Cell, 1996 Feb 9, 84(3), 481 - 90 Characterization of the active intermediate of a GroEL-GroES-mediated protein folding reaction; Weissman JS et al.; Recent studies of GroE-mediated protein folding indicate that substrate proteins are productively released from a cis ternary complex in which the nonnative substrate is sequestered within the GroEL channel underneath GroES . Here, we examine whether protein folding can occur in this space . Stopped-flow fluorescence anisotropy of a pyrene-rhodanese-GroEl complex indicates that addition of GroES and ATP (but not ADP) leads to a rapid change in substrate flexibility at GroEL . Strikingly, when GroES release is blocked by the use of either a nonhydrolyzable ATP analog or a single-ring GroEL mutant, substrates complete folding while remaining associated with chaperonin . We conclude that the cis ternary complex, in the presence of ATP, is the active state intermediate in the GroE-mediated folding reaction: folding is initiated in this state and for some substrates may be completed prior to the timed release of GroES triggered by ATP hydrolysis. Biochim Biophys Acta, 1996 Feb 9, 1289(1), 5 - 9 A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli; Yoshioka K et al.; A novel fluorescent derivative of glucose was synthesized by reacting D-glucosamine and NBD-Cl . The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (lambda Ex = 475 nm, lambda Em = 550 nm) . The obtained novel fluorescent product, which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by 1H-NMR and FAB-MS spectrometries, was applied to the assessment of the glucose uptake activity of Escherichia coli B . 2-NBDG accumulated in living cells and not in dead cells . The uptake of 2-NBDG was competitively inhibited by D-glucose and not by L-glucose, which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG . 2-NBDG taken into the cytoplasma of E . coli cells was supposedly converted into another derivative in the glucose metabolic pathway. Biochem Pharmacol, 1996 Feb 9, 51(3), 313 - 9 7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes; Yamazaki H et al.; 7-Ethoxycoumarin O-deethylation has been used widely as a marker activity for assessing substrate specificities of cytochromes P450 (P450) in liver microsomes of mammals, and extensive studies have shown that in rats and mice the major catalysts are P450 1A1, 1A2, and 2B enzymes . In contrast to findings in experimental animal models, P450 2E1 has been reported to be a principal enzyme involved in 7-ethoxy-coumarin O-deethylation in human livers . In this study, we further examined the roles of individual forms of human P450 involved in 7-ethoxycoumarin O-deethylation using microsomes from different human liver samples and from human lymphoblastoid cells expressing human P450 enzymes and purified P450 enzymes isolated from the membrane of Escherichia coli expressing modified P450 proteins . Kinetic analysis showed that there were at least two different enzymes involved in 7-ethoxycoumarin O-deethylation in different human samples . Samples that contained high amounts of P450 2E1 in liver microsomes showed biphasic curves for O-deethylation with relatively high turnover numbers, whereas P450 1A2-rich samples tended to have low Km values with low Vmax values . Anti-human P450 2E1 antibodies inhibited markedly (P < 0.05) the 7-ethoxycoumarin O-deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 microM) . However, we also found that anti-P450 1A2 antibodies suppressed O-deethylation activities only at a low substrate concentration (10 microM) . Recombinant human P450 1A2 was found to have a low Km value for 7-ethoxycoumarin O-deethylation, whereas P450 2E1 showed a high Km value . Of the P450 enzymes examined, P450 1A1 gave the highest O-deethylation activities with a low Km value, although this enzyme is reported to be expressed extrahepatically in humans . Other human P450 enzymes, including P450 2A6, 2C10, 2D6, 3A4, and 3A5, did not show significant O-deethylation activities except that P450 2B6, a minor P450 component in human livers, was found to have a Vmax value similar to that of P450 1A2 and a Km value similar to that of P450 2E1 . These results suggest that P450 1A2 is a low Km enzyme for 7-ethoxycoumarin O-deethylation in human liver microsome, although it has a low Vmax value than P450 2E1. Int J Cancer, 1996 Feb 8, 65(4), 437 - 41 Overexpression of human mutT homologue gene messenger RNA in renal-cell carcinoma: evidence of persistent oxidative stress in cancer; Okamoto K et al.; Data regarding oxidatively modified DNA bases suggest that cancer cells are more exposed to oxidative stress than adjacent non-tumorous tissue . This novel concept may contribute to the understanding of certain aspects of tumor biology such as activated transcription factors, genetic instability, chemotherapy-resistance and metastasis . We therefore tested this concept in human renal-cell carcinomas (RCCs) by evaluating the expression of hMTH1, an enzyme preventing the misincorporation into DNA of 8-oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate), an oxidized form of dGTP in the nucleotide pool . The expression of hMTH1 messenger RNA (mRNA) in RCC was significantly higher than that in adjacent non-tumorous kidney . Moreover, advanced-stage tumors showed significantly higher hMTH1 mRNA expression than early-stage tumors, and there was a modest linear correlation between hMTH1 expression and c-myc expression . The results provide logical support for the concept of "persistent oxidative stress in cancer" and suggest a role of hMTH1 mRNA level as a prognostic marker. Nature, 1996 Feb 8, 379(6565), 511 - 8 The structure of the Escherichia coli EF-Tu.EF-Ts complex at 2.5 A resolution; Kawashima T et al.; The crystal structure of the EF-Tu.EF-Ts complex from Escherichia coli has been determined to a resolution of 2.5 A . The complex contains two subunits of each of the elongation factors . The two EF-Ts molecules form a tight dimer, but there is little contact between the two EF-Tu molecules . The interaction of EF-Ts with EF-Tu results principally in the disruption of the Mg2+ ion binding site, thereby reducing the affinity of EF-Tu for guanine nucleotides. Carbohydr Res, 1996 Feb 7, 281(1), 155 - 60 Structural studies of the Escherichia coli O26 O-antigen polysaccharide; Manca MC et al.; The structure of the O-specific side chain of the E . coli O26 lipopolysaccharide has been investigated . Based on sugar and methylation analyses, and 2D NMR spectroscopy employing HMBC experiments, it is concluded that the polysaccharide is composed of trisaccharide repeating units having the following structure . -->3)-alpha-L-Rhap-(1-->4)-alpha-L-FucpNAc-(1-->3)-beta-D-Gl cpNAc-(1--> Biochim Biophys Acta, 1996 Feb 7, 1305(1-2), 7 - 10 Cell line-specific transcriptional activation of the promoter of the human guanylyl cyclase C/heat-stable enterotoxin/receptor gene; Mann EA et al.; The guanylyl cyclase C protein, expressed primarily in the intestine, is the receptor for the heat-stable enterotoxin of Escherichia coli . We have isolated and sequenced the promoter region and the first exon of human guanylyl cyclase C and determined the major site of transcription initiation . Transfection of a -1973/+124 promoter/luciferase gene fusion construct in the Caco-2 intestinal cell line resulted in a high level of expression; results with deletion constructs indicate the presence of multiple positive-acting sequence elements . These promoter elements were not active upon transfection into NIH/3T3 and LLC-PK1 cell lines which do not express GC-C. Biochim Biophys Acta, 1996 Feb 7, 1305(1-2), 19 - 24 Nucleotide sequence of the nar beta gene encoding assimilatory nitrate reductase from the cyanobacterium Oscillatoria chalybea; Unthan M et al.; The nucleotide sequence of the structural gene of nitrate reductase (n ar beta) has been determined from the filamentous, non-heterocystous cyanobacterium Oscillatoria chalybea . The nar beta gene encodes a protein of 737 amino acid residues, which shows 61% identity to nitrate reductase of the unicellular cyanobacterium Synechococcus sp . PCC 7942 and only weak homologies to different bacterial molybdoenzymes, such as nitrate reductases or formate dehydrogenases. Biochim Biophys Acta, 1996 Feb 7, 1305(1-2), 11 - 4 Molecular cloning of murine folylpoly-gamma-glutamate synthetase; Spinella MJ et al.; Folylpoly-gamma-glutamate synthetase (FPGS) is essential for mammallian cell survival and is a major determinant of cytotoxicity and selectivity for folate antimetabolites . Here we describe the cloning of a cDNA encoding murine FPGS isolated from L1210 leukemia cells . The amino acid sequence of murine FPGS is 82% identical to human FPGS+{1} with identical discrete regions of up to 41 residues . Murine FPGS contains two AUG initiation codons, shown to be responsible for mitochondrial and cytosolic forms of the enzyme in human cells {2} Previous studies indicated species, tissue, and tumor specific differences in mammalian FPGS . The availability of murine FPGS expands the knowledge and understanding of the spectrum of these variations. Biochemistry, 1996 Feb 6, 35(5), 1692 - 9 Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants; Poyner RR et al.; High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345 . Two different proposals for general base catalysis have emerged from these structural studies . In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base {Lebioda, L., & Stec, B . (1991) Biochemistry 30, 2817-2822} . In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis {Wedekind, J . E., Poyner, R . R., Reed, G . H., & Rayment, I . (1994) Biochemistry 33, 9333-9342} . Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared . The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form . All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity . Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle . The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction . For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction . K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP . Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3 . This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product . All three mutant proteins are depressed in their abilities to carry out this reaction . In single-turnover assays, the activities vary in the order K345A > E168Q >> E211Q . These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction. Biochemistry, 1996 Feb 6, 35(5), 1681 - 91 Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-affinity and one low-affinity interaction; Philo JS et al.; Although there is considerable evidence that signaling by the erythropoietin (EPO) receptor is initiated when it is dimerized by binding EPO, it has been previously reported that the soluble extracellular domains of the EPO receptor (sEPOR) are not dimerized in the presence of EPO and are able to form only 1:1 complexes with EPO . We have now shown unambiguously by light scattering, sedimentation equilibrium, and titration calorimetry that two molecules of sEPOR can bind to a single EPO monomer but that the binding of the second sEPOR is approximately 1000-fold weaker than that of the first . Because this second binding interaction is quite weak (Kd of approximately 1 microM), the 2:1 sEPOR.EPO complexes are easily dissociated during chromatography (forming the 1:1 complexes reported previously) and cannot be isolated in pure form . Global analysis of the sedimentation equilibrium data has enabled us to determine the binding constants and is consistent with a model in which EPO has two independent binding sites for sEPOR but cannot exclude anticooperative or sequential binding models . The influence of glycosylation of EPO and/or sEPOR on the binding affinities has also been investigated . Titration calorimetry is consistent with the sedimentation data and shows that the weaker binding site has a more negative delta H . The relation of these results to the binding of EPO to membrane-bound receptors and to the phenomenon of apparent high-affinity and low-affinity classes of receptors is discussed. Biochemistry, 1996 Feb 6, 35(5), 1653 - 63 Engineering specificity for folate into dihydrofolate reductase from Escherichia coli; Posner BA et al.; Despite several similarities in structure and kinetic behavior, the bacterial and vertebrate forms of the enzyme dihydrofolate reductase (DHFR) exhibit differential specificity for folate . In particular, avian DHFR is 400 times more specific for folate than the Escherichia coli reductase . We proposed to enhance the specificity of the E . coli reductase for folate by incorporating discrete elements of vertebrate secondary structure . Two vertebrate loop mutants, VLI and VLII containing 3-7 additional amino acid insertions, were constructed and characterized by using steady-state kinetics, spectrofluorimetric determination of ligand equilibrium dissociation constants, and circular dichroism spectroscopy . Remarkably, the VLI and VLII mutants are kinetically similar to wild-type E . coli reductase when dihydrofolate is the substrate, although VLII exhibits prolonged kinetic hysteresis . Moreover, the VLI dihydrofolate reductase is the first mutant form of E . coli DHFR to display enhanced specificity for folate {(kcat/Km)mutant/(kcat/Km)wt = 13} . A glycine-alanine loop (GAL) mutant was also constructed to test the design principles for the VLI mutant . In this mutant of the VLI reductase, all of the residues from positions 50 to 60, except the strictly conserved amino acids Leu-57 and Arg-60, were converted to either glycine or alanine . A detailed kinetic comparison of the GAL and wild-type reductases revealed that the mutations weaken the binding by both cofactor and substrate by up to 20-fold, but under saturating conditions the enzyme exhibits a kcat value nearly identical to that of the wild type . The rate of hydride transfer is reduced by a factor of 30, with a compensating increase in the dissociation rate for tetrahydrofolate . Although key stabilizing interactions have been sacrificed (it shows no activity toward folate), the maintenance of the correct register between key residues preserves the activity of the enzyme toward its natural substrate . Collectively, neither specific proximal point site mutations nor larger, more distal secondary structural substitutions are sufficient to confer a specificity for folate reduction that matches that observed with the avian enzyme . This is consistent with the hypothesis that the entire protein structure must contribute extensively to the enzyme's specificity. Biochemistry, 1996 Feb 6, 35(5), 1571 - 80 Identification of the Zn(II) site in the copper-responsive yeast transcription factor, AMT1: a conserved Zn module; Farrell RA et al.; The N-terminal metal-binding domains of the copper-activated yeast transcription factors, ACE1 and AMT1, bind to specific DNA sequences in a Cu-dependent fashion . Recombinant AMT1 and ACE1 metal-binding domains are isolated as Cu4Zn1-protein complexes . Site-directed mutagenesis of AMT1 was used in this study to map the ligands of the Cu(I) and Zn(II) ions . The results are consistent with the N-terminal halves of AMT1 and ACE1 consisting of two independent submodules, one binding a single Zn(II) ion and the second binding the tetracopper cluster . The basis of this conclusion is, first, that mutations of two cysteinyl codons and a histidyl codon in the first 42 residues of AMT1 do not alter DNA binding . In contrast, serine substitutions at four cysteine positions at codons 43, 61, 90, and 98 abolish DNA binding . We demonstrated previously that population of the Zn(II) site in AMT1 does not alter the ability of the protein to bind DNA but bound Cu(I) ions are essential for DNA binding {Thorvaldsen, J . L., et al . (1994) Biochemistry 33, 9566-9577} . Second, mutations in the N-terminal 42 residue segment reduce the Zn(II) content of purified mutant AMT1 molecules . Third, a synthetic peptide consisting of the N-terminal 42 residues in AMT1 forms a stable Zn(II) complex and substitution with Co(II) reveals an electronic spectrum identical to that of the Co-substituted intact Cu4AMT1 protein . 113Cd(II) NMR studies reveal that the divalent metal site consists of ligands provided by three cysteinyl thiolates and a single histydyl imidazole . The sequence homology between AMT1, ACE1, and MAC1 in the N-terminal 42 residues suggests that ACE1 and MAC1 will, likewise, contain N-terminal Zn modules . A 42-residue ACE1 synthetic peptide gives identical metal binding properties to the corresponding AMT1 synthetic peptide . Thus, AMT1 and likely ACE1 consist of two contiguous modules, residues 1-42 forming an independent Zn(II) module and residues 43-110 enfolding a tetracopper cluster. Biochemistry, 1996 Feb 6, 35(5), 1417 - 22 Biochemical evidence for the formation of a covalent acyl-phosphate linkage between UDP-N-acetylmuramate and ATP in the Escherichia coli UDP-N-acetylmuramate:L-alanine ligase-catalyzed reaction; Falk PJ et al.; In the peptidoglycan biosynthesis pathway in Escherichia coli, UDP-N-acetylmuramate:L-alanine ligase (MurC) catalyzes the formation of UDP-N-acetylmuramyl-L-alanine . A peptide bond is formed in this reaction and an ATP molecule is hydrolyzed concomitantly to produce ADP and orthophosphate . A biochemical approach was devised to elucidate the role of ATP in this reaction . A fusion construct pMAL::murC was prepared and the maltose binding protein--UDP-N-acetylmuramyl:L-alanine ligase fusion protein was overproduced in E . coli/pMal::murC upon isopropyl beta-thiogalactoside induction . The fusion protein was purified to > or = 90% homogeneity by a single-step affinity chromatography . Subsequently, the ligase was released from the maltose binding protein by proteolytic cleavage and was purified to > or = 95% homogeneity by an ion-exchange chromatographic step . The kinetic parameters of the regenerated ligase are comparable to those of the purified native enzyme . This ligase was used to investigate the role that ATP plays in the formation of UDP-N-acetylmuramyl-L-alanine . UDP-N-acetyl{18O}muramate (with 18O located at the carboxylate function only) was prepared by a combination of chemical and enzymatic processes and was used as the substrate of the ligase to probe the reaction mechanism . All reaction products were purified and subjected to liquid chromatographic-mass spectrometric analysis . A single {18O}oxygen was transferred from UDP-N-acetyl{18O}muramate to the orthophosphate produced in the reaction . No {18O}oxygen was detected in the adenosine nucleotides recovered from the reaction . These results strongly suggest that this ligase-catalyzed peptide formation proceeds through an activated acyl-phosphate linkage during the reaction process . ATP therefore assists in the process of the peptide bond formation by donating its gamma-phosphoryl group to activate the carboxyl group of UDP-N-acetylmuramic acid. Biochemistry, 1996 Feb 6, 35(5), 1408 - 16 Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage, and DNA supercoiling; Bates AD et al.; Binding of the nonhydrolyzable ATP analogue 5'-adenylyl-beta, gamma-imidodiphosphate (ADPNP) to Escherichia coli DNA gyrase can lead to a limited noncatalytic supercoiling of DNA . Here we examine the efficiency of coupling between ADPNP binding and the change in linking number either of positively or negatively supercoiled plasmid DNA or of small DNA circles . The coupling efficiency varies from 100% (delta Lk = -2 per gyrase tetramer, a stoichiometry of 1) with positively supercoiled substrates under certain reaction conditions to an undetectably low value with moderately negatively supercoiled substrates (sigma = -0.046) or small circular substrates . Furthermore, the rate of ADPNP binding to the gyrase-DNA complex is also dependent on the topological state of the DNA; the previously observed slow binding of ADPNP to the complex of gyrase with linear DNA is accelerated 16-fold when the substrate DNA is negatively supercoiled, suggesting a functional interaction between the nucleotide-binding and DNA-binding domains which is independent of the strand-passage process . The implications for the normal ATP-dependent supercoiling reaction of the enzyme are considered and the results discussed in terms of current mechanistic models for DNA gyrase action and the possible in vivo roles of the enzyme. Biochemistry, 1996 Feb 6, 35(5), 1358 - 66 Anti-human FSH receptor monoclonal antibodies: immunochemical and immunocytochemical characterization of the receptor; Vannier B et al.; The extracellular domain of the human FSH receptor was expressed in Escherichia coli as a fusion protein with ubiquitin . It was tagged with a poly-His tract which was used for its purification . Immunization of mice allowed the preparation of high affinity antireceptor monoclonal antibodies . The latter fell into two categories: some of them were inhibited hormone binding and adenylate cyclase activation whereas others were devoid of these properties . None of the antibodies had agonistic activity (i.e., stimulated adenylate cyclase) . Immunoaffinity chromatography allowed us to purify the native receptor in a single step either from a permanently transfected L cell line (75% recovery) or from human ovaries (33% recovery) . Immunoblotting of the receptor in human ovaries showed the presence of a major band of 87 kDa and of a minor band of 81 kDa . Endoglycosidase digestion and pulse-chase experiments showed the former to be the mature receptor and the latter the precursor containing mannose-rich carbohydrates . Thus, as in the case for the LH receptor, there was an accumulation (albeit to a lower degree) of the precursor in target cells . We did not detect variant forms of the protein corresponding to the alternative mRNA transcripts previously described . Additive binding to the receptor of several antibodies, but not of the same antibody, allowed us to establish a sandwich-type ELISA for the receptor (sensitivity approximately 1 fmol) and to obtain evidence against the existence of previously described oligomeric forms of the protein . All monoclonal antibodies were able to label the receptor immunocytochemically in transfected cells, and two of them were also able to detect it at the markedly lower physiological concentrations, i.e., in human Sertoli and granulosa cells. Biochemistry, 1996 Feb 6, 35(5), 1342 - 51 (E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB); Lees WJ et al.; UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-isomers of enolbutyryl-UDP-GlcNAc, C4 analogs of the physiological C3 enolpyruvyl substrate, to UDP-methyl-N-acetylmuramic acid in the presence of NADPH . The X-ray crystal structure of the (E)-enolbutyryl-UDP-GlcNAc-MurB complex is similar to that of the enolpyruvyl-UDP-GlcNAc-MurB complex . In both structures the groups thought to be involved in hydride transfer to C3 and protonation at C2 of the enol ether substrate are arranged anti relative to the enol double bond . The stereochemical outcome of reduction of (E)-enolbutyryl-UDP-GlcNAc by NADPD in D2O is thus predicted to yield a (2R,3R)-dideuterio product . This was validated by conversion of the 2,3-dideuterio-UDP-methyl-N-acetylmuramic acid product to 2,3-dideuterio-2-hydroxybutyrate, which was shown to be (2R) by enzymatic analysis and (3R) by NMR comparison to authentic (2R,3R)- and (2R,3S)-2,3-dideuterio-2-hydroxybutyrate . Remarkably, the (E)-enolbutyryl-UDP-GlcNAc was found to partition between reduction to UDP-methyl-N-acetylmuramic and isomerization to the (Z)-substrate isomer in the MurB active site, indicative of a C2 carbanion/enol species that is sufficiently long-lived to rotate around the C2-C3 single bond during catalysis. Biochem Biophys Res Commun, 1996 Feb 6, 219(1), 213 - 8 Reversible red-ox reactions of the diiron site in the mouse ribonucleotide reductase R2 protein; Davydov A et al.; The red-ox reactions of the dinuclear iron center of mouse R2 protein upon interaction with different reductants (dithionite alone and with mediators) and oxidants (PES, methylene blue, hydrogen peroxide and para-benzoquinone) have been studied by EPR and optical spectroscopy . The obtained results indicate that the transitions between Fe(III)Fe(III), Fe(II)Fe(III) and Fe(II)Fe(II) states of the dinuclear iron center are reversible and the mu-oxo-bridge may be formed upon oxidation by non-oxygen oxidants . In contrast to the case for the E . coli R2 protein, dithionite alone reduces the tyrosyl radical and diiron center in mouse R2 protein. Biochem Biophys Res Commun, 1996 Feb 6, 219(1), 173 - 9 Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity; Weiss S et al.; PrP27-30 represents the protease-resistant core of the prion protein and was found to be the main component in Scrapie prion preparations . Recombinant (r) PrP27-30 corresponding to aa 90-231 from the Syrian golden hamster prion protein was expressed as a fusion with GST in E . coli and secreted from insect cells infected with recombinant baculoviruses, GST::rPrP27-30 isolated from either system was purified to homogenity by glutathione-Sepharose chromatography . rPrP27-30 from both systems was generated by direct cleavage of GST::rPrP27-30 in the presence of thrombin revealing a molecular weight of 17 kDa . GST::rPrP27-30 as well as the authentic protein rPrP27-30 were identified by immunoblotting employing a polyclonal antibody directed against a peptide corresponding to aa 95-110 of the Syrian golden hamster prion protein . In contrast to scrapie prior PrP27-30, the recombinant proteins GST::rPrP27-30 and rPrP27-30 were both sensitive towards proteinase K, suggesting that the molecules lack infectivity. Biochem Biophys Res Commun, 1996 Feb 6, 219(1), 168 - 72 Identification of the lysosomal membrane glycoprotein Lamp-1 as a receptor for type-1-fimbriated (mannose-specific) Escherichia coli; Karlsson A et al.; The presence of several glycosylated sites with high-mannose oligosaccharides on the lysosome-associated membrane glycoproteins (Lamps)_ combined with the fact that neutrophil Lamps are present in mobilizable organelles inspired us to investigate their ability to bind type-1 fimbriated (mannose-binding) Escherichia coli and subsequently define a potential function for the Lamps in human neutrophils . Bacterial binding to Lamps purified from chronic myeloic leukemia cells was investigated by separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transfer to a blotting membrane and overlay with type-1-fimbriated bacteria . The overlays were developed by growth . The bacteria bound readily to Lamp-1 while there was almost no binding to Lamp-2 . Hence, we state that a possible function for neutrophil Lamp-1 is bacterial binding. Biochem Biophys Res Commun, 1996 Feb 6, 219(1), 134 - 9 DNA-helicase activity from sea urchin mitochondria; Roberti M et al.; As a step toward the characterization of the main components of mitochondrial DNA replication apparatus in sea urchin, we report the identification of a DNA-helicase activity in Paracentrotus lividus mitochondria . The activity was detected in a protein fraction obtained by fractionating on DEAE-Sephacel a lysate of gradient purified mitochondria from paracentrotus lividus eggs . The mitochondrial helicase unwound, in the presence of ATP and Mg++, a 39-base oligonucleotide annealed to single-stranded M13mp18 (+) DNA . Its direction of movement is 3' to 5' with respect to the single stranded portion of the partial duplex DNA substrate . This polarity is similar to that exhibited by the Escherichia coli rep helicase and by the helicase from bovine brain mitochondria . These features suggest that the sea urchin mitochondrial helicase could function in enabling the polymerization of the H-strand during mitochondrial DNA replication. Biochem Biophys Res Commun, 1996 Feb 6, 219(1), 116 - 21 cDNA sequence analysis and expression of cardiotoxins from Taiwan Cobra; Chang LS et al.; The cDNAs encoding cardiotoxins I, III and N were constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription-polymerase chain reaction . A high degree of sequence homology was observed with the three cardiotoxins . The cardiotoxins were subcloned in the expression vector pET 20b(+) and transformed in BL 21(DE3) E . coli strain . The expressed protein was isolated from the inclusion bodies of E . coli and purified by reversed phase high performance liquid chromatography . The purified recombinant cardiotoxin III showed an immunoreactivity with anti-cardiotoxin III antibodies as revealed by immunoblot analysis. Biochem Biophys Res Commun, 1996 Feb 6, 219(1), 111 - 5 Glu-416 of beta-galactosidase (Escherichia coli) is a Mg2+ ligand and beta-galactosidases with substitutions for Glu-416 are inactivated, rather than activated, by MG2+; Roth NJ et al.; Glu-416 of beta-galactosidase (E . coli) was replaced with Gln and Val using site-directed mutagenesis . The substituted enzymes displayed a greatly decreased sensitivity to Mg2+ . Equilibrium dialysis studies indicated that wild-type beta-galactosidase bound Mg2+ tightly, whereas E416V-beta-galactosidase did not . In addition, the pH profile of E416V-beta-galactosidase was unaffected by the presence or absence of 1 mM Mg2+ . Surprisingly, both substituted enzymes were inactivated, rather than activated, by Mg2+ but high amounts of Mg2+ were needed (1 mM) . E416Q-beta-galactosidase was unstable when stored in the presence of Mg2+ . The substituted enzymes displayed a dramatically lowered affinity for the synthetic substrate, ONPG, and for IPTG (a substrate analog inhibitor) in both the presence and the absence of Mg2+. Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1340 - 5 Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle; Gong MC et al.; The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-{gamma-thio}triphosphate (GTP{gamma-S})-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle . A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein . The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100 . ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP {in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles}, by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP{gamma-S} at constant Ca2+ concentrations . AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-{beta-thio}diphosphate (GDP{beta-S}) and by EDIN . EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum . ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization . We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase. Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1292 - 7 Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli; Mellon I et al.; To improve our understanding of the mechanism that couples nucleotide-excision repair to transcription in expressed genes, we have examined the effects of mutations in several different DNA repair genes on the removal of cyclobutane pyrimidine dimers from the individual strands of the induced lactose operon in UV-irradiated Escherichia coli . As expected, we found little repair in either strand of the lactose operon in strains with mutations in established nucleotide excision-repair genes (uvrA, uvrB, uvrC, or uvrD) . In contrast, we found that mutations in either of two genes required for DNA-mismatch correction (mutS and mutL) selectively abolish rapid repair in the transcribed strand and render the cells moderately sensitive to UV irradiation . Similar results were found in a strain with a mutation in the mfd gene, the product of which has been previously shown to be required for transcription-coupled repair in vitro . Our results demonstrate an association between mismatch-correction and nucleotide-excision repair and implicate components of DNA-mismatch repair in transcription-coupled repair . In addition, they may have important consequences for human disease and may enhance our understanding of the etiology of certain cancers which have been associated with defects in mismatch correction. Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1226 - 31 Isolation of an oxygen-sensitive FNR protein of Escherichia coli: interaction at activator and repressor sites of FNR-controlled genes; Melville SB et al.; The Escherichia coli fnr gene product, FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth . FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state . To investigate this process, a fnr mutant that encodes an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis . In vivo, the mutant behaved like a wild-type strain under anaerobic conditions but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth . The altered fur gene was overexpressed in E . coli and the resultant FNR protein was purified to near homogeneity by using anaerobic chromatography procedures . An in vitro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein . The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mol of Fe per FNR monomer . In vitro DNase I protection studies were performed to establish the locations of the FNR-binding sites at the narG, narK, dmsA, and hemA promoters that are regulated by either activation or repression of their transcription . The sizes of the DNA footprints are consistent with the binding of two monomers of FNR that protect the symmetrical FNR-recognition sequence TTGAT-nnnnATCAA . Exposure of the FNR protein or protein-DNA complex to air for even short periods of time (approximately 5 min) led to the complete loss of DNA protection at a consensus FNR recognition site . A model whereby the FNR protein exists in the cell as a monomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed. Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1173 - 7 Transcriptional activation by protein-induced DNA bending: evidence for a DNA structural transmission model; Parekh BS et al.; Integration host factor (IHF) is a DNA-bending protein that binds to an upstream activating sequence (UAS1) and, on a negatively supercoiled DNA template, activates transcription from the ilvPG promoter of the ilvG-MEDA operon of Escherichia coli . The transcriptional initiation site of the ilvGMEDA operon is located 92 bp downstream of UAS1 . Activation is still observed when the orientation of the upstream IHF binding site is reversed . This manipulation places the IHF binding site on the opposite face of the DNA helix, directs the IHF-induced DNA bend in the opposite direction, and presents the opposite face of the nonsymmetrical, heterodimeric, IHF molecule to the downstream RNA polymerase . Lymphoid enhancer-binding factor, LEF-1, is a DNA-bending, lymphoid-specific, mammalian transcription factor that shares no amino acid sequence similarity with IHF . When the IHF site in UAS1 is replaced with a LEF-1 site, LEF-1 activates transcription from the downstream ilvPG promoter in E . coli as well as it is activated by its natural activator, IHF . These results suggest that specific interactions between IHF and RNA polymerase are not required for activation . The results of DNA structural studies show that IHF forms a protein-DNA complex in the UAS1 region that, in the absence of RNA polymerase, alters the structure of the DNA helix in the -10 hexanucleotide region of the downstream ilvPG promoter . The results of in vitro abortive transcription assays show that IIIF also increases the apparent rate of RNA polymerase isomerization from a closed to an open complex . We suggest, therefore, that IHF activates transcription by forming a higher-order protein-DNA complex in the UAS1 region that structurally alters the DNA helix in a way that facilitates open complex formation at the downstream ilvPG promoter site. Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1141 - 5 Differential functions of the two Src homology 2 domains in protein tyrosine phosphatase SH-PTP1; Pei D et al.; SH-PTP1 (also known as PTP1C, HCP, and SHP) is a non-transmembrane protein tyrosine phosphatase (PTPase) containing two tandem Src homology 2 (SH2) domains . We show here that the two SH2 (N-SH2 and C-SH2) domains in SH-PTP1 have different functions in regulation of the PTPase domain and thereby signal transduction . While the N-terminal SH2 domain is both necessary and sufficient for autoinhibition through an intramolecular association with the PTPase domain, truncation of the C-SH2 domain {SH-PTP1 (delta CSH2) construct} has little effect on SH-PTP1 activity . A synthetic phosphotyrosine residue (pY) peptide derived from the erythropoietin receptor (EpoR pY429) binds to the N-SH2 domain and activates both wild-type SH-PTP1 and SH-PTP1 (delta CSH2) 60- to 80-fold . Another pY peptide corresponding to a phosphorylation site on the IgG Fc receptor (Fc gamma RIIB1 pY309) associates with both the C-SH2 domain (Kd = 2.8 microM and the N-SH2 domain (Kd = 15.0 microM) and also activates SH-PTP1 12-fold . By analysis of the effect of the Fc gamma RIIB1 pY309 peptide on SH-PTP1 (delta CSH2), SH-PTP1 (R30K/R33E), SH-PTP1 (R30K/R136K), and SH-PTP1 (R136K) mutants in which the function of either the N- or C-SH2 domain has been impaired, we have determined that both synthetic pY peptides stimulate SH-PTP1 by binding to its N-SH2 domain; binding of pY ligand to the C-SH2 domain has no effect on SH-PTP1 activity . We propose that the N-terminal SH2 domain serves both as a regulatory domain and as a recruiting unit, whereas the C-terminal SH2 domain acts merely as a recruiting unit. Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1006 - 11 A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity; Tamura T et al.; We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441 . Cells were grown in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 microM {75Se}selenite . A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose . The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively . The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3 . It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein . The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx) . The specific activity was determined to be 31 units/mg by DTNB assay . Apparent Km values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 microM, respectively . DTNB reduction was inhibited by 0.2 mM arsenite . Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays . The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR. Mol Gen Genet, 1996 Feb 5, 250(2), 237 - 9 Binding specificity and tissue-specific expression pattern of the Arabidopsis bZIP transcription factor TGA2; de Pater S et al.; The binding specificity and tissue-specific expression pattern of TGA2 (AHBP-1b), an Arabidopsis bZIP transcription factor have been determined . Filter-binding and gel-shift assays showed that TGA2 has high affinity for C-boxes (ATGACGTCAT) . In this respect TGA2 is similar to other members of the Arabidopsis TGA family (such as TGA1, TGA3 and OBF4) and to tobacco TGA1a . Genomic Southern blot analysis confirmed that TGA2 is a member of the gene family . Northern blot analysis showed that the gene is expressed at similar levels in root, stem, leaf and flower t at somewhat lower levels in siliques . TGA3 was also found to be expressed at the same level throughout the plant, whereas genes encoding TGA1 and OBF4 have relatively high RNA expression levels in root . The differential expression of these genes suggests that they have distinct functions. FEBS Lett, 1996 Feb 5, 379(3), 295 - 8 Altered voltage sensitivity of mutant OmpC porin channels; Bishop ND et al.; Single OmpC porin channels have been reconstituted in planar bilayer membranes . Wild-type OmpC forms trimers which are largely insensitive to voltages below 250 mV.A single-point mutation of the ompC gene has been prepared resulting in replacement of Trp56 by Cys in the eyelet region of the channel wall in a highly conserved segment of the polypeptide . The monomeric channels of which the trimer is composed have smaller conductivity in 1 M NaCl (400 +/- 20 pS, mean and S.E.M., n=30) and increased voltage sensitivity by comparison with the wild-type under similar conditions, whereas other (Dex) mutants form larger channels and display different behaviour . Further, by treatment in SDS solutions at different temperatures, the W56C mutant has been shown to be less stable than either the wild-type or the Dex mutants. Gene, 1996 Feb 2, 168(1), 43 - 8 Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menE gene from Escherichia coli; Sharma V et al.; In Escherichia coli, the biosynthesis of the electron carrier menaquinone (vitamin K2) involves at least seven identified enzymatic activities, five of which are encoded in the men cluster . One of these, the conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid, requires the formation of o-succinylbenzoyl-CoA (OSB-CoA) as an intermediate . Formation of the intermediate is mediated by OSB-CoA synthetase encoded by the menE locus known to be located either 5' of menB, or 3' of menC . A DNA fragment overlapping the 3' end of menC in shown by enzymatic complementation to elevate OSB-CoA synthetase activity . Nucleotide sequence analysis of the fragment identified a 1.355-kb open reading frame (ORF) which, when deleted at either the 5' or 3' end, failed to generate increased enzymatic activity . The ORF is preceded by a consensus ribosome-binding site, but no apparent sigma-70 promoter . An oppositely transcribed unidentified gene cluster follows the menE ORF . The region 5' of menB contains an an additional ORF of unknown function (orf241) and establishes the order of genes in the men cluster as menD, orf241, menB, menC and menE . All loci are transcribed counter-clockwise. Gene, 1996 Feb 2, 168(1), 37 - 41 A tightly regulated expression system in Escherichia coli with SP6 RNA polymerase; Sagawa H et al.; A tightly regulated gene-expression system was developed using SP6 RNA polymerase (RpoSP6) . The RpoSp6-encoding gene (rpoSP6) was inserted into a mini-F plasmid (mini-F) and expression was controlled by the lactose promoter (P(lac)) and operator (O(lac)) on the plasmid . Therefore, a controlled expression system for the target genes can easily be constructed in various host strains by co-transformation of the system plasmid pFSP6 with other vector plasmids containing the genes linked to the SP6 promoters (P(SP6)) . Using the lac gene linked to P(SP6) as a reporter, we evaluated the regulation of expression in this system in various host strains . Low-level expression of lac was detected in Escherichia coli harboring this expression system when RpoSP6 was uninduced, although very low activities of beta-galactosidase (beta-Gal) were observed which were independent of the presence of pFSP6 . This basal level of beta-Gal activity was possibly derived, because the P(SP6) element has very weak activity for E . coli RNA polymerase (Rpo) . These results showed that RpoSP6 seemed to be produced at very low levels in uninduced cells . Beta-GAl activity increased about 18-32-fold when the expression of rpoSP6 was induced, as compared with the beta-Gal activity when uninduced . The tight regulation of this system is superior to that of other known systems and it has a considerable advantage for gene expression in E . coli. Gene, 1996 Feb 2, 168(1), 15 - 21 Sequence and expression of an isocitrate dehydrogenase-encoding gene from a polycyclic aromatic hydrocarbon oxidizer, Sphingomonas yanoikuyae B1; Wang Y et al.; An 18.5-kb DNA fragment was cloned from Sphingomonas yanoikuyae (Sy) B1 (previously Beijerinckia B1) . Analysis of a 4.3-kb sequence revealed an isocitrate dehydrogenase (Idh)-encoding gene (idhA), an unidentified open reading frame (ORF) and a partial glucosamine synthetase-encoding ORF (glmS) . As in a number of bacteria, Tn7 insertion was found specifically at a site past the stop codon of glmS . The predicted 406-amino-acid sequence of IdhA shows, for the first time, an extensive sequence identity (66%) with an eukaryotic NADP+-specific Idh . The idhA gene was expressed in Escherichia coli . Identical restriction fragments carrying idhA were found in B1, Sy IFO15102 and Sy Q1 (formerly S . paucimobilis Q1), indicating a well-conserved idh gene. J Biol Chem, 1996 Feb 2, 271(5), 2856 - 62 CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases; Bucurenci N et al.; CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues . The protein exhibits little overall sequence similarities with other known NMP kinases . However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases . The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties . The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution . The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment . Substrate specificity studies showed that CMP kinase from E . coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors . This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides . Binding of CMP enhanced the affinity of E . coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate. J Biol Chem, 1996 Feb 2, 271(5), 2844 - 50 cDNA cloning, expression, and mutagenesis study of leukotriene B4 12-hydroxydehydrogenase; Yokomizo T et al.; Leukotriene B4 12-hydroxydehydrogenase catalyzes the conversion of leukotriene B4 into its biologically less active metabolite, 12-oxo-leukotriene B4 . This is an initial and key step of metabolic inactivation of leukotriene B4 in various tissues other than leukocytes . Here we report the cDNA cloning for porcine and human enzymes from kidney cDNA libraries . A full-length cDNA of the porcine enzyme contains an open reading frame consisting of 987 base pairs, corresponding to 329 amino acids . The human enzyme showed a 97.1% homology with the porcine enzyme . Northern blotting of human tissues revealed its high expression in the kidney, liver, and intestine but not in leukocytes . The porcine enzyme was expressed as a glutathione S-transferase fusion protein in Escherichia coli, which exhibited similar characteristics with the native enzyme . Because the enzymes have a homology, in part, with NAD(P)(+)-dependent alcohol dehydrogenases, a site-directed mutagenesis study was carried out . We found that three glycines at 152, 155, and 166 have crucial roles in the enzyme activity, possibly by producing an NADP+ binding pocket. J Biol Chem, 1996 Feb 2, 271(5), 2812 - 6 Cardiac myotrophin exhibits rel/NF-kappa B interacting activity in vitro; Sivasubramanian N et al.; Myotrophin is a soluble-12 kilodalton protein isolated from hypertrophied spontaneously hypertensive rat and dilated cardiomyopathic human hearts . We have recently cloned the gene coding for myotrophin and expressed it in Escherichia coli . In the present study, the expression of myotrophin gene was analyzed, and at least seven transcripts have been detected in rat heart and in other tissues . We have further analyzed the primary structure of myotrophin protein and identified significant new structural and functional domains . Our analysis revealed that one of the ankyrin repeats of myotrophin is highly homologous specifically to those of myotrophin is highly homologous specifically to those of I kappa B alpha/rel ankyrin repeats . In addition, putative consensus phosphorylation sites for protein kinase C and casein kinase II, which were observed in I kappa B alpha proteins, were identified in myotrophin . To verify the significance of these homologies, kappa B gel shift assays were performed with Jurkat T cell nuclear extract proteins and the recombinant myotrophin . Results of these assays indicate that the recombinant myotrophin has the ability to interact with NF-kappa B/rel proteins as revealed by the formation of ternary protein-DNA complexes . While myotrophin-specific antibodies inhibited the formation of these complexes, rel-specific p50 and p65 antibodies supershifted these complexes . Thus, these results clearly indicate that the myotrophin protein to be a unique rel/NF-kappa B interacting protein. J Biol Chem, 1996 Feb 2, 271(5), 2762 - 8 DNA binding and dimerization of the Fe-S-containing FNR protein from Escherichia coli are regulated by oxygen; Lazazzera BA et al.; The transcription factor FNR from Escherichia coli regulates transcription of genes in response to oxygen deprivation . To determine how the activity of FNR is regulated by oxygen, a form of FNR had to be isolated that had properties similar to those observed in vivo . This was accomplished by purification of an FNR fraction which exhibited enhanced DNA binding in the absence of oxygen . Iron and sulfide analyses of this FNR fraction indicated the presence of an Fe-S cluster . To determine the type of Fe-S cluster present, an oxygen-stable mutant protein LH28-DA154 was also analyzed since FNR LH28-DA154 purified anoxically contained almost 3-fold more iron and sulfide than the wild-type protein . Based on the sulfide analysis, the stoichiometry (3.3 mol of S2-/FNR monomer) was consistent with either one {4Fe-4S} or two {2Fe-2S} clusters per mutant FNR monomer . However, since FNR has only four Cys residues as potential cluster ligands and an EPR signal typical of a 3Fe-4S cluster was detected on oxidation, we conclude that there is one {4Fe-4S} cluster present per monomer of FNR LH28-DA154 . We assume that the wild type also contains one {4Fe-4S} cluster per monomer and that the lower amounts of iron and sulfide observed per monomer were due to partial occupancy . Consistent with this, the Fe-S cluster in the wild-type protein was found to be extremely oxygen-labile . In addition, molecular-sieve chromatographic analysis showed that the majority of the anoxically purified protein was a dimer as compared to aerobically purified FNR which is a monomer . The loss of the Fe-S cluster by exposure to oxygen was associated with a conversion to the monomeric form and decreased DNA binding . Taken together, these observations suggest that oxygen regulates the activity of wild-type FNR through the lability of the Fe-S cluster to oxygen. J Biol Chem, 1996 Feb 2, 271(5), 2599 - 603 Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist; Proudfoot AE et al.; Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist . RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases . When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases . This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1 . However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1 alpha (MIP-1 alpha) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines . T cell chemotaxis was similarly inhibited . The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced responses in these cells . Met-RANTES can compete with both {125I}RANTES and {125I}IMP-1 alpha binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1 . These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation. J Biol Chem, 1996 Feb 2, 271(5), 2589 - 93 Sequence determinants of C-terminal substrate recognition by the Tsp protease; Keiler KC et al.; Cytochrome b562 is not cleaved by the tail-specific protease Tsp in vitro or in the periplasm of Escherichia coli but becomes a good substrate when the C-terminal sequence WVAAA is added . Following randomization of the final three residue positions of this substrate, 54 different mutants with single residue substitutions were recovered . The steady-state expression levels of cytochrome variants bearing these mutant tails were similar in an E . coli strain deleted for the tsp gene but differed markedly in a strain containing Tsp . Wild-type cytochrome b562 and seven variants, displaying a range of intracellular expression levels, were purified . These proteins were found to have the same Tm values in thermal denaturation experiments but to be cleaved by Tsp at rates differing by as much as 30-fold . Overall, the rates of Tsp cleavage of these proteins in vitro correlate with their rates of cleavage in vivo as determined by pulse-chase experiments . These results indicate that the C-terminal sequence of the cytochrome-b562 variants is important in determining their proteolytic fate in the cell and show that this degradation is mediated predominantly by Tsp . There are different selectivity rules at each of the three C-terminal positions . The identity of the C-terminal residue of the substrate, where small, uncharged residues (Ala, Cys, Ser, Thr, Val) are preferred, is most important in determining the rate of substrate cleavage by Tsp . Non-polar residues are also preferred at the second and third positions, but larger and more hydrophobic side chains are also acceptable at these positions in good substrates. J Biol Chem, 1996 Feb 2, 271(5), 2578 - 88 High complexity in the expression of the B' subunit of protein phosphatase 2A0 . Evidence for the existence of at least seven novel isoforms; Csortos C et al.; Association of the catalytic subunit (C2) with a variety of regulatory subunits is believed to modulate the activity and specificity of protein phosphatase 2A (PP2A) . In this study we report the cloning and expression of a new family of B-subunit, the B', associated with the PP2A0 form . Polymerase chain reactions and cDNA library screening have identified at least seven cDNA isotypes, designated alpha, beta 1, beta 2, beta 3, beta 4, gamma, and delta . The different beta subtypes appear to be generated by alternative splicing . The deduced amino acid sequences of the alpha, beta 2, beta 3, beta 4 and gamma isoforms predict molecular weights of 57,600, 56,500, 60,900, 52,500, and 68,000, respectively . The proteins are 60-80% identical and differ mostly at their termini . Two of the isoforms, B' beta 3 and B' gamma, contain a bipartite nuclear localization signal in their COOH terminus . No homology was found with other B- or B- related subunits . Northern analyses indicate a tissue-specific expression of the isoforms . Expression of B' alpha protein in Escherichia coli generated a polypeptide of approximately 53 kDa, similar to the size of the B' subunit present in the purified PP2A0 . The recombinant protein was recognized by antibody raised against native B' and interacted with the dimeric PP2A (A.C2) to generate a trimeric phosphatase . The deduced amino acid sequences of the B' isoforms show significant homology to mammalian, fungal, and plant nucleotide sequences of unknown function present in the data bases . Notably, a high degree of homology (55-66%) was found with a yeast gene, RTS1, encoding a multicopy suppressor of a rox3 mutant . Our data indicate that at least seven B' subunit isoforms may participate in the generation of a large number of PP2A0 holoenzymes that may be spatially and/or functionally targeted to different cellular processes. J Biol Chem, 1996 Feb 2, 271(5), 2557 - 62 Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli; Rech S et al.; The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of a high affinity molybdate uptake system . Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a periplasmic binding protein based on the presence of a leader-like sequence at its N terminus . To examine the properties of the ModA protein, the modA structural gene was overexpressed, and its product was purified . The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock . The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell . The apparent size of ModA is 31.6 kDa as determined by gel sieve chromatography, whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel electrophoresis . A ligand-dependent protein mobility shift assay was devised using a native polyacrylamide gel electrophoresis protocol to examine binding of molybdate and other anions to the ModA periplasmic protein . Whereas molybdate and tungstate were bound with high affinity (approximately 5 microM), sulfate, chromate, selenate, phosphate, and chlorate did not bind even when tested at 2 mM . A UV spectral assay revealed apparent Kd values of binding for molybdate and tungstate of 3 and 7 microM, respectively . Strains defective in the modA gene were unable to transport molybdate unless high levels of the anion were supplied in the medium . Therefore the modA gene product is essential for high affinity molybdate uptake by the cell . Tungstate interference of molybdate acquisition by the cell is apparently due in part to the high affinity of the ModA protein for this anion. J Biol Chem, 1996 Feb 2, 271(5), 2491 - 6 Beta*, a UV-inducible shorter form of the beta subunit of DNA polymerase III of Escherichia coli . II . Overproduction, purification, and activity as a polymerase processivity clamp; Skaliter R et al.; Control elements located inside the coding sequence of dnaN, the gene encoding the beta subunit of DNA polymerase III holoenzyme, direct the synthesis of a shorter and UV-inducible form of the beta subunit (Skaliter, R., Paz-Elizur, T., and Livneh, Z . (1996) J . Biol . Chem . 271, 2278-2281, and Paz-Elizur, T., Skaliter, R., Blumenstein, S., and Livneh, Z . (1996) J . Biol . Chem . 271, 2282-2290) . The protein, termed beta*, was overproduced using the phage T7 expression system, leading to its accumulation as inclusion bodies at 5-10% of the total cellular proteins . beta* was purified in denatured form, followed by refolding to yield a preparation > 95% pure . Denatured beta* had a molecular mass of 26 kDa and contained two isoforms when analyzed by two-dimensional gel electrophoresis . The major isoform had a pI of 5.45, and comigrated with cellular beta* . Size exclusion high performance liquid chromatography under nondenaturing conditions and chemical cross-linking experiments indicate that beta* is a homotrimer . DNA synthesis by DNA polymerase III* was stimulated up to 10-fold by beta*, primarily due to an increase in the processivity of polymerization . It is suggested that beta* functions as an alternative sliding DNA clamp in a process associated with DNA synthesis in UV-irradiated cells. J Biol Chem, 1996 Feb 2, 271(5), 2482 - 90 Beta*, a UV-inducible smaller form of the beta subunit sliding clamp of DNA polymerase III of Escherichia coli . I . Gene expression and regulation; Paz-Elizur T et al.; The 40.6-kDa beta subunit of DNA polymerase III of Escherichia coli is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity (Stukenberg, P . T., Studwell-Vaughan, P . S., and O'Donnell, M . (1991) J . Biol . Chem . 266, 11328-11334) . UV irradiation of E . coli induces a smaller 26-kDa form of the beta subunit, termed beta*, that, when overproduced from a plasmid, increases UV resistance of E . coli (Skaliter, R., Paz-Elizur, T., and Livneh, Z . (1996) J . Biol . Chem . 271, 2478-2481) . Here we show that this protein is synthesized from a UV-inducible internal gene, termed dnaN*, that is located in-frame inside the coding region of dnaN, encoding the beta subunit . The initiation codon and the Shine-Dalgarno sequence of dnaN* were identified by site-directed mutagenesis . The dnaN* transcript was shown to be induced upon treatment with nalidixic acid, and transcriptional dnaN*-cat gene fusions were UV inducible, suggesting induction of dnaN* at the transcriptional level . Analysis of translational dnaN*-lacZ gene fusions revealed that UV induction was abolished in strains carrying the recA56, lexA3, or delta rpoH mutations, indicating involvement of both SOS and heat shock stress responses in the induction process . Expression of dnaN* represents a strategy of producing several proteins with related functional domains from a single gene. J Biol Chem, 1996 Feb 2, 271(5), 2478 - 81 A smaller form of the sliding clamp subunit of DNA polymerase III is induced by UV irradiation in Escherichia coli; Skaliter R et al.; The beta subunit of DNA polymerase III holoenzyme of Escherichia coli is a 40.6-kDa protein that functions as a sliding DNA clamp (Stukenberg, P . T., Studwell-Vaughan, P . S., and O'Donnell, M . (1991) J . Biol . Chem . 266, 11328-11334) . It is responsible for tethering the polymerase to DNA and endowing it with the high processivity required for DNA replication . Here and in a companion study (Paz-Elizur, T., Skaliter, R., Blumenstein, S., and Livneh, Z . (1996) J . Biol . Chem . 271, 2482-2490) we report that the dnaN gene, encoding the beta subunit, contains an internal in-frame gene, termed dnaN*, that encodes a smaller form of the beta subunit . The novel 26-kDa protein, termed beta*, is UV-inducible, and when overexpressed from a plasmid under an inducible promoter, it increases up to 6-fold the UV resistance of E . coli cells . These findings suggest that the beta* protein functions in a reaction associated with DNA repair or recovery of DNA replication in UV-irradiated cells. J Mol Biol, 1996 Feb 2, 255(4), 604 - 16 The elastic I-band region of titin is assembled in a "modular" fashion by weakly interacting Ig-like domains; Politou AS et al.; The vertebrate striated muscle protein titin is thought to play a critical role in myofibril assembly and passive tension . The recently determined complete primary structure of titin revealed a modular architecture that opens the way to a structural characterisation and the understanding of essential properties of this molecule through dissection into units that are structurally and/or functionally relevant . To understand the assembly process of titin, and ultimately the molecular basis of its elastic behaviour, we studied the thermodynamic properties of module pairs, the smallest structural unit that includes a module-module interface . Thus, selected module pairs and their component single modules from the I-band part of the titin molecule were expressed in Escherichia coli and their heat-induced and denaturant-induced unfolding was investigated with a combination of techniques (circular dichroism, fluorescence spectroscopy and nuclear magnetic resonance) . The stabilities of single modules and pairs were determined from denaturation experiments . The module interface was also modelled on the basis of the sequence alignment of all approximately 40 immunoglobulin like modules from the I-band and the known structure of one of them . Our results show that all modules and module pairs examined are independently folded in solution . When covalently linked, although weakly interacting, they still behave as autonomous co-operative units upon unfolding . These observations lead us to suggest that folding of titin in vitro is a hierarchical event and that weak interactions between its adjacent modules must only partly account for its presumed elastic function. J Mol Biol, 1996 Feb 2, 255(4), 559 - 63 Direct measurement of the association of a protein with a family of membrane receptors; Evans LJ et al.; A specific receptor is a requirement for most protein toxins and OmpF, a trimeric porin, was previously considered to be the unique membrane-receptor for colicin N . We show by qualitative in vivo analysis that the related porins OmpC or PhoE act as much less effective receptors . To elucidate receptor function, the in vitro binding of the 42 kDa toxin to each of the 120 kDa porin trimers was determined quantitatively using isothermal titration calorimetry . Colicin N binds to OmpF with Ka approximately 5 x 10(5) M-1 and a stoichiometry consistent with about three per trimer but it also binds to PhoE and OmpC with surprisingly similar affinities and stoichiometry . However, thermodynamic analysis of these hitherto unmeasured interactions suggests an unexpected entropic difference between these protein import receptors. Tokai J Exp Clin Med, 1996 Feb, 21(1), 25 - 31 Infected aneurysms--clinical study of 5 cases; Fujimura T et al.; The infected aneurysm has been assumed to be a disease with a poor prognosis due to the occurrence of aneurysmal ruptures and sepsis, in contrast to the outcome of atherosclerotic aneurysms . In the present study, we conducted surgical treatment on five patients with infected aneurysms (infected abdominal aortic aneurysm in three cases and iliac artery aneurysm in two cases) . In particular, two of the three patients suffering from infected abdominal aortic aneurysms underwent extra-anatomic bypass and the remaining one case underwent vascular graft replacement in situ . In the two patients who underwent an extra-anatomic bypass, an aneurysm was found at the site of aortic stump closure . In the patient who underwent in situ replacement, wrapping was carried out using the omentum after vascular graft replacement, and the postoperative course was uneventful . Accordingly, we consider that the optimum primary therapeutic intervention for infected aneurysms is in situ revascularization followed by wrapping with the omentum after removal of the aneurysm and debridement of the surrounding infected tissue to the maximum extent possible. Mol Divers, 1996 Feb, 1(2), 97 - 108 Directed evolution studies with combinatorial libraries of T4 lysozyme mutants; Patten PA et al.; Gene duplication with divergence to new functions has been an important mechanism in protein evolution . However, the questions of how many new functions can arise from a particular ancestral gene and how many mutational steps are typically required to generate new functions have been difficult to approach experimentally . We have addressed these questions using T4 lysozyme as a model system by synthesizing two combinatorial libraries of > 10(7) mutant T4 lysozyme genes: one library with an average of 14 missense mutations spread throughout the gene and one library in which 13 active site residues have been simultaneously randomized . These libraries were placed under selection in lacZ or pheA deficient strains of E . coli to investigate whether they sample sufficient diversity to contain mutants with acquired beta-galactosidase or prephenate dehydratase activities . Although neither selection yielded T4 lysozyme mutants with these new activities, a novel E . coli locus was cloned that weakly complements these mutants, allowing them to form 1 mm colonies in 4-6 weeks . This growth rate corresponds to a turnover number of approximately 1000 or 25 min-1 for the lacZ or pheA complementation systems, respectively, thus defining the limits of evolved enzymatic activity detectable in these selections . Thus, the strong selective pressure uncovered an unexpected solution to the biochemical blocks, a frequently observed phenomenon in selection experiments . The characterization of this locus will allow its elimination from future E . coli complementation schemes. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1996 Feb, 18(1), 60 - 5 {The study of BL21(DE3)/PET-11 as an expression system of human IL-2}; Gong M et al.; The coding region of hIL-2 cDNA is specifically amplified by PCR . A Nde I site is introduced into its 5'-end and its 3'-non coding region is partly deleted . After filling-in by Klenow enzyme, the DNA fragment is cloned into Sma I site of PUC18 . Eight white colonies are screened and one (C1) contains Nde I site . DNA sequencing shows that non-specific mutation has not been found in its coding region . The DNA fragment digested by BamH I, Nde I of C1 plasmid is recovered and subcloned into PET-11 and transfered into BL21 (DE3) . After IPTG induction, the Bacteria lysate is run directly on SDS-PAGE . Western blot test shows a specific rhIL-2 McAb binding band . The molecular weight is about 17000 . Laser scanning indicates that the absorption area of IL-2 is about 5% of the total area. Neurobiol Dis, 1996 Feb, 3(1), 76 - 86 Direct intracerebral nerve growth factor gene transfer using a recombinant adenovirus: effect on basal forebrain cholinergic neurons during aging; Castel-Barthe MN et al.; Gene therapy in the nervous system offers an attractive strategy for the administration of therapeutic factors in a potentially region-specific, sustained, and well-tolerated manner . We tested the trophic effect of a recombinant adenovirus encoding nerve growth factor (AdNGF) in vivo on basal forebrain cholinergic neurons of aged rats, a neuronal population affected during normal and pathological aging . Three weeks after unilateral injection of the recombinant adenovirus into the nucleus basalis magnocellularis, a significant increase in the somal areas of cholinergic neurons ipsilateral to the injection was observed . No increase was detected in animals receiving a recombinant adenovirus carrying the Escherichia coli Lac Z reporter gene . Injected animals did not lose weight, an adverse effect usually described after intracerebroventricular infusion of NGF, and no tissue loss or massive local inflammatory response was observed around injection sites . Thus, a single intracerebral injection of AdNGF produces trophic effects similar to those resulting from chronic intracerebroventricular high levels of NGF . These findings indicate that recombinant adenoviruses encoding growth factors are potentially powerful tools for improving neuronal deficits associated with degenerative processes. Protein Expr Purif, 1996 Feb, 7(1), 92 - 103 Negative effect of sequential serine codons on expression of foreign genes in Escherichia coli; Bula C et al.; Herpes simplex virus encodes a 1298-residue protein designated ICP4 that regulates transcription of viral genes . Structural and functional analyses of ICP4 have been facilitated by production of portions of ICP4 in Escherichia coli . We previously observed that expression of most truncated forms of ICP4 in E . coli was relatively efficient, with the exception of portions of the ICP4 gene approximately between codons 160 and 220 . We have now localized the portion of ICP4 that inhibits expression to a serine-rich region from position 176 to 199 . Our experimental results suggest that codons within the serine-rich domain do not induce termination of transcription, do not alter the intrinsic stability of mRNA, and do not create a proteolytically sensitive site in this portion of ICP4 . Silent mutations that alter codon usage of many of the 19 serine codons in this region had no effect on expression . However, we observed that the level of protein expression was inversely proportional to the number of serine codons in this region . The results are consistent with a model in which the serine-rich domain induces premature termination of translation . This effect is not due to any specific secondary structure in the mRNA or lack of sufficient seryl-tRNA synthetase . It remains to be determined whether premature termination can result from insufficient seryl-charged tRNAs . Our results suggest that foreign genes with more than 20 consecutive serine codons may be poorly expressed in E . coli. Protein Expr Purif, 1996 Feb, 7(1), 58 - 66 High-level expression and purification of a human "mini"-hexokinase; Bianchi M et al.; Human hexokinase type I is a 100-kDa enzyme with the catalytic site located in the C-terminal domain . We had previously expressed this domain in Escherichia coli, however only a small amount of the recombinant enzyme was catalytically active . To overcome this problem we have now expressed the "mini"-hexokinase using the pET expression system . An average of 1000 U of enzyme per liter of culture was obtained . The recombinant enzyme was purified to homogeneity by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography . The enzyme was unstable under ultrafiltration; thus, a multicolumn purification procedure was developed in order to avoid the ultrafiltration steps . The recombinant "mini"-hexokinase was found to have the same kinetic properties as the entire enzyme . Using the method described, the enzyme can be obtained in sufficient quantities for biophysical and biochemical investigations. Protein Expr Purif, 1996 Feb, 7(1), 51 - 7 Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP; McPherson DT et al.; By constructing a basic gene unit encoding (GVGVP)10, it was possible to build concatemer genes with as many as 25 repeats of the monomer unit encoding a protein-based polymer with a molecular weight of greater than 100,000 Da . This employed the use of terminal cloning adaptor oligonucleotides as chain terminators to enhance the desired polymer gene size distribution . These genes have been expressed in Escherichia coli and the products have been purified from the culture lysates using a simple centrifugation method which relies upon the inverse temperature transitional properties of these elastomeric protein-based polymers . At 4 degrees C, the polymers are soluble; on raising the temperature above 26 degrees C, the onset temperature (Tt) for the (GVGVP)251 inverse temperature transition, the polymer separates out as the more dense phase . Upon shifting the temperature between 4 and 37 degrees C, the recombinant elastomeric protein-based polymers undergo reversible phase transitions from soluble (4 degrees C) to insoluble (37 degrees C) allowing their separation from other cellular components by several cycles of centrifugations at alternate transitional states . Additional centrifugation, at a temperature just below Tt, allows for dramatic lowering of endotoxin levels . Furthermore, many ways of varying the value of Tt, such as adding salt to lower Tt or changing the degree of ionization in polymers with functional side chains, can be used to achieve purification of more complex polymers. Protein Expr Purif, 1996 Feb, 7(1), 27 - 32 Single-step purification of recombinant wild-type and mutant HIV-1 reverse transcriptase; Fletcher RS et al.; We have devised a single-step method that enables purification of HIV-1 recombinant reverse transcriptase directly from bacterial lysates in less than 2 h . Clarified lysates are applied to commercial Q- and S-matrix cartridge columns connected in series . The columns are washed with low-salt buffer to remove unbound protein, then the Q column is removed and reverse transcriptase is eluted from the S column using a salt gradient . The purification has been carried out with both medium-pressure and high-pressure chromatographic systems . Purifications are carried out at room temperature near neutral pH, providing enzyme with high DNA polymerase specific activity . A crucial aspect of the procedure is the use of Tris buffer, a buffer that is normally incompatible in cation-exchange methods . The method is applicable for the purification of the p51/p66 heterodimer and the p5l and p66 homodimer forms of reverse transcriptase . We have used this method to purify wild-type reverse transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance. Genes Cells, 1996 Feb, 1(2), 201 - 8 Roles of cysteine residues of DsbB in its activity to reoxidize DsbA, the protein disulphide bond catalyst of Escherichia coli; Kishigami S et al.; BACKGROUND: DsbA, a periplasmic protein, catalyses the disulphide bond formation of other cell surface proteins in E . coli . Reoxidation of DsbA for catalytic turn over is assured by DsbB, a membrane protein with four essential cysteine residues facing the periplasm . We and others previously reported that the reactive Cys30 residue of DsbA forms a mixed disulphide with DsbB in the absence of its partner Cys33 residue . RESULTS: Under the medium condition in which the DsbA mutant lacking Cys33 forms a mixed disulphide only with DsbB, we examined cysteine mutants of epitope-tagged DsbB for their ability to form the complex . It was shown that Cys104 of DsbB is absolutely required while other three cysteines are also required for maximum interaction . Examination of the redox states of cysteines in wild-type and mutant DsbB suggested that Cys104 and Cys130 form a disulphide bond which will be transferred to DsbA . In agreement with this notion, DsbB mutants lacking one of the N-terminally located cysteines retain weak DsbB activity in vivo . The primary role of the N-terminally located thioredoxin-like motif of DsbB is probably to reoxidize Cys104 and Cys130 . CONCLUSIONS: We propose the following reaction cycle . DsbB is initially oxidized (State A in Summary Figure) . Disulphide interaction between Cys30 of DsbA and Cys104 of DsbB should then trigger the recycling reaction of DsbA (State B), allowing over all electron transfer from newly secreted protein via DsbA (Cys30/Cys33) to DsbB in which intrachain electron flow from Cys104/Cys130 (State C) to Cys41/Cys44 (State D) may occur. Genes Cells, 1996 Feb, 1(2), 189 - 99 Dissociation kinetics of RepA dimers: implications for mechanisms of activation of DNA binding by chaperones; Chattoraj DK et al.; BACKGROUND: The replication initiator of plasmid P1, RepA, binds DNA as monomer . The binding is stimulated by the chaperones DnaJ, DnaK and GrpE of Escherichia coli . Two models of chaperone action have been proposed . (i) Chaperones dissociate RepA dimers, which are inactive in DNA binding, into active monomers . (ii) The dissociation occurs spontaneously but the monomeric products require the chaperones for refolding into the active form . The latter model was based on the observation that RepA diluted 1000-fold below the K(D) for dimer dissociation, still required the chaperones for DNA binding . RESULTS: We have confirmed that under the condition of DNA binding experiments, the RepA dimers dissociate reversibly into monomers with a K(D) value of 1.1 +/- 0.1 microM . In the vicinity of this concentration, the sedimentation coefficient of RepA was concentration dependent, allowing estimation of s(20,w) coefficients for the RepA monomer (2.95 S) and dimer (4.01 S) . Dynamic light scattering experiments indicated an increase of the monomer fraction within 5 min of RepA dilution . Circular dichroism (CD) measurements were consistent with these results . CONCLUSION: RepA monomerization is efficient without the mediation of chaperones . They are required to activate RepA most likely because they are needed to re-fold RepA monomers. Genes Cells, 1996 Feb, 1(2), 179 - 88 Histone-like protein HU as a specific transcriptional regulator: co-factor role in repression of gal transcription by GAL repressor; Aki T et al.; BACKGROUND: Transcription initiation from the two overlapping promoters of the gal operon in Escherichia coli is negatively regulated by binding of Gal repressor (GalR) to bipartite operators, which encompass the promoters . Coordinated repression of the two promoters requires GalR binding to both operators . In a purified system, GalR, nevertheless, fails to show the coordinated repression, predicting the participation of an additional factor(s) in the regulation in vivo . RESULTS: We have purified a protein that restored the expected GalR-mediated repression for the in vitro system and have identified this factor to be the bacterial histone-like protein HU . In vitro transcription assays in the presence of GalR and HU show that, just as in vivo, the coordinated repression of the two gal promoters requires GalR binding to both operators and is sensitive to the inducer, D-galactose . The GalR and HU dependent repression also requires supercoiled DNA template and prevents open complex formation . CONCLUSION: We propose that HU, acting as a co-factor, brings about the GalR-mediated repression by forming a distinct nucleoprotein complex of higher order structure . Although how HU participates in the assembly process is unknown, there may be a cooperative effect in the formation of the repression complex. Genes Cells, 1996 Feb, 1(2), 171 - 8 Differential thermoregulation of two highly homologous cold-shock genes, cspA and cspB, of Escherichia coli; Etchegaray JP et al.; BACKGROUND: The major cold-shock protein in Escherichia coli is CspA, a 7.4 kDa protein . A CspA family has been found which consists of four additional proteins, CspB, CspC, CspD and CspE . The expression of cspB, unlike the other homologues, is cold-shock inducible like cspA . RESULTS: We examined the cold-shock induction of CspA and CspB at various temperatures . The cspA induction is observed by temperature shift from 37 to 30 degrees C and high levels of CspA production are observed between 24 and 10 degrees C . In contrast, CspB production occurs only by temperature shift to below 20 C, with maximum induction at 15 degrees C . Both cspA and cspB expressions were found to be induced at the level of transcription as determined by primer extension . CONCLUSIONS: These results show that cspA and cspB expressions are differentially regulated at low temperature indicating that E . coli contains at least two different biothermostats or thermoregulators that are likely to play important roles in cellular adaptation to low temperature . The cspB promoter shows sequence similarity to the cspA promoter . Furthermore, both cspA and cspB mRNAs have unusually long 5' untranslated regions (159 and 161 bases, respectively), both of which are able to form similar extensive secondary structures . These features are considered to contribute to the nature of the thermostats for cspA and cspB. Genes Cells, 1996 Feb, 1(2), 139 - 45 MutT-related error avoidance mechanism for DNA synthesis; Sekiguchi M; Mutator mutants that show an increased frequency of spontaneous mutation have led to the elucidation of the multiple pathways of spontaneous mutagenesis . 8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is formed in the nucleotide pool of a cell during normal cellular metabolism, and when it is incorporated into DNA causes mutation . MutT protein of Escherichia coli and related mammalian enzymes specifically degrade 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing occurrence of transversion mutation . The gene encoding the human enzyme, designated MTH1 (for mutT homologue 1), maps to chromosome 7p22 . These proteins may be responsible for genomic stability. Hybridoma, 1996 Feb, 15(1), 49 - 53 Monoclonal antibody to thyroid transcription factor-1: production, characterization, and usefulness in tumor diagnosis; Holzinger A et al.; Thyroid transcription factor-1 (TTF-1), a member of the NKx2 family of homeodomain transcription factors, is expressed in epithelial cells of the thyroid gland and the lung . To produce monoclonal antibodies specific for TTF-1, the polypeptide was expressed in E . coli and purified utilizing affinity chromatography of a polyhistidine-tagged TTF-1 fusion protein . Splenocytes from BALB/c mice immunized with recombinant TTF-1 were fused with P3x/63Ag8.653 myeloma cells to produce hybridomas . Tissue culture supernatant was screened for anti TTF-1 activity by ELISA employing recombinant TTF-1 as antigen . Hybridomas producing high-affinity antibodies were subcloned by limiting dilution . Antibodies from tissue culture fluid from an IgG1 clone (8G7G3/1) that stained the nuclei of paraffin-embedded human thyroid tissues were precipitation-purified and further characterized . The antibody stained a single 40-kDa polypeptide in immunoblots of nuclear extracts or lysates of cell lines known to express TTF-1 mRNA . MAb 8G7G3/1 also stained nuclei of tissue in a highly specific manner consistent with the pattern of expression obtained with an established polyclonal TTF-1 antibody and by in situ hybridization . MAb 8G7G3/1 was used for TTF-1 immunohistochemistry of human adenocarcinomas of the lung, colon, and breast as well as small cell carcinomas of the lung . TTF-1 was detected in primary lung adenocarcinomas and small cell carcinomas and was absent in colon and breast carcinomas . These findings demonstrate that anti-TTF-1 MAb 8G7G3/1 specifically binds TTF-1 in cell extracts and tissues and can be used to distinguish between lung and nonlung origin of a tumor. Biosci Biotechnol Biochem, 1996 Feb, 60(2), 309 - 15 Isolation and characterization of the heat-responsive genes in Escherichia coli; Utsumi R et al.; The ompC gene expression is induced by increasing temperature as well as osmotic pressure . In this study, a mutant (TD2) defective in this thermoresponse was isolated with transposon Tn10; the mutation was complemented by pMAN55 or pMAN56 containing micF and mapped at 48 min on Escherichia coli K-12 . Furthermore, a new gene (hrsA) that suppressed the mutation was cloned . Its nucleotide sequence was analyzed and it was located close to the suc operon at 16.7 min corresponding to #18F11 (Kohara bank) on E . coli genome . In TD2 containing the hrsA on a multicopy plasmid, the ompC expression was induced and dependent on OmpR with increased temperature . The HrsA was found to have Enzyme IIA, IIB, and IIC domains that are homologous to Enzyme II, involved in the fructose-specific PTS (phosphotransferase system) . The putative phosphorylation sites (His87 and Cys192) were also conserved in HrsA. Biosci Biotechnol Biochem, 1996 Feb, 60(2), 277 - 83 A cyanobacterial gene that interferes with the phosphotransfer signal transduction involved in the osmoregulatory expression of ompC and ompF in Escherichia coli; Hirokawa K et al.; Synechococcus sp . PCC7942 is a phototrophic cyanobacterium . In this study we cloned a Synechococcus gene that has a striking effect on the production of the Escherichia coli outer membrane proteins, OmpC and OmpF, provided that this gene was introduced by a multicopy plasmid into the heterologous cells . This multicopy gene in E . coli cells was able to specifically shut off the production of both OmpC and OmpF at the level of transcription . The nucleotides were sequenced for this gene, named sis, and its gene product was purified from E . coli to near homogeneity . A computer-aided search found that the deduced amino acid sequence consisting of 138 residues is novel, with no significant similarity to any other protein in the databases . Since the transcription of ompC and ompF is regulated by the regulatory factors EnvZ and OmpR, through phosphotransfer signal transduction, we explored the inhibitory effect of Sis in various genetic backgrounds as to envZ and ompR . In particular, the inhibitory effect of Sis was observed even in an DeltaenvZ background, but was not observed in a certain background in which the ompC and ompF transcription was supported by a mutant OmpR that can function in a phosphorylation-independent manner . These results suggested that the EnvZ kinase may not be the direct target of Sis, but rather that the process(es) concerning the phosphorylation and/or dephosphorylation of the OmpR protein may be affected by Sis . However, no direct effect of Sis was seen in an in vitro OmpR-phosphorylation assay with the purified OmpR and Sis proteins . Based on these results, possible functions of Sis are discussed with special reference to the phosphotransfer signal transduction in E . coli. Vet Microbiol, 1996 Feb, 48(3-4), 243 - 55 Characterization of Escherichia coli isolated from foals; Holland RE et al.; Serotype, biotype, antibiogram, hemolysin production, fimbrial hemagglutinins, select toxin genes (STb, STaP, LT, slt1 and slt2) and the attaching effacing (eae) gene were determined for 99 foal strains of E . coli . E . coli from diarrheic and normal foals could not be distinguished by serotype, biotype, or antibiogram . Differences (P < or = 0.05) were observed in hemolysin production (11.5% vs 0%) and the expression of mannose-resistant hemagglutinins (23% vs 13%) among E . coli from diarrheic and healthy foals, respectively . Three of the E . coli strains from diarrheic foals were positive with probes for slt genes and one was positive for STb and LT genes . One strain from a healthy foal possessed the STb gene . As determined by the polymerase chain reaction, 8 strains possessed the eae gene . Seven of the 8 strains were from diarrheic foals and one eae-positive strain was from a healthy foal . The slt-positive strains did not possess eae genes and the eae-positive strains did not possess slt genes . These results indicate that enterotoxigenic strains of E . coli are not implicated in any substantial degree in sporadic foal diarrhea . However, the identification of slt-positive and eae-positive strains in foal feces indicate the presence of potentially virulent strains among foals. Vet Microbiol, 1996 Feb, 48(3-4), 231 - 41 Fimbriae extracts from enterotoxigenic Escherichia coli strains of bovine and porcine origin with K99 and/or F41 antigens; Vazquez F et al.; Fimbriae extracts obtained using the thermal shock method, from bovine and porcine enterotoxigenic Escherichia coli strains with K99 and/ or F41 antigens, were analyzed by SDS-PAGE, immunoblotting and haemagglutinating activity . Three major protein bands with molecular weights 17 kDa, 29.3 kDa and 30.9 kDa were detected depending on the strain assayed . A 17 kDa band was identified as the fimbrial subunit for K99 fimbriae and was detected in strains of bovine and porcine origin . The 30.9 kDa band was identified as the fimbriae subunit for F41 fimbriae and was detected in all porcine strains with F41 antigen and only in the bovine strains of serogroups O9 and 0101 that proved positive for F41 antigen . The 29.3 kDa band was shown to be antigenically related to F41 and K88, and was only detected in bovine strains of serogroups O8 (5 strains) and O20 (a single strain) . We speculate that the 29.3 kDa band may be related to the CS31A antigen. J Lipid Res, 1996 Feb, 37(2), 237 - 49 Isoproterenol decreases LDL receptor expression in rat adipose cells: activation of cyclic AMP-dependent proteolysis; Kraemer FB et al.; The low density lipoprotein (LDL) receptor is part of a family of proteins that mediate the uptake of lipoproteins into cells . In this paper we have demonstrated the over-expression in E . coli of a rat LDL receptor fusion protein that contains the region of the receptor sharing homology with the EGF precursor . The fusion protein was utilized to immunize rabbits and successfully generate antibodies that recognize the intact LDL receptor . These anti-LDL receptor/fusion protein antibodies were used to examine the effects of cyclic AMP on the expression of LDL receptors in isolated rat adipocytes . Incubation of adipocytes with isoproterenol caused a dose-dependent diminution in intact LDL receptors in the plasma membrane with the concomitant appearance of smaller immunoreactive proteins . Pulse-chase experiments demonstrated that isoproterenol rapidly shortened the initial half-life of intact, immunoprecipitable LDL receptors in the plasma membrane . The effects of isoproterenol on LDL receptor expression were mimicked by forskolin, by an analog of cyclic AMP, and by ACTH . In contrast, incubation with propranolol blocked the effects of isoproterenol on LDL receptor expression . While antioxidants and several different protease inhibitors had no effects, N-acetyl-leucine-leucine-methionine (ALLM) was able to prevent the isoproterenol-induced effects on LDL receptors . Thus, it appears that agents acting via cyclic AMP cause a rapid decrease in LDL receptors in the plasma membranes of isolated adipose cells due to the apparent stimulation of an ALLM-sensitive protease that degrades the LDL receptor . These results suggest a novel mechanism for the posttranscriptional regulation of LDL receptor expression in adipocytes. Protein Eng, 1996 Feb, 9(2), 231 - 8 Destabilizing interactions between the partners of a bifunctional fusion protein; Blondel A et al.; Hybrid MalE-GVP is a bifunctional protein in vitro since it binds maltose as protein MalE of Escherichia coli and since it is dimeric and specifically binds single-stranded DNA as protein GVP of phage M13 . The oxidation rate of a unique cysteine residue was used to compare the stabilities of GVP in its free and hybrid forms, under conditions where MalE was either folded or unfolded by a denaturing agent . The results showed that both the covalent link and tertiary non-covalent interactions between MalE and GVP destabilized GVP in MalE-GVP . To test whether GVP had identical structures in its free and hybrid forms, mutations were used as local conformational probes . The effects of these mutations on the capabilities of MalE-GVP to dimerize and to bind single-stranded DNA were assayed in vitro . They were compatible with the effects of the same mutations on the global activity of free GVP in vivo and with the effects that could be predicted from the known data on free GVP, in particular its crystal structure . Thus, one partner of a hybrid protein can be destabilized by the other partner while maintaining its structural and functional characteristics. Protein Eng, 1996 Feb, 9(2), 223 - 30 Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module; Riley LG et al.; Human c-Jun and c-Fos leucine zipper domains were examined for their ability to serve as autonomous dimerization domains as part of a heterologous protein construct . Schistosoma japonicum glutathione S-transferase (GST) was fused to recombinant Jun leucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains . SDS-PAGE 'snapshot' analyses based on disulphide linkage of monomers demonstrated the ability of rJunLZ to function as a dimerization motif in a foreign protein environment . Steric hindrance prevented formation of rJunLZ-GST::rFosLZ-GST heterodimers whereas rJunLZ-GST::rFosLZ and rJunLZ:: rFosLZGST formed readily . Furthermore, rJunLZGST generated homodimers suggesting fusion protein heterodimers interact differently to homodimers . Gel filtration chromatography confirmed that GST is a dimer in solution and that attachment of a leucine zipper domain allows further interactions to take place . Sedimentation equilibrium analyses showed that GST is a stable dimer (K(a) > 10(6) M(-1)) with no higher multimeric forms . rFosLZ-GST weakly associates beyond a dimer (K(a) approximately 4 x 10(4) M(-1)) and rJunLZ-GST associates indefinitely (K(a) approximately 4 x 10(5) M(-1)) {corrected}, consistent with an isodesmic model of association . The interaction of these leucine zippers independently of GST association demonstrates their utility in the modification of proteins when multimer formation is desired. Protein Eng, 1996 Feb, 9(2), 213 - 23 Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential; Wnendt S et al.; The blood clotting enzyme thrombin plays a central role in the aetiology of occlusive disorders such as stroke and acute myocardial infarction . During fibrinolytic therapy with plasminogen activators, thrombin is neutralized by anticoagulative drugs . In order to combine plasminogen-activating and thrombin-inhibitory activities we constructed chimeric derivatives of recombinant single-chain, urokinase-type plasminogen activator (rscu-PA) which comprise the kringle and protease domain of rscu-PA fused via a linker sequence to a thrombin-inhibitory domain . The inhibitory domain contains a sequence element directed to the active site of thrombin and a sequence taken from either hirudin or the human thrombin receptor both binding to the fibrinogen recognition site of thrombin . Analysing different sets of point mutants showed that the linker between the protease domain and the active site-directed sequence is contributing significantly to the thrombin-inhibitory potential . Kinetic analysis of thrombin inhibition revealed that most of the chimeras tested competitively inhibit the thrombin-mediated cleavage of a peptide substrate in a concentration-dependent manner; however, in two examples the insertion of one glycine residue into the active site directed-sequence abolished the blockade of the active site . This supports the conclusion that the chimeras with high thrombin-inhibitory potential interact with the active site and the fibrinogen recognition site of thrombin. Protein Eng, 1996 Feb, 9(2), 203 - 11 Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion; Kipriyanov SM et al.; In antigen-antibody interactions, the high avidity of antibodies depends on the affinity and number of the individual binding sites . To develop artificial antibodies with multiple valency, we have fused the single-chain antibody Fv fragments to core streptavidin . The resulting fusion protein, termed scFv::strep, was found after expression in Escherichia coli in periplasmic inclusion bodies . After purification of the recombinant product by immobilized metal affinity chromatography, refolding and size-exclusion FPLC, tetrameric complexes resembling those of mature streptavidin were formed . The purified tetrameric scFv::strep complexes demonstrated both antigen- and biotin-binding activity, were stable over a wide range of pH and did not dissociate at high temperatures (up to 70 degrees C) . Surface plasmon resonance measurements in a BIAlite system showed that the pure scFv::strep tetramers bound immobilized antigen very tightly and no dissociation was measurable . The association rate constant for scFv::strep tetramers was higher than those for scFv monomers and dimers . This was also reflected in the apparent constants, which was found to be 35 times higher for pure scFv::strep tetramers than monomeric single-chain antibodies . We could also show that most of biotin binding sites were accessible and not blocked by biotinylated E.coli proteins or free biotin from the medium . These sites should therefore facilitate the construction of bispecific multivalent antibodies by the addition of biotinylated ligands. Chem Pharm Bull (Tokyo), 1996 Feb, 44(2), 328 - 32 Partially-hydrophobized polymer particles derived from N,N-dimethylaminopropylacrylamide for endotoxin removal from acidic protein solution; Sakata M et al.; Novel copolymeric adsorbents for the selective removal of endotoxin from an acidic protein solution were prepared . The adsorbents comprise spherical copolymers derived from N,N-dimethylaminopropylacrylamide (DMAPAA) and divinylbenzene (DVB) . When the molar ratio of DMAPAA to DVB was 80/20 (amino-group content: 5.1 meq/g) and the pore size (molecular mass exclusion of polysaccharide, Mlim) was 4000 to 10000, DMAPAA/DVB showed high endotoxin-adsorbing activity at pH 5.0 to 9.0 and ionic strengths of mu = 0.05 to 0.4 . The capacity of the adsorbent (Mlim: 4000) was 390 micrograms of endotoxin (lipopolysaccharide purified from E . coli O111:B4) per ml of the adsorbent using the batchwise method . The apparent dissociation constant between endotoxin and the adsorbent was 2.2 x 10(-12) M . On the other hand, the adsorption of bovine serum albumin, an acidic protein, by the adsorbent increased with an increase in Mlim from 4000 to 10000, but decreased with an increase in ionic strength (mu) from 0.05 to 0.2 . As a result, DMAPAA/DVB (80/20) (Mlim: 4000) selectively removed endotoxin from various acidic protein solutions at pH 7.0 and mu = 0.05 . The residual concentration of endotoxin in the protein solution always decreased to a concentration lower than 0.1 ng/ml, and recovery of the protein was more than 97%. Bioseparation, 1996 Feb, 6(1), 17 - 23 Flocculation of Esch . coli with cationic polymers: a model for the dose curve based on charge; Cumming RH et al.; Continuously grown cells from a glucose limited chemostat were flocculated with four different cationic polymers . The polymer was added to the cells either dropwise or as a slug at the start of the flocculation period . Dose curves for each polymer type and using each method of polymer addition were constructed . It was evident that classical overdose was possible with all four polymers if slug addition was used . Continuous addition produced dose curves overdose was possible with all four polymers if slug addition was used . Continuous addition produced dose curves with no overdosing except when low molecular weight, low charge density polymer was used . The dose curves could be combined if they were based on amount of charge added . The dose curve was not linear, but fitted a logarithmic model well . Charge density was much more important than MW of the polymer. Immunol Cell Biol, 1996 Feb, 74(1), 96 - 104 Antigen-specific apoptosis in immortalized T cells by soluble MHC class II-peptide complexes; Arimilli S et al.; The recognition of T cell receptors (TCR) by purified major histocompatibility complex (MHC) class II-peptide complexes in the absence of costimulatory signals leads to the induction of T cell non-responsiveness or anergy . In a recent study using human T cell clones, it was observed that prolonged incubation of resting T cells with soluble MHC II-peptide complexes appears to result in T cell apoptosis . The present study shows that the engagement of TCR by soluble MHC II-peptide complexes also results in antigen-specific apoptosis in immortalized T cells . Apoptosis was demonstrated in a herpes saimiri virus (HSV) transformed human T cell clone (SS8T) restricted for HLA-DR2 in association with an epitope from the myelin basic protein {MBP(84-102)} . A dose- and time-dependent T cell death was observed upon incubation of SS8T cloned T cells with purified complexes of native human HLA-DR2 and MBP(83-102)Y83 peptide . The specificity of T cell apoptosis was demonstrated by exposing SS8T cells with DR2 alone and DR2 bound to another high affinity epitope {MBP(124-143)} from the same MBP . Recently, we have shown that the complexes of HLA-DR2 and {MBP(83-102)Y83} can be reconstituted by refolding Escherichia coli expressed individual DR2 alpha and beta (B5*0101) polypeptide chains lacking the transmembrane region . When SS8T cloned T cells were exposed to purified reconstituted rDR2.MBP(83-102)Y83 complexes, similar apoptosis of T cells was observed . Agarose gel analysis of T cells incubated with complexes showed a degradation of celluar deoxyribonucleic acid (DNA) to oligonucleosomal bands, a characteristic of apoptosis . The quantitative detection of DNA strand breaks was performed by pulsing T cells with 5-bromo-2'-deoxyuridine (BrdU) followed by the detection of BrdU-labelled DNA fragments using an antibody sandwich enzyme-linked immuno assay (ELISA) . The fragmentation of DNA was also measured by double fluorescence flow cytometry by 3' end labelling of fragmented DNA with biotinylated-deoxyuridine triphosphate (dUTP) in the presence of terminal deoxynucleotide transferase (TdT) enzyme . The expression of the bcl-2 protein in SS8T cells following TCR engagement by soluble MHC II-peptide complexes was monitored by chemiluminescence blot analysis using anti-bcl-2 monoclonal antibody . Finally, the nucleosomal condensation of T cells following complex treatment, characteristics of typical apoptosis, was demonstrated by transmission electron microscopy . These results suggest that the binding of soluble MHC class II-peptide complexes to TCR induces antigen-specific apoptosis in transformed CD4 positive T cells in vitro . Such induction of apoptosis by soluble MHC II-peptide complexes may provide a novel therapeutic strategy to delete autoreactive T cells in various autoimmune diseases. Microbiology, 1996 Feb, 142 ( Pt 2), 389 - 400 The second aconitase (AcnB) of Escherichia coli; Bradbury AJ et al.; The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kanR mutant lacking AcnA, and the corresponding polypeptide identified by activity staining and weak cross-reactivity with AcnA antiserum . The acnB gene was located at 2 center dot 85 min (131 center dot 6 kb) in a region of the chromosome previously assigned to two unidentified ORFs . Aconitase specific activities were amplified up to fivefold by infection with lambdaacnB phages from the Kohara lambda-E . coli gene library, and up to 120-fold (50% of soluble protein) by inducing transformants containing a plasmid (pGS783) in which the acnB coding region is expressed from a regulated T7 promoter . The AcnB protein was purified to > or = 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of Mr 100 000 (SDS-PAGE) and 105 000 (gel filtration analysis) compared with Mr 93 500 predicted from the nucleotide sequence . The sequence identity between AcnA and AcnB is only 17% and the domain organization of AcnA and related proteins (1-2-3-linker-4) is rearranged in AcnB (4-1-2-3). Microbiology, 1996 Feb, 142 ( Pt 2), 367 - 75 Escherichia coli RNase II: characterization of the promoters involved in the transcription of rnb; Zilhao R et al.; The rnb gene encodes ribonuclease II (RNase II), one of the two major Escherichia coli exonucleases involved in mRNA degradation . In this paper, the rnb transcript is characterized regarding its promoter and terminator regions . The combined results from S1 nuclease protection analysis, DNase I footprinting and gene fusions with lacZ have shown that rnb is expressed from two promoters . S1 nuclease protection analysis and DNA footprinting have shown that rnb has two promoters, P1 and P2 . Transcriptional and translational lacZ reporter fusions, constructed to the rnb gene, revealed that P2, the rnb proximal promoter, is stronger than P1 . However, P2 is not transcribed in vitro, suggesting that an additional factor is required in vivo . The 3' end of the rnb transcript mapped to a stem-loop structure immediately after the translated region. Microbiology, 1996 Feb, 142 ( Pt 2), 359 - 65 The defective phosphoribosyl diphosphate synthase in a temperature-sensitive prs-2 mutant of Escherichia coli is compensated by increased enzyme synthesis; Post DA et al.; An Escherichia coli strain which is temperature-sensitive for growth due to a mutation (prs-2) causing a defective phosphoribosyl diphosphate (PRPP) synthase has been characterized . The temperature-sensitive mutation was mapped to a 276 bp HindIII-BssHII DNA fragment located within the open reading frame specifying the PRPP synthase polypeptide . Cloning and sequencing of the mutant allele revealed two mutations . One, a G --> A transition, located in the ninth codon, was responsible for the temperature-conditional phenotype and resulted in a serine residue at this position . The wild-type codon at this position specified a glycine residue that is conserved among PRPP synthases across a broad phylogenetic range . Cells harbouring the glycine-to-serine alteration specified by a plasmid contained approximately 50% of the PRPP synthase activity of cells harbouring a plasmid-borne wild-type allele, both grown at 25 degrees C . The mutant enzyme had nearly normal heat stability, as long as it was synthesized at 25 degrees C . In contrast, there was hardly any PRPP synthase activity or anti-PRPP synthase antibody cross-reactive material present in cells harbouring the glycine to serine alteration following temperature shift to 42 degrees C . The other mutation was a C --> T transition located 39 bp upstream of the G --> A mutation, i.e . outside the coding sequence and close to the Shine-Dalgarno sequence . Cells harbouring only the C --> T mutation in a plasmid contained approximately three times as much PRPP synthase activity as a strain harbouring a plasmid-borne wild-type prs allele . In cells harbouring both mutations, the C --> T mutation appeared to compensate for the G --> A mutation by increasing the amount of a partially defective enzyme at the permissive temperature. Microbiology, 1996 Feb, 142 ( Pt 2), 269 - 75 The dnrM gene in Streptomyces peucetius contains a naturally occurring frameshift muta