J Bacteriol, 1999 Nov, 181(22), 7014 - 20 Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions; Maurer J et al.; The current model for autodisplay suggests a mechanism that allows a passenger protein to be translocated across the outer membrane by coordinate action of a C-terminal beta-barrel and its preceding linking region . The passenger protein, linker, and beta-barrel are together termed the autotransporter, while the linker and beta-barrel are here referred to as the translocation unit (TU) . We characterized the minimal TU necessary for autodisplay with the adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter . The assumed beta-barrel structure at the C terminus of the AIDA-I autotransporter was studied by constructing a set of seven AIDA-I-cholera toxin B subunit fusion proteins containing various portions of AIDA-I . Surface exposure of the cholera toxin B moiety was assessed by dot blot experiments and trypsin accessibility of the chimeric proteins expressed in Escherichia coli JK321 or UT5600 . Export of cholera toxin B strictly depended on a complete predicted beta-barrel region . The absolute necessity for export of a linking region and its influence on expression as an integral part of the TU was also demonstrated . The different electrophoretic mobilities of native and denatured chimeras indicated that the proposed beta-barrel resides within the C-terminal 312 amino acids of AIDA-I . Together these data provide evidence for the predicted beta-barrel structure and support our formerly proposed model of membrane topology of the AIDA-I autotransporter. J Bacteriol, 1999 Nov, 181(22), 6958 - 68 Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein; Arias P et al.; The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes . The target regions for the ActII-ORF4 protein were located within the act cluster . These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator . The mutant phenotype could be made to revert with extra copies of the wild-type actII-ORF4 gene but not with the actII-ORF4-177 mutant . His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified from Escherichia coli cultures; both showed specific DNA-binding activity for the actVI-ORF1-ORFA and actIII-actI intergenic regions . DNase I footprinting assays clearly located the DNA-binding sites within the -35 regions of the corresponding promoters, showing the consensus sequence 5'-TCGAG-3' . Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the act genes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the act biosynthetic genes. J Bacteriol, 1999 Nov, 181(22), 6898 - 906 Partition of the linear plasmid N15: interactions of N15 partition functions with the sop locus of the F plasmid; Ravin N et al.; A locus close to one end of the linear N15 prophage closely resembles the sop operon which governs partition of the F plasmid; the promoter region contains similar operator sites, and the two putative gene products have extensive amino acid identity with the SopA and -B proteins of F . Our aim was to ascertain whether the N15 sop homologue functions in partition, to identify the centromere site, and to examine possible interchangeability of function with the F Sop system . When expressed at a moderate level, N15 SopA and -B proteins partly stabilize mini-F which lacks its own sop operon but retains the sopC centromere . The stabilization does not depend on increased copy number . Likewise, an N15 mutant with most of its sop operon deleted is partly stabilized by F Sop proteins and fully stabilized by its own . Four inverted repeat sequences similar to those of sopC were located in N15 . They are distant from the sop operon and from each other . Two of these were shown to stabilize a mini-F sop deletion mutant when N15 Sop proteins were provided . Provision of the SopA homologue to plasmids with a sopA deletion resulted in further destabilization of the plasmid . The N15 Sop proteins exert effective, but incomplete, repression at the F sop promoter . We conclude that the N15 sop locus determines stable inheritance of the prophage by using dispersed centromere sites . The SopB-centromere and SopA-operator interactions show partial functional overlap between N15 and F . SopA of each plasmid appears to interact with SopB of the other, but in a way that is detrimental to plasmid maintenance. FEBS Lett, 1999 Nov 5, 460(3), 554 - 8 Second transmembrane segment of FtsH plays a role in its proteolytic activity and homo-oligomerization; Makino S et al.; The FtsH (HflB) protein of Escherichia coli is a membrane-bound ATP-dependent zinc protease . The role(s) of the N-terminal membrane-anchoring region of FtsH were studied by fusion with a maltose-binding protein (MBP) at five different N-termini of FtsH . The MBP-FtsH fusions were expressed in the cytoplasm of E . coli, and were purified as soluble proteins . The four longer constructs, which have a second transmembrane segment and the C-terminal cytoplasmic region in common, retained ATP-dependent protease activity toward heat-shock transcription factor sigma(32), and were found to be homo-oligomers . In contrast, the shortest construct which has the C-terminal cytoplasmic region but not the second transmembrane segment showed neither protease activity nor oligomerization . Therefore, the second transmembrane segment, which neighbors the C-terminal cytoplasmic region of the FtsH, participates in not only its membrane-anchoring, but also its protease activity and homo-oligomerization. Protein Eng, 1999 Oct, 12(10), 863 - 72 The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis; Jeffery CJ et al.; The Escherichia coli aspartate receptor is a dimer with two transmembrane sequences per monomer that connect a periplasmic ligand binding domain to a cytoplasmic signaling domain . The method of 'hydrophobic-biased' random mutagenesis, that we describe here, was used to construct mutant aspartate receptors in which either the entire transmembrane sequence or seven residues near the center of the transmembrane sequence were replaced with hydrophobic and polar random residues . Some of these receptors responded to aspartate in an in vivo chemotaxis assay, while others did not . The acceptable substitutions included hydrophobic to polar residues, small to larger residues, and large to smaller residues . However, one mutant receptor that had only a few hydrophobic substitutions did not respond to aspartate . These results add to our understanding of sequence specificity in the transmembrane regions of proteins with more than one transmembrane sequence . This work also demonstrates a method of constructing families of mutant proteins containing random residues with chosen characteristics. Fed Regist, 1999 Feb 1, 64(20), 4884 - 5 Government-owned inventions; availability for licensing . National Institutes of Health, Public Health Service, DHHS . Notice; Nonequilibrium mechanism of transcription termination from observations of single RNA polymerase molecules; Department of Biochemistry, Brandeis University, Waltham, MA 02454-9110, USACessation of transcription at specific terminator DNA sequences is used by viruses, bacteria, and eukaryotes to regulate the expression of downstream genes, but the mechanisms of transcription termination are poorly characterized . To elucidate the kinetic mechanism of termination at the intrinsic terminators of enteric bacteria, we observed, by using single-molecule light microscopy techniques, the behavior of surface-immobilized Escherichia coli RNA polymerase (RNAP) molecules in vitro . An RNAP molecule remains at a canonical intrinsic terminator for approximately 64 s before releasing DNA, implying the formation of an elongation-incompetent (paused) intermediate by transcription complexes that terminate but not by those that read through the terminator . Analysis of pause lifetimes establishes a complete minimal mechanism of termination in which paused intermediate formation is both necessary and sufficient to induce release of RNAP at the terminator . The data suggest that intrinsic terminators function by a nonequilibrium process in which terminator effectiveness is determined by the relative rates of nucleotide addition and paused state entry by the transcription complex. Plant Physiol, 1999 Nov, 121(3), 813 - 820 Sucrose and Cytokinin Modulation of WPK4, a Gene Encoding a SNF1-Related Protein Kinase from Wheat; Ikeda Y et al.; WPK4, a gene encoding a putative protein kinase, was initially identified in wheat (Triticum aestivum) and shown to be up-regulated by light, nutrient deprivation, and cytokinins . To confirm that WPK4 has protein kinase activity, the protein was produced in Escherichia coli as a fusion protein with glutathione S-transferase . The purified protein exhibited autophosphorylation activity and phosphorylated both myelin basic protein and a peptide fragment of rice 3-hydroxy-3-methylglutaryl-coenzyme A reductase . Levels of WPK4 transcripts in wheat seedlings were increased and decreased by the removal and addition of sucrose (Suc), respectively, to the culture medium . The introduction of the N-terminal kinase region of WPK4 into the yeast snf1 mutant cells, which cannot utilize Suc as a carbon source, rescued growth in Suc-containing medium . Cytokinins up-regulated the accumulation of WPK4 transcripts, but their effects were cancelled by the addition of Suc . Our results suggest that Suc negatively regulates the signaling pathway in which transcriptional activation of WPK4 is mediated by cytokinins. Genes Dev, 1999 Nov 1, 13(21), 2889 - 903 Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo; Motamedi MR et al.; DNA double-strand-break repair (DSBR) is, in many organisms, accomplished by homologous recombination . In Escherichia coli DSBR was thought to result from breakage and reunion of parental DNA molecules, assisted by known endonucleases, the Holliday junction resolvases . Under special circumstances, for example, SOS induction, recombination forks were proposed to initiate replication . We provide physical evidence that this is a major alternative mechanism in which replication copies information from one chromosome to another generating recombinant chromosomes in normal cells in vivo . This alternative mechanism can occur independently of known Holliday junction cleaving proteins, requires DNA polymerase III, and produces recombined DNA molecules that carry newly replicated DNA . The replicational mechanism underlies about half the recombination of linear DNA in E . coli; the other half occurs by breakage and reunion, which we show requires resolvases, and is replication-independent . The data also indicate that accumulation of recombination intermediates promotes replication dramatically. Biotechnol Bioeng, 1999, 66(1), 61 - 7 Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway; Schmidt M et al.; The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition . This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies . It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence . Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene . The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time . It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source . It is postulated that the increased dissimilation of the carbon substrate by lon-deficient Bl21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon . Biotechnol Bioeng, 1999, 66(1), 17 - 34 Cutinase: from molecular level to bioprocess development; Carvalho CM et al.; This review analyzes the role of cutinases in nature and their potential biotechnological applications . The cloning and expression of a fungal cutinase, Fusarium solani f . pisi, in Escherichia coli and Saccharomyces cerevisiae hosts are described . The three-dimensional structure of this cutinase is also analyzed and its function as a lipase is discussed and compared with other lipases . The biocatalytic applications of cutinase are described taking into account the preparation of different cutinase forms and the media in which the different types of reactions have been performed, namely hydrolysis, esterification, transesterification, and resolution of racemic mixtures . The stability of cutinase preparations is discussed and, in particular, the cutinase stability in anionic reversed micelles is analyzed considering the role of hexanol as a substrate, a cosurfactant, and a stabilizer . Process development, based on the operation of cutinase reactors, is also reviewed . J Endocrinol, 1999 Nov, 163(2), 213 - 20 Increased responsiveness to intravenous lipopolysaccharide challenge in steers grazing endophyte-infected tall fescue compared with steers grazing endophyte-free tall fescue; Filipov NM et al.; Fescue toxicosis in cattle occurs as a result of consumption of ergot alkaloids in endophyte-infected (E+, Neotyphodium coenophialum) tall fescue (Festuca arundinacea) . The condition is characterized by pyrexia, decreased weight gains, rough hair coats, and decreased calving rates . The objective of this experiment was to investigate whether steers grazing E+ fescue have altered host response to lipopolysaccharide (endotoxin, LPS) challenge compared with steers grazing endophyte-free (E-) fescue . Angus steers (n=8) had continuously grazed either E+ (n=4) or E- (n=4) tall fescue grass for 8 months prior to the experiment . The E+ steers had lower body weight, depressed average daily gain, and decreased basal serum prolactin compared with the E- steers prior to LPS administration . Each steer received a single bolus i.v . injection of LPS (0.2 microgram/kg body weight; Escherichia coli; 026:B6) dissolved in sterile saline, and blood was serially collected every 30 min for 4 h and at 24 h post LPS administration . LPS increased serum tumor necrosis factor-alpha (TNF-alpha), cortisol, and haptoglobin but decreased plasma glucose and IGF-I . Importantly, however, TNF-alpha, cortisol, and IGF-I responses to LPS were greater in E+ compared with E- steers . These results indicated that animals grazing E+ fescue had altered integrated metabolic host response compared with animals grazing E- fescue . Potentially, combined exposure to E+ fescue and a bacterial LPS could have greater deleterious effects on the animal compared with exposure to only one of the two and would likely lead to increased catabolism. FEMS Microbiol Lett, 1999 Nov 15, 180(2), 345 - 9 Characterization of Escherichia coli 50S ribosomal protein L31; Eistetter AJ et al.; The published C-terminal sequence of Escherichia coli 50S ribosomal protein L31, ellipsisRFNK (Brosius, J . (1978) Biochemistry 17, 501-508), differs from that predicted by the gene sequence, ellipsisRFNKRFNIPGSK (GenBank accession no . X78541) . This discrepancy might be due to post-translational processing of the protein . To examine this possibility, we have isolated L31 from E . coli strain MRE600 and sequenced the C-terminal tryptic peptide . We find the sequence to be FBIPGSK . Size comparisons of L31 from several E . coli strains demonstrate that all are identical in size to the protein isolated from MRE600 and larger than the previously described protein, indicating that ellipsisRFNKRFNIPGSK represents the true C-terminus of L31 . In addition, we show that the failure to identify L31 in many ribosome preparations is probably due to the protein's loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels. FEMS Microbiol Lett, 1999 Nov 15, 180(2), 271 - 7 Analysis of Escherichia coli 4.5S RNA binding affinity to Ffh and EF-G; Suzuma S et al.; Escherichia coli 4.5S RNA is a member of the signal recognition particle RNA family that binds to Ffh and EF-G proteins in vivo . To assess the binding affinity of E . coli 4.5S RNA, wild-type Ffh and a series of amino terminal truncated EF-G mutants with a histidine tag were over-expressed in Escherichia coli and purified . Among them, EF-G mutants with a deletion of all upstream sequences up to and including the second or the third GTP binding sequence element were expressed at high levels and bound with the same activity as wild-type EF-G . Nitrocellulose filter binding assays revealed that the binding affinity values (M(1/2)) for Ffh and EF-G, defined as the concentration giving half-maximal binding, were 0.15 microM and 1.5 microM, respectively . Moreover, we also show that very little EF-G can form a stable complex with 4.5S RNA in vivo, whereas almost all Ffh binds to 4.5S RNA. J Immunol Methods, 1999 Oct 29, 229(1-2), 141 - 53 Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphoma vaccination; Osterroth F et al.; An expression system for rapid and standardized production of human recombinant immunoglobulin Fab fragments in E . coli was developed . Functional folding of the Fab fragments was accomplished by the dicistronic expression vector pFab.gammakappa containing specialized leader sequences to direct the immunoglobulin heavy and light chains to the periplasmic bacterial space . A C-terminal hexahistidine tag of the Fd chain facilitated metal affinity chromatography and purification to homogeneity as assessed by SDS PAGE and silver staining . Specific antigen recognition by a hybridoma-derived Fab fragment was indistinguishable from that of the corresponding monoclonal antibody . This protocol may be useful for analysis of the antigen specificity of human B cells and for convenient production of lymphoma-derived idiotype protein for vaccination strategies . To obtain unmodified immunoglobulin cDNA sequences from small human biopsies for insertion into pFab.gammakappa, oligo(dG)-tailed cDNA was amplified with an oligo(dC)- and nested mu or kappa constant region-specific primers . Using single sets of primers for each class of immunoglobulin transcripts, the products of this anchored PCR reflected the relative abundance of the starting cell population and permitted reliable identification of clonal, lymphoma-derived sequences for subsequent expression cloning. Biochim Biophys Acta, 1999 Nov 10, 1413(3), 159 - 71 Effect of NBD chloride (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) on the pyridine nucleotide transhydrogenase of Escherichia coli; Bragg PD et al.; Pyridine nucleotide transhydrogenases of bacterial cytosolic membranes and mitochondrial inner membranes are proton pumps in which hydride transfer between NADP(+) and NAD(+) is coupled to proton translocation across cytosolic or mitochondrial membranes . The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two subunits (alpha and beta) . Three domains are recognized . The extrinsic cytosolic domain 1 of the amino-terminal region of the alpha subunit bears the NAD(H)-binding site . The NADP(H)-binding site is present in domain 3, the extrinsic cytosolic carboxyl-terminal region of the beta subunit . Domain 2 is composed of the membrane-intrinsic carboxyl-terminal region of the alpha subunit and the membrane-intrinsic amino-terminal region of the beta subunit . Treatment of the transhydrogenase of E . coli with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) inhibited enzyme activity . Analysis of inhibition revealed that several sites on the enzyme were involved . NBD chloride modified two (betaCys-147 and betaCys-260) of the seven cysteine residues present in the transhydrogenase . Modification of betaCys-260 in domain 2 resulted in inhibition of enzyme activity . Modification of residues other than cysteine residues also resulted in inhibition of transhydrogenation as shown by use of a cysteine-free mutant enzyme . The beta subunit was modified by NBD chloride to a greater extent than the alpha subunit . Reaction of domain 2 and domain 3 was prevented by NADPH . Modification of domain 3 is probably not associated with inhibition of enzyme activity . Modification of domain 2 of the beta subunit resulted in a decreased binding affinity for NADPH at its binding site in domain 3 . The product resulting from the reaction of NBD chloride with NADPH was a very effective inhibitor of transhydrogenation . In experiments with NBD chloride in the presence of NADPH it is likely that all of the sites of reaction described above will contribute to the inhibition observed . The NBD-NADPH adduct will likely be more useful than NBD chloride in investigations of the pyridine nucleotide transhydrogenase. Mutat Res, 1999 Oct 22, 435(2), 141 - 50 The influence of formamidopyrimidine-DNA glycosylase on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZ alpha gene; Kuipers GK et al.; Base excision repair (BER) is a very important repair mechanism to cope with oxidative DNA damage . One of the most predominating oxidative DNA damages after exposure to ionizing radiation is 7, 8-dihydro-8-oxoguanine (8oxoG) . This damage is repaired by formamidopyrimidine-DNA glycosylase (Fpg), a DNA glycosylase which is part of BER . Correct repair of 8oxoG is of great importance for cells, because 8oxoG has strong miscoding properties . Mispairing of 8oxoG with adenine instead of cytosine results in G:C to T:A transversion mutations . To determine the effect of a Fpg-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene, double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target, was irradiated with gamma-rays in aqueous solution under oxic conditions . Subsequently, the DNA was transfected into a wild-type Escherichia coli strain (JM105) and an isogenic Fpg-deficient E . coli strain (BH410) . Although the overall spontaneous mutation spectra between JM105 and BH410 seemed similar, remarkable differences could be observed when the individual base pair substitutions were viewed . The amount of C to A transversions, which are most probably caused by unrepaired 8oxoG, has increased 3 . 5-fold in the spontaneous BH410 spectrum . When the gamma-radiation-induced mutation spectra of JM105 and BH410 were compared, there was even a larger increase of C to A transversions in the BH410 strain (7-fold) . We can therefore conclude that the straightforward approach used in this study confirms the importance of Fpg in repair of gamma-radiation-induced damage, and most probably especially in the repair of 8oxoG. Biochim Biophys Acta, 1999 Sep 21, 1451(2-3), 334 - 42 A strategy to make constitutively active MAP kinase by fusing with constitutively active MAP kinase kinase; Miyata Y et al.; Classical mitogen-activated protein kinases (MAPKs) play a pivotal role in a variety of cellular signal transduction pathways . MAPKs are activated by phosphorylation at specific threonine and tyrosine residues catalyzed by upstream MAPK kinases (MAPKKs) . Mutations of these two activation phosphorylation sites into acidic amino acids, however, do not convert MAPKs into constitutively active forms . Here, we report an approach to make a molecule with constitutive MAPK activity . The nuclear export signal-disrupted, constitutively active MAPKK was fused to the N-terminal end of wild-type MAPK . When the resulting fusion protein was expressed in Escherichia coli, the MAPK moiety became phosphorylated and the fusion protein was constitutively active as MAPK . Moreover, when expressed in mammalian cultured cells, the fusion protein was also activated as MAPK and was able to induce marked morphological changes in NIH-3T3 cells . These results suggest that the fusion protein can work as constitutively active MAPK and that this approach may be applicable to other members of the MAPK family to make constitutively active forms. Biochim Biophys Acta, 1999 Sep 21, 1451(2-3), 288 - 96 Nuclear localization conferred by the pocket domain of the retinoblastoma gene product; Zacksenhaus E et al.; The tumor suppressor Rb is a nuclear phosphoprotein that controls cell growth and differentiation by modulating the activity of certain transcription factors . Transport of Rb to the nucleus is affected by both a bipartite nuclear localization signal (NLS) in the C-terminus of the protein and a central domain, termed A/B or pocket, through which Rb interacts with transcription factors and viral oncoproteins . Mutations in either the A or B subdomains of the pocket render a NLS-deficient Rb completely cytoplasmic . Fusing the A/B domain of Rb to the Escherichia coli beta-galactosidase, to create betagal-A/B, confers nuclear localization upon this bacterial protein . Moreover, co-expression with the adenovirus oncoprotein, E1A, further augments nuclear localization of betagal-A/B . These findings provide direct evidence that the pocket domain of Rb is not only required but also sufficient to induce nuclear transport by a 'piggyback' mechanism . Thus, nuclear localization of Rb is dictated by two independent and autonomous domains: (i) the bipartite NLS and (ii) the pocket domain . We suggest that via these domains, Rb chaperons and co-compartmentalizes with its associated factors and preempts their activity prior to nuclear transport. Biochim Biophys Acta, 1999 Sep 14, 1434(1), 103 - 13 Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein; Jensen PY et al.; We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively . The domain(s) have been characterised using circular dichroism (CD) and fluorescence spectroscopy, and their copper(I) binding properties have been determined . Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet . The tryptophan fluorescence maximum is blue-shifted in the constructs containing two and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct . Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions . We show that the copper(I) binding to constructs containing two and six domains is cooperative, with Hill coefficients of 1.5 and 4, respectively . The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively . Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration . The regulation does not occur as an 'all-or-none' cooperativity, suggesting that the copper(I) binding domains have a basal low affinity for binding and release of copper(I) at low concentrations but are able to respond to higher copper levels by increasing the affinity, thereby contributing to prevent the copper concentration from reaching toxic levels in the cell. Biochim Biophys Acta, 1999 Sep 14, 1434(1), 58 - 63 The role of His(143) for the pH-dependent stability of plasminogen activator inhibitor-1; Sui GC et al.; Using site-directed mutagenesis, His(143) on the alpha-helix F of PAI-1 was substituted by Lys, Asp, Phe and Thr, respectively . The generated single-site changed plasminogen activator inhibitor-1 (PAI-1) mutants were expressed in Escherichia coli and purified by heparin-Sepharose and anhydrotrypsin agarose chromatographies . When compared with wild-type (wtPAI-1), the PAI-1 mutants His143Asp and His143Phe had shorter half-lives at pH 7.5 (1.1 and 1.4 h, respectively, vs . 2 h for wtPAI-1) . They also exhibited less pH dependency of their stability, with half-lives at pH 5.5 of 2.5 and 2.2 h, respectively, vs . 18 h for wtPAI-1 . However, the PAI-1 mutants His143Lys and His143Thr had similar properties as wtPAI-1 in this respect . In conclusion, our results suggest that His(143) in one way or another might be involved in the pH-dependent stability of PAI-1 . However, it seems that the protonation of this particular residue is of less importance . The PAI-1 mutants His143Asp and His143Phe only displayed about 20% of the specific activity obtained for wtPAI-1, because they, to a large extent, act as substrates for tissue-type plasminogen activator. Biochim Biophys Acta, 1999 Sep 14, 1434(1), 31 - 43 Conformational dynamics and molecular interaction reactions of recombinant cytochrome p450scc (CYP11A1) detected by fluorescence energy transfer; Lepesheva GI et al.; Bovine adrenocortical cytochrome P450scc (P450scc) was expressed in Escherichia coli and purified as the substrate bound, high-spin complex (16.7 nmol of heme per mg of protein, expression level in E . coli about 400-700 nmol/l) . The recombinant protein was characterized by comparison with native P450scc purified from adrenal cortex mitochondria . To study the interaction of the electron transfer proteins during the functioning of the heme protein, recombinant P450scc was selectively modified with fluorescein isothiocyanate (FITC) . The present paper shows that modified P450scc, purified by affinity chromatography using adrenodoxin-Sepharose to remove non-covalently bound FITC, retains the functional activity of the unmodified enzyme, including its ability to bind adrenodoxin . Based on the efficiency of resonance fluorescence energy transfer in the donor-acceptor pair, FITC-heme, we calculated the distance between Lys(338), selectively labeled with the dye, and the heme of P450scc . The intensity of fluorescence from the label dramatically changes during: (a) denaturation of P450scc; (b) changing the spin state or redox potential of the heme protein; (c) formation of the carbon monoxide complex of reduced P450scc; (d) as well as during reactions of intermolecular interactions, such as changes of the state of aggregation, complex formation with the substrate, binding to the electron transfer partner adrenodoxin, or insertion of the protein into an artificial phospholipid membrane . Selective chemical modification of P450scc with FITC proved to be a very useful method to study the dynamics of conformational changes of the recombinant heme protein . The data obtained indicate that functionally important conformational changes of P450scc are large-scale ones, i.e . they are not limited only to changes in the dynamics of the protein active center . The results of the present study also indicate that chemical modification of Lys(338) of bovine adrenocortical P450scc does not dramatically alter the activity of the heme protein, but does result in a decrease of protein stability. Biochim Biophys Acta, 1999 Sep 14, 1434(1), 6 - 17 Calcium and phospholipid activation of a recombinant calcium-dependent protein kinase (DcCPK1) from carrot (Daucus carota L.); Farmer PK et al.; A calmodulin-like domain protein kinase (DcCPK1, previously designated CDPK431) cloned from carrot (Daucus carota L.) was expressed at high levels in Escherichia coli and partially purified . Ca(2+)-induced gel mobility shift and (45)Ca(2+) ligand binding assays confirmed that recombinant DcCPK1 binds Ca(2+) through its calmodulin-like domain and undergoes a significant conformational change . Ca(2+) activated the kinase activity of recombinant DcCPK1 (K(0.5)=1.7 microM) up to 20-fold . Ca(2+) combined with certain lipids, including phosphatidic acid, phosphatidylserine and phosphatidylinositol, but not diolein or lysophosphatidylcholine, provided even greater Ca(2+)-dependent protein kinase activity . DcCPK1 phosphorylated casein and histone III-S, and a variety of peptide substrates containing a hydrophobic and a basic residue situated P-5 and P-3 amino acids N-terminal to a Ser or Thr residue . The calmodulin and protein kinase inhibitors, W-7 and staurosporine, inhibited CDPK activity . The similarities between DcCPK1 and mammalian protein kinase C (PKC) in substrate specificity, sensitivity to inhibitors, and activation by Ca(2+) and phospholipid suggest that various CDPK isoforms may be responsible for some PKC-like activities in plant cells. J Immunol Methods, 1999 Aug 31, 228(1-2), 97 - 108 Selection of phage antibodies to surface epitopes of Phytophthora infestans; Gough KC et al.; Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments . The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated . Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns . Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli . These antibody fusion proteins bound to P . infestans germlings and to mycelial homogenates from various Phytophthora species . The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower . Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings . The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions. FEBS Lett, 1999 Nov 5, 460(3), 485 - 90 A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis; Miller B et al.; Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria . For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs . Subsequent hybridization screening of a genomic cosmid library of S . leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S . leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing . The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis. FEBS Lett, 1999 Nov 5, 460(3), 457 - 61 Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli; Yamaguchi T et al.; Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily . CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively . Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products . More importantly, we found a cross-reaction between CHS and STS, i.e . resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites. Mamm Genome, 1999 Nov, 10(11), 1054 - 61 Identification and characterization of the mouse MutS homolog 5: Msh5; Her C et al.; We have identified and characterized the complete cDNA and gene for the mouse MutS homolog 5 (Msh5), as a step toward understanding the molecular genetic mechanisms involved in the biological function of this new MutS homologous protein in mammals . The Msh5 cDNA contains a 2502-bp open reading frame (ORF) that encodes an 833-amino acid protein with a predicted molecular weight of 92.6 kDa, which shares 89.8% amino acid sequence identity with the human hMSH5 protein . Northern blot analysis demonstrated the presence of a Msh5 mRNA approximately 2.9-kb in length, most abundantly expressed in mouse testis . Yeast two-hybrid analysis indicated that the mouse Msh5 protein positively interacted with the human hMSH4 protein-suggesting that Msh5 shares common functional properties with its human counterpart . Sequence and structural analyses show that the mouse gene Msh5 spans approximately 18 kb and contains 24 exons that range in length from 36 bp for exon 7 to 392 bp for exon 1 . Structural comparison with the human hMSH5 gene revealed that all of the Msh5 internal exons, but not introns, are conserved in length with the human hMSH5 . The Msh5 gene is located on mouse Chromosome (Chr) 17 in a location that is syntenic to the region of human Chr 6 harboring the hMSH5 gene . The identification and characterization of Msh5 will facilitate studies of the potential functional roles of this new member of the MutS family. Arch Environ Contam Toxicol, 2000 Jan, 38(1), 1 - 6 Oxidative cell damage in Kat-sod assay of oxyhalides as inorganic disinfection by-products and their occurrence by ozonation; Ueno H et al.; Nine oxyhalides as possible inorganic disinfection by-products were tested for oxidative cell damage by Kat-sod assay with E . coli mutant strains deficient in the active oxygen-scavenging enzymes . Chlorine dioxide, chlorite, and iodate were highly cytotoxic, whereas in the presence of cysteine, bromate (BrO3-) and metaperiodate (IO4-) showed more growth inhibition toward the superoxide dismutase-deficient strains than the wild strain . BrO3- also showed oxidative mutagenicity with cysteine or glutathione ethyl ester in S . typhimurium TA 100 . To identify oxyhalides formed by ozonation of raw water containing sea water, the occurrence of ozonation by-products of bromide and iodide was investigated . The results indicate that BrO3- is toxicologically one of the most remarkable oxyhalides detectable in drinking water because IO4- was not detected in the ozonated solution of iodide, and the ozonation condition to lower BrO3- is to keep it neutral in the presence of ammonium ion. Blood, 1999 Nov 1, 94(9), 2955 - 62 Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections; Roos D et al.; We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia . The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal . In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate . Activated neutrophils showed a decreased respiratory burst . Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient . Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement . Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation . This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters . Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele . Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability. Development, 1999 Dec, 126(23), 5387 - 98 Control of DAF-7 TGF-(alpha) expression and neuronal process development by a receptor tyrosine kinase KIN-8 in Caenorhabditis elegans; Koga M et al.; KIN-8 in C . elegans is highly homologous to human ROR-1 and 2 receptor tyrosine kinases of unknown functions . These kinases belong to a new subfamily related to the Trk subfamily . A kin-8 promoter::gfp fusion gene was expressed in ASI and many other neurons as well as in pharyngeal and head muscles . A kin-8 deletion mutant was isolated and showed constitutive dauer larva formation (Daf-c) phenotype: about half of the F(1) progeny became dauer larvae when they were cultivated on an old lawn of E . coli as food . Among the cells expressing kin-8::gfp, only ASI sensory neurons are known to express DAF-7 TGF-(beta), a key molecule preventing dauer larva formation . In the kin-8 deletion mutant, expression of daf-7::gfp in ASI was greatly reduced, dye-filling in ASI was specifically lost and ASI sensory processes did not completely extend into the amphid pore . The Daf-c phenotype was suppressed by daf-7 cDNA expression or a daf-3 null mutation . ASI-directed expression of kin-8 cDNA under the daf-7 promoter or expression by a heat shock promoter rescued the dye-filling defect, but not the Daf-c phenotype, of the kin-8 mutant . These results show that the kin-8 mutation causes the Daf-c phenotype through reduction of the daf-7 gene expression and that KIN-8 function is cell-autonomous for the dye-filling in ASI . KIN-8 is required for the process development of ASI, and also involved in promotion of daf-7 expression through a physiological or developmental function. J Mol Biol, 1999 Nov 19, 294(1), 163 - 80 Selection for improved protein stability by phage display; Jung S et al.; A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling . The library was based on the already well folding and stable scFv fragment 4D5Flu . In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability . Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride . Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process . It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type . The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display . J Mol Biol, 1999 Nov 19, 294(1), 67 - 77 Designed hyperstable Lac repressor.DNA loop topologies suggest alternative loop geometries; Mehta RA et al.; Lac repressor (LacI) forms DNA loops which are critical for efficient operator binding and transcriptional repression . Designed DNA loops formed on three constructs with lac operators bracketing phased A-tract bends were characterized by mobility shift, footprinting, and DNA cyclization and topology . Operator dyad axes point either in or out relative to the sequence-induced curvature . Possible conformations suggested from X-ray structures of LacI and LacI.DNA include "wrapping away" (WA), "simple loop" (SL), and "wrapping toward" (WT) models . The WA loop should be preferentially stabilized by the outward operators, the SL and WT loops by the inward operators . Competition experiments demonstrated increased loop stability for all the bent constructs, with the SL/WT construct supporting hyperstable loops (t1/2 of days) . This offers a general approach to stabilizing multi-protein DNA complexes on short DNA . DNA cyclization of loops gave minicircle products with altered topologies . WA constructs afforded relaxed and positive topoisomers, and cyclization kinetics indicated slow interconversion of precursors to the two topoisomers . The SL/WT construct gave a relaxed topoisomer, with a small amount of negative supercoil . These results suggest that while it is possible to force the WA loop to form (as in a model proposed from the LacI.DNA structure), the most stable loop geometry is different, probably a U-shape around an extended LacI tetramer . The topological results show how a protein-induced positive supercoil can be reconciled with LacI's preference for binding negatively supercoiled DNA . We suggest that looping proteins can affect the assembly of subsequent proteins by controlling loop topology . J Mol Biol, 1999 Nov 19, 294(1), 49 - 65 Replication control of a small cryptic plasmid of Escherichia coli; Burian J et al.; The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated . The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n'-pasH, which were essential for efficient plasmid replication . The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2 . RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number . An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD . Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency . Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA . Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication . Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication . When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell) . In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained . J Mol Biol, 1999 Nov 19, 294(1), 35 - 48 Transcription-modulated repair in Escherichia coli evident with UV-induced mutation spectra in supF; Li BH et al.; We have determined several mutation spectra with the supF sequence after UV mutagenesis in Escherichia coli . The cells were either mfd(+) or mfd(-) and grown in defined or complex medium . The tRNA supF gene was expressed from the plasmid pZ189 or pLS1D (similar to pLS189, a variant of pZ189, but with a tac promoter for supF) . Most of the mutations with either plasmid could be attributed to possible targeting photoproducts at dipyrimidine sites in the transcribed (TS) or non-transcribed (NTS) DNA strand with differential characteristics relevant to the repair process "mutation frequency decline" (MFD): (1) with pZ189, targeting sites in TS were favored over sites in NTS in all conditions except after an explicit MFD incubation with mfd(+) cells, when there was a majority in NTS; (2) with pLS1D (tac promoter), there was always a marked bias for targeting sites in TS and this was not altered by an MFD incubation; and (3) with pLS1D, spectra with mfd(-) cells vis-a-vis wild-type indicated a notable shift in the position of a hot-spot (both targeting sites in TS) and an increase in deletion mutations . The results support the Selby-Sancar idea that transcription-coupled nucleotide excision repair (TCR) at tRNA genes accounts for MFD and can be inhibited by rapid transcription . During interference of TCR by rapid transcription, however, the presence or absence of functional Mfd protein (transcription-repair coupling factor) can still influence the pattern of mutation, e.g . alter the position of a hot-spot in pLS1D . Only when a tRNA promoter is modulated by an MFD condition is transcription at a rate conducive to TCR . There were several deletion mutations with pLS1D between direct repeats (not present in pZ189) and a model for their production by UV damage is suggested . The spectra with pZ189 in E . coli had similarities with those published for UV mutagenesis in human cells, e.g . mutations at positions approximately 124 and 156 . J Mol Biol, 1999 Nov 19, 294(1), 21 - 34 Linearization and transposition of circular molecules of insertion sequence IS3; Sekine Y et al.; IS3 transposase has been shown to promote production of characteristic circular and linear IS3 molecules from the IS3-carrying plasmid; IS3 circles have the entire IS3 sequence with terminal inverted repeats, IRL and IRR, which are separated by a three base-pair sequence originally flanking either end in the parental plasmid, whereas linear IS3 molecules have three nucleotide overhangs at their 5' ends . Here, we showed that a plasmid carrying an IS3 derivative, which is flanked by different sequences at both ends, generated IS3 circles and linear IS3 molecules owing to the action of transposase . Cloning and sequencing analyses of the linear molecules showed that each had the same 5'-protruding three nucleotide overhanging sequences at both ends, suggesting that the linear molecules were not generated from the parental plasmid by the two double-strand breaks at both end regions of IS3 . The plasmid carrying IS3 with a two base-pair mutation in the terminal dinucleotide, which would be required for transposase to cleave the 3' end of IS3, could still generate linear molecules as well as circles . Plasmids bearing an IS3 circle were cleaved by transposase and gave linear molecules with the same 5'-protruding three nucleotide overhanging sequences . These show that the linear molecules are generated from IS3 circles via a double-strand break at the three base-pair intervening sequence . Plasmids carrying an IS3 circle with the two base-pair end mutation still were cleaved by transposase, though with reduced efficiencies, suggesting that IS3 transposase has the ability to cleave not only the 3' end of IS3, but a site three nucleotides from the 5' end of IS3 . IS3 circles also were shown to transpose to the target plasmids . The end mutation almost completely inhibited this transposition, showing that the terminal dinucleotides are important for the transfer of the 3' end of IS3 to the target as well as for the end cleavage . J Theor Biol, 1999 Nov 21, 201(2), 141 - 56 Variations of the mononucleotide and short oligonucleotide distributions in the genomes of various organisms; Haring D et al.; We calculated the variation coefficients of the mononucleotide and short oligonucleotide distributions in over 1700 long genomic sequences originating from six organisms to demonstrate that the human and Escherichia coli genomic sequences were the least and the most uniform, respectively . The most non-random genomic distributions were exhibited by the four canonical nucleotides, followed by the strong and weak nucleotides, while the distributions of purine or pyrimidine nucleotides and especially the distributions of (A+C) and (G+T) were significantly more uniform even in the human genome . In the human and mouse genomes, the highest coefficients of variation were further observed with the oligonucleotides where CG was combined with the strong nucleotides while its combination with the weak nucleotides significantly decreased the variation which, however, was still very high . High variation was also exhibited by the remaining oligonucleotides composed exclusively of the strong nucleotides or those containing only weak nucleotides . On the other hand, the distributions of oligonucleotides containing similar and especially the same numbers of the strong and weak nucleotides, but no CG or TA dinucleotide, were the most uniform . The information following from the present analysis will be useful not only in the identification of important genomic regions but also in computer simulations of the genomic nucleotide sequences in order to trace and reproduce the pathways of genome evolution . Biochemistry, 1999 Nov 9, 38(45), 15009 - 16 Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH; Uversky VN et al.; Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins . The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding . Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH . This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule . The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues . It is possible that such conformational changes may be associated with the protein function. Biochemistry, 1999 Nov 9, 38(45), 14906 - 15 ClpA and ClpP remain associated during multiple rounds of ATP-dependent protein degradation by ClpAP protease; Singh SK et al.; The Escherichia coli ClpA and ClpP proteins form a complex, ClpAP, that catalyzes ATP-dependent degradation of proteins . Formation of stable ClpA hexamers and stable ClpAP complexes requires binding of ATP or nonhydrolyzable ATP analogues to ClpA . To understand the order of events during substrate binding, unfolding, and degradation by ClpAP, it is essential to know the oligomeric state of the enzyme during multiple catalytic cycles . Using inactive forms of ClpA or ClpP as traps for dissociated species, we measured the rates of dissociation of ClpA hexamers or ClpAP complexes . When ATP was saturating, the rate constant for dissociation of ClpA hexamers was 0.032 min(-1) (t(1/2) of 22 min) at 37 degrees C, and dissociation of ClpP from the ClpAP complexes occurred with a rate constant of 0 . 092 min(-1) (t(1/2) of 7.5 min) . Because the k(cat) for casein degradation is approximately 10 min(-1), these results indicate that tens of molecules of casein can be turned over by the ClpAP complex before significant dissociation occurs . Mutations in the N-terminal ATP binding site led to faster rates of ClpA and ClpAP dissociation, whereas mutations in the C-terminal ATP binding site, which cause significant decreases in ATPase activity, led to lower rates of dissociation of ClpA and ClpAP complexes . Dissociation rates for wild-type and first domain mutants of ClpA were faster at low nucleotide concentrations . The t(1/2) for dissociation of ClpAP complexes in the presence of nonhydrolyzable analogues was >/=30 min . Thus, ATP binding stabilizes the oligomeric state of ClpA, and cycles of ATP hydrolysis affect the dynamics of oligomer interaction . However, since the k(cat) for ATP hydrolysis is approximately 140 min(-1), ClpA and the ClpAP complex remain associated during hundreds of rounds of ATP hydrolysis . Our results indicate that the ClpAP complex is the functional form of the protease and as such engages in multiple rounds of interaction with substrate proteins, degradation, and release of peptide products without dissociation. Biochemistry, 1999 Nov 9, 38(45), 14810 - 9 Insights into the mechanism of Escherichia coli methionine aminopeptidase from the structural analysis of reaction products and phosphorus-based transition-state analogues; Lowther WT et al.; In an effort to differentiate between alternative mechanistic schemes that have been postulated for Escherichia coli methionine aminopeptidase (eMetAP), the modes of binding of a series of products and phosphorus-based transition-state analogues were determined by X-ray crystallography . Methionine phosphonate, norleucine phosphonate, and methionine phosphinate bind with the N-terminal group interacting with Co2 and with the respective phosphorus oxygens binding between the metals, interacting in a bifurcated manner with Co1 and His178 and hydrogen bonded to His79 . In contrast, the reaction product methionine and its analogue trifluoromethionine lose interactions with Co1 and His79 . The interactions with the transition-state analogues are, in general, very similar to those seen previously for the complex of the enzyme with a bestatin-based inhibitor . The mode of interaction of His79 is, however, different . In the case of the bestatin-based inhibitor, His79 interacts with atoms in the peptide bond between the P(1)' and P(2)' residues . In the present transition-state analogues, however, the histidine moves 1.2 A toward the metal center and hydrogen bonds with the atom that corresponds to the nitrogen of the scissile peptide bond (i.e., between the P(1) and P(1)' residues) . These observations tend to support one of the mechanistic schemes for eMetAP considered before, although with a revision in the role played by His79 . The results also suggest parallels between the mechanism of action of methionine aminopeptidase and other "pita-bread" enzymes including aminopeptidase P and creatinase. Cell, 1999 Oct 29, 99(3), 271 - 81 Novel functions of nanos in downregulating mitosis and transcription during the development of the Drosophila germline; Deshpande G et al.; It has previously been shown that germ cells in embryos derived from nos mutant mothers do not migrate to the primitive gonad and prematurely express several germline-specific markers . In the studies reported here, we have traced these defects back to the syncytial blastoderm stage . We show that pole cells in nos embryos fail to establish/maintain transcriptional quiescence; the sex determination gene Sex-lethal (Sxl) and the segmentation genes fushi tarazu and even-skipped are ectopically activated in nos- germ cells . We show that nos- germ cells are unable to attenuate the cell cycle and instead continue dividing . Unexpectedly, removal of the Sxl gene in the zygote mitigates both the migration and mitotic defects of nos- germ cells . Supporting the conclusion that Sxl is an important target for nos repression, ectopic, premature expression of Sxl protein in germ cells disrupts migration and stimulates mitotic activity. J Biol Inorg Chem, 1999 Oct, 4(5), 593 - 600 Double-strand DNA hydrolysis by dilanthanide complexes; Branum ME et al.; Although there has been progress in developing artificial hydrolytic DNA cleaving agents, none of these has been shown to carry out the double-strand hydrolysis of DNA . We demonstrate that La(III) or Ce(IV) combined with the ligand 1,3-diamino-2-hydroxypropane-N,N,N', N'-tetraacetate (HPTA) in a 2 : 1 ratio can efficiently cleave supercoiled plasmid DNA at 55 degrees C within a 3-h period . Analysis of end-labeled restriction fragments cleaved by these complexes reveals 3'- and 5'-ends consistent with a hydrolytic mechanism . Unlike for other polydentate carboxylate complexes, plasmid DNA cleavage by La(2)(HPTA) or Ce(2)(HPTA) affords a significant amount of linear DNA with a considerable fraction of the supercoiled form still remaining . This result implies that La(2)(HPTA) and Ce(2)(HPTA) can carry out double-strand cleavage of plasmid DNA . La(2)(HPTA) and Ce(2)(HPTA) represent the first metal complexes demonstrated to be capable of double-strand hydrolytic cleavage of plasmid DNA. Intensive Care Med, 1999 Oct, 25(10), 1160 - 4 Acute effects of vitamin A on the kinetics of endotoxin in conscious rabbits; Iversen MH et al.; BACKGROUND: Vitamin A reduces the pathophysiological effects of endotoxin in animals, but the mechanism and the lowest effective dose are not clear . METHODS: An intravenous bolus of endotoxin 20 microg . kg(-1) was given to 30 rabbits . In 10 of them, 1000 IE . kg(-1) retinyl palmitate was injected intravenously 1 h before the endotoxin and in another 10 rabbits 1 h after the endotoxin . A one-compartment open model was fitted to the time-concentration profile of endotoxin in plasma . RESULTS: The half-life of endotoxin was half as long when vitamin A was given for prophylaxis (median 35 min) and for treatment (33 min) than in the controls (67 min; p < 0.004) . The plasma concentrations of immunoglobulin G and M endotoxin-core antibodies, the leucocyte count and the acid-base balance did not differ between the groups during the experiment, but the pyrogenic reaction was more pronounced in the controls . CONCLUSION: A fairly low dose of vitamin A reduced the half-life of endotoxin. J Biol Chem, 1999 Nov 12, 274(46), 33105 - 13 Twelve species of the nucleoid-associated protein from Escherichia coli . Sequence recognition specificity and DNA binding affinity; Azam TA et al.; The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid . We expressed and purified 12 species of the DNA-binding protein, i.e . CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td(-) phenotype A) . The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays . Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities . Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA . From the apparent dissociation constant (K(d)) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nM (HU binding to the non-curved DNA) to 250 nM (Hfq binding to the non-curved DNA), under the assay conditions employed. J Biol Chem, 1999 Nov 12, 274(46), 33043 - 9 Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain; Wang Y et al.; Membrane type (MT) matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn(2+)- and Ca(2+)-dependent MMPs . To investigate the proteolytic capabilities of human MT4-MMP (i.e . MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library . Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies in Escherichia coli, was purified to homogeneity and refolded in the presence of Zn(2+) and Ca(2+) . While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-{2-mercapto-4-methylpentanoyl}-LG-OEt and Mca-PLGL-Dpa-AR-NH(2) with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-alpha converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH(2) . Catalytic efficiency with the pro-tumor necrosis factor-alpha converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(a3) = 6.2, pK(a2) = 8.3, and pK(a1) = 10.6) . MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin . Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form . EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity . MT4-MMP is, therefore, a competent Zn(2+)-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A. J Biol Chem, 1999 Nov 12, 274(46), 32909 - 14 The bifunctional active site of s-adenosylmethionine synthetase . Roles of the active site aspartates; Taylor JC et al.; S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction . Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release . The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues . Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine . Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency . The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers . However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively . In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold . The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme . In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants . Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme . This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the smaller Mg(2+). J Biol Chem, 1999 Nov 12, 274(46), 32875 - 80 Product of side-chain cleavage of cholesterol, isocaproaldehyde, is an endogenous specific substrate of mouse vas deferens protein, an aldose reductase-like protein in adrenocortical cells; Lefrancois-Martinez AM et al.; Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown . We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells . The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities . Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors . For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far . In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors . We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5-6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment . In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD(50) . In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide . These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. J Biol Chem, 1999 Nov 12, 274(46), 32667 - 71 The mechanism of ATP hydrolysis at the noncatalytic sites of the transcription termination factor Rho; Kim DE et al.; Escherichia coli transcription termination factor rho is a hexamer with three catalytic subunits that turnover ATP at a fast rate and three noncatalytic subunits that turnover ATP at a relatively slow rate . The mechanism of the ATPase reaction at the noncatalytic sites was determined and was compared with the ATPase mechanism at the catalytic sites . A sequential mechanism for ATP binding or hydrolysis that was proposed for the catalytic sites was not observed at the noncatalytic sites . Pre-steady-state pulse-chase experiments showed that three ATPs were tightly bound to the noncatalytic sites and these were simultaneously hydrolyzed at a rate of 1.8 s(-1) at 18 degrees C . The apparent bimolecular rate constant for ATP binding was determined as 5.4 x 10(5) M(-1) s(-1) in the presence of poly(C) RNA . The ATP hydrolysis products dissociated from the noncatalytic sites at 0.02 s(-1) . The hydrolysis of ATP at the noncatalytic sites was at least 130 times slower, and the overall ATPase turnover was 1500 times slower than that at the catalytic sites . These results from studies of the rho protein are likely to be general to hexameric helicases . We propose that the ATPase activity at the noncatalytic site is too slow to drive translocation of the protein on the nucleic acid or to provide energy for nucleic acid unwinding. Naunyn Schmiedebergs Arch Pharmacol, 1999 Oct, 360(4), 435 - 44 Tetramethylpyradizine prevents inducible NO synthase expression and improves survival in rodent models of endotoxic shock; Wu CC et al.; This study is to investigate the possible mechanism of beneficial effects of tetramethylpyrazine (TMP) on endotoxic shock which we showed in our preliminary study (Liao et al . 1998; Proc Natl Sci Counc Repub China B 22:46-54) . Here, we have confirmed the beneficial effects of TMP on the hypotension, vascular hyporeactivity to noradrenaline (NA), release of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in a rat model of circulatory shock induced by bacterial endotoxin (E . coli lipopolysaccharide, LPS) . In addition, we further examined the expression of inducible NO synthase in the lung and in the aorta from these rats and evaluated the effect of TMP on the 36-h survival rate in a murine model of endotoxaemia . Male Wistar-Kyoto rats were anaesthetised and instrumented for the measurement of mean arterial pressure (MAP) and heart rate (HR) . Injection of LPS (10 mg kg(-1), i.v.) resulted in an acute fall followed by a substantial fall in MAP within 4 h and an increase in HR . In contrast, animals pretreated with TMP (10 mg kg(-1), i.p.; at 30 min prior to LPS) maintained a significantly higher MAP but the tachycardia was further enhanced at 1-2 h when compared to rats given only LPS (LPS rats) . The pressor effect of NA (1 microg kg(-1), i.v.) was also significantly reduced after the treatment of rats with LPS . Similarly, the thoracic aorta obtained from rats at 4 h after LPS showed a significant reduction in the contractile responses elicited by NA (1 microM) . Pretreatment of LPS rats with TMP partially, but significantly, prevented this LPS-induced hyporeactivity to NA in vivo and ex vivo . The injection of LPS resulted in a bell-shaped change in plasma TNF-alpha level which reached a maximum at 1 h, whereas the effect of LPS on the plasma level of nitrate (an indicator of NO formation) was increased in a time-dependent manner . This increment of both TNF-alpha and nitrate levels was significantly reduced in LPS rats pretreated with TMP . Endotoxaemia for 4 h caused a significantly increased protein expression of iNOS in the lung and the aorta . In LPS rats pretreated with TMP, iNOS protein expression in lung and aorta homogenates was attenuated by 75+/-3% and 57+/-6%, respectively . In addition, the lack of evidence of pressor effect of TMP on rats with endotoxaemia for 4 h suggested that TMP inhibits the induction of iNOS rather than directly inhibiting NOS activity . Treatment of conscious ICR mice with a high dose of endotoxin (60 mg kg(-1), i.p.) resulted in a survival rate of only 15% at 36 h (n=20) . However, therapeutic application of TMP (10 mg kg(-1), i.p.; at 0, 6, 15 and 24 h after LPS) increased the 36 h survival rate to 55% (n=20) . Thus, TMP inhibits the expression of iNOS and mitigates the delayed circulatory failure caused by endotoxic shock in the rat . In addition, TMP also improves survival in a murine model of severe endotoxaemia. Oral Microbiol Immunol, 1999 Oct, 14(5), 321 - 5 Use of the Actinobacillus actinomycetemcomitans leukotoxin promoter to drive expression of the green fluorescent protein in an oral pathogen; Lippmann JE et al.; The gene for the green fluorescent protein, gfp, was cloned, under the control of the Actinobacillus actinomycetemcomitans leukotoxin (ltx) promoter, in the A . actinomycetemcomitans shuttle vector, pSU20 . A actinomycetemcomitans containing the ltx-gfp construct emitted bright green fluorescence in the standard invasion assay using epifluorescence microscopy . These data demonstrate that the green fluorescent protein will be a useful tool for the live analysis of A . actinomycetemcomitans interactions with host cells, and that the ltx promoter can be used to drive the expression of non-A . actinomycetemcomitans genes. J Allergy Clin Immunol, 1999 Nov, 104(5), 1084 - 92 Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida; Wagner B et al.; BACKGROUND: Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy . OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy . METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis) . PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3 . The 5'-end sequence was obtained by specific amplification of cDNA ends . The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein . Immunoblotting and inhibition assays were performed to characterize the recombinant allergen . RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained . In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group . The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3 . Hev b 3 is related to Hev b 1 by a sequence identity of 47% . Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3 . CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB . The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy. J Allergy Clin Immunol, 1999 Nov, 104(5), 991 - 9 Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins; Karamloo F et al.; BACKGROUND: Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables . The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced . Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods . OBJECTIVE: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties . METHODS: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region . The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography . Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein . IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2 . RESULTS: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants . On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients . IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes . A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits . A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs . Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy . In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1 . CONCLUSIONS: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy . Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions. J Biol Inorg Chem, 1999 Oct, 4(5), 614 - 20 Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli; Mulliez E et al.; The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced {4Fe-4S}(+) form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical . The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mossbauer spectroscopies . The {4Fe-4S}(+) form is shown to be extremely sensitive to oxygen and converted to {4Fe-4S}(2+), {3Fe-4S}(+/0), and to the stable {2Fe-2S}(2+) form . It is remarkable that the oxidized protein retains full activity . This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active {4Fe-4S} center . This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air . Furthermore, the {2Fe-2S} form of the protein can be converted into a {3Fe-4S} form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster . This is the first report of a 2Fe to 3Fe cluster conversion. J Biol Inorg Chem, 1999 Oct, 4(5), 568 - 78 Ferredoxin-NADP(+) reductase uses the same site for the interaction with ferredoxin and flavodoxin; Martinez-Julvez M et al.; The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions . The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface . In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E . All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics . Their abilities for complex formation with the ET proteins have also been tested . The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase . In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld. Arch Virol, 1999, 144(10), 2013 - 22 The human homologue of the bovine leukemia virus receptor BLVRcp1 is the delta-subunit of adaptor-related AP-3 protein that does not bind the BVLgp51; Ban J et al.; The relationship between the putative bovine leukemia virus receptor gene (BVLRcp) and the susceptibility of human cells to BLV infection was studied . Three cDNA clones encoding different portions of the human equivalent of bovine BLVRcp1 were isolated by DNA-DNA hybridization by comparison of the human cDNA clones to bovine BLVRcp1 . Amino acid sequence indicated that the human sequence encodes the delta subunit of the AP-3 adaptor-related protein . When the recombinant human homologue BLVRcp2 was expressed in E . coli, it failed to bind the BLVgp51 . However, the BVLVgp51 binding ability was restored when the chimerical BLVRcp molecule was prepared by exchanging 5' ends between bovine and human BLVRcp cDNAs . This finding implies that this BLVgp51 binding site is present only on the bovine BLVRcp and therefore its human homologue cannot be recognized by BLVgp51 . This might also explain the resistance of human cells to BLV infection. Arch Virol, 1999, 144(10), 1947 - 60 Immunological characterization of Toscana virus proteins; Di Bonito P et al.; The genome of Toscana virus (Bunyaviridae family, Phlebovirus genus) consists of three single stranded RNA segments (L, M, S), with negative polarity . The L and M segments contain a single ORF in viral complementary sense and the S segment contains two ORFs in "ambisense" orientation . The M segment codes for three proteins in 3'-5' genomic orientation: a 30 kDa non structural protein and two 65 kDa glycoproteins, GN, and GC . In this paper we report the expression in E . coli of the S segment ORFs and of three regions of the L ORF . The expressed proteins were used to produce monospecific polyclonal antibodies in mice . By using these antibodies the N and the NSs proteins were unequivocally assigned to the S viral-complementary and viral-sense ORFs, respectively, and the L protein to the L ORF . We have found that like N and L proteins, NSs protein is associated with the viral nucleocapsids in mature virions, suggesting its possible involvement in early events of viral replication . NSs protein was also found associated with cellular polysomes . In virus-infected cells the anti-L antibodies recognized proteins shorter than the full-length L protein, possibly products of L subgenomic segments . Interestingly these defective products were not found in mature virions, suggesting specific mechanisms in virion assembly. Differentiation, 1999 Oct, 65(2), 73 - 88 Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum; Agarwal AK et al.; Starvation for amino acids initiates the developmental cycle in the cellular slime mold, Dictyostelium discoideum . Upon starvation one of the earliest developmental events is the selective loss of the ribosomal protein mRNAs from polysomes . This loss depends upon sequences in the 5' non-translated leader of the ribosomal protein (r-protein) mRNAs . Here evidence is presented which indicates that those cells which will become prestalk cells express the ribosomal protein genes during development under starvation conditions . Cells which enter the prespore pathway shut off r-protein synthesis . The promoter and 5' non-translated leader sequences from two ribosomal protein genes, the rp-L11 and the rp-S9 genes, are fused to the Escherichia coli beta-galactosidase reporter gene . While beta-galactosidase enzyme activity is detected in situ in most growing cells, by 15 h of development beta-galactosidase enzyme activity is largely lost from the prespore cells although strong beta-galactosidase enzyme activity is present in the prestalk cells . These observations suggest the possibility that the ribosomal protein mRNAs are excluded from polysomes in a cell-type-specific manner. Arch Microbiol, 1999 Nov, 172(5), 295 - 302 Negatively charged phospholipids influence the activity of the sensor kinase KdpD of Escherichia coli; Stallkamp I et al.; Synthesis of the high-affinity K(+)-translocating Kdp-ATPase of Escherichia coli, encoded by the kdpFABC operon, is regulated by the membrane-bound sensor kinase KdpD and the soluble response regulator KdpE . K(+) limitation or a sudden increase in osmolarity induces the expression of kdpFABC . Due to the importance of K(+) to maintain turgor, it has been proposed that KdpD is a turgor sensor . Although the primary stimulus that KdpD senses is unknown, alterations in membrane strain or the interaction between KdpD and membrane components might be good candidates . Here, we report a study of the influence of the membrane phospholipid composition on the function of KdpD in vivo and in vitro using various E . coli mutants defective in phospholipid biosynthesis . Surprisingly, neither the lack of the major E . coli phospholipid phosphatidylethanolamine nor the drastic reduction of the phosphatidylglycerol/cardiolipin content influenced induction of kdpFABC expression significantly . However, in vitro reconstitution experiments with synthetic phospholipids clearly demonstrated that KdpD kinase activity is dependent on negatively charged phospholipids, whereas the structure of the phospholipids plays a minor role . These results indicate that electrostatic interactions are important for the activity of KdpD. Clin Diagn Lab Immunol, 1999 Nov, 6(6), 861 - 7 Development of a recombinant antigen for antibody-based diagnosis of Mycoplasma bovis infection in cattle; Brank M et al.; Mycoplasma bovis induces various clinical manifestations in cattle, such as mastitis, arthritis, and pneumonia . We have evaluated the immunoreactivity of three variable surface proteins (Vsps) of M . bovis, namely VspA, VspB, and VspC, with sera collected from herds with mycoplasmosis or from cattle experimentally infected with M . bovis . Western blot analysis revealed that the Vsps are the predominant antigens recognized by the host humoral response during M . bovis infection . The immunoreactivity of VspA, VspB, and VspC with host antibodies was independent of the clinical manifestations, the geographical origin of the M . bovis isolates, the mode of infection, and the animal's history . Moreover, the results showed that Vsp-specific host antibodies can be detected about 10 days after experimental infection and for up to several months . The full-length or truncated versions of the VspA product were overexpressed in Escherichia coli as fusion proteins (FP-VspA) . Recombinant products showed strong immunoreactivity with the Vsp-specific monoclonal antibodies 1A1 and 1E5, with the corresponding epitopes localized at the VspA N-terminal and C-terminal ends, respectively . Anti-M . bovis sera of cattle naturally or experimentally infected also strongly recognized the full-length FP-VspA . The seroreactivity of sera collected from cattle between 6 and 10 days after experimental infection was weaker with truncated versions of VspA lacking the 1E5 epitope than with the full-length VspA or the truncated versions lacking the 1A1 epitope . Overall, the results indicate that the Vsps, despite their inter- and intraclonal variability, may be applied as target antigens in serodiagnostic assays for epidemiological studies. Nature, 1999 Oct 21, 401(6755), 818 - 22 NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP; Sun C et al.; The inhibitor-of-apoptosis (IAP) family of proteins, originally identified in baculoviruses, regulate programmed cell death in a variety of organisms . IAPs inhibit specific enzymes (caspases) in the death cascade and contain one to three modules of a common 70-amino-acid motif called the BIR domain . Here we describe the nuclear magnetic resonance structure of a region encompassing the second BIR domain (BIR2) of a human IAP family member, XIAP (also called hILP or MIHA) . The structure of the BIR domain consists of a three-stranded antiparallel beta-sheet and four alpha-helices and resembles a classical zinc finger . Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were found to be critical for inhibiting caspase-3 . The absence or presence of these residues may explain the differences in caspase inhibition observed for different truncated and full-length IAPs . Our data further indicate that these residues may bind to the active site and that the BIR domain may interact with an adjacent site on the enzyme. Protein Sci, 1999 Oct, 8(10), 2186 - 93 Design of highly stable functional GroEL minichaperones; Wang Q et al.; GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinant proteins . With the aim of enhancing and widening their use, we have created two highly stable functional variants of minichaperone GroEL(193-345) . A sequence alignment of 130 members of the chaperonin 60 (Cpn60) family was used to design 37 single mutations . Two small-to-large mutations, A223T, A223V and one similar-size mutation, M233L, all located in the hydrophobic core were found to stabilize the protein by more than 1 kcal mol(-1) each . Six stabilizing mutations were combined, yielding two multiple mutants that were 6.99 and 6.15 kcal mol(-1) more stable than wild-type protein . Even though some of the substituted residue pairs are close to each other in the protein structure, the energetic effects of mutation are approximately additive . In particular, the stabilizing substitution A223T is unexpected and would have been missed by purely structural analysis . In the light of previously reported successes employing similar methods with several other proteins, our results show that a homology based approach is a simple and efficient method of increasing the stability of a protein. Protein Sci, 1999 Oct, 8(10), 2166 - 76 Cooperative effects of potassium, magnesium, and magnesium-ADP on the release of Escherichia coli dihydrofolate reductase from the chaperonin GroEL; Clark AC et al.; Previous investigation has shown that at 22 degrees C and in the presence of the chaperonin GroEL, the slowest step in the refolding of Escherichia coli dihydrofolate reductase (EcDHFR) reflects release of a late folding intermediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 268:512-525) . In this paper, we investigate the effects of potassium, magnesium, and MgADP on the release of the EcDHFR late folding intermediate from GroEL . The data demonstrate that GroEL consists of at least two conformational states, with apparent rate constants for EcDHFR release that differ by four- to fivefold . In the absence of potassium, magnesium, and ADP, approximately 80-90% of GroEL resides in the form with the faster rate of release . Magnesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium-induced transition is observed only in the presence of potassium . MgADP at low concentrations (0-50 microM) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium dependent . Nearly identical results were obtained with a GroEL mutant that forms only a single ring, demonstrating that these effects occur within a single toroid of GroEL . In the presence of saturating magnesium, potassium, and MgADP, the apparent rate constant for the release of EcDHFR from wild-type GroEL at 22 degrees C reaches a limiting value of 0.014 s(-1) . For the single ring mutant of GroEL, the rate of EcDHFR release under the same conditions reaches a limiting value of 0.024 s(-1), suggesting that inter-ring negative cooperativity exists for MgADP-induced substrate release . The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a conformational change leading to more rapid release of substrate protein. Protein Sci, 1999 Oct, 8(10), 2151 - 7 Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin; Chen G et al.; A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1) . For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column . The maltose moiety was removed by Factor Xa cleavage . Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC . This labeling was concentration dependent . In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM) . Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner . SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate . These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1 . Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site. Protein Sci, 1999 Oct, 8(10), 2085 - 9 A novel method for increasing production of mature proteins in the periplasm of Escherichia coli; Liu XQ et al.; A novel strategy to obtain high-level production of mature proteins exported to the periplasm of Escherichia coli is described . It is based on a modified signal sequence generated by insertion of a coding sequence of the polypeptide precursor of interest at the BamHI site of the commercial vector pQE-30 resulting in an addition of a dodeca-peptide (MRGSH6GS) at the N-terminus of the precursor . The modification does not affect correct processing of the modified signal nor proper folding of the target protein, resulting in an untagged native product . The method is simple for avoiding onerous optimization of translation initiation and screening of host stains . The usefulness of this method is illustrated by overexpression of DsbC and DsbA . Induced by 0.01 mM IPTG at 37 degrees C, proteins were overproduced to comprise 20-30% of the total cellular proteins, and more than 95% of the expressed proteins were correctly processed and exported into the periplasm with yields of more than 100 mg per liter culture. Nat Genet, 1999 Nov, 23(3), 305 - 8 Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome; Rabinowicz PD et al.; The genomes of higher plants and animals are highly differentiated, and are composed of a relatively small number of genes and a large fraction of repetitive DNA . The bulk of this repetitive DNA constitutes transposable, and especially retrotransposable, elements . It has been hypothesized that most of these elements are heavily methylated relative to genes, but the evidence for this is controversial . We show here that repeat sequences in maize are largely excluded from genomic shotgun libraries by the selection of an appropriate host strain because of their sensitivity to bacterial restriction-modification systems . In contrast, unmethylated genic regions are preserved in these genetically filtered libraries if the insert size is less than the average size of genes . The representation of unique maize sequences not found in plant reference genomes is also greatly enriched . This demonstrates that repeats, and not genes, are the primary targets of methylation in maize . The use of restrictive libraries in genome shotgun sequencing in plant genomes should allow significant representation of genes, reducing the number of reactions required. Nat Biotechnol, 1999 Nov, 17(11), 1109 - 11 A microfabricated fluorescence-activated cell sorter; Fu AY et al.; We have demonstrated a disposable microfabricated fluorescence-activated cell sorter (microFACS) for sorting various biological entities . Compared with conventional FACS machines, the microFACS provides higher sensitivity, no cross-contamination, and lower cost . We have used microFACS chips to obtain substantial enrichment of micron-sized fluorescent bead populations of differing colors . Furthermore, we have separated Escherichia coli cells expressing green fluorescent protein from a background of nonfluorescent E . coli cells and shown that the bacteria are viable after extraction from the sorting device . These sorters can function as stand-alone devices or as components of an integrated microanalytical chip. J Mol Biol, 1999 Oct 22, 293(2), 199 - 213 Transcription activation by catabolite activator protein (CAP); Busby S et al.; Transcription activation by Escherichia coli catabolite activator protein (CAP) at each of two classes of simple CAP-dependent promoters is understood in structural and mechanistic detail . At class I CAP-dependent promoters, CAP activates transcription from a DNA site located upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at these promoters, transcription activation involves protein-protein interactions between CAP and the RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex . At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex; and (ii) protein-protein interactions between CAP and RNAP alpha subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open complex . Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic transcription activation by multiple molecules of CAP, or by CAP and other activators . Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits "anti-activation" by negative regulators . Basic features of transcription activation at class I and class II CAP-dependent promoters appear to be generalizable to other activators . Biol Pharm Bull, 1999 Oct, 22(10), 1122 - 6 Casein kinase II (CK-II)-mediated stimulation of HIV-1 reverse transcriptase activity and characterization of selective inhibitors in vitro; Harada S et al.; The physiological significance of the casein kinase II (CK-II)-mediated phosphorylation of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on its three enzymatic activities {RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP) and ribonuclease H (RNase H)} was investigated in vitro . It was found that (i) the purified recombinant RT (rRT) functioned as an effective phosphate acceptor for CK-II; (ii) the RDDP, DDDP and RNase H activity of rRT was stimulated about 2.8-, 4.1- and 3.9-fold, respectively, after full phosphorylation by CK-II; and (iii) this stimulation was selectively inhibited by potent CK-II inhibitors, such as neocarzinostatin-chromophore (NCS-chrom) and three polyphenol-containing anti-oxidant compounds {quercetin, epigallocatechin gallate (EGCG) and 8-chloro-3',4',5,7-tetrahydroxyisoflavone (8C-3',4',5,7-THI)} . These results suggest that (i) CK-II may be responsible for activation of RT in HIV-1-infected cells; and (ii) the selective inhibition of CK-II-mediated activation of HIV-1 RT by potent CK-II inhibitors may be involved in the mechanism of their anti-HIV-1 effects at the cellular level. Biol Pharm Bull, 1999 Oct, 22(10), 1068 - 72 Construction of the single-chain Fv from 196-14 antibody toward ovarian cancer-associated antigen CA125; Hashimoto Y et al.; The variable regions of heavy- and light-chains of mouse monoclonal antibody 196-14 toward ovarian cancer-associated antigen CA125 were linked with a peptide linker (GSTSGSGKSSEGKG) and a histidine tag was attached at the carboxyl terminal . This single-chain Fv (scFv) with a histidine tag was expressed in Escherichia coli as an inclusion body . The inclusion body was solubilized with guanidium chloride, followed by purification on nickel nitrilotriacetic acid agarose column and refolding into the active form . The scFv thus obtained bound to the Siso cells, which express CA125, and may recognize the same epitope as the parental 196-14 antibody IgG does. Neuroreport, 1999 Sep 29, 10(14), 3071 - 4 The L392V mutation of presenilin 1 associated with autosomal dominant early-onset Alzheimer's disease alters the secondary structure of the hydrophilic loop; Gantier R et al.; Autosomal dominant early-onset Alzheimer's disease results mainly from mutations of the presenilin 1 (PSEN1) gene, which codes for an integral membrane protein of 467 amino acids . The hydrophilic loop (amino acids 263-407) of PSEN1, in which many pathogenic mutations have been localized, appears to be crucial for the protein function since it includes the binding domains to different PSEN1 partners . Using circular dichroism (CD) we analyzed the structural effects of the pathogenic L392V mutation and compared them with those of the E318G substitution . This study revealed that, the L392V mutation, in a phospholipidic medium which mimics the in vivo membrane environment, reduces the alpha helix content of the PSEN1 loop, whereas the E318G substitution, considered as a polymorphism, does not . These results suggest that the pathogenic effect of some PSEN1 mutations within the hydrophilic loop could be the alteration of the interaction to the different binding proteins through a disruption of the secondary structure. Neuroreport, 1999 Sep 29, 10(14), 3061 - 5 Tolerance to lipopolysaccharide is related to the nitric oxide pathway; Almeida MC et al.; Repeated administration of lipopolysaccharide (LPS) induces a refractory state to its usual pyrogenic effects which is called endotoxin tolerance . We tested the hypothesis that nitric oxide (NO) participates in the endotoxin tolerance . Single injection of LPS resulted in an elevation in body temperature (Tb), whereas a significant reduction of the thermoregulatory response to LPS was observed to repeated administration of LPS (administered at 48 h intervals) . Intracerebroventricular (i.c.v.) injection of L-NAME (a non-selective NO inhibitor of nitric oxide synthesis) markedly enhanced the febrile response to LPS in tolerant rats . The data suggest that NO pathway in the central nervous system plays a role in endotoxin tolerance. Arch Pharm Res, 1999 Oct, 22(5), 520 - 3 HIV integrase inhibitory activity of Agastache rugosa; Kim HK et al.; We have been screening anti-HIV integrase compounds from Korean medicinal plants by using an in vitro assay system which is mainly composed of recombinant human immunodeficiency virus type 1 integrase and radiolabeled oligonucleotides . From the above screening, the aqueous methanolic extract of the roots of Agastache rugosa exhibited a significant activity . Bioactivity-guided chromatographic fractionation of the methanolic extract resulted in the isolation of rosmarinic acid . The structure of the compound was determined by spectroscopic data and by the comparison with the reported values . The IC50 of the rosmarinic acid was approximately 10 microg/ml against HIV integrase. Novartis Found Symp, 1999, 223, 132 - 45; discussion 146-9 Diversity and variability of terpenoid defences in conifers: molecular genetics, biochemistry and evolution of the terpene synthase gene family in grand fir (Abies grandis); Bohlmann J et al.; This review focuses on the molecular genetics, biochemistry and evolution of terpenoid synthases relevant to terpenoid defences in conifers . In grand fir (Abies grandis) biosynthesis of terpenoids of the three classes of monoterpenes, sesquiterpenes and diterpenes is inducible by stem wounding at the level of gene activation and increase of enzyme activity of the respective terpene synthases . The monoterpene, sesquiterpene and diterpene synthases utilize prenyl diphosphates of appropriate size as substrates to generate the large diversity of carbon skeletons characteristic of the terpenoid resin of conifers . A large and diverse gene family of grand fir terpene synthases has been cloned and cDNAs are actively expressed in Escherichia coli for enzyme characterization . The monophyletic group of grand fir monoterpene, sesquiterpene and diterpene synthases represents both constitutively expressed and inducible genes encoding single product and multiple product enzymes . Several events of gene duplication and functional specialization of new synthases occurred during the evolution of terpenoid biosynthesis in grand fir, and gave rise to the enormous diversity and variability of this ancient and successful plant defence against herbivores and pathogens . The review concludes with a perspective of the biotechnological applications of terpenoid synthases for the genetic engineering of agricultural crops and forest trees. Mol Cell, 1999 Oct, 4(4), 563 - 71 Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5; Hymowitz SG et al.; Formation of a complex between Apo2L (also called TRAIL) and its signaling receptors, DR4 and DR5, triggers apoptosis by inducing the oligomerization of intracellular death domains . We report the crystal structure of the complex between Apo2L and the ectodomain of DR5 . The structure shows three elongated receptors snuggled into long crevices between pairs of monomers of the homotrimeric ligand . The interface is divided into two distinct patches, one near the bottom of the complex close to the receptor cell surface and one near the top . Both patches contain residues that are critical for high-affinity binding . A comparison to the structure of the lymphotoxin-receptor complex suggests general principles of binding and specificity for ligand recognition in the TNF receptor superfamily. Mol Cell, 1999 Oct, 4(4), 519 - 28 Transfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis; Nomanbhoy TK et al.; Misactivation of amino acids by aminoacyl-tRNA synthetases can lead to significant errors in protein synthesis that are prevented by editing reactions . As an example, discrete sites in isoleucyl-tRNA synthetase for amino acid activation and editing are about 25 A apart . The details of how misactivated valine is translocated from one site to the other are unknown . Here, we present a kinetic study in which a fluorescent probe is used to monitor translocation of misactivated valine from the active site to the editing site . Isoleucine-specific tRNA, and not other tRNAs, is essential for translocation of misactivated valine . Misactivation and translocation occur on the same enzyme molecule, with translocation being rate limiting for editing . These results illustrate a remarkable capacity for a specific tRNA to enhance amino acid fine structure recognition by triggering a unimolecular translocation event. Indian J Biochem Biophys, 1999 Feb, 36(1), 55 - 8 Chemical characterization of the lipopolysaccharides from enteropathogenic Escherichia coli O142 and O158; Datta AK et al.; Lipopolysaccharides (LPS) from two enteropathogenic strains of E . coli O142 and O158 were isolated by hot phenol-water extraction procedure . Polyacrylamide gel electrophoretic pattern of the LPS showed the typical ladder like pattern of smooth type of LPS . The LPS of E . coli O158 was found to contain L-rhamnose, D-glucose and N-acetyl-D-galactosamine as major constituents together with D-galactose, N-acetyl-D-glucosamine, L-glycero-D-manno-heptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) whereas LPS from E . coli O142 contained L-rhamnose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as major constituents together with D-glucose, D-galactose, N-acetyl-D-glucosamine, L-glycero-D-mannoheptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) . LPS was degraded by mild acid hydrolysis to yield a degraded polysaccharide fraction and an insoluble lipid-A fraction . The main fatty acids of the lipid-A fraction of the LPS were C12:O, C14:O, and 3-OH C14:O for O158 strain whereas E . coli O142 lipid-A consisted of C12:O, C14:O, 3-OH C14:O, and C16:O . The degraded polysaccharide fraction on gel permeation chromatography gave a high moleculer weight O-chain fraction and a core oligosaccharide and a fraction containing degraded sugars . The chemical composition of LPS and its fragmented products are reported in this communication. Nucleosides Nucleotides, 1999 Sep, 18(9), 1935 - 44 Identification of a good c-myc antisense oligodeoxynucleotide target site and the inactivity at this site of novel NCH triplet--targeting ribozymes; Giles RV et al.; A region of c-myc mRNA was identified which permitted very efficient antisense effects to be achieved in living cells using chimeric methylphosphonate--phosphodiester antisense effectors . Novel inosine--containing ribozymes (which cleave after NCH triplets) were directed to an ACA triplet within this region and delivered into living cells . No ribozyme intracellular activity could be identified . Very low