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| Biochemistry, 1996 Jan 30, 35(4), 1232 - 41 Thermodynamics of the membrane insertion process of the M13 procoat protein, a lipid bilayer traversing protein containing a leader sequence; Soekarjo M et al.; For the first time, the standard free energy change, delta Gzero, of a membrane-inserting protein with a leader sequence has been determined experimentally, using M13 procoat protein as an example . The partition coefficient for the distribution of the procoat protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of gamma = 6.5 x 10(5) M-1, corresponding to a delta Gzero of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC . For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: gamma = 1 x 10(5) M-1 (delta Gzero = -9.3 kcal/mol) . Proteinase K digestion experiments have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region . By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estimated to be -14.8 kcal/mol . In order to distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coefficient of the mutant procoat protein OM30R {which contains a positively charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)} was determined, yielding gamma = 0.3 x 10(5) M-1 (delta Gzero = -8.6 kcal/mol) . Previously reported in vivo experiments have shown that the OM30R mutant protein is not translocated across Escherichia coli membranes but only binds to the inner surface . The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to delta Gzero, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein . Neither the leader sequence nor the mature coat protein alone yields this free energy gain . For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the crystalline lipid phase . Furthermore, by using lipid bilayers with negative membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coefficients of the same order of magnitude as for the phosphatidylcholine lipid membrane. Biochemistry, 1996 Jan 30, 35(4), 1179 - 86 Accurate kinetic modeling of alkaline phosphatase in the Escherichia coli periplasm: implications for enzyme properties and substrate diffusion; Martinez MB et al.; Alkaline phosphatase in the periplasm of Escherichia coli presents many of the complex factors that may influence enzymes in vivo . These include an environment that contains a high enzyme concentration, is densely populated with other macromolecules, and is separated from other compartments by a partial diffusion barrier . A previous study provided a partial description of this situation and developed a model that utilized kinetic behavior to estimate the permeability of the outer membrane {Martinez, M . B., et al., (1992) Biochemistry 31, 11500} . This study extends that description to provide a complete model for the enzyme at all substrate levels . Some of the parameters needed for complete modeling include the following: outer membrane permeability to the substrate and product, catalytic efficiency of the enzyme, number of enzymes per cell, and effects of the reaction product (an inhibitor) on the enzyme . The theoretical model fit the data quite well over a wide range of values for each of these parameters . The best fit of theory with experimental data required that the rate constant for product escape from the periplasm was 4-fold greater than that for substrate entry . This correlated with the relative sizes of the substrate and product . The excellent fit of theory and results suggested that alkaline phosphatase and its substrate were unaffected by the solution conditions in the periplasm . That is, the catalytic parameters (kcat and KM), determined for the enzyme in dilute solution, appeared to be unchanged by the conditions in the periplasm . The major factor that altered the kinetic behavior was the combined effect of the permeability barrier and the dense population of enzyme molecules in the periplasm . Given the large impact of these parameters on reaction properties, the excellent fit of theory and results was striking . Overall, this study demonstrated that enzyme action in the complex biological environment can be accurately modeled, if all factors that influence enzyme behavior are known. Biochemistry, 1996 Jan 30, 35(4), 1162 - 72 Mode of selectivity in cyclic AMP receptor protein-dependent promoters in Escherichia coli; Pyles EA et al.; Escherichia coli cAMP receptor protein (CRP) controls more than 20 genes . There are significant differences in the promoter regions in these genes . Thus, an elucidation of the mechanism of CRP action requires knowledge about the mode of selectivity in these promoters . An earlier study {Heyduk, T., & Lee, J . C . (1990) Proc . Natl . Acad . Sci . U.S.A . 81, 1744-8} indicates that the CRP(cAMP)1 conformer exhibits the highest affinity for the lac PI site in the lac operon . It is conceivable that the CRP conformer that binds with the highest affinity to these other sites may not be CRP(cAMP)1 . To investigate this possibility, the binding of CRP to nine CRP binding sites was studied as a function of cAMP concentration . The CRP binding sites employed in this investigation were chosen to represent the primary promoter sites from class I (lac site PI) and class II (sites PI of gal and crp) as well as secondary CRP binding sites (crp site PII and cat PII) to further understand the molecular mechanism of CRP in controlling the transcription of these bacterial genes . The affinity of CRP for three synthetic CRP binding sites was also examined to explore the contribution of the inverted repeat region and sequences surrounding the recognition motifs . The synthetic sequences are gallac which contains the lac recognition motifs in the background of gal, modified cat PII which contains an 8-base pair spacer between the recognition motifs rather than the 7-base pair sequence naturally found in cat PII, and a random sequence that has no known similarity to any CRP binding site found in nature . The apparent affinities of these sequences for CRP were quantitatively determined to be biphasic in their cAMP dependence . The CRP(cAMP)1 conformer was found to have the highest affinity for all of the DNA sequences examined . No specific affinity was observed for these sequences with free CRP and CRP(cAMP)2 . The affinity of CRP for DNA was sequence-dependent and increased in the following order: random < cat site PII, modified cat site PII, crp sites PI and PII < gal site PI < lac site PI < gallac . These results indicate that the entire CRP binding site sequence and its natural variability provide information to CRP . These promoter sites which appear to have different mechanisms at the molecular level are transcriptionally controlled by the same CRP conformer, CRP(cAMP)1 . Thus, the regulation of transcription by CRP is more subtle than choosing different conformational forms of CRP . Using "physiological" concentrations of various components, a computer simulation study was conducted to illustrate the possible consequences of the thermodynamic parameters determined in this study . It is evident that the promoters of protein systems regulating the transport and metabolism of carbohydrates are responsive to low cAMP concentrations . However, the promoter for controlling the expression of CRP is highly regulated by the fluctuation of cAMP concentration. FEBS Lett, 1996 Jan 29, 379(2), 135 - 8 Expression of human inducible nitric oxide synthase in Escherichia coli; Fossetta JD et al.; We have expressed active full-length human inducible nitric oxide synthase (iNOS) in E . coli . Expression required co-expression with calmodulin, a particularly tight-binding cofactor . The extracts also required tetrahydrobiopterin to display activity . Specific activity of the purified recombinant iNOS was similar to iNOS purified from murine macrophages . This result indicates that no special processing events unique to eucaryotic cells are necessary for iNOS activity. FEBS Lett, 1996 Jan 29, 379(2), 122 - 6 Use of immobilized synthetic peptides for the identification of contact sites between human interleukin-6 and its receptor; Weiergraber O et al.; Synthetic peptides immobilized on cellulose membranes proved to be a powerful tool for the identification of sites in the cytokine IL-6 involved in receptor binding . Similarly, a region in the extracellular part of the IL-6 receptor which is important for interaction with its ligand was identified. J Chromatogr A, 1996 Jan 26, 722(1-2), 359 - 68 Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis; Arakawa H et al.; Analysis of single-strand conformation polymorphism (SSCP) by capillary electrophoresis (CE) was developed . The conformational change of single-strand DNA is caused by a mutation in a DNA fragment . The change is detected as mobility shift in CE . The effects of acrylamide gel concentration, running temperature and fragment size amplified by the polymerase chain reaction (PCR) were studied to develop the separation of SSCP . The model DNA used was the divE 42 gene carrying wild- and mutant-type (G-->A point mutation at the 141 site) . The results show that two single-strand DNA fragments that differ in one nucleotide can be separated by CE within minutes . This method was also applied to the separation of SSCP for N-ras gene including four kinds of mutations . All mutations tested in this study could be distinguished . CE is well suited for clinical analysis of SSCP because it is rapid and reproducible, allows on-line detection and is easy. Biochem Biophys Res Commun, 1996 Jan 26, 218(3), 902 - 7 Synthesis and purification of soluble ligand binding domain of the human vitamin D3 receptor; Craig TA et al.; We expressed and purified milligram quantities of the ligand binding domain of the human 1,25-dihydroxyvitamin D3 receptor using a glutathione-S-transferase (GST) fusion protein expression system . Amino acids 105-427 were expressed in E . coli as a GST fusion protein at a reduced (20 degrees C) temperature and purified on glutathione sepharose . The fusion protein adsorbed to glutathione sepharose was cleaved with thrombin to yield soluble 105-427 human 1,25-dihydroxyvitamin D3 receptor . The 105-427 human 1,25-dihydroxyvitamin D3 receptor was further purified by Mono Q ion exchange chromatography and was characterized as a single band on SDS-polyacrylamide gel electrophoresis . The 105-427 human 1,25-dihydroxyvitamin D3 receptor bound 1,25-dihydroxyvitamin D3 with high affinity (Kd approximately 10(-9)M) and with a binding capacity of 47 pmoles/nmole protein . Large scale expression of 105-427 human 1,25-dihydroxyvitamin D3 receptor will provide human 1,25-dihydroxyvitamin D3 receptor ligand binding domain suitable for structural studies. Biochem Biophys Res Commun, 1996 Jan 26, 218(3), 682 - 7 A stable phage-display system using a phagemid vector: phage display of hen egg-white lysozyme (HEL), Escherichia coli alkaline, phosphatase, and anti-HEL monoclonal antibody, HyHEL10; Maenaka K et al.; A stable expression system for displaying the pIII fusion protein on the surface of a filamentous phage was constructed . A phagemid pIII display vector, pLUCK, was constructed by inserting the gene encoding the pIII fusion protein in the opposite direction to that of the lac promoter of pTZ18U . Using this phage display system, two enzymes, hen egg-white lysozyme (HEL) and E . coli alkaline phosphatase, and the single-chain Fv fragment of anti-HEL monoclonal antibody HyHEL10, could be stably and functionally displayed . Northern and primer extension analyses showed that a small amount of the sense mRNA encoding pIII-fused HEL was transcribed from the minor phage promoter located in the region encoding the C-terminus of pIII . Repressed expression of the pIII fusion protein can lead to the display of a wide range of proteins on filamentous phages without the need for strict expression conditions. Int J Cancer, 1996 Jan 26, 65(3), 383 - 8 Immunochemical identification of novel high-molecular-weight protein isoforms of the adenomatous polyposis coli (APC) gene; Kraus C et al.; Mapping analyses of monoclonal antibodies (MAbs) directed against the amino-terminus of the adenomatous polyposis coli (APC) gene product revealed that epitopes recognized by the MAbs FE9, CF11 and AC4 constitute different peptide sequences encoded by the APC exons 1, 2 and 3, respectively . The protein pattern detected with these specificity-defined immunoreagents, however, differed depending on the particular antibody used on Western blots of cellular urea extracts . APC exon 15-positive "classic" p300apc polypeptide chains were identified by the MAb FE9, MAb CF11 and the C-terminus-specific MAb IE1, but only weak signals were obtained with the AC4 MAb, which defines an exon 3-encoded epitope . In contrast with this immunoreactivity, 2 novel high m.w . products of approx . 150/160 and 200 kDa were exclusively recognized by the AC4 MAb, which was shown to bind to the APC exon 3-encoded peptide sequence SRESTGYL . A molecular form of some 400 kDa was identified to represent a disulfide-bound oligomer of the p150/160apc molecules . The novel APC-related molecules did not contain exon 1- and exon 15-encoded epitopes, as confirmed with the help of the FE9 and IE1 MAbs, respectively . This observation was corroborated by the fact that these novel proteins were not truncated in a collection of familial adenomatous polyposis patients with stop mutations in exon 15 . We conclude, that APC MAb AC4-reactive p150/160 and p200 polypeptide chains represent novel genuine products of the APC gene devoid of exon 1- and exon 15-encoded protein interaction domains. J Mol Biol, 1996 Jan 26, 255(3), 425 - 34 Duplex destabilization in superhelical DNA is predicted to occur at specific transcriptional regulatory regions; Benham CJ; Analytic methods that accurately calculate the extent of duplex destabilization induced in each base-pair of a DNA molecule by superhelical stresses are used to analyze several genomic DNA sequences . Sites predicted to be susceptible to stress-induced duplex destabilization (SIDD) are found to be closely associated with specific transcriptional regulatory regions . Operators within the promoters of SOS-regulated Escherichia coli genes are destabilized by superhelical stresses, whereas closely related sequences present elsewhere on that genome are not . Analysis of genomic sequences from the budding yeast Saccharomyces cerevisiae finds a distinctive tripartite pattern, in which the 3' and 5' termini of genes are destabilized, but the sequence encoding the primary transcript is not . Three rDNA genes from higher eukaryotes exhibit a similar pattern . Implications of these results regarding possible mechanisms of activity of the regions involved are discussed . A strategy is presented for designing experiments in which the susceptibility to SIDD of a local region is altered without changing its local base sequence . The occurrence of the observed SIDD patterns provides a new approach to searching uncharacterized genomic sequences for transcriptionally active regions. J Mol Biol, 1996 Jan 26, 255(3), 373 - 86 Affinity selective isolation of ligands from peptide libraries through display on a lac repressor "headpiece dimer"; Gates CM et al.; DNA binding by the Escherichia coli lac repressor is mediated by the approximately 60 amino acid residue 'headpiece' domain . The dimer of headpiece domains that binds to the lac operator is normally formed by association of the much larger approximately 300 amino acid residue C-terminal domain . We have used in vitro selection to isolate 'headpiece dimer' molecules containing two headpiece domains connected via a short peptide linker . These proteins bind plasmid molecules with sufficient stability to allow association of a peptide epitope displayed at the C terminus of the headpiece dimer with the plasmid encoding that peptide . Libraries of peptides displayed on the C terminus of a headpiece dimer can be screened for specific receptor ligands by affinity enrichment of peptide-headpiece dimer-plasmid complexes using an immobilized receptor . After each round of enrichment, transformation of E . coli with recovered plasmids permits amplification of the selected population . After several rounds of enrichment, sequencing of individual clones reveals the structure of the selected peptides . Headpiece dimer libraries allow selection of peptide ligands of higher average affinity than similar libraries based on the intact lac repressor . Interestingly, the presence of the lac operator is not required for plasmid binding by the headpiece dimer protein. J Mol Biol, 1996 Jan 26, 255(3), 349 - 55 The N-terminal domain of the rne gene product has RNase E activity and is non-overlapping with the arginine-rich RNA-binding site; McDowall KJ et al.; The rne gene of Escherichia coli encodes a 118 kDa protein that has ribonuclease E (RNase E) activity and binds RNA . A functional rne gene product is essential for cell viability and for the processing and/or decay of a variety of RNA species, including 9 S RNA, mRNA and RNAI, the antisense RNA regulator of ColE1-type plasmid replication . By testing the ability of different segments of the Rne protein to catalyze RNA cleavage and to bind RNA, we found that the N-terminal half (residues 1 to 498) of Rne contains a catalytic function sufficient for site-specific cleavage of oligoribonucleotides and complex RNAs . The C-terminal half of the protein, which contains both an arginine-rich region (residues 597 to 684) that we show binds RNA and a segment that is essential for cell viability (residues 844 to 1061), had no detectable endoribonucleolytic activity . Our results, which map the catalytic domain of RNase E, indicate the existence of discrete functional domains within the multifaceted Rne protein. J Biol Chem, 1996 Jan 26, 271(4), 2249 - 54 Mapping and functional role of phosphorylation sites in the thyroid transcription factor-1 (TTF-1); Zannini M et al.; The phosphorylation of thyroid transcription factor-1 (TTF-1), is homeodomain-containing transcription factor that is required for thyroid-specific expression of the thyroglobulin and thyroperoxidase gene promoters, has been studied . Phosphorylation occurs on a maximum of seven serine residues that are distributed in three tryptic peptides . Mutant derivatives of TTF-1, with alanine sites, have been constructed and used to assess the functional relevance of TTF-1 phosphorylation . The DNA binding activity of TTF-1 appears to be phosphorylation-independent, as indicated also by the performance of TTF-1 purified from an overexpressing Escherichia coli strain . Transcriptional activation by TTF-1 could require phosphorylation only in specific cell types since in a co-transfection assay in heterologous cells both wild-type and mutant proteins show a similar transcriptional activity. J Biol Chem, 1996 Jan 26, 271(4), 2206 - 12 Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with P-32, a protein co-purified with splicing factor SF2 . Hyaluronic acid-binding protein as P-32 protein, co-purified with splicing factor SF2; Deb TB et al.; The purification of a 68-kDa hyaluronic acid-binding protein (HA-binding protein), a homodimer of 34 kDa that binds specifically to hyaluronic acid, has been reported earlier by us (Gupta, S., Batchu, R.B., and Datta, K . (1991) Eur . J . Cell Biol . 56, 58-67) . Here, we report the isolation of a partial cDNA clone from a lambda gt11 cDNA expression library of human skin fibroblast by immuno-screening with HA-binding protein antiserum . The internal polypeptide sequence (83 residues) of the purified hyaluronic acid-binding protein is identical to the predicted protein sequence derived from hyaluronic acid-binding protein cDNA, suggesting the authenticity of the clone . Interestingly, this hyaluronic acid-binding protein cDNA sequence has complete homology with the cDNA sequence of a protein P-32, co-purified with the human pre-mRNA splicing factor SF2 (Krainer, A.R., Mayeda, A., Kozak, D., and Binns, G . (1991) Cell 66, 383-394) . Furthermore, the data on the N-terminal sequence of hyaluronic acid-binding protein and the predicted polypeptide of P-32 revealed the identical coding sequence of 209 amino acids for both the proteins . As the identity and functional characterization of P-32 have not yet been reported, P-32 cDNA was expressed in Escherichia coli, and the recombinant P-32 protein was purified by hyaluronic acid-Sepharose affinity chromatography . The recombinant P-32 protein showed immunocross-reactivity with the polyclonal antibodies raised against HA-binding protein . The predicted amino acid sequence of the protein fulfilled the minimal criteria for binding to hyaluronic acid, i.e . two basic amino acids flanking a seven-amino acid stretch, as reported for other hyaluronic acid affinity of the recombinant P-32 protein was confirmed by biotinylated hyaluronic acid binding assay . The binding of recombinant P-32 protein to biotinylated hyaluronic acid binding assay . The binding of recombinant P-32 protein to biotinylated hyaluronic acid can be competed only with excess unlabeled hyaluronic acid, confirming its specificity toward hyaluronic acid . All these results suggest that both P-32, co-purified with the human pre-mRNA splicing factor SF2, and 34-kDa hyaluronic acid-binding protein reported by us are the same protein and that it is a new member of the hyaluronic acid-binding protein family, the "hyaladherins." J Biol Chem, 1996 Jan 26, 271(4), 2057 - 62 Functional expression of subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex and reconstitution of recombinant protein with three-subunit core complex; Chen YR et al.; Subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex was over-expressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-RSIV) using the expression vector, pGEX/RSIV . Maximum yield of soluble active recombinant fusion protein was obtained from cells harvested 3 h after induction of growth at 37 degrees C in LB medium . Subunit IV was released from the fusion protein by proteolytic cleavage with thrombin . When subjected to SDS-polyacrylamide gel electrophoresis, isolated recombinant subunit IV of R . sphaeroides cytochrome b-c1 complex . Although the isolated recombinant subunit IV is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1000 kDa . The addition of detergent deaggregates the isolated protein, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution . When the three-subunit core cytochrome b-c1 complex, purified from RS delta IV-adapted chromatophores containing a fraction of the wild type cytochrome b-c1 complex activity, was reacted with varying amounts of recombinant subunit IV, the activity increased as the subunit IV concentration increased . Maximum activity restoration was reached when 1 mol of subunit IV/mol of three-subunit core complex was used . The reconstituted cytochrome b-c1 complex is similar to the wild-type complex in molecular size, apparent Km for Q2H2, and inhibitor sensitivity, indicating that recombinant subunit IV is properly assembled into the active cytochrome b-c1 complex . A tryptophan residue in subunit IV was found to be involved in the interaction with the three-subunit core complex. J Biol Chem, 1996 Jan 26, 271(4), 1998 - 2004 Promoter selectivity of Escherichia coli RNA polymerase E sigma 70 and E sigma 38 holoenzymes . Effect of DNA supercoiling; Kusano S et al.; The functional specificity was compared between two sigma factors, sigma 70 (the major sigma at exponentially growing phase) and sigma 38 (the essential sigma at stationary growth phase), of Escherichia coli RNA polymerase . The core enzyme binding affinity of sigma 38 was less than half the level of sigma 70 as measured by gel filtration column chromatography or by titrating the concentration of sigma required for the maximum transcription in the presence of a fixed amount of core enzyme . In addition, the holoenzyme concentration required for the maximum transcription of a fixed amount of templates was higher for E sigma 38 than E sigma 70 . The transcription by E sigma 38 was, however, enhanced with the use of templates with low superhelical density, in good agreement with the decrease in DNA superhelicity in the stationary growth phase . We thus propose that the selective transcription of stationary-specific genes by E sigma 38 holoenzyme requires either a specific reaction condition(s) or a specific factor(s) such as template DNA with low superhelical density. J Biol Chem, 1996 Jan 26, 271(4), 1966 - 71 Strand displacement synthesis of the long terminal repeats by HIV reverse transcriptase; Fuentes GM et al.; According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo . We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer . The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation . Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not . The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis . Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA . This observation suggests that recombination is favored during strand displacement synthesis in vivo. J Biol Chem, 1996 Jan 26, 271(4), 1853 - 6 CTG triplet repeats from human hereditary diseases are dominant genetic expansion products in Escherichia coli; Ohshima K et al.; The relative ability of the 10 triplet repeat sequences to be expanded in Escherichia coli was determined . Surprisingly, CTG tracts are expanded at least 8 times more frequently than any of the other nine triplets . Low levels of expansion were found also for CGG, GTG, and GTC . Thus, the structure of the CTG repeats and/or their utilization by the DNA synthetic systems in vivo must be quite different from the other triplets . These data further validate this genetically defined system for elucidating molecular mechanisms of expansion and may explain why most triplet repeat hereditary neuromuscular and neurodegenerative disease genes contain CTG repeats. J Biol Chem, 1996 Jan 26, 271(4), 1833 - 6 Regulation of fatty acid elongation and initiation by acyl-acyl carrier protein in Escherichia coli; Heath RJ et al.; Long chain acyl-acyl carrier protein (acyl-ACP) has been implicated as a physiological inhibitor of fatty acid biosynthesis since acyl-ACP degradation by thioesterase overexpression leads to constitutive, unregulated fatty acid production . The biochemical targets for acyl-ACP inhibition were unknown, and this work identified two biosynthetic enzymes that were sensitive to acyl-ACP feedback inhibition . Palmitoyl-ACP inhibited the incorporation of {14C}malonyl-CoA into long chain fatty acids in cell-free extracts of Escherichia coli . A short chain acyl-ACP species with the electrophoretic properties of beta-hydroxybutyryl-ACP accumulated concomitant with the overall decrease in the amount of {14C}malonyl-CoA incorporation, indicating that the first elongation cycle was targeted by acyl-ACP . All of the proteins required to catalyze the first round of fatty acid synthesis from acetyl-CoA plus malonyl-CoA in vitro were isolated, and the first fatty acid elongation cycle was reconstituted with these purified components . Analysis of the individual enzymes and the pattern of intermediate accumulation in the reconstituted system identified initiation of fatty acid synthesis by beta-ketoacyl-ACP synthase III (fabH) and enoyl-ACP reductase (fabI) in the elongation cycle as two steps attenuated by long chain acyl-ACP. Nature, 1996 Jan 25, 379(6563), 311 - 9 The 2.0 A crystal structure of a heterotrimeric G protein; Lambright DG et al.; The structure of a heterotrimeric G protein reveals the mechanism of the nucleotide-dependent engagement of the alpha and beta gamma subunits that regulates their interaction with receptor and effector molecules . The interaction involves two distinct interfaces and dramatically alters the conformation of the alpha but not of the beta gamma subunits . The location of the known sites for post-translational modification and receptor coupling suggest a plausible orientation with respect to the membrane surface and an activated heptahelical receptor. Biochemistry, 1996 Jan 23, 35(3), 990 - 8 Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli; Sun J et al.; Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation {Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H . R . (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasco, N., & Kaback, H . R . (1984) Biochemistry 23, 3688} . In this paper, 4B1 binding to purified lactose permease is shown to exhibit a KD of about 5 x 10(-10) M by surface plasmon resonance . Furthermore, the combined use of mutants containing 6 contiguous His residues in each periplasmic loop in the permease and Cys-scanning mutagenesis in conjunction with chemical labeling demonstrates that 4B1 binds specifically to the periplasmic loop between helices VII and VIII and that Phe247 and Gly254 are the primary determinants . Remarkably, although 4B1 binding uncouples lactose and H+ translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an important role in the transport mechanism . Moreover, binding of avidin to biotinylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity . Therefore, the uncoupling effect of 4B1 involves highly specific interactions which in all likelihood exert a torsional effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pKas of residues involved in lactose-coupled H+ translocation. Biochemistry, 1996 Jan 23, 35(3), 948 - 54 Role of glutamate-104 in generating a transition state analogue inhibitor at the active site of cytidine deaminase; Carlow DC et al.; The 19F-NMR resonance of 5-{19F}fluoropyrimidin-2-one ribonucleoside moves upfield when it is bound by wild-type cytidine deaminase from Escherichia coli, in agreement with UV and X-ray spectroscopic indications that this inhibitor is bound as the rate 3,4-hydrated species 5-fluoro-3,4-dihydrouridine, a transition state analogue inhibitor resembling an intermediate in direct water attack on 5-fluorocytidine . Comparison of pKa values of model compounds indicates that the equilibrium constant for 3,4-hydration of this inhibitor in free solution is 3.5 x 10(-4) M, so that the corrected dissociation constant of 5-fluoro-3,4-dihydrouridine from the wild-type enzyme is 3.9 x 10(-11) M . Very different behavior is observed for a mutant enzyme in which alanine replaces Glu-104 at the active site, and kcat has been reduced by a factor of 10(8) . 5-{19F}Fluoropyrimidin-2-one ribonucleoside is strongly fluorescent, making it possible to observe that the mutant enzyme binds this inhibitor even more tightly (Kd = 4.4 x 10(-8) M) than does the native enzyme (Kd = 1.1 x 10(-7) M) . 19F-NMR indicates, however, that the E104A mutant enzyme binds the inhibitor without modification, in a form that resembles the substrate in the ground state . These results are consistent with a major role for Glu-104, not only in stabilizing the ES++ complex in the transition state, but also in destabilizing the ES complex in the ground state. Biochemistry, 1996 Jan 23, 35(3), 922 - 9 Preparation and characterization of a disulfide-linked bioconjugate of annexin V with the B-chain of urokinase: an improved fibrinolytic agent targeted to phospholipid-containing thrombi; Tanaka K et al.; A conjugate of annexin V and the B-chain of urokinase was prepared and its fibrinolytic properties were studied . First, a mutant of annexin V was constructed with an N-terminal extension of six amino acids (Met-Ala-Cys-Asp-His-Ser) and with Cys316 mutated to Ser; this molecule was expressed in Escherichia coli . The urokinase B-chain was prepared by limited reduction of the interchain disulfide bond between the A- and B-chains of urokinase . These two molecules were then then connected by a disulfide bond and purified to yield a 1:1 stoichiometric conjugate . The conjugate had the same catalytic activity as urokinase against a synthetic substrate, Glt-Gly-Arg-MCA, and a similar plasminogen activating activity . The conjugate showed the same binding affinity for phosphatidylserine-containing membranes as annexin V . The in vitro fibrinolytic activity of the conjugates on clots prepared from platelet-rich plasma was comparable to that of urokinase . However, the conjugate showed 3-4-fold stronger in vivo thrombolytic activity than urokinase in a rat pulmonary embolism model, while having essentially the same plasma clearance rate as urokinase or B-chain . These results show that annexin V is a useful agent for targeting plasminogen activators to phospholipid-containing thrombi. Biochemistry, 1996 Jan 23, 35(3), 743 - 8 Core packing defects in an engineered Cro monomer corrected by combinatorial mutagenesis; Mollah AK et al.; The crystal structure of an engineered monomer of the lambda Cro repressor shows unexpected expansion of the hydrophobic core of the protein and disorder of the five C-terminal residues {Albright et al . (1996) Biochemistry 35, 735-742} . This structural information has guided the construction of a second generation of monomeric Cro proteins by combinatorial mutagenesis of selected core and C-terminal residues . Clones were identified in a library of randomized cro genes by a genetic screen for protein accumulation in Escherichia coli . Sequencing of candidate genes followed by purification and analysis of their product proteins has identified alternative arrangements of hydrophobic core residues which result in substantial increases in thermal stability . In contrast, residue replacements at the C-terminus have minor effects on stability but may increase protein expression levels. FEBS Lett, 1996 Jan 22, 379(1), 94 - 6 Cloning, purification, and crystallization of Escherichia coli cystathionine beta-lyase; Laber B et al.; The metC gene coding for cystathionine beta-lyase of Escherichia coli has been cloned and used to construct an overproducing E . coli strain . An efficient purification scheme has been developed and the purified enzyme has been crystallized by the hanging drop vapour diffusion method using either ammonium sulfate or polyethyleneglycol 400 as precipitating agent . The crystals belong to the orthorombic space group C222 . Their unit cell parameters are a = 60.9 A, b = 154.7 A and c = 152.7 A . Consideration of the possible values of VM accounts for the presence of one dimer per asymmetric unit . The crystals are suitable for X-ray analysis and a complete native date set to 1.83 A resolution has been collected using synchrotron radiation. FEBS Lett, 1996 Jan 22, 379(1), 47 - 50 15N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH-(1-24); Uegaki K et al.; For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required . A direct over-expression system by E . coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells . We here developed an over-expression system by means of thioredoxin gene fusion system . The fused protein composed of thioredoxin and the objective peptide was expressed in E . coli and then the peptide part was released by enterokinase . This system was successfully applied for the production of 15N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis. FEBS Lett, 1996 Jan 22, 379(1), 107 - 11 Molecular cloning of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A; Tanabe O et al.; Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or delta) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library . The cDNA had an open reading frame encoding an M(r) 66,138 protein of 570 amino acids . Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit . The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72 . Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, and SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence . The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues. J Theor Biol, 1996 Jan 21, 178(2), 183 - 204 Evolution of transcriptional regulation system through promiscuous coupling of regulatory proteins with operons; suggestion from protein sequence similarities in Escherichia coli; Otsuka J et al.; As an advanced molecular study of the problems of the evolution of organisms, the transcriptional regulation system is studied by investigating the amino acid sequence similarities between the proteins in the regulation system of Escherichia coli in which the data of sequenced proteins as well as of regulator-regulon relationships are accumulated . The similarities between the proteins are calculated by the FASTA algorithm and their homology is also evaluated in terms of statistical significance with the use of the RDF2 program . This investigation reveals that the similarity between the regulatory protein and the regulated protein is hardly found, but many similarities are found between regulatory proteins and between regulated proteins . These similarity relations are compared with the regulator-regulon relationships ascertained experimentally . From this comparison, it is found that similar regulatory proteins rarely regulate the transcription of similar protein genes . As most of the highly similar proteins are considered to have diverged from a common ancestral protein, this finding strongly suggests the possibility that descendant regulatory proteins have been promiscuously coupled with descendant operons, independently of their ancestral regulator-regulon relationship, and that some of the couplings have been fixed by selection to form the present system of transcriptional regulation . The compatibility of such promiscuous coupling with regulatory organization is illustrated in the carbohydrate transport systems and the succeeding metabolic pathways, whose organization is comprehensive in sending nutritious substances to the central path of glycolysis under different environmental conditions . The benefit of flexibility in regulator-regulon relationships in evolutionary processes is also discussed in connection with the punctuational divergence of species in macroevolution and the cell differentiation in multicellular organisms. Hum Gene Ther, 1996 Jan 20, 7(2), 173 - 82 Defective HSV-1 vector expressing BDNF in auditory ganglia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration; Geschwind MD et al.; The neurotrophins are a family of growth factors that play an important role in the development and maintenance of the nervous system . Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that appears to participate in the maturation and function of mammalian auditory neurons . Forms of deafness due to varied injurious stimuli that are amenable to treatment with implantable prosthetic devices require the survival of these BDNF-responsive auditory neurons for effective outcome . To evaluate the feasibility of developing a gene therapy for deafness that may be used in conjunction with a prosthetic device, we constructed replication-defective herpes simplex virus (HSV) amplicon vectors that carry the human BDNF cDNA . Using these vectors, HSVbdnf and HSVbdnflac (expresses BDNF and Escherichia coli beta-galactosidase), we evaluated the expression and biological activity in established cell lines and explant cultures prepared from spiral ganglia of the murine ear . Gene transfer with HSVbdnf resulted in the efficient expression of human BDNF mRNA in murine fibroblasts . Using two BDNF-responsive cell lines, PC12trkB and MG87trkB, we demonstrate efficient secretion of biologically active BDNF . Finally, transduction of explanted spiral ganglia with HSVbdnflac elicited robust neuritic process outgrowth comparable to exogenously added BDNF . Overall, these data demonstrate that HSV vectors can efficiently transfer and express the BDNF gene in many cell types, including auditory neurons . Moreover, they suggest that similar vectors may be used to express the neurotrophin in auditory neurons in vivo and perhaps as adjunctive gene therapy for deafness. J Biol Chem, 1996 Jan 19, 271(3), 1726 - 31 Expression and biological activity of mouse fibroblast growth factor-9; Santos-Ocampo S et al.; Receptor specificity is an essential mechanism governing the activity of fibroblast growth factors (FGF) . To begin to understand the developmental role of FGF-9/glial activating factor, we have cloned and sequenced the murine FGF-9 cDNA and expressed the protein in mammalian cells and in Escherichia coli . We demonstrate that the FGF-9 protein is highly conserved between mouse and human . Receptor specificity was determined by direct binding to soluble and cell surface forms of FGF receptor (FGFR) splice variants and by the mitogenic activity on cells, which express unique FGF receptor splice variants . Our data demonstrate that FGF-9 efficiently activates the "c" splice forms of FGFR2 and FGFR3, receptors expressed in potential target cells for FGF-9 . Significantly, FGF-9 also binds to and activates the "b" splice form of FGFR3, thus becoming the first FGF ligand besides FGF-1 to activate this highly specific member of the FGF receptor family. J Biol Chem, 1996 Jan 19, 271(3), 1435 - 40 Monovalent cation activation and kinetic mechanism of inosine 5'-monophosphate dehydrogenase; Xiang B et al.; Human type II inosine 5'-monophosphate dehydrogenase has been purified to homogeneity from an Escherichia coli strain that express large quantities of the enzyme from the cloned gene . Steady state kinetic studies have been used to characterize the activation by monovalent cations, including Li+, Na+, K+, Rb+, Cs+, Tl+, NH4+, and N(CH3)4+ . The enzyme has less than 1% of the maximal activity in the absence of an added monovalent cation, such as K+, Na+, Rb+, Tl+, or NH4+ . The enzyme is activated by K+ and Tl+ at lower concentrations than those of other monovalent cations . Li+ and N(CH3)4+ do not activate the enzyme, nor do they inhibit the K(+)-activated enzyme, implying that ionic radius is important in binding selectivity . The Km values for both substrates and Vmax differ with different monovalent cations . Initial velocity and product inhibition kinetic data are consistent with an ordered steady state mechanism in which the enzyme binds K+ first, TMP second, and then NAD; the product NADH is released before xanthosine 5'-monophosphate . Substrate and product binding experiments support this mechanism and show the presence of one substrate binding site per subunit . Several rate constants were obtained from a computer simulation of the complete steady state rate equation. J Biol Chem, 1996 Jan 19, 271(3), 1416 - 23 Novel regulation of keratin gene expression by thyroid hormone and retinoid receptors; Tomic-Canic M et al.; Expression of keratin proteins, markers of epidermal differentiation and pathology, is uniquely regulated by the nuclear receptors for retinoic acid (RAR) and thyroid hormone (T3R) and their ligands: it is constitutively activated by unliganded T3R, but it is suppressed by ligand-occupied T3R or RAR . This regulation was studied using gel mobility shift assays with purified receptors and transient transfection assays with vectors expressing various receptor mutants . Regulation of keratin gene expression by RAR and T3R occurs through direct binding of these receptors to receptor response elements of the keratin gene promoters . The DNA binding "C" domain of these receptors is essential for both ligand-dependent and -independent regulation . However, the NH2-terminal "A/B" domain of T3R is not required for either mode of regulation of keratin gene expression . Furthermore, v-ErbA, an oncogenic derivative of cT3R, also activates keratin gene expression . In contrast to the previously described mechanism of gene regulation by T3R, heterodimerization with the retinoid X receptor is not essential for activation of keratin gene expression by unliganded T3R . These findings indicate that the mechanism of regulation of keratin genes by RAR and T3R differs significantly from the mechanisms described for other genes modulated by these receptors. J Biol Chem, 1996 Jan 19, 271(3), 1400 - 4 Evidence that transmembrane segment 2 of the lactose permease is part of a conformationally sensitive interface between the two halves of the protein; Jessen-Marshall AE et al.; A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), is found in a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane . This motif is located within the cytoplasmic side of transmembrane domain 2 (TM-2) and extends through the hydrophilic loop that connects transmembrane domains 2 and 3 . The motif is repeated again in the second half of the protein . In a previous study concerning the loop 2/3 motif (Jessen-Marshall, A . E., Paul, N . J., and Brooker, R . J . (1995) J . Biol . Chem . 270, 16251-16257), it was shown that the conserved aspartate at the fifth position in the motif is critical for transport activity since a variety of site-directed mutations were found to greatly diminish the rate of transport . In the current study, two of these mutations, in which the conserved aspartate was changed to threonine or serine, were used as parental strains to isolate second site suppressor mutations that restore transport function . A total of 10 different second site mutations were identified among a screen of 19 independent mutants . One of the suppressors was found within loop 1/2 in which Thr-45 was changed to arginine . Since the conserved aspartate and position 45 are at opposite ends of TM-2, these results suggest that the role of the conserved aspartate residue in loop 2/3 is to influence the topology of TM-2 . Surprisingly, the majority of suppressor mutations were found in the second half of the permease . All of these are expected to alter helix topology in either of two ways . Some of the mutations involved residues within transmembrane segments 7 and 11 that produced substantial changes in side chain volume: TM-7 (Cys-234-->Trp or Phe, Gln-241-->Leu, and Phe-247-->Val) and TM-11 (Ser-366-->Phe) . Alternatively, other mutations were highly disruptive substitutions at the ends of transmembrane segments or within hydrophilic loops (Gly-257-->Asp, Val-367-->Glu, Ala-369-->Pro, and a 5-codon insertion into loop 11/12) . It is hypothesized that the effects of these suppressor mutations are to alter the helical topologies in the second half of the protein to facilitate a better interaction with the first half . Overall, these results are consistent with a transport model in which TM-2 acts as an important interface between the two halves of the lactose permease . According to our tertiary model, this interaction occurs between TM-2 and TM-11. J Biol Chem, 1996 Jan 19, 271(3), 1349 - 56 Transcriptional regulation of the yeast DnaJ homologue SIS1; Zhong T et al.; The Saccharomyces cerevisiae SIS1 gene encodes an essential heat shock protein with similarity to the Escherichia coli DnaJ protein . In sis 1-85 and sis1-86 mutants, the sis1 RNA is induced to high levels at room temperature and without heat shock . The presence of wild type SIS1 in the sis1-85 mutant represses the overexpression of SIS1-85 protein . Furthermore, overexpression of wild type SIS1 reduces the beta-galactosidase activity expressed from a SIS1:lacZ fusion . These results suggest that SIS1 negatively regulates its own expression . The autoregulation of SIS1 transcription is mediated through a 39-base pair cis-element containing the SIS1 heat shock element plus additional flanking sequences on one side . Although SIS1 transcription is constitutive, it is transiently induced upon heat shock . In addition, SIS1 transcription is regulated by SSA (a class of HSP70 proteins) function . The elevated transcription of SIS1 in ssa1 ssa2 mutants is mediated solely through the SIS1 heat shock element . Therefore, the SIS1 autoregulatory element is different from the SSA-responsive element, suggesting that the mechanism involved in autoregulation of SIS1 is distinct from regulation of SIS1 by SSA proteins. J Biol Chem, 1996 Jan 19, 271(3), 1314 - 21 Folding-related dimerization of human cystatin C; Ekiel I et al.; With the aim to improve our understanding of the structural basis for protein self-association and aggregation, in particular in relationship to protein refolding and amyloid formation, folding-related processes for human cystatin C have been studied . Using NMR spectroscopy together with chromatographic and electrophoretic methods, a self-association process resulting in dimer formation for protein samples treated with denaturing agents as well as for samples subjected to low pH or high temperature conditions could be studied with amino acid resolution . In all three cases, the dimerization involves properly folded molecules and proceeds via the reactive site of the inhibitor, which leads to complete loss of its biological activity . This dimerization process has potential relevance for amyloid formation by the brain hemorrhage-causing Leu58-Gln variant of cystatin C . The results also indicate that cystatin C dimerization and inactivation may occur in acidified compartments in vivo, which could be relevant for the physiological regulation of cysteine proteinase activity. J Biol Chem, 1996 Jan 19, 271(3), 1285 - 94 Effects of assembly and mutations outside the active site on the functional pH dependence of Escherichia coli aspartate transcarbamylase; Yuan X et al.; Electrostatics are central to the function and regulation of Escherichia coli aspartate transcarbamylase, and modeling has suggested that long range electrostatic effects are likely to be important (Glackin, M . P., McCarthy, M . P., Mallikarachchi, D., Matthew, J . B., and Allewell, N . M . (1989) Proteins Struct . Funct . Genet . 5, 66-77; Oberoi, H., Trikha, J., Yuan, X., and Allewell, N . M . (1995) Proteins Struct . Funct . Genet., in press) . To investigate this possibility from an experimental standpoint, we have examined the effects both of assembly and of removing ionizable and polar side chains outside the active site (Glu-50, Tyr-165, and Tyr-240) on the pH dependence of the kinetic parameters of aspartate transcarbamylase . The holoenzyme (c6r6) assembles from three regulatory dimers (r2) and two catalytically active trimers (c3) . pH dependences of the enzyme kinetic parameters suggest that the mechanisms of productive binding of L-Asp to the binary complexes of the catalytic subunit (c3) and holoenzyme (c6r6) with carbamyl phosphate are different . In contrast, the Michaelis complex appears similar for both c3 and c6r6, except for pK shifts of approximately 1 pH unit . Results also indicate that the catalytic mechanism of the holoenzyme does not involve reverse protonation, as has recently been proposed for the catalytic trimer (Turnbull, J . L., Waldrop, G . L., and Schachman, H . K . (1992) Biochemistry 31, 6562-6569) . The tyrosines at positions 165 and 240 are part of a cluster of interactions that links the catalytic subunits in the T state (the cluc4 interface) and which is disrupted in the T --> R transition . The effects of mutating the two Tyr residues are quite different: Y240F has higher than wild-type activity and affinity over the entire pH range, while Y165F has activity and affinity an order of magnitude lower than wild-type . Removal of the regulatory subunits from Y165F increases activity and affinity and restores the pH dependence of the wild-type catalytic subunit . Like Y165F, E50A has low activity and affinity over the entire pH range . Linkage analysis indicates that there is long range energetic coupling among the active site, the ear subunit interfaces, and residue Y165 . The substantial quantitative difference between Y165F and Y240F, both of which are at the c1:c4 interface about 14-16 A from the closest active site, demonstrates specific path dependence, as opposed to general distance dependence, of interactions between this interface and the active site. Nature, 1996 Jan 18, 379(6562), 277 - 80 Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2; Eck MJ et al.; The domain organization of many signalling proteins facilitates a segregation of binding, catalytic and regulatory functions . The mammalian SH2 domain protein tyrosine phosphatases (PTPs) contain tandem SH2 domains and a single carboxy-terminal catalytic domain . SH-PTP1 (PTP1C, HCP) and SH-PTP2 (Syp, PTP2C, PTP1D) function downstream from tyrosine kinase-linked insulin, growth factor, cytokine and antigen receptors . As well as directing subcellular localization by binding to receptors and their substrates, the two SH2 domains of these PTPs function together to regulate catalysis . Here we report the structure of the tandem SH2 domains of SH-PTP2 in complex with monophosphopeptides . A fixed relative orientation of the two domains, stabilized by a disulphide bond and a small hydrophobic patch within the interface, separates the peptide binding sites by approximately 40 A . The defined orientation of the SH2 domains in the structure, and data showing that peptide orientation and spacing between binding sites is critical for enzymatic activation, suggest that spatial constraints are important in this multidomain protein-protein interaction. Nature, 1996 Jan 18, 379(6562), 225 - 32 Structure and mechanism of DNA topoisomerase II; Berger JM et al.; The crystal structure of a large fragment of yeast type II DNA topoisomerase reveals a heart-shaped dimeric protein with a large central hole . It provides a molecular model of the enzyme as an ATP-modulated clamp with two sets of jaws at opposite ends, connected by multiple joints . An enzyme with bound DNA can admit a second DNA duplex through one set of jaws, transport it through the cleaved first duplex, and expel it through the other set of jaws. Biochem Biophys Res Commun, 1996 Jan 17, 218(2), 461 - 5 Inhibition of the H+/peptide cotransporter in the human intestinal cell line Caco-2 by cyclic AMP; Muller U et al.; Treatment of Caco-2 cells with cholera toxin inhibits the activity of the H+/peptide cotransporter . The effect of cholera toxin is mimicked by E . coli heat-labile enterotoxin, forskolin and isobutylmethylxanthine and is associated with an increase in cAMP levels in the cells . The inhibition is due to a decrease in the maximal velocity of the transport system . Inhibitors of protein kinase A and protein kinase C block the effect of cholera toxin . Interestingly, the H+/peptide cotransporter in Caco-2 cells does not possess any putative site for phosphorylation by protein kinase A but does possess sites for phosphorylation by protein kinase C . It appears that the cAMP-dependent inhibition of the H+/peptide cotransporter in Caco-2 cells is mediated through activation of protein kinase C. Biochemistry, 1996 Jan 16, 35(2), 659 - 65 3'- and 5'-strand cleavage reactions catalyzed by the Fpg protein from Escherichia coli occur via successive beta- and delta-elimination mechanisms, respectively; Bhagwat M et al.; The Fpg protein from Escherichia coli is a multifunctional protein that excises damaged purine bases from DNA to generate aldehydic abasic sites and then catalyzes the successive cleavage of the phosphodiester bonds first on the 3'-side and then on the 5'-side of the abasic site to generate 5'- and 3'-phosphate ends, respectively, thereby excising the deoxyribose residue . The mechanisms of the 3'- and 5'-strand cleavage reactions have been studied by nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS) . The 3'-strand cleavage reaction is a beta-elimination reaction in which the 2'-hydrogen is abstracted and the 3'-phosphate is eliminated . The 5'-strand cleavage reaction is a delta-elimination reaction in which the 4'-hydrogen is abstracted and the 5'-phosphate is eliminated . Two types of experiments were performed to establish the occurrence of the sequential elimination reactions . First, when the reaction was performed in H2(18)O, 31P NMR demonstrated that neither phosphate group contained 18O . Second, the five-carbon product derived from the deoxyribose residue was stabilized by reduction with NaBH4 and characterized by GC-MS . The mass spectrum of the reduced product was identical to that of authentic 4-oxo-2-pentenal, the tautomerized product of successive beta- and delta-elimination reactions. Biochemistry, 1996 Jan 16, 35(2), 608 - 15 Evidence that specificity of microhelix charging by a class I tRNA synthetase occurs in the transition state of catalysis; Gale AJ et al.; Determinants for the identities of tRNAs are located in the acceptor stem and, commonly, in the anticodon as well . Although the anticodon is an important determinant for the identity of methionine tRNA, RNA microhelices whose sequences are based on the acceptor stem alone can be aminoacylated by the class I methionyl-tRNA synthetase . We show here that specific nucleotide substitutions in a microhelix significantly reduced its rate of aminoacylation . In contrast, affinity coelectrophoresis analysis showed that microhelix binding to the enzyme was not significantly affected by the same substitutions . These and additional experiments and considerations imply that specific determinants for microhelix aminoacylation are needed for orientation of the acceptor stem in the transition state of catalysis rather than for enhanced binding interactions . The effect of linking together acceptor stem interactions with those in the anticodon, as occurs in the whole tRNA molecule, was also evaluated . This analysis showed that linkage results in some of the favorable acceptor stem and anticodon interactions being used to offset the free energy cost of straining the structure of the enzyme-tRNA complex. Biochemistry, 1996 Jan 16, 35(2), 601 - 7 Aminoacyl-tRNA synthetases optimize both cognate tRNA recognition and discrimination against noncognate tRNAs; Sherman JM et al.; Specific protein--nucleic acid interactions are usually the product of sequence-dependent hydrogen bonding . However, in the crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln, leucine 136 (Leu136) stabilizes the disruption of the weak first (U1-A72) base pair in tRNAGln by stacking between A72 and G2 . We have demonstrated, by a combined in vivo and in vitro mutational analysis, that Leu136 is important for tRNA specificity despite making no hydrogen bonds with tRNAGln . Both more (L136F) and less (L136V, L136M, L136A, and L136T) mischarging mutants of GlnRS have been identified . GlnRS(L136F) is more mischarging and less specific than wild-type GlnRS in vivo, due not to an increased affinity for the noncognate tRNAs but to a decreased affinity for tRNAGln . Also, unlike other mischarging mutants of GlnRS that have been characterized, it does not exhibit generally relaxed tRNA specificity in vivo and mischarges only a subset of the tRNAs tested . A possible sequence preference for a Py1-Pu72/Pu2-Py71 combination is suggested . The L136A/M/T/V mutants are the first GlnRS variants, including wild-type, expressed on pBR322 which no longer mischarge tyrT(UAG) in vivo . We have shown that, while the L136A mutant is less mischarging than wild-type both in vivo and in vitro, it is not more specific as it also exhibits reduced affinity for its cognate glutamine tRNA . On the basis of these results, we suggest that the aminoacyl-tRNA synthetases have evolved to balance cognate tRNA recognition and discrimination against noncognate tRNAs. Biochemistry, 1996 Jan 16, 35(2), 545 - 53 Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains; Newton DL et al.; The gene for human angiogenin (Ang), a member of the ribonuclease superfamily, was fused to a gene encoding a single-chain antibody (sFv) against the human transferrin receptor . Three Ang single-chain immunofusion proteins (AngsFvs) were constructed with variations in the type of linker connecting the VL and VH chain {EGKSSGSGSESKEF, L1 or (GGGGS)3, L2} as well as with or without a spacer (FB) connecting the Ang and sFv (AngFBsFvL1 or L2; AngsFv(L2)} . Although the nature of the linker did not affect the enzymatic activity of the FB-containing fusion proteins, the fusion protein containing the L2 linker was 2.3-fold more effective than the L1 linker in competing with the labeled monoclonal IgG1 antibody for binding to the transferrin receptor . The fusion protein containing the L2 linker without the FB spacer exhibited a 13-fold decrease in binding to the transferrin receptor as well as a decrease in its capacity to degrade tRNA and to inhibit translation in the rabbit reticulocyte lysate compared to its counterpart containing the FB spacer . Binding of placental ribonuclease inhibitor (PRI) to Ang also was affected by the nature of the linker and by the presence or absence of a spacer . PRI bound to Ang and AngFBsFv(L2) and inhibited their ribonuclease activity . A 3-fold greater concentration of PRI, however, did not affect the activity of AngFBsFv(L1) or AngsFv(L2), suggesting that the conformation of these fusion proteins was altered . Binding of monoclonal and polyclonal anti-Ang antibodies to AngsFvs was also used to investigate conformational alterations of the fusion proteins . AngFBsFv(L2) was the least altered while AngFBsFv(L1) exhibited the greatest change in structure . Yet maximal concentrations of all AngsFvs elicited angiogenesis in the chick chorioallantoic membrane assay, demonstrating that Ang in all three fusion proteins remained functionally active . Consistent with all the activities, the fusion protein containing the FB spacer and L2 linker was the most cytotoxic to three different human tumor cell lines . The fusion protein lacking the FB spacer exhibited the least cytotoxicity . These data demonstrate that the linker connecting the VH-VL chains can affect the binding and cellular cytotoxicity of Ang immunofusions and that placement of a spacer between the antibody binding domains and Ang is necessary for optimal activity . Thus, a new class of targeted therapeutic agents containing Ang as the toxic moiety can be designed that potentially will be less immunogenic and less toxic than immunotoxins available currently. Biochemistry, 1996 Jan 16, 35(2), 433 - 43 Phosphotransfer and CheY-binding domains of the histidine autokinase CheA are joined by a flexible linker; Zhou H et al.; Multidimensional heteronuclear NMR techniques were applied to study a protein fragment of the histidine autokinase CheA from Escherichia coli . This fragment (CheA1-233) contains the phosphotransfer domain and the CheY-binding domain joined by a linker region . Comparison of chemical shift and NOE cross-peak patterns indicates that the structures of the two domains in CheA1-233 remain nearly the same as in the two individual domain fragments, CheA1-134 and CheA124-257 . Relaxation properties of the backbone 15N nuclei were measured to study the rotational correlations of the two domains and properties of the linker region . Dynamics data were analyzed both by an isotropic motional model and an anisotropic motional model . The experimental T1 and T2 values, the derived rotational correlation times, and motional anisotropy are significantly different for the two domains, indicating the two domains reorient independently and the linker region is highly flexible . Dynamics data of CheA1-233 were also compared with those of CheA1-134 . Our studies show that flexible domain linkers and extended and flexible terminal polypeptide chains can have significant effects on the motional properties of the adjacent structured regions . These observations suggest a model for the graded regulation of CheA autophosphorylation activity . In this model, the various activity states of the receptor are generated by controlling the access of the mean position of the kinase domain to the phosphotransfer domain . This would then modulate the diffusional encounter rate of the domains and hence activity over a wide and graded range of values. J Neurosci Res, 1996 Jan 15, 43(2), 235 - 45 Possible involvement of tyrosine kinase activation in lipopolysaccharide-induced expression of Ca(2+)-insensitive but calmodulin-coupling nitric oxide synthase in rat glial cells; Kitamura Y et al.; To clarify the properties of an inducible type of nitric oxide synthase (i-NOS) in the brain, we examined whether lipopolysaccharide (LPS) induces NOS in glial cells cultured from neonatal rats . NOS activities (NO2- accumulation and L-{14C}citrulline formation) were detected by treatment with LPS at 10 micrograms/ml for 6-72 hr . L-{14C}citrulline formation by LPS-induced i-NOS was inhibited by NG-monomethyl-L-arginine (a NOS inhibitor) and diphenyleneiodonium (a flavo-protein inhibitor) . The activity was not markedly changed in the presence or absence of Ca2+ . The induction of i-NOS by LPS was abolished by cycloheximide, actinomycin D, or dexamethasone . In addition, the induction was inhibited by herbimycin A (a tyrosine kinase inhibitor), but was not by staurosporine, W-7, or FK-506 . After LPS stimulation, 130 kDa proteins were reacted with anti-rat liver i-NOS antibody 5-72 hr . i-NOS induced from glial cells coupled tightly with endogenous calmodulin (CaM) even in the absence of Ca2+ . These results suggest that LPS induces expression of 130-kDa i-NOS through an activation of tyrosine kinase, after which i-NOS couples with CaM, and that NO is formed for 6-72 hr in glial cells. J Neurosci Res, 1996 Jan 15, 43(2), 146 - 60 Proximal regions of the olfactory marker protein gene promoter direct olfactory neuron-specific expression in transgenic mice; Walters E et al.; Olfactory marker protein (OMP) expression is highly restricted to mature olfactory neurons (ON) . Less than 0.3 kb of upstream 5' flanking sequence of the OMP gene directs lacZ expression preferentially to ON in several independently derived lines of transgenic mice . A larger transgene with 0.8 kb of upstream flanking sequence also gave lacZ expression in ON and in a few ectopic sites in the central nervous system (CNS) . In addition to the main olfactory epithelium, endogenous OMP is also expressed in chemosensory neurons of the vomeronasal and septal organs, and lacZ expression was detected in neurons of these sites as well . This confirmed the presence of regulatory sequences in the proximal portion of the OMP gene . Endogenous OMP expression in ON was normal in all transgenic lines . Strikingly, in several transgenic lines lacZ expression was restricted to subsets of ON . In one such line, ON axons were intensely stained for lacZ and projected to a subset of olfactory bulb glomeruli . Although identifiable subsets of ON and their termination fields have been described previously, this is the first demonstration of this phenomenon in transgenic mice . These lines of transgenic mice thus provide in vivo models for characterization of genetic elements regulating developmental and functional organization of the olfactory neuroepithelium. Structure, 1996 Jan 15, 4(1), 89 - 96 The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations; Bujacz G et al.; BACKGROUND: Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity . So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E . coli and human immunodeficiency virus 1 (HIV-1) RNase H . Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes . RESULTS: The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution . A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination . The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN . CONCLUSIONS: Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN . This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site . Only a single divalent cation was observed even at extremely high concentrations of the metals . We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein . The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism. Structure, 1996 Jan 15, 4(1), 67 - 77 The complex of the anti-cancer therapeutic, BW1843U89, with thymidylate synthase at 2.0 A resolution: implications for a new mode of inhibition; Stout TJ et al.; BACKGROUND: Thymidylate synthase (TS) is critical to DNA synthesis as it catalyzes the rate limiting step in the only biosynthetic pathway for deoxythymidine monophosphate (dTMP) production . TS is therefore an important target for anti-proliferative and anti-cancer drug design . The TS enzymatic mechanism involves the reductive methylation of the substrate, deoxyuridine monophosphate (dUMP), by transfer of a methylene group from the co-factor, methylenetetrahydrofolate (CH2H4folate), resulting in the production of deoxythymidine monophosphate (dTMP) and dihydrofolate (H2folate) . Previous drug design efforts based on co-factor analogues have produced good inhibitors of TS, but poor bioavailability and toxicity have limited their usefulness . BW1843U89, a folate analogue, is a recently developed compound which is an exceptionally strong inhibitor (Ki = 0.09 nM), has good bioavailability and in clinical trials thus far has not demonstrated significant toxicity . RESULTS: We report the crystal structure of E . coli TS in ternary complex with dUMP and BW1843U89 at 2.0 A resolution . Although the benzoquinazoline ring system of the inhibitor binds to TS in much the same manner as previously determined for H2folate and CB3717, the larger size of the ligand is accommodated by the enzyme through a local distortion of the active site, that is not strictly conserved in both monomers in the asymmetric unit . Several conserved waters that had been previously implicated in mechanistic roles have been displaced . CONCLUSIONS: BW1843U89 forms a ternary complex with dUMP and completes with CH2H4 folate at the active site . Inhibition of TS by BW1843U89 shows four unique aspects in its mechanism of action . BW1843U89 prevents the Michael addition of dUMP to Cys146, in contrast to the mechanisms implicated from crystallography of other quinazoline based inhibitors; displaces a catalytic water from the active site; reorders a peptide loop (Leu72-Trp83) in the active site; and is unique amongst the antifolates in inactivating TS at a stoichiometric ratio of one molecule per TS dimer . Thus, it exploits the principles of negative cooperativity that are increasingly being recognized in the catalytic mechanism of the enzyme per se . The structure suggests that this 'half-the-sites' effect is catalytic and not related to ligand binding . Therefore BW1843U89 is both a competitive inhibitor (at the binding site) and a non-competitive inhibitor at the other site. Rev Prat, 1996 Jan 15, 46(2), 189 - 95 {Travelers' diarrhea}; Chagnon A; Up to fifty per cent of travellers going from temperate countries to tropical or subtropical countries present a diarrhoea . Enterotoxigenic Escherichia coli remain the most frequent bacterial cause, being identified in 40 to 70% of cases . Laboratory investigations are reserved to grave or protracted cases or those which resist to empirical therapy . Careful selection of food and beverage can limit its incidence . In adults at high risk of complications (sick or aged people) or who cannot suffer an interruption of their activity, one can prefer to a chemoprophylaxis with possible occurrence of adverse effects the early administration of a fluoroquinolone, which besides is a first-choice treatment of serious cases. Eur J Biochem, 1996 Jan 15, 235(1-2), 438 - 43 Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides; Willows RD et al.; The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase . The Rhodobacter sphaeroides genes, bchI and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21 (DE3) . The 40-kDa BchI protein was synthesized in greater abundance compared to the 70-kDa BchD protein when both were expressed together from the same plasmid . The production of large amounts of the 140-kDa BchH protein in E . coli was accompanied by an accumulation of protoporphyrin IX . The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1:1 . All three recombinant proteins were soluble; BchH was monomeric, Bchl was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa . The BchH and BchI proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified . Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP . One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio . The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2+-ATP, and a metal-insertion step which in addition requires Mg2+, protoporphyrin IX, and BchH . The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion. Eur J Biochem, 1996 Jan 15, 235(1-2), 372 - 81 Human biliverdin IXalpha reductase is a zinc-metalloprotein . Characterization of purified and Escherichia coli expressed enzymes; Maines MD et al.; Biliverdin IXalpha reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin . BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH, NADPH) specificity . A cDNA clone encoding human BVR was isolated from a gamma library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA . This approach was taken because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed . The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296-amino-acid protein in which could be identified four peptides previously identified by micro-sequencing purified protein . The cDNA hybridized with a single message of approximately 1.2 kb in human kidney poly(A)-rich RNA, and appeared, by Southern blot analysis, to be the product of a single-copy gene . Sequence analysis indicated that the human reductase shows approximately 83% identity, at both the nucleotide and amino acid levels, with rat BVR . In some regions including the carboxyl terminus, protein sequence identity drops to 45% . Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat) . The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence . However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions . Western blot analysis and isoelectric focusing demonstrate that, although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants . Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio . That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli . Exogenous Zn also inhibits NADPH-dependent, but not NADH-dependent, activity . Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe-hematoporphyrin, however, inhibited both NADH- and NADPH-dependent activity. Eur J Biochem, 1996 Jan 15, 235(1-2), 256 - 61 Comparison of self-sustained sequence-replication reaction systems; Gebinoga M et al.; The 3SR (self-sustained sequence-replication) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences in vitro . This method requires three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H . We have modified the original protocol by using human immunodeficiency virus (HIV)-1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E . coli ribonuclease H . Comparison of the incorporation kinetics between the conventional three-enzyme 3SR and our two-enzyme 3SR shows differences in the kinetic behaviour . Furthermore, by the new two-enzyme 3SR, the amplified RNA is obtained in a purer form compared with the experiments with three-enzyme 3SR . The aim of our research is to adapt 3SR as a useful tool for darwinian evolutionary experiments. Eur J Biochem, 1996 Jan 15, 235(1-2), 248 - 55 Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI; Nakari-Setala T et al.; The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose-containing medium, was shown to encode a novel fungal hydrophobin . The encoded 97-amino-acid protein is cysteine-rich and has a typical signal sequence for secretion . Signal-sequence cleavage and putative proteolytic processing results in the mature HFBI protein of 75 amino acids . Antibodies raised against the HFBI protein expressed in Escherichia coli detected the T . reesei HFBI protein in the fungal cell wall and in the culture medium of submerged glucose-containing cultures . The identity of HFBI was verified by N-terminal and peptide sequencing of proteins purified both from the cell wall and culture medium . In the cell wall most of the HFBI formed SDS-insoluble complexes that could be extracted with trifluoroacetic acid . Bubbling or freezing of the culture medium caused HFBI to form aggregates that coprecipitated with a yellow pigment produced by the fungus. Eur J Biochem, 1996 Jan 15, 235(1-2), 215 - 24 Molecular characterisation of plant endoplasmic reticulum . Identification of protein disulfide-isomerase as the major reticuloplasmin; Coughlan SJ et al.; Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two-step centrifugation procedure . These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components . A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity-purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide-isomerase . Northern and Southern blot analysis showed that the mRNA from a single-copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein . Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide-isomerase catalytic activity, thus confirming identity of this protein. Eur J Biochem, 1996 Jan 15, 235(1-2), 207 - 14 Influence of the signal sequence and chaperone SecB on the interaction between precursor protein prePhoE and phospholipids; Van Raalte AL et al.; To investigate in a direct way the interaction between a precursor protein and phospholipids, monolayer studies were performed using the purified precursor of Escherichia coli outer-membrane protein PhoE . It was demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole2GroPGro) and dioleoylglycerophosphoethanolamine (Ole2GroPEtn), this insertion was mainly driven by hydrophobic forces . Compared with previous results obtained with PhoE signal peptide, the full-length precursor protein does not show the specific interaction with acidic lipids . PrePhoE inserted into a Ole2GroPGro monolayer occupies an area of 28 +/- 3 {corrected} nm2/molecule, which is approximately 10-fold larger than the area occupied by the PhoE signal peptide . The purified mature PhoE protein has a lower capacity to insert into Ole2GroPGro and Ole2GroPEtn monolayers and is, in contrast to prePhoE, fully accessible to proteinase K after interacting with a Ole2GroPGro monolayer . The results demonstrate that in the context of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers . It was found that PhoE, like prePhoE, can form in vitro a complex with the cytosolic chaperone SecB . Complexation with SecB increases the insertion of (pre)PhoE into acidic lipid monolayers . The high lipid affinity of prePhoE was also demonstrated by vesicle-binding experiments which showed that SecB dissociates from the SecB-prePhoE complex upon binding of the precursor to the bilayer . The implications of these findings for preprotein translocation are discussed and in addition some extrapolations to the insertion of PhoE into the outer membrane are made. Eur J Biochem, 1996 Jan 15, 235(1-2), 173 - 9 Sequence differences between human muscle and liver cDNAs for UDPglucose pyrophosphorylase and kinetic properties of the recombinant enzymes expressed in Escherichia coli; Duggleby RG et al.; UDP-Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glc1P and UDP-Glc plus MgPPi . Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library . Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5' region and we suggest that these arise from the use of a different first exon in the two transcripts . These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library {Peng, H.-L . & Chang, H.-Y . (1993) FEBS Lett . 329, 153-158} and it is proposed that these liver and muscle forms are derived from different genes . The cDNA for muscle form I, muscle form II, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichia coli and the kinetic properties of each enzyme were characterised . Muscle form I and the LacZ/liver fusion enzyme exhibit Michaelis-Menten kinetics towards all substrates while muscle form II has a sigmoidal dependence of rate upon the concentration of MgPPi . The liver form shows Michaelis-Menten kinetics towards MgUTP . For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration. Eur J Biochem, 1996 Jan 15, 235(1-2), 167 - 72 Redox properties of wild-type, Cys69Ala, and Cys69Ser Azotobacter vinelandii flavodoxin II as measured by cyclic voltammetry and EPR spectroscopy,; Steensma E et al.; This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478) . Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue . Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin . In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode . However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response . EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry . The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK . Instead, the presence of at least two redox-linked protonation sites is suggested: pKred.1 = 5.39 +/- 0.08, pKox = 7.29 +/- 0.14, and pKred.2 = 7.84 +/- 0.14 with Em.7 = -459 +/- 4 mV, and a constant redox potential at high pH of -485 +/- 4 mV . The dependence of the semiquinone/hydroquinone redox potential on temperature is -0.5 +/- 0.1 mV . K(-1), yielding delta H degrees = 28.6 +/- 1.5 kJ . mol(1) and delta S degrees = -50.0 +/- 6.2 J . mol(-1) . K(-1) . No significant differences in redox properties of wild-type, C69A, and C69S flavodoxin were observed . The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A . vinelandii flavodoxin II. Eur J Biochem, 1996 Jan 15, 235(1-2), 152 - 8 Isolation and characterisation of dhel II, a DNA helicase from Drosophila melanogaster embryos stimulated by Escherichia coli-type single-stranded-DNA-binding proteins; Thommes P et al.; We have purified a DNA helicase from Drosophila embryos by following unwinding activity during the purification of the cellular single-stranded DNA-binding protein dRP-A . This DNA helicase unwinds DNA 5' to 3', has a salt-tolerant activity, and has a preference for purine triphosphates as cofactors for the unwinding reaction . The purified enzyme consists of a single polypeptide of 120 kDa, which cosediments with the helicase activity . Sedimentation analysis suggests that this polypeptide exists as a monomer under high and low salt conditions . Dhel II is able to unwind long stretches of DNA, but with decreased efficiency . Addition of Escherichia coli-like single-stranded DNA-binding proteins stimulates the unwinding activity at least 10-fold on substrates greater than 200 nucleotides . In particular, the mitochondrial single-stranded DNA-binding protein isolated from Drosophila embryos is able to stimulate unwinding by dhel II . These properties show that the helicase described is different from another Drosophila helicase dhel I; it has thus has been classified as dhel II. Nucleic Acids Res, 1996 Jan 15, 24(2), 348 - 53 DNA detection by strand displacement amplification and fluorescence polarization with signal enhancement using a DNA binding protein; Walker GT et al.; Strand displacement amplification (9SDA) is an isothermal in vitro method of amplifying a DNA sequence prior to its detection . We have combined SDA with fluorescence polarization detection . A 5'-fluorescein-labelled oligodeoxynucleotide detector probe hybridizes to the amplification product that rises in concentration during SDA and the single- to double strand conversion is monitored through an increase in fluorescence polarization . Detection sensitivity can be enhanced by using a detector probe containing an EcoRI recognition sequence at its 5'-end that is not homologous to the target sequence . During SDA the probe is converted to a fully double-stranded form that specifically binds a genetically modified form of the endonuclease EcoRI which lacks cleavage activity but retains binding specificity . We have applied this SDA detection system to a target sequence specific for Mycobacterium tuberculosis. EMBO J, 1996 Jan 15, 15(2), 408 - 17 A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates; Szabo A et al.; The Escherichia coli heat-shock protein DnaJ cooperates with the Hsp70 homolog DnaK in protein folding in vitro and in vivo . Little is known about the structural features of DnaJ that mediate its interaction with DnaK and unfolded polypeptide . DnaJ contains at least four blocks of sequence representing potential functional domains which have been conserved throughout evolution . In order to understand the role of each of these regions, we have analyzed DnaJ fragments in reactions corresponding to known functions of the intact protein . Both the N-terminal 70 amino acid 'J-domain' and a 35 amino acid glycine-phenylalanine region following it are required for interactions with DnaK . However, only complete DnaJ can cooperate with DnaK and a third protein, GrpE, in refolding denatured firefly luciferase . As demonstrated by atomic absorption and extended X-ray absorption fine structure spectroscopy (EXAFS), the 90 amino acid cysteine-rich region of DnaJ contains two Zn atoms tetrahedrally coordinated to four cysteine residues, resembling their arrangement in the C4 Zn binding domains of certain DNA binding proteins . Interestingly, binding experiments and cross-linking studies indicate that this Zn finger-like domain is required for the DnaJ molecular chaperone to specifically recognize and bind to proteins in their denatured state. EMBO J, 1996 Jan 15, 15(2), 399 - 407 Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria; Komiya T et al.; Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria . Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner . Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70 . Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane . The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70-dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP . The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added . The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins. EMBO J, 1996 Jan 15, 15(2), 392 - 98 Preferential binding of an unfolded protein to DsbA; Frech C et al.; The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate . During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA . To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1 . We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol . This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions . The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds . Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 275 - 80 4-Phospho-hydroxy-L-threonine is an obligatory intermediate in pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12; Zhao G et al.; We show that thrB-encoded homoserine kinase is required for growth of Escherichia coli K-12 pdxB mutants on minimal glucose medium supplemented with 4-hydroxy-L-threonine (synonym, 3-hydroxyhomoserine) or D-glycolaldehyde . This result is consistent with a model in which 4-phospho-hydroxy-L-threonine (synonym, 3-hydroxyhomoserine phosphate), rather than 4-hydroxy-L-threonine, is an obligatory intermediate in pyridoxal 5'-phosphate biosynthesis . Ring closure using 4-phospho-hydroxy-L-threonine as a substrate would lead to formation of pyridoxine 5'-phosphate, and not pyridoxine, as the first B6-vitamer synthesized de novo . These considerations suggest that E . coli pyridoxal/pyridoxamine/pyridoxine kinase is not required for the main de novo pathway of pyridoxal 5'-phosphate biosynthesis, and instead plays a role only in the B6-vitamer salvage pathway. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 245 - 9 Comparison of cytosolic levels of calcium and G actin in diffuse and localised adherent Escherichia coli-infected HeLa cells; Karmaker S et al.; In the present study we compared the intracellular level of free calcium ({Ca2+}i) and monomeric (G)/total (G+F) actin ratio in HeLa cells infected with diffuse (DAEC) and localised adherent Escherichia coli (LAEC) . The level of {Ca2+}i was increased in both DAEC- and LAEC-infected HeLa cells . However, studies with EGTA- and dantrolene-treated cells and also suspension of cells in Ca(2+)-free buffer suggested that the rise of {Ca2+}i in DAEC-infected cells was due to the influx of Ca2+ from extracellular medium, whereas Ca2+ mobilisation from the intracellular stores was responsible for the enhancement of {Ca2+}i in LAEC-infected cells . It was also evident that the infection of HeLa cells with DAEC and LAEC caused alteration of G/G+F actin ratio as compared to that of control cells . The ratio was much lower in LAEC-infected cells than that of DAEC-infected ones . Moreover, cytochalasin B inhibited both DAEC and LAEC invasion to HeLa cells, suggesting further the role of microfilaments in the invasion process. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 237 - 43 A stable Escherichia coli-mycobacteria shuttle vector 'pSO246' in Mycobacterium bovis BCG; Matsumoto S et al.; The most widely used plasmid vector system in mycobacteria is based on pAL5000 from Mycobacterium fortuitum . The derivatives of the pAL5000-based shuttle vectors between Escherichia coli and mycobacteria, which we have utilized to secrete recombinant antigens, were generated . The stability of the vectors was assessed in Mycobacterium bovis BCG (BCG) . The plasmid vector pSO246 was stable in BCG for at least 50 generations. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 213 - 21 Identification of sequences important for recognition of vnf genes by the VnfA transcriptional activator in Azotobacter vinelandii; Woodley P et al.; To analyze regulation of the vanadium-dependent nitrogenase of Azotobacter vinelandii, plasmids carrying vnfE-, vnfH-, or vnfD-lacZ fusions were transferred to Escherichia coli . These genes were expressed only if VnfA was present . Deletions of the vnfE upstream region were constructed and comparison of a region necessary for expression with sequences upstream of other vnf genes indicated a substantially conserved motif, GTAC-N6-GTAC, hypothesized to be the binding site for VnfA . This motif was duplicated with 17 or 18 bases lying between each in the vnfH and vnfD promoters . Deletion analysis of the vnfH promoter indicated that both motifs were necessary for full expression . In footprinting experiments, VnfA significantly protected from methylation the guanine residues within or immediately adjacent to the proposed VnfA recognition motifs . The active form of VnfA is probably interacting dimers, a tetramer, or a higher order oligomer since two regions of dyad symmetry are required for its interaction with the DNA. Biochem J, 1996 Jan 15, 313 ( Pt 2), 589 - 96 Biosynthesis of dermatan sulphate . Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism; Hannesson HH et al.; The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ({GlcA(beta 1-->3)GalNAc(beta 1-->4)}n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues . Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis . Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units . A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate) . Vmax was about 4 times higher with dermatan than with the K4 substrate . A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-{5-3H}glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme . Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA . A corresponding incubation of dermatan yielded approx . 22% GlcA, which contained virtually all the 3H label . These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base . Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed. Biochem J, 1996 Jan 15, 313 ( Pt 2), 479 - 86 Administration of Escherichia coli endotoxin to rat increases liver mass and hepatocyte volume in vivo; Qian D et al.; We have established, in vivo, an increase in liver mass and hepatocyte volume after a single intraperitoneal administration, to fasted rats, of Escherichia coli lipopolysaccharide (0127:B8) at 3 mg/kg . The phenomenon was time- and dose-dependent and could be prevented by treatment with polyclonal antiserum against tumour necrosis factor-alpha (TNF-alpha) before the endotoxin injection . Endotoxin caused an increase of 26% in the hepatic mass compared with fasted controls at 24 h . An increase of 27% in the hepatic water content underlay the altered hepatic mass which could not be accounted for by a change in the volume of hepatic blood and/or interstitial fluid (measured in vivo), suggesting an expansion in the hepatocellular volume . This is supported by an increase of 25% in the K+ content of the endotoxic livers . Morphometric study confirmed a 15% increase in hepatocyte volume after endotoxin administration . The data are discussed in the light of possible metabolic effects of increased hepatocyte volume. Biochem J, 1996 Jan 15, 313 ( Pt 2), 415 - 21 Evidence for a covalent intermediate in the S-adenosyl-L-methionine-dependent transmethylation reaction catalysed by sirohaem synthase; Woodcock SC et al.; CysG, also known as uroporphyrinogen III methylase and sirohaem synthase (CysG; EC 2.1.1.107), is a multifunctional enzyme that is able to transform uroporphyrinogen III into sirohaem via two S-adenosyl-L-methionine (AdoMet)-dependent transmethylations, an NAD(+)-dependent dehydrogenation and a ferrochelation . The apparent tight binding of AdoMet to this multifunctional enzyme is investigated . The use of a rapid AdoMet binding assay demonstrates that CysG becomes labelled with both {methyl-3H}AdoMet and {carboxyl-14C}AdoMet . Further experiments show that the CysG-AdoMet complex is subsequently able to methylate uroporphyrinogen III . CysG remains associated with the labelled constituents of the AdoMet even after denaturation with urea and SDS/PAGE, suggesting that the AdoMet has become covalently linked to the protein . A rapid examination of some of the other transmethylases involved in corrin biosynthesis reveals that they bind the AdoMet in a similar fashion . A multistep transmethylation mechanism is proposed to explain the observed results. Biochem J, 1996 Jan 15, 313 ( Pt 2), 409 - 14 Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease; Doskeland AP et al.; The phosphorylation of human phenylalanine hydroxylase by cyclic AMP-dependent protein kinase was studied using recombinant enzyme expressed as a fusion protein in the pMAL system of Escherichia coli . Using the target sequence of the restriction protease enterokinase (Asp4-Lys) as the linker peptide, 100% full-length human phenylalanine hydroxylase was obtained on protease cleavage . The fusion protein and human phenylalanine hydroxylase were both phosphorylated at Ser-16 with a stoichiometry of 1 mol of Pi/mol of subunit . The rate of phosphorylation of human phenylalanine hydroxylase was inhibited about 40% by the cofactor tetrahydrobiopterin, and this inhibition was completely prevented by the simultaneous presence of L-phenylalanine (i.e . at turnover conditions) . Phosphorylated enzyme revealed a 1.6-fold higher specific activity than the non-phosphorylated enzyme form, and it also required a lower concentration of L-Phe for substrate activation . Pre-incubation with L-Phe increased the specific activity of phenylalanine hydroxylase 2- to 4-fold, L-Phe acting with positive cooperativity . Thus, the basic catalytic and regulatory properties of recombinant human phenylalanine hydroxylase, as well as those observed for the enzyme as a fusion protein, are similar to those previously reported for the rat liver enzyme . When the target sequence of the restriction protease factor Xa (Ile-Glu-Gly-Arg) was used as the linker between maltose-binding protein and human phenylalanine hydroxylase, cleavage of the fusion protein gave a mixture of full-length hydroxylase and a truncated form of the enzyme lacking the 13 N-terminal residues . Interestingly, phosphorylation of the fusion protein, before exposure to factor Xa, almost completely protected against secondary cleavage by this restriction protease at Arg-13 of phenylalanine hydroxylase. Mol Gen Genet, 1996 Jan 15, 250(1), 81 - 8 Escherichia coli cis- and trans-acting mutations that increase glyA gene expression; Lorenz E et al.; We used an Escherichia coli strain blocked in serine biosynthesis and carrying a partial glyA deletion to isolate strains with altered regulation of the glyA gene . The glyA deletion results in 25% of the normal serine hydroxymethyltransferase activity . Three classes of mutants with increased glyA expression were isolated on glycine supplemented plates . One class of mutations increased glyA expression 10-fold by directly altering the -35 consensus sequence of the glyA promoter . The two other classes increased glyA expression about 2- and 6-fold, respectively . The latter two classes of mutations also affected regulation of the metE gene of the folate branch of the methionine pathway, but not metA in the nonfolate branch of the methionine pathway, or the gcv operon, encoding the glycine cleavage enzyme system . The mutations were mapped to about minute 85.5 on the E . coli chromosome. J Clin Invest, 1996 Jan 15, 97(2), 359 - 65 A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide; Somerville JE Jr et al.; A unique screen was used to identify mutations in Escherichia coli lipid A biosynthesis that result in a decreased ability to stimulate E-selectin expression by human endothelial cells . A mutation was identified in the msbB gene of E . coli that resulted in lipopolysaccharide (LPS) that lacks the myristoyl fatty acid moiety of the lipid A . Unlike all previously reported lipid A mutants, the msbB mutant was not conditionally lethal for growth . Viable cells or purified LPS from an msbB mutant had a 1000-10,000-fold reduction in the ability to stimulate E-selectin production by human endothelial cells and TNF alpha production by adherent monocytes . The cloned msbB gene was able to functionally complement the msbB mutant, restoring both the LPS to its native composition and the ability of the strain to stimulate immune cells . Nonmyristoylated LPS acted as an antagonist for E-selectin expression when mixed with LPS obtained from the parental strain . These studies demonstrate a significant role for the myristate component of LPS in immune cell activation and antagonism . In addition, the msbB mutant allowed us to directly examine the crucial role that the lipid A structure plays when viable bacteria are presented to host defense cells. Arch Biochem Biophys, 1996 Jan 15, 325(2), 270 - 8 The sequencing expression, purification, and steady-state kinetic analysis of quinolinate phosphoribosyl transferase from Escherichia coli; Bhatia R et al.; The nadC gene from Escherichia coli was isolated and sequenced . The gene was then cloned into an expression vector and, following transformation, the resulting bacteria were able to produce quinolinate phosphoribosyl transferase as about 2% of the soluble protein . The enzyme was purified in five steps leading to a homogeneous preparation . The enzyme reaction shows an ordered binding mechanism where the magnesium ion complex of 5-phosphoribosyl-1-pyrophosphate binds first followed by quinolinic acid . The products are pyrophosphate CO2, and nicotinate mononucleotide . Product inhibition studies show that nicotinate mononucleotide is a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate while pyrophosphate is noncompetitive with respect to both 5-phosphoribosyl-1-pyrophosphate and quinolinic acid . Phthalic acid and fructose-1,6-bisphosphate were used as dead-end inhibitors . Phthalate was competitive with respect to quinolinic acid but uncompetitive with respect to 5-phosphoribosyl-1-pyrophosphate . Fructose-1,6-bisphosphate was a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate and noncompetitive with respect to quinolinic acid . The Km values for the substrates are 15.6 microM for 5-phosphoribosyl-1-pyrophosphate and 6.4 microM for quinolinic acid. FEBS Lett, 1996 Jan 15, 378(3), 286 - 90 Effects of N-terminal deletions on 1-aminocyclopropane-1-carboxylate synthase activity; Li N et al.; A series of nested N-terminal deletions were made on the full-length (wt) and C-terminal deleted (Cdel) 1-aminocyclopropane-1-carboxylate synthase cDNAs . These wt and mutant ACC synthases were over-expressed in a heterologous E . coli expression system . It was found that removal of an amino acid region (residues 2-12) from the non-conserved N-termini of wt and Cdel ACC synthases led to a slight increase in both in vivo ACC production and in vitro ACC synthase activity . Further deletion of 11 amino acids through Glu-23 from the N-termini of both wt and Cdel ACC synthases resulted in a substantial reduction in both in vivo ACC production and in vitro enzyme activity . Deletion of an amino acid region, residues 3 through 27, from the N-terminus of ACC synthase abolished enzyme activity completely . Kinetic analysis of a highly purified double-deletion mutant (NCdel-1) of ACC synthase demonstrated that the Km of this mutant is 42 microM, which is much smaller than that of the corresponding Cdel (280 microM) and closer to that of wt (22 microM) reported previously, suggesting a clear effect of the non-conserved N-terminal region on its ACC synthase function. J Immunol, 1996 Jan 15, 156(2), 778 - 85 Modulation of endotoxin-induced histamine synthesis by cytokines in mouse bone marrow-derived macrophages; Takamatsu S et al.; The aim of this study was to examine the roles of various cytokines in histamine synthesis by macrophages in bone marrow . Pure (> 99% nonspecific esterase-, CD14-, and Mac-1-positive) macrophage populations were obtained after long term culture of bone marrow cells (bone marrow-derived macrophages) . Culture of the cells in the presence of Escherichia coli LPS caused a slight, but dose-dependent, increase in histidine decarboxylase-associated histamine synthesis with a concomitant increase in the expression of CD14, a LPS receptor, as well as the Mac-1 Ag on their surface . Granulocyte/macrophage CSF (GM-CSF) or IL-3 strongly enhanced LPS-induced histamine formation and expression of CD14 on bone marrow-derived macrophages, without affecting the expression of Mac-1 Ag . GM-CSF and IL-3 also caused a marked accumulation of both histidine decarboxylase and IL-6 mRNAs in the cells . Macrophage CSF and IL-1-alpha also potentiated LPS-dependent histamine formation when it was stimulated with GM-CSF or IL-3 . In contrast to these cytokines, IFN-gamma suppressed LPS-induced histamine production regardless of whether it was stimulated by GM-CSF or IL-3, and inhibited CD14 expression . Neither IL-6 nor granulocyte CSF had any appreciable effect on LPS-induced histamine production even in combination with GM-CSF or IL-3 . These results suggest that GM-CSF and IL-3 enhance LPS-induced histamine production in bone marrow-derived macrophages and that macrophage CSF and IL-1 alpha augment the actions of GM-CSF and IL-3 . Possible implication of CD14 molecule in the reactions is discussed. Mol Cell Biochem, 1996 Jan 12, 154(1), 9 - 16 Biochemical and immunological identification and enrichment of poly(A) polymerase from human thymus; Kyriakopoulou C et al.; Human thymus poly(A) polymerase (EC 2.7.7.19) activity has been investigated using poly(A) and oligo(A) as initiators . All obtained fractions reveal more than one polypeptide as detected by immunoblotting after SDS-PAGE . In addition to the homogeneously purified (Tsiapalis et al., J Biol Chem 250: 4486-4496, 1975 and Wahle, J Biol Chem 266: 3131-3139, 1991), about 60 kDa polypeptide, a larger polypeptide, about 80 kDa, that comigrates in the region of poly(A) polymerase activity was detected, enriched and partially characterized; it appears having similar size with bovine poly(A) polymerase cloned in E . coli . Polyclonal antiserum produced against recombinant bovine poly(A) polymerase reacts more efficiently with the about 80 kDa polypeptide upon immunoblotting, and can precipitate the poly(A) polymerase activity . This enzyme form, from human tissue, is novel in terms of size and may reflect intact or physiological form of poly(A) polymerase in human thymus, and supports and substantiates recent reports on the enzyme from other sources. J Mol Biol, 1996 Jan 12, 255(1), 55 - 66 In vitro evolution of the DNA binding sites of Escherichia coli methionine repressor, MetJ; He YY et al.; The SELEX procedure was used to study the recognition between the E . coli methionine repressor (MetJ) and its DNA binding sites . DNA ligands with high affinity for either the holo-repressor or apo-repressor were isolated from a pool of molecules randomized over 20 base-pairs . Among 90 DNA ligands selected by holo-repressor binding, roughly 90% contain variations of two tandem, perfect eight base-pair Met-boxes, which are the consensus deduced from natural met operators . Base-pairs that are important, for specific interactions with the protein are highly conserved . The data also reveal the importance of the non-contacted operator base-pairs in facilitating the conformational changes in the operator which must occur for repressor binding . There are also effects due to the sequences of the base-pairs immediately flanking the operator site . DNA ligands selected by apo-repressor share a very similar, but not identical, consensus with that selected by holo-repressor, suggesting that the corepressor does not greatly alter the specificity of repressor binding. J Biol Chem, 1996 Jan 12, 271(2), 878 - 82 Monocyte chemotactic factor in rheumatoid arthritis synovial tissue . Probably a cross-linked derivative of S19 ribosomal protein; Nishiura H et al.; The extracts of rheumatoid arthritis-synovial lesions from seven patients possessed a strong chemotactic activity for monocytes and a negligible one for polymorphonuclear leukocytes . These results are consistent with a prominent histological feature of the synovial lesion, the mononuclear cell predominant infiltration . The major monocyte chemotactic factor in the synovial tissue extracts was purified to a single protein peak in reverse phase high performance liquid chromatography with a C4 column . NH2-terminal amino acid analysis of the initial 20 residues yielded a single sequence . Surprisingly, this sequence was completely identical to that of S19 ribosomal protein . The purified sample demonstrated two protein bands in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 34 and 68 kDa . These sizes were 2 and 4 times that of S19 ribosomal protein, suggesting that the chemotactic factor would be a dimer or tetramer of S19 ribosomal protein cross-linked by factor XIIIa . A recombinant human S19 ribosomal protein was prepared as a fusion protein with a maltose binding protein in Escherichia coli . After treatment with factor XIIIa, cross-linked recombinant S19 ribosomal protein exhibited the monocyte chemotactic activity, although the untreated recombinant protein did not. J Biol Chem, 1996 Jan 12, 271(2), 783 - 8 Substrate specificity of the Escherichia coli 4-aminobutyrate carrier encoded by gabP . Uptake and counterflow of structurally diverse molecules; Brechtel CE et al.; Transport of 4-aminobutyrate into Escherichia coli is catalyzed by gab permease (GabP) . Although published studies show that GabP is relatively specific, recognizing the common alpha-amino acids with low affinity, recent work from this laboratory indicates that a number of synthetic compounds are high affinity transport inhibitors (50% inhibition at 5-100 microM) . Here we present evidence that many of these structurally heterogeneous compounds not only inhibit transport but also function as alternative GabP substrates (i.e . a set of observations inconsistent with the idea that the core of the GabP transport channel exhibits rigid structural specificity for the native substrate, 4-aminobutyrate. J Biol Chem, 1996 Jan 12, 271(2), 653 - 62 Lipoyl domain-based mechanism for the integrated feedback control of the pyruvate dehydrogenase complex by enhancement of pyruvate dehydrogenase kinase activity; Ravindran S et al.; To conserve carbohydrate reserves, the reaction of the pyruvate dehydrogenase complex (PDC) must be down-regulated when the citric acid cycle is provided sufficient acetyl-CoA . PDC activity is reduced primarily through increased phosphorylation of its pyruvate dehydrogenase (E1) component due to E1 kinase activity being markedly enhanced by elevated intramitochondrial NADH:NAD+ and acetyl-CoA:CoA ratios . A mechanism is evaluated in which enhanced kinase activity is facilitated by the build-up of the reduced and acetylated forms of the lipoyl moieties of the dihydrolipoyl acetyltransferase (E2) component through using NADH and acetyl-CoA in the reverse of the downstream reactions of the complex . Using a peptide substrate, kinase activity was stimulated by these products, ruling out the possibility kinase activity is increased due to changes in the reaction state of its substrate, E1 (thiamin pyrophosphate) . Each E2 subunit contains two lipoyl domains, an NH2-terminal (L1) and the inward lipoyl domain (L2), which were individually produced in fully lipoylated forms by recombinant techniques . Although reduction and acetylation of the L1 domain or free lipoamide increased kinase activity, those modifications of the lipoate of the kinase-binding L2 domain gave much greater enhancements of kinase activity . The large stimulation of the kinase generated by acetyl-CoA only occurred upon addition of the transacetylase-catalyzing (lipoyl domain-free) inner core portion of E2 plus a reduced lipoate source, affirming that acetylation of this prosthetic group is an essential mechanistic step for acetyl-CoA enhancing kinase activity . Similarly, the lesser stimulation of kinase activity by just NADH required a lipoate source, supporting the need for lipoate reduction by E3 catalysis . Complete enzymatic delipoylation of PDC, the E2-kinase subcomplex, or recombinant L2 abolished the stimulatory effects of NADH and acetyl-CoA . Retention of a small portion of PDC lipoates lowered kinase activity but allowed stimulation of this residual kinase activity by these products . Reintroduction of lipoyl moieties, using lipoyl protein ligase, restored the capacity of the E2 core to support high kinase activity along with stimulation of that activity up to 3-fold by NADH and acetyl-CoA . As suggested by those results, the enhancement of kinase activity is very responsive to reductive acetylation with a half-maximal stimulation achieved with approximately 20% of free L2 acetylated and, from an analysis of previous results, with acetylation of only 3-6 of the 60 L2 domains in intact PDC . Based on these findings, we suggest that kinase stimulation results from modification of the lipoate of an L2 domain that becomes specifically engaged in binding the kinase . In conclusion, kinase activity is attenuated through a substantial range in response to modest changes in the proportion of oxidized, reduced, and acetylated lipoyl moieties of the L2 domain of E2 produced by fluctuations in the NADH:NAD+ and acetyl-CoA:CoA ratios as translated by the rapid and reversible E3 and E2 reactions. J Biol Chem, 1996 Jan 12, 271(2), 646 - 52 Initial binding of the elongation factor Tu.GTP.aminoacyl-tRNA complex preceding codon recognition on the ribosome; Rodnina MV et al.; The first step in the sequence of interactions between the ribosome and the complex of elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA, which eventually leads to A site-bound aminoacyl-tRNA, is the codon-independent formation of an initial complex . We have characterized the initial binding and the resulting complex by time-resolved (stopped-flow) and steady-state fluorescence measurements using several fluorescent tRNA derivatives . The complex is labile, with rate constants of 6 x 10(7) M-1 s-1 and 24 s-1 (20 degrees C, 10 mM Mg2+) for binding and dissociation, respectively . Both thermodynamic and activation parameters of initial binding were determined, and five Mg2+ ions were estimated to participate in the interaction . While a cognate ternary complex proceeds form initial binding through codon recognition to rapid GTP hydrolysis, the rate constant of GTP hydrolysis in the non-cognate complex is 4 orders of magnitude lower, despite the rapid formation of the initial complex in both cases . Hence, the ribosome-induced GTP hydrolysis by EF-Tu is strongly affected by the presence of the tRNA . This suggests that codon-anticodon recognition, which takes place after the formation of the initial binding complex, provides a specific signal that triggers fast GTP hydrolysis by EF-Tu on the ribosome. J Biol Chem, 1996 Jan 12, 271(2), 1133 - 7 Escherichia coli RNase T functions in vivo as a dimer dependent on cysteine 168; Li Z et al.; It was shown that Cys-168 is required for RNase T function and thermostability and that its hydrophobic properties are important for this role (Li, Z., Zhan, L., and Deutscher, M . P . (1996) J . Biol Chem . 271, 1127-1132) . To understand the molecular basis for these findings, further studies of Cys-168 and RNase T structure were carried out . Treatment of RNase T with the sulfhydryl-modifying agent 5,5'-dithiobis-(2-nitrobenzoic acid) leads not only to inactivation, but also to monomerization of the protein . Similarly, specifically converting Cys-168 to either serine or asparagine leads to loss of activity and to monomer formation at 37 degrees C . However, at 10 degrees C the serine mutant remains as a dimer and retains full RNase T activity, whereas the asparagine derivative shows only a low level of activity and of dimer formation . These data show a strong correlation between activity and the dimer form of RNase T . The importance of dimer formation was also shown in vivo using genetic studies . An inactive mutant of RNase T, termed HA2, which exists as a dimer at 37 degrees C in vitro, completely suppresses endogenous RNase T activity in vivo and in vitro when introduced into a RNase T+ cell on a multicopy phagemid, most likely as a consequence of inactive heterodimer formation . Introduction of the HA2 gene on a single-copy plasmid, as expected, leads to a proportionally smaller effect on endogenous activity . The dominant negative effect displayed by the HA2 protein can be relieved by an additional mutation in HA2 RNase T that abolishes its ability to dimerize . An inactive mutant asparagine derivative of Cys-168, which also does not dimerize, also shows little of the dominant negative phenotype . Thus, these data demonstrate that RNase T dimerizes in vivo, that the dimer form is required for RNase T activity, and that Cys-168 is needed for dimerization of the enzyme. J Biol Chem, 1996 Jan 12, 271(2), 1127 - 32 The role of individual cysteine residues in the activity of Escherichia coli RNase T; Li Z et al.; Escherichia coli RNase T, which is responsible for the 3' processing and end-turnover of tRNA and the maturation of 5 S RNA, is extremely sensitive to sulfhydryl reagents and to oxidation, suggesting a role for cysteine residues in its activity . Titration of homogeneous RNase T with 5,5'-dithiobis-(2-nitrobenzoic acid) revealed that the 4 cysteine residues present in each of the two protein subunits are in a reduced form and that 1 or 2 of them are important for activity . To identify these residue(s), each of the cysteines in RNase T was changed individually to either serine or alanine . The serine mutant at position 168 is greatly reduced in RNase T activity both in vivo and in vitro; likewise, the serine mutant at position 112 and the alanine mutants at positions 112 and 168 also display decreased RNase T activity . Mutations at the other cysteine positions show little or no change . Kinetic analyses of the mutant enzymes showed that the Km values of C168S and C168A are increased considerably, whereas their Vmax values are reduced only slightly compared to the wild type enzyme . The other mutant enzymes are little changed . Additional amino acid replacements at position 168 showed that the in vivo and in vitro activities of RNase T are in the order Cys approximately Val > Ala >> Ser >> Asn approximately Asp, which closely follows the relative hydrophobicity of these amino acid residues . However, the affinity for tRNA, determined by fluorescence quenching, is not altered in C168S, suggesting that Cys-168 is not directly involved in substrate binding . Interestingly, proteins altered at position 168 showed increased temperature sensitivity as the residue at that position became less hydrophobic . These data indicate that Cys-168 contributes a hydrophobic group that influences the structure and ultimately the catalytic activity of RNase T. J Biol Chem, 1996 Jan 12, 271(2), 1048 - 53 Overexpression, purification, and properties of Escherichia coli ribonuclease II; Coburn GA et al.; Ribonuclease II (RNase II) is a major exonuclease in Escherichia coli that hydrolyzes single-stranded polyribonucleotides processively in the 3' to 5' direction . To understand the role of RNase II in the decay of messenger RNA, a strain overexpressing the rnb gene was constructed . Induction resulted in a 300-fold increase in RNase II activity in crude extracts prepared from the overexpressing strain compared to that of a non-overexpressing strain . The recombinant polypeptide (Rnb) was purified to apparent homogeneity in a rapid, simple procedure using conventional chromatographic techniques and/or fast protein liquid chromatography to a final specific activity of 4,100 units/mg . Additionally, a truncated Rnb polypeptide was purified, solubilized, and successfully renatured from inclusion bodies . The recombinant Rnb polypeptide was active against both {3H}poly(A) as well as a novel (synthetic partial duplex) RNA substrate . The data show that the Rnb polypeptide can disengage from its substrate upon stalling at a region of secondary structure and reassociate with a new free 3'-end . The stalled substrate formed by the dissociation event cannot compete for the Rnb polypeptide, demonstrating that duplexed RNAs lacking 10 protruding unpaired nucleotides are not substrates for RNase II . In addition, RNA that has been previously trimmed back to a region of secondary structure with purified Rnb polypeptide is not a substrate for polynucleotide phosphorylase-like activity in crude extracts . The implications for mRNA degradation and the proposed role for RNase II as a repressor of degradation are discussed. J Biol Chem, 1996 Jan 12, 271(2), 1022 - 8 Important role of the amino acid attached to tRNA in formylation and in initiation of protein synthesis in Escherichia coli; Li S et al.; In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation . This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase . Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine . By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E . coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis . Thus, the G4: C69 base pair contributes toward formylation of the tRNA and protein synthesis in E . coli can be initiated with formyllysine . We also discuss the implications of these and other results on recognition of tRNAs by E . coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases. Science, 1996 Jan 12, 271(5246), 203 - 7 Structure of the heat shock protein chaperonin-10 of Mycobacterium leprae; Mande SC et al.; Members of the chaperonin-10 (cpn10) protein family, also called heat shock protein 10 and in Escherichia coli GroES, play an important role in ensuring the proper folding of many proteins . The crystal structure of the Mycobacterium leprae cpn10 (Ml-cpn10) oligomer has been elucidated at a resolution of 3.5 angstroms . The architecture of the Ml-cpn10 heptamer resembles a dome with an oculus in its roof . The inner surface of the dome is hydrophilic and highly charged . A flexible region, known to interact with cpn60, extends from the lower rim of the dome . With the structure of a cpn10 heptamer now revealed and the structure of the E . coli GroEL previously known, models of cpn10:cpn60 and GroEL:GroES complexes are proposed. Science, 1996 Jan 12, 271(5246), 195 - 7 Functional evidence for indirect recognition of G.U in tRNA(Ala) by alanyl-tRNA synthetase; Gabriel K et al.; The structural features of the G.U wobble pair in Escherichia coli alanine transfer RNA (tRNA(Ala)) that are associated with aminoacylation by alanyl-tRNA synthetase (AlaRS) were investigated in vivo for wild-type tRNA(Ala) and mutant tRNAs with G.U substitutions . tRNA(Ala) with G.U, C.A, or G.A gave similar amounts of charged tRNA(Ala) and supported viability of E . coli lacking chromosomal tRNA(Ala) genes . tRNA(Ala) with G.C was inactive . Recognition of G.U by AlaRS thus requires more than the functional groups on G.U in a regular helix and may involve detection of a helical distortion. Biochim Biophys Acta, 1996 Jan 11, 1273(1), 62 - 70 Reconstitution of the F1-ATPase activity from purified alpha, beta, gamma and delta or epsilon subunits with glutathione S-transferase fused at their amino termini; Shin Y et al.; Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established . The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography . Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate . By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained . The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography . These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography . The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins . These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex . The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly . The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly. Biochim Biophys Acta, 1996 Jan 11, 1273(1), 4 - 12 Estimation of the H+/H- ratio of the reaction catalysed by the nicotinamide nucleotide transhydrogenase in chromatophores from over-expressing strains of Rhodospirillum rubrum and in liposomes inlaid with the purified bovine enzyme; Bizouarn T et al.; Two strains of Rhodospirillum rubrum were constructed in which, by a gene dosage effect, the transhydrogenase activity of isolated chromatophores was increased 7-10-fold and 15-20-fold, respectively . The H+/H- ratio (the ratio of protons translocated per hydride ion equivalent transferred from NADPH to an NAD+ analogue, acetyl pyridine adenine dinucleotide), determined by a spectroscopic technique, was approximately 1.0 for chromatophores from the over-expressing strains, but was only approximately 0.6 for wild-type chromatophores . Highly-coupled proteoliposomes were prepared containing purified transhydrogenase from beef-heart mitochondria . Using the same technique, the H+/H- ratio was close to 1.0 for these proteoliposomes . It is suggested that the mechanistic H+/H- ratio is indeed unity, but that a low ratio is obtained in wild-type chromatophores because of inhomogeneity in the vesicle population. Biochim Biophys Acta, 1996 Jan 11, 1273(1), 21 - 30 Comparison of the inhibitory action of synthetic capsaicin analogues with various NADH-ubiquinone oxidoreductases; Satoh T et al.; Capsaicin is a new naturally occurring inhibitor of proton-pumping NADH-ubiquinone oxidoreductase (NDH-1), that competitively acts against ubiquinone . A series of capsaicin analogues was synthesized to examine the structural factors required for the inhibitory action and to probe the structural property of the ubiquinone catalytic site of various NADH-ubiquinone reductases, including non-proton-pumping enzyme (NDH-2), from bovine heart mitochondria, potato tuber (Solanum tuberosum, L) mitochondria and Escherichia coli (GR 19N) plasma membranes . Some synthetic capsaicins were fairly potent inhibitors of each of the three NDH-1 compared with the potent rotenone and piericidin A . Synthetic capsaicin analogues inhibited all three NDH-1 activities in a competitive manner against an exogenous quinone . The modification both of the substitution pattern and of the number of methoxy groups on the benzene ring, which may be superimposable on the quinone ring of ubiquinone, did not drastically affect the inhibitory potency . In addition, alteration of the position of dipolar amide bond unit in the molecule and chemical modifications of this unit did not change the inhibitory potency, particularly with bovine heart and potato tuber NDH-1 . These results might be explained assuming that the ubiquinone catalytic site of NDH-1 is spacious enough to accommodate a variety of structurally different capsaicin analogues in a dissimilar manner . Regarding the moiety corresponding to the alkyl side chain, a rigid diphenyl ether structure was more inhibitory than a flexible alkyl chain . Structure-activity studies and molecular orbital calculations suggested that a bent form is the active conformation of capsaicin analogues . On the other hand, poor correlations between the inhibitory potencies determined with the three NDH-1 suggested that the structural similarity of the ubiquinone catalytic sites of these enzymes is rather poor . The sensitivity to the inhibition by synthetic capsaicins remarkably differed between NDH-1 and NDH-2, supporting the notion that the sensitivity against capsaicin inhibition correlates well with the presence of an energy coupling site in the enzyme (Yagi, T . (1990) Arch . Biochem . Biophys . 281, 305-311) . It is noteworthy that several synthetic capsaicins discriminated between NDH-1 and NDH-2 much better than natural capsaicin. Biochemistry, 1996 Jan 9, 35(1), 7 - 13 Ionization equilibria for side-chain carboxyl groups in oxidized and reduced human thioredoxin and in the complex with its target peptide from the transcription factor NF kappa B; Qin J et al.; The pH dependence of the 13C chemical shifts of the side-chain carboxyl carbons of all Asp and Glu residues in the reduced and oxidized states of human thioredoxin and in a mixed disulfide complex of human thioredoxin with a target peptide from the transcription factor NF kappa B has been investigated by multidimensional triple-resonance NMR spectroscopy . While the titration curves for most of the side-chain carboxyl resonances exhibit simple Henderson-Hasselbalch behavior with pKa values not far from those found for model compounds, several side chains give rise to two- or three-step titration curves, indicative of the influence of multiple ionizations . In particular, the triad formed by Asp58, Asp60, and Asp61 forms such a complex network of titrating groups . The ionization behavior of Asp26 shows an abnormally high pKa value for an aspartate residue in all states of human thioredoxin, with pKa values of 9.9 in the reduced state, 8.1 in the oxidized state, 8.9 in the mixed disulfide complex, and 8.6 in an active site mutant in which Cys35 was replaced by Ala . The unambiguous determination of the pKa values of Asp26 for a variety of states of human thioredoxin presented in this paper is highly significant in view of two recent reports on Escherichia coli thioredoxin which presented contradicting pKa values for Asp26 and Cys35 {Wilson et al . (1995) Biochemistry 34, 8931-8939; Jeng et al . (1995) Biochemistry 34, 10101-10105} . The stabilization of the protonated side chain of Asp26 in human thioredoxin is achieved via a hydrogen-bonding network involving the hydroxyl group of the neighboring Ser28 which is then connected to the active site region (comprising Cys32 and Cys35) via bound water molecules . The coupling of the buried Asp26 to the active site is responsible for the influence of the Asp26 ionization behavior on the titration shifts of active site residues. Biochemistry, 1996 Jan 9, 35(1), 273 - 81 Folding topologies of human interleukin-6 and its mutants as studied by NMR spectroscopy; Nishimura C et al.; To understand the structure-function relationship in the human interleukin-6 (IL-6) system, comparative studies were performed on the basis of NMR data obtained using the wild-type IL-6 and six mutants . In each of the six mutants, either Leu152, Leu159, Leu166, Leu168, Leu175, or Leu182, which exist in the C-terminal receptor-binding region, was substituted with Val . The resonance assignments of Val, Ile, Leu, and Phe residues were made by using specific double-labeling and site-specific mutagenesis strategies . On the basis of chemical shift and NOE data collected for six IL-6 mutants and those for the wild-type IL-6, we analyzed the structural changes induced by the substitution of each of the six Leu residues . The NMR data showed that substitution of Leu182 with Val (L182V) induced no structural change in IL-6, suggesting that Leu182 is located on the surface of the IL-6 molecule . A significant decrease in receptor-binding activity was observed in the L182V mutant . It was concluded that the side chain of Leu182 is directly involved in receptor binding . Substitution of Leu175 with Val (L175V) was shown to induce a significant structural change in IL-6 . The NMR data are discussed on the basis of the location of four helix elements and an up-up-down-down helix topology of the predicted structure of IL-6 {Bazan, J.F . (1991) Neuron 7, 197-208} . It is possible that helix D bent more sharply toward helix B in the L175V mutant than in the wild-type IL-6 to maintain a closely packed and solvent-inaccessible core formed in the mutated region . It is suggested that the kink of helix D is related to the decrease in receptor-binding activity in the L175V mutant . On the basis of the observed NOE network, the folding topology of IL-6 was analyzed . A comparison of the folding topology of IL-6 with that of human granulocyte colony-stimulating factor determined by X-ray crystallography {Hill, C . P., Osslund, T . D., & Eisenberg, D . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 5167-5171} indicated that IL-6 has a significant similarity of folding topology to that of human granulocyte colony-stimulating factor. Biochemistry, 1996 Jan 9, 35(1), 224 - 8 Activation of myosin light chain kinase and nitric oxide synthase activities by engineered calmodulins with duplicated or exchanged EF hand pairs; Persechini A et al.; We have constructed three engineered calmodulins (CaMs) in which the two EF hand pairs have been substituted for one another or exchanged: CaMNN, the C-terminal EF hand pair (residues 82-148) has been replaced by a duplication of the N-terminal pair (residues 9-75); CaMCC, the N-terminal pair has been replaced by a duplication of the C-terminal pair; CaMCN, the two EF had pairs have been exchanged . Skeletal muscle myosin light chain kinase (skMLCK) activity is activated to 75% of the maximum level by CaMCC and to 45% of the maximum level by CaMCN and is not significantly activated by CaMNN; Kact or Ki values for the engineered CaMs are 2-3.5 nM . Smooth muscle myosin light chain kinase activity (gMLCK) is fully activated by CaMCN and is not significantly activated by either CaMNN or CaMCC; the Kact value for CaMCN is 2 nM and the Ki values for CaMNN and CaMCC are 10 and 40 nM, respectively . Cerebellar nitric oxide synthase activity (nNOS) is fully activated by CaMNN and CaMCN and is not significantly activated by CaMCC; the engineered CaMs have Kact or Ki values for this enzyme activity of 2-8 nM . These results indicate that the EF hand pairs contain distinct but overlapping sets of determinants for binding and activation of enzymes, with the greater degree of overlap in determinants for binding . Furthermore, while the structural changes associated with swapping the EF hand pairs do not affect activation of nNOS or gMLCK activities, they significantly reduce activation of skMLCK activity, indicating that this process requires specific determinants in CaM outside the EF hand pairs. Biochemistry, 1996 Jan 9, 35(1), 117 - 23 Conservation in evolution for a small monomeric phenylalanyl-tRNA synthetase of the tRNA(Phe) recognition nucleotides and initial aminoacylation site; Aphasizhev R et al.; We previously showed that yeast mitochondrial phenylalanyl-tRNA synthetase (MSF protein) is evolutionarily distant to the cytoplasmic counterpart based on a high degree of divergence in protein sequence, molecular mass, and quaternary structure . Using yeast cytoplasmic tRNA(Phe) which is efficiently aminoacylated by MSF protein, we report here the tRNA(Phe) primary site of aminoacylation and the identity determinants for MSF protein . As for the cytoplasmic phenylalanyl-tRNA synthetase (Sampson, J . R., Di Renzo, A . B., Behlen, L . S., & Uhlenbeck, O . C . (1989) Science 243, 1363-1366), MSF protein recognizes nucleotides from the anticodon and the acceptor end including base A73 and, as shown here, adjacent G1-C72 base pair or at least C72 base . This indicates that the way of tRNA(Phe) binding for the two phenylalanine enzymes is conserved in evolution . However, tRNA(Phe) tertiary structure seems more critical for the interaction with the cytoplasmic enzyme than with MSF protein, and unlike cytoplasmic phenylalanyl-tRNA synthetase, the small size of the monomeric MSF protein probably does not allow contacts with residue 20 at the top corner of the L molecule . We also show that MSF protein preferentially aminoacylates the terminal 2'-OH group of tRNA(Phe) but with a catalytic efficiency for tRNA(Phe)-CC-3'-deoxyadenosine reduced 100-fold from that of native tRNA(Phe), suggesting a role of the terminal 3'-OH in catalysis . The loss is only 1.5-fold when tRNA(Phe)-CC-3'-deoxyadenosine is aminoacylated by yeast cytoplasmic PheRS (Sprinzl, M., & Cramer, F . (1973) Nature 245, 3-5), indicating mechanistic differences between the two PheRS's active sites for the amino acid transfer step. Biochemistry, 1996 Jan 9, 35(1), 109 - 16 Covalent attachment of Arc repressor subunits by a peptide linker enhances affinity for operator DNA; Robinson CR et al.; By designing a recombinant gene containing tandem copies of the arc coding sequence with intervening DNA encoding the linker sequence GGGSGGGTGGGSGGG, the two subunits of the P22 Are repressor dimer have been covalently linked to form a single-chain protein called Arc-L1-Arc . The 15-residue linker joins the C-terminus of one monomer to the N-terminus of the second, a distance of approximately 45 A in the Arc-operator cocrystal structure . Arc-L1-Arc is expressed at high levels in Escherichia coli, with no evidence of degradation or proteolytic clipping of the linker, and is more active than wild-type Arc in repression assays . The purified Arc-L1-Arc protein has the molecular weight expected for the designed protein and unfolds cooperatively, reversibly, and with no concentration dependence in thermal-denaturation studies . Arc-L1-Arc protects operator DNA in a manner indistinguishable from that of wild-type Arc in DNase I and copper-phenanthroline footprinting studies, but the covalent attachment of the two monomers results in enhanced affinity for operator DNA . Arc-L1-Arc binds operator DNA half-maximally at a concentration of 1.7 pM, compared with the wild-type value of 185 pM, and also binds DNA fragments containing the left or right operator half-sites more tightly than wild type . Because wild-type Arc is monomeric at sub-nanomolar concentrations and must dimerize before binding to the operator, it was anticipated that Arc-L1-Arc would exhibit a lower half-maximal binding concentration . However, even when the change from a monomeric to a dimeric species is taken into account, the affinity of Arc-L1-Arc for operator and half-operator DNA is greater than the wild-type affinity . This tighter binding appears to result from slower dissociation, as Arc-L1-Arc DNA complexes with full or half-site operators dissociate at rates 5-10 times slower than the corresponding Arc--DNA complexes . Hence, the activity of the designed Arc-L1-Arc protein is substantially increased relative to wild-type Arc in a variety of assays. Biochemistry, 1996 Jan 9, 35(1), 1 - 6 Direct measurement of the aspartic acid 26 pKa for reduced Escherichia coli thioredoxin by 13C NMR; Jeng MF et al.; Because of interference from the pH-dependent behavior of nearby groups in the active site of Escherichia coli thioredoxin, the pKa of the buried carboxyl group of the aspartic acid at position 26 has been difficult to quantitate . We report a direct measurement of this pKa using an NMR method utilizing the correlation between the C beta H proton resonances and the 13CO of the titrating carboxyl group . The experiments show unequivocally that the pKa is 7.3-7.5, rather than the value of 9 or greater recently proposed by Wilson, N . A., et al . {(1995) Biochemistry 34, 8931-8939} . The assignment of the titrating resonances to Asp 26 is unambiguous: the values of the C beta H chemical shifts correspond exactly to those of Asp 26, and their titration in the pH range 5.7-10.0 is the same as that observed previously for the proton resonances alone . In addition, the chemical shift of the carboxyl 13C resonance at pH 5.7 is upfield of those of the other carboxyl and carboxamide resonances, diagnostic for a protonated carboxyl group . The resonances assigned to Asp 26 are the only ones that titrate in the pH range 5.7-10.5 . None of the other aspartate and glutamate residues in the molecule are titrated in this pH range, consistent with their positions on the surface of the molecule . The pKa measured for Asp 26 in reduced thioredoxin is identical within experimental error to that measured in the oxidized form of the protein . This is significant for the reductive mechanism of thioredoxin: the buried salt bridged/hydrogen-bonded side chains of Asp 26 and Lys 57 are likely to contribute to the facility of the reaction by providing a convenient source and sink for protons in the hydrophobic environment of the complex between thioredoxin and its substrates. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 76 - 80 Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli; Jones PG et al.; A 70-kDa protein was specifically induced in Escherichia coli when the culture temperature was shifted from 37 to 15 degrees C . The protein was identified to be the product of the deaD gene (reassigned csdA) encoding a DEAD-box protein . Furthermore, after the shift from 37 to 15 degrees C, CsdA was exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein in cells grown at 15 degrees C . The csdA deletion significantly impaired cell growth and the synthesis of a number of proteins, specifically the derepression of heat-shock proteins, at low temperature . Purified CsdA was found to unwind double-stranded RNA in the absence of ATP . Therefore, the requirement for CsdA in derepression of heat-shock protein synthesis is a cold shock-induced function possibly mediated by destabilization of secondary structures previously identified in the rpoH mRNA. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 71 - 5 Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting; Greiner DP et al.; We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli) . We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins . Comparison of the cleaved fragments of the subunits from the core enzyme (alpha 2 beta beta') and the holoenzyme (core+sigma 70) shows that absence of the sigma 70 subunit is associated with the appearance of several cleavage sites on the subunits beta (within 10 residues of sequence positions 745, 764, 795, and 812) and beta' (within 10 residues of sequence positions 581, 613, and 728) . A cleavage site near beta residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when sigma 70 binds . No differences are observed for the alpha subunit. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 326 - 30 The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter; Zou JX et al.; The transcriptional transactivator (Tas) of simian foamy virus type 1 strongly augments gene expression directed by both the promoter in the viral long terminal repeat and the newly discovered internal promoter located within the env gene . A region of 121 bp, located immediately 5' to the TATA box in the internal promoter, is required for transactivation by Tas . The present study aimed to identify the precise Tas-responsive target(s) in this region and to determine the role of Tas in transcriptional regulation . By analysis of both clustered-site mutations and hybrid promoters in transient expression assays in murine and simian cells, two separate sequence elements within this 121-bp region were shown to be Tas-dependent transcriptional enhancers . These targets, each < 30 bp in length and displaying no apparent sequence homology one to the other, are designated the promoter-proximal and promoter-distal elements . By means of the gel electrophoresis mobility-shift assays, using purified glutathione S-transferase-Tas fusion protein expressed in Escherichia coli, the target proximal to the TATA box exhibited strong binding to glutathione S-transferase-Tas, whereas the distal element appears not to bind . In addition, footprint analysis revealed that 26 bp in the promoter proximal element was protected by glutathione S-transferase-Tas from DNase I . We propose a model for transactivation of the simian foamy virus type 1 internal promoter in which Tas interacts directly with the proximal target element positioned immediately 5' to the TATA box . In this model, Tas attached to this element is presumed to interact with a component(s) of the cellular RNA polymerase II initiation complex and thereby enhance transcription directed by the viral internal promoter. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 226 - 30 Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets; Nashar TO et al.; The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D) . Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses . Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro . EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many of which were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon gamma . These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 111 - 5 Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers; Piller SC et al.; A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix . Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential . The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive . Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen . Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber . Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber . The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential . The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber . The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 101 - 5 Receptor-mediated gene transfer into macrophages; Ferkol T et al.; Gene transfer systems targeting various receptors have been developed to introduce functional genes into cells in culture and into intact animals . A synthetic molecular conjugate, consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor, was constructed and complexed to expression plasmids containing either the Photinus pyralis luciferase or Escherichia coli beta-galactosidase (lacZ) reporter genes . The DNA complexes were used to transfect murine macrophages isolated from peritoneal exudates in vitro . Luciferase and beta-galactosidase activity was found in transfected cells in culture, whereas complexes consisting of an irrelevant plasmid bound to mannosylated polylysine or the expression plasmid bound to galactosylated polylysine resulted in no detectable transgene expression . Gene transfer was inhibited by the addition of excess mannosylated bovine serum albumin to the culture medium before transfection . Reporter genes were also transferred into macrophages residing in the spleen and liver of adult animals using this system . Luciferase activity was maximal at 4 days after transfection and decreased to lower levels by 16 days . Transgene expression conformed to the distribution of cells that had nonspecific esterase, a cytochemical marker for macrophages . Thus, this system can be used to introduce functional genes into macrophages and may be an approach to the treatment of storage diseases that affect the reticuloendothelial system. Toxicology, 1996 Jan 8, 106(1-3), 207 - 19 Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies; Belloc C et al.; Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics . Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate . Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies . These antibodies were characterized for their specificity and sensitivity by immunoblots; anti-CYP3A4 was immunoinhibitor . These antibodies could be used in association with other means to identify the CYPs responsible for production of a given metabolite . The use of our human recombinant CYP1A2 as antigen and the corresponding specific antibody enabled us to quantify the CYP1A2 content in 43 human livers . The average level was 69 pmol of CYP1A2/mg of microsomal proteins . Finally, these antibodies were also used to evaluate the level of heme incorporation in human microsomal CYP expressed in yeasts. FEBS Lett, 1996 Jan 8, 378(2), 195 - 8 Molecular characterization of a cDNA encoding functional human deoxyhypusine synthase and chromosomal mapping of the corresponding gene locus; Bevec D et al.; Deoxyhypusine synthase is essentially required for the post-translational formation of hypusine, a modification of a specific lysine residue in eukaryotic initiation factor 5A, which appears to be pivotal for cell proliferation . From a human peripheral blood mononuclear cells cDNA library we isolated two independent sequences encoding biologically active deoxyhypusine synthase . DNA sequence analysis revealed a 369 amino acid protein with a molecular mass of 41.055 kDa . This recombinant deoxyhypusine synthase showed significant catalytic activity in synthesis of deoxyhypusine after in vitro transcription and translation as well as upon expression in Escherichia coli . Using a panel of somatic rodent-human cell hybrids we localized the deoxyhypusine synthase gene to human chromosome 19. FEBS Lett, 1996 Jan 8, 378(2), 171 - 6 Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence; Castellanos-Serra LR et al.; We report the expression in E . coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue . The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension . It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600) . This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide. FEBS Lett, 1996 Jan 8, 378(2), 131 - 4 An isochorismate hydroxymutase isogene in Escherichia coli; Muller R et al.; The pivotal step in enterobactin and menaquinone biosynthesis is the conversion of chorismate to isochorismate . Circumstantial evidence pointed to Escherichia coli isochorismate hydroxymutase isogenes being responsible for this conversion . While the gene involved in enterobactin synthesis (entC) was known, the corresponding gene for menaquinone biosynthesis (menF) was not but has now been identified and sequenced . The amino acid sequence of MenF is 23.5% identical and 57.8% similar to that of EntC. Biochem Biophys Res Commun, 1996 Jan 5, 218(1), 45 - 9 GTP hydrolysis by human tissue transglutaminase homologue; Fraij BM; Human tissue transglutaminase homologue cDNA was expressed in E . coli to analyze the catalytic characteristics . The transglutaminase homologue was purified by immunoaffinity chromatography . Specificity of GTP binding by the homologue was demonstrated by photoaffinity labeling in the absence or presence of GTP-gamma-S . The homologue had GTPase activity with an apparent Km value of 1.8 microM, several-fold lower than the reported Km values for the native tissue transglutaminase . GTPase activity was inhibited by guanine nucleotides in order GTP-gamma-S > GDP > GMP . The higher GTPase activity of the homologue may be related to the signaling events function. Biochem Biophys Res Commun, 1996 Jan 5, 218(1), 260 - 6 Expression of the carbohydrate recognition domain of bovine conglutinin and demonstration of its binding to iC3b and yeast mannan; Lim BL et al.; Bovine conglutinin is a collagenous C-type lectin (collectin) that is found in bovine serum . A recombinant protein, composed of the neck-region and the carbohydrate binding domain of bovine conglutinin, has been overexpressed in E . coli . The recombinant protein has been successfully renatured and showed the same sugar binding specificity as the native protein and was able to bind to yeast mannan and complement-activated immune complexes . The binding was calcium-dependent and was inhibited by N-acetylglucosamine . The concentration of N-acetylglucosamine required for 50% inhibition of binding to mannan was 1.77 mM for recombinant conglutinin and 0.71 mM for native conglutinin, respectively . The recombinant conglutinin should be useful in the assay and purification of circulating immune complexes and for therapeutic purposes involving the removal of immune complexes from patient's plasma. Biochem Biophys Res Commun, 1996 Jan 5, 218(1), 159 - 63 Islet fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase: isozymic form, expression, and characterization; Sakurai T et al.; Polymerase chain reaction analysis of the mRNA isolated from rat islets demonstrated that the major isozyme of Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase was the heart type enzyme, and that the liver type enzyme was not detectable . The islet enzyme was expressed in Escherichia coli and purified to homogeneity . The islet enzyme showed the highest Fructose 6-P,2-kinase activity (478 milliunits/mg) compared to the other isozymes and Fructose 2,6-Pase activity (39 milliunits/mg) . Fructose 6-P,2-kinase showed KmF6P = 17 microM, which is within the range of in vivo Fru 6-P concentrations in islets . 6-P-Gluconate was a potent inhibitor of Fructose 2,6-Pase . The data suggest that Fructose 6-P,2-kinase activity of the bifunctional enzyme was high and Fructose 2,6-Pase activity was inhibited under physiological variations of blood glucose concentration. Biochem Biophys Res Commun, 1996 Jan 5, 218(1), 137 - 41 Decrease in the linking number of plasmid DNA in dnaA mutants of Escherichia coli; Mizushima T et al.; We made use of agarose gel electrophoresis in the presence of chloroquine to examine the linking number of plasmids in temperature-sensitive dnaA mutants, including dnaA5 and dnaA46 mutants . The linking number of DNA prepared from dnaA mutants growing at 37 degrees C was lower than that from wild type cells yet there was no significant difference when cells were grown at 28 degrees C . Complementation analysis with a plasmid containing the wild type dnaA gene and phage P1-mediated transduction confirmed that mutations in the dnaA gene were responsible for the decrease in the linking number of DNA. J Biol Chem, 1996 Jan 5, 271(1), 68 - 76 Biochemical characterization of symmetric GroEL-GroES complexes . Evidence for a role in protein folding; Llorca O et al.; When chaperonins GroEL and GroES are incubated under functional conditions in the presence of ATP (5 mM) and K+ (150 mM), GroEL-GroES complexes appear in the incubation mixture, that are either asymmetric (1:1 GroEL:GroES oligomer ratio) or symmetric (1:2 GroEL:GroES oligomer ratio) . The percentage of symmetric complexes present is directly related to the {ATP}/{ADP} ratio and to the K+ concentration . Kinetic analysis shows that there is a cycle of formation and disappearance of symmetric complexes . A correlation between the presence of symmetric complexes in the incubation mixture and its rhodanese folding activity suggests some active role of these complexes in the protein folding process . Accordingly, under functional conditions, symmetric complexes are found to contain denatured rhodanese . These data suggest that binding of substrate inside the GroEL cavity takes place before the symmetric complex is formed. J Biol Chem, 1996 Jan 5, 271(1), 568 - 73 Expression and characterization of recombinant caveolin . Purification by polyhistidine tagging and cholesterol-dependent incorporation into defined lipid membranes; Li S et al.; Caveolin, a 22-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo . Caveolin has been proposed to function as a scaffolding protein to organize and concentrate signaling molecules within caveolae . Because of its unusual membrane topology, both the N- and C-terminal domains of caveolin remain entirely cytoplasmic and are not subject to luminal modifications that are accessible to other integral membrane proteins . Under certain conditions, caveolin also exists in a soluble form as a cytosolic protein in vivo . These properties make caveolin an attractive candidate for recombinant expression in Escherichia coli . Here, we successfully expressed recombinant full-length caveolin in E.coli . A polyhistidine tag was placed at its extreme C terminus for purification by Ni(2+)-nitrilotriacetic acid affinity chromatography . Specific antibody probes demonstrated that recombinant caveolin contained a complete N and C terminus . Recombinant caveolin remained soluble in solutions containing the detergent octyl glucoside and formed high molecular mass oligomers like endogenous caveolin . By electron microscopy, recombinant caveolin homo-oligomers appeared as individual spherical particles that were indistinguishable from endogenous caveolin homo-oligomers visualized by the same technique . As recombinant caveolin behaved as expected for endogenous caveolin, this provides an indication that recombinant caveolin can be used to dissect the structural and functional interaction of caveolin with other protein and lipid molecules in vitro . Recombinant caveolin was efficiently incorporated into lipid membranes as assessed by floatation in sucrose density gradients . This allowed us to use defined lipid components to assess the possible requirements for insertion of caveolin into membranes . Using a purified synthetic form of phosphatidylcholine (1,2-dioleoylphosphorylcholine), we observed that incorporation of caveolin into membranes was cholesterol-dependent; the addition of cholesterol dramatically increased the incorporation of caveolin into these phosphatidylcholine-based membranes by approximately 25-30-fold . This fits well with in vivo studies demonstrating that cholesterol plays an essential role in maintaining the structure and function of caveolae . Further functional analysis of these reconstituted caveolin-containing membranes showed that they were capable of recruiting a soluble recombinant form of G(i)2 alpha . This is in accordance with previous studies demonstrating that caveolin specifically interacts directly with multiple G protein alpha-subunits . Thus, recombinant caveolin incorporated into defined lipid membranes provides an experimental system in which the structure, function, and biogenesis of caveolin-rich membrane domains can be dissected in vitro. J Biol Chem, 1996 Jan 5, 271(1), 536 - 43 Characterization of three components of human adenovirus proteinase activity in vitro; Mangel WF et al.; Human adenovirus contains a virion-associated proteinase activity essential for the development of infectious virus . Maximal proteinase activity in vitro had been shown to require three viral components: the L3 23-kDa protein, an 11-amino acid cofactor (pVIc), and the viral DNA . Here, we present a quantitative purification procedure for a recombinant L3 23-kDa protein (recombinant endoproteinase (rEP)) expressed in Escherichia coli and the procedure that led to the purification and identification of pVIc as a cofactor . The cofactors stimulate proteinase activity not by decreasing Km, which changes by no more than 2-fold, but by increasing kcat . rEP alone had a small amount of activity, the kcat of which increased 355-fold with pVIc and 6072-fold with adenovirus serotype 2 (Ad2) DNA as well . Curves of Vmax of rEP.pVIc complexes with the substrate (Leu-Arg-Gly-NH)2-rhodamine as a function of pH in the absence and presence of Ad2 DNA indicate that the pKa values of amino acids that affect catalysis are quite different from those that affect catalysis by the cysteine proteinase papain . The pKa values in the absence of Ad2 DNA are 5.2, 6.4, 6.9, 7.5, and 9.4, and those in its presence are 5.2, 6.5, 7.4, and 8.8. J Biol Chem, 1996 Jan 5, 271(1), 521 - 7 Two hydrophobic subunits are essential for the heme b ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli; Nakamura K et al.; Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon . Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b556 in E . coli complex II . In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E . coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities . The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b556 . An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced . However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b556 in its CO reactivity and red shift of the alpha absorption peak to 557.5 nm at 77 K . Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinate-ubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells . In contrast, significantly higher amounts of cytochrome b556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquitnone oxidoreductase and succinate oxidase activities . These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b556 and are essential for the functional assembly of E . coli complex II in the membrane . Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E . coli complex II. J Biol Chem, 1996 Jan 5, 271(1), 367 - 71 Site-directed mutagenesis of the cysteine residues in the Neurospora crassa plasma membrane H(+)-ATPase; Mahanty SK et al.; A high-yield yeast expression system for site-directed mutagenesis of the Neurospora crassa plasma membrane H(+)-ATPase has recently been reported (Mahanty, S . K., Rao, U . S., Nicholas, R . A., and Scarborough, G . A . (1994) J . Biol . Chem . 269, 17705-17712) . Using this system, each of the eight cysteine residues in the ATPase was changed to a serine or an alanine residue, producing strains C148S and C148A, C376S and C376A, C409S and C409A, C472S and C472A, C532S and C532A, C545S and C545A, C840S and C840A, and C869S and C869A, respectively . With the exception of C376S and C532S, all of the mutant ATPases are able to support the growth of yeast cells to different extents, indicating that they are functional . The C376S and C532S enzymes appear to be non-functional . After solubilization of the functional mutant ATPase molecules from isolated membranes with lysolecithin, all behaved similar to the native enzyme when subjected to glycerol density gradient centrifugation, indicating that they fold in a natural manner . The kinetic properties of these mutant enzymes were also similar to the native ATPase with the exception of C409A, which has a substantially higher Km . These results clearly indicate that none of the eight cysteine residues in the H(+)-ATPase molecule are essential for ATPase activity, but that Cys376, Cys409, and Cys532 may be in or near important sites . They also demonstrate that the previously described disulfide bridge between Cys148 and Cys840 or Cys869 plays no obvious role in the structure or function of this membrane transport enzyme. J Biol Chem, 1996 Jan 5, 271(1), 343 - 8 Novel plant Ca(2+)-binding protein expressed in response to abscisic acid and osmotic stress; Frandsen G et al.; A cDNA corresponding to an mRNA which accumulates in germinating rice seeds in response to the phytohormone abscisic acid was isolated by differential hybridization . Northern blotting indicated that the mRNA also accumulates in vegetative tissues in response to treatment with abscisic acid and to osmotic stress . Sequencing identified a major open reading frame encoding a novel protein of 27.4 kDa . The identity of the open reading frame was confirmed by comparing the translation products of cellular, hybrid-selected, and in vitro transcribed RNAs and by immunoprecipitation . Western blotting of cellular extracts indicated that the protein is associated with microsomal or membrane fractions . Data base searches indicated that it contains a conserved Ca(2+)-binding, EF-hand motif and that related proteins are similarly expressed in Arabidopsis thaliana . A fusion protein purified from Escherichia coli containing the putative EF-hand region was shown to bind Ca2+ in blot binding assays . These data identify a novel gene family encoding proteins involved in the response of plants to abscisic acid and osmotic stress. J Biol Chem, 1996 Jan 5, 271(1), 32 - 9 Characterization of the essential gene glmM encoding phosphoglucosamine mutase in Escherichia coli; Mengin-Lecreulx D et al.; Two different approaches to identify the gene encoding the phosphoglucosamine mutase in Escherichia coli were used: (i) the purification to near homogeneity of this enzyme from a wild type strain and the determination of its N-terminal amino acid sequence; (ii) the search in data bases of an E . coli protein of unknown function showing sequence similarities with other hexosephosphate mutase activities . Both investigations revealed the same open reading frame named yhbF located within the leuU-dacB region at 69.5 min on the chromosome (Dallas, W . S., Dev, I . K., and Ray, P . H . (1993) J . Bacteriol . 175, 7743-7744) . The predicted 445-residue protein with a calculated mass of 47.5 kDa contained in particular a short region GIVISASHNP with high similarity to the putative active site of hexosephosphate mutases . In vitro assays showed that the overexpression of this gene in E . coli cells led to a significant overproduction (from 15- to 50-fold) of phosphoglucosamine mutase activity . A hexose 1,6-diphosphate-dependent phosphorylation of the enzyme, which probably involves the serine residue at position 102, is apparently required for its catalytic action . As expected, the inactivation of this gene, which is essential for bacterial growth, led to the progressive depletion of the pools of precursors located downstream from glucosamine 1-phosphate in the pathway for peptidoglycan synthesis . This was followed by various alterations of cell shape and finally cells were lysed when their peptidoglycan content decreased to a critical value corresponding to about 60% of its normal level . The gene for this enzyme, which is essential for peptidoglycan and lipopolysaccharide biosyntheses, has been designated glmM. J Biol Chem, 1996 Jan 5, 271(1), 319 - 23 Molecular cloning, heterologous expression, and characterization of human glyoxalase II; Ridderstrom M et al.; A clone encoding glyoxalase II has been isolated from a human adult liver cDNA library . The sequence of 1011 base pairs consists of a full-length coding region of 780 base pairs, corresponding to a protein with a calculated molecular mass of 28,861 daltons . Identities (50-60%) were found to partial 5' and 3' cDNA sequences from Arabidopsis thaliana as well as within a limited region of glutathione transferase I cDNA from corn . A vector was constructed for heterologous expression of glyoxalase II in Escherichia coli . For optimal yield of enzyme, silent random mutations were introduced in the 5' coding region of the cDNA . A yield of 25 mg of glyoxalase II per liter of culture medium was obtained after affinity purification with immobilized glutathione . The recombinant enzyme had full catalytic activity and kinetic parameters indistinguishable from those of the native enzyme purified from human erythrocytes. J Biol Chem, 1996 Jan 5, 271(1), 289 - 94 Substitutions of proline 42 to alanine and methionine 46 to asparagine around the RGD domain of the neurotoxin dendroaspin alter its preferential antagonism to that resembling the disintegrin elegantin; Lu X et al.; Previous studies have shown that the neurotoxin dendroaspin and the disintegrin kistrin, which show little overall sequence homology but similar residues around RGD (PRGDMP), preferentially inhibited platelet adhesion to fibrinogen . In contrast, the elegantin which has different amino acids around RGD (ARGDNP) preferentially inhibited platelet adhesion to fibronectin . To investigate further the role of amino acids around RGD in disintegrins, we have constructed the genes of a wild-type and of two mutant dendroaspins with substitutions around the RGD, namely {Asn46}- and {Ala42,Asn46}-dendroaspins . Proteins were expressed in Escherichia coli as glutathione S-transferase fusion recombinants and purified to homogeneity by affinity chromatography and reversed phase high performance liquid chromatography . Platelet aggregation studies revealed that wild-type dendroaspin showed an IC50 value similar to that of native dendroaspin, with {Ala42,Asn46}-dendroaspin showing an IC50 value similar to that of elegantin . Interestingly, in platelet adhesion assays, the mutants showed a progressive shift in inhibitory preference, in particular, {Ala42,Asn46}dendroaspin showed nearly identical behavior as elegantin when fibronectin was the immobilized ligand (IC50 = 0.33 microM and 0.6 microM, respectively, compared with 20 microM for native dendroaspin) . Native and recombinant wild-type dendroaspin bound to a single class of binding site exhibiting a Kd = 67 nM; {Asn46}- and {Ala42,Asn46}dendroaspins, however, both produced biphasic isotherms with Kd values = 87 nM and 361 nM for {Asn46}dendroaspin and 33 nM and 371 nM for {Ala42,Asn46}dendroaspin, which are close to those of elegantin (Kd values = 18 nM and 179 nM) . These studies prove that the amino acids flanking RGD provide an extended locus that regulate the affinity and selectivity of RGD protein dendroaspin. J Biol Chem, 1996 Jan 5, 271(1), 136 - 47 Crystal structure of S-adenosylmethionine synthetase; Takusagawa F et al.; The structure of S-adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6.) from Escherichia coli has been determined at 3.0 A resolution by multiple isomorphous replacement using a uranium derivative and the selenomethionine form of the enzyme (SeMAT) . The SeMAT data (9 selenomethionine residues out of 383 amino acid residues) have been found to have a sufficient phasing power to determine the structure of the 42,000 molecular weight protein by combining them with the other heavy atom derivative data (multiple isomorphous replacement) . The enzyme consists of four identical subunits; two subunits form a spherical tight dimer, and pairs of these dimers form a peanut-shaped tetrameric enzyme . Each pair dimer has two active sites which are located between the subunits . Each subunit consists of three domains that are related to each other by pseudo-3-fold symmetry . The essential divalent (Mg2+/Co2+) and monovalent (K+) metal ions and one of the product, Pi ions, were found in the active site from three separate structures. Biochim Biophys Acta, 1996 Jan 4, 1292(1), 47 - 52 Determination of the structural role of the N-terminal domain of human extracellular superoxide dismutase by use of protein fusions; Tibell LA et al.; The N-terminal domain, containing the 49 N-terminal amino-acid residues, of human extracellular superoxide dismutase (hEC-SOD) has been studied after construction of fusion proteins comprised of the defined domain and human carbonic anhydrase II (HCAII) . The specific advantage of this technique is that it allows characterization of properties that are intrinsic to the N-terminal domain of hEC-SOD, i.e., the results are not obscured by properties pertaining to the rest of the hEC-SOD molecule . Moreover, the fusion to HCAII allows a rapid and gentle one-step purification by affinity chromatography . When the N-terminal domain was fused to the N-terminal of HCAII ( = FusNN) a well defined structure was formed and the resulting protein was tetrameric . When the same hEC-SOD-derived domain was fused to the C-terminal of HCAII ( = FusNC), no defined structure of the fused domain could be observed, and the resulting protein was monomeric . It was concluded that a 'free' N-terminus is required for formation of the proper structure of the N-terminal domain. Biochim Biophys Acta, 1996 Jan 4, 1292(1), 168 - 76 Isolation and structural characterization of recombinant human neu differentiation factor expressed in Escherichia coli; Hara S et al.; Recombinant human neu differentiation factor produced in engineered E . coli was isolated and subject to structural characterization . The recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor . The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure . Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu differentiation factor preparation is properly folded and contains the correct disulfide structure . The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively. Biochim Biophys Acta, 1996 Jan 4, 1292(1), 156 - 62 Mouse coproporphyrinogen oxidase is a copper-containing enzyme: expression in Escherichia coli and site-directed mutagenesis; Kohno H et al.; We previously isolated cDNA for mouse coproporphyrinogen oxidase (CPO) and provided evidence for the induction of mRNA during differentiation of murine erythroleukemia cells (Kohno et al . (1993) J . Biol . Chem . 268, 21359-21363) . To better understand the structure and the mechanisms of reaction of the enzyme, we expressed mouse CPO in Escherichia . coli and purified it to a homogeneity . Analysis of the metal content revealed that the recombinant mouse CPO contains one copper atom per polypeptide chain . When the bacterial cells were treated with D-penicillamine, a copper chelator, formation of the active CPO was partially reduced . Addition of Cu2+ in minimal medium resulted in 6-fold higher level of CPO activity . These results suggest that expression of active mouse CPO in E . coli depended on the presence of Cu2+ in the culture medium . To elucidate the apparent involvement of Cu2+ in enzyme function, a series of mutant enzymes, whose highly conserved histidine and cysteine residues were individually converted to alanine residue, were prepared by site-directed mutagenesis . Mutant enzymes were expressed in E . coli and their activities examined . Mutation at histidine 158 resulted in a complete loss of enzyme activity, yet the enzyme protein was expressed at a comparable level . Concomitantly, only a trace amount of Cu2+ was detected in the purified H158A enzyme . We propose that mouse CPO is copper-containing enzyme and Cu2+ interacts with a conserved histidine residue. Biochim Biophys Acta, 1996 Jan 4, 1292(1), 133 - 9 Spectroscopical and functional characterization of the hemoglobin of Nostoc commune (UTEX 584 (Cyanobacterial); Thorsteinsson MV et al.; Structural analysis of a monomeric hemoglobin from the cyanobacterium Nostoc commune strain UTEX 584, cyanoglobin (Potts et al . (1992) Science 256, 1690-1692), is presented . Cyanoglobin binds molecular oxygen reversibly, with high oxygen affinity and non-cooperativity . There was no evidence for decreased stability of the pigment at 37 degrees C . Cyanoglobin-specific antibodies showed no cross-reactivity with two reference hemoglobins, leghemoglobin a and sperm whale myoglobin . The absorption spectral properties of cyanoglobin differ significantly from those of the two reference hemoglobins . The spectrum of oxy-cyanoglobin most closely resembles that of an oxy-hemoglobin from the protozoan Tetrahymena pyriformis, a hemoprotein that shares substantial amino-acid sequence identity with cyanoglobin . Met-cyanoglobin possesses spectral characteristics at pH 7.0-9.0 that resemble those of the alkaline met-hemoglobin (a putative hemichrome) of another protozoan, Paramecium caudatum . The spin-state character of met-cyanoglobin is pH-dependent . Met-cyanoglobin does not coordinate the strong-field ligands, cyanide and azide, at pH 7.0 . The capacity of cyanoglobin to coordinate cyanide increased with decreasing pH . Far-UV CD spectra of cyanoglobin are indicative of a protein with a significant amount of alpha-helical structure . Data from Soret-region CD spectra suggest that the orientations of the heme moieties in cyanoglobin and leghemoglobin a are similar to one another. EMBO J, 1996 Jan 2, 15(1), 162 - 71 Involvement of stress protein PspA (phage shock protein A) of Escherichia coli in maintenance of the protonmotive force under stress conditions; Kleerebezem M et al.; The expression of specific PhoE mutant proteins leads to induction of the expression of the psp operon of Escherichia coli and the export of various plasmid-encoded precursors is retarded in a pspA mutant strain . Here, we have investigated the specific role of various Psp proteins in the export process . PspB and PspC are both inner membrane proteins that are involved in the regulation of the transcription of the psp operon . Precursor PhoE translocation was retarded in a pspB mutant strain to a similar extent as in a pspA mutant strain . The reduced translocation efficiencies in the various psp mutants could be complemented by expression of PspA from a plasmid, indicating that only PspA is required for efficient translocation . Mutant prePhoE proteins that can be translocated independently of the deltamu H+ appeared to translocate equally efficiently in a wild-type and in a pspA mutant strain . Furthermore, quantitative in vivo determination of the deltamu H+ showed that it specifically decreased in a pspA mutant strain upon expression of plasmid-encoded (mutant) prePhoE protein . Apparently, the translocation defects observed in a psp mutant strain are caused by a decrease of the delta mu H+ and PspA functions by maintaining the delta mu H+ under these conditions. EMBO J, 1996 Jan 2, 15(1), 150 - 61 Role of Escherichia coli RNA polymerase alpha subunit in modulation of pausing, termination and anti-termination by the transcription elongation factor NusA; Liu K et al.; The alpha subunit (alpha) of RNA polymerase (RNAP) is critical for assembly of polymerase and positive control of transcription initiation in Escherichia coli . Here, we report that alpha also plays a role in transcription elongation, and this involves a direct interaction between alpha and NusA factor . During in vitro transcription without NusA, alpha interacts with the nascent RNA, as revealed by photocrosslinking . When NusA is present, RNA crosslinks to NusA rather than to alpha . We show that this NusA-RNA interaction is diminished during transcription with an RNAP mutant that lacks the C-terminus of alpha beyond amino acid 235, including the so-called alpha CTD . The absence of alpha CTD also affects NusA's ability to enhance transcription pausing, termination at intrinsic terminators and anti-termination by the phage lambda Q anti-terminator, but not anti-termination by the lambda N anti-terminator . NusA functions are not recovered even when transcription with mutant RNAP is done with excess NusA, a condition which does restore NusA-RNA crosslinking . By affinity chromatography, we show that NusA interacts directly with alpha, and also with beta and beta', but not with mutant alpha . Hence, alpha-NusA interaction is vital for the control of transcript elongation and termination. EMBO J, 1996 Jan 2, 15(1), 102 - 9 Phosphorylated BvgA is sufficient for transcriptional activation of virulence-regulated genes in Bordetella pertussis; Steffen P et al.; In Bordetella pertussis the expression of virulence factors is coordinately regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal transduction family, BvgS being the transmembrane sensor and BvgA the regulator . Activation of virulence gene expression requires phosphorylation of BvgA . On the basis of observed differences in the regulation of individual genes, the existence of accessory regulators has been postulated . They were supposed to be necessary for expression of genes encoding adenylate cyclase toxin (cya) and pertussis toxin (ptx), but not required for the expression of fha, encoding filamentous hemagglutinin . To clarify this issue we investigated the mechanism of activation of the BvgAS-controlled genes by performing in vitro run-off transcription experiments . We show, using purified RNA polymerase of B.pertussis, that phosphorylated BvgA is sufficient for transcriptional activation of the major virulence genes, thus providing good evidence that BvgA regulation operates directly with the transcription initiation machinery at the promoters of the virulence genes without a requirement for accessory activators . In addition, our results indicate that activation of the different promoters may involve distinct mechanisms . We suggest that the previously observed differences in regulation of individual virulence-associated genes reflect differences in the phosphorylation state of BvgA. FEBS Lett, 1996 Jan 2, 378(1), 37 - 42 Enzymatic characterization of purified NS3 serine proteinase of hepatitis C virus expressed in Escherichia coli; Mori A et al.; Non-structural protein 3 (NS3) of the hepatitis C virus (HCV) has been shown to be a serine proteinase which cleaves the HCV polyprotein thus activating its replicative machinery . To characterize enzymatic activities of NS3 serine proteinase, the proteinase region was expressed in Escherichia coli and purified . The purified proteinase specifically cleaved a purified fusion protein sandwiching the NS5A/5B cleavage sequence . In addition to serine proteinase inhibitors, some chelators also inhibited the cleavage activity . Metal ions were not required for its activity, suggesting that the proteinase may be a novel serine proteinase having a unique binding site for chelators. Mutat Res, 1996 Jan 2, 362(1), 65 - 74 Comparison of the rate of excision of major UV photoproducts in the strands of the human HPRT gene of normal and xeroderma pigmentosum variant cells; Tung BS et al.; Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer . Fibroblasts from such patients are extremely sensitive to mutations induced by UV radiation, and the spectrum of mutations induced in their hypoxanthine phosphoribosyltransferase (HPRT) gene differs significantly from that seen in normal cells . To determine if this UV hypermutability reflects abnormally slow excision repair of cyclobutane pyrimidine dimers (CPD) or 6-4 pyrimidine-pyrimidones (6-4s) in that gene, we synchronized XP variant and normal fibroblasts, irradiated them in early G1-phase, 12 or more hours prior to the scheduled onset of S phase, harvested them immediately or after allowing various times for repair, and analyzed the DNA for photoproducts in the HPRT gene, using quantitative Southern blotting . To incise the DNA at CPD, we used T4 endonuclease V; to incise at 6-4s, we first used photolyase and UV365nm to reverse CPD and then UvrABC excinuclease . Excision of CPD was rapid, preferential, and strand-specific, but there was no significant difference in rate between the two kinds of cells . The half life was 4 h in the transcribed strand of the gene and 6.5 h in the nontranscribed strand . For excision of CPD in the genome overall, this value is 12 h . Excision of 6-4s from either strand of the HPRT gene was extremely rapid and preferential in both kinds of cells, with a half life of approximately 30 min . The results indicate that the UV hypermutability of the XP variant cells cannot be caused by slower rates of repair of CPD and/or 6-4s in the target gene for mutagenesis. Mutat Res, 1996 Jan 2, 362(1), 61 - 4 Constitutive increase of RecA protein: its influence on pyrimidine dimer excision and survival of UV-irradiated Escherichia coli; Slezarikova V et al.; Transformation of E . coli with the plasmid pRA containing recA gene increased the constitutive level of RecA protein 50-67 fold . This slightly inhibited pyrimidine dimer excision and reduced cell survival in three investigated, UV-irradiated E . coli strains . Our data support the view that RecA protein prematurely present at a high level may mask the dimers . The masking subsequently reduces the dimer excision and switches off the inducing signal. Mutat Res, 1996 Jan 2, 362(1), 29 - 40 The in vitro more efficiently repaired cisplatin adduct cis-Pt.GG is in vivo a more mutagenic lesion than the relative slowly repaired cis-Pt.GCG adduct; Brandsma JA et al.; The toxic effect and the mutagenicity of two differentially repaired site-specific cis-diamminedichloroplatinum(II) (cis-DDP) lesions were investigated . Detailed analysis of the UvrABC-dependent repair of the two lesions in vitro showed a more efficient repair of the cis-Pt.GG adduct compared to that of the cis-Pt.GCG adduct (Visse et al., 1994) . Furthermore, previously, a dependency of cis-DDP mutagenesis on UvrA and UvrB, but not on UvrC was found (Brouwer et al., 1988) . To possibly relate survival and mutagenesis to repair, plasmids containing the same site-specific cis-DDP lesions as those that were used in the detailed repair studies were transformed into Escherichia coli . The results indicate that both lesions are very efficiently bypassed in vivo . Mutation analysis was performed using a denaturing gradient gel electrophoresis technique, which allows identification of mutations without previous selection . Although the cis-Pt.GG adduct is in vitro more efficiently repaired than the cis-Pt.GCG adduct, it appeared to be more mutagenic . We present a model in which this result is related to the previously observed dependency of the mutagenicity of cis-DDP lesions on the Uvr A and B proteins. Mutat Res, 1996 Jan 2, 362(1), 21 - 8 Overexpression of the natural recO sequence and its effects on DNA repair of Escherichia coli; Yang CL et al.; The recO gene is required for the RecF pathway of recombination and the repair of DNA daughter-strand gaps in Escherichia coli . In this work, the structural portion of recO was synthesized by the polymerase chain reaction (PCR) and cloned onto expression vectors at their Nco1 fusion cloning site, to eliminate the presence of mRNA leader sequence . While the plasmid carrying a Ptac promoter failed to overproduce RecO, the plasmid carrying a T7O10 promoter overproduced RecO in large quantity, indicating that the natural recO may be overexpressed . An increase of intracellular RecO, which may be due to the increased recO gene copies or to the induction of RecO synthesis, increased the UV resistance of recA, recF, and ssb cells, but did not increase the UV resistance of uvrB, uvrB recF, uvrB recA and uvrB ssb cells . We suggest that an increase of intracellular RecO may allow some recombination-deficient cells to perform more excision repair, thus increasing the survival . The possible causes for RecO overproduction on excision repair, and for the differential expression of recO by the Ptac and T7 promoter plasmids are discussed. Acta Physiol Hung, 1996, 84(3), 289 - 90 Do sodium nitroprusside and L-NAME affect pyrogen fever in rabbits? Gagalo IT, Hac EE, Korolkiewicz KZ, Matuszek MT, Szreder Z. Thermoregulatory responses after treatment with nitric oxide (NO) donor, sodium nitroprusside (SNP-3 mg/kg/h), or NO synthase inhibitor, NG-nitro-L-arginine methylester (L-NAME-100 mg/kg) were investigated in febrile rabbits (lipopolysaccharide E . coli-1 meg/kg) . Pretreatment with SNP attenuated pyrogen fever as well as metabolic rate . L-NAME also inhibited postpyrogen increases in metabolism; however, this effect did not lead to antipyresis. Cytobios, 1996, 87(351), 207 - 16 Effects of some saturated n-alkanes on the survival of Escherichia coli resting cells in sea water; Attrassi B et al.; The influence of some linear alkanes on the survival of Escherichia coli natural sea water was investigated . Alkanes with fifteen or more carbon atoms induced a large decrease in viability of E . coli cells in natural sea water . In this case and for concentrations higher than 100 ppm, the loss of viability followed an exponential relationship with the carbon chain length . In the presence of 500 mg l-1 heptane, the survival was 1.6 times higher than that of controls . The progressive disappearance of heptane from the survival medium with a low and temporary accumulation by cells, suggests that this alkane may have been responsible for the increase of cell viability. Biochem Cell Biol, 1996, 74(2), 241 - 8 Structural characterization of the O-antigenic polysaccharide of Escherichia coli serotype 017 lipopolysaccharide; Masoud H et al.; The structure of the O-polysaccharide component of the lipopolysaccharide produced by Escherichia coli 017 (ATCC 23512) was determined by the use of methylation, periodate oxidation, one- and two-dimensional nuclear magnetic resonance spectroscopy, and mass spectrometric methods . The O-polysaccharide was found to be a high molecular weight polymer of repeating branched pentasaccharide units composed of D-mannose, D-glucose, and 2-acetamido-2-deoxy-D-glucose residues (3:1:1) and had the structure (formula: see text). Bioseparation, 1996, 6(5), 265 - 71 Expanded bed adsorption for recovery of renatured human recombinant interleukin 8 from Escherichia coli inclusion bodies; Barnfield Frej AK; Expanded bed adsorption is a chromatography technique based on stable fluidization, designed for large scale recovery of proteins directly from particulate-containing feedstocks . Described in this article is the use of expanded bed adsorption for recovery of renatured recombinant human interleukin 8 from Escherichia coli inclusion bodies, using a cation exchange adsorbent . The yield of interleukin 8 from the expanded bed was approximately 100% and the purification factor was approximately 4 . Following each interleukin 8 purification a cleaning in place procedure was performed with a commercial cleaner . The results show that the adsorbent can be used at least 50 times without any significant loss of function. Acta Physiol Pharmacol Ther Latinoam, 1996, 46(3), 159 - 67 Water permeability properties of the human small intestine in vitro: effects of Escherichia coli heat-stable enterotoxin; Ibarra C et al.; The net absorptive water flux (Jw), the transepithelial potential difference (PD) and the short-circuit current (Isc) were simultaneously measured in the human small intestine in vitro with the following results: 1) An absorptive Jw was observed when the jejunum or the ileum were mounted between two identical standard solutions in the presence of an hydrostatic pressure gradient (delta P) of 13 cm of water (mucosal side positive) . 2) The absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (delta Osm) in both intestinal segments . The hydrostatic (Phydr) and osmotic (Posm) permeabilities to water for jejunum and ileum were: 0.349 +/- 0.049 cm/s vs . 0.156 +/- 0.022 cm/s and 0.0012 +/- 0.0001 cm/s vs . 0.0019 +/- 0.0003, respectively . 3) A fraction of this absorptive Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient and represented the transport associated to movement of water (Jwt) . 4) PD and Isc values were similar in the jejunum and in the ileum but the transepithelial resistance (Rt) was significantly greater in ileum than in jejunum . 5) 2 micrograms/ml of E . coli heat-stable enterotoxin (STa) caused a significant inhibition of the absorptive Jw without modification of Phydr, Posm or Isc . 6) After STa treatment, the absorptive Jwt reverted to a secretory one in the jejunum . In the ileum, STa action caused a 48% decrease in the absorptive Jwt values. Gene Expr, 1996, 6(4), 231 - 9 Isolation and characterization of a dihydrofolate reductase gene mutation in methotrexate-resistant Drosophila cells; Hao H et al.; Stepwise increases in methotrexate (MTX) concentration over a 4-year period led to the selection of a highly drug-resistant (2 x 10(-4) M MTX) Drosophila cell line . Uptake experiments with {3H}MTX showed a slightly lower level of intracellular MTX in the resistant S3Mtx cells than in the susceptible S3 parental cell line . Southern blot analysis demonstrated that the gene for the MTX target, dihydrofolate reductase (DHFR), was not significantly amplified in the resistant line . To determine the molecular basis for resistance, the DHFR cDNA sequence was amplified by polymerase chain reaction from both the resistant and susceptible cells . Sequence comparison revealed a single T to A base change at nucleotide 89, which resulted in the substitution of Gln for Leu at residue 30 in S3Mtx cells . Expression and purification of the wild-type and mutant DHFR from E . coli cells showed that the S3Mtx enzyme had a reduced binding affinity for the antifolates, MTX and trimethoprim, with 15-fold higher K{d} and K{i} values than those from the wild-type enzyme . Molecular modeling confirmed that the replacement of the hydrophobic Leu by the more polar Gln was in the substrate binding site and thus would decrease the binding of MTX . These results suggest that the high level of MTX resistance in the selected cell line can be attributed to the mutation in the DHFR gene and also provides a model for pesticide resistance in insects. Biodegradation, 1996-97, 7(6), 445 - 53 Cultivation of Escherichia coli with mixtures of 3-phenylpropionic acid and glucose: dynamics of growth and substrate consumption; Kovarova K et al.; In technical as well as natural ecosystems, pollutants are often mineralised in the presence of easily degradable carbon sources . A laboratory model system consisting of Escherichia coli ML 30 growing with mixtures of 3-phenylpropionic acid (3ppa, 'pollutant') and glucose (easily degradable substrate) was investigated in batch and carbon-limited continuous culture . Untypically, a linear growth pattern was observed during batch cultivation with 3ppa as the only carbon/energy source . When exposed to mixtures of both substrates in batch culture, E . coli utilised the two compounds sequentially . However, 3ppa and glucose were consumed simultaneously in continuous culture . Whereas a pulse of excess glucose to a batch culture growing with 3ppa led to the repression of 3ppa utilisation, an excess of glucose added into continuous culture did not inhibit the utilisation of 3ppa . During continuous cultivation the 3ppa-degrading enzyme system operated close to saturation. HPB Surg, 1996, 10(1), 7 - 10 The role of endotoxaemia in the development of renal disorders in experimental obstructive jaundice in rats; Dawiskiba J; In rats with 2-week obstructive jaundice the sensitivity to endotoxin was studied and the effect of a single dose of endotoxin on histological development in the kidney, liver and spleen was also investigated . We were tested the effect on accumulation and distribution within organs, of fibrinogen labelled with radioactive iodine I 125 . We showed an increased sensitivity to endotoxin in obstructive jaundice . The cause of death in most rats was acute circulatory failure during the course of endotoxic shock, without clinical features of disseminated intravascular coagulation . In the isotope study, after endotoxin administration there was a specific dynamic increase of fibrinogen accumulation in the kidneys of rats with obstructive jaundice . We proposed, that the cause of the kidney changes during the course of obstructive jaundice could be the local activation of intrarenal coagulation. Med Dosw Mikrobiol, 1996, 48(3-4), 177 - 81 {Occurrence of rota-I adenoviruses in diarrhea states of children}; Rybicka E et al.; 98 children aged 6 weeks to 5 years with diarrhoea were examined . Rotaviruses and adenoviruses were detected by latex tests Rotalex and Adenolex produced by Orion Diagnostica . Rotaviruses were found in 20.4% of cases, most frequently in children of the age from 6 to 18 months . Adenoviruses were found in 11.2% of cases, most frequently in children of the age from 18 months to 5 years, mainly in mixed infection by- and adenoviruses . Viruses infection were most rare in infants aged from 6 weeks to 6 months . In 4.1% studied cases the coexisting of viral and bacterial infection was observed (K . pneumoniae, E . coli). Verh K Acad Geneeskd Belg, 1996, 58(6), 711 - 38 {The involvement of prostaglandins in the inhibiting effect of endotoxin on the myoelectric activity of the gastrointestinal system in pigs}; Wechsung E; The probable involvement of prostaglandins in the myoelectrical response of the antrum pylori and small intestine to endotoxin (LPS) was studied in the piglet . In these experiments the influence of I.V . infusion of PGF2 alpha and PGE2 and of I.V . injection of LPS, without and with indomethacin (INDO) pretreatment, on myoelectrical activity of the antrum pylori, duodenum, jejunum and ileum as well as on some clinical and haematological parameters was studied . Infusion of the 2 PG's, especially PGE2, inhibited myoelectrical activity of the antrum pylori . PGE2 also reduced duodenal activity . PGF2 alpha was without effect on duodenal and jejunal activity, but stimulated ileal activity . Both PG's induced fever, nausea, vomiting and sedation or excitation . With the higher dose of PGE2 diarrhoea was also observed . Injection of LPS induced identical myoelectrical and clinical changes, as described for PGE2 . However, endotoxin did not induce diarrhoea . Depending on the dose, administration of LPS resulted in a leukocytosis or a leukopenia together with an increase in band neutrophils . Following pretreatment with INDO the effects of LPS on gastrointestinal electrical activity were reduced and its clinical symptoms were nearly completely inhibited . The haematological changes induced by LPS, however, were not influenced by INDO . These experiments suggest a possible involvement of the PG's in the clinical symptoms and in the initial inhibitory effect of LPS on myoelectrical activity especially of the antrum . However, the induced haematological changes are probably not mediated by the arachidonic acid pathway. Physiol Chem Phys Med NMR, 1996, 28(4), 239 - 53 Mechanism that regulates nitric oxide production by lipopolysaccharide-stimulated rat Kupffer cells; Ikeda K et al.; Mechanism that regulates nitric oxide (NO) production by stimulated Kupffer cells was studied . Lipopolysaccharide (LPS) stimulated NO production by primary-cultured Kupffer cells in a time- and dose-dependent manner . Twenty-four h after incubation with 1 microgram/ml of LPS, the nitrite concentration in the culture medium increased from 2.87 +/- 1.4 to 73.6 +/- 6.9 nmol/ml . NO production started to increase around 6 h after adding LPS and reached a maximum within 24 h . NO production was inhibited by 0.3 mM of NG-monomethyl-L-arginine and was reversed by adding 3 mM of L-arginine . When incubated with 1 microgram/ml of LPS for 24 h, NO synthase activity in Kupffer cells increased from 0.164 +/- 0.035 to 3.16 +/- 0.11 nmol/min/mg protein . Dexamethasone and prednisolone significantly inhibited the induction of NO synthase and NO production in Kupffer cells . Expression of mRNA for inducible type of NO synthase (iNOS) started to increase around 4 h after adding LPS (1 microgram/ml) and reached a maximum by about 20 h . The enhanced expression of iNOS mRNA was also suppressed by 1 microM dexamethasone . On the other hand, calcium ionophore A23187 significantly enhanced the induction of NO synthase and iNOS mRNA expression in LPS-stimulated Kupffer cells . In addition, a tyrosine kinase inhibitor, genistein (100 microM), significantly inhibited NO production by LPS-stimulated Kupffer cells . Neither phorbol 12-myristate 13-acetate, dibutyryl cAMP, indomethacin nor a protein kinase C inhibitor H-7 affected NO production by Kupffer cells . These results suggested that iNOS mRNA expression, NO synthase activity and NO production increased in LPS-stimulated Kupffer cells by a glucocorticoid-inhibitable mechanism and that Ca2+ and tyrosine phosphorylation might play important roles in LPS-dependent induction of NO synthase. Biochimie, 1996, 78(11-12), 1109 - 17 Mechanisms of primer RNA synthesis and D-loop/R-loop-dependent DNA replication in Escherichia coli; Masai H et al.; In DNA replication, DNA chains are generally initiated from small pieces of ribonucleotides attached to DNA templates . These 'primers' are synthesized by various enzymatic mechanisms in Escherichia coli . Studies on primer RNA synthesis on single-stranded DNA templates containing specific 'priming signals' revealed the presence of two distinct modes, ie immobile and mobile priming . The former includes primer RNA synthesis by primase encoded by dnaG and by RNA polymerase containing a sigma 70 subunit . Priming is initiated at a specific site in immobile priming . Novel immobile priming signals were identified from various plasmid replicons, some of which function in initiation of the leading strand synthesis . The latter, on the other hands involves a protein complex, primosome, which contains DnaB, the replicative helicase for E coli chromosomal replication . Utilizing the energy fueled by ATP hydrolysis of DnaB protein, primosomes are able to translocate on a template DNA and primase synthesizes primer RNAs at multiple sites . Two distinct primosomes, DnaA-dependent and PriA-dependent, have been identified, which are differentially utilized for E coli chromosomal replication . Whereas DnaA-dependent primosome supports normal chromosomal replication from oriC, the PriA-dependent primosome functions in oriC-independent chromosomal replication observed in DNA-damaged cells or cells lacking RNaseH activity . In oriC-independent replication, PriA protein may recognize the D- or R-loop structure, respectively, to initiate assembly of a primosome which mediates primer RNA synthesis and replication fork progression. Biochimie, 1996, 78(11-12), 1035 - 42 Differential contributions of two elements of rho-independent terminator to transcription termination and mRNA stabilization; Abe H et al.; The hallmark features of rho-independent transcription terminators are a G(+)C-rich dyad symmetry sequence followed by a run of T residues on a sense strand . Both of these structural elements are required for efficient transcription termination . Besides its primary function, rho-independent terminators are also known to enhance expression of an upstream gene by stabilizing RNA in a few cases . The Escherichia coli crp gene encoding cAMP receptor protein (CRP) contains a typical rho-independent terminator . To gain further insight into the roles of the G(+)C-rich dyad symmetry sequence and the poly(T) tract both in transcription termination and mRNA stabilization, we constructed a series of variant crp terminators and analyzed their abilities regarding these two functions . Disruption of the G(+)C-rich dyad symmetry sequence almost completely eliminated terminator activity while disruption of the poly(T) tract reduced terminator activity significantly but not completely . Thus, the contribution of the G(+)C-rich dyad symmetry sequence to transcription termination is larger than that of the poly(T) tract . Disruption of the G(+)C-rich dyad symmetry region reduced expression of the upstream crp gene by accelerating the rate of mRNA degradation . However, disruption of the poly(T) sequence had no effect on the stability of the crp mRNA, indicating that the poly(T) tract plays no role in mRNA stabilization . When the crp terminator was replaced by terminators derived from other genes, the fusion genes expressed the crp mRNA at the same level as did the native crp gene, suggesting that the mRNA stabilization effect is probably a general nature of rho-independent terminators. Biochimie, 1996, 78(11-12), 985 - 91 Structure and function of 10Sa RNA: trans-translation system; Muto A et al.; 10Sa RNA is a small stable bacterial RNA in which the 5'- and 3'-end sequences are folded into a tRNA-like structure . The RNA is aminoacylatable with alanine in vitro, and it interacts with 70S ribosomes in the cell . Recently, Escherichia coli 10Sa RNA has been shown to contain the sequence-encoding tag-peptides, which are found to attach to the C-termini of truncated peptides synthesized in vivo . We have found that the E coli 10Sa RNA stimulates incorporation of the tag-specific amino acids into proteins depending on the poly(U)-directed poly-phenylalanine synthesis in the in vitro translation system . Our finding supports the 'trans-translation' model proposed for the tag synthesis, in which alanyl-10Sa RNA enters the ribosome when translation stops at the 3'-end of the truncated mRNA lacking a stop codon, and translation of the tag-peptide occurs by switching the template from mRNA to 10Sa RNA . In this unique reaction, 10Sa RNA acts both as a tRNA and as an mRNA. Biochimie, 1996, 78(11-12), 979 - 83 Structural organization of Escherichia coli tmRNA; Felden B et al.; A secondary structure of Escherichia coli 10Sa RNA (tmRNA) recently proposed on the basis of a variety of chemical and enzymatic probing data combined with phylogenetic analysis (Felden et al, in press), indicates a highly folded structure . Several long-range interactions including pseudoknots are proposed based on comparative analysis of 10 tmRNA genes . Whereas most of the probing data support these predicted secondary structures, several atypical reactivities in specific domains of the molecule suggest structural dynamics, perhaps relating to the complex functions of the molecule as both tRNA and mRNA . The structure of tmRNA has three modular units; a tRNA-like domain, an mRNA-like domain and an intricate connecting unit probably responsible for correct orientation of the two functional parts of the molecule. Biochimie, 1996, 78(11-12), 971 - 8 Structural model for the selenocysteine-specific elongation factor SelB; Hilgenfeld R et al.; A structural model was established for the N-terminal part of translation factor SelB which shares sequence similarity with EF-Tu, taking into account the coordinates of the EF-Tu 3D structure and the consensus of SelB sequences from four bacteria . The model showed that SelB is homologous in its N-terminal domains over all three domains of EF-Tu . The guanine nucleotide binding site and the residues involved in GTP hydrolysis are similar to those of EF-Tu, but with some subtle differences possibly responsible for the higher affinity of SelB for GTP compared to GDP . In accordance, the EF-Tu epitopes interacting with EF-Ts are lacking in SelB . Information on the formation of the selenocysteyl-binding pocket is presented . A phylogenetic comparison of the SelB domains homologous to EF-Tu with those from EF-Tu and initiation factor 2 indicated that SelB forms a separate class of translation factors. Biochimie, 1996, 78(11-12), 953 - 8 Influence of the last amino acid in the nascent peptide on EF-Tu during decoding; Mottagui-Tabar S et al.; The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249-257; Bjornsson et al (1996) EMBO J 15, 1696-1704) . Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA(Trp) by varying the codons at positions-1 and -2 at the 5' side of UGA . Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J4, 1049-1052) . The effect on readthrough by changing these-1 and -2 codons is different on the two forms of tRNA(Trp) and is also dependent on the structure of EF-Tu . Readthrough by the tRNA(Trp)-derived suppressor, but not wild-type tRNA(Trp), is sensitive to the van der Waals volume of the last amino acid in the nascent peptide . Together with mutant EF-Tu, both forms of tRNA(Trp) are sensitive . The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex . This interaction is changed by mutation in tRNA(Trp) at position 24 or in EF-Tu at position 375 . No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found . Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509-1516) and ribosomal proteins S4 and S12 (Faxen et al (1988) J Bacteriol 170, 3756-3760) were found not be altered in sensitivity to the last two amino acids in the nascent peptide. Biochimie, 1996, 78(11-12), 945 - 52 The translational stop signal: codon with a context, or extended factor recognition element? Tate WP, Poole ES, Dalphin ME, Major LL, Crawford DJ, Mannering SA. Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal . Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element . A data base of natural termination sites from a wide range of organisms (148 organisms, approximately 40,000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4) . The nature of this base determines the efficiency of the stop signal in vivo, and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor 3 . Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly . Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2 . The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context. Mol Membr Biol, 1996 Jan-Mar, 13(1), 53 - 60 A method for determining transmembrane protein structure; Jones PC et al.; A simple and rapid protein chemical approach for determining the transmembrane structure of membrane proteins is described . The method involves single substitutions of consecutive amino acid residues, within putative transmembrane segments, to cysteine . This is followed by the analysis of their susceptibility to modification by maleimides with different physico-chemical properties . Fluorescein-5-maleimide (FM), being hydrophilic, modified only residues located in the aqueous environment, while the hydrophobic reagent, benzophenone-4-maleimide (BM) modified residues exposed to the lipid phase . These probes are large enough to cause an increase in the molecular weight of relatively small membrane proteins or polypeptide fragments, which is detectable by SDS-PAGE . Modification by much smaller probes, such as N-ethylmaleimide (NEM), could also be monitored indirectly by the ability to prevent SDS-solubilized protein from being modified with fluorescein-5-maleimide . The approach is demonstrated with the proteolipid complex of the vacuolar H(+)-ATPase expressed in yeast and with the putative Isk K(+)-channel expressed and radiolabelled in E . coli . The advantages of this approach are: (1)it is rapid, easy and inexpensive, (2) detection of the modification of engineered cysteines is simple, (3) it requires only minute quantities of the protein, (4) the protein does not require purification, (5) a broad range of maleimides with different physico-chemical properties can be used, (6) the structure can be investigated under native conditions and does not require protein reconstitution into artificial bilayers. Immunobiology, 1996-97, 196(5), 535 - 49 Recombinant Treponema pallidum antigens in syphilis serology; Gerber A et al.; Treponema pallidum, the etiological agent of syphilis, is characterized by a paucity of surface exposed outer membrane proteins and a high content of cytoplasma membrane associated lipoproteins . At all stages of infection intense antibody responses against lipoproteins are detectable . In order to provide antigens for syphilis diagnosis the highly immunogenic lipoproteins TpN17, TpN29-35 (TpD), TpN44.5 (TmpA), TpN47, and TpN35 (TmpC) and the membrane protein TpN39 (BMP) were cloned . Insertion of PCR amplified DNA into an E . coli expression vector resulted in high level expression of antigens . N-terminal hexahistidine sequence allowed efficient purification of fusion proteins by metal chelate affinity chromatography . The recombinant antigens were tested in enzyme-linked immunosorbent assays . TpN17, TpN47, and TpN44.5 antigens showed high antibody titers . Assays with the three antigens combined resulted in a further improvement of diagnostic sensitivity in comparison with single antigens . Antibodies were found in 17 of 18 patients in all stages of syphilis, whereas 42 normal human sera were nonreactive . No cross-reactivity was detected in 24 sera of patients with Lyme borreliosis. Hum Antibodies Hybridomas, 1996, 7(4), 167 - 74 Anti-gp210 antibodies in sera of patients with primary biliary cirrhosis . Identification of a 64 kD fragment of gp210 as a major epitope; Wesierska-Gadek J et al.; Patients with primary biliary cirrhosis (PBC) frequently produce autoantibodies against gp210, an integral glycoprotein of the nuclear pores . this protein consists of three main domains: a large glycosylated lumenal domain, a single hydrophobic transmembrane segment and a short cytoplasmic tail . It has been previously shown that autoantibodies from PBC patients exclusively react with the cytoplasmic tail when recombinant rat gp210 expressed in Escherichia coli was used as antigen . Using human gp210 isolated from HeLa cells we found the lumenal domain as the major target . The aim of this study was to further characterize the dominant autoepitopes of gp210 . Sera from 88 patients with autoimmune liver disease and 20 controls were used . Gp210 protein was digested with papain or endoglycosidase H and then subjected to immunoblotting . Autoantibodies against gp210 were detected in 12 of 43 (28%) PBC patients, but in none of the autoimmune hepatitis and control sera . Four of 12 (33%) anti-gp210 positive sera reacted with a fragment consisting of the cytoplasmic tail and 8 (66%) sera targeted an epitope located within the large lumenal domain . Furthermore, our data show that antigenic determinant is restricted to the 64 kD glycosylated amino-terminal fragment and that carbohydrate residues are an essential part of this novel epitope . We suggest that antigens possessing both epitopes namely; the glycosylated lumenal domain and the cytoplasmic tail should be used for screening tests in order to detect all sera with anti-gp210 specificity. Folia Microbiol (Praha), 1996, 41(4), 357 - 62 Effect of heat shock on DNA-dependent RNA polymerase from the cyanobacterium Synechococcus sp; Hammouda O; Purification of DNA-dependent RNA polymerase from exponentially growing cells of the cyanobacterium Synechococcus sp . is described in cultures grown at normal temperature (39 degrees C) and after heat shock (HS) (47 degrees C) . Polyethyleneimine precipitation followed by chromatography and gel filtration steps results in a 39% yield . The enzyme has a component of molar mass of 43 kDa, designated sigma, in addition to the typical procaryotic beta' beta alpha 2 and gamma . The results suggest that Synechococcus RNA polymerase is similar to that of cyanobacterial and E . coli RNA polymerases . Electrophoresis of the HS preparation showed that the enzyme has a component of 18 kDa . This suggests the existence of a functional relationship between this protein and the HS response of Synechococcus RNA polymerase, probably in salvaging denatured RNA polymerase or helping to regain its native structure. Folia Microbiol (Praha), 1996, 41(4), 315 - 9 Cloning of sucrase operon with mini-Mu and plasmid-mediated metabolism of sucrose; Grones J et al.; The in vivo cloning system based on mini-Mu derivatives was used for cloning the sucrase operon . We constructed several recombinant plasmids pJT21, pMM2324, pMM2325 with a complete sucrase operon . Two strains of Escherichia coli containing this plasmid replicon were able to grow on different concentrations of sucrose . The stability of recombinant plasmids was determined after cultivation under nonselective conditions . The stability after 5-d cultivation was higher than 75%. Acta Cardiol, 1996, 51(6), 535 - 40 Escherichia coli endocarditis of a native mitral valve with paravalvular pseudoaneurysm formation and fatal hemopericardium; Oosterbosch L et al.; Until recently only few cases have been described of acute infective endocarditis with E . Coli limited to a normal native mitral valve . Furthermore, mechanisms of so called abcess formation and rupture are still uncompletely understood . We report the case of an E . Coli endocarditis developing a rapidly progressive pseudoaneurysm of the mitral annulus . At necropsy diffuse infectious tissue weakening with pseudoaneurysm formation of the mitral ring and dissection into an hemorraghic pericard were seen . The authors further discuss the changing pattern of infectious agents causing acute infective endocarditis of the native mitral valve, transesophageal echocardiographic characteristics of paravalvular cavities and insights in mechanisms of pseudoaneurysm formation and dissection from clinicopathological findings. Neuropharmacology, 1996, 35(8), 1023 - 8 Phosphorylation of the predicted major intracellular domains of the rat and chick neuronal nicotinic acetylcholine receptor alpha 7 subunit by cAMP-dependent protein kinase; Moss SJ et al.; The predicted major intracellular domains of the chick and rat neuronal nicotinic acetylcholine receptor alpha 7 subunits were expressed in E . coli as glutathione-S-transferase fusion proteins . These proteins were then purified to near homogeneity by chromatography on immobilized glutathione . The intracellular domains of the alpha 7 subunit from both species were phosphorylated to high stoichiometry by cAMP-dependent protein kinase, but not by protein kinase C, cGMP-dependent protein kinase, or calcium/calmodulin-dependent protein kinase . Phosphorylation occurred on serine residues only within an identical single tryptic peptide for both proteins . This conserved phosphorylation site was identified as Ser 342 utilizing site-directed mutagenesis . These results demonstrate that the intracellular domain of the alpha 7 subunit is a substrate of PKA, and suggest a role for protein phosphorylation in mediating cellular regulation upon neuronal AChRs containing this subunit. Microbios, 1996, 88(354), 45 - 53 Statistical model fitting Escherichia coli survival data during starvation in artificial sea water; Utzet F et al.; The survival of Escherichia coli in artificial sea water was studied using linear and nonlinear regression analysis in order to fit the simplest model . The surviving population was evaluated in a rich medium, and two mathematical models, monophase and diphase linear, were considered . The diphase model gave the best fit, although both models can be adjusted to the results obtained . The criteria applied to decide which model was more statistically significant, were residuals, the residual sum of squares and the coefficient of determination . These models are discussed in the light of present day knowledge on survival. Rom J Physiol, 1996 Jan-Dec, 33(1-4), 75 - 81 Effects of thymectomy on blood neutrophils phagocytic activity and phagocytic response in mice; Baciu I et al.; The role of the thymus in maintenance of the basal phagocytosis of blood neutrophils and eliciting the phagocytic response, induced by i.v . Escherichia coli, was studied in 9 NMRI thymectomized and in 12 control mice . Thymectomy depresses the percentage of phagocyting neutrophils from 70.91 +/- 0.9 (mean +/- SEM) in controls, to 61.49 +/- 2.33 in the thymectomized rats . Phagocytic activity, as assessed by the number of bacteria engulfed by 100 neutrophils, was also lower in thymectomized mice (114.42 +/- 7.52) than in controls (163.71 +/- 4.53) . A phagocytic response to i.v . Escherichia coli could nevertheless be noted in thymectomized mice, their phagocytic activity rising from the basal activity of 114.42 +/- 7.52 to 142.19 +/- 5.40 three hours after injection of Escherichia coli, while in control animals this activity rose from 163.71 +/- 4.53 to 216.46 +/- 12.91 . These results may, at least partially, explain the recurrent infections and the septicemia occurring in children with Down's syndrome . It is suggested that the thymus, as an endocrine organ, may be involved in maintaining the basal phagocytic activity of blood neutrophils, while the phagocytic response is modulated by extrathymic mechanisms. Mol Biol Rep, 1996, 23(3-4), 147 - 51 Epitope analysis of chromo antigen and clinical features in a subset of patients with anti-centromere antibodies; Muro Y et al.; Heterochromatin protein 1 (HP1) is one of the nonhistone chromosomal components tightly associated with the pericentromeric heterochromatic region in Drosophila . The human homologue of HP1 is recognized by a subpopulation of anti-centromere antibodies (ACA) . Such autoantibodies recognize a group of several nuclear proteins with Mr of 23-25 kDa and have been termed 'anti-chromo antibodies (AChA)' because an evolutionarily conserved N-terminal half called the 'chromo domain' of HP1 is the epitope . In this study, 84 ACA sera were examined by immunoblotting with recombinant 25-kDa chromo protein (p25) . The p25 antigen was expressed as a glutathione S-transferase-fusion protein in E . coli and purified with glutathione-sepharose . Except for one serum specimen, AChA-positive sera reacted with the N-terminus (a.a 16-106) and/or the C-terminus (a.a . 83-191) of p25 . Autoimmune response against the N-terminus of p25 in 33 patients was significantly associated with systemic lupus erythematosus and significantly related to leukopenia, thrombocytopenia and elevated erythrocyte sedimentation rate; C-terminal reactivity in 30 patients was significantly associated with primary Sjogren's syndrome and related to leukopenia . The internal 64-amino acid stretch (a.a 43-106) with DNA-binding activity was not autoantigenic . p25 has two separate homologous regions to Drosophila HP1 at the N- and C-termini; the chromo domain and the chromo shadow domain . Patients with autoimmune response against these conserved domains might form a clinical subset of patients positive for ACA. Acta Biochim Pol, 1996, 43(4), 611 - 21 Expression and purification of 6xHis-tagged DNA binding domains of functional ecdysteroid receptor from drosophila melanogaster; Rusin A et al.; Two members of the nuclear receptor superfamily, EcR and Ultraspiracle (Usp) heterodimerize to form a functional receptor for 20-hydroxyecdysone-the key ecdysteroid controlling induction and modulation of morphogenetic events through Drosophila development . In order to study aspects of receptor function and ultimately the structural basis of the ecdysteroid receptor-DNA interaction, it is necessary to produce large quantities of purified EcR and Usp DNA-binding domains . Toward this end, we have expressed the EcR DNA-binding domain and the Usp DNA-binding domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expression vector pQE-30 . Under optimal conditions, elaborated in this study, bacteria can express the recombinant 6xHis-EcRDBD to the levels of 11% of total soluble proteins and the 6xHis-UspDBD to the levels of 16% . Both proteins were purified to homogeneity from the soluble protein fraction using combination of ammonium sulphate fractionation and affinity chromatography on Ni-NTA agarose . The gel mobility shift experiments demonstrated that the purified 6xHis-EcRDBD and the 6xHis-UspDBD interact specifically with an 20-hydroxyecdysone response element from the promoter region of the hsp 27 Drosophila gene. Adv Exp Med Biol, 1996, 409, 269 - 77 Molecular characterization of Hor v 9 . Conservation of a T-cell epitope among group IX pollen allergens and human VCAM and CD2; Astwood JD et al.; We have cloned, sequenced and expressed a recombinant group IX pollen allergen from barley (Hordeum vulgare) . Hor v 9 is a polypeptide of 313 amino acids . The Hor v 9 cDNA clone was engineered into the E . coli protein expression vector pMAL and expressed as a fusion of maltose binding protein and truncated Hor v 9 . Polyclonal antibodies to the fusion protein were raised in mice . Cross-reactive proteins, RNA and DNA homologues were found in many agricultural species including wheat, rye, triticale, oats, maize, sunflower and flax . The presence of group IX-like proteins in a variety of agricultural crops may represent a previously uncharacterized aeroallergenic occupational hazard . Sequence comparisons of the barley allergen, Hor v 9, with Poa p 9 and other cloned group IX pollen allergens revealed putative structural domains common to all . These include a signal peptide, two conserved immunoglobulin-like motifs, a 150 amino acid highly conserved carboxyterminal domain and a carboxyterminal transmembrane helix . This structural arrangement is also found in cell adhesion molecules . The highly conserved T-cell epitope previously characterized and mapped in group IX allergens (and present in Hor v 9) was found in several human cell adhesion molecule sequences (VCAM, NCAM and CD2) . This T-cell epitope corresponded to the most highly conserved amino acid residues common to all group IX homologues sequenced to date . CD2 and VCAM are known to play a role in allergic inflammation: VCAM is involved in the recruitment of lymphocytes to sites of inflammation, and cross-linking CD2 leads to T-cell activation . We anticipate that the similar structural arrangement of group IX allergens and human cell adhesion molecules, as well as the presence of a T-cell epitope common to group IX pollen allergens and cell adhesion molecules, will have important consequences in the natural history of the atopic immune response. Adv Exp Med Biol, 1996, 409, 251 - 4 Characterisation of recombinant isoforms of birch pollen allergen Bet v 1; Spangfort MD et al.; Three isoforms of the major birch pollen allergen, Bet v, 1 from Betula verrucosa have been expressed as recombinant proteins in E . coli and purified . The immunochemical properties of recombinant isoforms (rBet v 1) differed on immunoblots when compared using Mabs and birch pollen allergic patients serum IgE . 2-D gel analysis showed that recombinant isoforms with different epitope structure can focus under the same protein spot after electrophoresis . The structure of conformational epitopes can be distorted by amino acid substitutions even when T-cell epitopes are not affected as judged by T-cell proliferation studies. Adv Exp Med Biol, 1996, 409, 117 - 26 Biological and immunological importance of Bet v 1 isoforms; Breitenbach M et al.; In 2D-PAGE analysis of Bet v 1, the major birch pollen allergen, up to 12 isoforms can be demonstrated that differ in their isoelectric points from about pH 4.9 to pH 5.9 . The molecular weights of these isoforms seem to be rather similar, but minor variations can also be seen . Preliminary experiments with birch leaves seem to indicate that in aging leaves some isoforms can be found that do not occur in pollen . In birch cells cultured in vitro, Bet v 1 isoforms can be induced by bacterial infection that do not occur in pollen (Swoboda et al . (1995), Pant, Cell and Environment 18, 865-874) . In a recent paper (Swoboda et al (1995)., J . Biol . Chem . 270, 2607-2613) we show that in natural Bet v 1 from pollen the isoforms are due to different protein sequences . The derived protein sequences of 10 different isoforms (corresponding to 13 different cDNAs) were determined and confirmed by plasma desorption mass spectrometry of purified natural Bet v 1 after trypsin and endoproteinase Glu-C digestion . These experiments also showed that pollen Bet v 1 isoforms were reactive to patients' sera to different degrees and that common post-synthetic modifications (besides N-terminal methionine cleavage) did not occur on Bet v 1 . Recombinant isoforms were produced in E . coli, purified and tested with selected patients allergic to birch pollen (Ferreira et al., J . Exp . Med., in the press) . The pattern of IgE binding to Bet v 1 isoforms widely differs . Also, T-cell clones from individual patients in some cases are specific to peptides occurring only in certain isoforms . It was of particular interest that three of the naturally occurring pollen Bet v 1 isoforms do not or hardly bind IgE of untreated patients allergic to Bet v 1 . However, a comparison of IgE reactivity in patients before and after conventional immunotherapy with natural pollen extract clearly showed that this form of immunotherapy induced IgE to the isoforms that had been unreactive in untreated patients . One of these, Bet v 1d, showed a particularly strong potency towards T-cell stimulation . The isoform(s) that do not bind IgE in untreated patients but still show T-cell reactivity could be potentially utilized for a new form of immunotherapy that avoids the risk of anaphylaxis. Chin J Biotechnol, 1996, 12(3), 161 - 7 Amplification, cloning, and sequence comparison of the growth hormone gene for carp (Cyprinus carpio) by the polymerase chain reaction; Zhao X et al.; Total RNA was isolated from carp pituitary gland . The first strand cDNA was synthesized using oligo(dT) 12-18 as a primer, the total RNA as a template, and AMV reverse transcriptase . Next the polymerase chain reaction (PCR) was performed using the first strand cDNA as a template, the synthetic 29 oligonucleotides as primer and Taq DNA polymerase (94 degrees C, 60 s; 55 degrees C, 30 s; 72 degrees C, 50 s; 35 cycles) . After PCR amplification, the products were cloned into an E . coli expression vector (PBluescript II KS+/-) . The result of the sequence analysis and the restriction map shows that an open reading frame of the carp growth hormone gene contains 630 base pairs which code for a polypeptide of 210 amino acids including 22 amino acids of the signal peptide and 188 amino acids of the nature growth hormone . Nucleotide sequence and amino acid sequence of our carp growth hormone gene are the same as Koren's carp GH cDNA in the coded region . Compared with Chao's carp GH cDNA, the homology of nucleotide sequence and amino acid sequence for our carp growth hormone gene is 95.6% and 96.7%, respectively, in the coded region. Basic Life Sci, 1996, 64, 397 - 413 The determination of the in situ structure by nuclear spin contrast variation; Stuhrmann HB et al.; Polarized neutron scattering from polarized nuclear spins in hydrogenous substances opens a new way of contrast variation . The enhanced contrast due to proton spin polarization was used for the in situ structure determination of tRNA of the functional complex of the E . coli ribosome. Basic Life Sci, 1996, 64, 267 - 72 Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography; Timmins PA et al.; The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H2O/D2O contrast variation . If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering . The application of various constraints allows the resulting scattering length density map to be realistically modeled . The method has been applied to two different forms of the membrane protein porin . In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished. J Urol (Paris), 1996, 102(3), 127 - 9 {Emphysematous pyelonephritis . A case medically treated}; Labussiere AS et al.; Authors report on a case of emphysematous pyelonephritis in a woman affected with diabetes and renal failure . In order to avoid chronic dialysis, no nephrectomy was performed and the patient was treated only with drugs . Full recovery was obtained, without worsening of the renal function. Connect Tissue Res, 1996, 35(1-4), 393 - 9 Generation and use of recombinant human bone sialoprotein and osteopontin for hydroxyapatite studies; Stubbs JT 3rd; Bone sialoprotein (BSP) and osteopontin (OPN) are two extracellular bone matrix proteins that have the ability to modulate the growth of hydroxyapatite in vitro . Studies of BSP/OPN hydroxyapatite interactions in the past have been directed toward the identification of essential structural elements that allow these two proteins to modulate hydroxyapatite growth . However, these studies are limited by the finite quantities of purified extracellular matrix proteins . I have utilized a recombinant E . coli expression system to obtain milligram quantities of human bone sialoprotein and human osteopontin that may be used to study extracellular matrix protein interactions with hydroxyapatite. Yi Chuan Xue Bao, 1996, 23(6), 469 - 76 {Structure analysis of a DNA fragment containing strong promoter activity from Streptomyces mycarofaciens 1748}; Gu H et al.; A HindIII-HindIII 2.0kb DNA fragment containing promoter activity has been cloned from medicamycin producing strain (S . mycarofaciens 1748), using promoter -probe plasmid vector pIJ486 . Transformants of recombinant plasmid p4H2 containing this fragment were resistant to Km in the level above 500 micrograms/ml in MM medium . The result of subcloning deletion analysis indicated that different part of deletion of this fragment would show various extent of effect on the promoter activity . A rather complicated transcriptional regulatory mechanism was suggested . DNA sequence analysis revealed that this fragment consisted of 1984 nucleotides with 47.7% of G+C content . No typical ORF of Streptomyces gene was found . Certain regions showed some homologies with promoters of Streptomyces genes . In 520-570bp region, a sequence similar to the upstream activator sequence (UAS) of E.coli tRNA was discovered . This implied that a possible "enhancer" element was involved in Streptomyces gene transcription. Tsitologiia, 1996, 38(11), 1203 - 10 {The suppression of the repair of radio-induced DNA damage in Escherichia coli cells by indazolin derivatives and proksifein}; Zhestianikov VD et al.; A study was made of the postradiation effect of five indasoline derivatives and proxyfeine on the survival rate and repair of DNA single-strand breaks in E . coli exposed to gamma-irradiation . Some indasoline derivatives (three substances) and proxyfeine added in postradiation medium in nontoxic concentrations decreased the survival rate of radioresistant strains WP2 hcr+, Hr30 and Hs30, but do not influence survival rate of radiosensitive mutant Bs-1 . These substances inhibit the repair of DNA single-strand breaks in E . coli WP2 hcr+ . Substances, which do not inhibit the survival rate of radioresistant strains, do not inhibit the repair of breaks . Proxyfeine in non-toxic concentrations in non-irradiated cells induces DNA degradation . Indazoline derivatives do not induce DNA degradation . Data presented suggest that indasolines are new class inhibitors of DNA repair . It is possible that proxyfeine is too an inhibitor of DNA repair. Fold Des, 1996, 1(5), 335 - 46 Molecular dynamics simulations of apocytochrome b562--the highly ordered limit of molten globules; Laidig KE et al.; BACKGROUND: Cytochrome B562 is a heme-containing, four-helix bundle . It has been proposed that the apo form of the protein is a molten globule . We present a molecular dynamics study of apocytochrome b562 to investigate its structural and dynamic properties . RESULTS: Our simulations suggest that all four helices are essentially intact and confirm that the experimental difficulties of assigning helical NOEs in the C-terminal helix are not due to structural disorder . The increased 'moltenness' of the apoprotein is due to an increased mobility of the sidechains . The small observed increase in compressibility for the apoprotein is proposed to be the result of an increase in the intrinsic protein compressibility, which is opposed by the increase in the size of the protein hydration shell . CONCLUSIONS: Apocytochrome b562 is postulated to be near the highly ordered limit of the molten globule state, a structure whose molten character is due primarily to increased sidechain mobility with concurrent loss in tertiary contacts between the helices, rather than changes in the folding topology or substantially increased disorder of the secondary structure. Fold Des, 1996, 1(3), 167 - 78 Analysis of the effect of local interactions on protein stability; Munoz V et al.; BACKGROUND: Protein stability appears to be governed by non-covalent interactions . These can be local (between residues close in sequence) or non-local (medium-range and long-range interactions) . The specific role of local interactions is controversial . Statistical mechanics arguments point out that local interactions must be weak in stable folded proteins . However, site-directed mutagenesis has revealed that local interactions make a significant contribution to protein stability . Finally, computer simulations suggest that correctly folded proteins require a delicate balance between local and non-local contributions to protein stability . RESULT: To analyze experimentally the effect of local interactions on protein stability, each of the five Che Y alpha-helices was enhanced in its helical propensity . alpha-Helix-promoting mutations have been designed, using a helix/coil transition algorithm tuned for heteropolypeptides, that do not alter the overall hydrophobicity or protein packing . The increase in helical propensity has been evaluated by far-UV CD analysis of the corresponding peptides . Thermodynamic analysis of the five Che Y mutants reveals, in all cases, an increase in half urea ({urea}1/2) and in Tm, and a decrease in the sensitivity to chemical denaturants (m) . ANS binding assays indicate that the changes in m are not due to the stabilization of an intermediate, and the kinetic analysis of the mutants shows that their equilibrium unfolding transition can be considered as following a two-state model, while the change in m is found in the refolding reaction (m(k)f) . CONCLUSIONS: These results are explained by a variable two-state model in which the changes in half urea and Tm arise from the stabilization of the native state and the decrease in m from the compaction of the denatured state . Therefore, the net change in protein stability in aqueous solution produced by increasing the contribution of native-like local interactions in Che Y is the balance between these two conflicting effects . Our results support the idea that optimization of protein stability and cooperativity involve a specific ratio of local versus non-local interactions. Fold Des, 1996, 1(2), 77 - 89 Mutational effects on inclusion body formation in the periplasmic expression of the immunoglobulin VL domain REI; Chan W et al.; BACKGROUND: Inclusion body (IB) formation in bacteria is an important example of protein misassembly, a phenomenon which also includes folding-dependent aggregation in vitro and amyloid deposition in human disease . Previous studies of mutational effects in other systems implicate the stability of a folding intermediate-rather than the native state-as playing a key role in IB formation . To contribute to an understanding of the comparative biophysics of VL misassembly in different biological settings, we have studied mutation-dependent periplasmic IB formation by the VL domain REI in Escherichia coli . RESULTS: A series of mutants were produced in periplasmic IBs, where, in all cases, the signal peptide was removed . In addition, the intradomain disulfide was clearly formed before deposition into IBs . IB formation in these mutants does not correlate with monomer/dimer equilibrium constants, but does correlate with the thermodynamic stability of the native state . CONCLUSIONS: The results implicate a late, equilibrium folding intermediate in IB formation, in contrast to the apparent involvement of transient folding intermediates in other IB systems described to date . As equilibrium unfolding intermediates have also been implicated in light chain amyloidosis and deposition diseases, IB formation may prove a useful model for these human diseases. Fold Des, 1996, 1(1), 43 - 55 Structure of the transition state for folding of the 129 aa protein CheY resembles that of a smaller protein, CI-2; Lopez-Hernandez E et al.; BACKGROUND: Protein engineering analysis has been used as a tool to determine the structure of the transition state of two different proteins: CI-2 and barnase . CI-2 belongs to the group of small, globular proteins with no disulphide bonds that fold via a two-state mechanism . Barnase is a larger protein (110 aa) and displays a folding intermediate . The structure of the transition state of both proteins is quite different . Whereas in CI-2 no region is fully native and it looks like an expanded form of the folded state, in barnase several regions are folded and the rate-limiting step seems to be the consolidation of the hydrophobic core . On the basis of these results, a unified scheme for the transition state of protein folding has been presented . We decided to characterize the folding pathway, or pathways, present in the alpha/beta parallel family of proteins using one of the smallest members, CheY (129 aa), as a model case . RESULTS: The folding pathway of CheY contains, as does that of barnase, a kinetic intermediate . The picture obtained for CheY from the equilibrium and kinetic analyses of several mutations scattered throughout the whole protein is different from that found for barnase . On the basis of the experimental results and the structure of CheY, the protein can be divided into two subdomains (from beta-strand 1 to beta-strand 3 and from beta-strand 3 to the C terminus) . Whereas the structure of the first subdomain in the transition state resembles that found for the CI-2 protein, the second subdomain is compact but unstructured . The packing of the first alpha-helix against beta-strands 1 and 2 seems to be the nucleus around which the rest of the protein folds . CONCLUSIONS: Comparison of the transition state of barnase with those of CheY and CI-2 indicates that different proteins have different transition states, probably depending on the energetics and the position of the rate-limiting step in the folding pathway . CheY appears to fold through a nucleation/condensation mechanism as has been found for CI-2 . The rate-determining step in some multimodular proteins could be the formation of a stable domain, with the less stable domains folding after the major rate-determining step. Fold Des, 1996, 1(1), 21 - 7 Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation; Goldberg ME et al.; BACKGROUND: Attempts to renature proteins often yield aggregates rather than native protein . To minimize aggregation, low protein concentrations and/or solubilizing agents are used . Here, we test new solubilizing molecules, non-detergent sulphobetaines, to improve the renaturation of two very different enzymes, hen egg white lysozyme and bacterial beta-D-galactosidase . RESULTS: The renaturation was conducted in the presence of five different sulphobetaines and the yield of active enzyme was measured . The five sulphobetaines improved the yield of native lysozyme up to 12-fold . Some sulphobetaines improved the yield of galactosidase up to 80-fold, but one reduced it 100-fold . CONCLUSIONS: Non-detergent sulphobetaines strongly affect the balance between aggregation and folding . Their effect depends on their structure and on their interactions with folding intermediates . These results should serve as a basis for designing more efficient sulphobetaines; for designing improved renaturation protocols using existing sulphobetaines; and for characterizing folding intermediates that interact with sulphobetaines. Fold Des, 1996, 1(1), R9 - 15 Revisiting the Anfinsen cage; Ellis RJ; In the past five years, ideas about protein folding inside cells have changed as a results of experiments with the chaperonin family of molecular chaperones . The folding of at least some proteins is no longer regarded as a spontaneous energy-independent process, but as involving transient interactions with chaperonin ATPases that serve to increase the efficiency of correct folding within the highly crowded intracellular environment . This review discusses in an historical context one model for how the chaperonins function . This model suggests that proteins fold inside cells in the same way as they do in pure dilute solution, but that they do so inside macromolecular Anfinsen cages that serve as sequestration devices to prevent and reverse unproductive interactions. Hum Antibodies Hybridomas, 1996, 7(3), 97 - 105 Efficient in vitro affinity maturation of phage antibodies using BIAcore guided selections; Schier R et al.; Selection of higher affinity mutant phage antibodies has proven less than straightforward due to sequence dependent differences in phage antibody expression, toxicity to Escherichia coli, and difficulty in eluting the highest affinity phage . These differences lead to selection for increased levels of expression or decreased toxicity rather than for higher affinity . In this work, we demonstrate how surface plasmon resonance as employed in the BIAcore can be used to increase the efficiency of phage antibody selections, yielding greater increments in affinity from a single library . A mutant phage antibody library was created by randomizing nine amino acids located in the V(L) CDR3 of C6.5, a human scFv which binds the tumor antigen c-erbB-2 with a Kd of 1.6 x 10-8 M . The library was subjected to five rounds of selection in solution using decreasing concentrations of biotinylated c-erbB-2 . After each round of selection, polyclonal phage were prepared and the rate of binding to c-erbB-2 determined in a BIAcore under mass transport limited conditions . Determination of the rate of binding permitted calculation of the concentration, and hence percent, of binding phage present . Results were used to select the antigen concentration for the next round of selection . To determine the optimal eluent, polyclonal phage was injected in a BIAcore and eluted using one of five different solutions (10 mM HCl, 50 mM HCl, 100 mM HCl, 100 mM triethylamine, 2.6 M MgCl2) . Differences were observed in eluent efficacy, which was reflected in significant differences in the affinities of phage antibodies isolated from the library after a round of selection using the different eluents . Use of the BIAcore to determine the optimal eluent and guide the antigen concentration used for selection yielded a C6.5 mutant with a 16 fold reduction in Kd (Kd = 1.0 x 10-9 M) . This represents at least a twofold greater increment in affinity than previously obtained from a single library of phage antibodies which bind antigens. Protein Eng, 1996 Jan, 9(1), 85 - 93 A rapid and effective procedure for screening protease mutants; Venekei I et al.; We describe a simple and effective procedure to screen for active proteases among a large number of mutants . First, the mutants are genetically tested by the protease activity produced in the periplasm of transformed bacteria which supplies the cells with a nitrogen source by hydrolyzing a protein applied to plates . Then a less sensitive activity staining and an X-ray film digestion assay are used to verify and estimate the activity of the mutants that proved to be positive in the first step . Depending essentially on the level of periplasmic protease activity, the method can detect both the activity and the stability of the expressed enzymes . We calibrated the method with transformants that produce wild-type trypsin, chymotrypsin and trypsin mutants of known activity . Using this method we found two active revertants of the inactive Asn102 trypsin mutant, by screening approximately 4.4 x 10(4) random mutants that were generated by the polymerase chain reaction on a cDNA fragment . This procedure should be useful in searching for proteases of novel specificity and/or reaction chemistry engineered by random mutagenesis, and also for in vitro evolution studies. Protein Eng, 1996 Jan, 9(1), 69 - 75 Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase; Finer-Moore JS et al.; Invariant arginine 179, one of four arginines that are conserved in all thymidylate synthases (TS) and that bind the phosphate moiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP), can be altered even to a negatively charged glutamic acid with little effect on kcat . In the mutant structures, ordered water or the other phosphate-binding arginines compensate for the hydrogen bonds made by Arg179 in the wild-type enzyme and there is almost no change in the conformation or binding site of dUMP . Correlation of dUMP Kds for TS R179A and TS R179K with the structures of their binary complexes shows, that the positive charge on Arg179 contributes significantly to dUMP binding affinity . kcat/K(m) for dUMP measures the rate of dUMP binding to TS during the ordered bi-substrate reaction, and in the ternary complex dUMP provides a binding surface for the cofactor . kcat/K(m) reflects the ability of the enzyme to accept a properly oriented dUMP for catalysis and is less sensitive than is Kd to the changes in electrostatics at the phosphate binding site. Protein Eng, 1996 Jan, 9(1), 61 - 7 A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation; Morris AJ et al.; Lys146 of rabbit aldolase A {D-fructose-1,6-bis(phosphate): D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13} was changed to arginine by site-directed mutagenesis . The kcat of the resulting mutant protein, K146R, was 500 times slower than wild-type in steady-state kinetic assays for both cleavage and condensation of fructose-1,6-bis(phosphate), while the K(m) for this substrate was unchanged . Analysis of the rate of formation of catalytic intermediates showed K146R was significantly different from the wild-type enzyme and other enzymes mutated at this site . Single-turnover experiments using acid precipitation to trap the Schiff base intermediate on the wild-type enzyme failed to show a build-up of this intermediate on K146R . However, K146R retained the ability to form the Schiff base intermediate as shown by the significant amounts of Schiff base intermediate trapped with NaBH4 . In the single-turnover experiments it appeared that the Schiff base intermediate was converted to products more rapidly than it was produced . This suggested a maximal rate of Schiff base formation of 0.022 s-1, which was close to the value of kcat for this enzyme . This observation is strikingly different from the wild-type enzyme in which Schiff base formation is > 100 times faster than kcat . For K146R it appears that steps up to and including Schiff base formation are rate limiting for the catalytic reaction . The carbanion intermediate derived from either substrate or product, and the equilibrium concentrations of covalent enzyme-substrate intermediates, were much lower on K146R than on the wild-type enzyme . The greater bulk of the guanidino moiety may destabilize the covalent enzyme-substrate intermediates, thereby slowing the rate of Schiff base formation such that it becomes rate limiting . The K146R mutant enzyme is significantly more active than other enzymes mutated at this site, perhaps because it maintains a positively charged group at an essential position in the active site or perhaps the Arg functionally substitutes as a general acid/base catalyst in both Schiff base formation and in subsequent abstraction of the C4-hydroxyl proton. Protein Eng, 1996 Jan, 9(1), 51 - 9 A mixed helix-beta-sheet model of the transmembrane region of the nicotinic acetylcholine receptor; Ortells MO et al.; We have modelled the transmembrane region of the alpha 7 nicotinic acetylcholine receptor as a mixed alpha-helical/beta-sheet structure . The model was mainly based on the crystal structure of a pore-forming toxin, heat-labile enterotoxin . This is a pentameric protein having a central pore or channel composed of five alpha-helices, one from each of the 5 B subunits that form this pentamer . The remainder of this structure is beta-sheet, loops and a short alpha-helix, not included in the model . The model uses this channel as a template to build the transmembrane region, from M1 to the middle of M3 . The remainder of M3 and M4 were built de novo as alpha-helices . Great consideration was given to labelling data available for the transmembrane region . In general terms, the shape of the model agrees very well with that obtained independently by electron microscopic analysis and the secondary structure predicted by the model is in accord with that estimated independently by Fourier transform infrared spectroscopy . The M2 helical region of the model is only slightly kinked, contrary to what is inferred from electron microscopic analysis, but has the same overall shape and form . On the membrane face of the model, the presence of deep pockets may provide the structural basis for the distinction between annular and non-annular lipid binding sites . Also, the transmembrane region is clearly asymmetric in the direction perpendicular to the membrane, and this may have strong influence on the surrounding lipid composition of each leaflet of the cytoplasmic membrane. Protein Eng, 1996 Jan, 9(1), 101 - 6 Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli; Masuda K et al.; We have developed a high-level production system for the C-terminal domain of secretory leukoprotease inhibitor (SLPI) to investigate its pharmacological activities . A gene for the C-terminal domain of SLPI, (Asn55-Ala 107)SLPI, was constructed from chemically synthesized deoxyoligonucleotides . It was fused to a gene for the N-terminal portion of human growth hormone via a DNA sequence encoding Leu-Val-Pro-Arg, which can be cleaved by thrombin . The fused gene was expressed in Escherichia coli under the control of a trp promoter, and the fusion protein was obtained as an inclusion body . After sulfonation of the cysteine residues, the sulfonated fusion protein was cleaved at the desired site by thrombin . Sulfonated (Asn55-Ala107) SLPI was refolded in Tris buffer containing reduced and oxidized glutathione . The resulting (Asn55-Ala107) SLPI was purified by cation-exchange chromatography and reverse-phase high performance liquid chromatography . The final yield was 50 mg/I culture . (Asn55-Ala107) SLPI was as active against elastase as, but had less trypsin inhibitory activity than, native SLPI . This system is suitable for the large-scale production of the C-terminal domain of SLPI, which is an elastase-specific inhibitor. Acta Physiol Hung, 1996, 84(2), 157 - 70 Comparative study of the circulatory effects of aminoguanidine and N-nitro-L-arginine in hyperdynamic endotoxemia; Tarnoky K et al.; We have studied the effects of NG-nitro-L-arginine (NNA) a nitric oxide synthase (NOS) inhibitor, and aminoguanidine (AG) a diamine oxidase inhibitor, on hemodynamic parameters and plasma histamine level using a dog model in which a hyperdynamic circulatory response was elicited with a 2-hour infusion of a low dose (13.75 micrograms/kg) of E . coli 055:B5 endotoxin (ETX) . AG (50 mg/kg) or NNA (0.5 mg/kg) was administered intravenously as pretreatment . Hemodynamic variables were studied for 4 hours after the beginning of the ETX infusion . The ETX-elicited hyperdynamic response was abolished by NNA and partially inhibited by AG . AG prevented the increases in cardiac output and heart rate and delayed the early decrease in total peripheral resistance (TPR) . The plasma histamine concentration elevation was higher in animals receiving AG than in those receiving only ETX . In the group treated with ETX plus NNA the cardiac output was lower and the TPR was higher than in the ETX plus AG group . In future studies, AG should be considered as one of the possible therapeutic tools in sepsis, as its adverse effect on the compensatory hyperdynamic response is less than that of NOS inhibitors of the L-arginine analog type, while it may favourably influence the deleterious excessive activity of the inducible NOS in the later stages. Tsitologiia, 1996, 38(10), 1106 - 14 {The DNA-binding activity of the membrane protein annexin II and its interaction with antibodies to the chromatin nuclear ribonucleoprotein (alpha-RNP)}; Krutilina RI et al.; By screening with labeled Alu DNA, a clone was isolated from cDNA expression library, which appeared identical in sequence to the well-known Ca-phospholipid-binding protein annexin II . To evidence the DNA-binding activity of recombinant annexin II and its presence in the cell nucleus, we have expressed full-length mouse annexin II cDNA in bacteria with pGEMEX vector . The expressed protein was studied with electrophoretic mobility shift assay and for its reaction with polyclonal antibody to chromatin-associated ribonucleoprotein (alpha-RNP), which is one of the major acid-dissolvent components of the nucleus . The obtained results confirm the DNA-binding activity of recombinant annexin II . Annexin II reacts with polyclonal antibody to rat alpha-RNP . So, annexin II is a major nuclear DNA-binding protein in mammalian cells. Eisei Shikenjo Hokoku, 1996, (114), 13 - 5 {Purification of EcoO44I restriction endonuclease in Escherichia coli O44 isolated from an affected human}; Miyahara M et al.; A restriction endonuclases (ENase) designated EcoO44I was purified without non specific nucleases from enteropathogenic Escherichia coli O44 Hiromi strain of affected human origin . The yield was 1, 100 units/g of wet cells . The EcoO44I ENase recognized and cleaved the specific sequence of 5'-GGTCTC-3' (1/5) as was the case with Eco31I or BsaI ENase . Because of the stability and high yield, EcoO44I would be useful for recombinant DNA technology after isolation of EcoO44-positive, avirulent mutant strains of E . coli O44 Hiromi. Microbios, 1996, 87(351), 123 - 33 Phenylalanine utilization for protein synthesis in beta-phenylpyruvic acid treated Escherichia coli cells; Miseta A et al.; The possibility of coupling along the supply routes of phenylalanine from its uptake by the cell, through the charging of specific tRNAs, has been postulated . The experimental approach to testing this hypothesis has been to study the competition between endogenously synthesized and exogenously supplied amino acids, from which preferences for their incorporation into cellular proteins can be deduced . The results indicate that manipulation of the endogenous phenylalanine pool size, achieved by addition of its immediate precursor, beta-phenylpyruvate, does not cause the predicted changes in the incorporation of exogenous labelled phenylalanine into proteins . The evidence favours exogenous phenylalanine being preferentially delivered to the sites of protein synthesis. Basic Life Sci, 1996, 64, 359 - 67 Time-of-flight Laue fiber diffraction studies of perdeuterated DNA; Forsyth VT et al.; The diffractometer SXD at the Rutherford Appleton Laboratory ISIS pulsed neutron source has been used to record high resolution time-of-flight Laue fiber diffraction data from DNA . These experiments, which are the first of their kind, were undertaken using fibers of DNA in the A conformation and prepared using deuterated DNA in order to minimise incoherent background scattering . These studies complement previous experiments on instrument D19 at the Institut Laue Langevin using monochromatic neutrons . Sample preparation involved drawing large numbers of these deuterated DNA fibers and mounting them in a parallel array . The strategy of data collection is discussed in terms of camera design, sample environment and data collection . The methods used to correct the recorded time-of-flight data and map it into the final reciprocal space fiber diffraction dataset are also discussed . Difference Fourier maps showing the distribution of water around A-DNA calculated on the basis of these data are compared with results obtained using data recorded from hydrogenated A-DNA on D19 . Since the methods used for sample preparation, data collection and data processing are fundamentally different for the monochromatic and Laue techniques, the results of these experiments also afford a valuable opportunity to independently test the data reduction and analysis techniques used in the two methods. Basic Life Sci, 1996, 64, 309 - 23 High-level expression and deuteration of sperm whale myoglobin . A study of its solvent structure by X-ray and neutron diffraction methods; Shu F et al.; Neutron diffraction has become one of the best ways to study light atoms, such as hydrogens . Hydrogen however has a negative coherent scattering factor, and a large incoherent scattering factor, while deuterium has virtually no incoherent scattering, but a large positive coherent scattering factor . Beside causing high background due to its incoherent scattering, the negative coherent scattering of hydrogen tends to cancel out the positive contribution from other atoms in a neutron density map . Therefore a fully deuterated sample will yield better diffraction data with stronger density in the hydrogen position . On this basis, a sperm whale myoglobin gene modified to include part of the A c11 protein gene has been cloned into the T7 expression system . Milligram amounts of fully deuterated holo-myoglobin have been obtained and used for crystallization . The synthetic sperm whale myoglobin crystallized in P2(1) space group isomorphous with the native protein crystal . A complete X-ray diffraction dataset at 1.5A has been collected . This X-ray dataset, and a neutron data set collected previously on a protonated carbon-monoxymyoglobin crystal have been used for solvent structure studies . Both X-ray and neutron data have shown that there are ordered hydration layers around the protein surface . Solvent shell analysis on the neutron data further has shown that the first hydration layer behaves differently around polar and apolar regions of the protein surface . Finally, the structure of per-deuterated myoglobin has been refined using all reflections to a R factor of 17%. Kurume Med J, 1996, 43(4), 313 - 24 Cloning and expression of cDNA for soluble form of rat heme oxygenase-1; Hidaka T et al.; Heme oxygenase catalyzes the oxidation of heme to biliverdin and carbon monoxide . The gene encoding the truncated soluble rat heme oxygenase-1 (Metl-Pro267) was cloned . The enzyme protein was expressed in E . coli JM109 and purified to homogeneity . The molecular weight of the recombinant enzyme was 30 kDa as assessed by SDS-polyacrylamide gel electrophoresis . From a 3-L culture, about 90 mg of the purified enzyme was routinely obtained . The dependency of the heme oxygenase reaction catalyzed by the soluble enzyme on the NADPH-cytochrome P-450 reductase concentrations and the effect of catalase on the reaction were examined to compare with the purified membrane-bound form of heme oxygenase-1 (Yoshida and Kikuchi, 1978b) . The activity of the soluble enzyme was inhibited at high concentrations of NADPH-cytochrome P-450 reductase and the inhibition was not alleviated by addition of catalase unlike the membrane-bound form . The ferric iron of the heme-heme oxygenase complex was in a typical high spin state at acidic to neutral pH (pH 6.5-7.0) but conversion to low spin state was observed at basic pH (pH 9-10) . The heme bound to heme oxygenase was converted to biliverdin at a stoichiometric ratio of unity in the presence of NADPH-cytochrome P-450 reductase system . During the heme degradation of the heme-heme oxygenase complex under atmospheric oxygen, several intermediates, that is, oxygenated heme and verdoheme, were spectrally discriminated. J Neural Transm, 1996, 103(12), 1415 - 28 Regulation of N-terminus-deleted human tyrosine hydroxylase type 1 by end products of catecholamine biosynthetic pathway; Ota A et al.; The N-terminal 52-, 70-, and 157-amino acids-deleted mutants and wild-type tyrosine hydroxylases were expressed in Escherichia coli and utilized to investigate the roles of the N-terminus in the catecholamine inhibition on enzyme activity . Their lysate's supernatants were used as enzyme samples . Three catecholamines, namely dopamine, norepinephrine, and epinephrine, affected both wild-type and mutant enzymes after preincubation in the mode of mixed inhibition, and the most marked alteration among the kinetic parameters produced by the deletion was the increase in the inhibition constants . The deletions also abolished the catecholamine-induced shift of the pH profile of the enzyme activity toward a more acidic pH optimum . All three mutants responded to catecholamines almost in the same way . These results suggest that the three catecholamine end products exert their inhibition on tyrosine hydroxylase to the same extent and that the N-terminal 52 amino acid residues contain the key sequence in mediating the inhibitory action. J Cell Biochem Suppl, 1996, 25, 99 - 107 Mutational specificity and cancer chemoprevention; Curry J et al.; Mutational specificity describes the composite of all of the genetic alterations in a collection of mutations arising from a specific treatment . The information includes not only the nature of the genetic change (e.g., a base substitution or a frameshift), but also information about nucleotide position and hence the DNA context . As both the type of DNA damage and its position can be expected to reflect the nature of the chemical and physical mutagen, mutational specificity can be expected to provide insights into mechanisms of mutation . Conversely, mutational spectra should also provide insights into the identity of the mutagen . Indeed, the pioneering work on mutational specificity in Escherichia coli indicates that each physical or chemical treatment produces a unique spectrum of mutations . With the application of biotechnology to the field of genotoxicology, the database of sequenced mutations has become quite substantial . Both in vitro and in vivo data has been obtained following exposure to a variety of agents . In this communication we will critically assess whether the reality of mutational specificity has fulfilled the expectations and to examine what potential remains to be explored, especially in the area of monitoring human populations . The usefulness of both mutational spectra analysis and population monitoring with regards to chemoprevention are discussed. Methods Enzymol, 1996, 275, 122 - 33 Expression and purification of retroviral HIV-1 reverse transcriptase; Stahlhut MW et al.; Modern molecular biology techniques have provided valuable tools which allow for the expression of large amounts of enzyme in E . coli . For potential therapeutic targets such as HIV-1 reverse transcriptase, it is desirable that the enzyme studied is pure and correlates to the active form of the enzyme found in vivo . This poses a particular challenge for those researchers studying HIV-RT since a significant degree of heterogeneity is introduced by nonspecific proteolytic cleavage of the p66 subunit by E . coli proteases . The advantage of the purification protocol presented here is that the association of monomers is facilitated by mixing an excess of p51 subunit, which is truncated at a site that is N-terminal to known bacterial cleavage sites, with p66 protein . This avoids enzymatic processing of the larger subunit since the formation of heterodimeric RT is rapid and the dimer is stable against proteolytic cleavage . Therefore, it is possible to isolate a pure homogeneous p66/p51 heterodimer . An enzyme prepared in this manner yields crystals that defract to a 3.2-A resolution . It has also been used to study both sensitivity of HIV-1 RT mutants to azidothymidine triphosphate and the kinetics of a potent nonnucleoside RT inhibitor (L-743,726) . Finally, it is interesting to note the similarity of HIV-1 RT with reverse transcriptases from other lentiviruses (FIV and EIAV RT) . Both of these enzymes consist of heterodimers of p66 and p51 subunits and share other biophysical characteristics . Purification of these reverse transcriptases can, in all likelihood, be optimized by using methods similar to those described in this chapter. Chirality, 1996, 8(8), 545 - 50 Synthesis and analysis of the enantiomers of calmidazolium, and a 1H NMR demonstration of a chiral interaction with calmodulin; Edwards AJ et al.; Calmidazolium {R24571, 1-{bis(4-chlorophenyl)methyl}-3-{2-(2,4-dichlorophenyl)-2-{(2,4- dichlorophenyl)methoxy}ethyl}-1H-imidazolium chloride} is a potent calmodulin inhibitor . This paper describes the synthesis and properties of the enantiomers of calmidazolium from the enantiomers of miconazole {1(N)-(2-(2,4-dichlorobenzyloxy)-2-(2,4 dichlorophenyl))-ethyl imidazole}, prepared from the racemate by chiral preparative scale high performance liquid chromatography . Overlap between ligand and protein resonances in the aromatic region of the 1H NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by preparation of the protein with all of its nine phenylalanine rings deuterated (Phe-d5 calmodulin) . This has been accomplished by the overexpression of calmodulin derived from Trypanosoma brucei rhodiesiense in E . coli in a medium supplemented with ring-deuterated phenylalanine . The kinetics of binding of each enantiomer are slow on the 1H NMR time scale as judged by the behaviour of the H2 resonance of Histidine-107, which is clearly visible under the sample conditions used . The aromatic spectral regions of the protein-bound (+) and (-) enantiomers contrast strikingly, reflecting differences in bound environment and/or conformation. Microbiol Immunol, 1996, 40(12), 949 - 53 The UL2 open reading frame of bovine herpesvirus 1 encodes a uracil-DNA glycosylase; Chung YT et al.; Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence . To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence down-stream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli . Upon induction with isopropyl beta-D-thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence . A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay . These results provide evidence that the BHV-1 UL2 gene does encode a UDGase. Acta Oncol, 1996, 35(7), 889 - 94 Lethal effect of K-shell absorption of intracellular phosphorus on wild-type and radiation sensitive mutants of Escherichia coli; Maezawa H et al.; The present study was conducted to clarify the lethality of Auger cascades induced by the K-shell photoabsorption of phosphorus in Escherichia coli . Killing of wild-type and radiation-sensitive mutants of E . coli was examined . Three x-ray energies were chosen for irradiation; at 2.153 keV: the resonance peak of K-shell photoabsorption of phosphorus; at 2.146 and 2.160 keV: off-peak . Enhancement ratio, which was defined as the ratio of the killing sensitivity of 2.153 keV to that at 2.146 keV, were 1.32 to 1.54 for examined strains . Increment of absorbed energy calculated in entire cells for 2.153 keV radiation could not explain the degree of observed enhancement of killing . Lethality of Auger cascades depended on the killing sensitivity with x-rays which did not induce Auger cascades . The lethality for wild-type was lower than that for recombination repair-deficient mutants . It was concluded that one part of damages produced by Auger cascades was repaired in wild-type strains. Microbiol Immunol, 1996, 40(5), 345 - 52 Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction; Yamasaki S et al.; To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed . Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced . The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants . The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection. Mol Biochem Parasitol, 1996 Jan, 75(2), 231 - 40 Molecular cloning of a developmentally regulated protein isolated from excretory-secretory products of larval Dirofilaria immitis; Frank GR et al.; Three proteins isolated from the excretory-secretory products (ES) of larval Dirofilaria immitis have been previously characterized and termed the 20, 22L and 22U kDa proteins . Two of the proteins (20 and 22L) were produced and released around the time of the third molt and were specifically recognized by immune dog sera . An amino acid sequence common to both proteins was used to synthesize a DNA probe to molecularly clone these molecules from a 48-h third stage larval cDNA library . The DNA sequence of the isolated clones encoded a 17.5 kDa protein with a 21 amino acid hydrophobic leader sequence that when removed yielded a 15.3 kDa protein starting with the N-terminal sequence obtained from the 20 kDa protein and containing all sequences obtained from tryptic peptides of the 20 and 22L kDa proteins . It was hypothesized that the 20 and 22L kDa proteins were the same, differing only by a 21 amino acid hydrophobic leader sequence which was later cleaved . The calculated molecular masses were consistent with those determined by reducing Tris-tricine SDS-PAGE . Expression of the protein without the leader sequence was accomplished in Escherichia coli . Antiserum raised against the expressed protein demonstrated the presence of the protein in L3 and L4, but not in adults or microfilariae . Expression of the protein with the leader sequence using a baculovirus system demonstrated processing of the signal sequence at the same time as found in larval D . immitis ES . Sera from dogs immune to infection were reactive with the D . immitis proteins expressed in either E . coli or insect cells. Fiziol Zh, 1996, 42(1-2), 25 - 30 {The effect of myelopid on the ultrastructural changes in the rat myocardium induced by an endotoxin}; Kharlanova NG et al.; Ultrastructural changes in the myocardial atria and ventricles have been studied in experiments on rats in the intermediate period of the endotoxin shock, on the stage of late endotoxemia and its correction with myelopid . It was established that 5 hours after endotoxin injection contractile disturbances and intracellular myocytolysis take place and growth of secretory activity is observed in atrial cardiomyocytes . Simultaneously vascular permeability increases . Only the endocrine heart function is restored 3 days later, while dystrophic changes and glycogen depletion are maintained . The myelopid administration beginning from the intermediate period of the endotoxin shock and then once during 3 days, removes most of ultrastructural damages or attenuates their expression. Environ Mol Mutagen, 1996, 28(4), 465 - 70 A new transgenic mouse mutagenesis test system using Spi- and 6-thioguanine selections; Nohmi T et al.; A new transgenic mouse mutagenesis test system has been developed for the efficient detection of point mutations and deletion mutations in vivo . The mice carry lambda EG10 DNA as a transgene . When the rescued phages are infected into Escherichia coli YG6020-expressing Cre recombinase, the phage DNA is converted into plasmid pYG142 carrying the chloramphenicol-resistance gene and the gpt gene of E . coli . The gpt mutants can be positively detected as colonies arising on plates containing chloramphenicol and 6-thioguanine . The EG10 DNA carries a chi site along with the red and gam genes so that the wild-type phages display Spi- (sensitive to P2 interference) phenotype . Mutant phages lacking both red and gam genes can be positively detected as plaques that grow in P2 lysogens of E . coli . These mutant phages are called lambda Spi- . The spontaneous gpt mutation frequencies of five independent transgenic lines were 1.7 to 3.3 x 10(-5) in bone marrow . When the mice were treated with ethylnitrosourea (single i.p . treatments with 150 mg/kg body weight; killed 7 days after the treatments), mutation frequencies were increased four- to sevenfold over the background in bone marrow . The average rescue efficiencies were more than 200,000 chloramphenicol-resistant colonies per 7.5 micrograms bone marrow DNA per packaging reaction . In contrast to gpt mutation frequencies, spontaneous Spi- mutation frequencies were 1.4 x 10(-6) and 1.1 x 10(-6) in bone marrow and sperm, respectively . No spontaneous Spi- mutants have been detected so far in spleen, although 930,000 phages rescued from untreated mice were screened . In gamma-ray-treated animals, however, induction of Spi- mutations was clearly observed in spleen, at frequencies of 1.4 x 10(-5) (5 Gy), 1.2 x 10(-5) (10 Gy), and 2.0 x 10(-5) (5O Gy) . These results suggest that the new transgenic mouse "gpt delta" could be useful for the efficient detection of point mutations and deletion mutations in vivo. Environ Mol Mutagen, 1996, 28(4), 447 - 58 Parameters affecting the use of the lac repressor system in eukaryotic cells and transgenic animals; Wyborski DL et al.; Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals . A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences . In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction . Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene . In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent . After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary . In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes . Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures . The applicability and challenges of the system for regulation of gene expression are discussed. Environ Mol Mutagen, 1996, 28(4), 430 - 3 Mutational spectra of tamoxifen-induced mutations in the livers of lacI transgenic rats; Davies R et al.; Tamoxifen, an important drug in breast cancer treatment, causes liver cancer in rats . The standard range of in vitro tests have failed to show that it causes DNA damage, but 32P-postlabelling and DNA-binding studies have shown that tamoxifen forms DNA adducts in rat liver . In 1995 a transgenic rat (Big Blue; Stratagene, La Jolla, CA) became available which harbours the bacterial lacI gene, thereby allowing the in vivo study of tamoxifen mutagenesis . Recently, we {Styles JA et al . (1996): Toxicologist 30; 161} showed that tamoxifen caused on increase in the mutation frequency at the lacI gene in these transgenic rats . In this study, we report on our preliminary analysis of the mutational spectra of 33 control and 38 tamoxifen-induced mutant lacI genes . Plasmid DNA containing the lacI gene was isolated from the mutant phages and its DNA sequence determined . In the control animal group, 81% of the mutant lacI genes were point mutations, whilst in the tamoxifen-treated group, 62% of the mutant lacI genes were point mutations . Of the tamoxifen-induced mutants, 43% were GC-->TA transversions and 70% of point mutations . In the control group, GC-->TA transversions were 19% of all mutations and 24% of point mutations . Thus, compared with control animals, tamoxifen treatment had significantly increased the proportion of GC-->TA transversions. Environ Mol Mutagen, 1996, 28(4), 424 - 9 Assessment of 1,3-butadiene mutagenicity in the bone marrow of B6C3F1 lacI transgenic mice (Big Blue): a review of mutational spectrum and lacI mutant frequency after a 5-day 625 ppm 1,3-butadiene exposure; Recio L et al.; 1,3-Butadiene (BD) is a carcinogen that is bioactivated to at least two genotoxic metabolites . In the present article, we review briefly our previous studies on the in vivo mutagenicity and mutational spectra of BD in bone marrow and extend these studies to examine the effect of exposure time (5-days vs . 4-week exposure to 625 ppm BD used in previous studies) on the lacI mutant frequency in the bone marrow . Inhalation exposure to BD at 625 ppm and 1,250 ppm mutagenic in vivo, inducing an increase in the transgene mutant and mutation frequency in the bone marrow . Analysis of the mutational spectrum in BD-exposed and air control mice demonstrated that BD exposure induced an increased frequency of mutations at A:T base pairs . There was no difference in the lacI mutant frequency determined in the bone marrow between a short-term exposure to BD (5 days) and a longer-term exposure (4 weeks) . These data taken together demonstrate that inhalation exposure to BD induces in vivo somatic cell mutation. Environ Mol Mutagen, 1996, 28(4), 418 - 23 Spectrum of mutations in kidney, stomach, and liver from lacI transgenic mice recovered after treatment with tris(2,3-dibromopropyl)phosphate; de Boer JG et al.; The flame retardant tris(2,3-dibromopropyl)phosphate (TDBP), once used in cotton sleep wear for children, is presently banned from commerce . It produces tumors in rodents in both a sex- and tissue-specific manner . The kidney is the main target for tumor formation in male and female rats, as well as in male mice . In contrast, tumors are formed in the liver of female animals . We have used lacI transgenic male B6C3F1 mice (Big Blue) to examine the induction of mutation in kidney, liver, and stomach after exposure to 150 mg/kg (2 days), 300 mg/kg (4 days), and 600 mg/kg (4 days) of TDBP . At the highest dose, the mutant frequency was approximately 50% above control values in the kidney (P < 0.01) . A smaller increase was observed in the liver (P = 0.07), while no increase was seen in the stomach (P = 0.28) . Sequence analysis of the recovered mutants showed a TDBP-specific change in mutation spectrum in kidney, which was not observed in liver and stomach . In kidney, a dose-dependent decrease in G:C-->A:T transitions, including at 5'-CpG-3' sites, was observed . This was accompanied by an increase in the loss of single G:C base pairs from approximately 3% to 15% . These results illustrate both the sensitivity and specificity of the lacI transgenic system in the analysis of tissue-specific mutation . This study also reinforces the importance of examining mutational spectra when mutant induction levels are low. Environ Mol Mutagen, 1996, 28(4), 393 - 6 An efficient laboratory protocol for the sequencing of large numbers of lacI mutants recovered from Big Blue transgenic animals; Erfle HL et al.; Mutational spectra provide a powerful approach to investigate both the mutagenic potential and the mechanism of action of suspected mutagens and carcinogens . Recently, transgenic techniques have made it possible to generate mutational spectra in animals . Such a spectrum may consist of 50 to 200 mutants depending on the nature of the mutations, and many spectra can be generated depending on the design of the experiment . This report describes a practical approach for the processing and sequencing of large numbers of lacI mutants recovered from Big Blue animals. Environ Mol Mutagen, 1996, 28(4), 385 - 92 The genetic analysis of lacI mutations in sectored plaques from Big Blue transgenic mice; Stuart GR et al.; The Big Blue lacI transgenic rodent assay, which uses the lambda LIZ/lacI gene as the target for mutation, provides a convenient short-term assay for the study of mutation in vivo {Kohler et al . (1991): Proc Natl Acad Sci USA 88:7958-7962; Provost et al . (1993): Mutat Res 288:133-149) . However, the interpretation of data from transgenic animal assays is sometimes complicated by mutants that appear as sectored mutant lambda plaques . These mutants can form a significant fraction of the mutant plaques {Hayward et al . (1995): Carcinogenesis 16:2429-2433} . Thus, in order to accurately determine in vivo mutant frequencies and mutational specificities, it is necessary to score sectored plaques and partition them from the rest of the data . In this study, the specificity of mutation in sectored plaques recovered from untreated and UVB-treated Big Blue mouse skin was analyzed and compared to mutations recovered from lambda LIZ/lacI grown on the Escherichia coli host . The mutational spectra of sectored plaques from untreated and UVB-treated mice were remarkably similar to each other and resembled those recovered from the lambda LIZ/lacI phage plated directly on E . coli . Both the sectored mutants and those recovered in lambda LIZ/lacI phage differed from the spectra of spontaneous mutants in E . coli and in Big Blue mouse skin . While sectored mutants from UVB-treated mouse skin and lambda LIZ/lacI mutants were also different from spontaneous mutants recovered from Big Blue liver, these was little difference between sectored mutants from untreated mouse skin and spontaneous liver mutants (P = 0.07) . The mutational spectra of sectored plaques is thus largely consistent with their origin as spontaneous mutations arising in vitro during growth of the lambda LIZ/lacI shuttle vector DNA on the E . coli host, although the potential contribution from lesions in mouse DNA being expressed ex vivo in the E . coli host cannot be excluded. Environ Mol Mutagen, 1996, 28(4), 376 - 84 Mutagenic response of the endogenous hprt gene and lacI transgene in benzo{a}pyrene-treated Big Blue B6C3F1 mice; Skopek TR et al.; Big Blue (BB) and generic B6C3F1 mice were given one to three i.p . injections of 50 mg/kg benzo{a}pyrene (B{a}P) in DMSO every other day to achieve cumulative doses of 50 to 150 mg/kg . Three weeks after treatment, the mutation frequency at the endogenous hprt gene and lacI transgene was measured in splenic T cells . Generic mice given 50, 100, and 150 mg/kg B{a}P displayed induced hprt frequencies (observed hprt frequency minus control frequency) of 5.5 +/- 1.0, 11 +/- 2.0, and 19 +/- 2.6 x 10(-6), respectively (average +/- SEM) . In contrast, BB mice given 50 and 150 mg/kg B{a}P displayed induced hprt frequencies of 0.9 +/- 0.6 and 9.1 +/- 1.5 x 10(-6) . 32P postlabelling revealed that the lower hprt response in BB mice correlated with lower amounts of BP-DNA adducts in spleen, liver, and lung 24 hours after B{a}P exposure . Western blot analysis of liver samples from B{a}P-treated mice suggests that the reduced adduct load in turn may be due to lower P450 1A1 levels in BB mice . The frequency of induced, nonsectored blue plaques (observed blue plaque frequency minus control frequency) in BB mice receiving 50 and 150 mg/kg B{a}P was 41 +/- 9 and 134 +/- 10 x 10(-6) (15- to 40-fold higher than the induced hprt frequency in the same treated animals) . Sectored plaques were observed in both control and B{a}P groups but their frequency showed no relationship to dose (sectored frequency in all groups was approximately 20 x 10(-6)) . To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified lambda-LIZ DNA was treated in vitro with B{a}P diol epoxide (BPDE), packaged, and plated on E . coli lawn cells . Treatment with BPDE did not produce significant increases in homogeneous blue plaques, suggesting that the majority of mutants obtained from B{a}P-treated BB mice occurred in vivo . These results indicate that B{a}P exposure produces many more mutations at the lacI transgene than at the endogenous hprt locus. Environ Mol Mutagen, 1996, 28(4), 354 - 62 Evaluation of the in vivo genotoxic potential of three carcinogenic aromatic amines using the Big Blue transgenic mouse mutation assay; Suter W et al.; Three genotoxic mouse carcinogens, 4-chloro-o-phenylenediamine (4-C-o-PDA), 2-nitro-p-phenylenediamine (2-N-p-PDA), and 2,4-diaminotoluene (2,4-DAT), were tested in the Big Blue transgenic mouse mutation assay . Each experiment consisted of a vehicle control group with ten Big Blue C57BL/6 mice, five of either sex, and an equally sized group treated with a high dose of the test chemical . In addition, four animals were treated with the vehicle and six animals with the test compound for the measurement of bromodeoxyuridine (BrdU) incorporation to determine cellular proliferation . Prior to the mutagenicity experiments, the maximally tolerated dose of each compound was determined using nontransgenic C57BL/6 mice . Based on these results the doses used in the main study were 200 mg/kg/day for 4-C-o-PDA, 150 mg/kg/ day for 2-N-p-PDA, and 80 mg/kg/day for 2,4-DAT . Animals were treated for 10 days over a 2 week period and were killed 10 days after the ast treatment . In an additional experiment with 2,4-DAT, animals were killed 28 days after treatment . Since all three chemicals are liver carcinogens in the mouse, the DNA of the liver was analyzed using the standard procedures for the Big Blue assay . Hepatocyte proliferation was assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and, in some studies, by measuring BrdU incorporation . 4-C-o-PDA and 2-N-p-PDA did not induce an increase in PCNA expression when measured 10 days after the last treatment . There was no increase in BrdU incorporation immediately after treatment with 4-C-o-PDA or with 2,4-DAT . However, 10 days after the last treatment with 2,4-DAT, a strong mitogenic effect was found with both techniques, i.e., in the PCNA and BrdU assays . 4-C-o-PDA, a liver carcinogen in both genders of mice, induced a small, statistically significant increase of the mutant frequencies in females . No increase was found in males . 2-N-p-PDA, which has been reported to induce liver tumors only in females, was found positive in males and was clearly negative in females . 2,4-DAT, a liver carcinogen in female mice, was positive in females and negative in males when the animals were killed 10 days after the last treatment . After an expression time of 28 days, 2,4-DAT induced a statistically significant increase in both sexes . The effect in females was marginally stronger than after 10 days' expression time and almost identical to the effect observed in males under these test conditions . In conclusion, the experiments showed that the Big Blue assay detects the genotoxicity of the three carcinogenic monocyclic aromatic amines tested . However, it seems that the sex specificity of the carcinogenic effects of these compounds is not reflected by the mutagenicity data in Big Blue mice. Environ Mol Mutagen, 1996, 28(4), 342 - 7 Mutagenic response to benzene and tris(2,3-dibromopropyl)-phosphate in the lambda lacI transgenic mouse mutation assay: a standardized approach to in vivo mutation analysis; Provost GS et al.; The genotoxic response of benzene and tris(2,3-dibromopropyl)-phosphate (TDBP) have been evaluated in several tissues using the standardized lambda/lacI (Big Blue) transgenic mouse mutation assay . Separate groups of four to five male B6C3F1 transgenic lambda/lacI mice were given oral administrations of benzene or TDBP at varying concentrations . Tissues evaluated include lung, bone marrow, and spleen in benzene-treated animals, and liver, kidney, and stomach in TDBP-treated animals . Significant increases in lacI mutations were observed in the spleen and bone marrow of benzene treated mice, and the kidneys of TDBP-treated mice . Where applicable, mutagenesis patterns of tissue sensitivity were consistent with what has been observed previously in other assays . In addition, mutagenicity in tissues not traditionally evaluated for mutations correlated to sites of carcinogenicity for the chemicals tested. Environ Mol Mutagen, 1996, 28(4), 325 - 33 Spontaneous and ethylnitrosourea-induced mutation fixation and molecular spectra at the lacI transgene in the Big Blue rat-2 embryo cell line; Zimmer DM et al.; Big Blue Rat-2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea {ENU}-induced) . Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined . Exposures were 100, 200, 400, 600, and 1,000 micrograms ENU/ml . The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 X 10(5) . Dose-related increases in mutant frequency were observed following ENU treatment . Kinetics (time course) of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 micrograms ENU/ml . Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C-->A:T transitions (five at CpG sites), six (23%) G:C-->T:A transversions, three (11.5%) G:C-->C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion+insertion) mutation . Among 46 ENU-induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C-->A:T transitions (four at CpG sites), five (13.5%) A:T-->G:C transitions, four (10.8%) G:C-->T:A transversions, 11 (30%) A:T-->T:A transversions, and two (5.4%) A:T-->C:G transversions . Nearly 50% of the base substitutions in the ENU-treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found . Both the spontaneous and the ENU-induced mutational spectra were similar to that reported in vivo and for other cells . An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 micrograms/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity. Chin J Biotechnol, 1996, 12(2), 131 - 6 Cloning and function determination of promoter region of glucoamylase gene from Aspergillus niger T21; Tang G et al.; A 850 bp fragment of the 5' flanking region of the glucoamylase gene (glaA) was synthesized from A . niger T21 genome using PCR . The function detection vector was constructed by fusing this fragment to E . coli hygromycin B phosphotransferase gene (hph) and was used to transform A . niger . The high hygromycin resistance transformants thus obtained verified that the synthesized fragment functioned as a promoter in filamentous fungi . Southern blot analysis showed the hph gene has been integrated into genomic DNA of A . niger transformant. Chin J Biotechnol, 1996, 12(2), 99 - 109 Construction of a stable expression vector carrying sop genes {ZJ1}; Du X et al.; Mini-F, the fifth fragment of F plasmid from EcoRI digestion, is known to carry an efficient partitioning function . Two pBR322 plasmid derivatives, pDMC32 and pDMC311, have been constructed from this fragment . The plasmid pDMC32 carries all the relative genes for plasmid stability, ccd, repD, and sop genes (sopA, sopB, and sopC), along with oriS and oriV, while pDMC311 carries only sop genes (sopA, sopB, and sopC) . The plasmid maintenance proportions for pDMC32 and pDMC311 in E . coli were 93% and 100%, respectively, after 100 generations continuous cultivation of cells harboring the derivatives, MI32 (pDMC32) and MI311 (pDMC311), in a phosphate-limited basal medium . As a control, the maintenance proportion of plasmid pBR322 dropped down to a low of 10% at generation 55 of continuous cultivation of E . coli MIR322 (pBR322) in the same medium . In order to make a stable expression vector that carries only sop genes, plasmid pDMC40 was constructed by adding a trp promoter from pDR720 to pBR322 . The stable expression vector pDMC48 was then derived from pDMC40 by inserting sop genes into it from pDMC311 . The maintenance proportion of plasmid pDMC48 in E . coli was still 100% after 100 generations of continuous cultivation of cells harboring the plasmid in phosphate-limited basal medium. Planta, 1996, 200(1), 64 - 70 Enzymatic properties of chorismate synthase isozymes of tomato (Lycopersicon esculentum Mill.); Braun M et al.; Three plastidic chorismate synthase isozymes (CS1, CS2 and CS2 delta) of tomato were identified by isolation of the corresponding cDNAs . These three cDNAs are derived from only two genes (LeCS1 and LeCS2) . This additional complexity results from differential splicing of the primary transcript of one of the genes (LeCS2) giving rise to two different transcripts (CS2 and CS2 delta transcripts) . All three isozymes were individually expressed in Escherichia coli both as precursor proteins with N-terminal transit peptides and as mature proteins . Only the mature but not the precursor isozymes CS1 and CS2 were enzymatically active . The enzyme CS2 delta was unstable in E . coli . Both CS1 and CS2 were purified to near homogeneity and their enzymatic properties were analyzed . They differ substantially in their Km values for the substrate 5-enol-pyruvylshikimate 3-phosphate (11 and 80 microM for the mature forms of CS1 and CS2, respectively) . The two isozymes appear to be active only as oligomers, and the potential physiological implications of this result are discussed. Planta, 1996, 200(1), 13 - 9 Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis; Osakabe Y et al.; The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNA1 (Osakabe et al., 1995, Plant Sci . 105: 217-226), isolated from Populus kitakamiensis (P . sieboldii x P . grandidentata), was expressed in Escherichia coli cells . The polypeptide was purified and an antiserum raised against it . The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein and the partially purified PAL protein from P . kitakamiensis . Moreover, the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P . kitakamiensis proteins and this protein had PAL activity . Furthermore, the antibody inhibited PAL activity of extracts from stem tissues . These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P . kitakamiensis . Immunolocalization studies of P . kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves. J Reprod Fertil Suppl, 1996, 50, 63 - 7 Binding of recombinant pig zona pellucida protein 1 (ZP1) to acrosome-reacted spermatozoa; Tsubamoto H et al.; In an immunofluorescent study, recombinant pig zona pellucida protein 1 (pZP1) produced in Escherichia coli bound to acrosome-reacted spermatozoa from five different mammals . The location of the binding site moved from the equatorial region to the midpiece and tail regions during the incubation . This finding suggests that pig ZP1 acts as a secondary sperm-binding receptor in an interspecies manner and assists penetration of the spermatozoa through the zona pellucida . The recombinant pZP1 bound to 55 kDa protein (proacrosin) and 40 kDa protein of the boar spermatozoa as revealed by immunoblotting. J Reprod Fertil Suppl, 1996, 50, 127 - 34 Strategies for designing an immunocontraceptive vaccine based on zona pellucida synthetic peptides and recombinant antigen; Kaul R et al.; Immunization of female bonnet monkeys (Macaca radiata) with pig zona pellucida glycoprotein ZP3 using adjuvants permissible for human use leads to infertility . Fifty per cent of the animals conceived after antibody titres declined . Animals that failed to regain their fertility did not show any obvious ovarian changes . As part of the attempt to design an effective immunocontraceptive vaccine based on synthetic peptides, the epitopes recognized by monoclonal antibodies (mAb) against pig ZP3 alpha and ZP3 beta and possessing contraceptive efficacy in vitro were mapped . These studies identified an N-blocked decapeptide from the N-terminus corresponding to 23-32 amino acids of the precursor protein of pig ZP3 beta . As an alternative approach to using synthetic peptides, DNA encoding bonnet monkey ZP3 was cloned and sequenced . The deduced primary amino acid sequence revealed an overall similarity of 93.9% with human ZP3 . Bonnet monkey ZP3 corresponding to an internal 975 nucleotide fragment excluding the N-terminus signal sequence and the C-terminus transmembrane domain has been expressed in Escherichia coli . These results will assist in the design of an immunocontraceptive vaccine based on either synthetic peptides or recombinant glycoproteins or proteins corresponding to ZPPIP: Previous studies have demonstrated the efficacy, in mice, of synthetic peptides derived from zona pellucida (ZP) glycoprotein in blocking fertility without ovarian dysfunction . This study used bonnet monkeys (closely related to humans in the primate evolutionary tree and less susceptible to summer amenorrhea than rhesus monkeys) to explore the design of an immunocontraceptive vaccine based on synthetic peptides, recombinant glycoproteins, or proteins corresponding to ZP . Immunization of female monkeys with pig ZP3 glycoprotein using adjuvants permissible for human use produced infertility . Although only half the animals conceived after antibody titres declined, monkeys that failed to conceive did not show any obvious ovarian changes . Mapping of the epitopes recognized by monoclonal antibodies against ZP3 alpha and beta and possessing contraceptive efficacy in vitro identified an N-blocked decapeptide from the N-terminus corresponding to 23-32 amino acids of the precursor protein of pig ZP3 beta . When DNA encoding bonnet monkey ZP3 was cloned and sequenced, the deduced primary amino acid sequence showed a 93.9% similarity with human ZP3 . Bonnet monkey ZP3 corresponding to an internal 975 nucleotide fragment excluding the N-terminus signal sequence and the C-terminus transmembrane domain has been expressed in Escherichia coli . Planta, 1996, 200(3), 326 - 36 Comparison of the expression of a plastidic chaperonin 60 in different plant tissues and under photosynthetic and non-photosynthetic conditions; Schmitz G et al.; A partial cDNA which codes for the beta-subunit of a plastidic chaperonin 60 (cpn60-beta) from rye (Secale cereale L.) leaves was identified and sequenced, except for 46 amino acids of the N-terminus of the mature protein and the transit sequence . This is the first cpn60-beta sequence determined for a monocotyledonous plant . Specific antibodies against cpn60-beta were affinity-purified from an antiserum raised against the total soluble protein fraction of ribosome-deficient plastids . The localization of cpn60-beta in chloroplasts or non-green plastids was confirmed by immunodetection in Percoll gradient-purified organelles . The expression and occurrence of cpn60-beta was analysed by immunoblotting with the specific antibodies and Northern hybridization . The cpn60-beta protein was constitutively expressed in various green and non-green tissues . It was evenly distributed along the major part of a rye leaf, while highest transcript levels occurred in the youngest and oldest leaf sections . The expression of the cpn60-beta protein was not enhanced by a heat-shock treatment at 42 degrees C . The cpn60-beta transcript and protein were more strongly expressed in various non-green, for instance etiolated, 70S-ribosome-deficient 32 degree C-grown, or herbicide-bleached tissues, than in green leaves of rye . A rapid increase in the cpn60-beta transcript level was also observed when green leaves were transferred from light to darkness while the protein level was not affected . The dark-induced increase in the cpn60-beta transcript was totally suppressed in the presence of 2% sucrose . Inhibitor treatments suggested that the change in cpn60-beta transcript level was not related to changes of the ATP supply of the tissue . While the large subunit of the photosynthetic protein ribulose-1,5-bisphosphate carboxylase was largely degraded during ripening of tomato fruits, high levels of cpn60-beta were detected in tomato chromoplasts and in the yellow flower petals of Narcissus . Low levels of cpn60-beta were detected in root tissue. Mol Biol Rep, 1996, 23(2), 109 - 17 Positive and negative modulation of H-ras transforming potential by mutations of phenylalanine-28; Ricketts MH et al.; Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ({V12}p21), and built into SV40-driven expression vectors . The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells . Non-conservative mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of {V12}p21 . The conservative mutation of phenylalanine-28 to tryptophan ({V12W28}p21) was also still transforming . Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ({W28}p21) was weakly transforming while, in contrast, {V12D28}p21 was unable to transform Rat-1 cells and retarded cell growth . Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that {V12D28}p21 and {D28}p21 do not bind GTP . The dissociation rate of both GTP and GDP bound to {W28}p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells . These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21H-ras . The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins. Scand J Clin Lab Invest Suppl, 1996, 226, 47 - 56 Molecular basis for amyloidosis related to hereditary brain hemorrhage; Abrahamson M; The aim of the project has been to elucidate molecular events leading to amyloidosis in Hereditary Cystatin C Amyloid Angiopathy (HCCAA) patients, to enable simple diagnosis of the disease and with the ultimate goal to understand the amyloid formation process in detail, in order to develop inhibitors to the process . At the DNA level, a point mutation segregating with HCCAA was identified in the cystatin C gene on chromosome 20, after basic characterization of cDNA and gene for the wildtype protein . The mutation results in the amino acid substitution Leu-68-Gin (L68Q) and abolishes a recognition site for Alu I . This information was used to design a PCR based assay for simple and rapid mutation detection in DNA from blood samples to allow routine diagnosis of HCCAA . Studies at the protein level, allowed through E . coli expression of wildtype and L68Q mutated cystatin C genes, revealed that both protein variants effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation: 0.4 and 0.3 nM, respectively), but differ considerably in their tendency to dimerize and form aggregates . The initial dimerization of L68Q-cystatin C results in complete loss of biological activity and is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate . This result might be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates . The three-dimensional structure of normal cystatin C, crystallized in a complex with cathepsin B, was elucidated by X-ray analysis and subsequent refinement of the structure to 3.0 A resolution . Besides pinpointing the cystatin C structures resulting in efficient target enzyme inhibition, the results demonstrated that the Leu-68 residue is buried in the hydrophobic core of the protein . Studies of the three-dimensional solution structure of wildtype cystatin C by NMR spectroscopy revealed that cystatin C dimers can be formed as a result of slight, localized structural changes under conditions preceding complete defolding and denaturation of the protein . Dimers of L68Q-cystatin C are likely similar but are formed at temperatures nearly 30 degrees C lower than needed for the wildtype protein, indicating that the Leu-68-Gln substitution lowers the transition temperature for unfolding . Thus, the results presented suggest that cystatin C provides a system where decreased stability of a mutant protein correlates with its amyloidogenic nature . The NMR results furthermore imply that the hydrophobic proteinase-binding region of cystatin C is directly involved in dimer formation and that compounds designed to interact with this region could serve as inhibitors to the dimerization, and likely also the subsequent amyloid formation process, of cystatin C in HCCAA patients. Gene Expr, 1996, 6(2), 101 - 12 A base substitution in the amino acid acceptor stem of tRNA(Lys) causes both misacylation and altered decoding; Pagel FT et al.; In 1984, our laboratory reported the characterization of the first misacylated tRNA missense suppressor, a mutant Escherichia coli lysine tRNA with a C70 to U base change in the amino acid acceptor stem . We suggested then that the suppressor tRNA, though still acylated to a large extent with lysine, is partially misacylated with alanine . The results reported in this article demonstrate that is the case both in vitro and in vivo . For the in vitro studies, the mutant tRNA species was isolated from the appropriate RPC-5 column fractions and shown to be acylatable with both lysine and alanine . For the in vivo demonstration, use was made of a temperature-sensitive alaS mutation, which results in decreasing acylation with Ala as the temperature is increased, resulting ultimately in lethality at 42 degrees C . The alaSts mutation was also used to demonstrate that the ability of the same missense suppressor, lysT(U70), to suppress a trpA frameshift mutation is not affected by the Ala-acylation deficiency . We conclude that the misacylation and altered decoding are two independent effects of the C70 to U mutation in tRNA(Lys) . The influence of an alteration in the acceptor stem, which is in contact with the large (50S) ribosomal subunit, on decoding, which involves contact between the anticodon region of tRNA and the small (30S) ribosomal subunit, may occur intramolecularly, through the tRNA molecule . Alternatively, the U70 effect may be accomplished intermolecularly; for example, it may alter the interaction of tRNA with ribosomal RNA in the 50S subunit, which may then influence further interactions between the two subunits and between the 30S subunit and the anticodon region of the tRNA . Preliminary evidence suggesting some form of the latter explanation is presented . The influence of a single nucleotide on both tRNA identity and decoding may be related to the coevolution of tRNAs, aminoacyl-tRNA synthetases, and ribosomes. Cancer Surv, 1996, 28, 155 - 67 Mutation in resting cells: the role of endogenous DNA damage; Bridges BA; In E coli, new spontaneous mutations can arise in bacteria that are non-dividing and in which there is little or no DNA synthesis . These mutations are almost invariably those that enable the cell to resume growth, a phenomenon that has been termed directed or adaptive mutation . Evidence is accumulating from studies with DNA repair deficient strains that damage produced by endogenous mutagens may be an important source of such mutations . A DNA lesion that can miscode can explain the apparent adaptive behaviour since if a "mutant" RNA transcript confers sufficient advantage that the cell is triggered into a cycling state, the ensuing round of DNA replication will be likely to fix the mutation by means of a DNA miscoding event . The most important lesion in this respect appears to be 8-oxoG, which can pair equally well with adenine or cytosine and so give rise to G to T transversions . It is responsible for almost half the G to T transversions arising in non-growing repair proficient bacteria . Alkylations contribute to the production of both transitions and transversions but only those at A:T base pairs are important in repair proficient bacteria . There is also a report of a lesion susceptible to UvrA,B,C dependent excision repair, but whether it is important in bacteria possessing excision repair has not been addressed . Data on mammalian cells are almost non-existent, but there is evidence that point mutations can occur in vivo in postmitotic neurons . The underlying assumption that there is little or no DNA synthesis in non-dividing bacteria has been challenged by recent data suggesting that there may be extensive cryptic DNA turnover. Cancer Surv, 1996, 28, 117 - 40 Damage inducible mutagenesis: recent insights into the activities of the Umu family of mutagenesis proteins; Woodgate R et al.; Despite our advances, much remains to be elucidated about the molecular mechanisms of damage inducible mutagenesis . Although some aspects of the intricate protein interactions that lead to the formation of the mutasome have been determined, the precise nature of the interactions between pol III and the UmuD'C-RecA proteins remains entirely speculative . Further progress will depend on the development of a completely reconstituted, cell free lesion bypass system in vitro, as well as structural modelling of the mutasome in its entirety before we understand how error prone translesion DNA synthesis is achieved . Even if a structural homologue of the Escherichia coli mutasome does not exist in higher organisms, it seems quite likely that a functionally homologous mutagenic pathway will indeed be found . Such an active mutagenic process in animal cells would certainly have significant implications for our understanding of the mechanisms of genetic instability, as well as of human carcinogenesis and other pathologies associated with the formation of mutations in DNA. Cancer Surv, 1996, 28, 47 - 68 Mismatch repair and cancer; Jiricny J; A common form of human malignancy, hereditary non-polyposis colorectal cancer (HNPCC), as well as some sporadic human cancers, has been shown to exhibit frequent alterations in microsatellite sequences . This phenotype was ascribed to a defect in replication error correction, and indeed several tumour derived cell lines are deficient in mismatch repair . To date, four HNPCC loci, on chromosomes 2p, 2q, 3p and 7q, have been linked with genes designated hMSH2, hPMS1, hMLH1 and hPMS2, respectively, which encode proteins that display an extensive degree of sequence similarity to polypeptides involved in postreplicative mismatch correction in Escherichia coli and Saccharomyces cerevisiae . We have recently identified a new protein, GTBP, that is essential for mismatch repair in human cells . GTBP mutations are not associated with the profound MI commonly encountered in hereditary colon cancers . The roles of the proteins encoded by the individual mismatch repair genes in postreplicative mismatch correction and genome instability are discussed, with a view to assessing the potential utility of these findings in diagnosis of cancer predisposition and therapy. Cancer Surv, 1996, 28, 33 - 46 Patterns of mutation in cancer cells; Meuth M; The discovery of powerful mutator phenotypes in a subset of colon cancers provides direct support for the hypothesis that destabilization of replication fidelity and repair drive the accumulation of mutations in tumour suppressor or proto-oncogenes . Nevertheless, many important questions remain . The tumour cell lines in which these mutator genes were characterized have many other mutations that may contribute to the mutator phenotype and the characteristic pattern of mutations found in these cells . Thus, mismatch repair deficiency may be necessary for the mutator phenotype, but is it sufficient? Certainly, changes in DNA replication fidelity or cell cycle checkpoint controls may contribute to the mutator phenotype . This question also has important implications for the effect of mismatch repair deficiency on tumour development . Does the mutator phenotype in HNPCC patients arise as a very early event resulting from the loss of the wild type allele or does it arise in later stages only after alterations of cell cycle controls or replication fidelity? Given that eukaryotic cells have numerous homologues of the mismatch repair genes, what are the roles of all these genes? Are these involved in the repair of very specific types of replication errors or do they have other roles in cells? Finally, what mechanisms underlie the accumulation of mutations in other types of tumours? Given the rapid progress made since the isolation of the human homologues of the E coli mismatch repair genes less than 3 years ago, we can look forward to the answers to many of these questions in the near future. Arch Virol, 1996, 141(11), 2129 - 38 Mapping of antigenic sites on the major inner capsid protein of avian rotavirus using an Escherichia coli expression system; Ito H et al.; The cDNA encoding the VP6 gene of avian rotavirus PO-13 strain was inserted into the bacterial expression vector pET-3a . Upon isopropyl-1-thio-beta-D-galactoside induction, the E . coli BL21 (DE3) harboring the vector containing cDNA of the VP6 gene produced an approximately 45-kDa polypeptide, which reacted with rabbit serum against PO-13 strain in Western blotting . To study the antigenic sites on VP6, various deletion mutants were constructed, expressed in E . coli and the reactivity with antigenic site I- and II-specific MAbs analyzed by Western blotting . Site I, which is shared with all group A mammalian and avian rotaviruses except for chicken rotavirus, was found to be located at amino acid positions 45 to 65, and site II, which probably contributes to an authentic group A antigen common to both mammalian and avian rotaviruses, at amino acid positions 134 to 142. Horm Res, 1996, 45(3-5), 197 - 201 Sex hormone-binding globulin: gene organization and structure/function analyses; Hammond GL et al.; Human plasma sex hormone-binding globulin (SHBG) and testicular androgen-binding protein (ABP) are homodimeric glycoproteins with a single steroid-binding site . They share the same primary structure and differ only with respect to the types of oligosaccharides attached to them . Both are products of a single gene (Shbg), the expression of which has been detected in several tissues including liver, testis, placenta, brain and endometrium . The transcript encoding SHBG in hepatocytes and ABP in Sertoli cells is identical . Several other transcripts result from differential exon utilization in sex steroid-responsive tissues, but their biological significance remains obscure . Wild-type and mutant forms of human SHBG have been produced in mammalian cells and Escherichia coli, and have provided insight into the structural and functional properties of SHBG and its related gene products. Intervirology, 1996, 39(1-2), 85 - 92 Chimeric potyvirus-like particles as vaccine carriers; Jagadish MN et al.; Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses . Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs) . The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly . Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs . Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant . Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles . This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier. Vet Res, 1996, 27(6), 569 - 78 Chronic exposure of pigs to airborne dust and endotoxins in an environmental chamber: technical note; Urbain B et al.; A new experimental setup was developed to expose pigs to dust and airborne endotoxins in an environmental chamber, at levels liable to be encountered in pig farm buildings . The following parameters were evaluated in a chamber containing two pigs of 10 kg body-weight: inhalable and respirable dust gravimetric concentrations were measured using area samplers and expressed as mg/m3 . The respirable dust concentration was also measured using a "TM digital microP respirable dust-measuring instrument', which has been shown to give similar results to the gravimetric method . The endotoxin concentration was evaluated using the Limulus-assay and expressed as ng/m3 of air containing the inhalable or respirable dust or as ng/mg of inhalable and respirable dust . Feed flour dust was introduced into the chamber to obtain different concentrations of inhalable and respirable dust ranging from 3.62 to 76.66 mg/m3 and from 0.24 to 1.40 mg/m3, respectively . The endotoxin concentration was modulated by mixing the feed flour with Escherichia coli endotoxins before blowing it into the chamber . The endotoxin concentrations in the air containing inhalable or respirable dust ranged from 28.9 to 270.0 ng/m3 and from 2.22 to 36.38 ng/m3, respectively, depending on the amount of endotoxins added to the dust . Data were also obtained in a piggery . The experimental setup detailed in this paper could be used to study the significance of air contaminants in the development of pig respiratory diseases. Biochimie, 1996, 78(7), 597 - 604 Yeast tRNA(Met) recognition by methionyl-tRNA synthetase requires determinants from the primary, secondary and tertiary structure: a review; Senger B et al.; The primordial role of the CAU anticodon in methionine identity of the tRNA has been established by others nearly a decade ago in Escherichia coli and yeast tRNA(Met) . We show here that the CAU triplet alone is unable to confer methionine acceptance to a tRNA . This requires the contribution of the discriminatory base A73 and the non-anticodon bases of the anticodon loop . To better understand the functional communication between the anticodon and the active site, we analysed the binding and aminoacylation of tRNA(Met) based anticodon and acceptor-stem minihelices and of tRNA(Met) chimeras where the central core region of yeast tRNA(Met) is replaced by that of unusual mitochondrial forms lacking either a D-stem or a T-stem . These studies suggest that the high selectivity of the anticodon bases in tRNA(Met) implies the L-conformation of the tRNA and the presence of a D-stem . The importance of a L-structure for recognition of tRNA(Met) was also deduced from mutations of tertiary interactions known to play a general role in tRNA(Met) folding. Biochimie, 1996, 78(7), 555 - 67 Translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and ribosomal proteins L20 and L35; Chiaruttini C et al.; The Escherichia coli IF3-L35-L20 operon encodes translation initiation factor IF3 and the ribosomal proteins L35 and L20, respectively . The expression of the genes encoding the two ribosomal proteins is negatively regulated at the translational level by L20, which acts at an operator located within the IF3 gene and just upstream of the L35 gene . We have previously shown that L20 directly represses the expression of the L35 gene, and indirectly that of the L20 gene, via translational coupling . On the basis of mutational analysis and in vitro RNA structure probing experiments, we proposed that a large secondary structure in which the translation initiation site of the L20 gene is sequestered by base-pairing, is responsible for coupling . The ribosome binding site of the L20 gene becomes available when the secondary structure is melted by ribosomes translating the L35 mRNA . Here we describe that this secondary structure forms in vivo by showing that single mutations in either strand reduce coupling and that compensatory mutations that re-establish pairing also re-establish coupling . In vitro 'toeprinting' analysis enabled us to show that the wild-type inhibitory secondary structure directly blocks ribosome binding to the ribosome binding site of rpIT. Biochimie, 1996, 78(7), 543 - 54 Molecular recognition governing the initiation of translation in Escherichia coli . A review; Schmitt E et al.; Selection of the proper start codon for the synthesis of a polypeptide by the Escherichia coli translation initiation apparatus involves several macromolecular components . These macromolecules interact in a specific and concerted manner to yield the translation initiation complex . This review focuses on recent data concerning the properties of the initiator tRNA and of enzymes and factors involved in the translation initiation process . The three initiation factors, as well as methionyl-tRNA synthetase and methionyl-tRNA(f)Met formyltransferase are described . In addition, the tRNA recognition properties of EF-Tu and peptidyl-tRNA hydrolase are considered . Finally, peptide deformylase and methionine aminopeptidase, which catalyze the amino terminal maturation of nascent polypeptides, can also be associated to the translation initiation process. Eur J Hum Genet, 1996, 4(5), 274 - 82 A systematic analysis of the mutations of the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria; Fontanellas A et al.; Congenital erythropoietic porphyria (CEP) or Gunther's disease is an inborn error of heme biosynthesis, transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase activity (UROIIIS) . The molecular defects observed in CEP are mainly heterogeneous, except for one missense mutation, C73R (Cys to Arg substitution at codon 73) which represents nearly 40% of the disease alleles . A convenient strategy was designed to establish a rapid diagnosis at the genetic level in samples from patients with CEP . As a first step, the most frequent mutation is searched for by restriction analysis from genomic . DNA amplified by PCR . Next, the nine coding exons and intron-exon boundaries are sequenced from genomic DNA . As an alternative, the mutation can be determined by sequencing the UROIIIS cDNA of the patient, using the RT-PCR technique on RNAs when a lymphoblastoid cell line can be established . Finally, for each new mutation in UROIIIS coding sequence, the corresponding mutant protein is expressed in Escherichia coli, in order to demonstrate the pathological significance of the mutation . This work describes the analysis of UROIIIS gene mutations in 10 new families with CEP and summarizes the data from 20 unrelated families studied in our laboratory . Three new missense mutations of UROIIIS coding sequence (H173Y, Q187P and P248Q) have been observed together with 8 known mutations . The significance of three intronic base changes (476 -31 T-->C; 562 -4 A-->T; 562 -23 A-->G) is discussed . In 6 alleles out of 40 (15%), the mutation remains undetermined. Semin Thromb Hemost, 1996, 22(4), 357 - 66 The influence of C1-esterase inhibitor substitution on coagulation and cardiorespiratory parameters in an endotoxin-induced rabbit model of hypercoagulability; Scherer RU et al.; In a short-time model of endotoxin-induced (lipopolysaccharide from Escherichia coli, 120 micrograms kg-1 i.v.) hypercoagulability in rabbits, the therapeutic effects of C1-esterase inhibitor (C1I) substitution (bolus 400 U kg-1 i.v . followed by continuous infusion of 400 U kg-1 4 h-1 i.v.) were studied . When compared to endotoxin-challenged untreated animals, C1I substitution significantly stabilized mean arterial pressure (p < 0.01), increased central venous oxygen saturation (p < 0.05), prevented the decrease of antithrombin III (p < 0.05), and reduced fibrin deposition in the microcirculation of the liver and the lungs to approximately 30% of that observed in the untreated animals (p < 0.01) . Although C1I substitution did not reduce systemic procoagulant turnover, the improvement of blood pressure and blood flow and local inhibitory actions in the coagulation and complement cascade prevented fibrin deposition in the microcirculation of vital organs . This study supports the beneficial role of C1I substitution during early disseminated intravascular coagulation. Neuropharmacology, 1996, 35(7), 923 - 31 Time resolved kinetics of direct G beta 1 gamma 2 interactions with the carboxyl terminus of Kir3.4 inward rectifier K+ channel subunits; Doupnik CA et al.; The direct interaction of recombinant G beta 1 gamma 2 proteins with the carboxyl terminal domain of a G protein-gated inward rectifier K channel subunit, Kir3.4 (GIRK4), was measured in real time using biosensor chip technology . The carboxyl terminus of Kir3.4 (a.a . 186-419) was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, GST-Kir3 . 4ct . GST-Kir3.4ct was immobilized to the surface of a biosensor chip by high affinity binding of the GST domain to a covalently attached anti-GST antibody . The association and dissociation rates of G beta 1 gamma 2 dimers with the immobilized Kir3.4ct domain were temporally resolved as a change in refractive index detected by surface plasmon resonance . Specific binding of G beta 1 gamma 2 dimers to Kir3.4ct was characterized by a dissociation rate (kd) of approximately 0.003 s-1 . Association kinetics were dominated by a concentration-independent component (time constant approximately 50 s) which complicates models of binding and may indicate conformational changes during binding of G beta 1 gamma 2 to Kir3.4ct . The estimated equilibrium dissociation binding constant (Kd) was approximately 800 nM . These studies demonstrate that G beta gamma dimers interact directly with the Kir3.4 channel subunit, and suggest interesting details in the interaction with the major cytosolic carboxyl terminal domain . The slow G beta 1 gamma 2 dissociation rate measured on the sensor chip is similar in magnitude to a slow component of channel deactivation measured electrophysiologically in Xenopus oocytes expressing Kir3.1/3.4 multimeric channels and a G protein-coupled receptor . Biosensor-based experiments such as those described here will complement electrophysiological studies on the molecular basis of G protein interactions with Kir channels and other ion channel proteins. Neuropharmacology, 1996, 35(7), 915 - 21 Interaction of Ca2(+)-activated K+ channels with refolded charybdotoxins mutated at a central interaction residue; Naini AA et al.; Charybdotoxin is a small peptide blocker of K+ channels, rigidly held in active conformation by three disulfide bonds . The toxin blocks K+ channels by binding to a receptor site located at the external "vestibule", and thus physically occluding the outer opening of the K+ conduction pore . In the blocked complex, K27, a residue on the toxin's molecular surface, projects its epsilon-amino group into the K(+)-selective pore . The results here show that CTX, produced by heterologous expression in E . coli, may be manipulated to place unnatural positively charged residues at position 27 . The toxin folds faithfully to its native conformation when the crucial lysine at position 27 is replaced by a cysteine residue, a maneuver that allows specific chemical modification of this side-chain . Replacements of K27 by side-chains slightly shorter or slightly longer than lysine yield active toxins . The toxin variant with ornithine at this position interacts much less strongly with K+ ions in the pore of slowpoke-type Ca2(+)-activated K+ channels than does wild-type toxin . This result argues that the epsilon-amino group of K27 in bound toxin lies only a few angstroms away from a K+ ion occupying the blocked pore . The peptide folds with high efficiency to form the correct disulfides even in the presence of strong denaturants. Free Radic Biol Med, 1996, 21(7), 975 - 93 Role of Escherichia coli rpoS and associated genes in defense against oxidative damage; Eisenstark A et al.; The first phenotype described for mutations in the Escherichia coli rpoS gene was hypersensitivity to near-ultraviolet radiation and to its oxidative photoproduct, hydrogen peroxide . Initially named nur, this gene is now known to code for a sigma factor, and has acquired new names such as katF and rpoS . The role of its protein product (sigma-38) is to regulate a battery of genes as cells enter and rest in stationary phase . Some of the gene products are involved in protection against oxidants (e.g., catalases) and repair of oxidative damage (e.g., exonuclease III) . Sigma-38 may also modulate transcription of certain growth phase genes, including hydroperoxidase I and glutathione reductase . Sigma-38 activity is regulated at transcriptional, translational, and protein stabilization levels . This review describes the complex mechanisms whereby sigma-38 controls various genes, the interaction of sigma-38 with other regulators, and a possible role of sigma-38 in bacterial virulence. Urol Res, 1996, 24(5), 273 - 7 Construction and expression of single-chain Fv antibody against human bladder carcinoma; Yu LZ et al.; We designed two sets of oligonucleotide primers to amplify the immunoglobulin heavy- and light-chain variable-region genes from genomic DNA by polymerase chain reaction (PCR) . The genomic DNA was extracted from hybridoma BDI-1 cells, which secreted a monoclonal antibody (mAb) against human bladder carcinoma . The primers contained special restriction sites that allowed the variable-region genes to be easily cloned for sequencing and expression . The recombinants were sequenced by Sanger's method . It was proved that the full lengths of the VH and VK genes were 366 and 324 bp, respectively . Compared with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from the rearrangement of VH, Dsp2.2 and JH4 . The VK gene was VK subgroup IV and from VK and JK4 . The VH and VK genes was inserted expression vector pWAI80 . By inducement, the ScFv antibodies were expressed and secreted from Escherichia coli . Binding activities against the bladder carcinoma cells were detected . We suggest that ScFv antibody recognized the antigen specifically. Gene Ther, 1996 Jan, 3(1), 59 - 66 Construction of recombinant vaccinia virus expressing GM-CSF and its use as tumor vaccine; Qin HX et al.; We made several generic plasmids for construction of recombinant vaccinia virus (rvv) expressing foreign proteins in high yield . Rvvs expressing biologically active Escherichia coli beta-galactosidase (rvv-lacZ) and the cytokine murine GM-CSF (rvv-mGM-CSF) were constructed by using these plasmids . To obtain attenuated rvv, cDNA for these proteins was inserted in the thymidine kinase gene of vaccinia virus . Their expression was controlled by vaccinia early/late promoter, 7.5 K so that these proteins could be expressed in the infected cells throughout the life cycle of the virus . Female C57BL/6 mice were immunized subcutaneously with B16-F10 melanoma cells infected with rvv, and 2 weeks later challenged with viable B16 cells . Mice immunized with rvv-mGM-CSF showed delay in tumor development, smaller tumor volumes and longer survival time compared with unimmunized mice, as well as mice immunized with rvv-lacZ . Mice immunized with rvv-mGM-CSF followed by a booster injection after 1 week responded slightly better than those immunized once, but this difference was not statistically significant . These results suggested that rvv-mGM-CSF could be a promising vaccine for cancer therapy. Chem Res Toxicol, 1996 Jan-Feb, 9(1), 277 - 83 Relative contribution of cytosine deamination and error-prone replication to the induction of propanodeoxyguanosine-->deoxyadenosine mutations in Escherichia coli; Fink SP et al.; The role of cytosine deamination as a possible mechanism for PdG-->A transitions induced by propanodeoxyguanosine (PdG) was investigated by site-specific mutagenesis techniques . PdG was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into a gapped-duplex derivative of the vector . Unmodified and PdG-modified M13MB102 genomes containing either uracil or thymine in the (+)-strand were transformed into Escherichia coli strains differing in their ability to excise uracil bases from DNA . After replication of the site specifically modified M13MB102, base-pair substitutions were detected by in situ hybridization using {32P}-labeled probes containing each of the possible mismatched bases opposite position 6256 in the (+)-strand . The ratio of PdG-->A and PdG-->T was unchanged in strains defective in the repair of uracil residues, which suggests that uracil is not an intermediate in the generation of PdG-->A mutations . Similar results were obtained when PdG-M13MB102 was incubated for 14 days prior to transformation in an attempt to increase the extent of deamination . As a control experiment to test the sensitivity of the assay to detect deaminations opposite PdG, uracil-containing M13MB102 with a PdG.T mismatch at position 6256 was transformed into E . coli JM105 . Hybridization analysis indicated that approximately 80% of the phage plaques generated after genome replication contained T in the (+)-strand at position 6256 . Thus, any deamination of cytosine to uracil would have been easily detected . Adducted and unadducted genomes were also transformed into E . coli LM114 or LM113, which carry a mutant umuD or umuC gene, respectively . Significant and comparable reductions in PdG-->A and PdG-->T were observed, suggesting that both mutations require the active participation of the UmuD and the UmuC proteins in the replication complex . The results of our experiments suggest that the PdG-->A mutations induced by PdG are not caused by cytosine deamination, but arise coincident with PdG-->T mutations during replication of the PdG-containing genomes . Also, the uracil-containing (+)-strand does not appear to be degraded, as is commonly assumed in site-specific mutagenesis experiments, and serves as a template for DNA synthesis when replication of the (-)-strand is blocked by an adduct such as PdG. Acta Biochim Pol, 1996, 43(3), 497 - 501 Biosynthesis and distribution of leucocyte elastase inhibitor . Production of recombinant inhibitor; Kasza A et al.; The horse leucocyte elastase inhibitor (HLEI), present in neutrophils, monocytes and bone marrow cells, is apparently a cytoplasmic protein which is not released from cells even in response to stimulation with lipopolysaccharide, phorbol ester, tumour necrosis factor alpha, interleukin-1 or elastin degradation products . Although no expression of the inhibitor was detected in neutrophils, both monocytes and bone marrow cells were efficient in its synthesis . Using a new expression vector pREST5d, recombinant inhibitor was produced in a large quantity in a soluble form, with a yield of 88 mg per 10 litres of E . coli culture . A two-step purification procedure, consisting of ion-exchange chromatography and gel filtration, yielded 36 mg of the recombinant inhibitor of a purity higher than 95%, as judged by SDS/PAGE . The recombinant protein had physicochemical and kinetic properties indistinguishable from those of the natural one, including irreversible elastase inhibition with an association rate constant kass > 10(7) M-1s-1 . Both proteins were eliminated from rat circulation at the same ratio, and within the first 20 min 70% of the protein was removed . Such a short half-life in the circulation suggests that local delivery of HLEI directly to lungs in the form of aerosol could be a more efficient therapeutic approach than its intravenous injection. Growth Factors, 1996, 13(3-4), 181 - 91 Human betacellulin, a member of the EGF family dominantly expressed in pancreas and small intestine, is fully active in a monomeric form; Seno M et al.; Betacellulin (BTC) was found to be expressed mainly in human pancreas and small intestine . This finding suggests that BTC possesses some specific function distinguished from the other members of epidermal growth factor (EGF) family . To clarify this function, the released form of human BTC has been expressed in E.coli, purified, and characterized . The recombinant human BTC was produced as an inclusion body . This material was dissolved in guanidine-HCl under reducing conditions, refolded, and purified through sequential liquid chromatography . Purified BTC was electrophoresed under reducing conditions and a molecular size of 18 kDa was determined, which is the supposed size of a dimer of the peptide . However, chemical analysis failed to show a covalently linked dimer . The molecular mass of BTC analyzed by mass spectrometry revealed it to be 9 kDa, which is consistent with theoretical value for a monomer . Recombinant BTC showed growth promoting activity for mouse fibroblasts and rat aortic smooth muscle cells which was equivalent to EGF On the other hand, BTC was found to exhibit a growth inhibitory effect on the cells overexpressing EGF receptor. Biochimie, 1996, 78(6), 416 - 24 Multiple degradation pathways of the rpsO mRNA of Escherichia coli . RNase E interacts with the 5' and 3' extremities of the primary transcript; Hajnsdorf E et al.; The degradation process of the rpsO mRNA is one of the best characterised in E coli . Two independent degradation pathways have been identified . The first one is initiated by an RNase E endonucleolytic cleavage which allows access to the transcript by polynucleotide phosphorylase and RNase II . Cleavage by RNase E gives rise to an rpsO message lacking the stabilising hairpin of the primary transcript; this truncated mRNA is then degraded exonucleolytically from its 3' terminus . This pathway might be coupled to the translation of the message . The second pathway allows degradation of polyadenylated rpsO mRNA independently of RNase II, PNPase and RNase E . The ribonucleases responsible for degradation of poly(A) mRNAs under these conditions are not known . Poly(A) tails have been proposed to facilitate the degradation of structured RNA by polynucleotide phosphorylase . In contrast, we believe that removal of poly(A) by RNase II stabilises the rpsO mRNA harbouring a 3' hairpin . In addition to these two pathways, we have identified endonucleolytic cleavages which occur only in strains deficient for both RNase E and RNase III suggesting that these two endonucleases protect the 5' leader of the mRNA from the attack of unidentified ribonuclease(s) . Looping of the rpsO mRNA might explain how RNase E bound at the 5' end can cleave at a site located just upstream the hairpin of the transcription terminator. Arch Immunol Ther Exp (Warsz), 1996, 44(2-3), 97 - 101 Tumor necrosis factor and interferon production by peripheral blood leukocytes of patients with alcoholic cirrhosis; Daniluk J et al.; Sixteen male and female patients with alcoholic cirrhosis and 13 healthy controls were included in the study . The level of interferon (IFN) and tumor necrosis factor (TNF) activity was examined in the sera and also in cultures in peripheral blood leukocytes (PBL) after in vitro stimulation with the cytokine inducers . In sera of patients with alcoholic cirrhosis higher IFN and TNF levels were detected than in controls . However, after induction with Newcastle disease virus (NDV), phytohemagglutinin M (PHA) and lipopolysaccharide from E . coli (LPS), PBL from cirrhotic patients produced lower IFN levels in comparison to controls . In contrast to depressed ability to produce IFNs, TNF production was higher in PBL of cirrhotic patients induced by PHA and a low dose of LPS (1 microgram/ml) . NDV induced comparable levels of TNF in both groups . It appears likely that cells of cirrhotic patients were suppressed by an unknown factor or were hyporeactive for IFN production, but synthesis and release of TNF was enhanced, suggesting that cells producing TNF were preactivated in vivo . Mechanism of such preactivation is discussed. Cell Motil Cytoskeleton, 1996, 35(3), 200 - 9 Site-directed mutagenesis of the phosphorylation site of cofilin: its role in cofilin-actin interaction and cytoplasmic localization; Nagaoka R et al.; It has been demonstrated that the activity of ADF and cofilin, which constitute a functionally related protein family, is markedly altered by phosphorylation, and that the phosphorylation site is Ser 3 in their amino acid sequences {Agnew et al., 1995: J . Biol . Chem . 270:17582-17587; Moriyama et al., 1996: Genes Cells 1:73-86} . In order to clarify the function of the phosphorylated and unphosphorylated forms of cofilin in living cells especially in the process of cytokinesis, we generated analogs of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser 3) of cofilin to Ala and Asp, respectively . The mutated proteins were produced in an Escherichia coli expression system, and conjugated with fluorescent dyes . In in vitro functional assay, labeled A3-cofilin retained the authentic ability to bind to and sever F-actin, while labeled D3-cofilin failed to interact with actin . They were then injected into living cells to examine their cellular distribution . They exhibited distinct localization patterns in the cytoplasm; A3-cofilin was highly concentrated at the membrane ruffles and cleavage furrow, where endogenous cofilin is also known to be enriched . In contrast, D3-cofilin showed only diffuse distribution both in the cytoplasm and nucleus . These results suggest that the subcellular distribution of cofilin as well as its interacting with actin in vivo is regulated by its phosphorylation and dephosphorylation. Probl Tuberk, 1996, (1), 43 - 6 {The isolation and characteristics of monoclonal antibodies to Mycobacterium smegmatis and M . kansasii}; Kulikovskaia NV et al.; Immunization of BALB/c mice with sonicates of M . smegmatis and kansasii and fusion of splenocytes of the immunized mice with cells of syngeneic myeloma P3X63Ag8653 yielded hybrid clones synthetizing antibodies to these antigens . The selection of hybrid clones and analysis of the antibodies specificity were performed at ELISA using the immunization antigens and antigens from other mycobacteria and E . coli . To define immunoglobulin class of the antibodies the authors used antiglobulin sera of class A, M, G . The monoclonal antibodies belonged to IgG . Molecular mass of polypeptides carrying the epitope against which the antibodies were directed was measured at immunoblotting . Two antibodies reacted only with M . smegmatis, 38 and 10 kD proteins . The other 2 cross-reacted with other mycobacteria and were directed against epitopes on polypeptides varying in molecular mass. J Biochem (Tokyo), 1996 Jan, 119(1), 173 - 9 Escherichia coli phosphorylates 1,5-Anhydroglucitol and releases 1,5-Anhydroglucitol 6-phosphate when glucose is absent in the medium; Shiga Y et al.; The cyclic polyol 1,5-anhydro-D-glucitol (AG) is detected in most organisms, but little is known about its metabolism and physiological roles . Our previous study demonstrated that Escherichia coli C600 synthesizes AG when glucose is exhausted in the medium and that it temporarily releases AG into and then takes it back from the medium, thus forming a sharp peak in AG concentration in the medium a few hours after reaching stationary growth phase . The present study demonstrates that when glucose is absent in the culture medium, E . coli C600 takes up and phosphorylates AG and releases a large portion of it back into the medium in the form of a phosphate ester . {U-13C}AG was added to the medium after the exhaustion of glucose and the resulting {U-13C}AG phosphate was partially purified by several steps of anion exchange chromatography and identified as AG 6-phosphate by 13C-NMR . The identity of the phosphate ester was also confirmed by GC-MS analysis after further purification. Annu Rev Microbiol, 1996, 50, 645 - 77 rRNA transcription and growth rate-dependent regulation of ribosome synthesis in Escherichia coli; Gourse RL et al.; The synthesis of ribosomal RNA is the rate-limiting step in ribosome synthesis in bacteria . There are multiple mechanisms that determine the rate of rRNA synthesis . Ribosomal RNA promoter sequences have evolved for exceptional strength and for regulation in response to nutritional conditions and amino acid availability . Strength derives in part from an extended RNA polymerase (RNAP) recognition region involving at least two RNAP subunits, in part from activation by a transcription factor and in part from modification of the transcript by a system that prevents premature termination . Regulation derives from at least two mechanistically distinct systems, growth rate-dependent control and stringent control . The mechanisms contributing to rRNA transcription work together and compensate for one another when individual systems are rendered inoperative. Planta, 1996, 200(2), 159 - 66 Feed-back regulation of gibberellin biosynthesis and gene expression in Pisum sativum L; Martin DN et al.; Treatment of tall and dwarf (3 beta-hydroxylase impaired) genotypes of pea (Pisum sativum L.) with the synthetic, highly active gibberellin (GA), 2,2-dimethyl GA4, reduced the shoot contents of C19-GAs, including GA1, and increased the concentration of the C20-GA, GA19 . In shoots of the slender (la crys) mutant, the content of C19-GAs was lower and GA19 content was higher than in those of the tall line . Metabolism of GA19 and GA20 in leaves of a severe (na) GA-deficient dwarf mutant was reduced by GA treatment . The results suggest feed-back regulation of the 20-oxidation and 3 beta-hydroxylation reactions . Feed-back regulation of GA 20-oxidation was studied further using a cloned GA 20-oxidase cDNA from pea . The cDNA, Ps074, was isolated using polymerase chain reaction with degenerate oligonucleotide primers based on pumpkin and Arabidopsis 20-oxidase sequences . After expression of this cDNA clone in Escherichia coli, the product oxidized GA12 to GA15, GA24 and the C19-GA, GA9, which was the major product . The 13-hydroxylated substrate GA53 was similarly oxidized, but less effectively than GA12, giving mainly GA44 with low yields of GA19 and GA20 . Ps074 hybridized to polyadenylated RNA from expanding shoots of pea . Amounts of this transcript were less in the slender genotype than in the tall line and were reduced in GA-deficient genotypes by treatment with GA3, suggesting that there is feed-back regulation of GA 20-oxidase gene expression. Haemostasis, 1996, 26 Suppl 1, 83 - 91 Role of tissue factor and factor VIIa in the coagulant and inflammatory response to LD100 Escherichia coli in the baboon; Taylor FB Jr; Anti-tissue factor antibody and active site-inhibited factor VIIa inhibit both the disseminated intravascular coagulant response to LD100 Escherichia coli and its lethal inflammatory effects . In contrast, active site inhibited factor Xa while completely inhibiting disseminated intravascular coagulant does not protect baboons from LD100 E . coli . This review examines the role of tissue factor and factor VIIa in initiating the disseminated intravascular coagulant response and in amplifying the inflammatory response. Methods Enzymol, 1996, 274, 445 - 55 Reading of DNA sequence logos: prediction of major groove binding by information theory; Schneider TD; DNA sequences to which the OxyR protein binds under oxidizing conditions were analyzed by the sequence logo method, a quantitative graphic technique based on information theory . A sequence logo shows both the sequence conservation and the frequencies of bases at each position in a site . Unlike the consensus sequence, the sequence logo analysis revealed that OxyR should bind to four major grooves of DNA . This was later confirmed by experiments . Detailed interpretation of the sequence logo also allowed the prediction of likely major and minor groove OxyR-DNA base contacts, consistent with available experimental results . Because the sequence logo shows the original base frequencies in a clear, easily interpreted graphic that does not distort the data, highly refined analysis of binding site contacts becomes easy . Not only can these methods be applied to any DNA sequence binding site, they can also be applied to sites on RNA and proteins.
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