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