 kDa nuclear protein.

Biochem J, 1993 Sep 1, 294 ( Pt 2), 521 - 7
Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing; Morris SA et al.; Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer . Electrospray mass spectrometry was used to confirm that the recombinant S . erythraea acyl-carrier protein over-expressed in E . coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct . The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography . The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S . erythraea.

Biochem J, 1993 Sep 1, 294 ( Pt 2), 373 - 80
Molecular cloning and heterologous expression of an alternatively spliced human Mu class glutathione S-transferase transcript; Ross VL et al.; Two cDNA clones encoding a new Mu class glutathione S-transferase (GST) have been isolated from a human testis cDNA library . Both clones are incomplete and appear to result from alternative splicing . One clone is missing the sequence encoding exon 4 and the other is missing exon 8 . The complete sequence of the previously undescribed isoenzyme can be deduced from the two cDNA clones . This is the first report of alternative splicing in a GST transcript and may represent either a novel form of regulation in this multigene family or illegitimate transcription and experimental alternative splicing as part of the evolutionary process . By combining components from each clone a complete cDNA has been constructed and the encoded protein expressed in Escherichia coli . In general, the recombinant enzyme has relatively low activity when compared with all the previously described human Mu class GST isoenzymes.

Arch Biochem Biophys, 1993 Sep, 305(2), 606 - 10
Inhibitory effect of the polyanionic drug suramin on the in vitro HIV DNA integration reaction; Carteau S et al.; An obligatory step in retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host . Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely the processing of the LTR ends and the strand transfer reaction . Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-1 U5 LTR as substrate and supercoiled pSP65 DNA as target, we have investigated the effect of the polyanionic drug suramin on the catalytic activity of the IN protein . It was found that at stoichiometric suramin to protein ratios, suramin displays a strong inhibitory effect on both the processing and strand transfer reactions . This inhibitory effect is related to the decrease of IN protein binding efficiency to the LTR end DNA fragment.

Arch Biochem Biophys, 1993 Sep, 305(2), 588 - 94
Interaction of indoleacrylic acid with Trp aporepressor from Escherichia coli; Hu D et al.; Trans-beta-Indoleacrylic acid (IAA) binds with moderately high affinity to the dimeric protein, trp aporepressor from Escherichia coli . IAA is itself nonfluorescent, and, upon binding, IAA quenches approximately 95% of the intrinsic tryptophan fluorescence of the protein . Since there is an overlap between the absorbance of IAA and the emission of tryptophan, this quenching appears to be due to resonance energy transfer . To adequately fit binding isotherms, it was necessary to use a model in which the binding of IAA to one subunit is able to quench fluorescence from both subunits of the protein . This model also assumes that binding to the two subunits occurs independently . Binding data were obtained as a function of the concentration of trp aporepressor, over the range of 1 x 10(-8) to 5 x 10(-5) M, in order to test the possibility that protein subunit association or dissociation occurs . When analyzed in terms of the above model, no significant protein concentration dependence is seen for the IAA association constant, indicating that either (i) the protein does not undergo changes in oligomerization over this concentration range or that (ii) different states of aggregation bind IAA with similar affinity . The affinity of IAA was found to increase at higher ionic strength and at lower pH . Also, evidence is presented for a photoinduced change in the configuration of IAA.

Arch Biochem Biophys, 1993 Sep, 305(2), 499 - 508
Dihydrofolate reductase from the pathogenic fungus Pneumocystis carinii: catalytic properties and interaction with antifolates; Margosiak SA et al.; Dihydrofolate reductase (DHFR) from the fungus Pneumocystis carinii (pcDHFR), a target for antifolate inhibitors, has been compared with host enzyme, human DHFR (hDHFR), and with DHFR from Escherichia coli . Among the results of the considerable structural differences between pcDHFR and the other two enzymes is a much higher turnover number (kcat, 136 s-1) for pcDHFR . This is due to rapid hydride transfer from NADPH to dihydrofolate (rate constant 402 s-1), very rapid dissociation of NADP from the product complex (rate constant, k(off), > 1000 s-1), and after NADPH binding, rapid dissociation of tetrahydrofolate (k(off), 216 s-1) . Cycling of pcDHFR is almost exclusively by this pathway . The high kcat contributes to a high Km for NADPH (9 microM) and an unusually high Km for dihydrofolate (2.5 microM) . Nevertheless, the efficiency of pcDHFR is greater than DHFR from E . coli and about 25% that of hDHFR . Of seven clinically relevant inhibitors investigated, only one (trimethoprim) had a slightly lower Ki for pcDHFR than for hDHFR . The therapeutic value of trimethoprim-sulfa treatment of P . carinii infections indicates that other factors play an important role, but the results are consistent with the frequency of complications due to toxicity of trimethoprim.

Arch Biochem Biophys, 1993 Sep, 305(2), 460 - 6
Role of the highly conserved histidine residues in rat liver mitochondrial aldehyde dehydrogenase as studied by site-directed mutagenesis; Zheng CF et al.; One histidine residue (H235) is conserved in all known aldehyde dehydrogenases (ALDH), from Escherichia coli to human, except for those from P . oleovorans and rat hepatoma . Kinetic studies with horse liver mitochondrial ALDH indicated that a group with a pKa of 7 may be involved in the active site . Using site-directed mutagenesis, the four conserved histidine residues of the six histidines in rat liver mitochondrial ALDH were converted to alanines . Only modification of H235 and H29 caused alterations in properties of the enzyme . H29A had a decreased pI suggesting that this residue may normally be protonated . Its Vmax increased, as did the Km for NAD+, while the Km for propionaldehyde decreased . H235A had the same pI as the native enzyme but the Vmax decreased by 50% . Like native enzyme, H235A was active in Hepes and Mops buffer as well as in phosphate buffer . Purified H235A was thermally less stable than was native enzyme . H235 was also changed to F, Y, E, K, and Q . All of these substitutions resulted in the formation of insoluble aggregates or inclusion bodies when they were expressed in E . coli . It appears then that the highly conserved histidine residues may not be functioning as a general base in the deacylation step as we originally suggested . Instead, both H29 and H235 may be of structural importance and the presence of a histidine residue at position 235 may be required for the newly synthesized peptide to fold and/or assemble into the native conformation of ALDH.

Am J Obstet Gynecol, 1993 Sep, 169(3), 531 - 7
Effects of endotoxins and cytokines on the secretion of platelet-activating factor-acetylhydrolase by human decidual macrophages; Narahara H et al.; OBJECTIVE: The aim was to clarify the role of platelet-activating factor in parturition, preterm labor, and premature rupture of membranes . STUDY DESIGN: Decidual macrophage populations were obtained by enzymic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting . The effects of endotoxins and cytokines on platelet-activating factor-acetylhydrolase secretion by these cells were examined . RESULTS: Lipopolysaccharide inhibited the platelet-activating factor-acetylhydrolase secretion by decidual macrophages . The inhibition was partially reversed by interleukin-1 receptor antagonist or by neutralizing antibodies against interleukin-1 alpha, interleukin-1 beta, or tumor necrosis factor-alpha . Tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-1 beta also decreased the enzyme secretion . The inhibitory actions of tumor necrosis factor-alpha and interleukin-1 beta were specifically neutralized by the corresponding antibodies . The effect of interleukin-1 alpha or interleukin-1 beta on the secretion was abolished by interleukin-1 receptor antagonist . CONCLUSION: It is suggested that platelet-activating factor is involved in the pathogenesis of preterm labor or premature rupture of membranes caused by endotoxins and the subsequent activation of cytokine network.

Genes Dev, 1993 Sep, 7(9), 1810 - 23
Functional interaction of adenovirus E1A with holo-TFIID; Boyer TG et al.; The activation domains of several regulatory transcription factors have been shown to bind directly in vitro to the TATA box-binding protein (TBP) . Yet TBP must also interact with multiple associated polypeptides, called TAFs, for these same activators to stimulate transcription . These findings raise the question of how TBP can interact with so many proteins, both activators and TAFs, simultaneously . Here, we show that the activation domain of the adenovirus large E1A protein can bind specifically and stably to isolated holo-TFIID, the multisubunit protein complex consisting of TBP plus TAFs . Consequently, the surface of TBP that interacts with E1A must be exposed in the holo-TFIID complex . To assess the functional significance of this interaction, we established an in vitro transcription system responsive to the E1A activation domain . The addition of excess E1A to this system inhibits (squelches) both E1A-dependent and E1A-independent transcription by sequestering a target factor required for E1A activation . From among the component activities that collectively reconstitute E1A-responsive transcription in this system, holo-TFIID alone is singularly capable of reversing the inhibition of transcription mediated by excess E1A, indicating that holo-TFIID is the direct functional target of the E1A activation domain.

Genes Dev, 1993 Sep, 7(9), 1766 - 78
Transcription-dependent recombination induced by triple-helix formation; Kohwi Y et al.; The homologous recombination between direct repeat sequences separated by either 200 or 1000 bp was induced by active transcription of the downstream gene when poly(dG)-poly(dC) sequences exist between the two direct repeats . This dG tract-mediated and transcription-induced recombination was RecA independent, and the frequency of recombination was dependent on both the length and the orientation of the poly(dG)-poly(dC) sequences relative to the gene . An intramolecular dG.dG.dC triplex formation was detected in Escherichia coli cells in a length-dependent manner when the transcription of the downstream gene was activated . We suggest that the negative superhelical strain generated by active transcription of the downstream gene induces poly(dG)-poly(dC) sequences to adopt a triple-helix structure in vivo and that this structure brings two remote sequences together to stimulate homologous recombination.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8229 - 33
Evidence that the 60-kDa protein of 17S U2 small nuclear ribonucleoprotein is immunologically and functionally related to the yeast PRP9 splicing factor and is required for the efficient formation of prespliceosomes; Behrens SE et al.; Small nuclear ribonucleoprotein (snRNP) U2 functions in the splicing of mRNA by recognizing the branch site of unspliced mRNA . The binding of U2 snRNP and other components to pre-mRNA leads to the formation of a stable prespliceosome . In HeLa nuclear extracts, U2 snRNP exists either as a 17S form (under low salt conditions) or a 12S form (at higher salt concentrations) . We have recently shown that the purified 17S U2 snRNP contains nine proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa in addition to the common snRNP proteins and the U2 proteins A' and B" that are found in the 12S U2 snRNP form . By using antibodies against the PRP9 protein from Saccharomyces cerevisiae (a protein required for the addition of U2 to prespliceosomes in yeast), we have shown that the 60-kDa protein specific to human U2 snRNP particles is structurally related to the yeast PRP9 protein . Interestingly, anti-PRP9 antibodies strongly inhibit prespliceosome formation in HeLa nuclear splicing extracts, resulting in a complete inhibition of the mRNA splicing reaction in vitro . This indicates that the U2 60-kDa protein may also be functionally related to its yeast counterpart PRP9 . Most importantly, the addition of purified 17S U2 snRNPs, but not of 12S U2 snRNPs, to HeLa splicing extracts in which the endogeneous U2 snRNPs have been functionally neutralized with anti-PRP9 antibodies fully restores the mRNA-splicing activity of the extracts . These data suggest further that the 17S form is the functionally active form of U2 snRNP in the spliceosome.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8174 - 8
Crystal structure of yeast TATA-binding protein and model for interaction with DNA; Chasman DI et al.; The C-terminal 179-aa region of yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP), phylogenetically conserved and sufficient for many functions, formed crystals diffracting to 1.7-A resolution . The structure of the protein, determined by molecular replacement with coordinates from Arabidopsis TBP and refined to 2.6 A, differed from that in Arabidopsis slightly by an angle of about 12 degrees between two structurally nearly identical subdomains, indicative of a degree of conformational flexibility . A model for TBP-DNA interaction is proposed with the following important features: the long dimension of the protein follows the trajectory of the minor groove; two rows of basic residues conserved between the subdomains lie along the edges of the protein in proximity to the DNA phosphates; a band of hydrophobic residues runs down the middle of the groove; and amino acid residues whose mutation alters specificity for the second base of the TATA sequence are juxtaposed to that base.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8169 - 73
Targeting of the UmuD, UmuD', and MucA' mutagenesis proteins to DNA by RecA protein; Frank EG et al.; In addition to its critical role in genetic recombination, the Escherichia coli RecA protein plays a pivotal role in SOS-induced mutagenesis . This role can be separated genetically into three steps: (i) depression of the SOS regulon by mediating the posttranslational cleavage of the LexA repressor, (ii) activation of UmuD'-like proteins by mediating cleavage of the UmuD-like proteins, and (iii) a direct step, possibly to interact with and to target the Umu-like mutagenesis proteins to lesions in DNA . We have analyzed RecA's third role biochemically using protein affinity chromatography and an agarose-based DNA mobility-shift assay . RecA730 protein from a crude cell extract was specifically retained on UmuD and UmuD' protein affinity columns, suggesting that these proteins physically interact . Normally, neither UmuD nor UmuD' shows any affinity for DNA . In the presence of RecA protein, however, UmuD and UmuD' were targeted to DNA . RecA1730 protein, which is defective for UmuD' but proficient for MucA'-promoted mutagenesis, showed a dramatically reduced capacity to target UmuD' to DNA but was able to target a significant portion of MucA' to DNA . These data support the suggestion that the direct role of RecA protein in SOS-induced mutagenesis is to interact with and target the Umu-like mutagenesis proteins to DNA.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8108 - 12
Production of unmodified human adult hemoglobin in Escherichia coli; Shen TJ et al.; We have constructed a plasmid (pHE2) in which the synthetic human alpha- and beta-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters . The Hbs were expressed in E . coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b . Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed alpha and beta chains of component a, respectively, have the expected masses . The remaining 10% of the beta chain in component a corresponds in mass to the beta chain plus methionine . In component b, both alpha and beta chains have the correct masses without detectable N-terminal methionine (< 2%) . These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the alpha and beta chains of these two recombinant Hb (rHb) samples . rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A) . Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values . 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the beta chain . These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form . Thus, our E . coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8068 - 72
Control analysis of the dependence of Escherichia coli physiology on the H(+)-ATPase; Jensen PR et al.; The H(+)-ATPase plays a central role in Escherichia coli free-energy transduction and hence in E . coli physiology . We here investigate the extent to which this enzyme also controls the growth rate, growth yield, and respiratory rate of E . coli . We modulate the expression of the atp operon and determine the effect on said properties . When quantified in terms of control coefficients, we find that, in the wild-type cell growing on glucose in minimal medium, this key enzyme (H(+)-ATPase) exerts virtually no control on growth rate (magnitude of C < 0.01), a minor positive control on growth yield (C = 0.15), and a small but negative control on respiration rate (C = -0.25) . The control the enzyme exerts on the consumption rate of the carbon and free-energy substrate is negative (C = -0.15) . We also studied how the control coefficients themselves vary with the expression of the atp operon . As the level of expression of the atp operon was reduced, the control exerted by the H(+)-ATPase on growth rate and growth yield increased slightly; the control on growth rate passed through a maximum (C = 0.1) and disappeared when the atp operon was not expressed at all, reflecting that with this substrate there are alternative routes for ATP synthesis . At elevated levels of the H(+)-ATPase compared to the wild type, the control exerted by the enzyme on growth rate became negative . The evolutionary context of the absence of control by the atp operon on growth rate is discussed.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8000 - 4
Inhibition of human immunodeficiency virus type 1 replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS; Lisziewicz J et al.; We are investigating a strategy for somatic gene therapy to treat human immunodeficiency virus type 1 (HIV-1) infection by intracellular expression of an RNA decoy and a ribozyme . The RNA decoy, consisting of polymeric Tat activation response elements (TARs), is designed to compete for Tat binding in an equilibrium with viral TAR RNA, thereby inhibiting viral replication . The expression of polymeric TAR is regulated by the HIV long terminal repeat (LTR) and transcriptional activation is dependent on the presence of HIV Tat . Our initial studies indicated that plasmids expressing up to 50 tandem copies of TAR RNA (50TAR) inhibited tat-mediated gene expression by > 90% in a transient transfection assay . A HIV LTR-driven 50TAR construct was subcloned into a replication-defective retroviral vector to ensure high-efficiency gene transfer into T lymphocytes . In addition, a gag RNA-specific ribozyme gene was introduced into the 50TAR containing retroviral vector to enhance the inhibitory effect of the construct (designated TAR-Rib) . A human T-cell line (Molt3) was infected (transduced) with the TAR-Rib recombinant retrovirus and challenged with either HIV-1 or simian immunodeficiency virus (SIV) . HIV-1 replication was inhibited by 99% in the TAR-Rib-transduced T cells and was maintained over a 14-month period, suggesting that this antiviral strategy represses the formation of escape mutants . Interestingly, the TAR-Rib also inhibited SIV replication in transduced T cells, which suggests that polymeric TAR is a general inhibitor of primate lentiviruses; therefore, the macaque model could be used for further in vivo testing of this antiviral gene therapy strategy.

J Bacteriol, 1993 Sep, 175(17), 5706 - 9
The chimeric VirA-tar receptor protein is locked into a highly responsive state; Turk SC et al.; The wild-type VirA protein is known to be responsive not only to phenolic compounds but also to sugars via the ChvE protein (G . A . Cangelosi, R . G . Ankenbauer, and E . W . Nester, Proc . Natl . Acad . Sci . USA 87:6708-6712, 1990, and N . Shimoda, A . Toyoda-Yamamoto, J . Nagamine, S . Usami, M . Katayama, Y . Sakagami, and Y . Machida, Proc . Natl . Acad . Sci . USA 87:6684-6688, 1990) . It is shown here that the mutant VirA(Ser-44, Arg-45) protein and the chimeric VirA-Tar protein are no longer responsive to sugars and the ChvE protein . However, whereas the chimeric VirA-Tar protein was found to be locked in a highly responsive state, the VirA(Ser-44, Arg-45) mutant protein appeared to be locked in a low responsive state . This difference turned out to be important for tumorigenicity of the host strains in virulence assays on Kalanchoe daigremontiana.

J Bacteriol, 1993 Sep, 175(17), 5642 - 7
Escherichia coli mutants lacking NADH dehydrogenase I have a competitive disadvantage in stationary phase; Zambrano MM et al.; We have previously characterized mutant strains of Escherichia coli that are able to take over stationary-phase cultures . Here we describe two insertion mutations that prevent such strains from expressing this phenotype . Both insertions were mapped to min 51, and sequence analysis revealed that both mutated genes encode proteins homologous to subunits of mitochondrial NADH dehydrogenase I . Crude extracts prepared from both mutant strains were able to oxidize NADH but lacked the enzymatic activity needed to oxidize deamino-NADH, a substrate specific for NADH dehydrogenase I . This is the first identification of genes encoding subunits of NADH dehydrogenase I in E . coli . The significance of the inability of these mutant strains to compete in stationary-phase cultures is discussed.

J Bacteriol, 1993 Sep, 175(17), 5628 - 35
Escherichia coli rpiA gene encoding ribose phosphate isomerase A; Hove-Jensen B et al.; The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library . Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences . This DNA fragment was sequenced and shown to harbor an open reading frame of 219 codons, sufficient to encode a polypeptide with an M(r) of 22,845 . The synthesis of the rpiA-encoded polypeptide was detected by analysis of minicells, which established the subunit M(r) as 27,000 . The assignment of the correct reading frame was confirmed by amino-terminal analysis of partially purified ribose phosphate isomerase A . Our data indicate that the enzyme is composed of two identical subunits . The 5' end of the rpiA-specified transcript was analyzed by primer extension, which revealed a well-conserved -10 region 34 bp upstream of the presumed translation start codon . Analysis of the 3' end of the transcript by S1 nuclease mapping showed that transcription termination occurred within an adenylate-rich sequence following a guanylate-cytidylate-rich stem-loop structure resembling a rho factor-independent transcription terminator . Host strains harboring the rpiA gene in a multicopy plasmid contained up to 42-fold as much ribose phosphate isomerase A activity as the haploid strain.

J Bacteriol, 1993 Sep, 175(17), 5604 - 10
Identification, isolation, and overexpression of the gene encoding the psi subunit of DNA polymerase III holoenzyme; Carter JR et al.; The gene encoding the psi subunit of DNA polymerase III holoenzyme, holD, was identified and isolated by an approach in which peptide sequence data were used to obtain a DNA hybridization probe . The gene, which maps to 99.3 centisomes, was sequenced and found to be identical to a previously uncharacterized open reading frame that overlaps the 5' end of rimI by 29 bases, contains 411 bp, and is predicted to encode a protein of 15,174 Da . When expressed in a plasmid that also expressed holC, holD directed expression of the psi subunit to about 3% of total soluble protein.

J Bacteriol, 1993 Sep, 175(17), 5529 - 38
Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer; Hagege J et al.; pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens . During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain . The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined . Seven putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traSA (306 amino acids {aa}), orf84 (84 aa), spdA (224 aa), spdB (58 aa), spdC (51 aa), spdD (104 aa), and korSA (259 aa) . traSA is essential for pSAM2 intermycelial transfer and pock formation . It could encode a protein with similarities to the major transfer protein, Tra, of pIJ101 . TraSA protein contains a possible nucleotide-binding sequence and a transmembrane segment . spdA, spdB, spdC, and spdD influence pock size and transfer efficiency and may be required for intramycelial transfer . A kil-kor system similar to that of pIJ101 is associated with pSAM2 transfer: the korSA (kil-override) gene product could control the expression of the traSA gene, which has lethal effects when unregulated (Kil phenotype) . The KorSA protein resembles KorA of pIJ101 and repressor proteins belonging to the GntR family . Thus, the integrating element pSAM2 possesses for transfer general features of nonintegrating Streptomyces plasmids: different genes are involved in the different steps of the intermycelial and intramycelial transfer, and a kil-kor system is associated with transfer . However, some differences in the functional properties, organization, and sizes of the transfer genes compared with those of other Streptomyces plasmids have been found.

J Bacteriol, 1993 Sep, 175(17), 5505 - 9
Degradation of individual chromosomes in recA mutants of Escherichia coli; Skarstad K et al.; Rapidly growing wild-type Escherichia coli cells contain two, four, or eight fully replicated chromosomes after treatment with rifampin, reflecting that all replication origins are initiated simultaneously . Cells with defects in the timing of the initiation of replication may contain three, five, six, or seven fully replicated chromosomes after such treatment . This phenotype, termed the asynchrony phenotype, is also seen in recombination-deficient recA mutants . It is shown here that for recA strains, the phenotype can be explained by a selective and complete degradation of individual chromosomes . The selective degradation is largely recD dependent and is thus carried out by the RecBCD exonuclease.

J Bacteriol, 1993 Sep, 175(17), 5384 - 94
Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides; Jayaratne P et al.; In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V . Stout and S . Gottesman, J . Bacteriol . 172:659-669, 1990) . Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E . coli O9:K30:H12 . This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide . The rcsB gene from E . coli K30 (rcsBK30) is identical to the rcsB gene from E . coli K-12 (rcsBK-12) . rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein . To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E . coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30 . Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E . coli K30 . However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30 . K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis . To determine whether the involvement of the rcs system in E . coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules . All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined . It is has been suggested that E . coli strains with group I capsules can be subdivided based on K antigen structure . For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid . Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30 . Group IB capsular polysaccharides all contain amino sugars . In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.

J Bacteriol, 1993 Sep, 175(17), 5339 - 43
Subunit association in acetohydroxy acid synthase isozyme III; Sella C et al.; Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure . The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity . The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits . The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits . We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme . This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts . The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form . Reexamination of an E . coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed . As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation . AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.

J Bacteriol, 1993 Sep, 175(17), 5324 - 8
Escherichia coli produces linoleic acid during late stationary phase; Rabinowitch HD et al.; Escherichia coli produces linoleic acid in the late stationary phase . This was the case whether the cultures were grown aerobically or anaerobically on a supplemented glucose-salts medium . The linoleic acid was detected by thin-layer chromatography and was measured as the methyl ester by gas chromatography . The linoleic acid methyl ester was identified by its mass spectrum . Lipids extracted from late-stationary-phase cells generated thiobarbituric acid-reactive carbonyl products when incubated with a free radical initiator . In contrast, extracts from log-phase or early-stationary-phase cells failed to do so, in accordance with the presence of polyunsaturated fatty acid only in the stationary-phase cells.

Chest, 1993 Sep, 104(3), 825 - 30
The effects of inhalation of grain dust extract and endotoxin on upper and lower airways; Clapp WD et al.; To characterize the short-term effects of grain dusts on pulmonary function, mucosal inflammation, and systemic responses, four women and three men inhaled nebulized corn and soybean dust extracts, endotoxin diluted with Hanks' balanced salt solution (HBSS), and HBSS . Subjects were volunteers recruited via newspaper advertisement and were required to be healthy, nonasthmatic, nonatopic never-smokers . The mean age was 26.9 years (range, 19 to 36 years) . Using a randomized, double-blind, crossover design, each subject was challenged with each of the 4 substances with at least 10 days between challenges . Serial spirometry, peripheral blood leukocyte and differential cell counts, and 24-h postchallenge nasal lavages were performed . Extracts were produced by mixing 3 g of the corn or soybean dust with 30 ml HBSS followed by shaking for 60 min, centrifugation, then filter sterilization . The endotoxin solution was produced by mixing lyophilized Escherichia coli endotoxin (serotype 0111:B4) with HBSS to attain a final concentration of 7 mg/L, which was the same as the concentration of endotoxin in both grain dust solutions . The pH of all solutions and unmixed HBSS was adjusted to 5.8, which was the native pH of the soybean dust extract . Subjects were challenged with 0.08 ml/kg of each substance, resulting in a range of endotoxin doses of 30 to 60 micrograms, similar to that which a worker might inhale over the course of one workshift . The lowest mean percentage baseline FEV1 (+/- SD) after inhalation challenge was 99.2 +/- 2.1 for HBSS, and it was significantly lower for endotoxin (90.1 +/- 8.5, p = 0.03), corn dust extract (93.1 +/- 4.3, p = 0.02), and soybean dust extract (96.2 +/- 3.7, p = 0.03) . In addition, a peripheral blood leukocytosis developed after exposure to all three endotoxin-containing solutions (p < 0.05), yet a lower peripheral blood lymphocyte count was found only after inhalation of corn dust extract (p = 0.02) . Interestingly, this was associated with a higher nasal lavage lymphocyte count after inhalation of corn dust extract (p = 0.03) . Neither the decrease in peripheral blood lymphocytes nor the increase in nasal lymphocytes were found after inhalation of soybean dust extract or endotoxin . Our results indicate that extracts of grain dusts have physiologic effects similar to endotoxin . However, in spite of the same endotoxin levels, the effects of corn dust extract appear to have different biologic activity than either soybean dust extract or endotoxin.

Am J Cardiol, 1993 Sep 1, 72(7), 518 - 24
Open, noncontrolled dose-finding study with a novel recombinant plasminogen activator (BM 06.022) given as a double bolus in patients with acute myocardial infarction; Tebbe U et al.; The novel recombinant plasminogen activator (r-PA) (BM 06.022) is a mutant of tissue-type plasminogen activator expressed in escherichia coli which can be given as a bolus because of a prolonged half-life . The primary objective of this trial was to determine the efficacy of an intravenous r-PA double bolus (first bolus of 10 MU followed by 5 MU after 30 minutes) in patients with acute myocardial infarction . All patients received heparin intravenously and acetylsalicylic acid orally . Efficacy was assessed from infarct artery patency by coronary angiography (Thrombolysis in Myocardial Infarction trial perfusion grades 2 or 3) in 50 patients . Ninety minutes after administration of the first r-PA bolus, the infarct-related coronary artery was patent in 39 of 50 patients (78%; 95% confidence interval 64 to 88%) . An angiographically confirmed reocclusion occurred in 1 patient between 90 minutes and 24 to 48 hours . The reocclusion rate was influenced by 8 interventions and 1 angiogram missing at 24 to 48 hours . Measurements of hemostatic parameters showed a decrease in fibrinogen to 37% of baseline value . There were 3 clinical reinfarctions before discharge and 2 major puncture site hemorrhages . No further serious bleeding and no serious adverse event with lethal outcome occurred . The 10 + 5 MU r-PA double bolus regimen appears to be effective with regard to patency and the success of thrombolysis . The incidence of reocclusion is very low . From the limited number of patients treated in this study, one need not be concerned about the safety profile of r-PA.

J Neurochem, 1993 Sep, 61(3), 997 - 1005
The balance between tau protein's microtubule growth and nucleation activities: implications for the formation of axonal microtubules; Brandt R et al.; The microtubule-associated protein tau is found primarily in neuronal tissues and is highly enriched in the axon . It promotes microtubule assembly in vitro and stabilizes microtubules in cells . To study how tau protein might be involved in the unique features of axonal microtubules, we have analyzed the effect of E . coli-synthesized tau protein using an in vitro centrosome-mediated microtubule regrowth assay over a wide range of tau/tubulin ratios . We report that microtubule assembly promoted by tau protein exhibits characteristic changes dependent on the tau/tubulin ratio . Above a threshold level, nucleation of new microtubules is favored over growth of existing ones . tau isoform variation does not change this phase transition in microtubule assembly . We discuss how tau might participate in the elaboration of axonal morphology based on our results and present evidence that the phase transition from microtubule growth to nucleation is critical for axonal development.

Infect Immun, 1993 Sep, 61(9), 3843 - 53
Borrelia burgdorferi outer surface lipoproteins OspA and OspB possess B-cell mitogenic and cytokine-stimulatory properties; Ma Y et al.; Sonicated Borrelia burgdorferi was previously reported to possess both B-cell mitogenic and interleukin-6 (IL-6) stimulatory activities . In this report, two outer surface lipoproteins, OspA and OspB, were purified from B . burgdorferi and assessed for the presence of these functions . OspA was purified from two strains, an OspB-deficient variant of HB19 and N40, while OspB was purified from the N40 strain . All lipoprotein preparations were free of endotoxin contamination, and polymyxin B failed to inhibit responses, indicating that media contamination was not contributing to biological assays . All three preparations were able to stimulate proliferation of mononuclear cells from naive C3H/HeJ and BALB/c mice . Depletion experiments indicated that the responding cells were B lymphocytes and not T lymphocytes . Purified OspA and OspB stimulated immunoglobulin M production by splenocyte cultures from naive mice, a property also previously attributed to sonicated B . burgdorferi . OspA and OspB also stimulated the production of IL-6 and tumor necrosis factor alpha by bone marrow-derived macrophages from BALB/c and C3H/HeJ mice . Cytokine production was enhanced by the presence of gamma interferon in the cultures, indicating that the magnitude of responses to these lipoproteins may be modulated by cytokines in the microenvironment of infected tissues . Human endothelial cells produced IL-6 when incubated with OspA and OspB, indicating that non-hematopoietic lineage cells can respond to the lipoproteins . Purified OspA and OspB had approximately equal activity, with responses detected in the range of 10 ng of lipoprotein per ml to 1 microgram of lipoprotein per ml . Comparison with published dose responses for lipoproteins purified from Escherichia coli indicates that OspA and OspB purified from B . burgdorferi are much more potent . The high potency of the B . burgdorferi lipoproteins and the ability of the spirochete to invade tissues and persist argue that they could be important in the localized events contributing to the pathology of Lyme disease.

Infect Immun, 1993 Sep, 61(9), 3583 - 9
CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin; Betsou F et al.; Bordetella pertussis adenylate cyclase-hemolysin (AC-Hly), encoded by the cyaA gene, belongs to the RTX family of toxins with extensive glycine-rich repeats in the carboxy-terminal portion . AC-Hly possesses both adenylate cyclase toxic and hemolytic activities that depend on a posttranslational modification mediated by the product of the cyaC gene . An improved system for AC-Hly synthesis and activation in Escherichia coli was developed . The results show that with purified AC-Hly (i) increased expression of the cyaC gene leads to a higher proportion of activated AC-Hly, (ii) the increase in protective activity of the activated recombinant AC-Hly correlates with the increase in its invasive and hemolytic activities, and (iii) the activated recombinant AC-Hly, but not the nonactivated recombinant AC-Hly, is a protective antigen against B . pertussis infection in a murine respiratory model . This suggests that possibly an immunodominant epitope required for protective activity is linked to the CyaC-mediated modification . Surprisingly, the protective and hemolytic activities of activated recombinant AC-Hly were lower than those of AC-Hly produced by B . pertussis, while its invasive activity was higher . This indicates that the modification of AC-Hly in B . pertussis and that in E . coli may differ.

Exp Hematol, 1993 Sep, 21(10), 1366 - 70
Development and application of a sensitive radioimmunoassay for human granulocyte-macrophage colony-stimulating factor able to measure normal concentrations in blood; Mortensen BT et al.; A radioimmunoassay (RIA) for human granulocyte-macrophage colony-stimulating factor (GM-CSf) was developed based on antibodies from rabbits immunized with glycosylated recombinant human (rh) GM-CSF . The antibodies are specific for human GM-CSF and do not crossreact with other human hematopoietic growth factors or mouse GM-CSF . The antibodies also react with nonglycosylated rhGM-CSF, so E . coli-derived rhGM-CSF can be assayed as well . The RIA has a measuring range of about 10 to 200 pg/mL . Normal blood was found to contain 13 to 24 pg/mL (95% limits) with a mean of 18.5 pg/mL (n = 34) . Monoclonal antibodies against GM-CSF could remove GM-CSF from normal human serum, thus ensuring that the GM-CSF measured in serum is real and does not represent nonspecific reactivity with our polyclonal rabbit antibodies . While previously published methods have been unable to measure GM-CSF in human serum under normal conditions, our more sensitive RIA does confirm the presence of small amounts of GM-CSF in serum or plasma and can therefore be used to detect fluctuations of GM-CSF in health and in disease.

Virology, 1993 Sep, 196(1), 309 - 18
A transgenic model of transactivation by the Tax protein of HTLV-I; Bieberich CJ et al.; The human T-lymphotropic virus type I (HTLV-I) Tax protein is a transcriptional regulatory protein that has been suggested to play a causal role in the development of several HTLV-I-associated diseases . Tax regulates expression of its own LTR and of certain cellular promoters perhaps by usurping the function of the host transcriptional machinery . We have established a transgenic mouse model system to define the spectrum of tissues in vivo that are capable of supporting Tax-mediated transcriptional transactivation . Transgenic mice carrying the HTLV-I LTR driving expression of the Escherichia coli beta-galactosidase (beta gal) gene were generated, and this LTR-beta gal gene was transcriptionally inactive in all tissues . When LTR-beta gal mice were mated to transgenic mice carrying the same LTR driving expression of the HTLV-I tax gene, mice that carried both transgenes showed restricted expression of the beta gal reporter gene in several tissues including muscle, bone, salivary glands, skin, and nerve . In addition, a dramatic increase in the number of beta gal-expressing cells was seen in response to wounding . These observations provide direct evidence for viral transactivation in vivo, delimit the tissues capable of supporting that transactivation, and provide a model system to study the mechanism of gene regulation by Tax.

Mol Cell Biol, 1993 Sep, 13(9), 5524 - 37
Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein; Brazas RM et al.; The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene . A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified . The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA . This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo . A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified . Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography . In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively . Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts . SWI5 and GRF10 interactions were not detected in the absence of DNA . The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.

J Cell Biol, 1993 Sep, 122(5), 1043 - 52
Molecular characterization of mammalian cylicin, a basic protein of the sperm head cytoskeleton; Hess H et al.; The cytoskeletal calyx structure surrounding part of the nucleus of the mammalian sperm head contains two major kinds of basic proteins, i.e., the approximately 60-kD calicin and a group of very basic (IEP > pH 10) polypeptides ranging in size from approximately 58 to approximately 100 kD ("multiple band proteins," MBPs) . We have produced MBP-specific mAbs and have isolated a bovine and a human cDNA clone encoding one of these proteins, termed "cylicin" (from the Greek word c eta kv lambda l zeta for cup or beaker) . Bovine cylicin I of a calculated molecular weight of 74,788 contains a high proportion (29%) of positively charged amino acids, resulting in an IEP of 10.55, numerous KKD tripeptides, and is characterized by an organization of the central part of the molecule in nine repeating units of maximally 41 amino acids each of which according to prediction analysis should tend to form an alpha helix . The identity of the polypeptide has been proven by direct amino acid sequencing of > 14 different fragments and by experiments using antibodies raised against a partial cDNA-derived protein segment produced in E . coli . By Northern blot analysis we have identified the 2.4-kb cylicin I mRNA only in testis . The unusual cytoskeletal protein cylicin is compared with other proteins and its possible architectural role during spermiogenesis is discussed.

J Virol, 1993 Sep, 67(9), 5198 - 205
Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells; Bai M et al.; The adenovirus penton base protein has a cell rounding activity and may lyse endosomes during virus entry into the cytoplasm . We found that penton base that was expressed in Escherichia coli also caused cell rounding and that cells adhered to polystyrene wells that were coated with the protein . Mutant analysis showed that both properties required an Arg-Gly-Asp (RGD) sequence at residues 340 to 342 of penton base . In flat adherent cells, virus mutants with amino acid substitutions in the RGD sequence were delayed in virus reproduction and in the onset of viral DNA synthesis . In nonadherent or poorly spread cells, the kinetics of mutant virus reproduction were similar to those of wild-type adenovirus type 2 . Expression of the mutant phenotype exclusively in the flat cells that we tested supports a model in which penton base interacts with an RGD-directed cell adhesion molecule during adenovirus uptake or uncoating.

J Exp Med, 1993 Sep 1, 178(3), 1085 - 90
Passive immunization of mice against D factor blocks lethality and cytokine release during endotoxemia; Block MI et al.; D factor, also known as leukemia inhibitory factor, is a pleiotropic cytokine whose role during acute injury and inflammation is not known . Intraperitoneal administration of Escherichia coli endotoxin induced D factor gene expression in mice, and passive immunization against D factor protected them from the lethal effects of endotoxin and blocked endotoxin-induced increases in serum levels of interleukin 1 and 6 . Peak levels of tumor necrosis factor and interferon gamma were not affected . These results indicate that D factor is an essential early mediator of the inflammatory cytokine response and therefore may be important in the pathogenesis of the many inflammatory conditions, such as sepsis, arthritis, allograft rejection, and cancer immunotherapy.

J Urol, 1993 Sep, 150(3), 1030 - 3
Events leading to septic death from experimental acute pyelonephritis in the monkey; Roberts JA et al.; Experimental acute pyelonephritis in monkeys led to death in some of the animals following renal E . coli inoculation . It was found that both the inflammatory response and cytokine activation were much more severe in these monkeys as compared with others that survived . IL-1 was decreased just before death, and there were early increases in IL-2 and IL-6 serum concentrations, but no significant increase in TNF values . The data suggest that death in sepsis is due in part to excessive cytokine release because of a decrease in the protective activity of IL-1.

Res Microbiol, 1993 Sep, 144(7), 529 - 37
The importance of the binding-protein-dependent Mgl system to the transport of glucose in Escherichia coli growing on low sugar concentrations; Death A et al.; Glucose limitation in chemostats derepressed the binding-protein-dependent Mgl transport system, which is strongly repressed during growth in batch culture with high glucose levels . The limitation-induced Mgl activity was higher than that of batch cultures "fully induced" for the Mgl system after growth on glycerol plus fucose . Mgl- strains were impaired compared to Mgl+ bacteria in removing glucose from sugar-limited chemostats and were outcompeted in mixed continuous culture on limiting glucose . The influence of Mgl was not observed on growth with limiting maltose or non-carbohydrates, and thus was specific for glucose, a known substrate of the Mgl system . In the absence of the two glucose-specific membrane components of the phosphoenolpyruvate:sugar phosphotransferase system, non-PTS-dependent growth on glucose was observed in continuous culture, but only under sugar-limited conditions derepressing the Mgl system and not in glucose-rich batches or continuous culture . Hence growth of Escherichia coli on glucose at micromolar concentrations involves a significant contribution of a binding-protein-dependent transport system . The participation of multiple transporters in glucose transport can account for the complex non-hyperbolic dependence of growth-rate on glucose concentration and for discrepancies in studies attempting to describe growth on glucose purely in terms of phosphotransferase kinetics.

Eur J Epidemiol, 1993 Sep, 9(5), 489 - 96
Serotypes and colonization factors of enterotoxigenic Escherichia coli isolated in various countries; Blanco J et al.; One hundred and six enterotoxigenic E . coli (ETEC) isolated from many geographical areas were serotyped and investigated for the presence of colonization factor antigens CFA/I and CFA/II, the expression of mannose-resistant haemagglutination (MRHA) and the levels of surface hydrophobicity . CFA/I was found in 6 (17%) of 36 LT+STa+ strains and in 15 (54%) of 28 STa+ strains; CFA/II was found in 16 (44%) of 36 LT+STa+ strains . None of 42 LT+ strains showed CFA/I or CFA/II . CFA/I was found in ETEC of serotypes O63:K-:H-, O78:K80, O128:K67 and O153:K:H45, whereas CFA/II was found in serotypes O6:H-, O6:K15:H16 and O6:K?:H40 . Of the 69 CFA/I- CFA/II- ETEC strains, 9 (13%) showed MRHA with some of the seven erythrocyte species used and 21 (30%) were hydrophobic . Among the 21 hydrophobic strains CFA-negative we have detected: (i) 6 LT+ strains of serogroup O25 negative for MRHA, (ii) 5 strains O159 (4 LT+ and 1 LT+ STa+) also negative for MRHA, and (iii) 3 STa+ strains of serotype O27:K-:H7 that haemagglutinated calf and sheep erythrocytes when grown on Minca-Is . The 106 ETEC strains belonged to 20 different O serogroups . However, 77 (73%) were of one of nine serogroups (O6, O8, O25, O27, O78, O148, O153, O159 and O167) . E . coli strains belonging to O6 and O153 groups predominated among ETEC isolated in Spain, O159 strains in the Central African Republic, O25 and O148 strains in Japan, and O15 and O78 strains in India.

Int J Parasitol, 1993 Sep, 23(6), 785 - 92
The 3' terminal region of a gene encoding a cysteine-rich surface protein in Giardia duodenalis; Upcroft JA et al.; DNA derived from chromosome band 3 of the cloned Giardia duodenalis line, WB-1B was used to construct a cloned library in E . coli . One of these clones, C3/23, has been identified as the 3' coding region of a G . duodenalis cysteine-rich variable surface protein (CRVSP) gene by homology with other published CRVSPs and also contains 720 bp of the 3' flanking region . The sequence of C3/23, was derived from genomic DNA independently of cDNA, or expression copies of the CRVSP genes . The 3' flanking region is not homologous to the 3' untranslated regions of published CRVSPs which probably reflects its genomic origin . Subclones of C3/23 were used to show that the 3' flanking region was conserved in all strains examined in this study and was repeated many times in the genome . The 3' flanking repeats were located on three chromosome bands and were not always associated with the coding sequence of C3/23 which was represented, although not equally, on all chromosome bands . The highly conserved nature of the 3' flanking region and its multiple representation in the genome emphasize the probable role of this sequence in the localization or regulation of expression of the CRVSPs in G . duodenalis.

J Investig Allergol Clin Immunol, 1993 Sep-Oct, 3(5), 237 - 44
Abnormal migratory activity of peripheral neutrophils from asthmatic patients and its modulation by inhaled glucocorticoids; Paggiaro PL et al.; In order to investigate if bronchial asthma is associated with enhanced markers of activation in peripheral neutrophils, the migratory capacity of neutrophils in venous blood was measured by means of the Boyden chamber technique in 29 subjects with bronchial asthma of differing severity . Random migration (random motility), but not locomotion toward 10% Escherichia coli supernatant as chemoattractant (chemotaxis), was increased in asthmatic subjects with respect to 11 normal subjects (98 +/- 20 microns vs . 85 +/- 6 microns; p < 0.05) . When asthmatic subjects were subdivided into groups of different disease severity, subjects with mild and mild to moderate asthma showed significantly higher values for random motility and chemotaxis than normal subjects; on the other hand, subjects with more severe disease showed the lowest values for migratory activity . No correlation was found between migratory activity and clinical findings of asthma, except for baseline FEV1 (% of the predicted value), which showed a slight but significant positive correlation with chemotaxis (r = 0.44, p < 0.05) . Subjects with atopic or occupational asthma had higher values for migratory activity than subjects with nonallergic asthma . Thirteen asthmatic subjects repeated all evaluations after 1 month of treatment with high doses of inhaled glucocorticoids (beclomethasone dipropionate 1500 micrograms/day) . Random motility (95 +/- 24 microns vs . 75 +/- 15 microns; p < 0.05) and chemotaxis (130 +/- 22 microns vs . 105 +/- 25 microns; p < 0.05) were significantly reduced after treatment, as well as the symptom score; on the other hand, symptom score but not bronchial hyperresponsiveness to methacholine challenge significantly changed.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Res Toxicol, 1993 Sep-Oct, 6(5), 681 - 9
Construction of Escherichia coli vectors containing deoxyadenosine and deoxyguanosine adducts from (+)-anti-dibenz{a,j}anthracene diol epoxide at a defined site; Gill RD et al.; Dibenz{a,j}anthracene (DB{a,j}A) is a carcinogenic polycylic aromatic hydrocarbon, which is metabolically activated through the formation of bay region diol epoxides . Site-specifically modified M13mp19-based vectors containing a single (+)-anti-dibenz{a,j} anthracene diol epoxide {(+)-anti-DB{a,j{A-DE}-deoxyguanosine (dGuo) or -deoxyadenosine (dAdo) adduct were constructed . Four-base oligonucleotides, 5'-HOTGCA-3' and 5'-HOCATG-3', corresponding to the central four base pairs in the PstI and SphI restriction endonuclease sites, respectively, in the multiple cloning region of M13mp19, were reacted in solution with (+/-)-anti-DB{a,j}A-DE . The resulting adducted oligonucleotides were separated and purified using reverse-phase HPLC . Several different singly adducted oligonucleotides were isolated, consisting of the various cis and trans addition products of the (+) and (-) enantiomers of the diol epoxide bound to dGuo or dAdo in the oligonucleotides . 5'-HOTGCA-3' containing the (+)-anti-DB{a,j}A-trans-N2-dGuo adduct {T(DB{a,j}A-N2)GCA} and 5'-HOCATG-3' containing the (+)-anti-DB{a,j}A-trans-N6-dAdo adduct {C(DB{a,j}A-N6)ATG) were selected for subsequent ligation into M13mp19 vectors that had been constructed with a corresponding four base gap in the minus strand . Both unmodified and adducted oligonucleotides were successfully ligated into the M13mp19 vectors, {yields: unmodified -TGCA-M13mp19 (approximately 32%) and -CATG- M13mp19 (approximately 42%); adducted T(DB{a,j}A-N2)GCA-M13mp19 (approximately 13%) and C(DB{a,j}A-N6)ATG-M13mp19 (approximately 12%)} . The dAdo adduct-containing vector was characterized . The presence of a dAdo-DNA adduct at the recognition site of SphI inhibited restriction by SphI.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Res Toxicol, 1993 Sep-Oct, 6(5), 625 - 9
Quantitation of base substitutions and deletions induced by chemical mutagens during DNA synthesis in vitro; Shibutani S; An experimental system has been developed by which base substitutions and frameshift deletions can be quantitated in vitro, using two-phase 20% polyacrylamide gel electrophoresis . Oligodeoxynucleotides, modified site-specifically, were used as templates in primer extension reactions catalyzed by DNA polymerase alpha, polymerase beta, and the Klenow fragment of Escherichia coli DNA polymerase I, with and without 3'-->5' exonuclease activity . Lesions studied included 7,8-dihydro-8-oxodeoxyguanosine, 7,8-dihydro-8-oxodeoxyadenosine, O6-methyldeoxyguanosine, N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene, and N-(deoxyguanosin-8-yl)-2- aminofluorene . Products of translesional synthesis contained dC, dA, dG, or dT opposite the lesion or one- and two-base deletions and were separated using a two-phase polyacrylamide gel system . When a template containing 8-oxoguanine was used, dAMP and/or dCAMP was incorporated opposite the lesion, the relative amounts depending on the DNA polymerase used . In contrast, the nonmutagenic base, dTMP, was incorporated exclusively opposite 8-oxodA in reactions catalyzed by Klenow fragment and pol alpha . The improved resolution provided by the two-phase gel system revealed misincorporation of dGMP opposite 8-oxodA in reactions catalyzed by pol beta . dTMP and small amounts of dCMP were incorporated opposite the lesion on an O6MedG-modified template . The bulky adduct, dG-C8-AAF, principally produced deletions; in contrast, dG-C8-AF promoted incorporation of dCMP, a nonmutagenic base . This experimental system should prove useful for establishing the miscoding potential of defined lesions in DNA templates and in correlating this information with the mutagenic properties of DNA adducts observed in cells.

Chem Res Toxicol, 1993 Sep-Oct, 6(5), 616 - 24
Unwinding and hydrodynamic flow linear dichroism characteristics of supercoiled DNA covalently modified with two isomeric methylchrysene diol epoxides of different biological activities; Balasta L et al.; Adducts derived from the covalent binding of two positional monomethyl-substituted isomers of a bay region chrysene diol epoxide to supercoiled pIBI30 DNA (2926 base pairs/genome) were prepared, and their characteristics were investigated by a combination of gel electrophoresis and flow linear dichroism techniques . The 5- and 6-methyl derivatives of trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene {(+)-5- and (+)-6-MeCDE, respectively}, both with 1R,2S,3S,4R stereochemistry, are characterized by significant differences in their biological activities {Melikian et al . (1988) Cancer Res . 48, 1781-1787} . When covalently bound to plasmid DNA, these two molecules give rise to striking differences in the gel electrophoretic and flow hydrodynamic characteristics of the modified supercoiled DNA . The hydrodynamic flow linear dichroism of linearized DNA molecules (obtained by EcoRI enzyme digestion of covalently closed supercoiled pIBI30 DNA), modified covalently with the highly tumorigenic and mutagenic (+)-5-MeCDE derivative, indicates that flexible joints, bends, or kinks are formed at the site of binding of (+)-5-MeCDE . Slab gel data, as well as ethidium bromide-titration tube agarose gel electrophoresis data, indicate that the formation of (+)-5-MeCDE-DNA lesions causes the removal of superhelical turns with an unwinding angle of 13 +/- 3 degrees per covalently bound polycyclic aromatic residue . In contrast, the biological inactive (+)-6-MeCDE does not significantly alter the characteristics of supercoiled DNA, the unwinding angle is only 2.7 +/- 1 degrees, and the changes in persistence lengths detected by the flow linear dichroism technique are significantly smaller than in the case of (+)-5-MeCDE-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Mikrobiol Virusol, 1993 Sep-Oct, (5), 31 - 4
{Expression of the gag-precursor of the human T-cell leukemia virus type I in Escherichia coli: study of the stability and antigenic properties of virus-specific hybrid proteins}; Sankov MN et al.; A set of recombinant plasmids containing sequences of HTLV-I viral gag-gene has been constructed on the basis of pUR290-pUR292 vector plasmids . The resulting hybrid proteins containing different fragments of GAG-precursor in the C-end of beta-galactosidase differed to a large extent in stability in Escherichia coli cells . The presence of an N-end fragment of GAG-precursor in the recombinants decreases drastically their resistance to bacterial proteases . Elimination of the fragment resulted in obtaining the recombinant plasmid pGdN coding for high rate synthesis (up to 30% of total cellular protein) of gag-specific hybrid polypeptide in Escherichia coli HB101 cells . This 145 kDa protein efficiently interacts with HTLV-I positive sera . It can be used in diagnostic test-systems for indicating HTLV-I infected persons.

Mol Gen Mikrobiol Virusol, 1993 Sep-Oct, (5), 26 - 9
{Expression of the vif gene of human immunodeficiency virus type 1 in Escherichia coli and study of the immunoreactivity of the vif protein}; Karaseva NG et al.; Full-sized gen vif of human immunodeficiency virus has been synthesized and cloned into plasmid pGEX-2T . Vif-gene expression was found in Escherichia coli cells resulting in production of a hybrid GST-protein . The recombinant protein studied by the immunoblotting technique reacted with 8 of 22 probes of human HIV-positive sera . The recombinant protein is specifically cut by thrombin in two proteins corresponding to GST and VIF-proteins in molecular mass.

JPEN J Parenter Enteral Nutr, 1993 Sep-Oct, 17(5), 449 - 53
Hepatobiliary complications in healthy, intra-abdominally infected, and high-output fistula rats receiving total parenteral nutrition; Mok KT; This study examines the pathophysiology of hepatobiliary complications induced by total parenteral nutrition (TPN) by using animal models that underwent cecal ligation to produce intra-abdominal infection and received an enterostomy to mimic a high-output fistula, which causes the interruption of enterohepatic circulation of bile salt . Aspartate transaminase was elevated after TPN (p < .05) . Alkaline phosphatase was increased in animals receiving TPN plus an enterostomy (p < .05) . Serum albumin was significantly decreased in animals receiving TPN plus undergoing cecal ligation or enterostomy (p < .05) . Liver weight and liver protein and water content decreased in animals receiving TPN alone (p < .05) . Liver water content increased in animals receiving TPN plus undergoing cecal ligation (p < .05) . Liver lipid content increased after TPN and to a significant degree in rats receiving TPN plus undergoing cecal ligation or enterostomy (p < .05) . Bile flow diminished after TPN and to a level reaching significance in animals receiving TPN plus undergoing cecal ligation or enterostomy (p < .05) . Reduction of bile flow, decrease of biliary cholesterol secretion, and increase of biliary bilirubin secretion, which may be the cause of TPN-induced bilirubinate stones, were most significant in animals receiving TPN plus undergoing cecal ligation (p < .05) . In conclusion, TPN can induce hepatic dysfunction and bilirubinate stones, but these complications are more common in animals with associated intra-abdominal infection or high-output fistula.

G Chir, 1993 Sep, 14(7), 349 - 50
Spontaneous rupture of the liver; Celoria G et al.; A case of spontaneous hepatic rupture associated with bacterial cholangiohepatitis, an entity not previously described as an etiologic factor, is reported and the literature reviewed . Awareness of the pathophysiology of this process and avoidance of certain procedures are important parts of the surgical armamentarium.

J Biochem (Tokyo), 1993 Sep, 114(3), 421 - 31
Secondary structure and protein folding of recombinant chloroplastic thioredoxin Ch2 from the green alga Chlamydomonas reinhardtii as determined by 1H NMR; Lancelin JM et al.; The recombinant form of the chloroplastic thioredoxin Ch2 from the green alga Chlamydomonas reinhardtii {Jacquot et al . (1992) Nucleic Acids Res . 20, 617} that preferentially activates the NADP dependent malate dehydrogenase {EC 1.1.1.82} (m-type thioredoxin) through a light promoted reductive system, has been subjected to an extensive two-dimensional 1H NMR analysis . A complete 1H NMR assignment of the resonance lines in both the oxidized and the reduced states at pH 5.8 has been obtained allowing the recognition of the secondary structure patterns and the global protein folding . The single polypeptide chain, made of 106 residues plus one additional Met located at the N-terminal position (11.6 kDa) due to the protein expression system, folds into a pattern characteristic of the open twisted alpha/beta structures already found for Escherichia coli and human thioredoxins for which the protein shares 46 and 20% of sequence identity, respectively . The open alpha/beta structure is made of 5 beta-sheets associated in a parallel (beta 1 to beta 3) and anti parallel manner (beta 3 to beta 5) and surrounded by 4 helices . This represents the first structural exploratory study of the ubiquitous oxido-reductase thioredoxins in a photosynthetic living system.

J Biochem (Tokyo), 1993 Sep, 114(3), 393 - 7
Identification of an active dimeric form of aspartase as a denaturation intermediate; Murase S et al.; The guanidine-HCl (Gu-HCl)-induced denaturation of a tetrameric enzyme, aspartase from Escherichia coli has been studied by size-exclusion chromatography, and circular dichroism and fluorescence spectroscopies . The size-exclusion analysis showed that in the presence of 0.4 M Gu-HCl, the enzyme has a dimeric structure with 45% of the native activity . The fluorescence and CD studies showed that only a small change occurred in the secondary and tertiary structures in 0.4 M Gu-HCl . In the range of 0.4 to 1 M Gu-HCl, decrease in the activity was observed as the secondary and tertiary structures were disrupted, whereas the dimeric enzyme did not dissociate into inactive monomer until 1 M Gu-HCl . When the enzyme was denatured in less than 1 M Gu-HCl, more than 90% of the original activity was recovered from the renaturation reaction, indicating that the dissociation process from tetramer to dimer is reversible . In contrast, the renaturation yield was 43% when the enzyme was diluted from more than 1 M Gu-HCl, indicating that the process of dissociation into monomer is not reversible . Thus, we identified an active dimeric form as a denaturation intermediate in this study, although the intermediates (including dimer) that were detected in renaturation experiments at low temperature were inactive, as reported previously {Imaishi, H., Yumoto, N., & Tokushige, M . (1989) Physiol . Chem . Phys . Med . NMR 21, 221-228}.

J Crit Care, 1993 Sep, 8(3), 161 - 9
Infusion of ultrafiltrate from endotoxemic pigs depresses myocardial performance in normal pigs; Grootendorst AF et al.; We previously showed a beneficial effect of hemofiltration on hemodynamics of endotoxic shock pigs . To test the hypothesis that this effect of hemofiltration is caused by convective removal of factors that adversely affect hemodynamics during endotoxemia, we infused ultrafiltrate from endotoxic shock pigs into healthy pigs . Their hemodynamics were compared with those of pigs who were infused with ultrafiltrate from healthy pigs . Twelve anesthetized and ventilated pigs were hemodynamically monitored for 150 minutes following the infusion of 2 L of ultrafiltrate from 12 donor pigs . The acceptor pigs were randomly divided into two groups; group 1 received ultrafiltrate from pigs who were hemofiltered after the infusion of 0.5 mg/kg endotoxin over 30 minutes; group 2 served as a control group, receiving ultrafiltrate from healthy donor pigs . Group 1 showed a decrease in mean arterial pressure of 28 +/- 7 mm Hg (mean +/- SEM) versus an increase of 17 +/- 3 mm Hg in group 2 (P < 0.4) . Mean pulmonary artery pressure increased more in group 1 than in group 2 (9 +/- 2 mm Hg versus 1 +/- 1 mm Hg, P < .04) . The decrease in cardiac output in group 1 was greater than in group 2 (3.3 +/- 0.2 L/min v 0.3 +/- 0.3 L/min, P < .02) and was due to a decrease in stroke volume . The decrease in right ventricular ejection fraction was also greater (0.15 +/- 0.02 v 0.01 +/- 0.00, P < .01) . Systemic vascular resistance, right atrial pressure, right ventricular end-diastolic volume, pulmonary wedge pressure and heart rate did not differ between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Plant Microbe Interact, 1993 Sep-Oct, 6(5), 573 - 81
A gene encoding a host-specific elicitor protein of Phytophthora parasitica; Kamoun S et al.; Extracellular elicitor proteins (elicitins) from Phytophthora species induce local and distal defense responses specifically in plants of the Solanaceae and Cruciferae . Based on elicitin amino acid sequences, elicitin-coding sequences from P . parasitica were amplified by the polymerase chain reaction . A genomic clone containing a complete elicitin gene, parA1, was isolated and sequenced . Elicitin was confirmed to be encoded as a precursor protein containing a 20-amino acid signal peptide that is processed before secretion . Bacterial expression of the cloned elicitin gene as a translational fusion protein containing glutathione S-transferase yielded a biologically active protein capable of inducing a hypersensitive response in tobacco, suggesting that fungus-specific postranslational modifications of elicitin are not required for its activity . Southern blot analysis indicated that elicitin genes occur as a multigene family (at least two to 10 copies) in P . parasitica, P . capsici, P . citricola, P . citrophthora, P . cryptogea, P . drechsleri, P . megasperma, and P . palmivora . Some isolates of P . parasitica that did not produce elicitins still contained elicitin-coding sequences but did not accumulate elicitin mRNA.

Microb Pathog, 1993 Sep, 15(3), 169 - 76
Characterization of non-toxic mutant toxins of Vero toxin 1 that were constructed by replacing amino acids in the A subunit; Ohmura M et al.; Three mutant toxins of Vero toxin 1 (VT1) produced by Escherichia coli strains carrying mutant VT1 genes that were constructed by oligonucleotide-directed site-specific mutagenesis were purified to homogeneity . They are E167Q (glutamic acid at position 167 from the N-terminus of the A subunit was replaced by glutamine), R170L (arginine at position 170 from the N-terminus of the A subunit was replaced by leucine) and E167Q-R170L (glutamic acid at position 167 and arginine at position 170 were replaced by glutamine and leucine, respectively) . The purified E167Q and E167Q-R170L had markedly decreased activities, such as inhibition of protein synthesis, cytotoxicity to Vero cells and mouse lethality . The decrease in the R170L activities was less than those of the other two mutant VT1s . Neither additive nor synergistic decreases of the activities were observed in the double mutant E167Q-R170L in which two amino acids were replaced . Ouchterlony double gel diffusion showed that antiserum against the purified E167Q-R170L gave a line of identity between mutant VT1s and VT1 . Moreover, anti-E167Q-R170L antiserum showed similar activity to that of anti-VT1 antiserum in neutralizing the Vero cell cytotoxicity and mouse lethality of VT1 . The data suggest that these mutant VT1s, especially E167Q and E167Q-R170L, may be candidate toxoids that protect against diseases caused by Verocytotoxin-producing E . coli.

Dev Comp Immunol, 1993 Sep-Oct, 17(5), 407 - 18
Isolation and characterization of a hemagglutinin with affinity for lipopolysaccharides from plasma of the crayfish Pacifastacus leniusculus; Kopacek P et al.; A hemagglutinin with a high specific activity against trypsinized rabbit erythrocytes was identified in plasma of the freshwater crayfish Pacifastacus leniusculus . The activity of this crayfish hemagglutinin could be inhibited by sialoglycoproteins such as porcine stomach mucin, bovine submaxillary mucin, fetuin, and ovalbumin . However, the involvement of sialic acid in its binding specificity could not be unambiguously proven . Furthermore, the hemagglutinating activity in the crayfish plasma could be specifically inhibited by lipopolysaccharide from E . coli K-235, which might indicate a recognition role for this hemagglutinin . This hemagglutinin, which accounts for less than 0.01% of the total plasma protein, was purified to near homogeneity using affinity chromatography on a Fetuin-Sepharose 4B column . The molecular mass of the unreduced protein as revealed by sodium dodecyl sulphate electrophoresis in polyacrylamide gel was found to be 420,000 Da . Upon reduction with dithiothreitol the hemagglutinin dissociated to several subunits with masses ranging from 65,000 to 80,000 Da . Affinoblotting with peroxidase labelled lectins indicated that the hemagglutinin was likely to be a glycoprotein.

Vet Pathol, 1993 Sep, 30(5), 410 - 7
Clinical signs and lesions in gnotobiotic pigs inoculated with Shiga-like toxin I from Escherichia coli; Dykstra SA et al.; Gnotobiotic pigs were used as a model to study the contribution of Shiga-like toxin I to natural disease caused by enterohemorrhagic Escherichia coli in calves and human beings . Eleven 2- to 7-day-old gnotobiotic pigs of either sex, obtained by closed hysterotomy, were injected intramuscularly with graded doses of partially purified Shiga-like toxin I derived from a lysogenized Escherichia coli strain . Four other gnotobiotic pigs were injected with a mock toxin preparation obtained from a nonlysogenized culture of the same E . coli strain . All toxin-injected pigs developed diarrhea, and three displayed signs of neurologic disease . Pigs either died or were euthanatized 2 to 4 days post-inoculation . Necrosis of muscle was grossly evident at the site of injection in all toxin-inoculated pigs . Hemorrhage in the lumen of the small and large intestines and blood in the feces were also evident in two toxin-inoculated pigs . Microscopically, severe necrotizing myositis at the injection site, multifocal encephalomalacia, and mucosal infarcts and hemorrhage in the small and large intestines were seen . In small vessels at lesion sites, endothelial cells were frequently swollen or necrotic . Pigs inoculated with mock toxin did not develop diarrhea or exhibit signs of neurologic disease, and the only apparent lesion was mild microscopic myositis at the injection site in 1/4 pigs . The results of this study indicate that Shiga-like toxin I causes vascular damage and ischemic necrosis in the intestines and brains of gnotobiotic pigs . These lesions are similar to those seen in the intestines of calves and human beings with hemorrhagic colitis and in the brains of human beings with thrombotic thrombocytopenic purpura.

Biochem Mol Biol Int, 1993 Sep, 31(1), 95 - 103
Altered lead(II)-cleavage pattern of free Phe-tRNAPhe and Phe-tRNAPhe in ternary complex with EF-Tu:GTP; Otzen DE et al.; Pb2+ ions in sub-millimolar concentrations are known to cleave internucleotide bonds of phenylalanine-specific transfer RNA (tRNAPhe) from Saccharomyces . cerevisiae specifically between nucleotides D17 and G18 in the D-loop, with additional minor cleavages after D16 and G15 . This makes lead(II) a sensitive structural probe for correct folding of tRNAPhe . In the present paper we use Pb2+ ions as a functional probe to determine whether this part of tRNA is protected by the Escherichia coli elongation factor EF-Tu in the ternary complex formed between Phe-tRNAPhe and EF-Tu.GTP . Our results show that for tRNA in complex with EF-Tu:GTP, the phosphodiester bond after D17 is cleaved, yet the phosphodiester bonds after D16 and G15 are not . To our knowledge, this is the first time that Pb2+ ions, bound at a specific site in tRNA, have been used both to investigate the correct folding of tRNA in complex, and to footprint a functional complex with components whose individual structures are known.

Biochem Mol Biol Int, 1993 Sep, 31(1), 1 - 11
Phage lambda beta protein, a component of general recombination, is associated with host ribosomal S1 protein; Muniyappa K et al.; We have purified phage lambda beta protein produced by a recombinant plasmid carrying bet gene and confirm that it forms a complex with a protein of relative molecular mass 70 kDa . Therefore, beta protein, a component of general genetic recombination, is associated with two functionally diverse complexes; one containing exonuclease and the other 70 kDa protein . Using a number of independent methods, we show that 70 kDa protein is the ribosomal S1 protein of E . coli . Further, the association of 70 kDa protein with beta protein is biologically significant, as the former inhibits joining of the terminal ends of lambda chromosome and renaturation of complementary single stranded DNA promoted by the latter . More importantly, these findings initiate an understanding of an important mode of host- virus interaction in general with specific implication(s) in homologous genetic recombination.

EMBO J, 1993 Sep, 12(9), 3619 - 26
DbpA: a DEAD box protein specifically activated by 23s rRNA; Fuller-Pace FV et al.; The Escherichia coli protein DbpA is a member of the 'DEAD box' family of putative RNA-dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp, together with several other conserved elements . We have investigated DbpA expression under conditions where an endogenous promoter is used . In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in-frame GUG . Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism . Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA-dependent manner . This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA . This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA.

EMBO J, 1993 Sep, 12(9), 3587 - 98
Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA; Andino R et al.; The structure of a ribonucleoprotein complex formed at the 5'-end of poliovirus RNA was investigated . This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf-like structure and interact with both uncleaved 3CD, the viral protease-polymerase precursor, and a 36 kDa ribosome-associated cellular protein . The cellular protein is required for complex formation and interacts with unpaired bases in one stem-loop of the cloverleaf RNA . Amino acids within the 3C protease which are important for RNA binding were identified by site-directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein . The physiologic importance of the ribonucleic-protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability . Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5'-end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA.

EMBO J, 1993 Sep, 12(9), 3409 - 15
A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli; Nishiyama K et al.; A novel factor, which is a membrane component of the protein translocation machinery of Escherichia coli, was discovered . This factor was found in the trichloracetic acid-soluble fraction of solubilized cytoplasmic membrane . The factor was purified to homogeneity by ion exchange column chromatographies and found to be a hydrophobic protein with a molecular mass of approximately 12 kDa . The factor caused > 20-fold stimulation of the protein translocation when it was reconstituted into proteoliposomes together with SecE and SecY . SecE, SecY, SecA and ATP were essential for the factor-dependent stimulation of the activity . The factor stimulated the translocation of all three precursor proteins examined, including authentic proOmpA . Stimulation of the translocation of proOmpF-Lpp, a model presecretory protein, was especially remarkable, since no translocation was observed unless proteoliposomes were reconstituted with the factor . Partial amino acid sequence of the purified factor was determined . An antibody raised against a synthetic peptide of this sequence inhibited the protein translocation into everted membrane vesicles, indicating that the factor is playing an important role in protein translocation into membrane vesicles . The partial amino acid sequence was found to coincide with that deduced from the reported DNA sequence of the upstream region of the leuU gene . Cloning and sequencing of the upstream region revealed the presence of a new open reading frame, which encodes a hydrophobic protein of 11.4 kDa . We propose that the factor is a general component of the protein translocation machinery of E . coli.

EMBO J, 1993 Sep, 12(9), 3391 - 8
PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains; Osborne RS et al.; The SecY protein of Escherichia coli and its homologues in other organisms, are integral components of the cellular protein translocation machinery . Suppressor mutations that alter SecY (the prlA alleles) broaden the specificity of this machinery and allow secretion of precursor proteins with defective signal sequences . Twenty-five prlA alleles have been characterized . These suppressor mutations were found to cluster in regions corresponding to three distinct topological domains of SecY . Based on the nature and position of the prlA mutations, we propose that transmembrane domain 7 of SecY functions in signal sequence recognition . Results suggest that this interaction may involve a right-handed supercoil of alpha-helices . Suppressor mutations that alter this domain appear to prevent signal sequence recognition, and this novel mechanism of suppression suggests a proofreading function for SecY . We propose that suppressor mutations that alter a second domain of SecY, transmembrane helix 10, also affect this proof-reading function, but indirectly . Based on the synthetic phenotypes exhibited by double mutants, we propose that these mutations strengthen the interaction with another component of the translocation machinery, SecE . Suppressor mutations were also found to cluster in a region corresponding to an amino-terminal periplasmic domain . Possible explanations for this unexpected finding are discussed.

Protein Eng, 1993 Sep, 6(7), 779 - 85
Expression of deletion constructs of bovine beta-1,4-galactosyltransferase in Escherichia coli: importance of Cys134 for its activity; Boeggeman EE et al.; Bovine beta-1,4-galactosyltransferase (beta-1,4-GT; EC 2.4.1.90) belongs to the glycosyltransferase family and as such shares a general topology: an N-terminal cytoplasmic tail, a signal anchor followed by a stem region and a catalytic domain at the C-terminal end of the protein . cDNA constructs of the N-terminal deleted forms of beta-1,4-GT were prepared in pGEX-2T vector and expressed in E . coli as glutathione-S-transferase (GST) fusion proteins . Recombinant proteins accumulated within inclusion bodies as insoluble aggregates that were solubilized in 5 M guanidine HCl and required an 'oxido-shuffling' reagent for regeneration of the enzyme activity . The recombinant beta-1,4-GT, devoid of the GST domain, has 30-85% of the sp . act . of bovine milk beta-1,4-GT with apparent Kms for N-acetylglucosamine and UDP-galactose similar to those of milk enzyme . Deletion analyses show that both beta-1,4-GT and lactose synthetase activities remain intact even in the absence of the first 129 residues (pGT-d129) . The activities are lost when either deletions extend up to residue 142 (pGT-d142) or Cys134 is mutated to Ser (pGT-d129C134S) . These results suggest that the formation of a disulfide bond involving Cys134 holds the protein in a conformation that is required for enzymatic activity.

Protein Eng, 1993 Sep, 6(7), 771 - 7
Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states; Herberg FW et al.; The catalytic subunit of mouse cAMP-dependent protein kinase expressed in Escherichia coli was separated into three distinct species using Mono-S ion exchange chromatography . These isoenzymes corresponded to three isoelectric variants with pIs of 6.4 (30%), 7.2 (60%) and 8.2 (10%) . The Stokes' radius of each form was 27.7, 27.1 and 26.3 A respectively . Using electrospray mass spectroscopy the differences between the isozymes were shown to be due to phosphorylation, with each form differing by 80 mass units corresponding to a single phosphate . The fully phosphorylated recombinant enzyme contained four phosphates while the dominant isozyme contained only three . Since the enzyme is not phosphorylated when active site mutations are introduced into the C-subunit, these phosphates are incorporated in an autocatalytic mechanism and are not due to E . coli protein kinases . When the recombinant enzyme was compared with the mammalian porcine heart enzyme significant differences in post-translational modifications were observed . The mammalian enzyme could also be separated into two isozymes . However, in contrast to the recombinant enzyme, the mammalian isozymes displayed an identical mass of 40 840 . This correlated with two different post-translational modifications: two phosphates and an N-terminal myristyl moiety . The importance of post-translational modifications, and in particular the phosphorylation state, for the expression of eukaryotic proteins in E . coli is discussed.

Protein Eng, 1993 Sep, 6(7), 763 - 70
Overproduction of bovine beta-casein in Escherichia coli and engineering of its main chymosin cleavage site; Simons G et al.; A cDNA clone containing the entire coding region for bovine beta-casein A3 flanked by 53 base pairs of 5' non-coding and 358 base pairs of 3' non-coding sequences was isolated from a bovine mammary cDNA phagemid library . The coding segment for mature beta-casein was subcloned into the T7 expression system, in which the expression of recombinant beta-casein was controlled by the T7 gene 10 promoter and ribosome binding site . High level expression of Met-beta-casein to approximately 20% of the total soluble proteins was obtained in Escherichia coli within 2 h after induction of T7 RNA-polymerase synthesis . In an attempt to induce secretion the coding segment for mature beta-casein was coupled to the ompA translational initiation signal and signal peptide coding sequence but no secretion of the fusion protein and no processing of the signal peptide from the fusion protein was observed . Instead, the Met-beta-casein could be isolated in a soluble form from E.coli cells after an osmotic shock, indicative of a periplasmic location . This procedure did not lyse the cells . The protein was purified to homogeneity after a pH 4.8 isoelectric precipitation followed by reversed-phase high-performance liquid chromatography . The beta-casein cDNA was altered to change the main chymosin cleavage site in beta-casein at position 192-193 in two ways, namely from Leu-Tyr to Pro-Pro and to Leu-stop.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Eng, 1993 Sep, 6(7), 745 - 54
Strong inhibition of fibrinogen binding to platelet receptor alpha IIb beta 3 by RGD sequences installed into a presentation scaffold; Lee G et al.; In order to probe the structural constraints on binding of RGD sequences to the platelet receptor alpha IIb beta 3 we have used recombinant DNA techniques to install the RGD sequence into 'presentation scaffolds', small proteins of known 3-D structure chosen to present guest sequences in constrained orientations . Using Escherichia coli expression systems we made sequence variants in which loop residues of the immunoglobulin VL domain REI and of human interleukin-1 beta were replaced (without changing polypeptide length) by the RGD sequence at positions predicted, based on small molecule studies, to orient the RGD moiety into an active conformation . These variants do not compete for fibrinogen binding to alpha IIb beta 3 up to almost 1 mM concentration . Unfolded or proteolytically fragmented forms of these same proteins do compete, however, showing that the RGD sequences in the mutants must be prohibited from binding by constraints imposed by scaffold structure . To suppress the effects of such structural constraints we constructed two sequence variants in which RGD-containing sequences 42-57 or 44-55 from the snake venom platelet antagonist kistrin were inserted (this increasing the length of the loop) into the third complementarity determining loop of REI . Both of these variants compete strongly for fibrinogen binding with IC50s in the nM range . These results, plus data on kistrin-related peptides also presented here, suggest that the molecular scaffold REI is capable of providing to an installed sequence a structural context and conformation beneficial to binding . The results also suggest that in order to bind well to alpha IIb beta 3, RGD sequences in protein ligands must either project significantly from the surface of the scaffold and/or retain a degree of conformational flexibility within the scaffold . Molecular scaffolds like REI should prove useful in the elucidation of structure-function relationships and the discovery of new active sequences, and may also serve as the basis for novel therapeutic agents.

Protein Eng, 1993 Sep, 6(7), 739 - 44
Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45; Grunert HP et al.; Using point mutated overproducing strains of E . coli, ribonuclease T1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp, Tyr45Trp or Trp59Tyr and the corresponding double substitutions Tyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr . Steady state kinetics of the transesterification reaction for the two dinucleoside monophosphate substrates guanylyl-3',5'-cytidine and guanylyl-3',5'-adenosine indicate that the tryptophan can be introduced in different positions within the ribonuclease T1 molecule without abolishing enzymatic activity . The Trp59Tyr exchange even enhances catalysis of the cleavage reaction (kcat/Km) relative to the wild type enzyme and similar effects are found with single tyrosine to tryptophan substitutions . For the pH dependencies of the guanylyl-3',5'-cytidine transesterification reaction of wild type ribonuclease T1 and of the variants, typically bell-shaped curves are observed with a plateau in the range pH 4.5-7.0 . Their shapes and slopes indicate that the enzymes are comparable in their macroscopic pKa values . At pH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-fold higher transesterification activity for guanylyl-3',5'-adenosine and a 2-fold increase for guanylyl-3',5'-cytidine compared to the wild type enzyme, i.e . this variant catalyses the transesterification of the substrate guanylyl-3',5'-adenosine with the same or better efficiency as guanylyl-3',5'-cytidine.

Mol Biol (Mosk), 1993 Sep-Oct, 27(5), 1100 - 12
{Cloning of the gene for thermostable Thermus aquaticus YT1 DNA polymerase and its expression in Escherichia coli}; Patrushev LI et al.; Using the phasmid vector pSL5, the genomic DNA fragment of T . aquaticus YT1 which contained the thermostable DNA polymerase (Taq-polymerase) gene was cloned . The BglII fragment of this genome locus was subcloned in the BamHI site of the pUC19 plasmid . To optimize the Taq-polymerase gene expression in E . coli cells, the gene was cloned in the correct reading frame regarding the initiation ATG codon of the pPR-TGATG-1 expression vector . The gene expression in this vector was controlled by the phage lambda PR promoter and the temperature-sensitive phage lambda repressor . We used PCR to amplify the short 5'-end fragment of the Taq-polymerase gene coding for the part into which an artificial SacI site was introduced . This site has been used for cloning the PCR product into the pPR-TGATG-1 vector, and the missing gene part was cloned into the KpnI site of the PCR product from the natural cloned gene . The cells of the E . coli PVG-A1 strain, which was obtained in the end, expressed efficiently the Taq-polymerase gene at the nonpermissive temperature . The content of the recombinant Taq-polymerase in the cells was about 1-2% of total proteins . The purified nearly homogeneous Taq-polymerase amplified efficiently in the PCR DNA fragments up to 5.5 kb long and was useful in DNA sequencing the by Sanger method . The half-life of the purified Taq polymerase was about 60 min at 95 degrees C, it was active for at least 65 standard PCR circles . The specific activity of recombinant enzyme preparations was about 180-200,000 units per mg of protein . The E . coli PVG-A1 strain enables one to isolate up to 500,000 units of purified enzyme from 2 l of bacterial culture.

Mol Biol (Mosk), 1993 Sep-Oct, 27(5), 1085 - 93
{Study of the structure of Escherichia coli RNA polymerase and its complex with the lacUV5-promotor using protein-protein and DNA-protein crosslinks, formed by formaldehyde}; Brodolin KL et al.; The protein-protein and DNA-protein crosslinks produced by formaldehyde were used to investigate the intersubunit and subunit-DNA interactions for free RNA polymerase and for an open complex of RNA polymerase with the lacUV5 promoter . In both cases the contacts between beta,beta' and beta', sigma subunits were observed, while there were no contacts between beta and sigma subunits . Only one of beta or beta' subunits and a sigma subunit crosslink to promoter DNA . We have chosen the conditions for fixing the RNA polymerase-DNA complexes on different stages of transcription initiation . The possibility to use limited fixation with low concentrations of formaldehyde to study specific DNA-protein interactions was shown.

Mol Biol (Mosk), 1993 Sep-Oct, 27(5), 1014 - 22
{Expression of the human alpha-1-antitrypsin gene in Escherichia coli}; Strakhova MI et al.; The cDNA, containing the complete human alpha-1-antitrypsin (AT) sequence starting from codon 2, was used to construct bacterial strains producing AT . The fusion protein was obtained by junction of the AT cDNA to the fragment of an Escherichia coli ompF gene . We have also modified the AT cDNA's 5'-terminal part to construct DNAs containing ATG-codon and cDNA sequences starting from codons 1 or 2 . These DNAs were inserted into E . coli expression vectors pBR322/trpII-8 and pKK223-3 that allow transcription from efficient trp- and tac-promoters . This construction resulted in the induction of a 46 kDa protein . The polypeptide produced was recognized by an antiserum raised against human alpha-1-antitrypsin protein . Truncation of the gene at its 5'-end or synthesis as a fusion OmpF-AT protein increases expression up to 10-fold, to a level of approximately 1% . On the contrary, no dependence on the promoter type has been observed . Physical properties of the recombinant proteins are discussed.

Microbiol Rev, 1993 Sep, 57(3), 683 - 702
Mechanisms of genome propagation and helper exploitation by satellite phage P4; Lindqvist BH et al.; Temperate coliphage P2 and satellite phage P4 have icosahedral capsids and contractile tails with side tail fibers . Because P4 requires all the capsid, tail, and lysis genes (late genes) of P2, the genomes of these phages are in constant communication during P4 development . The P4 genome (11,624 bp) and the P2 genome (33.8 kb) share homologous cos sites of 55 bp which are essential for generating 19-bp cohesive ends but are otherwise dissimilar . P4 turns on the expression of helper phage late genes by two mechanisms: derepression of P2 prophage and transactivation of P2 late-gene promoters . P4 also exploits the morphopoietic pathway of P2 by controlling the capsid size to fit its smaller genome . The P4 sid gene product is responsible for capsid size determination, and the P2 capsid gene product, gpN, is used to build both sizes . The P2 capsid contains 420 capsid protein subunits, and P4 contains 240 subunits . The size reduction appears to involve a major change of the whole hexamer complex . The P4 particles are less stable to heat inactivation, unless their capsids are coated with a P4-encoded decoration protein (the psu gene product) . P4 uses a small RNA molecule as its immunity factor . Expression of P4 replication functions is prevented by premature transcription termination effected by this small RNA molecule, which contains a sequence that is complementary to a sequence in the transcript that it terminates.

Microbiol Rev, 1993 Sep, 57(3), 623 - 54
Colibri: a functional data base for the Escherichia coli genome; Medigue C et al.; Several data libraries have been created to organize all the data obtained worldwide about the Escherichia coli genome . Because the known data now amount to more than 40% of the whole genome sequence, it has become necessary to organize the data in such a way that appropriate procedures can associate knowledge produced by experiments about each gene to its position on the chromosome and its relation to other relevant genes, for example . In addition, global properties of genes, affected by the introduction of new entries, should be present as appropriate description fields . A data base, implemented on Macintosh by using the data base management system 4th Dimension, is described . It is constructed around a core constituted by known contigs of E . coli sequences and links data collected in general libraries (unmodified) to data associated with evolving knowledge (with modifiable fields) . Biologically significant results obtained through the coupling of appropriate procedures (learning or statistical data analysis) are presented . The data base is available through a 4th Dimension runtime and through FTP on Internet . It has been regularly updated and will be systematically linked to other E . coli data bases (M . Kroger, R . Wahl, G . Schachtel, and P . Rice, Nucleic Acids Res . 20(Suppl.):2119-2144, 1992; K . E . Rudd, W . Miller, C . Werner, J . Ostell, C . Tolstoshev, and S . G . Satterfield, Nucleic Acids Res . 19:637-647, 1991) in the near future.

Microbiol Rev, 1993 Sep, 57(3), 522 - 42
Regulation of fatty acid biosynthesis in Escherichia coli; Magnuson K et al.; Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years . Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E . coli has been studied in some detail . Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms . In fact, E . coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics . In this review, we concentrate on four major areas of research . First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail . The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway . Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed . Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E . coli are described . Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined . In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered . Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth.

Can J Microbiol, 1993 Sep, 39(9), 892 - 4
MutY repair is mutagenic in mutT- strains of Escherichia coli; Vidmar JJ et al.; We have determined the numbers and types of mutations that occur in strains of Escherichia coli defective in mutT and (or) mutY repair . High rates of C.G to A.T mutations in mutY- cells are unaffected by the status of mutT . However, mutT-/mutY+ strains have higher rates of A.T to C.G mutations than mutT-/mutY- strains . This result indicates that the high rates of A.T to C.G mutations seen in mutT- strains of E . coli are due in part to the activity of the mutY repair system . We conclude that mutY repair is mutagenic in a mutT- background.

Biophys J, 1993 Sep, 65(3), 1295 - 306
Two-dimensional crystallization of Escherichia coli-expressed bacteriorhodopsin and its D96N variant: high resolution structural studies in projection; Mitra AK et al.; Highly ordered two-dimensional (2-D) crystals of Escherichia coli-expressed bacteriorhodopsin analog (e-bR) and its D96N variant (e-D96N) reconstituted in Halobacterium halobium lipids have been obtained by starting with the opsin protein purified in the denaturing detergent sodium dodecyl sulfate . These crystals embedded in glucose show electron diffraction in projection to better than 3.0 A at room temperature . This is the first instance that expressed bR or a variant has been crystallized in 2-D arrays showing such high order . The crystal lattice is homologous to that in wild-type bR (w-bR) in purple membranes (PM) and permit high resolution analyses of the structure of the functionally impaired D96N variant . The e-bR crystal is isomorphous to that in PM with an overall averaged fractional change of 12.7% (26-3.6-A resolution) in the projection structure factors . The projection difference Fourier map e-bR-PM at 3.6-A resolution indicates small conformational changes equivalent to movement of approximately < 7 C-atoms distributed within and in the neighborhood of the protein envelope . This result shows that relative to w-bR there are no global structural rearrangements in e-bR at this 3.6 A resolution level . The e-D96N crystal is isomorphous to the e-bR crystal with a smaller (9.2%) overall averaged fractional change in the structure factors . The significant structural differences between e-D96N and e-bR are concentrated at high resolution (5-3.6 A); however, these changes are small as quantified from the 3.6 A resolution e-D96N-e-bR Fourier difference map . The difference map showed no statistically significant peaks or valleys within 5 A in projection from the site of D96 substitution on helix C . Elsewhere within the protein envelope the integrated measure of peaks or valleys was < approximately 3 C-atom equivalents . Thus, our results show that for the isosteric substitution of Asp96 by Asn, the molecular conformation of bR in its ground state is essentially unaltered . Therefore, the known effect of D96N on the slowed M412 decay is not due to ground-state structural perturbations.

Cell Growth Differ, 1993 Sep, 4(9), 731 - 43
rel/NF-kappa B nuclear complexes that bind kB sites in the murine c-rel promoter are required for constitutive c-rel transcription in B-cells; Grumont RJ et al.; The c-rel protooncogene, a member of a transcription factor family that includes NF-kappa B, displays a complex pattern of gene expression . To understand the basis of this expression, the regulatory region upstream of the murine c-rel transcription start sites has been cloned and characterized . Transcription of the murine c-rel gene initiates at multiple sites downstream of a GC-rich region conserved in the chicken c-rel promoter . This conserved region contains consensus transcription factor binding sites for SP-1 and NF-kappa B (kB3 site) and is sufficient for basal expression in Jurkat T-cells . In contrast, two additional NF-kappa B-like sites (kB1 and kB2) and an octamer consensus binding site, all located upstream of the conserved region, are required for expression of promoter-reporter gene constructs in the B-cell line I29B . NF-kappa B sites kB1 and kB3 bind p50/65 and p50 homodimers, whereas kB2 binds a distinct complex . The consensus octamer site, although only able to bind Oct1 and Oct2 with low affinity, appears to overlap with a binding site for a novel protein(s) expressed in I29B cells . Cotransfection studies show that p75-c-rel and a carboxyl-terminal truncated c-rel protein that lacks the known trans-activating domain both up-regulate the c-rel promoter in I29B cells via a mechanism independent of the NF-kappa B motifs, whereas a mutant c-rel protein lacking the DNA binding domain has no effect . Together, these findings suggest that, in this B-cell line, trans-activation of the c-rel promoter by rel proteins is via an indirect mechanism.

Bioessays, 1993 Sep, 15(9), 617 - 23
Relating biochemistry to biology: how the recombinational repair function of RecA protein is manifested in its molecular properties; Cox MM; The multiple activities of the RecA protein in DNA metabolism have inspired over a decade of research in dozens of laboratories around the world . This effort has nevertheless failed to yield an understanding of the mechanism of several RecA protein-mediated processes, the DNA strand exchange reactions prominent among them . The major factors impeding progress are the invalid constraints placed upon the problem by attempting to understand RecA protein-mediated DNA strand exchange within the context of an inappropriate biological paradigm-namely, homologous genetic recombination as a mechanism for generating genetic diversity . In this essay I summarize genetic and biochemical data demonstrating that RecA protein evolved as the central component of a recombinational DNA repair system, with the generation of genetic diversity being a sometimes useful byproduct, and review the major in vitro activities of RecA protein from a repair perspective . While models proposed for both recombination and recombinational repair often make use of DNA strand cleavage and transfer steps that appear to be quite similar, the molecular and thermodynamic requirements of the two processes are very different . The recombinational repair function provides a much more logical and informative framework for thinking about the biochemical properties of RecA and the strand exchange reactions it facilitates.

Artif Organs, 1993 Sep, 17(9), 775 - 81
Extracorporeal endotoxin removal by immobilized polyethylenimine; Mitzner S et al.; The neutralization of bacterial endotoxins (ET) is still an unsolved problem in therapeutic medicine . The efficacy of anti-endotoxin antibodies or receptor antagonists and other substances interfering with the endotoxin-induced pathomechanisms is dependent on an intact cellular degradation system of the host . However, the phagocytosis function of that system seems to be impaired regularly in patients with intense or long-lasting endotoxemia or septic shock and in patients undergoing hemodialysis . Extracorporeal adsorption of ET might well be an effective support in the anti-ET therapy by lowering the amount of circulating ET and thus relieving the defense system of the body . In this work a new ET-adsorbent based on macroporous cellulosic beads with immobilized polyethylenimine (PEI) was tested for its ET-removal capacity in vitro . A test solution with 100 ng/ml ET from Escherichia coli 055:B5 was recirculated in a system containing the adsorbent beads . Polymyxin B immobilized to the same carrier was used for comparison . PEI as well as polymyxin B showed complete removal of ET from plasma and water as was measured by the Limulus Amebocyte Lysate (LAL) test (Chromogenix) . The biocompatibility of the PEI absorber was superior to that of polymyxin B . The results indicate that the PEI absorber is of high efficacy and possibly of interest for the treatment of endotoxemia.

Am J Vet Res, 1993 Sep, 54(9), 1517 - 22
Comparison of flunixin, prednisolone, dimethyl sulfoxide, and a lazaroid (U74389F) for treating endotoxemic neonatal calves; Semrad SD; Saline (0.9% NaCl) solution, flunixin meglumine (1.1 mg/kg), prednisolone sodium succinate (1.1 mg/kg), U74389F (1.5 mg/kg), and dimethyl sulfoxide (0.5 g/kg) were each administered i.v . to 5 neonatal calves 15 minutes after the start of a 3-hour infusion of Escherichia coli lipopolysaccharide (LPS; 2 micrograms/kg/hr) . Four additional calves were given a 3-hour i.v . infusion of saline solution alone . Only flunixin significantly suppressed eicosanoid production and mitigated clinical signs associated with endotoxemia . Prednisolone provided partial protection against LPS-induced hypotension and lacticemia . Pronounced hypoglycemia and lacticemia were observed in U74389F-treated calves; LPS-induced hypotension and hypoglycemia were marked in dimethyl sulfoxide-treated calves.

Am J Vet Res, 1993 Sep, 54(9), 1511 - 6
Comparative efficacy of flunixin, ketoprofen, and ketorolac for treating endotoxemic neonatal calves; Semrad SD; Saline (0.9% NaCl) solution or 1 of 3 nonsteroidal anti-inflammatory drugs (NSAID) was administered i.v . to 5 neonatal calves 15 minutes after the start of a 3-hour i.v . infusion of Escherichia coli lipopolysaccharide (LPS; 2 micrograms/kg/h) . Four additional calves were given a 3-hour i.v . infusion of saline solution alone . Clinical attitude, mean arterial blood pressure, PCV, WBC, and plasma lactate, glucose, and eicosanoid concentrations (thromboxane B2, 6-keto-PGF1 alpha) were monitored for 12 hours . Flunixin meglumine (1.1 mg/kg of body weight, i.v.), ketoprofen (2.2 mg/kg, i.v.), and ketorolac tromethamine (1.1 mg/kg, i.v.) each ameliorated the clinical signs of endotoxemia and LPS-induced lacticemia, but failed to significantly alter the degree of leukopenia or hypoglycemia associated with infusion of LPS . Although the 3 NSAID prevented eicosanoid production, they provided only partial protection against LPS-induced hypotension . Each NSAID modified the response to LPS, but none was clearly superior to the others in modulating the clinical signs or physiologic alterations induced by infusion of LPS in neonatal calves.

Am J Vet Res, 1993 Sep, 54(9), 1462 - 70
Purification of surface-exposed integral outer membrane proteins of Actinobacillus pleuropneumoniae and their role in opsonophagocytosis; Thwaits RN et al.; Previously identified 39-, 50-, and 76-kd integral outer membrane proteins (IOMP) of Actinobacillus pleuropneumoniae, a respiratory tract pathogen, were separated by electroelution of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-obtained fragments and their role in opsonophagocytosis by porcine leukocytes was investigated by flow cytometry of fluorescein-labeled A pleuropneumoniae . Using specific antisera, immunoblot analysis indicated that the 3 proteins were antigenically distinct . Antibodies against each IOMP have an important role as opsonins for phagocytosis by porcine leukocytes . The effect of using a combination of all 3 of the specific antisera was minimal . Antiserum absorbed against intact A pleuropneumoniae and Escherichia coli organisms indicated that the antibodies to the 39-, 50-, and 76-kd IOMP were specific for A pleuropneumoniae antigens . Nonheat-treated antiserum did not increase phagocytosis, compared with heat-inactivated antiserum, indicating that complement may not have a major role in opsonization of A pleuropneumoniae.

Am J Vet Res, 1993 Sep, 54(9), 1411 - 4
Serum interleukin-6 concentrations in endotoxin-infused neonatal foals; Robinson JA et al.; Serum interleukin-6 (IL-6) concentration was measured in 11 colostrum-fed (CF) and 8 colostrum-deprived (CD) 2- to 3-day-old foals after foals were infused with lipopolysaccharide (LPS; Escherichia coli O55:B5 endotoxin, 0.5 microgram/kg of body weight in sterile saline {0.9% NaCl} solution) . Four CF and 2 CD foals were given saline solution alone . Serum IL-6 concentration was estimated by use of an in vitro proliferative bioassay, using the IL-6 dependent B.13.29 clone 9 cells . Interleukin-6 concentration increased in all LPS-infused foals, and geometric mean serum IL-6 concentration was significantly higher in CF than CD foals 30 and 90 minutes after infusion . Both LPS-infused groups had multiple spikes of mean IL-6 concentration that peaked at 120 minutes in CF foals and 150 minutes in CD foals . Results indicated that IL-6 is produced in neonatal foals in response to LPS infusion . Furthermore, colostrum deprivation resulted in longer times to peak mean serum IL-6 concentration and tended to reduce serum IL-6 concentration in neonatal foals.

Am J Vet Res, 1993 Sep, 54(9), 1404 - 10
Serum tumor necrosis factor alpha concentrations and clinical abnormalities in colostrum-fed and colostrum-deprived neonatal foals given endotoxin; Allen GK et al.; We examined the effect of infusion of lipopolysaccharide (LPS) on serum tumor necrosis factor alpha (TNF alpha) concentration and clinical attitude in 2- 3-day-old colostrum-fed (CF) and colostrum-deprived (CD) foals . Eleven CF and 8 CD neonatal foals were given a bolus i.v . infusion of Escherichia coli O55:B5 lipopolysaccharide (0.5 microgram/kg of body weight) in sterile saline (0.9% NaCl) solution . Four CF and 2 CD foals were given saline solution alone . Serum IgG concentration and serum anti-LPS IgG(T) antibody titer were determined for each foal prior to infusion . A depression index was used to score clinical abnormalities . Serum TNF alpha concentration was estimated by use of an in vitro cytotoxicity bioassay that used WEHI 164 clone 13 cells as targets . The cytotoxic serum factor was identified as TNF alpha by immunoprecipitation with caprine antisera raised against the 15 NH2-terminal amino acids of human TNF alpha . Tumor necrosis factor alpha was not detected in any preinfusion serum samples nor in any samples from foals given saline solution alone . Serum TNF alpha concentration increased in all LPS-infused foals and peaked between 60 and 90 minutes after infusion . Serum TNF alpha concentrations, expressed as mean percentage of peak serum TNF alpha concentration, persisted longer in CD foals given LPS than in CF foals given LPS . All LPS-infused foals displayed clinical signs of endotoxemia, but mean depression index scores of the CF and CD foals given LPS were not significantly different at any time . Serum TNF alpha concentrations were correlated with depression index scores in both LPS-infused groups.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bone Miner Res, 1993 Sep, 8(9), 1127 - 36
Osteoblast-specific expression of the alpha 2(I) collagen promoter in transgenic mice: correlation with the distribution of TGF-beta 1; D'Souza RN et al.; To begin to assess the transcriptional mechanisms that regulate type I collagen gene expression in differentiating osteoblasts, we have sought to determine the minimal promoter sequences that confer osteoblast-specific expression to the alpha 2(I) collagen gene during murine development . Transgenic mice were generated harboring DNA constructs in which the -2000, -500, and -350 to +54 regions located upstream of the start of transcription were linked to the Escherichia coli beta-galactosidase reporter gene (LacZ) . Histochemical staining using X-gal indicated that the -2000 lacZ transgene was strongly expressed in newly differentiated and fully functional osteoblasts at intramembranous and endochondral sites of ossification . The promoter was also active in osteocytes in regions of bone remodeling within alveolar bone . The temporal and spatial activity of this region of the promoter closely resembled the developmental patterns of expression of the endogenous alpha 2(I) collagen gene as determined by in situ hybridization . The cis-acting elements within the 500 and 350 bp segments of the alpha 2(I) collagen promoter also drove reporter gene expression in forming osteoblasts, although levels of transgene expression were not as marked as that seen with the 2000 bp promoter . Furthermore, the synthesis and secretion of TGF-beta 1 in osteogenic zones coincided with areas where the alpha 2(I) collagen promoter constructs were transcriptionally active . Since a nuclear factor 1 binding site present at -300 has been shown to mediate the effects of TGF-beta 1 on the alpha 2(I) collagen promoter, these data support a role for TGF-beta 1 in the control of this gene during development.

Virus Res, 1993 Sep, 29(3), 281 - 303
Comparative analysis of the conserved region of the orthopoxvirus genome encoding the 36K and 12K proteins; Ryazankina OI et al.; Genes encoding virus-specific proteins with molecular masses of 36 kDa and 12 kDa were mapped in HindIII-P and HindIII-U DNA fragments of vaccinia strain LIVP and ectromelia strain K-1 viruses, respectively, by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation . The 36K translation initiation codon was detected in the HindIII-J fragment . The nucleotide sequences of corresponding genes from vaccinia, ectromelia, cowpox and variola virus genomes were determined . The 12K protein has similarity to mammalian glutaredoxins . The derived amino acid sequence of the 36K polypeptide was compared with the protein bank PIR . No homology was found between the 36K protein and known structures of proteins . The 36K protein genes of vaccinia and ectromelia viruses were cloned in pUR290, which led to the production of E . coli chimeric proteins, consisting of the sequence of beta-galactosidase and the viral protein on their C-ends . The chimeric proteins were shown to possess viral antigenic specificity . To identify the protein product of the 36K gene monospecific antisera to chimeric proteins were obtained . The late 36K protein is associated with virosomes but is not incorporated into the virions of orthopoxviruses.

Med Microbiol Immunol (Berl), 1993 Sep, 182(4), 167 - 75
A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes; Bhakdi S et al.; Depending on the size of the pores one wishes to produce in plasma membranes, the choice will probably fall on one of the three toxins discussed above . S . aureus alpha-toxin should be tried first when pores of 1-1.5 nm diameter are required . This is generally the case when Ca2+ and nucleotide dependence of a given process is being studied . If alpha-toxin does not work, this is probably due to the fact that the toxin either does not produce pores, or that the pores are too small . In this case, high concentrations of alpha-toxin should be tried . If this still does not work, we recommend the use of HlyA . When very large pores are to be created, e.g . for introduction of antibodies into the cells, SLO or another member of this toxin family are the agents of choice . SLO preparations need to be checked for presence of protease contaminants . Tetanolysin currently offers advantages since it is protease-free, and the size of the pores can probably be controlled by varying the toxin dose . Methods for assessing the size of pores created by such agents have been published in the recent literature, and the appropriate papers can be consulted whenever the need arises.

Lipids, 1993 Sep, 28(9), 837 - 40
Phospholipids from the free-living nematode Caenorhabditis elegans; Satouchi K et al.; The phospholipid and the fatty chain compositions of diacyl, alkylacyl and alkenylacyl glycerophospholipids of the free-living nematode, Caenorhabditis elegans, were investigated . The phospholipids were comprised of 54.5% ethanolamine glycerophospholipid (EGP), 32.3% choline glycerophospholipid (CGP), 8.1% sphingomyelin and 5.1% others . The most abundant fatty acid in CGP was eicosapentaenoic acid (20:5n-3) . The fatty acids in CGP were more unsaturated than those in EGP . Alkenylacyl and alkylacyl subclasses accounted for 1.0 and 2.6%, respectively, of CGP and 14.0 and 19.6%, respectively, of EGP . At least 80% of the alkenyl and alkyl groups were 18:0 chains and the remaining were odd numbered chains . The potential presence of platelet-activating factor (PAF) was examined by bioassay, but PAF-like activity was not detected in the extracts of this nematode.

Masui, 1993 Sep, 42(9), 1302 - 5
{Effects of DbcAMP on tumor necrosis factor and interleukin-1 production in human monocytes}; Sato W et al.; Recent reports have shown that dibutyryl cAMP (DbcAMP) blocks endotoxin-induced lung injury . To determine whether DbcAMP suppresses the production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) in human monocytes, we measured the levels of TNF and IL-1 in response to E . Coli lipopolysaccharide (40 micrograms.ml-1) in vitro . We now show that DbcAMP suppressed dose-dependently the production of TNF in human monocytes, and DbcAMP totally suppressed it at the dose above 5 x 10(-4) M . However, DbcAMP did not suppress the production of IL-1 even at the dose of 5 x 10(-3) M in human monocytes . These data suggest that the productive mechanism of IL-1 may be different from that of TNF . Further, suppression of TNF by DbcAMP may contribute to the beneficial effects in animal models of septic shock or lung injury and this may have clinical implications.

J Biochem Biophys Methods, 1993 Sep, 27(2), 95 - 103
One-step immunoaffinity purification of human beta 1 thyroid hormone receptor with DNA and hormone binding activity; Park JB et al.; An efficient and versatile method to purify large amounts of active human beta 1 thyroid hormone receptor (h-TR beta 1) was developed . Using a T7 expression system, h-TR beta 1 was overexpressed in Escherichia coli . Approx . 80% of the expressed receptor protein was concentrated in the insoluble inclusion bodies and approximately 20% was in the soluble form (h-TR beta 1-S) . h-TR beta 1-S was conveniently purified by one immunoaffinity chromatographic step . From 1 l of cell culture, approx . 0.1 mg of purified h-TR beta 1-S was obtained . The purified h-TR beta 1-S binds to 3,3',5-triiodo-L-thyronine with a Ka = 2 x 10(9) M-1 and exhibits analog specificity . The purified h-TR beta 1-S also binds to T3 response elements (TRE) with different orientation in the half-sites with differential activity . In addition, binding of h-TR beta 1-S to TREs was enhanced by retinoid X receptor . These results indicate that the purified h-TR beta 1-S retains its hormone and DNA binding activity . The purified h-TR beta 1-S is suitable for structural and functional studies . This method could be used to purify h-TR beta 1 or rat TR beta 1 expressed in insect cells or yeast.

J Dairy Sci, 1993 Sep, 76(9), 2613 - 8
Blood polymorphonuclear leukocyte chemotaxis during experimental Escherichia coli bovine mastitis; Kremer WD et al.; The relationship between the severity of experimental Escherichia coli mastitis and the chemotactic response of blood polymorphonuclear leukocytes was investigated before and during mastitis . Experimental E . coli mastitis was induced in 10 healthy cows by inoculation of the rear right quarters with 10(3) cfu of E . coli . Cows were classified into two groups based on the severity of the mastitis . Bacterial growth in the inoculated quarter was used as parameter that indicated severity . Before and during experimental mastitis, the chemotactic response and the number of circulating polymorphonuclear leukocytes were greater for the moderately diseased cows than for the severely diseased cows . During the first 24 h of the experimental mastitis, the chemotactic response of polymorphonuclear leukocytes decreased in both groups . Recovery of the chemotactic response of white blood cells was more rapid in moderately diseased cows than in severely diseased cows . Possibly, the larger proportion of band neutrophils (the less chemotactically active band neutrophils) partially accounts for the lower chemotactic response of the circulating polymorphonuclear leukocytes during experimental mastitis in the severely diseased cows.

J Dairy Sci, 1993 Sep, 76(9), 2589 - 96
Relations between on-line electrical conductivity and daily milk production on a low somatic cell count farm; Nielen M et al.; To study the relation between on-line electrical conductivity and daily milk production, data of 1389 cow days were analyzed . After correction for cow effects and DIM, a rise of 1 mS of the mean electrical conductivity caused a decline of .88 kg/d in milk production . A rise of 1 ln(SCC) unit was associated with an additional decline of .54 kg/d in milk production . In cows without clinical mastitis during the test period, the losses associated with mean electrical conductivity and ln(SCC) were 1.06 and .45 kg/d of milk production, respectively . Electrical conductivity and SCC were associated with daily production loss; the effects were additive . Therefore, electrical conductivity and SCC can be utilized as indirect tests of subclinical mastitis.

J Dairy Sci, 1993 Sep, 76(9), 2579 - 88
Bovine acute mastitis: effects of intravenous sodium salicylate on endotoxin-induced intramammary inflammation; Morkoc AC et al.; Effects of the nonsteroidal antiinflammatory agent sodium salicylate on endotoxin-induced mastitis were evaluated in lactating cows . Escherichia coli endotoxin was administered to a mammary quarter 1 h after initiation of a 12-h i.v . infusion of sodium salicylate . Milk SCC, BSA concentrations in milk, mammary inflammation, rectal temperature, appetite, milk production, and plasma and lymph PGF2 alpha were monitored . Gross mammary inflammation was not reduced by salicylate infusion, nor did sodium salicylate prevent increased milk SCC or BSA concentrations in milk, although treatment tended to decrease the magnitude of these responses . Sodium salicylate decreased subcutaneous abdominal vein PGF2 alpha metabolite, and PGF2 alpha metabolite tended to be reduced in lymph during the acute phase of inflammation . The increased rectal temperature after endotoxin infusion was reduced in cows treated with sodium salicylate . Appetite was reduced after endotoxin infusion in untreated cows and those treated with sodium salicylate . Milk production declined after endotoxin challenge in all cows . Although sodium salicylate did not substantially reduce mammary inflammation, it had an antipyretic effect and reduced PGF2 alpha metabolite in mammary blood.

J Clin Pathol, 1993 Sep, 46(9), 840 - 5
Use of recombinant human parvovirus B19 antigens in serological assays; Cubie HA et al.; AIMS--To compare the sensitivity, specificity, and practicality of recombinant proteins in serological tests for the detection of human parvovirus B19 IgG and IgM . METHODS--Indirect enzyme linked immunosorbent assays using B19 structural proteins expressed in Escherichia coli were developed for the detection of B19 specific IgG and IgM (rELISA-G and rELISA-M) . Cells infected with baculovirus expressing B19 structural proteins were also used in an indirect immunofluorescence assay for IgG and IgM antibodies (IFA-G and IFA-M) . Antibody capture radioimmunoassays for IgG and IgM (GACRIA and MACRIA) were used as comparative assays . RESULTS--Twenty nine pools of intravenous immunoglobulin were clearly positive for B19 IgG by rELISA-G and contained low IgG titres by GACRIA . From 113 samples tested by all methods, sensitivities of 92% (77/84) and 97% (68/70) were obtained for ELISA and immunofluorescence, respectively, when compared with GACRIA . One hundred and sixteen samples from patients presenting with rash or arthralgia were compared by MACRIA, rELISA-M, and IFA-M . Sensitivities of both recombinant tests were more than 95% . Despite pretreatment to remove IgG or rheumatoid factor, false positive results were a problem in the rELISA-M but were not seen with the IFA-M . CONCLUSIONS--The limited supply of native antigen has severely restricted the wide application of serology for parvovirus B19 . The use of recombinant antigens permitted the introduction of local screening tests which had many advantages, including quicker results and relief of the burden on the Reference Laboratory . The use of rELISA-M for sensitivity and IFA-M for specificity and confirmation proved a useful and practical combination for diagnosis of recent infection with B19, and rELISA-G allowed the immune response to be determined in selected populations.

J Cell Biochem, 1993 Sep, 53(1), 1 - 12
Multitude of inverted repeats characterizes a class of anchorage sites of chromatin loops to the nuclear matrix; Boulikas T et al.; In order to understand the nature of DNA sequences that organize chromatin into domains or loops, we have cloned the nuclear matrix DNA (1.7% of the total DNA) from human myelogenous leukemia cells in culture . Nuclear matrix is formed by interactions between specific stretches of DNA of about 0.1 to 5.0 kb with protein transcription factors, nuclear enzymes, and structural proteins . Nuclear matrix is believed to be the exclusive nuclear microenvironment in which initiation of DNA replication, transcription, and repair take place . The matrix attachment regions (MARs) of DNA have transcriptional enhancer activity, harbor the origins of replication of the human genome, and define the borders between neighboring chromatin loops . In this study we report the sequence of the human MAR fragment 19.2 of a size of 542 bp . Hum . MAR 19.2 is composed of TG-, CA-, CT-, and GA-rich blocks and shows 8 perfect and imperfect inverted repeats . Thus, we have identified a novel class of MARs with sequence characteristics divergent from the AT-rich class of MARs . The inverted repeats of the 19.2 sequence might be stabilized into their cruciform configuration by torsional strain and by specific transcription/replication protein factors . This MAR might function in the initiation of replication of the flanking chromatin domain and in the regulation of the transcriptional activity of the gene(s) that reside in this domain.

Gastrointest Endosc, 1993 Sep-Oct, 39(5), 674 - 9
Serial cholangiographic appearances in recurrent pyogenic cholangitis; Khuroo MS et al.; From December 1982 to December 1991, cholangiograms were obtained in 227 patients with recurrent pyogenic cholangitis . Cholangiographic abnormalities included biliary dilation, calculi, sludge, excessive branching, and arrowhead formation of intrahepatic ducts and biliary strictures . In 21 patients, previous evidence of biliary ascariasis was seen . Repeat cholangiograms were performed in 55 patients in a follow-up period of 18.0 +/- 1 months . Of these patients, 12 treated conservatively continued to get recurrent cholangitis and revealed worsening abnormalities on repeat cholangiograms . Another 25 patients had successful endoscopic sphincterotomy and extraction of biliary calculi . These patients remained free of symptoms on follow-up, with significant resolution of abnormalities on repeat cholangiograms . The remaining 18 patients with failed surgical or endoscopic interventions continued to get recurrent episodes of cholangitis and worsening of abnormalities on repeat cholangiograms . This retrospective study indicates that the natural course of recurrent pyogenic cholangitis is a progressive, destructive cholangiopathy . Ascaris lumbricoides invasion of the biliary tree is an initiating event in a sub-group of patients.

Electrophoresis, 1993 Sep, 14(9), 847 - 51
Electroblotting proteolytic products from native gel for direct N-terminal sequence analysis: an approach for studying protein-protein interaction; Wang F et al.; Proteins which are electroblotted from native gels onto polyvinylidene difluoride (PVDF) membranes are suitable for detailed structural analysis . This method, in conjunction with limited proteolysis and N-terminal sequencing, has been used to study the molecular interactions between native protein molecules . The interaction between recombinant interleukin-2 (rIL-2) and its receptor (rIL-2R alpha) was examined as a model system . The working strategy consists of (i) proteolysis of rIL-2R alpha and rIL-2R alpha/rIL-2 complex, (ii) separation of the major proteolytic products by native polyacrylamide gel electrophoresis followed by electroblotting onto PVDF membrane, and (iii) sequence analysis of the blotted protein bands for the identification of peptide regions sensitive to proteolysis . Results have indicated that the exon 3 encoded region in rIL-2R alpha is sensitive to proteolysis regardless whether it is complexed with rIL-2 or not . This suggests that no major conformational changes occur in rIL-2R alpha during interaction with rIL-2 . This electroblotting approach is, therefore, useful for studying protein-protein interaction in solution.

Curr Genet, 1993 Sep, 24(3), 248 - 55
A gene involved in the biogenesis of c-type cytochromes is co-transcribed with a ribosomal protein gene in wheat mitochondria {corrected}; Gonzalez DH et al.; Sequence analysis of a transcribed region of the wheat mitochondrial (mt) genome revealed two open reading frames (orfs) coding for proteins of 589 and 174 amino acids . Both genes are co-transcribed in a 2.6-kb RNA . The largest orf codes for a hydrophobic protein which bears similarity to a bacterial protein involved in the biogenesis of c-type cytochromes . Its corresponding RNA sequence is fully edited at 34 positions . The second orf encodes a protein homologous to the amino-terminal third of E . coli ribosomal protein S1, corresponding to the ribosome-binding domain of this protein . Its RNA sequence is edited at four positions, one of the edits creating a stop codon . The presence of both proteins in wheat mitochondria was demonstrated using specific antibodies raised against fusion proteins obtained in E . coli from the corresponding cDNAs.

Curr Genet, 1993 Sep, 24(3), 229 - 40
Structure, expression, and phylogenetic relationships of a family of ypt genes encoding small G-proteins in the green alga Volvox carteri; Fabry S et al.; In addition to the previously described gene yptV1 encoding a small G-protein we have now identified and sequenced four more ras-related ypt genes (yptV2-yptV5) from the green alga Volvox carteri . The four new genes encode polypeptides consisting of 203 to 217 amino-acid residues that contain the typical sequence elements (GTP-binding domains, effector domain) of the ypt/rab subgroup of the Ras superfamily . Comparison of the derived amino-acid sequences from the V . carteri ypt gene products and their Ypt homologs from other species revealed similarity values ranging from 60% to 85%, whereas intraspecies similarities were found to approach only 55% . The coding sequences are interrupted by 5-7 introns of variable size (70-1000 nucleotides) occupying different positions in the genes . Reverse-transcribed samples of stage-specific RNAs were PCR-amplified with primers specific to yptV1, yptV3, yptV4, and yptV5 to determine if yptV transcription might be restricted to either cell type or to a specific stage of the life cycle . These experiments demonstrated that each of these genes is expressed throughout the entire Volvox life cycle and in both the somatic and the reproductive cells of the alga . The transcription start sites of yptV1 and yptV5 were mapped by primer extension . Expression of recombinant yptV cDNA in E . coli yielded recombinant proteins that bound GTP specifically, demonstrating a property which is typical for small G-proteins . The derived YptV polypeptide sequences were used to group them into four distinct classes of Ras-like proteins . These are the first proteins of the Ras superfamily to be identified in a green alga . We discuss the possible role of the YptV-proteins in the intracellular vesicle transport of Volvox.

Biotechniques, 1993 Sep, 15(3), 502 - 5
Directional cloning of blunt-ended PCR products; Weiner MP; A method that allows the directional cloning of blunt-ended polymerase chain reaction (PCR) fragments is described . One PCR primer must be 5' phosphorylated . Extra bases are not required on either PCR primer . A linearized vector is enzymatically processed to contain a single 5'-terminal phosphate . The monophosphorylated vector is amenable to recombinant-insertion during ligation when the fragment is in the correct orientation . Increased recombinant yield results from incubating the monophosphorylated vector with a restriction enzyme (SrfI) that relinearizes nonrecombinant plasmids during the ligation reaction.

Biotechniques, 1993 Sep, 15(3), 498 - 501
PCR-assisted large insertion/deletion mutagenesis; Tessier DC et al.; A mutagenesis protocol is presented that allows the exchange or simultaneous insertion/deletion (INDEL) of large fragments of DNA . The technique uses long single-stranded DNA molecules synthesized by asymmetric PCR . Insertion of large DNA fragments (> 300 bp) was accomplished using these single-stranded DNAs without the need for restriction sites or for subcloning . We have used the method to exchange large DNA fragments in the study of DNA regulatory regions and protein domain function.

Biotechniques, 1993 Sep, 15(3), 448 - 50, 452
A simple two-step method for efficient blunt-end ligation of DNA fragments; Damak S et al.; The formation of recombinant plasmids results from ligation between one end of the linearized vector and one end of the insert (favored by high DNA concentration), followed by self-ligation of the newly created hybrid molecule (favored by low DNA concentration) . Standard protocols recommend an average DNA concentration at which both events may occur . Since this DNA concentration is not optimum for both ligation events, efficient blunt-end ligation is compromised . We describe a method for blunt-end ligation starting at a high DNA concentration for 1 h then at 1/20 the initial DNA concentration overnight . The number of recombinant plasmids obtained with this method is about 10-fold higher than with standard protocols . Restriction digestion and agarose gel electrophoresis of 10 recombinant plasmids obtained with the two-step ligation method showed that all plasmids contained one copy of the insert.

Appl Environ Microbiol, 1993 Sep, 59(9), 3134 - 7
Gene cloning, sequencing, and biochemical characterization of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI; Lee YE et al.; The gene encoding endoxylanase (xynA) from Thermoanaerobacterium saccharolyticum B6A-RI was cloned and expressed in Escherichia coli . A putative 33-amino-acid signal peptide, which corresponded to the N-terminal amino acids, was encoded by xynA . An open reading frame of 3,471 bp, corresponding to 1,157 amino acid residues, was found, giving the xynA gene product a molecular mass of 130 kDa . xynA from T . saccharolyticum B6A-RI had strong similarity to genes from family F beta-glycanases . The temperature and pH optimum for the activity of the cloned endoxylanase were 70 degrees C and 5.5, respectively . The cloned endoxylanase A was stable at 75 degrees C for 60 min and displayed a specific activity of 227.4 U/mg of protein on oat spelt xylan . The cloned xylanase was an endo-acting enzyme.

Am J Physiol, 1993 Sep, 265(3 Pt 2), R653 - 8
Kinetics of systemic and intrahypothalamic IL-6 and tumor necrosis factor during endotoxin fever in guinea pigs; Roth J et al.; The time course of activity of interleukin-6 (IL-6) and tumor necrosis factor (TNF) was measured in blood plasma and hypothalamic push-pull perfusates during the febrile response to intramuscular injection of bacterial endotoxin (Escherichia coli, 20 micrograms/kg) in 24 guinea pigs . Injection of endotoxin caused a dramatic increase of IL-6 activity in plasma . The logarithmic values of plasma IL-6 activities showed a linear correlation to the febrile change in body temperature (r = 0.898) during the whole time course of fever . IL-6 activity in hypothalamic perfusates increased 12-fold in the first hour after pyrogen application and declined slowly despite the further increase in body temperature . Hypothalamic IL-6 activity did not correlate with the febrile increase in body temperature (r = -0.048) . TNF activity in plasma, not detectable before pyrogen application, had its peak in the first hour after endotoxin injection and rapidly declined to 15-20% of the peak activity within the next 2 h and to an undetectable value 5 h after injection . In the hypothalamus TNF was not detectable before endotoxin injection, but it could be monitored in most animals after pyrogen application without a clear correlation to the fever response . These results taken together indicate that endotoxin fever represents a physiological situation in which production and release of cytokines in the peripheral immune system and in the hypothalamus are regulated and stimulated in independent patterns.

Am J Physiol, 1993 Sep, 265(3 Pt 1), C851 - 6
Imaging of reconstituted biological channels at molecular resolution by atomic force microscopy; Lal R et al.; Using atomic force microscopy (AFM), we obtained high-resolution surface images of the bacterial outer membrane channels Escherichia coli OmpF porin and Bordetella pertussis porin that were reconstituted in artificial bilayer membranes as two-dimensional crystalline arrays . These porins were chosen because they are among the most extensively studied proteins of this type and are known for their well-defined crystalline nature in the native membrane . Such reconstituted membrane proteins are ideal specimens to assess the suitability and resolution of AFM for imaging biomembranes and associated proteins . Although OmpF porin often showed a mixed pattern of rectangular and hexagonal arrays with approximately 8.4 x 9.8- and approximately 7.2-nm-spacings, respectively, B . pertussis porin showed mostly a rectangular pattern with an approximately 7.9 x 13.8-nm spacing . The packing patterns of the E . coli OmpF porin in the membrane are very close to those found in electron-microscopic studies . When B . pertussis porin was imaged in a buffer solution, its trimeric subunits were apparently resolved, and the surface of each monomer revealed beadlike structures . This is the first report of such a high-resolution structural analysis of B . pertussis porin by any imaging method . We also imaged the lipid bilayer itself as an internal control for imaging and to further ascertain the resolution . Individual polar head groups of bilayer lipid molecules were resolved, suggesting the intrinsic resolution of AFM for bioimaging.

Hinyokika Kiyo, 1993 Sep, 39(9), 837 - 9
{A case of infected renal cyst: the usefulness of magnetic resonance imaging for preoperative diagnosis}; Takashima M et al.; A 22-year-old woman was admitted to our hospital with complaints of fever and left flank and back pain . Laboratory examinations showed leukocytosis and high C-reactive protein level . On the upper pole of the left kidney, two renal cysts were disclosed by ultrasonography and computerized tomography . A simple renal cyst and an infected renal cyst could be distinguished by magnetic resonance images (MRI), because the infected renal cyst was less intense than the simple renal cyst on T2 weighted MRI . Percutaneous puncture and drainage of the cyst were performed under the guidance of ultrasonography . Bacterial culture of the infected cyst fluid was positive for E . coli . Two months after treatment, computerized tomography showed no evidence of recurrence of the infected renal cyst.

Southeast Asian J Trop Med Public Health, 1993 Sep, 24(3), 420 - 4
Enterotoxigenic Escherichia coli and other causes of infectious pediatric diarrheas in Jakarta, Indonesia; Subekti D et al.; A hospital stool survey of Indonesian children less than 5 years of age determined the prevalence of diarrhea caused by enterotoxigenic Escherichia coli (ETEC) and other bacterial enteropathogens, compared to non-diarrheic control patients . ETEC were the second most frequent cause of diarrhea, isolated from 16 of 194 (8.2%) of patient's stools compared to 2 of 97 (2.1%) of control stools . The highest prevalence was in infants 12 to 23 months of age (17.9%).

Cytokine, 1993 Sep, 5(5), 506 - 11
Recombinant expression of rat and human Gro proteins in Escherichia coli; Konishi K et al.; A full-length rat gro cDNA containing the signal sequence was inserted to a plasmid/phage vector pTD-lacs which had the Escherichia coli alkaline phosphatase leader sequence down-stream of the lac promoter . After removal of the gro signal sequence by site-directed mutagenesis, the vector was introduced to E . coli JM109 . The cells grown in the presence of isopropyl beta-D-thiogalactopyranoside were found to contain the recombinant mature rat Gro protein in the periplasmic space . The protein was released from the cells by osmotic shock, and could be purified to homogeneity from the periplasmic fluid by a single-step procedure using reverse phase high performance liquid chromatography . By similar procedures, recombinant human Gro alpha could be obtained . In each case, about 10 mg of purified cytokine were obtained from 1 litre of bacterial culture.

Cytokine, 1993 Sep, 5(5), 498 - 505
Effects of recombinant human cytokines on mitogen-induced bovine peripheral blood mononuclear cell proliferation; Olsen SC et al.; The effects of interleukin 1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and insulin growth factor-1 (IGF-1) on proliferation of mitogen-stimulated bovine peripheral blood mononuclear cells (PBMC) were determined . Four concentrations of each cytokine were incubated 48 or 96 h with PBMC alone or with PBMC and three concentrations of three different mitogens . The mitogens included concanavalin A (Con A) phytohemagglutinin (PHA), and Escherichia coli 055:B5 lipopolysaccharide (LPS) . Proliferation of unstimulated PBMC was not affected by IL-1 alpha, IL-1 beta, IL-3, IL-5, IL-6, IL-8, and IGF-1 whereas IL-2, IL-4, and IL-7 increased proliferation . Incubation of PBMC with Con A plus IL-1 alpha, IL-2, IL-4, IL-7 or IL-8 increased proliferation when compared to PBMC that were incubated with either Con A or the cytokine alone . Interleukin-1 beta, IL-3, IL-5, IL-6 and IGF-1 did not influence proliferation of Con A-stimulated PBMC . Proliferation of PHA-stimulated PBMC was increased when PBMC were cultured with IL-2, IL-4 or IL-7, but not when cultured with IL-1 alpha, IL-1 beta, IL-3, IL-5, IL-6, IL-8 or IGF-1 . Similar results occurred with LPS-stimulated PBMC in that proliferation induced by LPS was enhanced by IL-2 or IL-7, but not by any of the other cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)

Vojnosanit Pregl, 1993 Sep-Oct, 50(5), 468 - 71
{An epidemic of acute enterocolitis caused by enterotoxigenic Escherichia coli}; Cobeljic M et al.; In an epidemic of enterocolitis on a military ship which lasted two days, 13 (54%) out of 24 soldiers became ill . The clinical picture was mild with predomination of diarrhoea and abdominal pain, and the troubles in all ill persons ended within 48 hours with administration of symptomatic therapy . Since routinely examined causative agents of infectious diseases were not detected, detailed examination of 13 Escherichia coli strains isolated from stools of the ill persons as well as 8 strains from stools of healthy but exposed soldiers, revealed that 7 of them produced enterotoxins; 5 strains produced heat-labile (LT) and heat-stabile (ST) enterotoxins, and 2 strains ST only . Enterotoxigenic strains were isolated from stools of 5 (38.5%) ill persons and 2 (25%) healthy persons . According to epidemiological investigations, infection was transmitted by secondary contaminated food due to its mishandling . The presented results prove that enterotoxigenic E . coli play a role as a causative agent in epidemics of acute enterocolitis in adults.

Biull Eksp Biol Med, 1993 Sep, 116(9), 249 - 53
{The role of the leukocytes in the mast cell reaction in an inflammatory focus}; Klimenko NA; Models of acute infectious and aseptic peritonitis in rats with vinblastine-induced leukopenia have shown the regulatory influence of leukocytes on mast cells consisting in antimicrobic and activating effects . While infectious inflammation is characterized by increased mast cell degranulation both before and after marked leukocyte accumulation in the inflammatory focus and blood . Aseptic inflammation is followed by retardation of mast cell reaction to the significant leukocyte influx into inflammatory focus and blood and increased mast cell degranulation after leukocyte influx.

Biull Eksp Biol Med, 1993 Sep, 116(9), 244 - 6
{The production by bone marrow cells of humoral factors in extreme exposures of different origins}; Gol'dberg ED et al.; The intensification of cytokine-producing activity by mouse bone marrow cells forming the hemopoiesis-inducing microenvironment (HIM) was demonstrated to be an obligatory component of adaptation processes developing in the body under the influence of different extreme factors (immobilization, inflammation, cytostatics, irradiation) . As a rule, the first stress reaction of adhesive and nonadhesive cells consists in interleukin-1 and interleukin-3 secretion with following erythropoietic and colony-stimulating activity induction . For all this, the levels and periods of production of hemopoiesis humoral regulators are comparable under hemopoiesis-stimulating or -suppressing influences . Finally Activation of HIM functional properties expresses the interaction of central and local regulating systems, which favours the formation of an optimal response of the blood system to stress.

Eur Cytokine Netw, 1993 Sep-Oct, 4(5), 343 - 9
Characterisation of monoclonal antibodies to human IL-4: application in an IL-4 ELISA and differential inhibition of IL-4 bioactivity on B cells and T cells; van der Pouw Kraan T et al.; Five murine monoclonal antibodies, raised against E . coli derived human IL-4, were established . All mAb were also reactive with natural IL-4 . Competition ELISA experiments revealed that mAb 1,2 and 4 recognized a related epitope on IL-4 . mAb 5 and mAb 6 recognized another epitope . Two non-competing mAb were used to develop a sandwich IL-4 ELISA . mAb 5 was used for coating and biotinylated mAb I was used as the second antibody . Intra- and interassay-coefficients were 3.3 and 10.1% respectively . The ELISA is specific for IL-4, rapid and sensitive (the detection limit is 2 pg/ml) . The capacities of the antibodies to inhibit IL-4 activity were tested in B cell and T cell assays . All antibodies inhibited IL-4 dependent IgE production by human B lymphocytes . A similar inhibition of IL-4 driven T cell proliferation by the antibodies was observed, with the exception that mAb 4 did not affect the activity of IL-4 on T cells . These results led to the suggestion that B cells make use of another (additional) IL-4 receptor chain.

In Vivo, 1993 Sep-Oct, 7(5), 431 - 4
Effects of tricyclic compounds on membrane binding of bivalent cations, activities of acetylcholinesterase and some tissue proteases; Molnar J et al.; A tricyclic compound tetrahydroaminoacridine is known to improve the cognitive function in Alzheimer's disease . The possible mechanism of action of acridine and structurally related tricyclic compounds was studied on the bivalent cation content of bacterial membrane, rat brain acetylcholinesterase and some tissue proteases in model experiments . Acridine orange and disubstituted chlorpromazine (CPZ) derivatives lowered Ca2+ and Mg2+ binding and membrane polarization in the simplest biological membrane (E . coli), as revealed by reactor neutron activation analysis . Acetylcholinesterase (AChE) was inhibited by CPZ, 3,7,8-trihydroxy-CPZ, acridine orange partially saturated desipramine, imipramine, trans-clopenthixol and tetrahydrocannabidiolic at 10(-4) to 10(-5) . A metalloproteinase, MMP-7-ase, was inhibited by tetrahydrocannabidiolic acid, 3,7,8-trihydroxy-CPZ, acridine orange but other tissue proteinases, ATN-ase and cathepsin B, were less sensitive to these compounds . (ATN-ase is an acetyltyrosine-p-nitroanilide splitting enzyme, a serine protease) . The chelate complex forming ability and electron donor capacity of the compounds may play a role in the biological effects tested . It is assumed that compounds which do not displace bivalent cations in membranes may exert an inhibitory effect on AChE, and that metalloproteinase enzymes may be promising for the treatment of degenerative brain diseases.

Int J Radiat Biol, 1993 Sep, 64(3), 311 - 8
On the cytotoxicity of irradiated media . To what extent are stable products of radial chain reactions in physiological saline responsible for cell death?
Saran M, Bertram H, Bors W, Czapski G.
In a previous publication (Czapski et al . 1992) we reported that HOCl accounts for the toxicity of irradiated phosphate-buffered saline towards Escherichia coli bacterial cells . We have now investigated the respective toxicities towards lambda phage and mammalian cells . For phage, as with bacteria, cytotoxicity of the irradiated media seems to derive from HOCl without detectable contribution of H2O2 . Mammalian cells (V79 CHO), in contrast, are more sensitive to H2O2 than to HOCl . Both agents, however, are not able to account quantitatively for the toxicity of irradiated solutions towards V79 cells; a hitherto unidentified chlorine/oxygen derivative--being formed in the sub-micromolar concentration range--is suggested to be responsible for toxicity in the case of eukaryotes.

Circ Shock, 1993 Sep, 41(1), 26 - 34
Hemodynamic and regional blood flow responses of neonatal and adult dogs to endotoxin challenge; Horton JW; Our recent studies showed developmental differences in the functional responses of the myocardium to isoproterenol and exogenous calcium, suggesting that decreased cardiovascular reserves in the immature heart contribute to the greater morbidity/mortality reported in neonatal compared to adult endotoxin shock . In this study, hemodynamic, hormonal and regional blood flow responses to Escherichia coli endotoxin challenge (1 mg/kg, 055:35 B lipopolysaccharide) were compared in adult (N = 12) and neonatal (19-21 days of age, N = 38) dogs . Compared to neonates, adult dogs had higher baseline mean arterial pressures (78 +/- 3 vs . 127 +/- 7 mmHg, P = 0.01), while values were lower for cardiac output (635 +/- 30 vs . 130 +/- 7 ml/kg, P = 0.01), left ventricular +dP/dt max (4,500 +/- 200 vs . 2,700 +/- 190 mmHg/sec, P = 0.02), heart rate (138 +/- 3 vs . 86 +/- 6 beats/min, P = 0.02), coronary flow (3.46 +/- 0.18 vs . 1.19 +/- 0.15 ml/min/g, P = 0.01), serum glucose (136 +/- 6 vs . 113 +/- 4 mg%, P = 0.01), insulin (20 +/- 3 vs . 15 +/- 2 mU/ml, P = 0.05), and glucagon (518 +/- 98 vs . 172 +/- 27 pg/ml, P = 0.01) . Endotoxin caused progressive cardiocirculatory dysfunction and metabolic acidosis, regardless of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Sci, 1993 Sep, 2(9), 1452 - 60
Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase; Daubner SC et al.; Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases . Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not . This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains . To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized . One protein contains the first 158 amino acids of rat tyrosine hydroxylase . The second lacks the first 155 amino acid residues of this enzyme . The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase . The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity . The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids . The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.

Plant Mol Biol, 1993 Sep, 22(6), 1087 - 100
A modified Escherichia coli chaperonin (groEL) polypeptide synthesized in tobacco and targeted to the chloroplasts; Wu HB et al.; The coding region for the Escherichia coli groEL (chaperonin-60) polypeptide was fused downstream of a pea rubisco small subunit transit peptide coding sequence under the control of a tandem 35S CaMV promoter . Transgenic tobacco plants (Nicotiana tabacum cv . Xanthi) containing this modified groEL gene were produced . The modified groEL polypeptide was correctly imported into chloroplasts and accumulated to high or low levels in different plants . The majority of the modified groEL polypeptide was processed correctly to the mature form within the chloroplasts . Approximately 20% of the imported polypeptides retained a portion of the N-terminal transit peptide (TPgroEL) . Both groEL and TPgroEL polypeptides assembled into tetradecameric species in the chloroplasts . In plants accumulating high levels of these products, the majority of the plant chaperonin-60 polypeptides in the chloroplast were present in novel hybrid tetradecameric species containing both bacterial and plant chaperonin-60 polypeptides . In plants accumulating low levels of groEL, the predominant species present appeared to be authentic plant cpn60(14) and authentic bacterial groEL14 . The growth and development of transgenic and control tobacco plants were indistinguishable.

J Clin Invest, 1993 Sep, 92(3), 1168 - 73
CD18-independent neutrophil and mononuclear leukocyte emigration into the peritoneum of rabbits; Winn RK et al.; The CD18 mAb 60.3 and the CD49d mAb HP1/2 were given at the time of intraperitoneal instillation of either protease peptone or live Escherichia coli bacteria and at 12 h . Leukocyte emigration was evaluated at 4 and 24 h . PMN emigration 4 h after protease peptone instillation and injection of both mAbs was 10% of that in saline treatment . It was 15% of that in saline treatment after mAb 60.3 alone and unchanged by mAb HP1/2 . At 24 h PMN emigration in response to protease peptone was not prevented by either CD18 or CD49d mAbs, however, when given together emigration was 10% of saline-treated animals . Mononuclear cell emigration to protease peptone was enhanced at 4 h by both CD18 and CD49d mAbs . The CD18 mAb did not augment mononuclear emigration in response to live bacteria . At 24 h, neither the CD18 nor the CD49d mAb alone blocked emigration of mononuclear cells, but the combination of the two did . These studies demonstrate that: (a) early (4 h) PMN emigration is CD11/CD18 dependent; (b) late (24 h) PMN emigration is CD11/CD18 independent; and (c) mononuclear cells utilize the integrins CD18 and CD49d.

J Bacteriol, 1993 Sep, 175(18), 6038 - 40
Escherichia coli K-12 can utilize an exogenous gamma-glutamyl peptide as an amino acid source, for which gamma-glutamyltranspeptidase is essential; Suzuki H et al.; Escherichia coli K-12 can utilize a gamma-glutamyl peptide as an amino acid source, for which gamma-glutamyltranspeptidase (EC 2.3.2.2) is essential . We suggest that the gamma-glutamyl linkage of a gamma-glutamyl peptide is hydrolyzed by gamma-glutamyltranspeptidase located in the periplasmic space, and the released amino acid is taken up and utilized by E . coli.

Infect Immun, 1993 Sep, 61(9), 3673 - 7
The K88 fimbrial adhesin of enterotoxigenic Escherichia coli binds to beta 1-linked galactosyl residues in glycosphingolipids; Payne D et al.; The K88 fimbrial adhesin enables certain strains of enterotoxigenic Escherichia coli to adhere to the porcine small intestine . In this study, the ability of the K88 adhesin to bind to glycosphingolipids was monitored by modified enzyme-linked immunosorbent assay and thin-layer chromatography overlay binding analysis . The binding of the K88 adhesin to glycosphingolipid-coated microtiter plates was saturable, with 50% maximal binding occurring with gangliotriaosylceramide, gangliotetraosylceramide, and lactosylceramide at 67 +/- 21, 117 +/- 21, and 73 +/- 22 pM, respectively . Thin-layer chromatography overlay binding analysis demonstrated that serotype O8:K87:K88ab:H19 E . coli bound to hydroxylated galactosylceramide, gangliotriaocylceramide, gangliotetraosylceramide, and lactosylceramide but not to globotriaosylceramide, nonhydroxylated galactosylceramide, glucosides, glucosylceramide, or a mixture of ceramides . The K88 adhesin did not bind by either assay to globoside, the Forssman glycolipid, GM1, GM2, GM3, GD1a, GD2, GD3, GQ1b, or GT1b . The binding pattern observed with the K88 adhesin suggests that beta 1-linked galactosyl residues are the minimum determinant required for binding, provided they are presented correctly . It is suggested that beta 1-linked galactose residues may form the molecular basis of both glycoprotein and glycolipid receptors for the K88 fimbrial adhesin in the porcine small intestine.

Mol Microbiol, 1993 Sep, 9(6), 1255 - 65
Regulation and sequence of the structural gene for cytochrome c552 from Escherichia coli: not a hexahaem but a 50 kDa tetrahaem nitrite reductase; Darwin A et al.; The structural gene, nrfA, for cytochrome c552, which is the terminal reductase of the formate-dependent pathway for nitrite reduction to ammonia, has been located at co-ordinate 4366 on the physical map of the Escherichia coli chromosome . The DNA sequence of nrfA encodes a tetrahaem c-type cytochrome with a predicted M(r) for the unprocessed product of 53,788 . Cleavage of the putative signal peptide at Ala-26 would result in a mature, periplasmic cytochrome of M(r) 50,580 rather than a larger hexahaem cytochrome, as has been widely reported previously . A cytochrome of this size was detected by staining SDS-polyacrylamide gels for covalently bound haem . This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome c552 by difference spectroscopy . Similar cytochromes were detected in five other E . coli strains including strain ST 249, which was used previously to purify and characterize the protein . A plasmid with an in-phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass . In-phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping . Expression from the FNR-dependent nrfA promoter was almost totally repressed during aerobic growth, partially induced during anaerobic growth in the absence of nitrite or in the presence of nitrate, but fully induced only during anaerobic growth in the presence of nitrite . No nitrate repression was detected in a narL mutant, but nitrite induction was unaffected, indicating that the nitrite-sensing mechanism is independent of the NarL protein . Expression from the nrfA promoter was subject to glucose repression but regulation was independent of the CRP-cAMP complex.

Mol Microbiol, 1993 Sep, 9(6), 1193 - 201
Rubisco but not Rubisco activase is clustered in the carboxysomes of the cyanobacterium Synechococcus sp . PCC 7942: Mud-induced carboxysomeless mutants; Friedberg D et al.; The Mud technology of Groisman and Casadaban was adapted to the cyanobacterium Synechococcus sp . PCC 7942 . A new high-CO2-requiring (hcr) mutant, hcr Mu28 was isolated following the integration of the Mud element 89 bp upstream of ORFI, at the 5'-flanking region of the rbc operon, which encodes RuBP carboxylase/oxygenase (Rubisco) . The integration involved a 7 bp duplication that formed a direct repeat at the integration site, as previously shown in Escherichia coli . The mutant was devoid of apparent carboxysome bodies, which are considered to be important for the availability of CO2 for Rubisco . Immunolabelling studies demonstrated that Rubisco was distributed throughout hcr Mu28 cells, while in the wild type (WT) and in the carboxysome aberrant mutant hcr O221, Rubisco was markedly associated with the carboxysomes . Rubisco activase, however, was evenly distributed throughout the cytosol of the hcr and WT cells, without any preferential association with the apparent carboxysomes.

Mol Microbiol, 1993 Sep, 9(5), 999 - 1009
Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxy-proximal secretion signals by the Escherichia coli alpha-haemolysin translocator; Sebo P et al.; The 1706-residue adenylate cyclase toxin (CyaA) of Bordetella pertussis is an RTX protein with extensive carboxy-proximal glycine and aspartate-rich repeats . CyaA does not have a cleavable amino-terminal signal peptide and can be secreted across both bacterial membranes of the Escherichia coli cell envelope by the alpha-haemolysin (HlyA) translocator (HlyBD/TolC) . We performed deletion mapping of secretion signals recognized in CyaA by this heterologous translocator . Truncated proteins with N-terminal and internal deletions were secreted at levels up to 10 times higher than intact CyaA and similar to HlyA . A secretion signal recognized by HlyBD/TolC was found within the last 74 residues of CyaA . However, secretion of CyaA was reduced but not abolished upon deletion of the last 75 or 217 residues, indicating that at least two additional secretion signals recognized by HlyBD/TolC are within CyaA . One of them was localized to the repeat sequence between residues Asp-1587 to Ile-1631 . Interestingly, a conserved 'acidic' motif (Glu/Asp)-(X)11-Asp-(X)3/5-(Glu/Asp)-(X)14-Asp was found in the C-terminal sequences of HlyA, CyaA and the two secreted CyaA derivatives . We speculate that the presence and spacing of acidic residues may be an important feature of secretion signals recognized by the haemolysin translocator.

Mol Microbiol, 1993 Sep, 9(5), 979 - 88
Targeted interruption of the psaA and psaB genes encoding the reaction-centre proteins of photosystem I in the filamentous cyanobacterium Anabaena variabilis ATCC 29413; Nyhus KJ et al.; The two reaction-centre proteins of the photosystem I (PSI) complex are encoded by two adjacent genes named psaA and psaB . We have performed targeted mutagenesis to insertionally inactivate each of these genes in the filamentous cyanobacterium Anabaena variabilis ATCC 29413 . The resulting mutant strains, termed psaA::NmR and psaB::NmR, were blue because of a high ratio of phycobilin to chlorophyll and were unable to grow in light . These mutant cells also lacked chemically reducible P700 (the reaction-centre chlorophylls of PSI) and as a consequence did not exhibit any PSI-mediated photochemical activity . However, their photosystem II (PSII) complexes were fully active . The loss of the PsaA and PsaB proteins and their associated chlorophyll molecules resulted in a five- to sevenfold decrease in the chlorophyll/PSII ratio in the mutant cells relative to the wild-type cells . Interestingly, the psaB::NmR and not the psaA::NmR mutant strain retained a small fluorescence peak (77K) at 721 nm originating from chlorophyll molecule(s) presumably bound to a small amount of the PsaA protein present in the psaB mutant . These results demonstrate that this organism is suitable for the manipulation of PSI reaction-centre proteins.

Mol Microbiol, 1993 Sep, 9(5), 1061 - 9
The role of the lipoprotein sorting signal (aspartate +2) in pullulanase secretion; Poquet I et al.; The analyses of hybrid proteins and of deletion and insertion mutations reveal that the only amino acid at the amino-proximal end of the cell surface lipoprotein pullulanase that is specifically required for its extracellular secretion is an aspartate at position +2, immediately after the fatty acylated amino-terminal cysteine . To see whether the requirement for this amino acid is related to its proposed role as a cytoplasmic membrane lipoprotein sorting signal, we used sucrose gradient floatation analysis to determine the subcellular location of pullulanase variants (with or without the aspartate residue) that accumulated in cells lacking the pullulanase-specific secretion genes . A non-secretable pullulanase variant with a serine at position +2 cofractionated mainly with the major peak of outer membrane porin . In contrast, most (55%) of a pullulanase variant with an aspartate at position +2 cofractionated with slightly lighter fractions that contained small proportions of both outer membrane porin and the cytoplasmic membrane marker NADH oxidase . Only 5% of this pullulanase variant cofractionated with the major NADH oxidase peak, while the rest (c . 40%) remained at the bottom of the gradient in fractions totally devoid of porin and NADH oxidase . When analysed by sedimentation through sucrose gradients, however, a large proportion of this variant was recovered from fractions near the top of the gradient that also contained the major NADH oxidase peak . When this peak fraction was applied to a floatation gradient the pullulanase activity remained at the bottom while the NADH oxidase floated to the top.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1993 Sep, 9(5), 1051 - 9
Regulation of mobilization of the broad-host-range plasmid pTF-FC2; Rohrer J et al.; The mobilization region of the broad-host-range plasmid pTF-FC2 consists of an oriT and five genes arranged in two operons that are divergently transcribed from the oriT . The transcriptional starts of both operons were identified and the quantity of transcript from the mobC-mobE promoter (P1) was at least 10-fold greater than that from the mobA-mobB promoter (P2) . A translational fusion between the first protein of each operon and a lacZ reporter gene was constructed and used to demonstrate that mob gene expression was autoregulated . Analysis of the oriT resulted in the detection of a putative integration host factor (IHF)-binding site and an intrinsically bent region . In the absence of IHF, the mobilization frequency and expression from P1 were reduced . The presence of ssi sites on both strands within the oriT region was demonstrated by using an M13 phage mutant, defective in its mechanism for priming DNA replication . Initiation of DNA synthesis at the oriT did not require a plasmid-encoded primase.

Afr J Med Med Sci, 1993 Sep, 22(3), 13 - 5
Cloning and restriction mapping of the L-sorbose utilization genes from a clinical isolate of Escherichia coli (1); Olukoya DK; About 30% of clinical isolates of Escherichia coli tested utilized L-sorbose as a carbon and energy source . Escherichia coli K-12 is naturally sorbose negative . The genes for L-sorbose utilization (sor+) is being used as a prototype for studying variable genes amongst bacterial pathogens . The sor+ genes from seven isolates were transferable to E . coli K-12 . The (sor+) region was cloned into plasmid pBR322 to give pDOK1 . Plasmid pDOK1 is approximately 20kb in size . A restriction endonuclease map of pDOK1 is presented.

Biosci Biotechnol Biochem, 1993 Sep, 57(9), 1536 - 40
Mutational analysis of the amino acid residues essential for the cis and trans cleavage activity of the potato virus Y 50-kDa protease; Yoshida Y et al.; To examine the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP) . For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction . The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E . coli . By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease . Introduction of His234-->Tyr, Asp269-->Asn, or Cys339-->Gly substitution in the putative catalytic triad of the protease abolished its activity . However, the mutated protease with Cys339-->Ser replacement retained a reduced proteolytic activity.

Biotechnol Prog, 1993 Sep-Oct, 9(5), 548 - 54
Effects of the par locus on the growth rate and structural stability of recombinant cells; Kim JY et al.; A decreased growth rate of recombinant cells is observed when a cloned gene protein encoded in a multicopy plasmid is induced from a strong promoter . This negative effect on the cell growth rate may be the consequence of alternate use or reallocation of energy, precursor metabolites, and protein synthesizing machinery . In order to analyze the causes for this adverse effect, we have studied how genetic elements present in multicopy plasmids affect the growth of recombinant Escherichia coli (K12 delta H1 delta trpEA) . Turning on of the PL promoter, whether or not protein-coding sequences were present downstream of the promoter, decreased the growth of the recombinant cells by 15-50% . This result suggests that the essential factor(s) for cell growth may be sequestered by the genetic elements present in the plasmids and thus may cause the decreased growth . The negative effect of the de-repressed PL promoters on cell growth was partially reversed when the par sequence from pSC101 was inserted into the same plasmid . In each case when the plasmid carried only the PL promoter, the PL promoter followed by the N-terminal part of the protein-coding region, or the PL promoter followed by complete sequences encoding a protein, the par sequence exhibited a positive effect on the growth rate of the recombinant cells, which usually grow at a slower rate than the host cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnol Prog, 1993 Sep-Oct, 9(5), 443 - 9
Specialized ribosomes in Escherichia coli; Leipold RJ et al.; Specialized ribosomes, developed by de Boer and co-workers at Genentech, carry a mutation in the anti-Shine-Dalgarno region of 16 S ribosomal RNA, the site of messenger RNA binding . A complementary mutation in the ribosome binding site (the Shine-Dalgarno region) of a particular messenger RNA results in specific and efficient translation of that messenger RNA on the specialized ribosomes . With this system, a fraction of the cell's ribosomes can be dedicated to the translation of a single messenger RNA; the remaining wild-type ribosomes carry out the normal cellular translation processes . Specialized ribosomes have been used for the overproduction of proteins, analysis of the feedback regulation of ribosomal RNA synthesis, and mutational analysis of 16 S ribosomal RNA . Experimental results related to each of these topics are summarized and discussed, and other potential applications are described . In particular, we propose a novel technique for mutational analysis of 23 S ribosomal RNA, the primary RNA constituent of the large ribosomal subunit, using the specialized ribosome approach.

Lett Appl Microbiol, 1993 Sep, 17(3), 139 - 43
Adaptation of Escherichia coli growth rates to the presence of pBR322; McDermott PJ et al.; Changes in the growth rate of Escherichia coli K12 J62-1 in response to the presence of plasmid pBR322 have been investigated . Plasmid-free and plasmid-containing strains were grown in batch culture and their maximum specific growth rate (mu max) determined . The acquisition of pBR322 by the host resulted in a decreased mu max . Following repeated subculturing of the plasmid-containing strain on selective medium, restoration in mu max was observed . The copy number and structure of the plasmid were not significantly altered during the experiment . Growth rate measurements for a series of strains constructed using a combination of host cells and plasmids with and without culture histories, indicated that the site of the adaptive mutation was located on the host chromosome rather than on the plasmid.

APMIS, 1993 Sep, 101(9), 695 - 702
Rapid detection and characterization of sialic acid-specific lectins of Helicobacter pylori; Lelwala-Guruge J et al.; A particle agglutination assay (PAA) using fetuin (Ft) covalently coupled to carboxylate-modified latex (CML) particles was evaluated for rapid detection of sialic acid-specific haemagglutinins/lectins (SALs) of Helicobacter pylori isolates which bind sialoglycoconjugates . Sixty-three percent (20/32) of the isolates examined gave a positive PAA test . Cell-bound SALs were extracted by washing the bacteria with deionized water or isotonic saline, and their expression was influenced by pH and culture conditions . The Ft-CML reactivity of the PAA-positive isolates was inhibited by bovine submaxillary mucin, transferrin, fetuin, orosomucoid, vitronectin and lactoferrin in a manner which suggested that the isolates contain a lectin recognizing the alpha(2-6) linkage of terminal sialic acid . Western blots of strain NCTC 11637 SALs probed with horseradish peroxidase (HRP)-labelled Ft identified three bands (MW 64 kD, 62 kD, 56 kD) which also reacted with HRP-labelled mucin, transferrin, lactoferrin, orosomucoid, vitronectin and laminin . Sera from patients with a H . pylori infection and one polyclonal rabbit antiserum (strain NCTC 11637) also reacted with the SALs . Immunogold labelling of a polyclonal rabbit antiserum raised against the 64 kD protein of strain NCTC 11637 that reacted strongly with Ft-CML showed that abundant SALs were loosely cell-associated with the cell surface of both spiral and coccoidal forms of H . pylori . SALs were also present in low amounts on the surface of strain NCTC 11638 and 66, a clinical isolate that did not react with Ft-CML.

J Virol Methods, 1993 Sep, 44(1), 117 - 127
In vitro detection of bovine immunodeficiency-like virus using monoclonal antibodies generated to a recombinant gag fusion protein; Wannemuehler Y et al.; An Escherichia coli recombinant fusion protein containing the major core protein of bovine immunodeficiency-like virus (BIV) was used to immunize mice for generation of monoclonal antibodies to BIV p26 . Eight hybridomas specific for BIV p26 were identified and two antibodies, designated 104 and 142, were further characterized . Both 104 and 142 antibodies were isotyped as IgG1; they reacted specifically with both BIV p26 and the recombinant fusion protein in Western immunoblot analyses . However, the epitope specificity of the antibodies was different . Immunoperoxidase assays were used to determine if antibodies 104 and/or 142 could detect BIV replication in cell culture . Both antibodies were found to react with BIV-induced syncytia and individual BIV-infected cells . The antibodies were also used successfully in a focal immunoassay for quantitation of BIV-infected cells . These antibodies will provide valuable reagents for detection and quantitation of BIV replication in studies of viral pathogenesis and immunity.

Radiat Res, 1993 Sep, 135(3), 405 - 10
Reduction of radiation cytotoxicity by human apurinic endonuclease in a radiation-sensitive Escherichia coli mutant; Chen DS et al.; Ionizing radiation produces a variety of DNA damage through active oxygen species such as the superoxide radical (O2.-), the hydroxyl radical (OH.), and hydrogen peroxide (H2O2) . The removal of alkylation-induced apurinic (AP) sites and 3'-blocking deoxyribose fragments by exonuclease III (xth) and endonuclease IV (nfo) has been well demonstrated in E . coli . Very little information on the repair of radiation-induced DNA damage by human apurinic endonuclease is available . We examined the biological roles of the human AP endonuclease in the repair of radiation-induced DNA damage . An expression vector was constructed with human APE cDNA and transformed into radiation-sensitive E . coli mutants (xth- and nfo-) . The radiation cytotoxicity was assayed by cell survival curves . Expression of human AP endonuclease in E . coli confirmed that AP endonuclease could complement exonuclease III functionally to diminish radiation cytotoxicity . In contrast, AP endonuclease was not able to increase resistance to H2O2, owing to a poor 3'-termini repair . We also tested whether AP endonuclease is a limiting factor for radiation cytotoxicity by using a plasmid nicking assay . Cell extracts from mutant cells with or without AP endonuclease expression were added to irradiated supercoiled plasmid DNA . The inability to convert supercoiled plasmid DNA to relaxed or linear forms suggested that there were large accumulations of AP sites in the mutant cell extracts . The AP endonuclease activities estimated from the plasmid nicking assays are 20-fold lower in the cell extracts of AP endonuclease-deficient mutant than in AP endonuclease-expressing cells . Therefore, AP endonuclease is a limiting step of base excision repair for the radiation-sensitive E . coli mutant, BW528 . Our results conclude that AP endonuclease is responsible for the removal of AP sites from gamma-radiation-induced base damage in E . coli.

Mutat Res, 1993 Sep, 303(1), 1 - 9
Mutagenicity of 3-azido-1,2-propanediol and 9-(3-azido-2-hydroxypropyl)-adenine in repair deficient strains of Escherichia coli; Gruz P et al.; The mutagenicity of two non-aromatic organic azido compounds, 3-azido-1,2-propanediol (AG) and 9-(3-azido-2-hydroxypropyl)-adenine (AHPA), was studied in E . coli repair deficient strains uvrA6, uvrA6 + umuC36, uvrA6+ umuC122::Tn5, polA1, tagA1+ alkA1, ada and dam3 . The mutagenicity of both agents was markedly enhanced by defects of UvrABC excinuclease (uvrA6) and was independent of umuC function of the SOS error-prone pathway . Neither azido compound promoted umuDC operon expression . The mutagenicity of AG in tag A1, alkA1 and ada mutants does not differ from that found in the wild-type strain . The expression of both ada and alkA genes was not elevated by AG . Experiments on polA1 and dam3 mutants suggest that DNA polymerase I as well as the mutHLS mismatch repair pathway does not contribute to the removal of putative DNA lesions induced by AG.

J Gen Virol, 1993 Sep, 74 ( Pt 9), 1871 - 7
Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase; Lankinen H et al.; The large subunits of herpes simplex virus types 1 and 2 ribonucleotide reductases contain unique amino-terminal regions comprising 311 and 318 residues respectively, which are not found in ribonucleotide reductases from other sources . We report the mapping of the epitope recognized by monoclonal antibody 1026, which is specific for the large subunit (R1) of HSV-1, and then deduce the structural relationship of the amino-terminal region of R1 with the rest of the protein . A panel of 10 fusion proteins containing sequences spanning the entire R1 subunit were constructed . They were used together with proteolytic fragments of R1 and several synthetic peptides to show that the epitope is discontinuous and appears to be a loop structure centered on a previously located trypsin-sensitive site at residue 305 . The existence of the loop was suggested by the observation that reactivity of the antibody with R1 could be blocked by peptides corresponding to residues 289 to 303 and 308 to 313 which flank the trypsin-sensitive site . Our results suggest that the unique amino-terminal region of R1 consists of a structurally distinct domain which is linked to the conserved carboxy region by an exposed loop.

J Clin Invest, 1993 Sep, 92(3), 1181 - 7
Defect of a complement receptor 3 epitope in a patient with systemic lupus erythematosus; Witte T et al.; Complement receptor 3 (CR3) is expressed on cells of the reticuloendothelial system and involved in the clearance of immune complexes . In this article a patient with a deficiency of the C3bi binding site of this receptor is described . Clinically this patient exhibited predominantly cutaneous manifestations of a systemic lupus erythematosus with an immune vasculitis and panniculitis . The deficiency of the CR3 epitope was demonstrated using flow cytometry . The functional relevance of this defect was demonstrated in a rosetting assay with C3bi-loaded erythrocytes . C3bi binding was found to be significantly decreased . Furthermore, there was an impairment of phagocytosis of opsonized Escherichia coli . The CR3 defect is not due to an autoantibody but is assumed to have a genetic basis . These data suggest that the defect of the CR3 may be involved in the pathogenesis of the immune vasculitis in this patient.

J Bacteriol, 1993 Sep, 175(18), 5791 - 7
Limited differential mRNA inactivation in the atp (unc) operon of Escherichia coli; Lagoni OR et al.; Individual subunits of ATP synthase, encoded by the eight genes of the atp operon (atpA through atpH), have been found to be synthesized at a 10-fold range in molar amounts (D.L . Foster and R.H . Fillingame, J . Biol . Chem . 257:2009-2015, 1982; K . von Meyenburg, B.B . Jorgensen, J . Nielsen, F.G . Hansen, and O . Michelsen . Tokai J . Exp . Clin . Med . 7:23-31, 1982) . We have determined the functional half-lives at 30 degrees C of mRNAs transcribed from these genes either during constitutive expression in a partial diploid strain or after induced expression from a plasmid . Accurate decay kinetics of the relative mRNA levels were determined by monitoring the rates of synthesis of the individual ATP synthase subunits by radioactive pulse labeling at different times after blocking transcription initiation with rifampin . The mRNA transcribed from the atp operon was found to be inactivated about twice as fast as the bulk mRNA in E . coli . Exceptions are the mRNA from the promoter-proximal atpB gene, which was inactivated about three times as fast as the bulk mRNA, and atpC mRNA, the inactivation rate of which was comparable to that of the bulk mRNA . These moderate differences in the kinetics of functional decay explain only a minor part of the differences in expression levels of the atp genes . We conclude, therefore, that the individual atp mRNAs must be translated with widely different efficiencies . The present analysis further revealed that mRNA degradation is sensitive to heat shock; i.e., after incubation at 39 degrees C for 5 min followed by a shift back to 30 degrees C, the decay rate of the bulk mRNA was decreased by 30%.

Eur J Biochem, 1993 Sep 1, 216(2), 369 - 75
A modified uridine in the first position of the anticodon of a minor species of arginine tRNA, the argU gene product, from Escherichia coli; Sakamoto K et al.; The argU (dnaY) gene product, a minor tRNA(Arg), from Escherichia coli has the anticodon N*CU with an unidentified modified nucleoside N* in position 34 {Kiesewetter, S., Fisher, W . & Sprinzl, M . (1987) Nucleic Acids Res . 15, 3184} . In the present study, argU tRNA was purified from E . coli A19 strain and nucleoside N* was characterized by the TLC and HPLC analyses . Nucleoside N* was found to be different from any naturally occurring modified nucleosides . From unfractionated E . coli tRNA species, nucleoside N* was prepared in an amount sufficient for 1H-NMR experiments . By the analyses of one-dimensional and two-dimensional NMR spectra, nucleoside N* was suggested to be 5-methylaminomethyluridine (mnm5U), which was confirmed by comparison with a chemically synthesized preparation of mnm5U . Thus, the occurrence of mnm5U in mature tRNA was found for the first time . Further, the modification of U(34) to mnm5U in this tRNA was found to contribute to the strict recognition of two degenerate codons terminating in A and G.

Virology, 1993 Sep, 196(1), 70 - 8
Stability governs the apparent expression of "particulate" hepatitis B e antigen by mutant hepatitis B virus core particles; Seifer M et al.; The p21.5 capsid or core protein of hepatitis B virus carries two distinct classes of epitopes . Core (HBc) epitopes are found exclusively on the surface of the 28-nm viral icosahedral capsids or core particles, while HBe epitopes are normally expressed only by subparticulate forms of the core protein . Recent studies have suggested that a "particulate" form of HBe is expressed on the surface of capsid particles assembled from p17, a truncated core protein that lacks the carboxy-terminal protamine-like region of p21.5 and hence the ability to bind and encapsidate RNA . In this report we have used epitope-specific ELISAs in conjunction with capsids assembled from a series of carboxy-terminally truncated core proteins to address the mechanistic basis for particulate HBe . Specifically, we sought to test the idea that particulate HBe expression might be linked to the loss of RNA binding . However, our results strongly suggest that expression of HBe by mutant core particles is a result of their intrinsic instability which increases sharply when RNA binding is lost . We show that core particles assembled from mutant core proteins lacking Cys residues also express HBe, again because of capsid instability . We report mild conditions that can induce the dissociation of the mutant capsids and discuss our findings in terms of the factors that control capsid stability.

J Pediatr, 1993 Sep, 123(3), 381 - 7
Hepatitis C virus infection in children with hemophilia: characterization of antibody response to four different antigens and relationship of antibody response, viremia, and hepatic dysfunction; Kanesaki T et al.; We studied hepatitis C virus (HCV) infection in children with hemophilia by characterizing the antibody responses to four different HCV antigens and investigating the relationship of the antibody response to viremia and hepatic dysfunction . Three antigens (core, nonstructural (NS) 3, and NS5) were expressed in Escherichia coli transfected with plasmids that contained fragments of the putative core and of the NS3 and NS5 regions of the HCV genome, respectively . Antibody responses to these three antigens and the commercially available C100 antigen were detected by enzyme-linked immunosorbent assay . In 45 children with hemophilia, the percentage of children with seropositivity for C100, core, NS3, and NS5 protein in one or more specimens was 82%, 91%, 91%, and 89%, respectively . The time course of changes in the antibody response to the four antigens was determined by using sera obtained from 44 of the 45 patients at intervals of 1 to 4 years . Antibodies to the core and NS3 antigens appeared earlier and persisted longer than those to C100 and NS5 after HCV infection . The relationship of antibody response to viremia and hepatic dysfunction was investigated in 27 children by using the polymerase chain reaction assay . Five children whose tests results were negative for all four antigens did not have viremia or hepatic dysfunction; 13 of the 16 children with positive results for the four antigens had both viremia and hepatic dysfunction . Five of the six children whose serum had the core and NS3 antibodies but not either C100 or NS5, or both, had viremia, and three of them also had hepatic dysfunction . These results suggest that detection of antibodies to the core and NS3 antigens is useful for the serologic diagnosis of HCV infection and that both antibodies are more related to viremia than are the antibodies to C100 and NS5 . In addition, viremia is strongly associated with hepatic dysfunction.

J Virol, 1993 Sep, 67(9), 5426 - 34
Endonucleolytic cleavages and DNA-joining activities of the integration protein of human foamy virus; Pahl A et al.; The bacterial expression plasmids, pET3b and pET16b, that contain the integrase domain of the human foamy virus (HFV) reverse transcriptase were constructed and expressed in Escherichia coli . The histidine-tagged HFV IN protein was purified to near homogeneity by single-step Ni2+ chelate affinity chromatography . HFV-specific proteins of 39 and 120 kDa from virus-infected cells reacted with antisera raised against the recombinant IN protein . Purified recombinant HFV IN protein was active as an endonuclease specifically cleaving two nucleotides from a 20-bp oligodeoxynucleotide substrate that mimics the authentic 5' ends of HFV DNA . Substrates with mutations relatively close to the cleavage site were less efficiently cleaved or not cleaved at all compared with the HFV U5 DNA end . The purified recombinant protein was active as integrase with double-stranded oligodeoxynucleotide substrates . The reverse reaction of DNA strand transfer, the disintegration activity, was shown by efficient cleavage of an intermediate Y-shaped oligodeoxynucleotide . In the presence of Mn2+ as the preferred divalent cation, oligodeoxynucleotides were specifically and efficiently cleaved . In contrast, endonucleolytic cleavages in the presence of Mg2+ ions led to a broad range of reaction products with the His-tagged HFV IN protein . After further purification of the HFV IN by cation-exchange chromatography, the unspecific degradation of oligonucleotide substrate in the presence of Mg2+ was not detectable.

Mol Microbiol, 1993 Sep, 9(6), 1131 - 42
PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids; He L et al.; The replication of ColE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication . We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI . In bacterial strains deleted for PcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5'-end processing by RNAase E, accumulates . This leads to a reduction in plasmid copy number . We show, using a GST-PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro . The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.

Mol Microbiol, 1993 Sep, 9(5), 1089 - 95
ColE1 multimer formation triggers inhibition of Escherichia coli cell division; Patient ME et al.; Multimer formation and consequent copy number depression are acknowledged causes of multicopy plasmid instability . Multimer resolution sites (among which ColE1 cer is best-characterized) have been identified in a variety of plasmids . They participate in the conversion of multimers to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss . We show that multimer resolution alone is insufficient to ensure stable maintenance of ColE1-like plasmids in a recombination-proficient host . The expression of Rcd, a transcript encoded within cer and expressed in multimer-containing cells, is also required . The appearance of Rcd correlates with the inhibition of division of multimer-containing cells, presumably allowing time for the conversion of multimers to monomers by site-specific recombination.

Biochemistry, 1993 Aug 31, 32(34), 8782 - 91
Proton NMR of Escherichia coli sulfite reductase: studies of the heme protein subunit with added ligands; Kaufman J et al.; The heme protein subunit of sulfite reductase (SiR-HP; M(r) 64,000) from Escherichia coli as isolated contains the isobacteriochlorin siroheme exchange-coupled to a {4Fe-4S} cluster in the 2+ oxidation state . SiR-HP in the presence of a suitable electron donor can catalyze the six-electron reductions of sulfite to sulfide and nitrite to ammonia . Paramagnetic 1H NMR was used to study the low-spin complexes of SiR-HP formed by binding the exogenous inhibitor cyanide or the substrates sulfite and nitrite . As a model, the cyanide complex of purified siroheme was also prepared . The NMR spectrum of isolated ferric low-spin siroheme-CN is consistent with spin density being transferred into the a2u molecular orbital, an interaction which is symmetry-forbidden in porphyrins . The pattern of proton NMR shifts observed for isolated ferric low-spin siroheme-CN is very similar to those obtained for the protein-cyanide complex . NMR spectra of the cyanide complex of SiR-HP were obtained in all three accessible redox states . The pattern of hyperfine shifts observed for the one-electron and two-electron reduced cyanide complexes is typical of those seen for {4Fe-4S} clusters in the 2+ and 1+ oxidation states, respectively . Resonances arising from the beta-CH2 protons of cluster cysteines have been assigned for all complexes studied utilizing deuterium substitution . The cyanide-, sulfite-, and nitrite-ligated states possessed an almost identically shifted upfield cluster cysteine resonance whose presence indicates that covalent coupling exists between siroheme and cluster in solution . Data are also presented for the existence of a secondary anion binding site, the occupancy of which perturbs the oxidized SiR-HP NMR spectrum, where binding occurs at a rate much faster than that of ligand binding to heme.

Biochem Biophys Res Commun, 1993 Aug 31, 195(1), 484 - 9
Phosphorylation of Mg(2+)-dependent protein phosphatase alpha (type 2C alpha) by casein kinase II; Kobayashi T et al.; In this study we show that rat Mg(2+)-dependent protein phosphatase alpha (MPP alpha) expressed in Saccharomyces cerevisiae cells was phosphorylated on serine residues in vivo . The recombinant rat MPP alpha purified from Escherichia coli cells harboring an expression vector was phosphorylated in vitro by casein kinase II, but not by casein kinase I, to 1.5 mol phosphate per mol enzyme protein . Analysis by phosphopeptide mapping and amino acid analysis suggested that the sites of both in vivo and in vitro phosphorylation were the same and involved only serine residues . These results suggest that the rat MPP alpha expressed in yeast cells is phosphorylated by yeast casein kinase II in vivo . It is further proposed that the phosphorylation sites are located in the carboxyl terminal region of the enzyme molecule.

Biochemistry, 1993 Aug 31, 32(34), 8863 - 70
Cytochrome P450 4A4: expression in Escherichia coli, purification, and characterization of catalytic properties; Nishimoto M et al.; Rabbit lung prostaglandin omega-hydroxylase (P450 4A4) was expressed in Escherichia coli using the isopropyl beta-D-thiogalactopyranoside (IPTG) inducible expression vector pCWori+, containing the full-length cDNA encoding the P450 4A4 . The first seven codons were changed to reflect E . coli codon bias {a modification of the method of Barnes et al . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 5597-5601}; only the second residue of P450 4A4 was altered (Ser to Ala), while the remaining mutations were silent . This strategy was adopted in order to minimize changes in the structure of the expressed enzyme . Induction by IPTG of the apoprotein peaked after 6 h, and by including the heme precursor delta-aminolevulinic acid, enzymatic activity peaked 12 h after addition of IPTG . The isolated membrane fraction, free of cell debris, contained 12-15 nmol of P450/L of media . The expressed enzyme was purified to electrophoretic homogeneity, and kinetic and spectrophotometric data indicate that this expressed, purified enzyme is equivalent to the enzyme purified from rabbit lung . The Km for PGE1 was determined to be 3.0 microM, which is the same as that obtained for the enzyme purified from lung {Williams et al . (1984) J . Biol . Chem . 259, 14600-14608} . The CO-reduced difference spectrum of purified P450 4A4 exhibited a lambda max at 450 nm, and the absolute absorbance spectrum of the pyridine hemochromogen revealed a typical b type heme . To characterize P450 4A4 further, the catalytic activities with prostaglandin E1 (PGE1), arachidonate, 15-hydroxyeicosatetraenoic acid (15-HETE), and palmitate were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Aug 31, 32(34), 8856 - 62
Apocytochrome P450cam is a native protein with some intermediate-like properties; Pfeil W et al.; Holo- and apocytochrome P450cam were studied by differential scanning calorimetry (DSC), limited proteolysis, second-derivative spectroscopy, circular dichroism, and size-exclusion chromatography . The holoprotein shows three folding units (domains) in DSC . The prosthetic group is related to the most unstable domain, which has a thermal transition at 41.9 degrees C . Compared with the holoprotein, apocytochrome P450cam has a reduced helix content . The protein is compact as judged by the Stokes radius and is still able to undergo a two-state transition . However, the enthalpy change at thermal melting is reduced from 980 kJ/mol for the holoprotein to 135 kJ/mol for the apo form . Parts of the molecule have a destabilized tertiary structure . This is indicated by second-derivative spectroscopy, circular dichroism in the near-ultraviolet region, and a high susceptibility to proteolytic digestion . Apocytochrome P450cam is considered a native protein with the extremely low stability of delta G = 7.5 kJ/mol, thus showing at the same time intermediate-like properties . The importance of the properties for in vivo folding are discussed.

Biochemistry, 1993 Aug 31, 32(34), 8758 - 71
Structural basis for transfer RNA aminoacylation by Escherichia coli glutaminyl-tRNA synthetase; Perona JJ et al.; The structure of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNA2Gln and ATP refined at 2.5-A resolution reveals structural details of the catalytic center and allows description of the specific roles of individual amino acid residues in substrate binding and catalysis . The reactive moieties of the ATP and tRNA substrates are positioned within hydrogen-bonding distance of each other . Model-building has been used to position the glutamine substrate in an adjacent cavity with its reactive carboxylate adjacent to the alpha-phosphate of ATP; the interactions of the carboxyamide side chain suggest a structural rationale for the way in which the enzyme discriminates against glutamate . The binding site for a manganese ion has also been identified bridging the beta- and gamma-phosphates of the ATP . The well-known HIGH and KMSKS sequence motifs interact directly with each other as well as with the ATP, providing a structural rationale for their simultaneous conservation in all class I synthetases . The KMSKS loop adopts a well-ordered and catalytically productive conformation as a consequence of interactions made with the proximal beta-barrel domain . While there are no protein side chains near the reaction site that might function in acid-base catalysis, the side chains of two residues, His43 and Lys270, are positioned to assist in stabilizing the expected pentacovalent intermediate at the alpha-phosphate . Transfer of glutamine to the 3'-terminal tRNA ribose may well proceed by intramolecular catalysis involving proton abstraction by a phosphate oxygen atom of glutaminyl adenylate . Catalytic competence of the crystalline enzyme is directly shown by its ability to hydrolyze ATP and release pyrophosphate when crystals of the ternary complex are soaked in mother liquor containing glutamine.

Biochem Biophys Res Commun, 1993 Aug 31, 195(1), 229 - 36
Lack of evidence for a role of Cys-138 as a base catalyst in the skeletal muscle 6-phosphofructo-2-kinase reaction; Kurland IJ et al.; The role of Cys-138 in the catalysis of the skeletal muscle 6-phosphofructo-2-kinase reaction was investigated by mutating this residue to serine, glutamine and alanine, expressing the mutants in E . coli with a T7 RNA polymerase-based expression system, and analyzing their kinetic properties . The Cys138Ala mutant had greatly diminished activity, while the Cys138Ser and Cys138Gln mutants had maximal velocities 2-3 fold higher than the wild-type enzyme . It was concluded that Cys-138 does not act as a base catalyst in the kinase reaction, but that it plays a significant structural role in the enzyme's active site.

FEBS Lett, 1993 Aug 30, 329(3), 287 - 90
Requirement of phosphatidylglycerol for flagellation of Escherichia coli; Tomura A et al.; We report that phosphatidylglycerol is required for flagellation of Escherichia coli . Cells carrying the pgsA3 mutation did not form swarm rings in semisolid agar . P1 transduction experiments revealed that the potential for phosphatidylglycerol synthesis and for the formation of swarm rings was co-transducible . The pgsA3 mutant transformed with the wild type pgsA+ gene cloned into the R-plasmid vector had the potential for both phosphatidylglycerol synthesis and cell motility . Electromicroscopic and SDS-PAGE analyses showed that the pgsA3 mutation causes the lack of flagellation.

J Chromatogr, 1993 Aug 27, 646(1), 213 - 25
Structural characterisation of native and recombinant forms of the neurotrophic cytokine MK; Fabri L et al.; The retinoic acid (RA)-inducible midkine (MK) gene encodes a heparin-binding protein which can induce neurite outgrowth in cultured mammalian embryonic brain cells . This cytokine shares 65% amino acid sequence identity with another RA-inducible cytokine, pleiotropin (PTN) . Both proteins contain 10 conserved cysteine residues, all of which appear to be disulphide linked . MK and PTN are also rich in lysine and arginine residues rendering them susceptible to proteolysis during purification, and making large-scale preparation of these molecules inherently difficult . Recombinant MK has been expressed as a fusion protein using a pGEX vector transfected into E . coli . To enable refolding of MK, the fusion protein was stored in solution at 4 degrees C for 14 days in the presence of dithiothreitol (DTT) . Thrombin cleavage of the fusion protein, post storage, typically generated 5 mg of MK per litre of bacterial pellet . To establish the structural integrity of the recombinant product, we have analysed the refolding kinetics and compared the disulphide bond assignment of recombinant MK with that of native MK and native PTN . The synergistic use of micropreparative HPLC, to separate and recover in small eluant volumes enzymatically derived peptide fragments, with matrix assisted laser desorption mass spectrometry (MALD-MS) and N-terminal sequence analysis has allowed the unambiguous identification of the disulphide bonded fragments of native and recombinant MK . The disulphide bond assignment of MK is C12-C36, C20-C45, C27-C49, C59-C91 and C69-C101, and is equivalent to that of PTN.

Science, 1993 Aug 27, 261(5125), 1164 - 7
Repair of DNA methylphosphotriesters through a metalloactivated cysteine nucleophile; Myers LC et al.; The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteines . Upon methyl transfer, Ada acquires the ability to bind specific DNA sequences and thereby to induce genes that confer resistance to methylating agents . The amino-terminal domain of Ada, which comprises the methylphosphotriester repair and sequence-specific DNA binding elements, contains a tightly bound zinc ion . Analysis of the zinc binding site by cadmium-113 nuclear magnetic resonance and site-directed mutagenesis revealed that zinc participates in the autocatalytic activation of the active site cysteine and may also function as a conformational switch.

J Immunol Methods, 1993 Aug 26, 164(1), 131 - 5
Enzyme release assay of human NK cell activity using beta-galactosidase-expressing K562 target cell line; Ohmori H et al.; In the present report, we established a K562 cell line useful for an enzyme release assay of human natural killer (NK) activity . Human myelogenous leukemia cell line, K562, was transfected with a plasmid carrying Escherichia coli beta-galactosidase (beta-gal) gene . A colony that permanently expresses the enzyme activity was isolated, and designated K562/Zneo . Incubation of K562/Zneo cells (1 x 10(4)) with nonadherent human peripheral blood lymphocytes (PBL) resulted in the release of beta-gal activity depending on the incubation time and the number of effector cells . Released beta-gal activity was assayed sensitively by using 4-methylumbelliferyl-beta-D-galactoside, a fluorescent substrate . The cytolytic activity of PBL was augmented significantly when the cells were preincubated with interleukin-2 for 20 h . This enzyme release assay showed a comparable sensitivity to that of 51Cr release assay . Thus, K562/Zneo cell line is thought to be useful for the nonradioactive assay of human NK and lymphokine-activated killer activities.

J Biol Chem, 1993 Aug 25, 268(24), 18053 - 61
Inclusion body formation and protein stability in sequence variants of interleukin-1 beta; Chrunyk BA et al.; Inclusion body formation during recombinant protein expression in bacteria is of both fundamental interest and practical importance . To elucidate molecular mechanisms of this process, we are examining the in vitro folding and stability properties of a series of human interleukin-1 beta (IL-1 beta) sequence variants which exhibit widely differing tendencies to form inclusion bodies . Of 67 variants surveyed, nine, including wild type, were purified and their in vitro stability properties determined . One of these, a high inclusion body mutant, exhibited very low solubility in native buffer after purification and was not pursued further . For the other eight sequence variants, no strong correlations were observed between extent of inclusion body formation and either thermodynamic or thermal stability . In particular, a Lys97-->Val mutation produces substantially more IL-1 beta in inclusion bodies than the wild type (61 versus 8%) despite generating a protein more thermodynamically stable than wild type . Furthermore, the Lys97-->Val mutant forms substantial levels of inclusion bodies at 32 degrees C but requires incubation at temperatures greater than 48 degrees C for thermally induced aggregation in vitro . This and other data suggest that the tendency of at least some IL-1 beta variants to form inclusion bodies is most likely related to the stability or solubility of folding intermediates rather than native states . Implications of the structural locations of these mutations are also discussed.

Nucleic Acids Res, 1993 Aug 25, 21(17), 3977 - 80
A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA; Lai D et al.; A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmids, bacterial strains or reagents is described . Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessary and a high efficiency of mutation (58-97%) was obtained . If two synthetic oligonucleotide primers are used, two separate mutations can be simultaneously created in a single reaction tube.

Gene, 1993 Aug 25, 130(2), 233 - 9
An engineered PGK promoter and lac operator-repressor system for the regulation of gene expression in mammalian cells; Hannan GN et al.; Previous reports have demonstrated that the Escherichia coli lac repressor can operate effectively in mammalian cells to repress expression of genes driven by modified viral or metallothionein (MT) promoters . We have developed a more general expression system using the promoter from the PGK1 gene (encoding murine 3-phosphoglycerate kinase) which is widely expressed in almost all cell types, including early embryonic and ES (embryonic stem) cells . Firstly, we engineered the lac repressor to include a nuclear localisation signal and placed it under control of the PGK1 promoter . Efficient nuclear localisation of the repressor was demonstrated by mobility-shift assays and immunofluorescence detection . For the target vectors, we modified the wild-type (wt) PGK1 promoter to include lac operator (lacO) sites for binding of the lac repressor and compared a number of different lacO positions and arrangements based on proximity to the native start points for transcription (tsp) and translation . In the absence of repressor, we observed reduced expression of the neo reporter gene for some placements of the lacO, but wt expression for placements near the tsp . When both target and repressor were present in the cells, we observed that the expression of neo could be strongly suppressed and reversibly regulated by induction with IPTG . In particular, for a promoter which contained two spaced lacO replacing native sequence around the major tsp, we observed 90-95% repression by the lac repressor for the neo reporter gene and up to 98% repression for the cat reporter gene . Efficient derepression by IPTG was observed in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1993 Aug 25, 268(24), 18382 - 9
The multiple roles for ATP in the Escherichia coli UvrABC endonuclease-catalyzed incision reaction; Thiagalingam S et al.; The biochemical properties of the Escherichia coli UvrA tandem ATPase site mutants in nucleotide excision repair have been studied . In these and earlier studies it was found that ATP binding is required for protein-protein and nucleoprotein association reactions, whereas the dissociation reactions are driven by the hydrolysis of ATP . The self-association of UvrA to form the reactive dimeric species UvrA2 is driven by nucleotide binding, but its dissociation from DNA requires ATP hydrolysis . Similarly, ATP binding drives those allosteric changes in DNA topology during UvrA2-nucleoprotein formation (Oh, E.Y., and Grossman, L . (1986) Nucleic Acids Res . 14, 8557-8571) . The manifestation of the UvrB-associated cryptic ATPase requires UvrA and DNA in a helicase-catalyzed supercoiling reaction . The UvrA2B helicase activity requires ATP hydrolysis by the C-terminal ATPase site of UvrA in addition to UvrB . ATP hydrolysis by the C-terminal ATPase site of UvrA also participates in the localization of damaged sites contributing to the formation of damage-specific high affinity nucleoprotein complexes . The levels of complementation to UV survival by the ATPase site mutants of UvrA (Thiagalingam, S., and Grossman, L . (1991) J . Biol . Chem . 266, 11395-11403) correspond to its ability to self-bind and translocate in combination with the UvrB subunit in its search for damaged sites during the preincision mode of nucleotide excision.

J Biol Chem, 1993 Aug 25, 268(24), 18335 - 9
The anticodon and discriminator base are important for aminoacylation of Escherichia coli tRNA(Asn); Li S et al.; A gel shift assay that distinguishes the aminoacylated form from the deacylated form of tRNAs was used to study the requirements for aminoacylation of Escherichia coli tRNA(Asn) in vivo . tRNA(Asn) derivatives containing single base changes in their anticodons or discriminator bases were constructed, and the extent of in vivo aminoacylation was determined directly . Substitution of U35 with C35 or U36 with C36 abolished aminoacylation of the tRNA . Substitution of G34 with C34 converted tRNA(Asn) into a lysine acceptor . Thus, each of the anticodon nucleotides are important for aminoacylation of tRNA(Asn) . Substitution of discriminator base G73 with A73 affected the extent of aminoacylation in vivo indicating that the discriminator base also contributes to aminoacylation of tRNA(Asn).

J Biol Chem, 1993 Aug 25, 268(24), 18143 - 50
Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins; Ullrich O et al.; Rab proteins comprise a family of small GTPases that serve a regulatory role in membrane traffic . These proteins are in part cytosolic and in part associated with the membranes of specific exocytic and endocytic organelles . Smg p25A/rab3A GDI, a cytosolic protein which inhibits the dissociation of GDP from smg p25A/rab3A, Sec4p, and rab11, has also been found to prevent association of rab3A with the membrane . In this study, we have used Madin-Darby canine kidney cells permeabilized with the bacterial toxin streptolysin O to test the general activity of rab3A GDI in modulating the membrane association of various small GTP-binding proteins . Rab3A GDP dissociation inhibitor (GDI) removed from the membrane all rab proteins we have tested and inhibited the membrane binding of in vitro translated rab proteins . However, rab3A GDI had a limited effect on the membrane association of a mutant rab5 protein which contained a farnesylated cysteine motif . Finally, we found that, although rab3A GDI resides primarily in the cytosol, it is also associated with compartments of the exocytic and endocytic pathways . Since rab3A GDI can modulate the membrane association of various rab proteins, we propose to rename it rab GDI.

J Biol Chem, 1993 Aug 25, 268(24), 18083 - 7
The sequence-specific high mobility group 1 box of TCF-1 adopts a predominantly alpha-helical conformation in solution; van Houte L et al.; The High Mobility Group (HMG) 1 box is a protein motif that mediates DNA binding in a novel family of transcription-regulating proteins . Several members of this family, including the lymphoid-specific proteins TCF-1 and LEF-1 and the mammalian sex-determining factor SRY, carry a single HMG box with affinity for the minor groove of the heptamer motif AACAAAG or variations thereof . To initiate studies on the structural characteristics of the TCF-1 HMG box, we have expressed the 87-amino acid HMG box in milligram quantities in Escherichia coli and purified the soluble peptide to > 95% homogeneity . The peptide bound DNA with the same specificity as the complete protein and was capable of inducing DNA bending . Circular dichroism (CD) analysis revealed the TCF-1 HMG box to adopt an approximately 60% alpha-helix/40% random coil conformation in solution . In the presence of an equimolar amount of double-stranded DNA containing the cognate motif, the CD spectrum changed significantly, implying the induction of a structural modification upon DNA/protein association.

J Biol Chem, 1993 Aug 25, 268(24), 17850 - 60
A method to study complex enzyme kinetics involving numerical analysis of enzymatic schemes . The mannitol permease of Escherichia coli as an example; Lolkema JS; An analysis of complex kinetic mechanisms is proposed that consists of two steps, (i) building of an kinetic scheme from experimental data other than steady-state kinetics and (ii) numerical simulation and analysis of the kinetics of the proposed scheme in relation to the experimental kinetics . Procedures are introduced to deal with large numbers of enzymatic states and rate constants, and numerical tools are defined to support the analysis of the scheme . The approach is explored by taking the mannitol permease of Escherichia coli as an example . This enzyme catalyzes both the transport of mannitol across the cytoplasmic membrane and the phosphorylation of mannitol . The challenge is to deduce the transport properties of this dimeric enzyme from the phosphorylation kinetics . It is concluded that (i) the steady-state kinetic behavior is largely consistent with the proposed catalytic cycle of the monomeric subunit, (ii) the kinetics provide no direct support but also do not disprove a coupled translocation of the binding sites on the two monomeric subunits . The approach reveals the need for further experimentation where the implementation of experimental results in the scheme conflict with the experimental kinetics and where specific experimental characteristics do not show up in the simulations of the proposed kinetic scheme.

J Biol Chem, 1993 Aug 25, 268(24), 17844 - 9
Steady state kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system; Lolkema JS et al.; The kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the bacterial P-enolpyruvate-dependent phosphotransferase system are described for three different physical conditions of the enzyme, (i) embedded in the membrane of inside-out (ISO) oriented vesicles, (ii) solubilized and assayed above the critical micellular concentration (cmc) of the detergent, and (iii) solubilized and assayed below the cmc of the detergent . The kinetic characteristics of enzyme IImtl, after solubilization of cytoplasmic membranes or after purification from these membranes are comparable . The mannitol-dependent kinetics at saturating concentration of P-HPr were biphasic both for the solubilized enzyme assayed above the cmc and for the enzyme in ISO vesicles . In contrast, the mannitol-dependent kinetics was monophasic for the solubilized enzyme assayed below the cmc . In the latter case, the maximal rate was about twice as high as observed with the two other conditions . The contribution of the high affinity phase to the maximal rate is lower for enzyme IImtl in ISO vesicles than for the solubilized enzyme . At limiting concentrations of P-HPr, the kinetics is not according to the expected "ping-pong" mechanism.

J Biol Chem, 1993 Aug 25, 268(24), 17837 - 43
Cloning and site-directed mutagenesis of human ADP-ribosylarginine hydrolase; Takada T et al.; Mono-ADP-ribosylation of arginine is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the opposing reactions in the cycle . ADP-ribosylarginine hydrolases differ in their dithiothreitol (DTT) requirements . Rat and mouse hydrolases require DTT for maximal activity, but calf, guinea pig, and human hydrolases are DTT-independent . To define the molecular basis for these differences, brain ADP-ribosylarginine hydrolases were cloned . Deduced amino acid sequences of mouse and rat hydrolases were 94% identical with 5 conserved cysteines . The human hydrolase sequence was 83% identical to that of rat but contained only 4 cysteines with cysteine 108 in rat corresponding to serine 103 in human . To investigate the role of rat cysteine 108, human and rat wild-type hydrolases and mutants, in which serine 103 in human was replaced by cysteine (S103C) and cysteine 108 in rat was replaced by serine (C108S), were expressed in Escherichia coli . Affinity-purified anti-rat brain hydrolase antibodies reacted with recombinant wild-type rat hydrolase, but only weakly with the C108S mutant . They did not react with human wild-type or the S103C mutant . Human hydrolase and rat C108S were DTT-independent; human S103C was, however, DTT-dependent . These data clearly show that cysteine 108 in rat hydrolase plays a critical role in DTT dependence and may be important in immunoreactivity.

J Biol Chem, 1993 Aug 25, 268(24), 17803 - 10
Vaccinia virus ribonucleotide reductase expression and isolation of the recombinant large subunit; Slabaugh MB et al.; The vaccinia virus gene encoding the 87-kDa protein that comprises the large subunit of ribonucleotide reductase (vvR1) was cloned into a bacterial expression vector under the control of an inducible promoter . Culture of Escherichia coli cells harboring the recombinant plasmid under standard induction conditions (0.4 mM isopropyl beta-D-thiogalactopyranoside, 37 degrees C) resulted in synthesis of a completely insoluble product . Production of soluble vvR1 was achieved by growing bacteria at low temperature (15 degrees C) during the induction period, initiating induction at low cell density, and using a low concentration (0.05 mM) of the inducer isopropyl beta-D-thiogalactopyranoside . Hydroxyurea, an inhibitor of ribonucleotide reductase, increased production of soluble vvR1 in a dose-dependent manner . Recombinant vvR1 was purified from a high salt extract of the E . coli lysate in four steps, the last utilizing an affinity column consisting of the carboxyl-terminal seven amino acids of the vvR2 protein linked to an insoluble resin . Using purified recombinant vvR2 to reconstitute active enzyme, we determined that maximizing the rate of CDP reduction required pH 8.0-8.8, 50 mM dithiothreitol, and 2 mM ATP . Specific activity of purified vvR1 was 122 nmol/min/mg . Limited proteolysis of the vvR1 protein revealed protease-resistant fragments approximately 30 and 58 kDa in size . To our knowledge, this study represents the first expression, solubilization, and isolation of a recombinant "eukaryotic" form of ribonucleotide reductase large subunit.

J Biol Chem, 1993 Aug 25, 268(24), 17705 - 10
Primary structure of alpha-tocopherol transfer protein from rat liver . Homology with cellular retinaldehyde-binding protein; Sato Y et al.; alpha-Tocopherol transfer protein (alpha TTP) present in rat liver cytosol specifically binds this vitamin and enhances its transfer between separate membranes . We previously reported purification of alpha TTP and showed that two isoforms exist in rat liver, of which the isoelectric points are 5.0 and 5.1, respectively (Sato, Y., Hagiwara, K., Arai, H., and Inoue, K . (1991) FEBS Lett . 288, 41-45) . In the present paper, we have isolated a cDNA clone with 2194 base pairs encoding alpha TTP from a rat liver cDNA library . The amino acid sequence deduced from the cDNA contained all the sequences of the peptide fragments obtained by digestion of the purified protein with endoproteinase Lys-C . The isolated cDNA was found to encode the isoform with a pI of 5.0 on the basis of the cross-reactivity of the recombinant protein expressed in Escherichia coli with the isoform-specific monoclonal antibody . From the longest open reading frame of the cloned cDNA, one isoform of rat liver alpha TTP is predicted to be composed of 278 amino acid residues of calculated molecular weight 31,845 . Both Western and Northern blot analyses revealed that alpha TTP is expressed exclusively in the liver in rats . alpha TTP has been found to exhibit a structural homology with the cellular retinaldehyde-binding protein present only in visual tissues.

J Biol Chem, 1993 Aug 25, 268(24), 17669 - 71
Expression, characterization, and crystallization of oxygen-avid Ascaris hemoglobin domains; Kloek AP et al.; The Ascaris perienteric hemoglobin is 10(4) times more oxygen-avid than mammalian hemoglobins . Inspection of its primary structure fails to explain this extraordinary association with oxygen . The Ascaris hemoglobin gene encodes a 40-kDa, two-domain globin; the two domains (D1 and D2) are 63% identical, and each is capable of binding a single heme . The native protein is an octamer . At the end of D2 is a highly charged carboxyl-terminal extension containing four direct repeats of HKEE . We have expressed the two domains separately in E . coli . Both individual domains are extremely oxygen-avid . D2, with attached COOH-terminal tail, is capable of multimerization, whereas D1 remains a monomer . Recombinant D1 readily forms diffractable, red, prismatic crystals . We conclude that: 1) the basis of the hemoglobin's oxygen avidity rests in an isolated heme pocket and does not involve inter-domain interactions and 2) multimerization is mediated through sequences in the second domain, most probably via the charged COOH-terminal tail.

Nucleic Acids Res, 1993 Aug 25, 21(17), 4059 - 65
U-U and T-T cyclobutane dimers have different mutational properties; Gibbs PE et al.; We have examined the mutagenic properties in E . coli of single stranded vectors containing a uniquely placed cis-syn or trans-syn uracil-uracil cyclobutane dimer in the sequence 5' GCAAGUUGGAG 3', and compared these with the properties of the corresponding T-T dimers in the same sequence context . The frequencies with which U-U and T-T photoproducts were bypassed were similar in SOS induced cells, and each induced similar frequencies of mutations . However, although both U-U and T-T cis-syn dimers showed a preference for misincorporation in about 5-7% of the replication products, with T or G being incorporated in place of A, the ratios of these events differed, being > 4:1 for T-T cis-syn, but only 2:1 for U-U cis-syn . A shift towards G insertion opposite dimerized uracil was also found with the trans-syn dimers, but the difference was greater; T and G were misincorporated opposite the U-U trans-syn dimer in a ratio of 1:2, compared with 4:1 for its T-T counterpart . In addition, the U-U dimer induced only nucleotide substitutions, unlike the T-T photoproduct which induced single nucleotide deletions as well as substitutions . We conclude that even relatively minor differences in photoproduct structure, such as the presence of a methyl group at C-5, can alter mutational properties, and that such properties cannot depend only on the attributes of the DNA polymerase . Neither the efficiency of bypass, the error frequency nor the mutation spectrum of either U-U isomer is influenced by DNA uracil glycosylase . In vitro, the U-U cis-syn dimer is a substrate for DNA photolyase, but not for the glycosylase.

Nucleic Acids Res, 1993 Aug 25, 21(17), 4025 - 30
Role of the 1-72 base pair in tRNAs for the activity of Escherichia coli peptidyl-tRNA hydrolase; Dutka S et al.; Previous work by Schulman and Pelka (1975) J . Biol . Chem . 250, 542-547, indicated that the absence of a pairing between the bases 1 and 72 in initiator tRNA(fMet) explained the relatively small activity of peptidyl-tRNA hydrolase towards N-acetyl-methionyl-tRNA(fMet) . In the present study, the structural requirements for the sensitivity of an N-acetyl-aminoacyl-tRNA to Escherichia coli peptidyl-tRNA hydrolase activity have been further investigated . Ten derivatives of tRNA(fMet) with various combinations of bases at positions 1 and 72 in the acceptor stem have been produced, aminoacylated and chemically acetylated . The release of the aminoacyl moiety from these tRNA derivatives was assayed in the presence of peptidyl-tRNA hydrolase purified from an overproducing strain . tRNA(fMet) derivatives with either C1A72, C1C72, U1G72, U1C72 or A1C72 behaved as poor substrates of the enzyme, as compared to those with C1G72, U1A72, G1C72, A1U72 or G1U72 . With the exception of U1G72, it could be therefore concluded that the relative resistance of tRNA(fMet) to peptidyl-tRNA hydrolase did not depend on a particular combination of nucleotides at positions 1 and 72, but rather reflected the absence of a base pairing at these positions . In a second series of experiments, the unpairing of the 1 and 72 bases, created with C-A or A-C bases, instead of G-C in methionyl-tRNA(mMet) or in valyl-tRNA(Val1), was shown to markedly decrease the rate of hydrolysis catalysed by peptidyl-tRNA hydrolase . Altogether, the data indicate that the stability of the 1-72 pair governs the degree of sensitivity of a peptidyl-tRNA to peptidyl-tRNA hydrolase.

J Biol Chem, 1993 Aug 25, 268(24), 17775 - 80
Phosphate efflux through the channels formed by colicins and phage T5 in Escherichia coli cells is responsible for the fall in cytoplasmic ATP; Guihard G et al.; Previous studies have shown that channel formation in the cytoplasmic membrane of Escherichia coli by colicin A and phage T5 leads to an efflux of cytoplasmic potassium and to a membrane depolarization . Here we show that upon opening of these channels, the intracellular ATP concentration is decreased to 10% of its original value in < 5 min . ATP is not found in the external medium, but is hydrolyzed in the cytoplasm into ADP and AMP . The rate of ATP hydrolysis depends on the number of channels and on their activity . ATP hydrolysis takes place if the F1F0-ATPase is absent or inhibited . Depolarization of the inner membrane is not the main cause of ATP hydrolysis . Opening of the phage and colicin channels also leads to an efflux of inorganic phosphate . Conditions that prevent the efflux of phosphate (i.e . depolarization of the cells and high external phosphate concentration) prevent ATP hydrolysis . We propose that ATP is hydrolyzed as a consequence of a shift in the ATP equilibrium due to the efflux of phosphate through the channels . The consequences for the cells of this ATP depletion are discussed.

J Biol Chem, 1993 Aug 25, 268(24), 17665 - 8
Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation . Homology with human CD36; Abumrad NA et al.; A cDNA for an adipocyte membrane protein, implicated in the transport of long-chain fatty acids, was isolated by screening with a synthetic oligonucleotide derived from the amino terminal sequence of the protein . The 88-kDa adipocyte membrane protein was previously identified by covalent labeling with N-sulfosuccinimidyl esters of long-chain fatty acids which irreversibly inhibited fatty acid transport by 75% (Harmon, C . M., and Abumrad, N.A . (1993) J . Membr . Biol . 124, 261-268) . The cDNA (FAT, 2432 base pairs (bp)) contained 70 bp of 5'-untranslated sequence, an open reading frame encoding a 472-amino acid protein with a predicted molecular mass of 52466, and 940 bp of 3'-untranslated sequence with two polyadenylation signal sequences but with no polyadenylation tail . The deduced protein sequence predicted two transmembrane segments and 10 potential N-linked glycosylation sites . Extensive glycosylation most likely explains why the molecular mass of the isolated protein (88 kDa) is different from that deduced from the cDNA sequence (53 kDa) . The sequence of FAT is 85% homologous with that of glycoprotein IV (CD36) identified in human platelets and in lactating mammary epithelium . Consistent with this, a polyclonal antibody against CD36 reacted with adipocyte plasma membranes and detected a single band at 88 kDa . Northern blot analysis of RNA obtained from rat adipose tissue and probed with the cDNA identified two major transcripts of 4.8 and 2.9 kilobases which were abundant in heart, intestine, fat, muscle, and testis . The mRNAs were not detectable in cultured adipose cell lines (Ob1771, 3T3F442A) at the fibroblastic stage but were strongly induced during the differentiation process and by treatment of preadipocytes with dexamethasone, conditions that were also associated with an increase in oleate transport . In contrast, the fibroblastic cell lines 3T3-C2 and L929, which do not differentiate, did not express the mRNAs at all stages of culture . The data suggest that FAT and CD36 belong to a family of proteins that bind/transport long-chain fatty acids or function as regulators of these processes.

Biochemistry, 1993 Aug 24, 32(33), 8589 - 95
A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics; Caamano CA et al.; A recombinant system was developed for generation of steroid-receptor complexes in vitro . The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase . The identity of the expressed recombinant protein was confirmed by Western blot analysis . Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding . However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the {3H}aldosterone-binding activity gradually increased . This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein) . The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor . The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor . Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively . Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient . Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Aug 24, 32(33), 8693 - 701
Modulation of the stability of a Lac repressor mediated looped complex by temperature and ions: allosteric regulation by chloride; Brenowitz M et al.; The lactose repressor of Escherichia coli (LacI) associates to a bidentate tetramer in solution and can simultaneously bind two operators to form a protein-mediated "looped complex" . Studies have been conducted of the binding of LacI to two operators separated by approximately 11 helical turns of DNA . Quantitative DNase I footprint titration analysis of the stability of the LacI-mediated looped complex reveals that the Gibbs free energy of cyclization (delta Gzeroj) of the looped complex of 11.7 +/- 0.4 kcal/mol is invariant with temperature . van't Hoff analysis reveals a large and positive enthalpy of cyclization (delta H degrees = 12.3 +/- 2.4 kcal/mol) and an entropy that is small and positive (delta S degrees = 2.2 cal/deg) . Quantitative DNase I footprint titration and kinetic dissociation studies were also conducted as a function of counter-ion type and concentration . Increasing concentrations of KCl or potassium glutamate destabilize the looped complex, a result completely accounted for by increases in the intrinsic DNA-binding free energies . While the value of delta Gzeroj is invariant with ion concentration, chloride is a positive regulator . The value of delta Gzeroj decreases by 1.5 kcal/mol upon substitution of chloride for glutamate . Measurements of delta Gzeroj conducted as a function of chloride concentration at constant ionic strength reveal that approximately one chloride ion per tetramer is bound upon looped complex formation . These results demonstrate specific allosteric regulation of the formation of the LacI-mediated looped complex by a mechanism distinct from the regulation of the constituent protein--DNA interactions.

Biochemistry, 1993 Aug 24, 32(33), 8622 - 7
Mechanism of fluorescent fatty acid transfer from adipocyte fatty acid binding protein to membranes; Wootan MG et al.; Adipocyte fatty acid binding protein (A-FABP) is a 15-kDa protein found in high abundance in the cytosol of adipose cells . To better understand the role of this protein in intracellular free fatty acid (ffa) transport, the mechanism of ffa transfer from A-FABP to model membranes was examined by monitoring the transfer of fluorescent anthroyloxy ffa (AOffa) to small unilamellar phospholipid vesicles, using a resonance energy transfer assay . Structural features of ffa that increase aqueous solubility, such as shorter chain length and unsaturation, did not increase the AOffa transfer rate . In addition, solution conditions that increase the aqueous solubility of ffa, such as decreasing ionic strength and increasing pH, had little effect on AOffa transfer from A-FABP to membranes . These results suggest that AOffa do not transfer through the aqueous phase . The small entropic contribution to the free energy of the transfer process provides further evidence that AOffa may not travel through the surrounding aqueous environment when transferred from A-FABP to phospholipid membranes . Finally, the rate of AOffa transfer from A-FABP was directly dependent on the concentration of the acceptor membranes . These studies suggest that AOffa transfer from A-FABP to phospholipid vesicles may occur via transient collisional interactions between the protein and membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Aug 24, 32(33), 8534 - 9
Three-dimensional model for the membrane domain of Escherichia coli leader peptidase based on disulfide mapping; Whitley P et al.; We have mapped the interface between the two transmembrane alpha-helices in the membrane domain of the Escherichia coli enzyme leader peptidase by analyzing disulfides formed between pairs of cysteine residues introduced near their respective periplasmic ends . The interface is formed primarily from aliphatic amino acids, and the two helices appear to pack against each other in a left-handed supercoil . We suggest that disulfide mapping may be a generally applicable approach for the construction of models of helix-helix interfaces in membrane proteins.

Biochemistry, 1993 Aug 24, 32(33), 8525 - 33
Primary structure of and studies on Acanthamoeba actophorin; Quirk S et al.; We determined the amino acid sequence of the actin monomer binding/actin filament severing protein actophorin from Acanthamoeba castellanii by automated Edman degradation of peptide fragments and by sequencing of full-length cDNA . Actophorin consists of 138 amino acids (calculated molecular weight of 15,543) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially vertebrate cofilin, vertebrate actin depolymerizing factor/destrin, and echinoderm depactin . Actophorin is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins . Southern blot analysis indicates that actophorin is a single-copy gene; however, Northern blots show two distinct mRNA species of 1 and 0.9 kb in size . Homogeneous recombinant actophorin purified from Escherichia coli is indistinguishable from the native protein in its physical properties and in biochemical assays of its interaction with actin, but is less reactive with three monoclonal antibodies raised against the native protein . The NH2 terminus of native actophrin is blocked, while the initiating methionine residue is removed from recombinant actophorin . This difference has no measurable effect on activity . By fluorescent antibody staining of Acanthamoeba, actophorin colocalizes with actin filaments in the cortical cytoplasm, especially at the leading edge of the cell . Additionally, actophorin binds phosphatidylinositol 4',5'-bisphosphate . The recombinant actophorin forms X-ray diffraction quality crystals of superior quality in poly(ethylene glycol)/2-propanol and, like the native crystal form, belongs to space group P2(1)2(1)2(1).

Biochemistry, 1993 Aug 24, 32(33), 8429 - 38
The solution structures of mutant calbindin D9k's, as determined by NMR, show that the calcium-binding site can adopt different folds; Johansson C et al.; The complete 1H NMR assignments have been obtained for five mutant proteins of calbindin D9k and the three-dimensional solution structures determined for two of the mutants . The structures have been determined using distance geometry and simulated annealing, with distance constraints from NMR . All mutants have modifications in the first calcium-binding site of calbindin (the N-terminal site designated the pseudo-EF-hand) . The 3D structure of the mutant with the most extensive modifications in the pseudo-EF-hand shows that the site has turned inside-out and coordinates calcium as in the normal EF-hand (the C-terminal site) . In a pseudo-EF-hand loop the calcium is coordinated by main-chain carbonyls, whereas calcium in the normal EF-hand is coordinated by side-chain carboxylates . The 3D structures and 1H NMR assignments show that in order to accomplish a change in the coordinating ligands of the pseudo-EF-hand the loop must be 12 residues long and have glycine in the sixth position . It does, however, seem possible to have alanine instead of aspartic acid in the first calcium coordinating position . The overall global fold of the proteins has not been affected by the mutations in the calcium-binding site, as compared to the wild-type calbindin D9k {Kordel, J., Skelton, N . J., Akke, M., & Chazin, W . J . (1993) J . Mol . Biol . (in press)} . The structures consist of two helix-calcium-binding loop-helix motifs, the so called EF-hands, and the loops are connected by a short antiparallel beta-sheet . All helices are pairwise in an antiparallel orientation.

Biochemistry, 1993 Aug 24, 32(33), 8560 - 7
Hydrolysis of adenosine 5'-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion; Todd MJ et al.; The potassium-ion activation constant (Kact) for the ATPase activity of Escherichia coli chaperonin groEL is inversely dependent upon the ATP concentration over at least 3 orders of magnitude . The ATPase activity shows positively cooperative kinetics with respect to ATP and K+ . Both the K0.5 for ATP and cooperativity (as measured by the Hill coefficient) decrease as the K+ concentration increases . Equilibrium binding studies under conditions where hydrolysis does not occur indicate that MgATP binds weakly to groEL in the absence of K+ . In the absence of groES, the K(+)-dependent hydrolysis of ATP by groEL continues to completion . In the presence of groES, the time course for the hydrolysis of ATP by groEL becomes more complex . Three distinct kinetic phases can be discerned . Initially, both heptameric toroids turn over once at the same rate that they do in the absence of groES . This leads to the formation of an asymmetric binary complex, groEL14-MgADP7-groES7, in which 7 mol of ADP is trapped in a form that does not readily exchange with free ADP . In the second phase, the remaining seven sites (containing readily exchangeable ADP) turn over, or have the potential to turn over, at the same rate as they do in the absence of groES, so that the overall rate of hydrolysis is maximally 50% . These remaining sites of the asymmetric binary complex do not hydrolyze all of the available ATP . Instead, the second phase of hydrolysis gives way to a third, completely inhibited state, the onset of which is dependent upon the relative affinities of the remaining sites for MgATP and MgADP.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1993 Aug 23, 329(1-2), 55 - 8
A role for iron in transcriptional activation by FNR; Green J et al.; FNR is a transcriptional regulator which controls the expression of target genes in response to anoxia in Escherichia coli . The mechanism by which FNR senses and responds to anaerobiosis is unknown but indirect evidence suggests that an iron cofactor is involved . Using KMnO4 as a probe for DNA melting at active promoters, footprinting studies have now shown that the ferrous iron chelator, ferrozine, inhibits open complex formation in vivo, and that FNR with a high iron-content is essential for open complex formation in vitro . Since open complex formation is an essential pre-requisite for transcription, it is concluded that transcriptional activation by FNR is mediated by a ferrous iron cofactor.

FEBS Lett, 1993 Aug 23, 329(1-2), 25 - 8
A rapid and efficient purification method for recombinant annexin V for biophysical studies; Burger A et al.; Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro . We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity . We describe here a method to obtain very pure recombinant annexin V required for such studies . The initial step is the mild opening of the bacterial cells by an osmotic shock . In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes . In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.

FEBS Lett, 1993 Aug 23, 329(1-2), 223 - 6
Receptor phage . Display of functional domains of the human high affinity IgE receptor on the M13 phage surface; Scarselli E et al.; In this paper we demonstrate that phage display technology is a suitable system for studying the interaction between the high-affinity receptor for IgE (Fc epsilon RI) and IgE . The alpha subunit extracellular domains of the human receptor were expressed on the surface of filamentous phage M13 fused to the carboxyl-terminal part of the gene III protein (pIII) . Two constructs were made, the first with both the Ig-like domains of the receptor alpha chain and the second with only the C-terminal domain . The fusion genes were cloned in a phagemid vector to display monovalently the receptor on the phage surface . Our results indicate that the alpha receptor expressed on the phage is able to interact with IgE as demonstrated by an ELISA assay . In addition, by using the same system, we show that a single domain of the alpha receptor is sufficient for the interaction with IgE although with a binding affinity lower than that of the two-domain receptor.

J Theor Biol, 1993 Aug 21, 163(4), 527 - 48
The elements for a classification of units of genetic information with a combinatorial component; Collado-Vides J; An integrative approach to the study of the regulation of gene expression has been undertaken here . The main goal of this approach is to make explicit the common rules that govern the relative location of regulatory sites within operons and other units of genetic information (UGIs) . A classification that emphasizes the regulatory properties of UGIs can be achieved by partitioning UGIs into short sequences with defined properties . Such a classification scheme can be precisely defined as a Grammar with a component of combinatorial (rewriting) rules, and a dictionary component . Sequences have then to be grouped into classes such that any sequence of the same class can mutually substitute and produce novel regulatable UGIs . It is shown here that individual nucleotides cannot define such classes--they are far from equivalent to phonemes . Neither pairs, triplets or any short sequence with a defined number of nucleotides can define productive substitutions . Defined sequences like promoter, operator and activator binding sites are the smallest elements of combinatorial rules within the defined range of transcription initiation of sigma 70 Escherichia coli promoters.

Brain Res Dev Brain Res, 1993 Aug 20, 74(2), 245 - 52
Allocation of mouse cerebellar granule cells derived from embryonic ventricular progenitors--a study using a recombinant retrovirus; Miyake T et al.; Granule cells of the mammalian cerebellar cortex originate from embryonic progenitors present in the ventricular germinal layer . To investigate the allocation fate of these ventricular progenitors in the mouse, we labeled a few of them on embryonic day 13 with a recombinant retrovirus carrying lacZ which encodes E . coli beta-galactosidase (beta-gal), and the labeled cells in the postnatal cerebellar cortex were detected by beta-gal histochemistry . In the postnatal cerebellar cortex, the virally-labeled beta-gal+ granule cells formed discrete clusters . These clusters were not compactly packed with the beta-gal+ cells, and there was intermingling with beta-gal- granule cells . Neither beta-gal+ Purkinje cells nor glia were found to be included in the clusters . Most of the granule cell clusters were incompatible with the functional areas of the cortex . These results suggest: (1) granule cells derived from individual ventricular progenitors are allocated in clusters and are not extensively dispersed, (2) granule cells descended from one progenitor may mix with their neighbors that are descended from another progenitor, (3) the allocation fate of the ventricular progenitors of granule cells is not restricted to the functional areas of the cerebellar cortex.

J Mol Biol, 1993 Aug 20, 232(4), 1213 - 6
Crystallization of a 67 kDa fragment of Escherichia coli DNA topoisomerase I; Lima CD et al.; Escherichia coli DNA topoisomerase I is a well-studied type I DNA topoisomerase that catalyzes the breakage and rejoining of one DNA strand to allow passage of the other strand . We have cloned and over-expressed a 67 kDa amino-terminal fragment of the protein, and shown that it retains the ability of the intact enzyme to cleave single-stranded DNA . High-quality crystals of the purified 67 kDa fragment have been obtained . The crystals belong to space group P2(1)2(1)2(1), with cell dimensions a = 64.0 A, b = 79.9 A and c = 142.3 A . They diffract to at least 2.8 A at low temperature and, when cooled to cryogenic temperatures, to at least 1.9 A in a synchrotron source . A complete native data set and two derivative data sets have been collected . A multiple isomorphous replacement map to 3 A resolution shows clear secondary structural elements . Final structure determination is in progress.

J Mol Biol, 1993 Aug 20, 232(4), 1217 - 20
Trypanosoma cruzi trypanothione reductase . Crystallization, unit cell dimensions and structure solution; Zhang Y et al.; We have reproducibly crystallized recombinant trypanothione reductase from Trypanosoma cruzi . Yellow tetragonal crystals in the shape of elongated prisms have unit cell dimensions of a = 92.8 A, c = 156.6 A, Laue symmetry of 4/m and are suitable for a detailed structural analysis . Diffraction data to 2.7 A resolution have been recorded using synchrotron radiation at the Daresbury laboratory . The structure has been solved by molecular replacement calculations using this synchrotron data and our previously determined Crithidia fasciculata enzyme structure as a search model . The space group has been identified as P4(3) with a homodimer of approximate molecular mass of 108 kDa in the asymmetric unit . Diffraction beyond 2.5 A has been recorded when large freshly grown crystals are exposed to X-rays . Refinement of the structure is in progress.

Biochim Biophys Acta, 1993 Aug 20, 1158(1), 47 - 51
The absence of glyoxylate cycle enzymes in rodent and embryonic chick liver; Holmes RP; There have been several reports over the past decade of the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase, in animal tissues . Reaction products in these assays have been measured principally by chromatographic separation of isotopes or by colorimetric procedures . In this report more sensitive and accurate HPLC and HPCE analyses were used to detect enzymatic activity . Reversed phase HPLC revealed the absence of detectable isocitrate lyase activity in guinea pig, rat and chick embryonic liver . The formation of several other alpha-keto acids was detected and this may account for the previously reported activities . Using HPCE to monitor malate formation malate synthase activity was not detected in these tissues . These results indicate that when assaying enzyme activities in crude tissue homogenates specific methods for the identification of end products are required.

J Mol Biol, 1993 Aug 20, 232(4), 1169 - 75
Crystal structure of an anticholera toxin peptide complex at 2.3 A; Shoham M; Cholera toxin peptide 3 (CTP3) is a 15-residue peptide corresponding in sequence to an immunogenic loop on the surface of the B-subunits of both cholera toxin and the heat-labile toxin from Escherichia coli . TE33 is the Fab fragment of a monoclonal antibody elicited against CTP3 . The crystal structure of the TE33-CTP3 complex at 2.3 A resolution reveals an antigen-binding pocket, 13 A deep and 13 A wide, which is lined with many aromatic residues . The N-terminal portion of the peptide antigen CTP3 forms a type II beta-turn that fits snugly into this pocket . At gln7 the peptide backbone of CTP3 forms a kink followed by an extended C-terminal chain that seals off the cleft and buries the beta-turn underneath it . All six complementarity-determining regions of TE33 contribute to the binding of CTP3 . The antibody-peptide contacts include, in addition to van der Waals' interactions and hydrogen bonds, also one salt bridge and one water molecule, which mediates the interaction.

Biochim Biophys Acta, 1993 Aug 19, 1174(2), 218 - 20
Cloning, expression and characterization of horse L-ferritin in Escherichia coli; Takeda S et al.; Horse L-ferritin cDNA was cloned from horse liver, and the base sequence was determined . The L-ferritin was expressed using pTZ18U encoding lac promoter, and found to possess an additional 8-amino acid sequence at the N-terminus as compared with commercially obtained horse spleen (natural) ferritin . It was determined that there was Pro at position 94 in both the recombinant and natural L-ferritin, although it was previously reported that Leu was in this position in the natural species . Transmission electron microscopy showed that this recombinant ferritin formed a 24-mer shell.

Biochim Biophys Acta, 1993 Aug 19, 1174(2), 153 - 61
Regulation of the uncoupled GTPase activity of elongation factor G (EF-G) by the conformations of the ribosomal subunits; Nagel K et al.; The elongation factor G (EF-G) GTPase activity is induced by either 70S ribosomes or 50S ribosomal subunits . The GTPase activity induced by 50S ribosomal subunits is predominant at low concentrations of monovalent cations and decreases with increasing concentrations of K+ or NH4+ . Double-logarithmic plots of the data reveal straight lines with different slopes for low and high concentrations of monovalent cations, respectively, intersecting at the same concentration of monovalent cations where maximal EF-G GTPase activity is measured in the presence of both ribosomal subunits . Substantially the same curves are obtained when 50S ribosomal subunits are substituted by 50S CsCl-core particles partially reconstituted by addition of purified 50S split proteins L7/L12 . Intact 30S ribosomal subunits, but not 30S CsCl-core particles are able to associate with 50S ribosomal subunits and to modulate ribosome-dependent EF-G GTPase activity . Therefore, our data clearly show that the biphasic courses of the NH4+ and K+ curves of EF-G GTPase activity induced by 50S ribosomal subunits are not due to contaminations with 30S ribosomal subunits but result from different conformations of EF-G/50S ribosomal-subunit complexes at low and high concentrations of monovalent cations, respectively . CD spectra of 50S ribosomal subunits measured under different salt conditions have shown that the conformation of the 50S ribosomal subunits is strongly dependent on the concentration of monovalent cations . The conformation of 30S ribosomal subunits is, however, considerably stronger influenced by the Mg2+ than by the concentration of monovalent cations . The salt effects on the conformation of the 30S ribosomal subunits correspond to the salt effects on the association of ribosomal subunits and the modulation of EF-G GTPase activity by 30S ribosomal subunits . Since, in the presence of both ribosomal subunits, EF-G GTPase activity is maximal at the same concentration of monovalent cations where obviously a spontaneous conformation change of 50S ribosomal subunits takes place, we postulate that EF-G GTPase primarily acts on the ribosomes by changing the conformation of 50S ribosomal subunits . The resulting model is based on the assumption that EF-G GTPase activity is considerably more strongly induced by the 'substrate conformation' ('state I') than by the 'product conformation' of the 50S ribosomal subunits ('state II') . A spontaneous transformation of 'state II' to 'state I' is expected to occur in the absence of mRNA, aminoacyl-tRNA and EF-T especially under salt conditions favouring state I.

Biochim Biophys Acta, 1993 Aug 18, 1178(2), 194 - 200
Identification of the product of the murine ST2 gene; Takagi T et al.; The murine ST2 gene is expressed in growth-stimulated BALB/c-3T3 cells . This gene encodes a protein that is similar to the extracellular portions of the interleukin-1 receptors (types 1 and 2) . In this study, we prepared a polyclonal antibody against the recombinant ST2 protein produced in Escherichia coli . This antibody detected recombinant ST2 protein in the culture fluid of COS7 cells transfected with a mammalian expression vector (pEF-BOS) carrying ST2 cDNA . Using this antibody, we could detect the ST2 protein in the culture fluid of growth-stimulated BALB/c-3T3 cells, and in the medium of continuously growing cells, but not in that of growth-arrested cells . ST2 proteins produced in COS7 cells and BALB/c-3T3 cells were N-glycosylated as predicted from nine putative N-glycosylation sites in its deduced amino-acid sequence.

Biochemistry, 1993 Aug 17, 32(32), 8268 - 75
Interaction of polypyrimidine tract binding protein with the encephalomyocarditis virus mRNA internal ribosomal entry site; Witherell GW et al.; Translation of encephalomyocarditis virus (EMCV) mRNA occurs in a cap-independent manner, requiring instead a cis-acting element termed the internal ribosomal entry site (IRES) . Binding of a 57-kDa ribosome-associated protein (p57) to the EMCV IRES has been found to correlate with cap-independent translation . p57 has recently been reported to be very similar, if not identical, to the polypyrimidine tract binding protein (pPTB), a spliceosome-associated factor possibly involved in U2 snRNP/pre-mRNA complex formation of 3'-splice-site recognition . The interaction between purified pPTB and the EMCV IRES was characterized in this study using nitrocellulose filter binding and UV cross-linking assays . pPTB bound the EMCV IRES with high affinity (Kd = 40 nM at 25 degrees C, pH 5.5, 80 mM ionic strength) . pPTB also bound strongly to RNA fragments containing either the 5'-end, 3'-end, or an internal stem-loop of the IRES . The binding properties of 16 RNA variants derived from the IRES revealed however that purified pPTB bound with less specificity than pPTB in a mixture of cytoplasmic HeLa cell polypeptides . The addition of HeLa extract to purified pPTB increased the binding specificity, suggesting that factors within the extract alter the binding specificity of pPTB . The binding of pPTB to the full-length IRES and three IRES-derived fragments was studied in detail . Complex formation was optimal at low pH and was driven entirely by entropy . As many as four ion pairs are formed upon binding, with electrostatic interactions accounting for approximately 35% of the total free energy of complex formation.

Biochemistry, 1993 Aug 17, 32(32), 8251 - 8
Characterization of the {3Fe-4S} and {4Fe-4S} clusters in unbound PsaC mutants C14D and C51D . Midpoint potentials of the single {4Fe-4S} clusters are identical to FA and FB in bound PsaC of photosystem I; Yu L et al.; In a previous paper we showed that the C51D mutant of PsaC contains a {3Fe-4S} cluster in the FA site and a {4Fe-4S} cluster in the FB site and that the C14D mutant contains an uncharacterized cluster in the FB site and a {4Fe-4S} cluster in the FA site {Zhao, J . D., Li, N., Warren, P . V., Golbeck, J . H., & Bryant, D . A . (1992) Biochemistry 31, 5093-5099} . In this paper we describe the electrochemical and electron spin resonance properties of the recombinant C14D and C51D holoproteins after in vitro reinsertion of the iron-sulfur clusters . Unbound PsaC shows no significant resonances in the oxidized state, but the unbound C14D and C51D mutant proteins show an intense set of resonances at g approximately 2.02 and 1.99 characteristic of an oxidized {3Fe-4S}1+/0 cluster . The Em' values for the {3Fe-4S}1+/0 clusters in C14D (FB*) and C51D (FA*) are -98 mV, and both represent one-electron transfers . After reduction with dithionite at pH 10.0, wild-type PsaC shows a broad set of resonances resulting from the superposition of FA- and FB- characterized by a low-field peak at an apparent g value of 2.051 and a high-field trough at an apparent g value of 1.898 . The FB resonances in C51D were slightly narrower, with a low-field peak at an apparent g value of 2.039 and high-field trough at an apparent g value of 1.908.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Aug 17, 32(32), 8130 - 9
Energetics of intersubunit and intrasubunit interactions of Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein; Cheng X et al.; Escherichia coli cAMP receptor protein (CRP) regulates the expression of a large number of catabolite-sensitive genes . The mechanism of CRP regulation most likely involves communication between subunits and domains . A specific message, such as the activation of CRP, may be manifested as a change in the interactions between these structural entities . Hence, the elucidation of the regulatory mechanism would require a quantitative evaluation of the energetics involved in these interactions . Thus, a study was initiated to define the conditions for reversible denaturation of CRP and to quantitatively assess the energetics involved in the intra- and intersubunit interactions in CRP . The denaturation of CRP was induced by guanidine hydrochloride . The equilibrium unfolding reaction of CRP was monitored by three spectroscopic techniques, namely, fluorescence intensity, fluorescence anisotropy, and circular dichroism . The spectroscopic data implied that CRP unfolds in a single cooperative transition . Sedimentation equilibrium data showed that CRP is dissociated into its monomeric state in high concentrations of denaturant . Unfolding of CRP is completely reversible, as indicated by fluorescence and circular dichroism measurements, and sedimentation data indicated that a dimeric structure of CRP was recovered . The functional and other structural properties of renatured and native CRP have also been examined . Quantitatively identical results were obtained . Results from additional studies as a function of protein concentration and from computer simulation demonstrated that the denaturation of CRP induced by guanidine hydrochloride proceeds according to the following pathway: (CRP2)Native<-->2(CRP)Native<-->2(CRP)Denatured . The delta G values for dissociation (delta Gd) and unfolding (delta G(u)) in the absence of guanidine hydrochloride were determined by linear extrapolation, yielding values of 12.0 +/- 0.6 and 7.2 +/- 0.1 kcal/mol, respectively . To examine the effect of the DNA binding domain on the stability of the cAMP binding domain, two proteolytically resistant cAMP binding cores were prepared from CRP in the presence of cAMP by subtilisin and chymotrypsin digestion, yielding S-CRP and CH-CRP, respectively . Results from an equilibrium denaturation study indicated that the denaturation of both CH-CRP and S-CRP is also completely reversible . Both S-CRP and CH-CRP exist as stable dimers with similar delta Gd values of 10.1 +/- 0.4 and 9.5 +/- 0.4 kcal/mol, respectively . Results from this study in conjunction with crystallographic data {McKay, D . B., Weber, I . T., & Stietz, T . A . (1982) J . Biol . Chem . 257, 9518-9524} indicate that the DNA binding domain and the C-helix are not the only structural elements that are responsible for subunit dimerization.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydr Res, 1993 Aug 17, 246, 219 - 28
Structure of the K10 capsular antigen from Escherichia coli O11:K10:H10, a polysaccharide containing 4,6-dideoxy-4-malonylamino-D-glucose; Sieberth V et al.; The K10 antigen from Escherichia coli O11:K10:H10 consists of equimolar amounts of rhamnose and 4,6-dideoxy-4-malonylaminoglucose {Qui4NMal; 4-(2-carboxyacetamido)-4,6-dideoxyglucose} . Methylation analysis and 1 and 2D NMR spectroscopy showed that the K10 capsular polysaccharide has the structure {formula: see text}

Biochemistry, 1993 Aug 17, 32(32), 8341 - 7
Isoprenoid diphosphate utilization by recombinant human farnesyl:protein transferase: interactive binding between substrates and a preferred kinetic pathway; Pompliano DL et al.; The catalytic utilization of dimethylallyl, geranyl, farnesyl, and geranylgeranyl diphosphates in the reaction catalyzed by recombinant human farnesyl:protein transferase (hFPTase) has been examined in the presence of three different protein substrates, Ras-CVLS, Ras-CVIM, and Ras-CAIL . hFPTase catalyzed both farnesylation and geranylation of Ras-CVLS and of Ras-CVIM but not of Ras-CAIL . Geranylgeranylation was observed, but only when Ras-CVIM was the acceptor substrate . Steady-state initial velocity and dead-end inhibitor studies indicate that hFPTase-catalyzed geranylation, like bovine FPTase-catalyzed farnesylation, proceeds through a random order, sequential mechanism . Surprisingly, however, Michaelis constants for a given protein acceptor substrate varied depending upon which isoprenoid diphosphate was used as the donor substrate, showing that these substrates do not bind independently to the enzyme (under catalytic conditions) . In addition, at very high concentrations of Ras-CVIM, substrate inhibition was observed in the presence of both FPP and GPP . Isotope partitioning studies showed that, at high concentrations of Ras-CVIM, more than 80% of the bound farnesyl diphosphate (FPP) can be trapped as product, suggesting that the binary complex is catalytically competent and that the ternary complex proceeds to product faster than it releases FPP . The release rate of FPP from the binary complex was calculated to be 0.05 s-1, which is only about eight times greater than kcat . Thus, the binding of FPP to the enzyme in the presence of the protein substrate is not an equilibrium situation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Aug 17, 32(32), 8329 - 40
Novel heteronuclear methods of assignment transfer from a diamagnetic to a paramagnetic protein: application to rat cytochrome b5; Guiles RD et al.; 15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferricytochrome b5 have been obtained, using a combination of novel heteronuclear assignment transfer methods from the known assignments of the diamagnetic protein {Guiles, R . D., Basus, V . J., Kuntz, I . D., & Waskell, L . A . (1992) Biochemistry 31, 11365-11375} and computational methods which depend on an accurate determination of the orientation of the components of the susceptibility tensor . The transfer of amide proton resonance assignments takes advantage of the apparent insensitivity of amide 15N resonances to pseudocontact effects, evident in overlays of 15N-1H heteronuclear correlation spectra . Amide-proton resonance assignments tentatively transferred from the known diamagnetic assignments to the paramagnetic form of the protein were confirmed using conventional assignment strategies employing 600-MHz COSY, HOHAHA, and NOESY spectra of the oxidized protein . As was observed in rat ferrocytochrome b5, more than 40% of all residues exhibited NMR detectable heterogeneity due to the two different orientations of the heme . Complete assignment of both forms enabled accurate determination of the orientation of the susceptibility tensor for both conformations of the heme . The orientation of the z-component of the susceptibility tensors for the two forms are indistinguishable, while the in-plane components appear to differ by about 6 degrees . Differences in the orientation of the in-plane susceptibility components are undoubtedly due dominantly to the relative axial rotation of the heme of between 5 degrees and 10 degrees indicated by the NOESY contacts to the protein observed in the spectra of the ferrocytochrome {Guiles, R . D., Basus, V . J., Kuntz, I . D., & Waskell, L . A . (1992) Biochemistry 31, 11365-11375; Pochapsky, T . C., Sligar, S . G., McLachlan, S . J., & LaMar, G . N . (1990) J . Am . Chem . Soc . 112, 5258-5263}.

Biochemistry, 1993 Aug 17, 32(32), 8112 - 9
Catalysis of a protein folding reaction: thermodynamic and kinetic analysis of subtilisin BPN' interactions with its propeptide fragment; Strausberg S et al.; The in vivo folding of subtilisin is dependent on a 77 amino acid propeptide, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme (Ikemura et al., 1987) . We have cloned, expressed, and purified large quantities of the 77 amino acid subtilisin propeptide . This has enabled us to characterize its participation in the subtilisin folding reaction by spectroscopic and microcalorimetric methods . Unfolded subtilisin, when returned to native conditions, is kinetically isolated from its native state . Folding of subtilisin with the native calcium site-A is extremely slow even in the presence of a high concentration of isolated propeptide . The folding of a calcium-free mutant subtilisin, however, is readily catalyzed by the isolated propeptide . The propeptide-subtilisin folding reaction can be described as the following equilibrium: P(u) + S(u)<==>P-S<==>Pf-Sf<==>P(u) + Sf, where S(u) and P(u) are subtilisin and propeptide, respectively, which are largely unstructured at the start of the reaction; P-S is a collision complex of unfolded subtilisin and propeptide; Pf-Sf is the complex of folded subtilisin and propeptide; and Sf is folded subtilisin . The rate-limiting step in the folding reaction of calcium-free mutant subtilisin is formation of the initial collision complex, P-S . The rate at which P(u) and S(u) form a productive collision complex is approximately 500 M-1 s-1 . The collision complex appears to be an early folding unit which, once formed, results in rapid isomerization to the fully folded complex . The rate constant for isomerization of the collision complex to the folded complex is > or = 0.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Aug 17, 32(32), 8092 - 7
Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy; Goldberg JM et al.; The azomethine (Schiff base) linkage between the epsilon-amino group of active-site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'-phosphate (PLP) normally exhibits absorbance maxima at ca . 360 (high-pH form) or ca . 430 nm (low-pH form) . However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (lambda max = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F) . This spectral shift suggested that PLP binds to Y225F as the free aldehyde . The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions . (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine . (3) The Raman spectrum of Y225F is unchanged in H(2)18O, while peaks at ca . 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca . 30 cm-1 in H(2)18O.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1420 - 6
Fully methylated oriC with negative superhelicity forms an oriC-membrane complex before initiation of chromosome replication; Kataoka T et al.; In an in vitro assay, the oriC DNA has been shown to bind to the outer membrane fraction only when it is hemimethylated (G.B . Ogden et al., Cell, 54, 127-135,1988) . In this report, however, we demonstrated that a significant amount of the oriC DNA was recovered from the cells just before initiation with the oriC DNA being fully methylated . Formation of this preinitiation oriC-membrane complex and following initiation of chromosome replication were strongly inhibited by novobiocin, a DNA gyrase B subunit inhibitor, which reduced the superhelicity of the reporter plasmid in the cells . On the other hand, both reactions proceeded in the presence of nalidixic acid, a DNA gyrase A subunit inhibitor, which did not have the effect of reducing the superhelicity . These results suggest that the negative superhelicity of the DNA is required for preinitiation oriC-membrane complex formation and following initiation event of replication.

Gene, 1993 Aug 16, 130(1), 1 - 8
CRP-binding sites: evidence for two structural classes with 6-bp and 8-bp spacers; Barber AM et al.; While classifying protein binding DNA sequences of the type GTGNxCAC, based on the size of Nx {Shumilov, Mol . Biologya (Engl . Transl.) 21 (1987) 168-187}, we had previously found that the cyclic AMP receptor protein (CRP)-binding sites found in the Escherichia coli genome are of at least two classes: (i) those with a conventional 6-bp spacer (N6) and (ii) those with a potential 8-bp spacer (N8) {Barber and Zhurkin, J . Biomol . Struct . Dyn . 8 (1990) 213-232} . In this paper, we present the first experimental evidence that CRP binds to DNA with an N8 spacer with relatively high affinity, as measured by gel electrophoresis of CRP-DNA complexes . We have tested two types of N8 spacers: A+T-rich and G+C-rich . Compared with the affinity of CRP for a reference site with an N6 spacer, the binding strength of CRP toward an A+T-rich N8 sequence is lower and that toward a G+C-rich N8 site is comparable . Just like DNA sites with N6 spacers, those with N8 spacers utilize both halves of the symmetrical protein recognition sequences, TGTGA and TCACA . Because of the increased number of nucleotides in the N8 spacer, the two recognition sequences in DNA will have an increased distance and a helical twist between them . These would cause displacement of the two recognition sequences with respect to the two symmetrically located alpha-helices of the CRP dimer, if there is no change in the DNA conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1500 - 7
Monoclonal antibodies against human neurotrophin-3; Shintani A et al.; Hybridomas producing monoclonal antibodies (MoAbs) against human neurotrophin-3 (hNT-3) were established using recombinant hNT-3 produced in CHO cells and E . coli as immunogens . Of the five MoAbs obtained, MoAb 3w3 showed the highest antibody titer and also best neutralized NT-3 activity as measured by the survival of chick embryonic day-8 dorsal root ganglia neurons . A sandwich enzyme immunoassay (EIA) for NT-3 was established with solid phase MoAb 3W3 and the Fab' fragment of MoAb 3W3 conjugated to horseradish peroxidase . The detection limit was 2.7 pg/well of NT-3 and no cross-reactivity with nerve growth factor up to 100 ng/well was observed . Using this EIA system we have screened a variety of cell lines for NT-3 production . Among these tested, only human Burkitt's lymphoma Namalwa cells were found to be producing NT-3.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1466 - 74
Identification of ionizable groups essential for the enzyme catalysis on glutathione S-transferase P; Nishihara J et al.; The pH-Vmax/KmGSH plot of glutathione S-transferase P (GST-P) showed a bell-shaped profile, indicating bifunctional catalysis for glutathione (GSH) conjugation . The ionization constant (Ke) and the heat of ionization (delta He) of the essential ionizable group in the GSH binding site were measured and the value of pKe1 was 5.9 and that of pKe2, 8.4, while the delta He1 and delta He2 were -0.2 and 7.9 kcal/mole, respectively . In a solvent containing 25% ethanol, pKe1 and pKe2 shifted to the alkaline side by 0.47 and 0.2, respectively . These kinetic results indicated that carboxyl and phenolic groups were ionizable groups essential for the GSH conjugation . Chemical modifications using aminomethane sulfonic acid and N-acetylimidazole supported the results of the kinetic studies.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1453 - 9
Cloning a novel form of rat PDGF A-chain with a unique 5'-UT: regulation during development and in glomerulonephritis; Feng L et al.; An unique form of rat platelet-derived growth factor A-chain (PDGF A-chain), with a novel 5' UT region, was cloned from a rat macrophage cDNA library and expressed . In the 5' UT, the homology of the 79 bp sequence adjacent to the ATG codon between rat and human was 92%; however, the homology of the remainder in the 5' UT was less than 30% . RNase mapping indicated this form was differentially expressed during development and immune glomerular injury, and that it probably arose from alternative splicing . We propose that the variant mRNAs reflect different levels of the control of PDGF A-chain expression.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1440 - 5
The determinants of the dimeric structure of seminal ribonuclease are located in its N-terminal region; Di Donato A et al.; A chimeric cDNA was constructed, coding for the N-terminal region of BS-RNase (residues 1-49) and the C-terminal region of RNase A (residues 50-124) . The resulting chimeric DNA was expressed in Escherichia coli and found to code for RNase chains that spontaneously assembled into a covalently dimeric ribonuclease.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1288 - 91
A novel domain sequence of connectin localized at the I band of skeletal muscle sarcomeres: homology to neurofilament subunits; Maruyama K et al.; A cDNA of 4.0 kb was cloned from a chicken embryo skeletal muscle cDNA library, using an antibody to muscle elastic protein connectin (titin), the molecular mass of which is estimated to be 3,000 kDa . Immunoelectron microscopy revealed that the antiserum raised against the product of the cDNA expressed in E . coli bound to the epitopes of the connectin filament near the N2 line of chicken breast muscle sarcomeres . The predicted amino acid sequence contains eight immunoglobulin C2 motifs and regions highly homologous with the high and medium molecular weight subunits of neurofilament . In addition, there are regions homologous with desmoplakin, calsequestrin, and calpastatin.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1008 - 11
Crystallisation and preliminary crystallographic analysis of recombinant Xenopus laevis Cu,Zn superoxide dismutase b; Carugo KD et al.; The recombinant Cu,Zn superoxide dismutase from the South African frog Xenopus laevis, expressed in E . coli, has been crystallized in a form suitable for high resolution crystallographic investigations . The crystals grow from polyethylene glycol solutions, at pH 6.0, 28 degrees C, and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell edges a = 73.33, b = 68.86, c = 59.73 A, one protein dimer (32,000 M(r)) per asymmetric unit . Diffraction data have been collected to 3.0 A resolution, and a molecular replacement solution found for Xenopus laevis superoxide dismutase using the bovine enzyme as search model . The crystallographic R-factor corresponding to this solution is 0.412, in the 15.0-3.0 A resolution range.

Biochim Biophys Acta, 1993 Aug 16, 1144(1), 77 - 84
Membrane-associated redox cycling of copper mediates hydroperoxide toxicity in Escherichia coli; Rodriguez-Montelongo L et al.; We are studying the action of tert-butylhydroperoxide (t-BOOH) on Escherichia coli as a model system for peroxide toxicity . In our previous report (De la Cruz-Rodriguez, L.C., Farias, R.N . and Massa, E.M . (1990) Biochim . Biophys . Acta 1015, 510-516), the respiratory chain was identified as a major target of t-BOOH . In the present paper, we study further the effect of t-BOOH on the NADH oxidase of the E . coli respiratory chain to clarify the mechanism of damage, especially regarding the identity and role of the metal ion involved . The results are: (a) t-BOOH toxicity is mediated by membrane-bound copper ions; (b) a small pool of the membrane-bound copper is reduced from Cu(II) to Cu(I) in the presence of NADH and other respiratory substrates (succinate, D-lactate); (c) this reduction of copper occurs at 37 degrees C but not at 0 degrees C or when the membranes are inactivated by previous heating; (d) the Cu(I) generated by reduction of Cu(II) during membrane preincubation with NADH, is oxidized by t-BOOH with simultaneous inactivation of the NADH oxidase, whereas treatment with only t-BOOH (without NADH) has no effect on the oxidase . It is concluded that the effect of t-BOOH on the respiratory chain is mediated by redox cycling of copper . It is proposed that the damage results from activation of the hydroperoxide through its interaction with Cu(I) in a site-specific Fenton-type reaction.

Biochim Biophys Acta, 1993 Aug 16, 1144(1), 17 - 21
Functional stability of the a-subunit of the F0F1-ATPase from Escherichia coli is affected by mutations in three proline residues; Howitt SM et al.; Site-directed mutagenesis was used to investigate the roles of three proline residues (Pro-103, Pro-122 and Pro-143) in the a-subunit of the E . coli F0F1-ATPase . All three were found to have a role in stabilizing the a-subunit structure in that removal of the F1-ATPase from membranes prepared from each of the mutant strains resulted in the loss of passive proton translocation activity . Pro-103 is predicted to be within a transmembrane helix . Pro-122 and Pro-143 are located just outside the membrane and near two residues (Asp-124 and Arg-140) previously proposed to form a charge pair . The phenotype of mutants in which Pro-122 or Pro-143 were replaced by alanine was similar to previously isolated mutants affected in Asp-124 and Arg-140 . This suggested that the main effect of the mutations was to destroy the charge pair between Asp-124 and Arg-140 . Double mutants resulting from all possible combinations of these four mutations were constructed and, with the exception of P122A + D124A, had a similar phenotype to the single mutants . This is consistent with the idea that all four single changes had the same effect on a-subunit structure . In contrast, combining the P122A or P143A changes with another mutation which caused a similar phenotype (D44N) resulted in a complete loss of oxidative phosphorylation.

Gene, 1993 Aug 16, 130(1), 9 - 14
Histidine-tagged RNA polymerase: dissection of the transcription cycle using immobilized enzyme; Kashlev M et al.; A stretch of six histidine residues (His6) has been genetically fused to the C terminus of the beta' polypeptide of Escherichia coli RNA polymerase . The His6-tagged beta' subunit assembles into RNA polymerase molecules which perform all vital in vivo functions and behave qualitatively normally in vitro . The His6 tag permits rapid purification of the enzyme directly from crude cell extracts or from an in vitro reconstitution reaction by adsorption to Ni(2+)-chelating agarose resin, followed by elution with imidazole . The enzyme bound to the matrix remains transcriptionally active . The immobilized enzyme can withstand repeated buffer changes without substantial activity loss and permits controlled stepwise 'walking' of the transcriptional complex along the DNA template, and isolation of defined intermediates in the transcription cycle . The immobilized RNA polymerase provides a powerful experimental system for structural and functional analysis of RNA polymerase and its interaction with regulatory factors.

Gene, 1993 Aug 16, 130(1), 155 - 6
Sequence of the Escherichia coli K-12 edd and eda genes of the Entner-Doudoroff pathway; Carter AT et al.; A 3.5-kb DNA fragment from the Clarke and Carbon Escherichia coli genomic clone, pLC37-44, was sequenced on both strands . Part of the zwf gene, encoding glucose-6-phosphate dehydrogenase, and all of the edd and eda genes, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, respectively, of the Entner-Doudoroff pathway, were identified . These data are compared with those of Egan et al . {J . Bacteriol . 174 (1992) 4638-4646} and important differences were noted.

Gene, 1993 Aug 16, 130(1), 15 - 22
Construction and characterization of a novel cross-regulation system for regulating cloned gene expression in Escherichia coli; Chen W et al.; A novel cross-regulation system employing two pairs of interacting promoter-repressor systems was constructed using the tac-lacI and lambda pL-cI promoter-operator-repressor systems . In particular, transcription of the cat gene and the fused cI gene is regulated by the tac promoter, while transcription of the lacI gene is controlled by the lambda pL promoter . In order to compare CAT production from this new system with a currently employed transcription control configuration, a control expression vector utilizing the constitutive repressor synthesis configuration was also constructed . In this construct, cat is under the control of the tac promoter, and lac repressor is provided from a single copy of the lacIq allele included in the plasmid . Induction results using different copy number vectors indicate that induced cat expression levels are at least twofold higher using the cross-regulation system which has very low basal expression . These results match well with previous mathematical modeling predictions indicating excellent control of basal expression and also higher cloned-gene expression post-induction over a broad range of copy numbers for a cross-regulation control configuration . Induction of the cross-regulation system both up-regulated the activation pathway and down-regulated the inhibition pathway, shifting the system steady-state from lac repressor expression to cat and cI expression . The control strategy presented here should be equally applicable to regulate transcription in diverse hosts.

FEMS Microbiol Lett, 1993 Aug 15, 112(1), 43 - 8
Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis; Kupke T et al.; For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems . Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion . The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing . For EpiA detection, anti-EpiA antisera were raised . Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R-1 and I+1 . The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A(-4)-E-P-R(-1)- to -A(-4)-E-P-Q(-1)- by site-directed mutagenesis.

FEMS Microbiol Lett, 1993 Aug 15, 112(1), 19 - 24
Investigation of the role of the cydD gene product in production of a functional cytochrome d oxidase in Escherichia coli; Bebbington KJ et al.; The cydD gene is needed for the formation of a functional cytochrome d oxidase in the aerobic respiratory chain of Escherichia coli . In this paper we demonstrate that transcription from a cydA-lacZ gene fusion is not significantly affected in a cydD mutant . This, together with the finding that subunit I of cytochrome d is present in cytoplasmic membranes of a cydD mutant, rules out a role for CydD in the regulation of cytochrome d expression or the assembly of its polypeptides into the membrane . The activity of the haem d-containing catalase HP II was found to be similar in a cydD mutant and the wild-type . Therefore, if CydD has a role in haem d biosynthesis it must be in a unique step only associated with the synthesis of the haem d prosthetic group of cytochrome d.

Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7431 - 5
Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank; Zwaal RR et al.; To understand how genotype determines the phenotype of the animal Caenorhabditis elegans, one ideally needs to know the complete sequence of the genome and the contribution of genes to phenotype, which requires an efficient strategy for reverse genetics . We here report that the Tc1 transposon induces frequent deletions of flanking DNA, apparently resulting from Tc1 excision followed by imprecise DNA repair . We use this to inactivate genes in two steps . (i) We established a frozen library of 5000 nematode lines mutagenized by Tc1 insertion, from which insertion mutants of genes of interest can be recovered . Their address within the library is determined by PCR . (ii) Animals are then screened, again by PCR, to detect derivatives in which Tc1 and 1000-2000 base pairs of flanking DNA are deleted, and thus a gene of interest is inactivated . We have thus far isolated Tc1 insertions in 16 different genes and obtained deletion derivatives of 6 of those.

J Biol Chem, 1993 Aug 15, 268(23), 17471 - 7
Characterization of promoter recognition complexes formed by CRP and CytR for repression and by CRP and RNA polymerase for activation of transcription on the Escherichia coli deoP2 promoter; Mollegaard NE et al.; The structure of the cAMP-CRP-CytR repression complex and the cAMP CRP-RNA polymerase initiation complex at the deoP2 promoter of E . coli have been probed by DNase I and uranyl footprinting . In the CRP2-CytR complex all protein DNA-phosphate contacts at CRP-1 and CRP-2 are retained, and in addition two new minor groove contacts, ascribed to phosphate-CytR interactions, are observed at -60 between the CRP sites . The contacts are compatible with a model in which the promoter DNA is wrapped around a complex of two CRPs and one CytR . In the RNA polymerase-CRP complex, the CRP-1 phosphate contacts are almost identical to those seen in the repression complex and strong RNA polymerase contacts are seen in the -10 and in the +10 regions . Most noteworthy are minor groove contacts in the -60 region ascribed to RNA polymerase contacts upstream from the CRP . Furthermore, binding of CRP to the CRP-2 target does not seem to interfere with RNA polymerase binding . Thus, a model is suggested in which the DNA is wrapped around a complex of RNA polymerase and one CRP . Finally, the results show that CytR and RNA polymerase are rivals that compete for binding with CRP at deoP2 and that CytR functions as an antiactivator.

J Biol Chem, 1993 Aug 15, 268(23), 17185 - 9
Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins . II . Interaction of RuvB with DNA; Muller B et al.; Using recombination intermediates made by RecA protein, we have shown that the Escherichia coli RuvB protein can mediate the branch migration of Holliday junctions in vitro . The reaction is dependent on the presence of > or = 10 mM Mg2+ and stoichiometric amounts of RuvB . The presence of E . coli RuvA protein reduces the requirement for Mg2+ and also the stoichiometric requirement for RuvB (Muller, B., Tsaneva, I . R., and West, S . C . (1993) J . Biol . Chem . 268, 17179-17184) . To determine the roles of the two proteins during branch migration, we have investigated the interaction of RuvB with DNA in the absence or presence of RuvA, by (i) gel retardation of protein-DNA complexes, (ii) stimulation of the RuvB ATPase, and (iii) protection of DNA from DNase I . The interaction of RuvB with duplex DNA was Mg(2+)-dependent and correlated with the Mg2+ requirement of the RuvB-mediated branch migration reaction . RuvB also interacted with ssDNA, but the affinity was significantly lower than for duplex DNA . In contrast to RuvB, the interaction of RuvA with duplex DNA occurred in the absence of Mg2+ and was inhibited by Mg2+ in a concentration-dependent manner . At 5 mM Mg2+, RuvA protein facilitated the interaction of RuvB with DNA, leading to the formation of a complex containing RuvA, RuvB, and duplex DNA.

J Biol Chem, 1993 Aug 15, 268(23), 17179 - 84
Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins . I . Comparison of RuvAB- and RuvB-mediated reactions; Muller B et al.; The Escherichia coli RuvA and RuvB proteins mediate the branch migration of Holliday junctions in vitro . In the presence of stoichiometric amounts of RuvB (1 RuvB dimer/12 nucleotides), branch migration can occur without need for RuvA . However, RuvA is required when the RuvB concentration is reduced 4-fold or more . Under optimal conditions, we found the minimal protein requirement to be 1 RuvB dimer per 500-1100 nucleotides and 1 RuvA tetramer per 600-1200 nucleotides . To determine the roles of RuvA and RuvB in branch migration, we compared branch migration reactions mediated by RuvB only and by RuvA and RuvB . The time courses of the two reactions were similar, and both required ATP and Mg2+ . However, RuvB-mediated branch migration occurred at lower ATP concentrations (> or = 200 microM) and higher Mg2+ concentrations (> or = 10 mM MgCl2) than the reaction mediated by RuvA and RuvB (> or = 1 mM ATP, > or = 5 mM MgCl2) . The Mg2+ requirement for RuvB-mediated branch migration reflects the Mg2+ requirement of RuvB for DNA binding (Muller, B., Tsaneva, I.R., and West, S . C . (1993) J . Biol . Chem . 268, 17185-17189) and can be overcome by addition of RuvA . These results indicate that RuvA protein facilitates the interaction of RuvB with DNA.

J Biol Chem, 1993 Aug 15, 268(23), 17155 - 61
Molecular cloning and characterization of a 42-kDa protein phosphatase of Leishmania chagasi; Burns JM Jr et al.; Using rabbit serum raised against a potent T cell-stimulating antigen fraction of Leishmania chagasi promastigotes, we have cloned and expressed the Leishmania type 2C serine/threonine protein phosphatase, LcPP2C . LcPP2C was shown to be present as a 42-kDa protein in both the infective promastigote and tissue amastigote stages of L . chagasi and Leishmania amazonensis . DNA hybridization studies established the close conservation of LcPP2C among eight of eight geographically diverse species of Leishmania which cause a spectrum of human diseases . To support the relationship between LcPP2C and mammalian type 2C protein phosphatases observed through predicted amino acid sequence comparisons, we expressed enzymatically active rLcPP2C in Escherichia coli . We demonstrated that purified rLcPP2C readily dephosphorylated {32P}casein, an activity dependent on Mg2+ and insensitive to okadaic acid . In agreement with studies of rat liver PP2C, activity was maintained when Mg+2 was replaced with Mn+2 but not with Ca2+ . As these parameters are characteristic of the eukaryotic type 2C serine/threonine protein phosphatases, LcPP2C can be classified as a member of this protein family . We further showed that of the four major classes of eukaryotic serine/threonine protein phosphatases, PP2C-and PP1-like activities, are readily detectable in Leishmania.

J Biol Chem, 1993 Aug 15, 268(23), 16895 - 8
NAD glycohydrolase specifically induced by retinoic acid in human leukemic HL-60 cells . Identification of the NAD glycohydrolase as leukocyte cell surface antigen CD38; Kontani K et al.; Human leukemic HL-60 cells are differentiated into granulocytic cells by retinoic acid, and this differentiation is preceded by the induction of an ecto-enzyme of NAD glycohydrolase (NADase) . The NADase specifically induced by retinoic acid appeared to be encoded by human leukocyte cell surface antigen CD38 as follows . 1) There was an early expression of CD38 mRNA, together with the induction of the NADase activity, in the retinoic acid-treated HL-60 cells . 2) The time course of the expression of CD38 antigen on the cell surface was well correlated with that of the induction of NADase activity . 3) The NADase activity solubilized from the differentiated HL-60 cell membrane could be immunoprecipitated with an anti-CD38 monoclonal antibody . 4) Introduction of the CD38 cDNA into Escherichia coli cells resulted in the expression of an NADase, the activity of which was inhibited by dithiothreitol . The NADase activity in the differentiated cells was also inhibited by the reducing reagent . These results clearly indicated that the dithiothreitol-sensitive NADase activity induced by retinoic acid in HL-60 cells is attributed to the molecule of human leukocyte cell surface antigen CD38, which contains cysteine-rich cytoplasmic domain within its molecule.

Eur J Biochem, 1993 Aug 15, 216(1), 293 - 9
Expression of the laminin-A chain is down-regulated by a non-canonical polyadenylation signal; Gottschling C et al.; It is well accepted that 3' untranslated regions (UTR) are an essential part of mRNA . However, little is known in detail about the contribution of different regions of 3' UTR on synthesis, stability and translatability of their mRNA . In addition to the highly conserved hexanucleotide AAUAAA, some consensus sequences for 3'-end processing and polyadenylation have been characterized, but most of this work has been done with viral mRNA or beta-globin mRNA . We have studied the influence of the 3' UTR of the mRNA for the three chains A, B1, B2 of laminin on the expression of a reporter gene (galK) . Laminin is a large glycoprotein of basement membranes and all three polypeptide chains are needed in equal amounts for a functional molecule . The three 3' UTR of the laminin mRNA differ widely with respect to length, number of polyadenylation signals and other consensus sequences . Nevertheless, all three 3' UTR reduce the expression of the reporter gene at least three-fold, when the corresponding cDNA sequences are inserted downstream of the reporter gene instead of the 3' UTR of simian virus 40 early genes . The 3' UTR of laminin-A mRNA contains the non-canonical polyadenylation signal AUUAAA which seems to be responsible for the limiting amounts of laminin-A mRNA and protein compared to those for laminin B1 and B2 {Speth and Oberbaumer (1993) Exp . Cell Res . 204, 302-310} . Mutation of the laminin-A polyadenylation signal to the canonical form AAUAAA increases expression by a factor of 2.5.

Eur J Biochem, 1993 Aug 15, 216(1), 239 - 45
Recombinant soluble human interleukin-6 receptor . Expression in Escherichia coli, renaturation and purification; Stoyan T et al.; The recombinant soluble human interleukin-6 receptor (srhIL-6R) was expressed in Escherichia coli as a non-glycosylated protein comprising the first 339 amino acids after the signal peptide . The protein accumulated within the cells as insoluble protein aggregates (inclusion bodies) . After solubilization, 10% of the denatured srhIL-6R could be renaturated by an in vitro folding procedure using L-arginine and the glutathione-redox system . The native receptors were purified to near homogeneity by affinity chromatography on an IL-6-Sepharose column . The functional features of the recombinant soluble receptor were further analysed . A part of the extracellular domain (amino acids 145-345) of the human interleukin-6 receptor (IL-6R) was expressed in E . coli and the purified protein was used to raise antibodies in rabbits . Characterization of the antiserum obtained indicated that an epitope of 13 amino acids close to the transmembrane region is needed for recognition by the antibodies . Since the antiserum obtained did not interfere with IL-6 binding, it could be used to establish a cell-free IL-6-binding assay, In this assay, the srhIL-6R bound IL-6 with an affinity of Kd = 1.5 nM as measured by Scatchard-plot analysis . When 125I-IL-6 was chemically cross-linked to the purified srhIL-6R and analyzed by SDS/PAGE, several 125I-IL-6-containing bands were detected, indicating the possible existence of a multimeric structure of the natural IL-6/IL-6R complex . The srhIL-6R was shown to exhibit biological activity, i.e . it stimulated acute-phase protein synthesis in the recently established human hepatoma cell line HepG2-IL-6 which does not express the IL-6-binding subunit of the IL-6R complex on the cell surface.

Eur J Biochem, 1993 Aug 15, 216(1), 103 - 7
Zinc coordination in mammalian sorbitol dehydrogenase . Replacement of putative zinc ligands by site-directed mutagenesis; Karlsson C et al.; Rat sorbitol dehydrogenase was expressed in Escherichia coli and purified to homogeneity, resulting in a protein with a specific activity of 4.7 U/mg, close to that of the enzyme isolated from mammalian liver . A Glu residue has been postulated to replace the Cys of alcohol dehydrogenase as a ligand to the active-site zinc atom of sorbitol dehydrogenase . This Glu (position 155 in the rat enzyme) was mutated both to Cys, in order to mimic the alcohol dehydrogenase relationships, and to Ala, as a control . A third mutation, Cys164 to Ala, was also performed since Cys has also been considered as a possible zinc ligand . With Ala at position 155, an inactive enzyme was obtained, showing that correct active-site relationships have been destroyed . With Cys at position 155, the enzyme is still partly active, but rapidly looses activity unless stabilized by the addition of ZnSO4 . The catalytic efficiency in the oxidation of sorbitol is 120-fold less than that of the native form, and reduction of fructose is lost completely . In contrast, the activity of the Cys164Ala mutant is comparable with that of the native enzyme and, in fact, even increased in the oxidation of sorbitol . Combined, the results strongly suggest that Glu155 is a ligand to the active-site zinc atom . Zinc analysis of the different variants of sorbitol dehydrogenase establishes that all contain one atom of zinc/subunit, also when the catalytic function is lost . Apparently, zinc remains coordinated even after replacement with an amino acid residue (Ala) unable to ligand metal atoms.

Biochem J, 1993 Aug 15, 294 ( Pt 1), 79 - 86
Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms; Brown NF et al.; cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli . In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated . The precursor form of the enzyme was not proteolytically processed . Removal of 126 bp from the 5' end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive . cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E . coli . However, the impediment was overcome when the latter cDNA was ligated in-frame 3' to sequence encoding a glutathione S-transferase . This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active . In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves . Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase . Thus rat CPT II can be expressed in active form in E . coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule . An alternative expression system will be needed for similar studies on human CPT II.

Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7879 - 83
Targeted mutagenesis of DNA using triple helix-forming oligonucleotides linked to psoralen; Havre PA et al.; Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA . Here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome . Site-specific triplex formation delivers the psoralen to the targeted site in the lambda DNA, and photoactivation of the psoralen produces adducts and thereby mutations at that site . Mutations in the targeted gene were at least 100-fold more frequent than those in a nontargeted gene, and sequence analysis of mutations in the targeted gene showed that 96% were in the targeted region and 56% were found to be the same T.A to A.T transversion precisely at the targeted base pair . The ability to reproducibly and predictably target mutations to sites in intact duplex DNA by using modified oligonucleotides may prove useful as a technique for gene therapy, as an approach to antiviral therapeutics, and as a tool for genetic engineering.

Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7671 - 5
In vivo and in vitro evidence for slipped mispairing in mammalian mitochondria; Madsen CS et al.; Slipped mispairing between repeated sequences during DNA replication is an important mutagenic event . It is one of several suggested mechanisms thought to be responsible for generating polymorphic regions and large-scale deletions found in mammalian mitochondrial DNA . In the porcine mitochondrial genome, a domain carrying a 10-bp tandemly repeated sequence displays a unique in vivo pattern of repeat copy number polymorphs . Upon passage in Escherichia coli, a recombinant plasmid containing this domain also displays a unique polymorphic pattern that is different from that seen in the animal . To test the hypothesis that these polymorphisms were slippage induced and that the different polymorphic patterns reflected differences in modes of replication, we performed a series of in vitro primer extension reactions . By utilizing either single- or double-stranded templates containing the repeat domain we were able to correlate in vitro generated repeat polymorphism patterns with those seen in the mitochondria or the bacteria, respectively, thus providing experimental evidence that slippage replication is responsible for a major class of mammalian mutations.

J Biol Chem, 1993 Aug 15, 268(23), 17623 - 7
Isolation and characterization of a cDNA encoding rat liver cytosolic epoxide hydrolase and its functional expression in Escherichia coli; Knehr M et al.; A cDNA of 1992 base pairs encoding the complete rat liver cytosolic epoxide hydrolase has been isolated using a polymerase chain reaction-derived DNA fragment (Arand, M., Knehr, M., Thomas, H., Zeller, H . D., and Oesch, F . (1991) FEBS Lett . 294, 19-22) known to represent the 3'-end of the cytosolic epoxide hydrolase mRNA . Sequence analysis revealed an open reading frame of 1662 nucleotides corresponding to 554 amino acids (M(r) = 62,268) . The DNA sequence obtained did not display significant homology to the sequences of microsomal epoxide hydrolase or leukotriene A4 hydrolase or to any other DNA included in the EMBL Data Bank (release 32) . On Northern blotting of rat liver RNA, a single mRNA species was detected that was strongly induced on treatment of the animal with fenofibrate, a potent peroxisome proliferator . The most significant structure of the deduced protein is a modified peroxisomal targeting signal (Ser-Lys-Ile) at the carboxyl terminus that is regarded to be responsible for the unusual dual localization of the cytosolic epoxide hydrolase in peroxisomes as well as in the cytosol . In addition, a leucine zipper-like motif was identified at the amino terminus . Its possible implication for the observed dimeric structure of cytosolic epoxide hydrolase is discussed . The isolated cDNA was expressed in bacteria to yield a catalytically active enzyme . Specific activity of the crude lysate obtained exceeded that of rat liver cytosols from maximally induced animals by a factor of 8.

J Biol Chem, 1993 Aug 15, 268(23), 17602 - 12
Model of lactose repressor core based on alignment with sugar-binding proteins is concordant with genetic and chemical data; Nichols JC et al.; Using primary sequence similarity to arabinose-binding protein, D-glucose/D-galactose-binding protein, and ribose-binding protein (Vyas, N . K., Vyas, M . N., and Quiocho, F . A . (1991) J . Biol . Chem . 266, 5226-5237; Mowbray, S . L., and Cole, L . B . (1992) J . Mol . Biol . 225, 155-175), the core domain (residues 62-323) of the bacterial regulatory protein lac repressor has been aligned to these sugar-binding proteins of known structure . Although the sequence identity is not striking, there is strong overall homology based on two separate matrix scoring systems (minimum base change per codon (MBC/C) and amino acid homology per residue (AAH/R)) (mean score: MBC/C < 1.25, AAH/R > 5.50; random sequences: MBC/C = 1.45, AAH/R = 4.46) . Similarly, the predicted secondary structure of the repressor exhibits excellent agreement with the known secondary structures of the sugar-binding proteins . Using this primary sequence alignment, the tertiary structure of the core domain of the lac repressor has been modeled based on the known structures of the sugar-binding proteins as templates . While the structure deduced for the repressor is hypothetical, the model generated allows a comparison between the predicted tertiary arrangement and the wealth of genetic and chemical data elucidated for the repressor . Important residues involved in operator and sugar binding and in protein assembly have been identified using genetic methods, and placement of these residues in the model is consistent with their known function . This approach, therefore, provides a means to visualize the core domain of the lac repressor that allows interpretation of genetic and chemical data for specific residues and rational design of future experiments.

J Biol Chem, 1993 Aug 15, 268(23), 17253 - 60
Isolation and heterologous expression of cloned cDNAs for two rabbit nasal microsomal proteins, CYP2A10 and CYP2A11, that are related to nasal microsomal cytochrome P450 form a; Peng HM et al.; Nasal microsomal P450 form a (NMa), a major cytochrome P450 isozyme in rabbit olfactory and respiratory nasal mucosa with high activity toward a variety of odorants and environmental toxicants, was previously purified to electrophoretic homogeneity from rabbit nasal microsomes . In the present study, a cDNA library constructed from poly(A)+ RNA from rabbit respiratory nasal mucosa was screened with antibodies to P450 NMa, and five immunopositive clones were isolated and characterized . Sequence analysis indicated that the clones encode two highly similar P450s that contain 494 amino acid residues, with the first 20 corresponding to P450 NMa, and differ from each other in only 8 residues scattered throughout the polypeptide chains . On the basis of structural homology the two proteins are designated as CYP2A10 and CYP2A11 and are the first members of the P450 2A subfamily to be identified in nasal tissue . Genomic blot analysis indicated that 2A10 and 2A11 are apparently not allelic variants . Both genes are expressed in liver and lung as well as in nasal tissues, as judged by RNA blot analysis, but the relative levels of the two mRNAs differ . Both enzymes were partially purified after expression of the cDNAs in Escherichia coli and shown to catalyze the oxygenation of a variety of substrates, including ethanol and procarcinogens such as N-nitrosodiethylamine and phenacetin . P450 2A10 is generally more active than P450 2A11 and strikingly so in the conversion of testosterone to androstenedione.

J Biol Chem, 1993 Aug 15, 268(23), 17145 - 50
Generation and characterization of a competitive antagonist of human hepatocyte growth factor, HGF/NK1; Lokker NA et al.; Our previous studies have suggested that a derivative of hepatocyte growth factor (HGF), HGF/NK2, containing the coding sequences for the N-terminal hairpin and first two kringle domains, is sufficient to mediate high affinity binding to the HGF receptor . Here, we wished to test directly whether HGF/NK1 (N-terminal hairpin and first kringle domains) could bind the receptor and/or mediate receptor signaling . HGF/NK1 was expressed in Escherichia coli and purified to homogeneity using heparin-affinity and fast protein liquid cation-exchange chromatography . Biological characterization of HGF/NK1 showed that it can compete for binding to the HGF receptor on human lung carcinoma A549 cells and to a soluble form of the HGF receptor . HGF/NK1 is inefficient at promoting autophosphorylation of the HGF receptor, although some activity was detected at very high concentrations . HGF/NK1 fails to exhibit mitogenic properties even at very high concentrations . However, HGF/NK1 can act as a potent competitive antagonist in this assay . Our data demonstrate directly that a receptor binding determinant of HGF is located within the N-terminal 32-212 residues of HGF . HGF/NK1 will serve as a powerful tool for (i) generating neutralizing antibodies, (ii) in determining x-ray crystallographic and nuclear magnetic resonance structures, and (iii) for in vivo studies as an HGF antagonist.

J Biol Chem, 1993 Aug 15, 268(23), 17126 - 30
Charge pair interactions stabilizing ferredoxin-ferredoxin reductase complexes . Identification by complementary site-specific mutations; Brandt ME et al.; Ferredoxin reductase (Fd-reductase) supplies electrons to mitochondrial steroid hydroxylase cytochrome P450 enzymes via a {2Fe-2S} ferredoxin . Chemical labeling studies with bovine Fd-reductase have implicated Lys-243 as important in binding to bovine ferredoxin (Hamamoto, I., Kazutaka, K., Tanaka, S., and Ichikawa, Y . (1988) Biochim . Biophys . Acta 953, 207-213) . We have used site-directed mutagenesis to examine the role of charged residues in this region of human Fd-reductase in ferredoxin binding . Mutant proteins were expressed in Escherichia coli and were assayed for activity by ferredoxin-mediated electron transfer to cytochrome c . Replacement of Lys-242 (homologous to Lys-243 in bovine Fd-reductase) with Gln and replacement of Arg-241 with Ser had little effect (2.7- and 3.6-fold increased Km, respectively) . In contrast, mutants at positions 239 and 243 (R239S and R243Q) exhibited markedly lower affinity for ferredoxin (17.5- and 1,600-fold increased Km, respectively) . Studies were also carried out with two ferredoxin charge mutants shown previously to have lowered affinity for Fd-reductase (Coghlan, V . M., and Vickery, L . E . (1991) J . Biol . Chem . 266, 18606-18612) . Comparisons of the binding of ferredoxin mutants D76N and D79N to Fd-reductase mutants R239S and R243Q suggest that Arg-239 and Arg-243 of Fd-reductase each interact directly with both Asp-76 and Asp-79 of ferredoxin during formation of the complex between the two proteins . These results support the hypothesis that specific electrostatic interactions involving this region are important in stabilizing the ferredoxin-Fd-reductase complex.

J Biol Chem, 1993 Aug 15, 268(23), 17069 - 73
OmpF-Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol-signal sequence interaction during protein translocation across the Escherichia coli inner membrane; Phoenix DA et al.; Using inverted Escherichia coli inner membrane vesicles we have analyzed the phosphatidylglycerol dependence of translocation of an OmpF-Lpp fusion protein carrying a signal sequence with varying positive charge at the N terminus and a hydrophobic core of varying length . It is shown that there is a direct relationship between the phosphatidylglycerol requirement of translocation and the requirement within the translocation process for positive charges on the signal sequence . This provides further evidence that the negative head group of the lipid is required for functional interaction with the positively charged N terminus of the signal sequence.

J Biol Chem, 1993 Aug 15, 268(23), 16903 - 6
Human ABR encodes a protein with GAPrac activity and homology to the DBL nucleotide exchange factor domain; Heisterkamp N et al.; We have previously cloned a segment of a gene, ABR, homologous to the BCR gene, which encodes a protein consisting of three distinct functional domains . In the present study, genomic ABR sequences were used to isolate human ABR cDNAs . Surprisingly, the two types of ABR cDNAs identified differed only in their most 5' coding sequences . These are predicted to encode proteins of 93.5 and 92.3 kDa molecular mass . ABR showed a differential expression pattern in various mouse tissues, analogous to that of BCR, and the highest level was found in brain . Similar to BCR, ABR contains a region with homology to DBL, vav, and CDC24, which are likely to or have been shown to encode GTP exchange factors . A domain of ABR with similarity to GAPrho was expressed as a fusion protein in Escherichia coli and was shown to have GAP activity toward rac . Although both ABR and BCR have GAP activity, ABR lacks homology to the serine/threonine kinase domain of BCR . Therefore, ABR is likely to have cellular functions overlapping with but also distinct from those of BCR.

J Immunol, 1993 Aug 15, 151(4), 2318 - 25
Pentoxifylline attenuates neutrophil activation in experimental endotoxemia in chimpanzees; van Leenen D et al.; Costimulation of neutrophils and cytokines may play an important role in organ injury in sepsis . Pentoxifylline inhibits various neutrophil functions in vitro, and attenuates endotoxin-induced production of TNF in both in vitro and in vivo models . To assess the effect of pentoxifylline on neutrophil activation in endotoxemia, nine adult chimpanzees (Pan troglodytes) were i.v . injected with saline (n = 2), Escherichia coli endotoxin (4 ng/kg; n = 4), or E . coli endotoxin (4 ng/kg) in combination with pentoxifylline (500 mg/3 h, starting 30 min before the endotoxin injection; n = 3) . Serial blood samples were obtained for measurements of leukocyte counts and the granulocytic proteinases elastase complexed with alpha 1-antitrypsin and lactoferrin, and cytokines during the next 5 h . No changes were observed in the saline-treated chimpanzees . Endotoxin induced a marked leukocytosis and neutrophilia, which were slightly reduced by pentoxifylline . In contrast, pentoxifylline almost completely prevented endotoxin-induced neutrophil degranulation: peak elastase-alpha 1-antitrypsin was 164 +/- 21 ng/ml (mean +/- SE) after endotoxin alone, vs 71 +/- 7 ng/ml after endotoxin with pentoxifylline (t = 3 h; p < 0.05); peak lactoferrin was 329 +/- 15 and 182 +/- 5 ng/ml, respectively (t = 5 h; p < 0.05) . Pentoxifylline also inhibited the endotoxin-induced release of TNF (271 +/- 26 vs 55 +/- 23 pg/ml at t = 1.5 h; p < 0.05) and IL-6 (225 +/- 42 vs 73 +/- 25 pg/ml at t = 2 h; p < 0.05) . IL-8 release was not significantly inhibited by pentoxifylline . In none of the animals activation of the C system could be detected . We conclude that pentoxifylline attenuates neutrophil activation in endotoxemia in chimpanzees, probably in part by inhibiting the release of TNF.

J Immunol, 1993 Aug 15, 151(4), 2105 - 15
Quantitation and kinetics of blood monocyte migration to acute inflammatory reactions, and IL-1 alpha, tumor necrosis factor-alpha, and IFN-gamma; Issekutz AC et al.; Monocytes migrate from the blood into acute inflammatory reactions, where they differentiate into macrophages, and after 12 to 24 h become the predominant histologic feature of the inflammatory infiltrate . The quantitation of monocyte migration into these reactions has been difficult . This report employs a novel combination of techniques to isolate highly purified monocytes from the blood of rats, and shows that these cells have a normal t1/2 of 26 h and migrate efficiently after i.v . injection into cutaneous acute inflammatory sites . Monocytes labeled with 51Cr accumulated in delayed-type hypersensitivity reactions, and sites injected with killed Escherichia coli, LPS, poly-inosine: cytosine, zymosan-activated serum (ZAS), a source of C5adesArg, and the cytokines IL-1 alpha, TNF-alpha, and IFN-gamma . Both radiolabeled monocytes and neutrophils migrated rapidly to E . coli, LPS, ZAS, IL-1 alpha, and TNF-alpha with a large increase in cell accumulation by 2 h . Neutrophil migration declined rapidly to undetectable levels by 3 to 4 h to all five stimuli, and monocyte migration to ZAS and IL-1 alpha also declined by this time . In contrast, E . coli, LPS, and TNF-alpha caused a sustained migration of monocytes for 5 to 6 h, long after neutrophils had stopped accumulating . Intradermal IFN-gamma did not recruit neutrophils but stimulated a prolonged monocyte migration from 1 to 6 h . Combinations of LPS, TNF-alpha, and IFN-gamma synergistically enhanced the late (> 5 h) but not the early phase of monocyte recruitment . In conclusion, purified monocytes isolated from rat blood can be used to quantify monocyte migration in vivo, and these cells migrate rapidly to cutaneous inflammation and in response to chemotactic factors, IL-1 alpha, TNF-alpha, and IFN-gamma, with initial kinetics similar to those of neutrophils . However, monocyte-selective mechanisms are induced by IFN-gamma and also appear to be involved in prolonged monocyte migration to TNF-alpha, LPS, and E . coli.

Arch Biochem Biophys, 1993 Aug 15, 305(1), 78 - 83
Protein-lipid interactions of the proteolipid c subunit of the Escherichia coli proton-translocating adenosinetriphosphatase; Ksenzenko SM et al.; Interactions between Escherichia coli membrane phospholipids and the hydrophobic c subunit of the F1F0 proton-translocating ATPase were characterized . Extraction of E . coli membranes with a neutral mixture of chloroform and methanol and subsequent separation steps produced several protein-containing fractions . The protein-containing fraction most soluble in organic solvents contained subunit c and a lipid fraction enriched in phosphatidylglycerol compared to total E . coli membrane phospholipids . Other ATPase subunits and some additional proteins extracted from the membranes by this procedure could be separated from the c subunit by subsequent extraction . The purified and delipidated c subunit contained fatty acids which were released upon treatment with boron trifluoride methanol . Furthermore, deleting and restoring the genes for the F0 subunits changed the composition of extractable membrane phospholipid and fatty acids, indicating that the F0 plays a significant structural role in the membrane.

Arch Biochem Biophys, 1993 Aug 15, 305(1), 123 - 31
Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme; Gillam EM et al.; A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transform Escherichia coli DH5 alpha cells . Little expression was seen with the native sequence; the highest level of expression (range of 40-110 membrane-bound nmol P450 liter-1) was achieved with a construct (NF14) in which residues 3-12 were deleted . In all of the constructs P450 was found primarily in the membranes . The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra . The protein was purified to electrophoretic homogeneity in a five-step procedure {nominally 23 nmol P450 (mg protein)-1} . For most purposes it was found to be more practical to purify the modified P450 3A4 to approximately 70% homogeneity {nominally 15 nmol P450 (mg protein)-1} in a simple two-step process . The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-alpha-dilauroyl- and L-alpha-dioleoyl-sn-glycero-3-phosphocholines, cholate, and cytochrome b5 suggested by others but also glutathione during the preincubation . Several other thiols were found not to substitute in this role . Good catalytic activity was seen for nifedipine oxidation, testosterone 6 beta-hydroxylation, and the 8,9-epoxidation and 3 alpha-hydroxylation of aflatoxin B1, reactions previously ascribed to the enzyme . These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.

Anal Biochem, 1993 Aug 15, 213(1), 147 - 51
Synthesis of alpha, beta-deuterated 15N amino acids using a coupled glutamate dehydrogenase-branched-chain amino acid aminotransferase system; Chanatry JA et al.; Gram amounts of various 15N-enriched L-amino acids have been synthesized using a coupled enzymatic system . Catalytic amounts of 15N-labeled L-glutamate are generated using (15NH4)2SO4, alpha-ketoglutarate, and glutamate dehydrogenase . The labeled glutamate in turn serves as an amine donor to an appropriate alpha-keto acid using the Escherichia coli branched-chain amino acid aminotransferase, the subcloning and overexpression of which is described . In order to drive the reaction to completion the redox cofactor NADPH is regenerated using a glucose dehydrogenase system . Since the amino-transferase catalyzes exchange of the alpha-hydrogen, carrying out this reaction in 2H2O gives rise to 2-2H,2-15N-labeled amino acids . Deuteration can be readily extended to the beta position as well by preexchanging the alpha-keto acids in basic 2H2O . The isotopically labeled amino acids are recovered in yields of 70-80%.

J Biol Chem, 1993 Aug 15, 268(23), 17504 - 12
Expression and characterization of the N-terminal domain of an oleosin protein from sunflower; Li M et al.; Oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins . Although oleosins are amphipathic proteins, they are unlike bilayer membrane proteins since they are associated with a single lipid:water interface at the oil body surface . Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain, flanked by relative polar C- and N-terminal domains . In the present study, we report the expression of the N-terminal domain of the 18-kDa oleosin isoform from sunflower as a recombinant fusion protein in Escherichia coli and the determination of its secondary structure using CD and Fourier transform infrared spectroscopy either as a purified but partially denatured peptide or reconstituted into liposomes . The structure derived from physical studies was then compared and assigned with those predicted from analysis of the primary sequence of the N-terminal domain . Based on data derived from CD spectroscopy analysis of purified and partially renatured N-terminal polypeptide, it contains about 10% alpha-helical structure, 20-30% beta-strand structure, approximately 8% beta-turn structure, and 60% random coil structure . However, analysis of the polypeptide reconstituted into liposomes showed an increased content of alpha-helical structure to about 20% and an increased beta-strand structure content to about 30-40% . Data derived from Fourier transform infrared spectroscopy studies and compared with the data predicted from the primary sequence showed the peptide is well structured with some antiparallel beta-strand structure from residues 2-9, parallel beta-strand structure from residues 30-37 and/or 42-49, and alpha-helical structure from residues 10-23 and/or 43-49 . There is potential amphipathic alpha-helix from residues 10-23 . Based on these results, the following model for the secondary structure of the N-terminal domain of sunflower oleosin can be proposed . Residues 2-9 would produce amphipathic antiparallel beta-strand structure . Residues 10-23 would produce an amphipathic alpha-helical structure . Residues 30-37 and/or 42-49 would give parallel beta-strand structure, or residues 42-49 could form a nonpolar alpha-helical structure that would insert into the oil matrix.

J Immunol, 1993 Aug 15, 151(4), 2062 - 9
Peritoneal gamma delta T cells induced by Escherichia coli infection in mice . Correlation between Thy-1 phenotype and host minor lymphocyte-stimulating phenotype; Takada H et al.; We found that gamma delta T cells increased in number in the peritoneal cavity after i.p . inoculation with Escherichia coli (ATCC 26) in mice . The increase of the gamma delta T cells was most prominent on day 5 after inoculation when the pathogens had been already eliminated from the hosts . Two-color flow cytometric (FCM) analysis revealed that these gamma delta T cells in infected C57BL/6 mice expressed Thy-1 Ag on their cell surface . On the other hand, gamma delta T cells induced by E . coli inoculation in C3H/He mice contained Thy-1-negative gamma delta T cells in addition to the Thy-1-positive gamma delta T cells . In both strains, irrespective of Thy-1 Ag expression, these gamma delta T cells were CD5 negative, CD44 positive, L-selectin negative, Ly-6C negative, and IL-2R low positive . Analyses of peritoneal exudate cells (PEC) from several other strains of mice after E . coli inoculation suggested that Thy-1-negative gamma delta T cells appear in mice carrying endogenous superantigen specific for V beta 3, especially mammary tumor virus-6 . These findings suggest that Thy-1 Ag expression on the gamma delta T cells appearing in the peritoneal cavity after i.p . E . coli inoculation is correlated to the Mls phenotype of the host mice.

Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7754 - 8
A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho; Geiselmann J et al.; Transcription termination protein Rho of Escherichia coli interacts with newly synthesized RNA chains and brings about their release from elongation complexes paused at specific Rho-dependent termination sites . Rho is thought to accomplish this by binding to a specific Rho "loading site" on the nascent RNA and then translocating preferentially along the transcript in a 5'-->3' direction . On reaching the elongation complex, Rho releases the nascent RNA by a 5'-->3' RNA.DNA helicase activity . These translocation and helicase activities are driven by the RNA-dependent ATPase activity of Rho . In this paper we propose a mechanism for these processes that is based on the structure and properties of the Rho protein . Rho is a hexamer of identical subunits that are arranged as a trimer of asymmetric dimers with D3 symmetry . The binding of ATP and RNA to Rho also reflects this pattern; the Rho hexamer carries three strong and three weak binding sites for each of these entities . The asymmetric dimers of Rho correspond to functional dimers that can undergo conformational transitions driven by ATP hydrolysis . We propose that the quaternary structure of Rho coordinates the ATP-driven RNA binding and release processes to produce a biased random walk of the Rho hexamer along the RNA, followed by RNA.DNA helicase activity and transcript release . The proposed model may have implications for other hexameric DNA.DNA, RNA.DNA, and RNA.RNA helicases that function in replication and transcription.

J Biol Chem, 1993 Aug 15, 268(23), 17392 - 6
Bovine seminal ribonuclease produced from a synthetic gene; Kim JS et al.; Bovine seminal ribonuclease (BS-RNase), a homolog of bovine pancreatic ribonuclease (RNase A), is isolated as a dimer in which the subunits are cross-linked by two disulfide bonds . In addition to this anomalous quaternary structure, the enzyme has extraordinary biological properties, such as antispermatogenic, antitumor, and immunosuppressive activities . The molecular bases for these properties are well-suited for exploration with the techniques of recombinant DNA . Accordingly, a gene encoding BS-RNase was designed based on criteria expected to maximize the translational efficiency of its mRNA in Escherichia coli . This gene was constructed from 12 synthetic oligonucleotides and expressed with the phage T7 system . The protein thus produced was insoluble and accumulated under optimal conditions to 15% of total cellular protein or 200 mg/liter of culture . Ribonuclease activity was generated by air oxidation of the reduced and denatured protein . Three forms of active BS-RNase were isolated by gel filtration chromatography: the well-characterized dimer and monomer and a previously uncharacterized form that migrated as a trimer . The ribonuclease activities of all three forms were equivalent to or higher than that of dimeric BS-RNase isolated from bull seminal plasma.

J Biol Chem, 1993 Aug 15, 268(23), 17051 - 6
Deletion analysis of the Escherichia coli rho-dependent transcription terminator trp t'; Zalatan F et al.; We have analyzed with deletion mutations the Escherichia coli rho-dependent transcription terminator trp t' . This site, 211 nucleotides in length, contains multiple sequence elements that are recognized by rho . Among these multiple elements are two that together appear to be important for full transcription termination efficiency in vitro and are similar to the rutA and rutB sequence elements in the lambda tR1 rho-dependent terminator previously described by Chen and Richardson (1987) . The results suggest for the first time that common functionally important elements are shared among rho-dependent terminators . Other regions of trp t' also appear to contribute to its functional efficiency, consistent with the idea that rho-dependent terminators contain dispersed and redundant recognition segments.

J Biol Chem, 1993 Aug 15, 268(23), 16907 - 16
Mechanisms of upstream activation of the rrnD promoter P1 of Escherichia coli; Sander P et al.; The rrn promoter regions of Escherichia coli contain a stretch of DNA, rich in An and Tn homopolymer sequences, which is located upstream of the tandem promoters P1 and P2 . We have studied the effects of the upstream sequence of the rrnD operon on promoter function, using deletion variants for in vitro transcription . The presence of the upstream activating sequence (UAS) was found to increase P1 promoter strength without influencing P2 . Two modes of P1 activation could be distinguished: a stimulation of P1 depending on the interaction of the factor of inversion stimulation (FIS) with the UAS (within the deletion boundaries of -50 and -112) and second, a factor-independent activation requiring the proximal part of the UAS (within the boundaries of -50 and -89) . Both modes of activation were previously observed in the case of the rrnB operon and were ascribed to increased constants of RNA polymerase binding to P1 (Leirmo, S., and Gourse, R . L . (1991) J . Mol . Biol . 220, 555-568; Zacharias, M., Theissen, G., Bradaczek, C., and Wagner, R . (1991) Biochimie 73, 699-712) . Our results show, however, that the mechanisms of upstream activation may vary with the reaction conditions . In a complex transcription system, originally designed for the use of cell extracts, FIS enhances first-order reactions that convert binary (open) complexes to transcribing complexes . Initiated complexes are stabilized by FIS . The factor-independent mode of P1 activation involves influences of the UAS on complex isomerizations as well as on binary complex formation . The results show that low molecular weight components of the complex transcription system change the function of the RNA polymerase at the rrn promoters (not at the reference promoter Ptac), so that the conversion of open to transcribing P1-complexes becomes dependent on the UAS and FIS.

Brain Res, 1993 Aug 13, 619(1-2), 195 - 8
High levels of quinolinic acid in brain of epilepsy-prone E1 mice; Nakano K et al.; Quinolinic acid (QUIN) may act.as an excitotoxin when it is abundant in the brain . We have shown previously that the activity of 3-hydroxyanthranilate 3,4-dioxygenase, a QUIN-synthesizing enzyme, was abnormally high in the brains of epilepsy-prone E1 mice as compared with that of ddY mice . Here, we estimated the QUIN contents in the brains of these mice . The results showed that the basal QUIN content in the cerebral cortex of E1 mice was twice as high as that of ddY mice . Systemic injection of 400 mumol/kg body weight of L-tryptophan (L-Trp) increased the cortical levels of QUIN in both E1 mice and ddY mice by 189% and 118%, respectively . Administration of 400 mumol/kg each of L-threonine and D,L-methionine had no appreciable effect on the L-Trp-caused increase in the cortical QUIN levels . Co-administration of 5-fluorotryptophan or 5-methyltryptophan, tryptophan analogs, with L-Trp did not reduce but rather enhanced the cortical QUIN levels (by 18% and 92%, respectively) . No significant change in the cortical QUIN concentrations was observed with injection of 2 mg/kg body weight of E . coli lipopolysaccharide (LPS) in E1 mice . However, injection of L-Trp in the LPS-treated E1 mice produced a more marked increase in the cortical QUIN levels than that injected with L-Trp alone . These results suggest that the brain QUIN contents of E1 mice are dependent not only on the activity of QUIN-synthesizing enzyme but also on the rate of flux of its substrate, L-Trp or its metabolite(s), in the brain.

Cell, 1993 Aug 13, 74(3), 483 - 92
The mitochondrial receptor complex: a central role of MOM22 in mediating preprotein transfer from receptors to the general insertion pore; Kiebler M et al.; The receptor complex in the mitochondrial outer membrane, which consists of at least seven different proteins, is responsible for the recognition and translocation of cytosolically synthesized preproteins . Two of its subunits, MOM19 and MOM72, function as surface receptors for preproteins . Four other subunits (MOM38, MOM30, MOM8, and MOM7) have been suggested to constitute the general insertion pore (GIP) . Here we report on the structure and function of MOM22 . MOM22 is anchored in the outer membrane by a single transmembrane segment . The highly negatively charged N-terminal domain is exposed to the cytosol and the C-terminal domain to the intermembrane space . MOM22 appears to be a central component of the receptor complex, required for the transfer of preproteins from the receptors to the GIP . We speculate that the negatively charged domain of MOM22 is involved in the transfer of positively charged signal sequences of preproteins.

Nucleic Acids Res, 1993 Aug 11, 21(16), 3659 - 65
DNA unwinding by replication protein A is a property of the 70 kDa subunit and is facilitated by phosphorylation of the 32 kDa subunit; Georgaki A et al.; Replication protein A (RP-A) is a heterotrimeric single-stranded DNA binding protein with important functions in DNA replication, DNA repair and DNA recombination . We have found that RP-A from calf thymus can unwind DNA in the absence of ATP and MgCl2, two essential cofactors for bona fide DNA helicases (Georgaki, A., Strack, B., Podust, V . and Hubscher, U . FEBS Lett . 308, 240-244, 1992) . DNA unwinding by RP-A was found to be sensitive to MgCl2, ATP, heating and freezing/thawing . Escherichia coli single stranded DNA binding protein at concentrations that coat the single stranded regions had no influence on DNA unwinding by RP-A suggesting that RP-A binds fast and tightly to single-stranded DNA . DNA unwinding by RP-A did not show directionality . Experiments with monoclonal antibodies strongly suggested that the 70kDa subunit is responsible for DNA unwinding . Phosphorylation of the 32kDa subunit of RP-A by chicken cdc2 kinase facilitated DNA unwinding indicating that this posttranslational modification might be important for modulating this activity of RP-A . Finally, DNA unwinding of a primer recognition complex for DNA polymerase delta which is composed of proliferating cell nuclear antigen, replication factor C and ATP bound to a singly-primed M13DNA slightly inhibited DNA unwinding . An important role for DNA unwinding by RP-A in processes such as initiation of DNA replication, fork propagation, DNA repair and DNA recombination is discussed.

Nucleic Acids Res, 1993 Aug 11, 21(16), 3875 - 84
Comparative DNA sequence features in two long Escherichia coli contigs; Cardon LR et al.; The recent sequencing of two relatively long (approximately 100 kb) contigs of E.coli presents unique opportunities for investigating heterogeneity and genomic organization of the E.coli chromosome . We have evaluated a number of common and contrasting sequence features in the two new contigs with comparisons to all available E.coli sequences (> 1.6 Mb) . Our analyses include assessments of: (i) counts and distributions of restriction sites, special oligonucleotides (e.g., Chi sites, Dam and Dcm methylase targets), and other marker arrays; (ii) significant distant and close direct and inverted repeat sequences; (iii) sequence similarities between the long contigs and other E.coli sequences; (iv) characterization and identification of rare and frequent oligonucleotides; (v) compositional biases in short oligonucleotides; and (vi) position-dependent fluctuations in sequence composition . The two contigs reveal a number of distinctive features, including: a cluster of five repeat/dyad elements with very regular spacings resembling a transcription attenuator in one of the contigs; REP elements, ERICs, and other long repeats; distinction of the Chi sequence as the most frequent oligonucleotide; regions of clustering, overdispersion, and regularity of certain restriction sites and short palindromes; and comparative domains of inhomogeneities in the two long contigs . These and other features are discussed in relation to the organization of the E.coli chromosome.

Nucleic Acids Res, 1993 Aug 11, 21(16), 3829 - 38
A quality control algorithm for DNA sequencing projects; White O et al.; Heterologous DNA sequences from rearrangements with the genomes of host cells, genomic fragments from hybrid cells, or impure tissue sources can threaten the purity of libraries that are derived from RNA or DNA . Hybridization methods can only detect contaminants from known or suspected heterologous sources, and whole library screening is technically very difficult . Detection of contaminating heterologous clones by sequence alignment is only possible when related sequences are present in a known database . We have developed a statistical test to identify heterologous sequences that is based on the differences in hexamer composition of DNA from different organisms . This test does not require that sequences similar to potential heterologous contaminants are present in the database, and can in principle detect contamination by previously unknown organisms . We have applied this test to the major public expressed sequence tag (EST) data sets to evaluate its utility as a quality control measure and a peer evaluation tool . There is detectable heterogeneity in most human and C.elegans EST data sets but it is not apparently associated with cross-species contamination . However, there is direct evidence for both yeast and bacterial sequence contamination in some public database sequences annotated as human . Results obtained with the hexamer test have been confirmed with similarity searches using sequences from the relevant data sets.

Nucleic Acids Res, 1993 Aug 11, 21(16), 3755 - 60
Site-specific mutagenesis induced by single O6-alkylguanines (O6-n-propyl, O6-n-butyl, O6-n-octyl) in vivo; Baumgart PM et al.; The mutagenic activity of a series of longer chain O6-n-alkylguanine residues (O6-n-propyl, O6-n-butyl, O6-n-octyl) has been analyzed using a plasmid molecule (pUC 9) in which single O6-alkylguanines were positioned in the unique Pstl recognition site by shot gun ligation (Nucleic Acids Res . 13, 3305-3316 (1985)) of overlapping synthetic oligonucleotides . After transfection of these vectors into E . coli cells having normal DNA repair systems, progeny plasmids were produced, of which 2.6%, 2.8% and 4.3% were mutated in their Pstl site when containing O6-n-propylguanine, O6-n-butylguanine, O6-n-octylguanine, respectively . DNA sequence analysis of mutant plasmid genomes revealed that O6-n-propylguanine and O6-n-butylguanine induced exclusively G-->A transitions located specifically at the preselected site . O6-n-octylguanine induced apart from G-->A transitions (70%) also targeted G-->T transversions (30%) . These results indicate that the mutation frequency of longer chain O6-alkylguanines can be substantial in cells with normal repair systems and that the mutation pattern depends on the nature of the alkyl group.

Nucleic Acids Res, 1993 Aug 11, 21(16), 3683 - 9
I-Sce III an intron-encoded DNA endonuclease from yeast mitochondria . Asymmetrical DNA binding properties and cleavage reaction; Schapira M et al.; We have previously discovered the new intron-encoded endonuclease I-Sce III by expressing, in E . coli, the ORF contained in the third intron of the yeast mitochondrial COX I gene . In this work, we analyzed the in vitro properties of partially purified I-Sce III and found that it is a very specific DNA endonuclease, tolerating relatively few base changes in its 20 base pair long target site . I-Sce III should be a useful molecular tool to analyze the structure of large genomes . Interestingly, I-Sce III is the first P1-P2 DNA endonuclease for which DNA binding properties could be analyzed by band-shift experiments . Clearly, the cleavage products corresponding to the upstream A3 exon and to the downstream A4 exon could compete with the substrate A3-A4 in forming a DNA-protein complex . However, the A3 exon competes more efficiently than the downstream A4 product . The cleavage of the two DNA strands is also asymmetric the top strand (non-transcribed strand) is cleaved faster than the bottom strand, a property found under various experimental conditions . These findings suggest that this intron-encoded DNA endonuclease may have role in the RNA splicing process of the intron.

Nucleic Acids Res, 1993 Aug 11, 21(16), 3667 - 70
Sigma s-dependent promoters in Escherichia coli are located in DNA regions with intrinsic curvature; Espinosa-Urgel M et al.; Expression of a number of genes during stationary phase in Escherichia coli is controlled by the alternative sigma factor sigma s (KatF) . Promoters recognized by sigma s do not present a well-defined consensus sequence in their -10 and -35 regions . By polyacrylamide gel electrophoresis of DNA fragments performed at different temperatures, and by computer prediction analyses, we have found that sigma s-regulated promoters are located in regions where DNA shows intrinsic curvatures . This feature does not appear in a stationary-phase-induced promoter which is not controlled by sigma s . We propose that DNA bending may help in recognition and/or binding of sigma s to stationary-phase-induced promoters.

What Is Activated Sludge?, What Is Yeast?, What Is Water Purification?, What Is Bioassay?, What Is Fermentation?, s, Bacteria, r, Microbes, r, Bacterium, s, Microbiology, i, Microorganisms, n, Escherichia coli, s, Bacillus, s, Xanthomonas, i, Erwinia, a, Staphylococcus, n, Neisseria, r, Microorganism, r, S. cerevisiae, r, Salmonella, s, S. cerevisiae, n, Staphylococcus aureus, i, Mueller hinton broth, e, Culture medium, c, Escherichia coli, n, Bacillus, i, Antibiotics, o, Cell cultures, e, Yeasts, i, S. cerevisiae, a, Waste treatment, o, Rhizobacter

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005