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| Fold Des, 1998, 3(6), 535 - 48 Functional analysis of the Escherichia coli genome for members of the alpha/beta hydrolase family; Zhang L et al.; BACKGROUND: Database-searching methods based on sequence similarity have become the most commonly used tools for characterizing newly sequenced proteins . Due to the often underestimated functional diversity in protein families and superfamilies, however, it is difficult to make the characterization specific and accurate . In this work, we have extended a method for active-site identification from predicted protein structures . RESULTS: The structural conservation and variation of the active sites of the alpha/beta hydrolases with known structures were studied . The similarities were incorporated into a three-dimensional motif that specifies essential requirements for the enzymatic functions . A threading algorithm was used to align 651 Escherichia coli open reading frames (ORFs) to one of the members of the alpha/beta hydrolase fold family . These ORFs were then screened according to our three-dimensional motif and with an extra requirement that demands conservation of the key active-site residues among the proteins that bear significant sequence similarity to the ORFs . 17 ORFs from E . coli were predicted to have hydrolase activity and their putative active-site residues were identified . Most were in agreement with the experiments and results of other database-searching methods . The study further suggests that YHET_ECOLI, a hypothetical protein classified as a member of the UPF0017 family (an uncharacterized protein family), bears all the hallmarks of the alpha/beta hydrolase family . CONCLUSIONS: The novel feature of our method is that it uses three-dimensional structural information for function prediction . The results demonstrate the importance and necessity of such a method to fill the gap between sequence alignment and function prediction; furthermore, the method provides a way to verify the structure predictions, which enables an expansion of the applicable scope of the threading algorithms. Biotechnol Appl Biochem, 1999 Feb, 29 ( Pt 1), 31 - 43 Oligonucleotide and plasmid DNA packaging into polyoma VP1 virus-like particles expressed in Escherichia coli; Braun H et al.; The drug delivery system described here is based on the properties of the capsoid or capsid-like structure resulting from the assembly of polyoma virus capsid protein VP1 expressed in Escherichia coli . The capsid protein VP1 was expressed as a fusion protein with a completely removable N-terminal His6 affinity tag . The pentameric morphology of the recombinant VP1 protein was confirmed by electron microscopy after affinity chromatography and factor Xa cleavage under conditions of low ionic strength . The self-assembly of VP1 capsoids can be induced from purified VP1 pentamers by increasing the ionic strength with (NH4)2SO4 . These VP1 capsoid particles were packed in vitro with anti-sense oligonucleotides and plasmid DNA . The loading with DNA was pH-dependent . We observed the highest efficiency at pH 5 . DNase I treatment of particles with encapsidated material showed that 37-55% of the bound oligonucleotides and fragments of 1.5-1.8 kb double-stranded DNA were protected against degradation. J Surg Res, 1999 Jan, 81(1), 81 - 6 Changes in intestinal transit and absorption during endotoxemia are dose dependent; Cullen JJ et al.; BACKGROUND: Septic patients are often intolerant of enteral feedings due to a combination of motility disturbances and impaired absorptive function . Our laboratory has previously demonstrated that endotoxemia results in rapid intestinal transit and decreased jejunal absorption of water, electrolytes, and glucose . We hypothesized that the changes in jejunal transit and absorption during endotoxemia may be dependent on the dose of endotoxin . MATERIALS AND METHODS: Under general anesthesia, rats underwent placement of an internal jugular line, a femoral arterial line, and a 20-cm jejunal Thiry-Vella loop . The jejunal segment was perfused with an isotonic solution containing polyethylene glycol . For 90 min, baseline measurements of blood pressure, heart rate, jejunal absorption of water, electrolytes, and glucose, and jejunal transit were made . Following this baseline period I, rats were given 0.9% NaCl (1 ml/kg) or one of three doses of Escherichia coli lipopolysaccharide (0.5, 1.0, or 5.0 mg/kg) . Studies were then repeated for an additional 90 min . RESULTS: Changes in blood pressure and heart rate were similar among the four groups of animals . Endotoxin decreased water and glucose flux, increased potassium flux, and quickened intestinal transit in a dose-dependent fashion . CONCLUSIONS: We conclude that endotoxemia causes dose-dependent changes in jejunal transit and absorption . The effects of increasing doses of endotoxin on jejunal absorptive and motor function do not appear to be mediated by changes in blood pressure or heart rate . Anal Biochem, 1999 Jan 15, 266(2), 192 - 7 A high-throughput radiometric assay for hepatitis C virus NS3 protease; Cerretani M et al.; A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed . The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein . The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions . To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A-NS4B junction . Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin . The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot . Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components . We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way . Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease . Anal Biochem, 1999 Jan 15, 266(2), 167 - 73 Highly efficient green fluorescent protein-based kinase substrates; Yang F et al.; We have developed a general strategy for designing efficient protein substrates of protein kinases by attaching a phosphorylatable peptide sequence to the C-terminus of His6-tagged green fluorescent protein (GFP) . We found that several C-terminal attachment sites in GFP allow for correct presentation of the phosphorylatable tail to a variety of protein kinases . Using this strategy, we have constructed highly efficient GFP-based substrates for Src, c-Abl, protein kinase A, and protein kinase C betaII protein kinases . The engineered GFP substrate for Src (GFP235IYGEFG) is 300 times more efficient than the protein most commonly used as a Src substrate-rabbit muscle enolase . Endocr Res, 1998 Aug-Nov, 24(3-4), 463 - 8 Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis; Arakane F et al.; Steroidogenic acute regulatory protein (StAR) facilitates delivery of cholesterol to the inner mitochondrial membranes . StAR is imported into mitochondria and processed to a mature form by cleavage of the N-terminal mitochondrial targeting sequence . We produced His-tagged (His-tag StAR) constructs lacking the N-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria . His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system . His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells, whereas wild-type StAR was localized to mitochondria . There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes . We established an assay system using mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in E . coli . Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations . A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein . This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria . Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import. Biochemistry, 1999 Jan 12, 38(2), 848 - 52 Tyrosine 146 of thymidylate synthase assists proton abstraction from the 5-position of 2'-deoxyuridine 5'-monophosphate; Liu Y et al.; Tyr 146 of TS has been proposed to assist in the removal of the proton from the 5-carbon of the pyrimidine in a steady-state intermediate {Hyatt, D . C., Maley, F., and Montfort, W . R . (1997) Biochemistry 36, 4585-4594} . We prepared a replacement set of mutations at position 146 of L . casei TS . The kcat and kcat/Km values of 15 mutants studied were significantly lower than wild-type TS . There was no effect on the Km of dUMP, and only moderate effects on the Km of the cofactor . We concluded that Y146 is not directly involved in substrate binding, but contributes significantly to catalysis . We also examined the Y146 mutants as catalysts for cofactor-independent dehalogenation of BrdUMP, a reaction which simulates early steps of the normal pathway up to and including enzyme-nucleotide covalent adduct formation . Many mutants had activity comparable to the wild-type enzyme, and we concluded that the effects of Tyr 146 mutations occur after the initial covalent adduct is formed . A covalent steady-state intermediate-containing enzyme, dUMP, and cofactor accumulated with Tyr 146 mutants, and could be isolated by SDS-PAGE . The complex was kinetically competent as an intermediate in dTMP formation . Using Y146D and F, it was shown that removal of the C-5 proton from the covalent intermediate was defective . We conclude that in the wild-type enzyme Tyr 146 assists in proton removal from the covalent intermediate . Mutants containing fluorinated tyrosines at position 146 showed an inverse linear correlation of activity versus acidity, again indicating that the basicity of the phenolic oxygen plays an important catalytic role . Speculations of how the poorly basic phenol group might assist proton removal are made in which Tyr 146 acts as a proton conduit to N5 of the cofactor or as a cohort of a water molecule serving as the direct general base catalyst. Biochemistry, 1999 Jan 12, 38(2), 813 - 9 Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli; Sahin-Toth M et al.; Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair {Venkatesan, P., and Kaback, H . R . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 9802-9807} . Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx . In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes . Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx . In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates . Surprisingly, however, no significant exchange or efflux activity is observed . Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax . Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside . Indirect binding assays utilizing substrate protection against {14C}-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG) . The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca . 80 microM) . The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity. Biochemistry, 1999 Jan 12, 38(2), 740 - 50 Magnetic circular dichroism used to examine the interaction of Escherichia coli cytochrome bd with ligands; Borisov V et al.; The interactions of the fully reduced and fully oxidized cytochrome bd from E . coli with ligands CO, NO, and CN- have been studied by a combination of absorption and magnetic circular dichroism (MCD) spectroscopy . In the reduced cytochrome bd, MCD resolves individual bands due to the high-spin heme b595 and the low-spin heme b558 components of the enzyme, allowing one to separately monitor their interactions along with ligand binding to the heme d component . The data show that at low concentrations, the ligands bind almost exclusively to heme d . At high concentrations, the ligands begin to interact with the low-spin heme b558 . At the same time, no evidence for significant binding of the ligands to the high-spin heme b595 is revealed in either the reduced or the fully oxidized cytochrome bd complex . The data support the model {Borisov, V . B., Gennis, R . B., and Konstantinov, A . A . (1995) Biochemistry (Moscow) 60, 231-239} according to which the two high-spin hemes d and b595 share a high-affinity ligand binding site with a capacity for only a single molecule of the ligand; i.e., there is a strong negative cooperativity with respect to ligand binding to these two hemes with cytochrome d having an intrinsic ligand affinity much higher than that of heme b595. Biochemistry, 1999 Jan 12, 38(2), 696 - 704 Structures of normal single-stranded DNA and deoxyribo-3'-S-phosphorothiolates bound to the 3'-5' exonucleolytic active site of DNA polymerase I from Escherichia coli; Brautigam CA et al.; The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer . In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen . To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli . Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively . They serve as standards for comparison with other Mn2+- and Zn2+-containing structures . In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J . Mol . Biol . 277, 363-377) . Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution . Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not . It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al . (1997) J . Am . Chem . Soc . 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis. Biochemistry, 1999 Jan 12, 38(2), 667 - 76 Chicken prion tandem repeats form a stable, protease-resistant domain; Marcotte EM et al.; Prion-linked diseases, such as mad cow disease, scrapie, and the human genetic disorder Creutzfeldt-Jakob disease, are fatal neurodegenerative diseases correlated with changes in the secondary structure of neural prion protein . We expressed recombinant chicken prion protein in Escherichia coli and purified the protein to homogeneity . Circular dichroism spectra of the 26 kDa recombinant protein closely resemble those of prion protein purified directly from healthy hamster brain . The chicken prion protein exists as a soluble, monodisperse monomer but can be forced to multimerize following lyophilization and resuspension . We analyzed the chicken prion protein domain structure by proteolysis and show that, unlike the mammalian homologues, the chicken prion protein N-terminal tandem amino acid repeats form a stable, protease-resistant domain . This domain probably represents a physiologically functional unit . As tested by both mass spectrometry and circular dichroism, the mature chicken prion protein does not bind copper, unlike synthetic peptides from the chicken prion N-terminus, suggesting that binding copper is not the physiological activity of the chicken prion . However, copper strongly destabilizes the prion protein and depresses the melting temperature by 30 degreesC, presumably by binding to the unfolded form of the prion protein . The chicken prion N-terminus may have evolved to fold without a cofactor, unlike mammalian prion proteins, whose N-termini are disordered without cofactors such as copper present . Chicken prion offers an alternative to intractable mammalian prions for structural studies of the amino-terminal domain. Biochemistry, 1999 Jan 12, 38(2), 636 - 42 Amino-terminal processing of chemokine ENA-78 regulates biological activity; Nufer O et al.; Epithelial cell-derived neutrophil-activating protein-78 (ENA-78) is a potent stimulator of neutrophils, inducing a variety of biological responses such as chemotaxis, enzyme release, up-regulation of surface receptors, and intracellular calcium mobilization . Proteolysis of ENA-78 with cathepsin G and chymotrypsin yielded a time-dependent increase in elastase-releasing activity, predicting the formation of truncation products with higher potency than native ENA-78 . To investigate the biological implications of progressive truncation of ENA-78, the N-terminal variants ENA(5-78), ENA(9-78), and ENA(10-78) were cloned and expressed in E . coli . When tested in the neutrophil elastase release assay, the variants ENA(5-78) and ENA(9-78) had a 2-3-fold higher potency than full-length ENA-78, while ENA(10-78) was 3-fold less potent . In the chemotaxis assay, the variant ENA(5-78) exhibited an 8-fold and ENA(9-78) a 2-fold higher potency than native ENA-78 . ENA(10-78), conversely, was 10-fold less potent, but reached a comparable efficacy to ENA-78 at 10(-)7 M concentration . In summary, the rank order in potency with respect to elastase release was ENA(9-78) > ENA(5-78) > ENA-78 > ENA(10-78), while for chemotaxis it was ENA(5-78) > ENA(9-78) > ENA-78 > ENA(10-78) . Variant ENA(5-78) had a higher overall potency and efficiency for chemotaxis than interleukin-8 (IL-8), while ENA(9-78) exhibited a higher efficiency at concentrations of 1-100 nM . The fact that neutrophil cathepsin G produces the stable ENA(9-78) variant in vitro strongly suggests a role for this N-terminal proteolysis during inflammatory processes in vivo. Biochemistry, 1999 Jan 12, 38(2), 589 - 95 Differences in DNA recognition and conformational change activity between boxes A and B in HMG2 protein; Yoshioka K et al.; High mobility group (HMG) 2 is a sequence-nonspecific DNA-binding protein consisting of a repeat of DNA-binding domains called HMG1/2 boxes A and B and an acidic C-terminal . To understand the mode of HMG2 interaction with DNA, we expressed various HMG2 peptides containing HMG1/2 box(es) in Escherichia coli cells and purified them . Gel retardation and DNA supercoiling assay indicated that the region essential for the preferential binding of HMG2 with negatively supercoiled DNA and DNA unwinding activity is located in box B, but not sufficient alone . The flanking C-terminal basic region or box A linked by a linker region is necessary to express activities . The SPR measurements certified that the intrinsic DNA binding affinity of box B is weaker (Kd = 170 microM), and these adjoining regions largely strengthen the affinity (Kd </= 1.2 microM) . In contrast, box A, even in the presence of the adjoining basic linker region, showed no such activities, indicating that boxes A and B are different in their DNA recognition mode . The computer modeling suggested that the side chain of Phe-102 in box B is inserted into the base stack to cause DNA conformational changes, while the side chain of Ala-16 in box A is too small to intercalate . These represent that boxes A and B have similar tertiary structures but their activities for DNA conformational changes obviously differ . Box B is the main region for DNA recognition and conformational changes, and box A must play an assistant to increase its DNA recognition. Biochemistry, 1999 Jan 12, 38(2), 532 - 9 Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4) pyrimidone photodimer by site-directed mutagenesis; Kobayashi H et al.; We have used site-directed mutagenesis to uncover the origin of the binding affinity differences exhibited by a series of monoclonal antibodies that recognize pyrimidine (6-4) pyrimidone photoproducts in the context of single- or double-stranded DNA . In this study, we have focused on two antibodies-64M3 and 64M5-that share the same binding specificity but differ in their affinities . We used single-chain Fv (scFv) derivatives for these studies since they can be easily expressed in Escherichia coli . To facilitate this, we also developed a simple, on-column refolding procedure for scFvs that is rapid and does not require high dilution . We took several precautions to ensure that the scFvs faithfully reflected the behavior of the parent monoclonal antibodies . Results obtained from chimeric scFvs constructed from 64M3 and 64M5 suggested that the higher affinity of the 64M5 antibody was mainly due to its VL region . Loop-grafting studies in which VH CDR loops of 64M3 were individually transplanted into 64M5 were consistent with this hypothesis . Since the VL sequences of 64M3 and 64M5 differ at only three positions (L30, L50, and L90), alanine-scanning mutagenesis was used to assess the importance of these three residues in DNA binding by 64M5 . These studies highlighted the importance of all three VL CDR loops; furthermore, they suggested that photoproduct binding involved conformational changes within the VL region. Atherosclerosis, 1998 Dec, 141 Suppl 1, S17 - 24 Molecular modelling and the biosynthesis of apolipoprotein B containing lipoproteins; Scott J et al.; APOBEC-1 is the cytidine deaminase . We show by sequence alignment, molecular modelling and mutagenesis, that it is related in crystal structure to the cytidine deaminase of Escherichia coli (ECCDA) . The two enzymes are both homodimers with composite active sites formed with loops from each monomer . In the sequence of APOBEC-1, three gaps compared to ECCDA match the size and contour of the minimal RNA substrate . We propose a model in which the asymmetric binding of one active site to the substrate cytidine which is positioned by the downstream binding of the product uridine and that this helps to target the other active site for deamination. Endocr Res, 1998 Aug-Nov, 24(3-4), 731 - 6 Identification of a novel secretogranin II-derived peptide in the adult and fetal human adrenal gland; Anouar Y et al.; Molecular cloning of secretogranin II (SgII) in different species has revealed the existence of a highly conserved 66-amino acid peptide (EM66) flanked by preserved pairs of basic residues . In the present study we have localized and characterized EM66 in the human adrenal gland . A fusion protein containing the human EM66 peptide was produced in E . coli and used to raise polyclonal antibodies in rabbits . Immunohistochemical staining of human adrenal slices revealed intense labeling of adrenochromaffin cells in the adult and fetal gland . HPLC analysis of adrenal extracts showed the presence of an immunoreactive peak exhibiting the same retention time as recombinant EM66 in both adult and fetus . These data demonstrate that post-translational processing of SgII actually generates a novel peptide in the human adrenal gland . The conservation of the sequence of EM66 in vertebrates and the occurrence of the mature peptide during early ontogenesis of the human adrenal gland strongly suggest that EM66 could exert physiological activities. Endocr Res, 1998 Aug-Nov, 24(3-4), 531 - 9 Structure of adrenodoxin and function in mitochondrial steroid hydroxylation; Bernhardt R et al.; The three-dimensional structure of a truncated mutant of bovine adrenodoxin has been resolved at 1.85 A resolution by MAD . The protein consists of a large core region and a more flexible hairpin loop bearing residues which have been previously described as being involved in redox partner recognition . To study the role of distinct protein domains and amino acids of adrenodoxin in interaction with adrenodoxin reductase (AdR), CYP11A1 and CYP11B1, as well as in electron transfer, mutants of adrenodoxin have been prepared by site-directed mutagenesis and produced in Escherichia coli, and their structural and functional properties have been characterized in detail . It could be demonstrated that Tyr82 is located at the edge of the flexible interaction loop of adrenodoxin participating in interactions with AdR and P450s . His56, being close to Tyr82, forms a bridge between the core region of adrenodoxin and the interaction loop . Its role in transmitting changes of the cluster region to the interaction site has also been supported by functional studies . Pro108 of adrenodoxin, the only proline residue contained in the protein and being conserved in this position among several other vertebrate-type ferredoxins, has been demonstrated to be of importance for the correct folding of this protein. Phytochemistry, 1998 Dec, 49(8), 2379 - 82 Alkyl peroxyl radical-scavenging activity of catechins; Nakao M et al.; Alkyl peroxyl radical (ROO.) generated from the reaction between 20 mM t-butyl hydroperoxide (t-BuOOH) and 200 microM hematin could kill E . coli . The minimum concentrations of catechins sufficient to rescue the bacteria treated with ROO . were found to be 70 microM for (-)-epicatechingallate, 100 microM for (-)-epicatechin and 125 microM for (+)-catechin . These values were comparable with the value of alpha-tocopherol, a typical ROO . scavenger . On the other hand, L-ascorbate and beta-carotene revealed about one tenth the scavenging activity of catechins . No scavenging activity was found for superoxide dismutase even at 86 mM . These facts indicate that catechins have high ROO . scavenging activity. Plasmid, 1999 Jan, 41(1), 78 - 81 New cloning vectors with temperature-sensitive replication; Phillips GJ; A series of cloning vectors with conditional, temperature-sensitive replication that are selectable with ampicillin, chloramphenicol, and kanamycin has been constructed . These vectors are derivatives of a pSC101 mutant that can replicate only at low temperatures . The cloning vectors carry a number of unique restriction sites and provide for screening of recombinant plasmids by alpha complementation . These vectors have proven useful for a variety of applications where conditional replication of a recombinant plasmid is desired . Plasmid, 1999 Jan, 41(1), 63 - 9 Instability of pUC19 in Escherichia coli transcription termination factor mutant, rho026; Sozhamannan S et al.; The higher copy number of pUC19, compared to its parent plasmid pBR322, is known to be due to deletion of rop, also known as rom, and to an ori mutation that impedes RNAI:RNAII interaction . pUC19, unlike pBR322, fails to transform E . coli rho mutant rho026 cells . Here we identify two features of pUC19 that contribute to this transformation defect . (1) The pUCori mutation is involved because replacing the pUCori with that of pBR322 restored transformation . (2) Transcription from the lac promoter in pUC19 is important, since deletion or inversion of the promoter or insertion of a transcription terminator (lambdat0) downstream of it restored transformation . Host RNase E activity is responsible for the transformation defect because introduction of an rne-1 allele into rho026 cells suppressed this defect, indicating that RNAI instability due to RNase E is aggravated in the rho026 strain . We suggest that in rho026 cells pUC19 RNAI:RNAII interaction is more impeded than in rho+ cells and Rop/Rom may confer stability by protecting RNAI against RNase E activity because expression of a rom gene inserted into pUC19 restored transformation . Plasmid, 1999 Jan, 41(1), 55 - 62 Allele-specific suppression of ColE1 high-copy-number mutants by a rpoB mutation of Escherichia coli; Yang YL et al.; We have isolated spontaneous rifampicin-resistant mutants from Escherichia coli that showed allele-specific suppression of the copy-number phenotype of ColE1 high-copy-number mutants in vivo . The key step in the regulatory circuitry of the initiation of ColE1 DNA replication is the formation of the persistent hybrid between the primer RNA and the DNA template around the replication origin . Three host-encoded enzymes, RNase H, DNA polymerase I, and RNA polymerase, are essential to the replication initiation in vitro . To decide whether the activity of RNA polymerase is involved directly in the formation of the persistent hybrid, we screened rifampicin-resistant colonies for suppressors of ColE1 copy-number mutants . Suppressor strain YY572 (rpoB572) changes the 572 residue of the beta subunit of RNA polymerase, encoded by the rpoB gene, from isoleucine to leucine . Another suppressor, YY513 (rpoB513), changes the 513 residue from glutamine to lysine . The other known rifampicin-resistant alleles located at residue 513, rpoB8 and rpoB101, did not show a significant suppression of the copy number of those ColE1 copy-number mutants as rpoB513 . The suppression by rpoB513 on different ColE1 copy-number mutants showed allelic specificity . The possible roles of RNA polymerase in control of ColE1 copy number are discussed . J Mol Biol, 1999 Jan 22, 285(3), 1179 - 94 Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli; Chaudhuri TK et al.; The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk . The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography . Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties . The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one . The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins . The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein . A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region . Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding . The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E . coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect . It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH . J Mol Biol, 1999 Jan 22, 285(3), 1067 - 80 Identification of the acidic residues in the active site of DNA polymerase III; Pritchard AE et al.; The mechanism of nucleotide addition by DNA polymerases involves two metal ions that are coordinated in the active site by conserved acidic residues . The three acidic residues that chelate Mg2+ in the active site of Escherichia coli DNA polymerase III have been identified as Asp401, Asp403, and Asp555 by site-directed mutagenesis . Candidates for mutagenesis were initially chosen based on absolute conservation of acidic residues in an alignment of more than 20 diverse DnaE sequences . Conservative Asp to Glu mutations at positions 401 and 403 reduced the activities of the mutant polymerases 2000 and 333-fold, respectively, from that of the wild-type . The third carboxylate was identified by a series of mutations for each critical candidate . With the exception of Glu, all of the mutations at Asp555 led to severely diminished polymerase activity, while each of the other candidates exhibited several relatively active mutant polymerases . Moreover, only the identified active site mutant polymerases displayed a significant enhancement of activity in Mn2+ compared with Mg2+ . These data suggest a direct involvement of the mutated amino acid in metal ion binding . Anal Biochem, 1999 Jan 1, 266(1), 16 - 22 A dilatancy assay for nucleoid denaturation: the centrifugation-dependent clumping of denatured spermidine nucleoids; Murphy LD et al.; The ability of low centrifugal forces to convert denatured spermidine nucleoids from Escherichia coli into nonsedimentable macroscopic clumps is the basis of a rapid and simple, nonisotopic assay for nucleoid denaturation. Anal Biochem, 1999 Jan 1, 266(1), 9 - 15 BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation; O'callaghan CA et al.; The enzyme BirA is a key reagent because of its ability to biotinylate proteins at a specific residue in a recognition sequence . We report a rapid, efficient, and economical method for the production, purification, and application of this enzyme . The method is easily scaled up and the protein produced is of high purity and can be stored for many months with retention of activity . We have used this enzyme to biotinylate the C termini of membrane proteins, allowing these proteins to be tetramerized by binding to streptavidin . Because of the specificity of the biotinylation at the C terminus, the orientation of the membrane proteins on the streptavidin is equivalent to that of the native protein on the cell surface . These tetrameric proteins can be used to study protein receptor-ligand interactions at the cell surface, and site-specific biotinylation can be used to study proteins in vitro using a defined orientation . Nat Struct Biol, 1999 Jan, 6(1), 80 - 8 Crystal structure of recombinant bovine neurocalcin; Vijay-Kumar S et al.; The crystal structure of calcium-bound unmyristoylated bovine neurocalcin from Escherichia coli has been determined at 2.4 A resolution . The three-dimensional structure reveals a highly compact structure consisting of: (i) two pairs of calcium-binding EF-hands (EF1-EF2 and EF3-EF4); (ii) a calcium ion bound at EF2, EF3 and EF4 sites; and (iii) an EF1-hand that is disabled from calcium-binding due to a Cys-Pro sequence in the Ca2+-binding loop . The crystal structure of neurocalcin resembles photoreceptor recoverin in overall topology, however its EF2- and EF4-hands differ . Recently, neurocalcin in the calcium-bound state has been shown to stimulate mammalian rod outer segment membrane guanylate cyclase . A possible site for cyclase activity based on the three-dimensional structure is discussed. Nat Struct Biol, 1999 Jan, 6(1), 56 - 63 Crystal structure of the outer membrane active transporter FepA from Escherichia coli; Buchanan SK et al.; Integral outer membrane receptors for iron chelates and vitamin B12 carry out specific ligand transport against a concentration gradient . Energy for active transport is obtained from the proton-motive force of the inner membrane through physical interaction with TonB-ExbB-ExbD, an inner membrane complex . Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 A resolution . Two distinct functional domains are revealed: (i) a 22-stranded beta-barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N-terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding . These loops could provide a signaling pathway between the processes of ligand recognition and TonB-mediated transport . The blockage of the pore suggests that the N-terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm. Am J Physiol, 1999 Jan, 276(1 Pt 1), L131 - 6 Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils; Cai GZ et al.; Human pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin with high binding specificity to alpha-D-glucosyl residues . It is composed of four regions: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain ("neck" region), and a COOH-terminal carbohydrate recognition domain (CRD) . Previous studies demonstrated that SP-D is chemotactic for inflammatory cells . To test which domain of SP-D might play a role in this function, a mutant that contains only neck and CRD regions was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose . A 17-kDa recombinant SP-D CRD was identified by two antibodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody . The recombinant SP-D CRD was confirmed by amino acid sequencing . Gel-filtration analysis found that 84% of CRD was trimeric and the rest was monomeric . Analysis of the chemotactic properties of the trimeric CRD demonstrated that the CRD was chemotactic for neutrophils (polymorphonuclear leukocytes), with peak activity at 10(-10) M equal to the positive control {formyl-Met-Leu-Phe (fMLP) at 10(-8) M} . The chemotactic activity was abolished by 20 mM maltose, which did not suppress the chemotactic response to fMLP . The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher concentration is required for peak activity (10(-10) vs . 10(-11) M) . The chemotactic response to CRD was largely prevented by preincubation of polymorphonuclear leukocytes with SP-D, and the response to SP-D was prevented by preincubation with CRD . These preincubations did not affect chemotaxis to fMLP . These results suggest that trimeric CRD accounts for the chemotactic activity of SP-D. J Immunol, 1999 Jan 1, 162(1), 195 - 202 Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants; Tomura T et al.; We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol . Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species . Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli . Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so . It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives . A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not . Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response. Microbiol Immunol, 1998, 42(11), 761 - 71 Studies on the rabies virus RNA polymerase: 2 . Possible relationships between the two forms of the non-catalytic subunit (P protein); Takamatsu F et al.; We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase . The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively . Double labeling experiments with {3H}leucine and {32P}orthophosphate demonstrated that p40 was much more phosphorylated than p37 . Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide . The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion . On the other hand, p40 was apparently detected only in the virion, and little detected in the cells . Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly . We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli . Incubation of 37-kDa products from E . coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin . From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s). Mol Plant Microbe Interact, 1999 Jan, 12(1), 68 - 73 Carbamoylation of azorhizobial Nod factors is mediated by NodU; D'Haeze W et al.; Lipochitooligosaccharides (LCOs) synthesized by Azorhizobium caulinodans ORS571 are substituted at the nonreducing-terminal residue with a 6-O-carbamoyl group . LCO biosynthesis in A . caulinodans is dependent on the nodABCSUIJZnoeC operon . Until now, the role of the nodulation protein NodU in the synthesis of azorhizobial LCOs remained unclear . Based on sequence similarities and structural analysis of LCOs produced by a nodU mutant, a complemented nodU mutant, and Escherichia coli DH5 alpha expressing the nodABCSU genes, NodU was shown to be involved in the carbamoylation step. Appl Biochem Biotechnol, 1998 Aug, 74(2), 95 - 103 Purification of recombinant proteins by chemical removal of the affinity tag; Rais-Beghdadi C et al.; The efficient removal of a N- or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine . Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation . Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification . It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and a Plasmodium falciparum antigen . The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag in Escherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag . Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins. Am J Hematol, 1999 Jan, 60(1), 6 - 11 Effects of low molecular weight heparin, alone or combined with antithrombin III, on mortality, fibrin deposits and hemostatic parameters in endotoxin-induced disseminated intravascular coagulation in rabbits; Hermida J et al.; The effect of low molecular weight heparin (LMWH) with or without antithrombin III (AT III) has been studied in a rabbit model of disseminated intravascular coagulation (DIC) induced by continuous infusion of 100 microg/kg/hr of Escherichia coli endotoxin for 6 hr . LMWH (5 and 10 IU/kg/hr/6 hr), alone or in combination with AT III (20 U/kg/hr/6 hr), or saline were administered simultaneously with endotoxin . Hemostatic markers at 0, 2, and 6 hr as well as kidney fibrin deposits and the mortality rate at 24 hr were determined . Rabbits receiving only endotoxin showed an impairment in hemostasis, as well as high kidney fibrin deposits and a high mortality rate . LMWH alone did not exert any effect . The simultaneous infusion of LMWH and AT III exerted a beneficial effect on the hemostatic markers and reduced the kidney fibrin deposits as well as the mortality rate in a LMWH dose-dependent manner . Fibrinogen and protein C consumption were significantly higher and renal fibrin deposits more intense in the rabbits that had died in the first 24 hr . There was also a significant positive correlation between kidney fibrin deposits and platelets, fibrinogen, and protein C consumption, taking the whole rabbit population . It is concluded that the simultaneous infusion of LMWH and AT III is useful in this DIC model and would make it possible to reduce significantly the AT III doses used when AT III is given alone. J Bacteriol, 1999 Jan, 181(2), 572 - 6 Effect of ppGpp on Escherichia coli cyclopropane fatty acid synthesis is mediated through the RpoS sigma factor (sigmaS); Eichel J et al.; Strains of Escherichia coli carrying mutations at the relA locus are deficient in cyclopropane fatty acid (CFA) synthesis, a phospholipid modification that occurs as cultures enter stationary phase . RelA protein catalyzes the synthesis of guanosine-3',5'-bisdiphosphate (ppGpp); therefore, ppGpp was a putative direct regulator of CFA synthesis . The nucleotide could act by increasing either the activity or the amount of CFA synthase, the enzyme catalyzing the lipid modification . We report that the effect of RelA on CFA synthesis is indirect . In vitro and in vivo experiments show no direct interaction between ppGpp and CFA synthase activity . The relA effect is due to ppGpp-engendered stimulation of the synthesis of the alternative sigma factor, RpoS, which is required for function of one of the two promoters responsible for expression of CFA synthase. Protein Expr Purif . 1998 Dec;14(3):IV. Papers to appear in forthcoming issues The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site O(B)3 but other KorB-binding sites form destabilizing complexes in the absence of O(B)3. School of Biological Sciences, The University of Birmingham, Edgbaston, UKThe sector of the genome of broad-host-range IncP plasmid RK2 from kb coordinate 54.0 to 60.0 confers an active partitioning phenotype, increasing the segregational stability of low-copy-number unstable plasmids . This Par region encodes the central control operon (korA, incC, korB, korF and korG) and the associated genes kfrA, upf54.8 and upf54.4 . Each ORF in this region was knocked out in turn and it was shown that only incC and korB are needed for the stability phenotype . incC encodes two polypeptides from alternative translational starts . A deletion of the start of the operon showed that only IncC2, the shorter product, is essential for partitioning . Directed mutation or deletion was used to inactivate in turn each of the three KorB-binding sites (O(B)s) which were candidate cis-acting sequences needed for stability . Only inactivation of O(B)3, which lies between upf54.4 and upf54.8, resulted in an increased rate of segregational loss . However, the rate of loss was significantly higher than the rate of loss of the test plasmid carrying none of this RK2 Par region . Either inactivation of korB or deletion of O(B)1 from this O(B)3 mutant resulted in restoration of the loss rate to that expected for the unstable test plasmid alone . Thus KorB can act on O(B)1 to create a complex that either inhibits replication or reduces the effective plasmid copy number, perhaps by promoting pairing between plasmid molecules . This implies that RK2 goes through a cycle of pairing and separation, akin to the mitotic cycle of eukaryotic chromosomes. FEBS Lett, 1998 Dec 18, 441(2), 327 - 30 Kinetic properties and stereospecificity of the monomeric dUTPase from herpes simplex virus type 1; Bergman AC et al.; Kinetic properties of the monomeric enzyme dUTPase from herpes simplex virus type 1 (HSV) were investigated and compared to those previously determined for homotrimeric dUTPases of bacterial and retroviral origins . The HSV and Escherichia coli dUTPases are equally potent as catalysts towards the native substrate dUTP with a kcat/K(M) of about 10(7) M(-1) s(-1) and a K(M) of 0.3 microM . However, the viral enzymes are less specific than the bacterial enzyme . The HSV and E . coli dUTPases show the same stereospecificity towards the racemic substrate analogue dUTPalphaS (2'-deoxyuridine 5'-(alpha-thio)triphosphate), suggesting that they have identical reaction mechanisms. FEBS Lett, 1998 Dec 18, 441(2), 247 - 50 Disruption of Escherichia coli transaldolase into catalytically active monomers: evidence against half-of-the-sites mechanism; Schorken U et al.; Disruption of the hydrogen bonding network at the interface of Escherichia coli transaldolase by substitution of R300 to a glutamic acid residue resulted in a monomeric enzyme at basic pH values, with almost no change in the kinetic parameters . The stability of the R300A and R300E mutants towards urea and thermal inactivation is similar to that of the wild-type enzyme . X-ray analysis showed that no structural changes occurred as a consequence of the side chain replacement . This indicates that the quaternary structure is not required for catalytic activity nor does it contribute significantly to the stability of the enzyme . The results are not consistent with a proposed half-of-the-sites reaction mechanism. FEBS Lett, 1998 Dec 18, 441(2), 242 - 6 Analysis of the conserved acidic residues in the regulatory domain of PhoB; Zundel CJ et al.; The PhoB protein from Escherichia coli is a member of the two-component signal transduction pathway that controls an adaptive response to limiting phosphate . Activation involves its phosphorylation on a conserved aspartate . Site-directed mutations were introduced at conserved acidic residues . The E9D, D10E, D10N, E11A, E11D and E11Q mutants were each able to induce alkaline phosphatase under low phosphate growth conditions whereas the E9A, D10A, D53A, D53E and D53N could not . The E9Q mutant was constitutively active . Phosphorylation assays showed that only the E9D, E11A, E11Q and E11D mutants were phosphorylated by acetyl phosphate . Most mutants also displayed defects in magnesium binding. FEBS Lett, 1998 Dec 18, 441(2), 195 - 9 Probing the potential metal binding site in Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (phenylalanine-sensitive); Sundaram AK et al.; The active site residues of the proposed metal binding site of DAH 7-P synthase (phe) were probed by site-directed mutagenesis of C61 to glycine and serine, H64 to glycine, and with the double mutant C61H/H64C . While C61S and C61H/ H64C were inactive, both C61G and H64G were active . All mutants, regardless of enzymatic activity, bound one equivalent of Fe2+ per monomeric unit . Even though C61 and H64 were shown not to be metal ligands for the DAH 7-P synthase (phe), they may provide some of the backbone interactions/secondary structural elements necessary to properly form the metal binding pocket. J Bacteriol, 1999 Jan, 181(2), 681 - 4 Control of Herbaspirillum seropedicae NifA activity by ammonium ions and oxygen; Souza EM et al.; The activity of a truncated form of Herbaspirillum seropedicae NifA in different genetic backgrounds showed that its regulatory domain is involved in nitrogen control but not in O2 sensitivity or Fe dependence . The model for nitrogen control involving PII could thus apply to the proteobacteria at large . NifA may have a role in controlling ADP-ribosylation of nitrogenase in Azospirillum brasilense. J Bacteriol, 1999 Jan, 181(2), 670 - 4 Isolation and characterization of the nikR gene encoding a nickel-responsive regulator in Escherichia coli; De Pina K et al.; Expression of the nickel-specific transport system encoded by the Escherichia coli nikABCDE operon is repressed by a high concentration of nickel . By using random transposon Tn10 insertion, we isolated mutants in which expression of the nik operon became constitutive with respect to nickel . We have identified the corresponding nikR gene which encodes a nickel-responsive regulator . Expression of nikR was partially controlled by Fnr through transcription from the nikA promoter region . In addition, a specific transcription start site for the constitutive expression of nikR was found 51 bp upstream of the nikR gene. J Bacteriol, 1999 Jan, 181(2), 662 - 5 S-methylmethionine metabolism in Escherichia coli; Thanbichler M et al.; Selenium-accumulating Astragalus spp . contain an enzyme which specifically transfers a methyl group from S-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid . Analysis of the amino acid sequence of this enzyme revealed that Escherichia coli possesses a protein (YagD) which shares high sequence similarity with the enzyme . The properties and physiological role of YagD were investigated . YagD is an S-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate . Mutants in yagD which also possess defects in metE and metH are unable to utilize S-methylmethionine for growth, whereas a metE metH double mutant still grows on S-methylmethionine . Upstream of yagD and overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in a metE metH genetic background . Since it displays sequence similarities with amino acid permeases it appears to be the transporter for S-methylmethionine . Methionine but not S-methylmethionine in the medium reduces the amount of yagD protein . This and the existence of four MET box motifs upstream of yfkD indicate that the two genes are members of the methionine regulon . The physiological roles of the ykfD and yagD products appear to reside in the acquisition of S-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis. J Bacteriol, 1999 Jan, 181(2), 563 - 71 The LysR homolog LrhA promotes RpoS degradation by modulating activity of the response regulator sprE; Gibson KE et al.; Synthesis of the OmpF porin of Escherichia coli is regulated in response to environmental and growth phase signals . In order to identify constituents of the various regulatory pathways involved in modulating ompF transcriptional expression, transposon insertion mutagenesis was performed and mutations that increased ompF'-lacZ activity were identified as previously described . Mutations mapping to a previously identified gene of unknown function, lrhA, were obtained . We found that LrhA, a LysR homolog, functions as a regulatory component in the RpoS-dependent growth phase repression of ompF . In addition to altered growth phase regulation of ompF, these lrhA mutants have pleiotropic stationary-phase defects as a result of decreased RpoS levels . We provide evidence that LrhA promotes degradation of RpoS by functioning within a genetic pathway that includes the response regulator SprE and the ClpXP protease . LrhA functions upstream of the other components in the pathway and appears to modulate the activity of SprE. J Bacteriol, 1999 Jan, 181(2), 521 - 30 Septal localization of FtsQ, an essential cell division protein in Escherichia coli; Chen JC et al.; Septation in Escherichia coli requires several gene products . One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain . We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes . The gfp-ftsQ gene complements a null mutation in ftsQ . Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site . Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting . GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains . In addition, the septal localization of ZipA apparently did not require functional FtsQ . Our results indicate that FtsQ is an intermediate recruit to the division site. J Bacteriol, 1999 Jan, 181(2), 477 - 82 Slipped misalignment mechanisms of deletion formation: in vivo susceptibility to nucleases; Bzymek M et al.; Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA . In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE) . Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands . We examined the influence of the 3' exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo . Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3' ends are a common intermediate in both mechanisms of slipped misalignments . Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I . If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms . Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system. J Bacteriol, 1999 Jan, 181(2), 462 - 8 BglF, the Escherichia coli beta-glucoside permease and sensor of the bgl system: domain requirements of the different catalytic activities; Chen Q et al.; The Escherichia coli BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities . In the absence of beta-glucosides, it phosphorylates BglG, a positive regulator of bgl operon transcription, thus inactivating BglG . In the presence of beta-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports beta-glucosides into the cell and phosphorylates them . BglF is composed of two hydrophilic domains, IIAbgl and IIBbgl, and a membrane-bound domain, IICbgl, which are covalently linked in the order IIBCAbgl . Cys-24 in the IIBbgl domain is essential for all the phosphorylation and dephosphorylation activities of BglF . We have investigated the domain requirement of the different functions carried out by BglF . To this end, we cloned the individual BglF domains, as well as the domain pairs IIBCbgl and IICAbgl, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo . We show here that the IIB and IIC domains, linked to each other (IIBCbgl), are required for the sugar-driven reactions, i . e., sugar phosphotransfer and BglG activation by dephosphorylation . In contrast, phosphorylated IIBbgl alone can catalyze BglG inactivation by phosphorylation . Thus, the sugar-induced and noninduced functions have different structural requirements . Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgl and IICbgl which occur upon the interaction of BglF with the sugar. J Bacteriol, 1999 Jan, 181(2), 401 - 10 Heat-induced synthesis of sigma32 in Escherichia coli: structural and functional dissection of rpoH mRNA secondary structure; Morita M et al.; The heat shock response in Escherichia coli depends primarily on the increased synthesis and stabilization of otherwise scarce and unstable sigma32 (rpoH gene product), which is required for the transcription of heat shock genes . The heat-induced synthesis of sigma32 occurs at the level of translation, and genetic evidence has suggested the involvement of a secondary structure at the 5' portion (nucleotides -19 to +247) of rpoH mRNA in regulation . We now present evidence for the mRNA secondary structure model by means of structure probing of RNA with chemical and enzymatic probes . A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo . These findings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions . The results indicated not only the primary importance of base pairings between the translation initiation region of ca . 20 nucleotides (the AUG initiation codon plus the "downstream box") and the internal region of rpoH mRNA but also the requirement of appropriate stability of mRNA secondary structures for characteristic thermoregulation, i.e., repression at a low temperature and induction upon a temperature upshift. Protein Expr Purif, 1998 Dec, 14(3), 395 - 402 High-level expression of rat farnesyl:protein transferase in Escherichia coli as a translationally coupled heterodimer; Zimmerman KK et al.; Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins . Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the alpha- and beta-subunits are coexpressed, large-scale overexpression of active enzyme has been challenging . We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/L Escherichia coli . Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid . The beta-subunit-coding sequence was placed upstream of the alpha-subunit coding sequence linked by overlapping beta-subunit stop and alpha-subunit start codons . Additionally, the initial 88 codons of the alpha-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E . coli . Since previous attempts at recombinantly expressing FPTase in E . coli from a translationally coupled system have demonstrated that initiation of translation of the alpha-subunit is poor, we propose that the optimization of the codons at the start of the alpha-subunit gene leads to the observed high level of recombinant expression . Protein Expr Purif, 1998 Dec, 14(3), 382 - 6 Green fluorescent protein purification by organic extraction; Yakhnin AV et al.; Green fluorescent protein (GFP) is widely used as an excellent reporter molecule in biochemistry and cell biology . Some biochemical and immunological assays require high-purity GFP . However, the majority of current procedures for GFP purification include multiple time-consuming chromatography steps with a low yield of the desired product or require tag-containing proteins . An alternative method is described for the GFP purification without affinity extensions using organic extraction yielding a highly homogeneous protein indistinguishable in spectroscopic properties from that purified by previous methods . Protein Expr Purif, 1998 Dec, 14(3), 371 - 81 Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli; Banerji S et al.; The CD44 molecule is a widely distributed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan . The ligand-binding site which is located in the membrane distal portion of the molecule encompasses a region of approximately 100 amino acids termed the Link domain, a structural unit that is conserved among members of the Hyaladherin superfamily which includes cartilage link protein, aggrecan, and tumor necrosis factor-stimulated gene-6 (TSG-6) . In contrast to these other Hyaladherins, however, the ligand-binding domain of CD44 appears to extend beyond the Link domain to involve additional basic residues located toward the membrane proximal region . Furthermore, recent molecular modeling studies indicate that within the CD44 Link domain itself, the spatial arrangement of critical residues involved in HA binding is likely to differ significantly from the prototypic TSG-6 Link module . In order to obtain material to solve the CD44 solution structure we have developed an optimized method for the expression and purification of functionally active CD44 ectodomains encompassing both the Link module and the additional downstream HA-binding residues in Escherichia coli . Here we describe the details of the method which involves solubilization of recombinant CD44 from inclusion bodies in 8 M urea, followed by refolding and purification of intact monomers using size-exclusion and reverse-phase chromatography . We show the method yields CD44 molecules that (1) retain reactivity with a panel of conformation-sensitive antibodies, (2) possess similar hyaluronan-binding characteristics to authentically folded CD44 molecules expressed in eukaryotic cells, and (3) display one-dimensional NMR spectra that indicate the presence of a single conformational species . This method should enable sufficient amounts of functional CD44 Link module to be produced for comprehensive structural analyses by multidimensional NMR spectroscopy . Protein Expr Purif, 1998 Dec, 14(3), 343 - 52 Recombinant human cytomegalovirus protease with a C-terminal (His)6 extension: purification, autocatalytic release of the mature enzyme, and biochemical characterization; Tomasselli AG et al.; Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics . To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed in Escherichia coli . The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site . The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence . In this design, the Ala-Ser bond at amino acids 256-257 constitutes a site naturally cleaved by the protease during capsid maturation . The 268-amino-acid polypeptide with the (His)6 tag was expressed at high levels in E . coli as inclusion bodies . After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography . The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids including the (His)6 tag from the C terminus of the protein . This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus . Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy . The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein in E . coli . In addition, the refolded CMV PR could be crystallized for X-ray diffraction . Protein Expr Purif, 1998 Dec, 14(3), 335 - 42 Production of leptin in Escherichia coli: a comparison of methods; Varnerin JP et al.; A procedure is described for gram-scale refolding of Escherichia coli-derived human leptin inclusion bodies . Refolding was achieved by gradually reducing denaturant using a diafiltration method . Refolded leptin is characterized by in vivo modulation of food intake, reduction in body weight, and lowering of insulin and glucose levels in ob/ob mice . In addition, refolded leptin is characterized by radioimmunoassay (RIA) and activation of the leptin receptor in a cell-based assay . For comparison we also refolded leptin by a simple dilution method and produced periplasmic derived leptin, which did not require ex vivo folding . Leptin produced by these three methods and leptin obtained from commercial sources were compared using the RIA and the cell-based assay and appeared to be of comparable quality and potency . Arch Biochem Biophys, 1999 Jan 15, 361(2), 302 - 8 Amino acid substitutions in the a subunit affect the epsilon subunit of F1F0 ATP synthase from Escherichia coli; Gardner JL et al.; Amino acid substitutions at many positions in the a subunit of F1F0 ATP synthase result in impaired proton translocation and altered catalytic activity . In this work, we demonstrate that amino acid substitutions in the a subunit affect the epsilon subunit . In mutant F1F0 ATP synthases, the epsilon subunit was studied by determining its sensitivity to proteolysis and by chemical crosslinking under conditions of active turnover and in quiescent enzyme . Like native F1F0 ATP synthase, the epsilon subunit in enzymes carrying either the aarg-210-->ile or agly-218-->asp substitutions proved resistant to trypsin digestion during ATP hydrolysis . In each case, the epsilon subunit was rapidly digested in the presence of a nonhydrolyzable ligand, but this did not result in the activation of hydrolytic activity typically seen in wild-type enzyme . In enzyme carrying the aala-217-->arg substitution, the trypsin digestion of the epsilon subunit occurred regardless of ligand and was accompanied by a limited hydrolytic activation . Relative to the native F1F0 ATP synthase, the aala-217-->arg substitution resulted in reduced efficiency of crosslinking between the epsilon and beta subunits using 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide . These observations indicate that the structural changes resulting from amino acid substitutions in the a subunit are propagated to the epsilon subunit and are specific to the individual substitutions . Arch Biochem Biophys, 1999 Jan 15, 361(2), 195 - 201 Identification and cloning of rat galectin-2: expression is predominantly in epithelial cells of the stomach; Oka T et al.; A complementary DNA clone preferentially expressed in the gastrointestinal tract was obtained from a rat stomach library . The protein coded by the clone had a single carbohydrate recognition domain having conserved motifs for beta-galactoside binding and showed 67% amino acid identity with human galectin-2 . The recombinant protein synthesized in Escherichia coli could bind to an asialofetuin column and was eluted with beta-galactopyranoside . From these observations, we named the protein rat galectin-2 coded by the cDNA . The rat galectin-2 was predominantly expressed in the epithelial cells of stomach . Thus this protein may form a mucin layer cross-linking with the beta-galactoside moiety of glycoproteins . Arch Biochem Biophys, 1999 Jan 1, 361(1), 106 - 12 Cloning, overproduction, and purification of native and mutant recombinant yeast orotate phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetime; Witte JF et al.; The gene for orotate phosphoribosyltransferase from Saccharomyces cerevisiae has been subcloned into an Escherichia coli overexpression vector and the enzyme has been produced in large quantities, thus simplifying the purification to one step . We were able to repeat the published (J . Victor, L . B . Greenberg, and D . L . Sloan J . Biol . Chem . 254, 2647-2655, 1979) . 32PPi/5-phosphorylribose 1-alpha-diphosphate exchange experiments and could demonstrate the exchange by magnetization inversion transfer NMR experiments as well . However, when contaminating orotidine 5'-monophosphate (OMP) was eliminated with OMP decarboxylase, any evidence of magnetization transfer vanished . Consequently, it is concluded that a ping pong mechanism is not operable and that a previously proposed oxocarbocation intermediate along the pathway to OMP does not persist long enough in the catalytic cycle of this enzyme to be recognized by NMR exchange experiments . Arch Biochem Biophys, 1999 Jan 1, 361(1), 94 - 100 Six putative IQ motifs of the recombinant chicken intestinal brush border myosin I are involved in calmodulin binding; Khoroshev MI et al.; Chicken brush border myosin I has up to six IQ sequence motifs at which it may bind calmodulin . To determine the relative contributions of these motifs to calmodulin binding, fusion deletion fragments were expressed in Escherichia coli and their ability to bind calmodulin was assessed by the gel overlay technique . The first three N-terminal IQ sites showed strong binding with calmodulin . Surprisingly, the last three incomplete IQ motifs also contributed substantial calmodulin binding . The first and fourth IQ sites bound calmodulin but tended to reduce binding in combination with other sites . The data indicate that interactions among all six IQ motifs contribute to the ability of myosin I to bind calmodulin . Anal Biochem, 1998 Dec 15, 265(2), 299 - 307 Purine nucleoside phosphorylase: its use in a spectroscopic assay for inorganic phosphate and for removing inorganic phosphate with the aid of phosphodeoxyribomutase; Nixon AE et al.; The kinetics of the phosphorolysis of 7-methylated guanosine analogues catalyzed by purine nucleoside phosphorylase has been analyzed to understand the use of this system as a "Pi mop" to remove Pi from solutions and as a spectroscopic assay for Pi at micromolar concentrations . An expression system was developed for the phosphorylase from Escherichia coli: this protein (subunit molecular mass 26 kDa) and one from a commercial source (29 kDa) were used in this study . Rates of >50 s-1 were obtained for the phosphorolysis at 30 degrees C, so that when the phosphorylase is coupled to the phosphatase being studied, rates of Pi release from the phosphatase can be measured close to this rate . The kinetic mechanism appears to obey the Michaelis-Menten model in the steady state with the bond cleavage rate limiting . Slow hydrolysis of ribose-1-phosphate to Pi catalyzed by the phosphorylase limits the efficiency of the Pi mop . To overcome this, phosphodeoxyribomutase was used to catalyze the conversion of ribose-1-phosphate to ribose-5-phosphate, enabling the Pi mop to remove large amounts of Pi quantitatively . Acyclovir diphosphate provides a simple method to switch off the Pi mop as it is a tight inhibitor (Kd 12 nM) of purine nucleoside phosphorylase . Environ Mol Mutagen, 1998, 32(4), 325 - 30 Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay; Zimmer DM et al.; We examined several experimental parameters of the lambda cI/cII transgenic mutation assay . In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue" transgenic mice . Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4 degrees C for up to 4 months did not affect either mutant frequency or titer . Storage of packaged phage for 28 days at 4 degrees C did not affect titer . The mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested . Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E . coli strains and incubated at 37 degrees C or 24 degrees C showed highest titers on G1217 at 37 degrees C . The fraction of the G1217, 37 degrees C titer seen in the other strains and conditions varied widely with the cII mutation. J Biol Chem, 1999 Jan 15, 274(3), 1449 - 57 Molecular recognition in a post-translational modification of exceptional specificity . Mutants of the biotinylated domain of acetyl-CoA carboxylase defective in recognition by biotin protein ligase; Chapman-Smith A et al.; We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase . We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro . This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins . Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants . The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly . Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance . Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins. J Virol, 1999 Feb, 73(2), 1649 - 54 Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli; Ferrari E et al.; Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents . In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents . Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B . Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others . For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity . Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner . Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases. J Virol, 1999 Feb, 73(2), 1609 - 16 A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement; Padidam M et al.; Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles . Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants . In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus . To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5) . The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA . The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA . The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication . ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms . In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei . We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection. J Virol, 1999 Feb, 73(2), 1399 - 410 Mutational analysis of the avian adenovirus CELO, which provides a basis for gene delivery vectors; Michou AI et al.; The avian adenovirus CELO is being developed as a gene transfer tool . Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein) . For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored . A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in trans for virus replication . A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent . Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette . Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector . The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors. J Virol, 1999 Feb, 73(2), 1392 - 8 Coxsackievirus and adenovirus receptor amino-terminal immunoglobulin V-related domain binds adenovirus type 2 and fiber knob from adenovirus type 12; Freimuth P et al.; The extracellular region of the coxsackievirus and adenovirus receptor (CAR) is predicted to consist of two immunoglobulin (Ig)-related structural domains . We expressed the isolated CAR amino-terminal domain (D1) and a CAR fragment containing both extracellular Ig domains (D1/D2) in Escherichia coli . Both D1 and D1/D2 formed complexes in vitro with the recombinant knob domain of adenovirus type 12 (Ad12) fiber, and D1 inhibited adenovirus type 2 (Ad2) infection of HeLa cells . These results indicate that the adenovirus-binding activity of CAR is localized in the amino-terminal IgV-related domain and confirm our earlier observation that Ad2 and Ad12 bind to the same cellular receptor . Preliminary crystallization studies suggest that complexes of Ad12 knob bound to D1 will be suitable for structure determination. J Virol, 1999 Feb, 73(2), 1046 - 53 A replication-incompetent adenovirus vector with the preterminal protein gene deleted efficiently transduces mouse ears; Moorhead JW et al.; Adenoviruses offer great potential as gene therapy agents but are limited by the strong inflammatory response that occurs in response to the recombinant virus . Since the degree of inflammation correlates in part with the potential of the viral vector for replication, we constructed a preterminal protein (pTP) deletion mutant adenovirus type 5 vector, Ad5dl308DeltapTPbeta-gal, that is replication incompetent due to deletion of the pTP gene and that has the E1 genes replaced by the Escherichia coli lacZ reporter gene under the control of the cytomegalovirus major immediate-early promoter . This virus was compared with a first-generation, replication-defective adenovirus vector, Ad5dl308beta-gal, that is isogenic except that it contains a wild-type pTP gene . To examine transduction efficiency and induction of inflammation, we developed a novel system involving intradermal injection of BALB/c mouse ears . Mouse ears can be accurately measured to determine the degree of edema as an indirect measurement of inflammation . Edema and inflammation were induced in a dose- and time-dependent manner by both viruses and correlated well . LacZ activity correlated inversely with edema and inflammation . The pTP-defective vector Ad5dl308DeltapTPbeta-gal transduced mouse ears much more efficiently and induced edema and inflammatory cell infiltration approximately 10-fold less efficiently than the first-generation vector Ad5dl308beta-gal . The diminished inflammatory response and increased efficiency of transduction observed with Ad5dl308DeltapTPbeta-gal indicate its promise as a gene therapy agent for other tissues . The results also demonstrate that the mouse ear model offers potential for the study of adenovirus-induced inflammation because of the ready access of the ears, the relative ease of continuous measurement, and the sensitivity to adenovirus transducing vectors. Lab Invest, 1998 Dec, 78(12), 1615 - 23 S19 ribosomal protein cross-linked dimer causes monocyte-predominant infiltration by means of molecular mimicry to complement C5a; Nishiura H et al.; S19 ribosomal protein is a component of the protein-producing machinery, ribosome . When recombinant S19 proteins were cross-linked intermolecularly by plasma transglutaminase (coagulation factor XIIIa), this homodimer newly exhibited monocyte chemotactic activity . This effect was specific to monocytes . The S19 protein homodimer shared the immunoreactivity with the complement-derived chemotactic factor, component 5a (C5a) . Monocytes pretreated with an anti-C5a receptor antibody or with a synthetic C5a receptor antagonist responded poorly in chemotaxis to this homodimer . These data indicate that the S19 protein homodimer possesses a 3-dimensional structure similar to C5a in terms of the immunologic epitope and the receptor ligand, although homology between their primary structures is only 4% . In contrast, the S19 protein homodimer did not attract polymorphonuclear leukocytes . In addition, the homodimer inhibited a chemotactic response of polymorphonuclear leukocytes to C5a in vitro and in vivo as a receptor antagonist . Furthermore, the S19 protein homodimer competitively inhibited the binding of radiolabeled C5a to polymorphonuclear leukocytes . The S19 protein homodimer with these opposite effects in the leukocyte chemotaxis seems to induce the monocyte/macrophage predominant infiltration in chronic inflammation. Glycoconj J, 1998 Jun, 15(6), 605 - 13 Inhibition of the type 1 fimbriae-mediated adhesion of Escherichia coli to erythrocytes by multiantennary alpha-mannosyl clusters: the effect of multivalency; Lindhorst TK et al.; Alpha-mannosyl glycoclusters and glycodendrimers were tested as multivalent inhibitors of the type 1 (mannose-specific) fimbriae of a recombinant E . coli HB101 strain . Inhibition of haemagglutination of guinea pig erythrocytes was determined on microtiter plates . The effect of multivalency is pronounced for up to three mannosyl residues in the molecule, whereas larger derivatives do not have an appreciable effect on binding to the fimbrial carbohydrate binding domain . The best glycoclusters tested reach the binding potency of the known potent inhibitor pNPMan (3) . The results support the idea of a monovalent recognition site at the adhesive protein FimH, which might best accommodate molecules with the size of a trisaccharide or those which expose up to three alpha-mannosyl residues, such as the glycocluster 8 . The results obtained with the thiourea-bridged alpha-mannosyl clusters, possessing defined sugar valencies, facilitate the development of high affinity inhibitors of the fimbrial lectin on type 1 fimbriae. Biol Pharm Bull, 1998 Dec, 21(12), 1282 - 5 Biochemical characterization of recombinant HIV-1 reverse transcriptase (rRT) as a glycyrrhizin-binding protein and the CK-II-mediated stimulation of rRT activity potently inhibited by glycyrrhetinic acid derivative; Harada S et al.; By means of successive Mono Q and glycyrrhizin (GL)-affinity column chromatography (HPLC), recombinant HIV-1 RT (rRT) was purified to apparent homogeneity from the Superdex 200 pg fraction of the crude protein extract of E . coli BL21 transfected with pET 21a(+)/HIV-1 PR-RT . It was found that (i) rRT functioned as an effective phosphate acceptor for recombinant human casein kinase II (rhCK-II) in vitro; (ii) this phosphorylation was inhibited by anti-HIV-1 substances {a glycyrrhetinic acid derivative (oGA) and quercetin} and a high dose (100 microM) of GL; (iii) RNA-dependent DNA polymerase (RDDP) activity was stimulated about 2.5-fold after full phosphorylation of rRT by rhCK-II; and (iv) oGA as well as NCS-chromophore effectively prevented the CK-II-mediated stimulation of RDDP activity . These results suggest that the anti-HIV-1 effect of oGA may be involved in the selective inhibition of the CK-II-mediated stimulation of HIV-1 RT at the cellular level. Plant J, 1998 Nov, 16(4), 515 - 22 An oligo selection procedure for identification of sequence-specific DNA-binding activities associated with the plant defence response; Wang Z et al.; Sequence-specific DNA-binding (SSDB) factors play central roles in transcription, DNA replication, recombination and repair . This report describes a simple procedure for high-throughput identification of SSDB activities without prior knowledge of their target genes or binding sequences . The procedure starts with a population of completely random oligo(nucleotide) sequences and selects for those oligo molecules that specifically bind to cellular SSDB factors by use of common gel-retardation assays and PCR . Amplification and subsequent cloning of these selected oligo molecules result in the establishment of oligo DNA libraries enriched in DNA molecules containing specific sequences recognized by SSDB factors . These oligo libraries can be rapidly screened to identify a large number of SSDB activities, including those that are differentially regulated by developmental and environmental signals . With identified oligo DNA as probes, the corresponding SSDB factors can be isolated and analysed with respect to their structures, regulation and functions . Using this procedure, we have identified approximately 100 SSDB activities from tobacco leaves, including seven that are differentially regulated during the tobacco mosaic virus-induced hypersensitive response in resistant tobacco plants. Plant J, 1998 Nov, 16(4), 421 - 31 cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana triflora; Fujiwara H et al.; Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less under-stood modification reactions during anthocyanin biosynthesis . Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins . A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) (EC 2.3.1.153) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme . The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52,736 . The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multi-functional acyltransferases (St-Pierre et al . (1998) Plant J . 14, 703-713) . The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT . The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT . Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G . triflora . RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes . Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol. Plant J, 1998 Nov, 16(3), 335 - 43 A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine; Guillen P et al.; Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines . It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity . We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA . This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol . The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii . Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates . However, the highest affinity was observed with 3-substituted benzaldehydes . Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin . This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback. J Biochem (Tokyo), 1999 Jan, 125(1), 186 - 93 An azide-insensitive superoxide dismutase from a hyperthermophilic archaeon, Sulfolobus solfataricus; Yamano S et al.; The superoxide dismutase (SOD) gene of Sulfolobus solfataricus, a hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized . When the protein was expressed in E . coli, it formed a homodimer that contained both Mn and Fe . Metal reconstitution experiments of the SOD with Fe or Mn showed that only the Fe-reconstituted SOD was active . Substitution of Tyr88 to Phe did not affect the metal specificity of the enzyme . The Fe-reconstituted SOD was extremely resistant to thermal denaturation; e.g . 96% of the initial activity was retained after heating at 95 degreesC for 2 h . Fe-reconstituted SOD was not inhibited by azide, but fluoride inhibition was observed . This suggests that some steric hindrance in the substrate funnel of the enzyme prevents the access of N3- but allows O2- and F- access to the active site. J Biochem (Tokyo), 1999 Jan, 125(1), 166 - 72 1,5-Anhydroglucitol promotes glycogenolysis in Escherichia coli; Shiga Y et al.; Glycogen is a storage compound that provides both carbon and energy, but the mechanism for the regulation of its metabolism has not been fully clarified . Recently, we found a new glycogenolytic pathway in rat liver in which glycogen is first metabolized to 1, 5-anhydrofructose (AnFru) and then to 1,5-anhydroglucitol (AnGlc-ol) . Because the amounts of glycogen and AnFru are closely related in various rat organs and the second reaction, AnFru to AnGlc-ol, is strongly inhibited in the presence of glucose, we expected that this pathway might play a regulatory role in glycogen metabolism . Here we evaluate the expected involvement of AnGlc-ol and AnFru in the regulatory mechanism in Escherichia coli C600 . Having established the presence of this new glycogenolytic pathway in E . coli C600, we further show that the conversion of AnFru to AnGlc-ol is activated only after the exhaustion of glucose in the medium, and that as little as 5 microM AnGlc-ol in the medium acutely accelerates the degradation of glycogen by 40% . We consider the role of AnGlc-ol in the mechanism that controls glycogen metabolism in E . coli to be as follows . When glucose is abundant, E . coli accumulate glycogen, a fraction of which is steadily degraded so that the amount of AnFru is about 1/1,000 of glycogen on a weight basis . When glucose is depleted and the demand for glycogen utilization is elevated, AnFru, which has accumulated mostly in the medium, is promptly taken up and converted to AnGlc-ol, which accelerates glycogen degradation . We also suggest the possibility that AnGlc-ol is one of the extracellular signaling molecules for bacteria. J Biochem (Tokyo), 1999 Jan, 125(1), 138 - 42 Characterization of the ubiquinol oxidation sites in cytochromes bo and bd from Escherichia coli using aurachin C analogues; Miyoshi H et al.; Natural aurachin C is the most potent inhibitor of oxidation of ubiquinols by cytochromes bo and bd from Escherichia coli . To probe the structural properties of the substrate oxidation site in the ubiquinol oxidases, we synthesized a systematic set of aurachin C analogues (N-hydroxy-4-quinolone derivatives) and examined how their structure affects their activity towards cytochromes bo and bd, which are structurally unrelated . We found that the presence of the 3-methyl group of the 2-n-decyl and 2-n-undecyl derivatives increased the inhibitory potency towards both enzymes, probably due to a local steric congestion that allows favorable interaction of the alkyl tail with the enzyme . Increase in the chain length of the 3-alkyl tail of the 2-n-undecyl derivatives decreased the inhibitory potency only in cytochrome bo, indicating that the binding site for the alkyl tails of cytochrome bo is smaller than that of cytochrome bd . Based on these findings, we discuss the differences in the molecular mechanism of substrate oxidation by these two terminal ubiquinol oxidases. J Biochem (Tokyo), 1999 Jan, 125(1), 24 - 6 Crystallization and preliminary X-ray diffraction studies of a replication initiator protein (RepE54) of the mini-F plasmid complexed with iteron DNA; Komori H et al.; A replication initiator protein (RepE54) complexed with iteron DNA at its binding site was crystallized by the hanging drop vapor diffusion method . The crystals belong to monoclinic space group C2 with unit cell dimensions of a = 108.4 A, b = 81.9 A, c = 73.9 A, and beta = 121.5 degrees, where one molecule of the protein-DNA complex exists per asymmetric unit . They diffract X-rays up to 2.6 A resolution with synchrotron radiation. J Neurosci, 1999 Jan 15, 19(2), 716 - 25 Lipopolysaccharide injected into the cerebral ventricle evokes fever through induction of cyclooxygenase-2 in brain endothelial cells; Cao C et al.; Activation of the arachidonic acid cascade is an essential step for the development of fever during brain inflammation . We investigated the brain sites where this activation takes place by use of a rat model of brain inflammation . Intracerebroventricular administration of lipopolysaccharide but not of its vehicle evoked fever . The fever was markedly suppressed when the rats had been pretreated with a cyclooxygenase-2-specific inhibitor . In situ hybridization and immunohistochemical studies revealed that cyclooxygenase-2 mRNA and its protein were induced by lipopolysaccharide in blood vessels near the cerebral ventricles and in those in the subarachnoidal space . Double immunohistochemical staining revealed that these cyclooxygenase-2-positive cells were mostly endothelial cells . The time course of fever and that of cyclooxygenase-2 induction in the endothelial cells were in parallel . Cyclooxygenase-2 mRNA in a certain type of telencephalic neurons was also upregulated by the intracerebroventricular administration, but this neuronal response occurred both in vehicle-injected rats and in lipopolysaccharide-injected ones to the same extent . Therefore, the neuronal response was not essential to the development of fever . These results suggest that brain endothelial cells play a crucial role in the development of fever during brain inflammation by activating their arachidonic acid cascade. J Biol Chem, 1999 Jan 15, 274(3), 1828 - 34 Isolation of a chinese hamster ovary (CHO) cDNA encoding phosphatidylglycerophosphate (PGP) synthase, expression of which corrects the mitochondrial abnormalities of a PGP synthase-defective mutant of CHO-K1 cells; Kawasaki K et al.; Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells . In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase . The gene for the isolated CHO cDNA was named PGS1 . Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase . A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y . (1993) J . Biol . Chem . 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities . The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL . In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria . When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth . These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL. J Biol Chem, 1999 Jan 15, 274(3), 1745 - 52 Cross-induction of glc and ace operons of Escherichia coli attributable to pathway intersection . Characterization of the glc promoter; Pellicer MT et al.; The metabolic pathways specified by the glc and ace operons in Escherichia coli yield glyoxylate as a common intermediate, which is acted on by two malate synthase isoenzymes: one encoded by glcB and the other by aceB . Null mutations in either gene exhibit no phenotype, because of cross-induction of the ace operon by glycolate and the glc operon by acetate . In this study, the regulation of the glc operon, comprising the structural genes glcDEFGB, was analyzed at the molecular level . This operon, activated by growth on glycolate, is transcribed as a single message and is under the positive control of GlcC encoded by a divergent gene . Expression of the glc operon is strongly dependent on the integration host factor (IHF) and is repressed by the global respiratory regulator ArcA-P . In vitro gel-shift experiments demonstrated direct binding of the promoter DNA to IHF and ArcA-P . Mutant analysis indicated that cross-induction of the glc operon by acetate is mediated by the GlcC protein that recognizes the compound as an alternative effector . The similar pattern of regulation of the Glc and Ace systems by IHF and ArcA-P ensures their effective cross-induction. J Biol Chem, 1999 Jan 15, 274(3), 1657 - 66 Molecular parameters of type IV alpha-internexin and type IV-type III alpha-internexin-vimentin copolymer intermediate filaments; Steinert PM et al.; During neuronal development, a dynamic replacement mechanism occurs in which the type VI nestin and type III vimentin intermediate filament proteins are replaced by a series of type IV proteins beginning with alpha-internexin . We have explored molecular details of how the type III to type IV replacement process may occur . First, we have demonstrated by cross-linking experiments that bacterially expressed forms of alpha-internexin and vimentin form heterodimer molecules in vitro that assemble into copolymer intermediate filaments . We show using a urea disassembly assay that alpha-internexin molecules are likely to be more stable than those of vimentin . Second, by analyses of the induced cross-links, we have determined the axial lengths of alpha-internexin homodimer and alpha-internexin-vimentin heterodimer molecules and their modes of alignments in filaments . We report that these dimensions are the same as those reported earlier for vimentin homopolymer molecules and, by implication, are also the same for the other neuronal type IV proteins . These data suggest that during neuronal development, alpha-internexin molecules are readily assimilated onto the pre-existing vimentin cytoskeletal intermediate filament network because the axial lengths and axial alignments of their molecules are the same . Furthermore, the dynamic replacement process may be driven by a positive equilibrium due to the increased stability of the alpha-internexin network. J Biol Chem, 1999 Jan 15, 274(3), 1342 - 8 Characterization and binding specificity of the monomeric STAT3-SH2 domain; Haan S et al.; Signal transducers and activators of transcription (STATs) are important mediators of cytokine signal transduction . STAT factors are recruited to phosphotyrosine-containing motifs of activated receptor chains via their SH2 domains . The subsequent tyrosine phosphorylation of the STATs leads to their dissociation from the receptor, dimerization, and translocation to the nucleus . Here we describe the expression, purification, and refolding of the STAT3-SH2 domain . Proper folding of the isolated protein was proven by circular dichroism and fluorescence spectroscopy . The STAT3-SH2 domain undergoes a conformational change upon dimerization . Using an enzyme-linked immunosorbent assay we demonstrate that the monomeric domain binds to specific phosphotyrosine peptides . The specificity of binding to phosphotyrosine peptides was assayed with the tyrosine motif encompassing Tyr705 of STAT3 and with all tyrosine motifs present in the cytoplasmic tail of the signal transducer gp130. J Biol Chem, 1999 Jan 15, 274(3), 1320 - 5 Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase, an enzyme not found in nature; Mouratou B et al.; Tyrosine phenol-lyase (TPL), which catalyzes the beta-elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the alpha-family of vitamin B6-dependent enzymes . To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL . Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs . Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme . The double mutation R100T/V283R of TPL increased the beta-elimination activity toward dicarboxylic amino acids at least 10(4)-fold . Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g . formate in the case of L-aspartate . The activity toward L-aspartate (kcat = 0.21 s-1) was two times higher than that toward L-tyrosine . beta-Elimination and transamination as a minor side reaction (kcat = 0.001 s-1) were the only reactions observed . Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity . Dicarboxylic amino acid beta-lyase is an enzyme not found in nature. J Biol Chem, 1999 Jan 15, 274(3), 1306 - 12 The Escherichia coli MutL protein physically interacts with MutH and stimulates the MutH-associated endonuclease activity; Hall MC et al.; All possible pairwise combinations of UvrD, MutL, MutS, and MutH were tested using the yeast two-hybrid system to identify potential interactions involving mismatch repair proteins . A two-hybrid screen previously identified a physical interaction between MutL and UvrD . Although several other known interactions were not observed, a novel interaction between MutL and MutH was detected . A series of truncations from the NH2 and COOH termini of MutL demonstrated that the COOH-terminal 218 amino acids were sufficient for the two-hybrid interaction with MutH . Removal of a small number of residues from either the NH2 or COOH termini of MutH eliminated the two-hybrid interaction with MutL . Protein affinity chromatography experiments confirmed that MutL, but not MutS, physically associates with MutH . Furthermore, MutL greatly stimulated the d(GATC)-specific endonuclease activity of MutH in the absence of MutS and a mispaired base . Stimulation of the MutH-associated endonuclease activity by MutL was dependent on ATP binding but not ATP hydrolysis . Further stimulation of this reaction by MutS required the presence of a DNA mismatch and a hydrolyzable form of ATP . These results suggest that MutL activates the MutH-associated endonuclease activity through a physical interaction during methyl-directed mismatch repair in Escherichia coli. J Biol Chem, 1999 Jan 15, 274(3), 1248 - 56 Human Rad51 protein can form homologous joints in the absence of net strand exchange; Gupta RC et al.; The eukaryotic homologs of RecA protein are central enzymes of recombination and repair, and notwithstanding a high degree of conservation they differ sufficiently from RecA to offer insights into mechanisms and biological roles . The yield of DNA strand exchange reactions driven by both Escherichia coli RecA protein and its human homolog HsRad51 protein was inversely related to the GC content of oligonucleotide substrates, but at any given GC composition, HsRad51 promoted less exchange than RecA . When 40% of bases were GC pairs, the rate constant for strand exchange by HsRad51 was unmeasurable, whereas the rate constants for homologous pairing were unaltered relative to more AT-rich DNA . The ability of HsRad51 to form joints in the absence of net strand exchange was confirmed by experiments in which heterologous blocks at both ends of linear duplex oligonucleotides produced joints that instantly dissociated upon deproteinization . These findings suggest that HsRad51 acting alone on human DNA in vivo is a pairing protein that cannot form extensive heteroduplex DNA. J Biol Chem, 1999 Jan 15, 274(3), 1203 - 6 The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a nudix hydrolase active on dinucleoside 5'-polyphosphates; Conyers GB et al.; ialA, one of two genes associated with the invasion of human red blood cells by Bartonella bacilliformis, the causative agent of several diseases, has been cloned and expressed in Escherichia coli . The protein, IalA, contains an amino acid array characteristic of a family of enzymes, the Nudix hydrolases, active on a variety of nucleoside diphosphate derivatives . IalA has been purified, identified, and characterized as an enzyme catalyzing the hydrolysis of members of a class of signaling nucleotides, the dinucleoside polyphosphates, with its highest activity on adenosine 5'-tetraphospho-5'-adenosine (Ap4A), but also hydrolyzing Ap5A, Ap6A, Gp4G, and Gp5G . In each case, a pyrophosphate linkage is cleaved yielding a nucleoside triphosphate and the remaining nucleotide moiety. J Biol Chem, 1999 Jan 15, 274(3), 1193 - 5 One protein, two enzymes; Dai Y et al.; Two enzymes, designated, E-2 and E-2', catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway . E-2 and E-2', overproduced in Escherichia coli from the same gene, have the same protein component . E-2 and E-2' are separable on an anion exchange column or a hydrophobic column . Their distinct catalytic and chromatographic properties result from binding different metals . The apo-enzyme, obtained after metal is removed from either enzyme, is catalytically inactive . Addition of Ni2+ or Co2+ to the apo-protein yields E-2 activity . E-2' activity is obtained when Fe2+ is added . Production in intact E . coli of E-2 and E-2' depends on the availability of the corresponding metals . These observations suggest that the metal component dictates reaction specificity. Plant Physiol, 1999 Jan, 119(1), 73 - 80 Overexpression of glutathione synthetase in indian mustard enhances cadmium accumulation and tolerance Liang Zhu Y, Pilon-Smits EA, Jouanin L, Terry N. An important pathway by which plants detoxify heavy metals is through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione . To identify limiting factors for heavy-metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian mustard (Brassica juncea) . The transgenic GS plants accumulated significantly more Cd than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to 3-fold higher . Moreover, the GS plants showed enhanced tolerance to Cd at both the seedling and mature-plant stages . Cd accumulation and tolerance were correlated with the gshII expression level . Cd-treated GS plants had higher concentrations of glutathione, phytochelatin, thiol, S, and Ca than wild-type plants . We conclude that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity. Vet Immunol Immunopathol, 1998 Dec 11, 66(3-4), 367 - 76 Kinetic and immunohistochemical characteristics of mitogen-induced cutaneous hypersensitivity in chickens selected for antibody responsiveness; Parmentier HK et al.; Mitogen-induced cutaneous hypersensitivity was evaluated in chickens selected for high and low antibody responses to SRBC, and in a random bred control line . Wing web swelling responses were found after subcutaneous administration of phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide (LPS), respectively, in all three lines . All mitogens induced significant acute 4 h wing web swelling responses, followed by a significant (classical) late 24 h wing web swelling response . The 4 h responses were significantly lower in the L line, whereas a tendency for lower responses at 24 h in the L line was found as well . Immunohistochemical evaluation of the early and late wing web swelling responses revealed extravascular localisation of leukocytes at 24 h after sensitization with mitogens, which consisted of CD4+ cells, CD8+ cells, TCR-1+ cells, and heterophils, but no B cells, whereas the 4 h swelling response was primarily characterized by oedema . Cutaneous hypersensitivity either initiated by T-cell mitogens as well as B-cell mitogens may depend for an important part on the rapid induction of local homing of lymphocytes towards the sensitizing agent, which may be mediated by an acute local expression of molecules with chemo-attractive capacities . Interpretation of cellular immunity responses in vivo such as delayed-type hypersensitivity should therefore incorporate oedema-initiating characteristics of sensitizing agents . The relationship between the magnitude of cutaneous hypersensitivity to mitogens and selection for antibody responsiveness is discussed. J Gen Virol, 1998 Dec, 79 ( Pt 12), 3155 - 61 Molecular characterization of Fiji disease fijivirus genome segment 9; Soo HM et al.; This is the first report of sequence from Fiji disease fijivirus (FDV), the type member of the genus Fijivirus of the family Reoviridae . FDV genome segment (S9) comprised 1843 nt and contained two non-overlapping ORFs, separated by a 57 nt intergenic region . S9 ORF 1 comprised 1008 nt and encoded a 335-amino-acid polypeptide (predicted molecular mass 38.6 kDa), while ORF 2 comprised 627 nt and encoded a 208-amino-acid polypeptide (predicted molecular mass 23.8 kDa) . The 5' and 3' non-coding regions were 49 and 102 nt, respectively . The S9 terminal sequences were 5' AAGUUUUU------UGUC 3', and located immediately adjacent to these sequences were 12 bp imperfect inverted repeats . The entire S9 ORF 1 and the hydrophilic regions of S9 ORF 2 were each expressed as a fusion protein with the maltose-binding protein in Escherichia coli . Antibodies produced against the ORF 1 fusion protein reacted strongly with a protein of approximately 39 kDa present in both crude extracts of FDV-infected sugarcane and partially purified FDV preparations . In contrast, antibodies raised against the modified ORF 2 fusion protein did not react with any proteins in the same samples . Further, polyclonal antibodies produced against partially purified FDV reacted with the ORF 1, but not the modified ORF 2, fusion protein . These results indicate that FDV S9 ORF 1 encodes a major structural protein, while ORF 2 probably encodes a non-structural protein. J Gen Virol, 1998 Dec, 79 ( Pt 12), 3123 - 7 VPg, coat protein and five non-structural proteins of potato A potyvirus bind RNA in a sequence-unspecific manner; Merits A et al.; The potato A potyvirus (PVA)-encoded proteins P1, HC-Pro, P3, CI, VPg, NIaPro, NIb and coat protein (CP) were expressed as 6 x His-tagged recombinant proteins in Escherichia coli and purified to homogeneity . RNA binding was tested using purified proteins in Northwestern and liquid assays . PVA proteins except P3 bound to positive- and negative-sense transcripts prepared from the nontranslated 5'- and 3'-regions of PVA genomic RNA and to full-length transcripts of PVA RNA . RNA binding by these proteins showed no sequence specificity since they also bound to various non-PVA control RNAs . Binding properties of P1, HC-Pro, CI and NIaPro are consistent with previous studies carried out on a few other potyviruses, but the binding of VPg, NIb and CP to RNA reveal novel interactions between RNA and potyvirus proteins . Furthermore, the RNA-binding properties of all major proteins of a potyvirus have not been reported previously. Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 177 - 87 Cloning and localisation of an avermectin receptor-related subunit from Haemonchus contortus; Delany NS et al.; Ivermectin is believed to exert its anthelminthic effects by binding to glutamate-gated chloride channels (Glu-Cl) and several cDNAs encoding subunits of Glu-Cl have been cloned from Caenorhabditis elegans . We report the cloning of cDNAs encoding a putative Glu-Cl subunit (HG4) from the parasite Haemonchus contortus . The HG4 cDNAs were isolated using RT-PCR and the sequence of the predicted polypeptide has 82% amino-acid identity with the C . elegans Glu-Cl beta subunit . Individual HG4 cDNAs showed up to 4% sequence variation at the nucleotide level, but the vast majority of these polymorphisms were translationally silent . A synthetic peptide corresponding to sequence near the N-terminus of the mature polypeptide was used to raise an antiserum that recognised the N-terminal domain of HG4 expressed in E . coli . Affinity purified antibodies reacted with motor neuron commissures in immuno-localisation studies: these commissures were limited to the anterior portion of the worms, from a region level with the nerve ring to just anterior of the vulva . Some possible nerve cord staining was also observed, but no expression of HG4 on pharyngeal muscle could be detected. Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 97 - 108 The major 36 kDa Neospora caninum tachyzoite surface protein is closely related to the major Toxoplasma gondii surface antigen; Sonda S et al.; The tachyzoites and the tissue cysts containing bradyzoites of Neospora caninum and Toxoplasma gondii, respectively, are difficult to distinguish morphologically . Specific antigens have been identified in T . gondii tachyzoites and bradyzoites, some of which are stage-specifically expressed, and different functions have been attributed to some of them . A tachyzoite stage-specifically expressed surface protein is the major surface antigen 1 (SAG1) which has been shown to be involved in host cell attachment and invasion . Previously we have identified a cell surface-associated glycoprotein (p36) in N . caninum tachyzoites . The full length coding sequence of the cDNA coding for p36 was determined, and analysis of the deduced amino acid sequence demonstrated that p36 is closely related to SAG1 . p36 is encoded by a single copy gene which produces a transcript of 1.4 kb . Immunogold labeling of resin-embedded parasites using polyclonal antibodies affinity-purified on a recombinant p36 fusion protein expressed in Escherichia coli showed that this protein is located exclusively on the tachyzoite cell surface . As SAG1 in T . gondii, p36 is expressed in the tachyzoite stage, but is absent from bradyzoites . p36 is recognized by antibodies present in sera of cows experimentally infected with N . caninum tachyzoites. Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 33 - 44 Molecular cloning and characterization of the genes encoding two isoforms of cysteine synthase in the enteric protozoan parasite Entamoeba histolytica; Nozaki T et al.; The enteric protozoan parasite Entamoeba histolytica was shown to possess cysteine synthase (CS) activity . The cDNA and genomic clones that encode two isoforms of the E . histolytica CS were isolated and characterized from a clonal strain of E . histolytica by genetic complementation of the cysteine-auxotrophic Escherichia coli NK3 with an E . histolytica cDNA library . The two types of the E . histolytica CS genes differed from each other by three nucleotides, two of which resulted in amino acid substitution . Deduced amino acid sequences of the E . histolytica CS, with a calculated molecular mass of 36721 Da and an isoelectric point of 6.39, exhibited 38-48% identity with CS of bacterial and plant origins . The absence of the amino-terminal transit peptide in the deduced protein sequences and the presence of the CS protein mainly in the supernatant fraction of the amoebic lysate after cellular fractionation suggested that the identified E . histolytica CS genes encoded cytosolic isoforms . Substrate specificity of the recombinant E . histolytica CS was similar to that of plant CS . Phylogenetic analysis indicates that the amoebic CS, first described in Protozoa, does not belong to any families of the CS superfamily, and represents a new family. Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 21 - 31 Transient expression of beta-galactosidase in differentiating sporozoites of Eimeria tenella; Kelleher M et al.; A transient transfection system has been developed for a member of the Apicomplexa, Eimeria tenella, using beta-galactosidase (betagal) from Escherichia coli as the reporter enzyme . Successfully expressed constructs contained sequences of the E . tenella microneme gene Etmic-1 fused to the coding region of lacZ . Transfectants expressing betagal were able to invade host cells and proceed through part of the life-cycle, forming schizonts from which merozoites were released . This indicated that transfectants could differentiate at least to first generation schizonts . However, this differentiation was delayed compared with unelectroporated sporozoites by approximately 15 h . Some merozoites arising from transfected sporozoites also expressed betagal . These results are encouraging for the development of a stable transfection system for E . tenella, using betagal as a reporter enzyme. Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 13 - 9 MGBG analogues as potent inhibitors of S-adenosylmethionine decarboxylase of Onchocerca volvulus; Da'dara AA et al.; Polyamines are essential for cell growth and differentiation and therefore, S-adenosylmethionine decarboxylase (SAMDC), a key regulatory enzyme of the polyamine biosynthesis, is considered as a potentially important target for chemotherapy of filarial infections . Recombinant Onchocerca volvulus SAMDC was expressed in Escherichia coli and characterised . The enzyme activity was found to be stimulated 15-fold by addition of 1 mM putrescine . The Km-value for S-adenosylmethionine was determined to be 36 microM . Furthermore, the efficiencies of SAMDC inhibitors were analysed: Berenil inhibits the enzyme activity competitively with a Ki-value of 0.1 microM . MDL 73811 acts as an irreversible inhibitor with a Ki-value of 1.4 microM . Recently synthesised aromatic methylglyoxal bis(guanylhydrazone) analogues demonstrated high efficacy as inhibitors of the SAMDCs . Some of these analogues exhibited Ki-values of 5 and 14 nM for the Onchocerca enzyme, a result which shows an up to 100-fold increase in specificity compared to the value of 0.47 microM for methylglyoxal bis(guanylhydrazone) . These inhibitors might have potential as drug candidates against filarial worms. J Cancer Res Clin Oncol, 1998, 124(12), 683 - 9 Adenovirus-mediated combined suicide gene and interleukin-2 gene therapy for the treatment of established tumor and induction of antitumor immunity; Ju DW et al.; The antitumor effect of the combined transfer of a suicide gene and a cytokine gene was evaluated in the present study . Adenoviruses expressing Escherichia coli cytosine deaminase (AdCD) and adenoviruses expressing murine interleukin-2 (AdIL-2) were utilized for the treatment of established tumors . The mice were inoculated s.c . with FBL-3 erythroleukemia cells and 3 days later received an intratumoral injection of AdCD in the presence or absence of AdIL-2 followed by intraperitoneal 5-fluorocytosine (5-FC) administration . The results demonstrated that tumor-bearing mice treated with AdCD/5-FC in combination with AdIL-2 showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdCD/5-FC, AdIL-2, adenovirus expressing beta-galactosidase/5-FC or phosphate-buffered saline . The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD4+ and CD8+ T cells infiltrating the tumor after combined therapy . The splenic natural killer and cytotoxic T lymphocyte activities increased significantly in the mice after combined therapy with AdCD/5-FC/AdIL-2 . Our results demonstrate that therapy combining a suicide gene and IL-2 gene can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently. Int J Dev Biol, 1998 Nov, 42(8), 1117 - 24 Expression of two even-skipped genes eve1 and evx2 during zebrafish fin morphogenesis and their regulation by retinoic acid; Brulfert A et al.; Growth and patterning during fin regeneration depend, like for fin development, on the integrated expression of homeogenes . In the present work we have studied, by in situ hybridization, the expression and regulation of two vertebrate homologs eve1 and evx2 of the Drosophila pair-rule even-skipped gene family . Upon amputation of pectoral and caudal fins, both genes, expressed transiently in the mesenchyme during early stages of fin development of these fins, are turned on . During the formation of the blastema they are transcribed first in the mesenchyme located underneath the wound epidermis and then, their expression is restricted to the regenerating rays regions . These expression patterns are developmentally regulated since both genes are no longer transcribed when the bony rays are differentiating . Exposure of the regenerates to retinoic acid (RA) modifies the boundaries of eve1 and evx2 expression: the signal is down-regulated in the ray region and up-regulated in the interray region . Moreover, expression is induced in the wound epidermis . These results indicate that eve1 and evx2 products are part of the molecular signals involved in pattern formation of the fin and fin rays in connection with outgrowth . RA might alter growth and morphogenesis of the regenerating fins by a fine regulation of these genes among others. Mol Cell Biochem, 1998 Dec, 189(1-2), 91 - 7 Recombinant bovine spleen myristoyl CoA: protein N-myristoyltransferase; Raju RV et al.; Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function . Recently, we have isolated full length cDNA encoding bovine spleen NMT {27} the full length cDNA was cloned and expressed in E . coli, resulting in the expression of functionally active 50 kDa NMT . Using the combination of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield . The spleen NMT (sNMT) fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE . Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity . The two synthetic peptide substrates based on the N-terminal sequence of pp60src (GSSKSKMR) and cAMP dependent protein kinase (GNAAAKKRR) have different kinetic parameters of Km values of 40 and 200 microM . Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 microM . The E . coli expressed sNMT was homogenous and showed enzyme activity. Strahlenther Onkol, 1998 Dec, 174 Suppl 4, 13 - 6 Relevance of oxygen in radiation oncology . Mechanisms of action, correlation to low hemoglobin levels; Molls M et al.; At the beginning of this century Schwarz (1909) and Holthusen (1921) observed the influence of oxygen on the radiosensitivity of cells . In 1951 Hollaender et al . described that E . coli needed 3-fold higher radiation doses when treated under anoxic conditions compared to normoxic conditions . This led to the evaluation of the oxygen enhancement ratio (OER) for photons (X-rays), neutrons and heavy ions . It was found that the OER for conventional radiation therapy (RT) with photons is much higher (about 3) than the OER for neutron-RT (only 1.5) or heavy ions . According to a hypothesis free radicals which are produced by radiation are fixed in the presence of oxygen . Radicals are interacting with DNA, macromolecules and membranes . The DNA lesion can be followed by cell death . There is some evidence that tumor cells respond to hypoxia with the expression of a variety of genes coding for oxygen regulated proteins such as c-jun, VEGF or p53 . Hypoxia also enhances the genetic instability of tumor cells . Oxygen tensions in malignant tumors can be determined under clinical routine conditions by using a computerized polarographic needle electrode system (Eppendorf, Hamburg, Germany) . Several studies in the past years showed that tumors are in general more hypoxic than the surrounding normal tissue and that a marked variability of intra- as well as intertumoral pO2 values exist (for review see Vaupel and Hockel 1998) . Moreover, it has been shown in different tumor entities that the oxygenation status influences the local control rate and overall survival . Furthermore, the oxygenation status obtained at one site (primary) is significantly related at other sites (lymph node metastases) in patients with squamous cell carcinoma of the head and neck (SCCHN) . In addition, there is a significant correlation between hemoglobin level and tumor oxygenation in patients with SCCHN . There is some evidence that the oxygenation status can be improved by the correction of a low hemoglobin level and consequently, the curative chance might rise. Biochim Biophys Acta, 1998 Dec 22, 1443(3), 275 - 84 Molecular cloning and characterization of a novel protein serine/threonine kinase highly expressed in mouse embryo; Kurioka K et al.; By a PCR-based screen for cDNA clones of protein kinases, we have isolated a cDNA clone encoding a novel protein kinase (referred to as EDPK) of 305 amino acids . EDPK has a catalytic domain of 271 amino acids that contains all conserved subdomains characteristic of the protein kinase family . Only short sequences are present at the N- and C-terminal ends outside the catalytic domain . EDPK expressed in Escherichia coli and in mammalian cells phosphorylated serine and threonine, but not tyrosine, residues in an exogenous substrate . The amino acid sequence similarity between EDPK and known serine/threonine kinases was less than 35% . Thus, the newly isolated protein kinase EDPK is a novel member of the serine/threonine kinase family . Northern blot analysis showed that the EDPK mRNA was highly expressed in various stages of mouse embryo development . The expression of the mRNA was also found in a variety of mouse adult tissues . These results suggest that EDPK plays a crucial role in intracellular signaling not only during mouse development but also in adult tissues. Biochim Biophys Acta, 1999 Jan 5, 1409(3), 154 - 64 The quinohemoprotein alcohol dehydrogenase of Gluconobacter suboxydans has ubiquinol oxidation activity at a site different from the ubiquinone reduction site; Matsushita K et al.; Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone . In addition to the reduction of ubiquinone, ADHs of Gluconobacter suboxydans and Acetobacter aceti were shown to have a novel function in the oxidation of ubiquinol . The oxidation activity of ubiquinol was detected as an ubiquinol:ferricyanide oxidoreductase activity, which can be monitored by selected wavelength pairs at 273 and 298 nm with a dual-wavelength spectrophotometer . The ubiquinol oxidation activity of G . suboxydans ADH was shown to be two times higher in 'inactive ADH', whose ubiquinone reductase activity is 10 times lower, than with normal 'active' ADH . No activity could be detected in the isolated subunit II or subunit I/III complex, but activity was detectable in the reconstituted ADH complex . Inactive and active ADHs exhibited a 2-3-fold difference in their affinity to ubiquinol despite having the same affinity to ubiquinone . Furthermore, the ubiquinol oxidation site in ADH could be distinguished from the ubiquinone reduction site by differences in their sensitivity to ubiquinone-related inhibitors and by their substrate specificity with several ubiquinone analogues . Thus, the results strongly suggest that the reactions occur at different sites . Furthermore, in situ reconstitution experiments showed that ADH is able to accept electrons from ubiquinol present in Escherichia coli membranes, suggesting the ubiquinol oxidation activity of ADH has a physiological function . Thus, ADH of acetic acid bacteria, which has ubiquinone reduction activity, was shown to have a novel ubiquinol oxidation activity, of which the physiological function in the respiratory chain of the organism is also discussed. Biochim Biophys Acta, 1999 Jan 4, 1426(1), 80 - 90 Characterisation of granulocyte stimulation by thionins from European mistletoe and from wheat; Stein GM et al.; Thionins are small basic peptides found in different plant species, which are known to exert cytotoxic properties . In addition, previous data indicated an activation of human granulocytes by thionins from European mistletoe (viscotoxins, VT) . To extend these latter findings, we investigated the influence of VT and from thionins from wheat flour (purothionin) on human granulocytes by flow cytometry and tried to characterise the involved molecular structures and mechanisms . Phagocytosis was determined by incorporation of FITC-labelled Escherichia coli and respiratory burst by oxidation of dihydrorhodamine 123 to rhodamine 123 . VT and purothionin significantly enhanced E . coli-stimulated phagocytosis and respiratory burst at 25 and 250 microgram/ml . Phagocytosis of damaged lymphocytes by granulocytes was detected by electron microscopy in the VT-stimulated (100 microgram/ml) but not in the control cultures . The poly-cationic structure of the intact molecule seems to be crucial, as evidenced by comparison of the burst and phagocytosis-enhancing effects induced by other poly-cationic (protamine sulphate, histone, poly-l-arginine, poly-l-lysine) and poly-anionic (poly-l-glutamic acid) peptides, while pore forming due to amphipathic properties seems to be less important . Ca2+ and Mg2+ could not inhibit VT-enhanced phagocytosis and, thus, could not inhibit binding of VT to granulocytes . In addition, verapamil at low concentrations inhibited VT activity, suggesting the involvement of Ca2+ channels for granulocyte activation by the VT . Similarly, thionins and histones in contrast to protamine sulphate induced cell death of granulocytes at 250 microgram/ml as demonstrated by an enhanced release of reactive oxygen intermediates in unstimulated granulocytes . From these data one may suggest that activity of VT is induced by strong unspecific ionic binding, probably followed by specific receptor binding, and thionins exhibit stimulatory and cytotoxic effects on immune cells, which have to be further characterised. J Struct Biol, 1998 Nov, 123(3), 248 - 59 At sixes and sevens: characterization of the symmetry mismatch of the ClpAP chaperone-assisted protease; Beuron F et al.; ClpAP, a typical energy-dependent protease, consists of a proteolytic component (ClpP) and a chaperone-like ATPase (ClpA) . ClpP is composed of two apposed heptameric rings, whereas in the presence of ATP or ATPgammaS, ClpA is a single hexameric ring . Formation of ClpAP complexes involves a symmetry mismatch as sixfold ClpA stacks axially on one or both faces of sevenfold ClpP . We have analyzed these structures by cryo-electron microscopy . Our three-dimensional reconstruction of ClpA at 29-A resolution shows the monomer to be composed of two domains of similar size that, in the hexamer, form two tiers enclosing a large cavity . Cylindrical reconstruction of ClpAP reveals three compartments: the digestion chamber inside ClpP; a compartment between ClpP and ClpA; and the cavity inside ClpA . They are connected axially via narrow apertures, implying that substrate proteins should be unfolded to allow translocation into the digestion chamber . The cavity inside ClpA is structurally comparable to the "Anfinsen cage" of other chaperones and may play a role in the unfolding of substrates . A geometrical description of the symmetry mismatch was obtained by using our model of ClpA and the crystal structure of ClpP (Wang et al., 1997, Cell 91, 447-456) to identify the particular side views presented by both molecules in individual complexes . The interaction is characterized by a key pair of subunits, one of each protein . A small turn (8.6(o) = 2pi/42; equivalent to a 4-A shift) would transfer the key interaction to another pair of subunits . We propose that nucleotide hydrolysis results in rotation, facilitating the processive digestion of substrate proteins. Biochem Biophys Res Commun, 1998 Dec 18, 253(2), 514 - 8 Expression of the mnb (dyrk) protein in adult and embryonic mouse tissues; Rahmani Z et al.; Mnb is a human homologue of the Drosophila minibrain gene which encodes a serine/threonine protein kinase that is required in distinct neuroblast proliferation centers during postembryonic neurogenesis . The high degree of homology of the human gene to the murine gene (dyrk) allowed us to use a human polyclonal anti-mnb antibody to study the expression pattern of the protein in adult and embryonic mouse tissues . Western blot analysis and immunohistochemical methods were used to define the detailed distribution of mnb in adult brain and 17 days mouse embryos . The results show a high expression in the cerebral cortex, the cerebellum, the hippocampus which is in accordance with previous reports of in situ hybridization studies using mRNA probes but also a very strong expression in the epithelial layers of the skin, the retina, the tongue, the intestine and the kidney which has not been described before . Since epithelial cells are highly mitotic cells and since mnb shares sequence similarities with the cdk kinases involved in the regulation of cell division, this result may indicate a important role of mnb in the cell cycle control . Biochem Biophys Res Commun, 1998 Dec 18, 253(2), 502 - 5 Overexpression of dnaK/dnaJ and groEL confers freeze tolerance to Escherichia coli; Chow KC et al.; Heat shock proteins not only can protect host cells against heat stress, they can also enable freeze tolerance as well . With respect to this unexpected feature, we are able to show that, at least in Escherichia coli, the heat shock proteins DnaK/DnaJ and GroEL play a very significant role . We found that the recovery rate of E . coli cultures that had been stored at -80 degreesC in the absence of any cryoprotectant was related to the abundance of these heat shock proteins accumulated before the freeze treatment . Before freezing, the DnaK in the bacterial cells was induced to accumulate to a level comparable to that produced in response to heat shock . After the freezing treatment, the recovery rate of the induced culture was very similar to that of the heat-shocked culture . Over production of GroEL was also protective but less effective . While freezing inevitably leads to protein denaturation, we propose that advance synthesis of DnaK/DnaJ and GroEL can accordingly prevent irreversible denaturation by chaperoning the unfolded polypeptides during freezing . Biochem Biophys Res Commun, 1998 Dec 18, 253(2), 415 - 22 cDNA of eight nuclear encoded subunits of NADH:ubiquinone oxidoreductase: human complex I cDNA characterization completed; Loeffen JL et al.; NADH:ubiquinone oxidoreductase (complex I) is an extremely complicated multiprotein complex located in the inner mitochondrial membrane . Its main function is the transport of electrons from NADH to ubiquinone, which is accompanied by translocation of protons from the mitochondrial matrix to the intermembrane space . Human complex I appears to consist of 41 subunits of which 34 are encoded by nDNA . Here we report the cDNA sequences of the hitherto uncharacterized 8 nuclear encoded subunits, all located within the hydrophobic protein (HP) fraction of complex I . Now all currently known 41 proteins of human NADH:ubiquinone oxidoreductase have been characterized and reported in literature, which enables more complete mutational analysis studies of isolated complex I-deficient patients . Biochem Biophys Res Commun, 1998 Dec 18, 253(2), 283 - 7 HSP90 has neurite-promoting activity in vitro for telencephalic and spinal neurons of chick embryos; Ishimoto T et al.; We purified a protein from the extract of denervated chick muscle . The protein had neurite-promoting activity in vitro for the chick telencephalic neurons and spinal neurons . Partial amino acid sequencing and immunoblotting revealed that the protein was chick heat shock protein 90 (HSP90) . Commercially available bovine HSP90 and recombinant chick HSP90 expressed in Escherichia coli also showed the same activity . Since HSP10 (GroES) and HSP60 (GroEL) exhibited no activity for neurite outgrowth in the same culture, this activity was specific to HSP90 among the heat shock proteins . J Magn Reson, 1998 Dec, 135(2), 487 - 99 Measurement of cross correlation between dipolar coupling and chemical shift anisotropy in the spin relaxation of 13C, 15N-labeled proteins; Ghose R et al.; We present a simple method for extracting interference effects between chemical shift anisotropy (CSA) and dipolar coupling from spin relaxation measurements in macromolecules, and we apply this method to extracting cross-correlation rates involving interference of amide 15N CSA and 15N-1H dipolar coupling and interference of carbonyl 13C' CSA and 15N-13C' dipolar coupling, in a small protein . A theoretical basis for the interpretation of these rates is presented . While it proves difficult to quantitatively separate the structural and dynamic contributions to these cross-correlation rates in the presence of anisotropic overall tumbling and a nonaxially symmetric chemical shift tensor, some useful qualitative correlations of data with protein structure can be seen when simplifying assumptions are made . J Mol Biol, 1999 Jan 15, 285(2), 675 - 87 Three-dimensional crystal structure of the transcription factor PhoB receiver domain; Sola M et al.; PhoB is the response regulator of the two-component signal transduction system activated under phosphate starvation conditions . This protein is a transcription factor that activates more than 30 genes of the pho regulon and consists of two domains: a DNA binding domain and a dimerization domain, the latter being homologous to the receiver domain described for two-component response regulators . Activation by phosphorylation induces dimerization of the protein and the consequent binding to the DNA direct repeat pho box, where it promotes the binding of RNA polymerase . In the absence of phosphorylation, the activating dimerization process can be mimicked by deletion of the DNA binding domain . The three-dimensional crystal structure of the receiver domain of PhoB from Escherichia coli has been solved by multiple anomalous diffraction using a gold derivative obtained by co-crystallization, and refined using data to 1.9 A resolution . The crystal structure reveals an alpha/beta doubly wound fold, similar to other known receivers, the most conspicuous difference being the displacement of helix alpha4 towards its N terminus . The active site includes the acidic triad Asp53 (the site of phosphorylation), Asp10 and Glu9 . Lys105, from loop beta5alpha5, and Glu88, from helix alpha4, interact with Asp53 via an H-bond and a water bridge, respectively . In the asymmetric unit of the crystal there are two molecules linked by a complementary hydrophobic surface, which involves helix alpha1, loop beta5alpha5 and the N terminus of helix alpha5, and is connected to the active site through the fully conserved residue Lys105 from loop beta5alpha5 . The possibility that this surface is the functional surface used for the activating dimerization is discussed . J Mol Biol, 1999 Jan 15, 285(2), 655 - 73 Crystal structure of dUTPase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex; Dauter Z et al.; The X-ray structures of dUTPase from equine infectious anaemia virus (EIAV) in unliganded and complexed forms have been determined to 1.9 and 2.0 A resolution, respectively . The structures were solved by molecular replacement using Escherichia coli dUTPase as search model . The exploitation of a relatively novel refinement approach for the initial model, combining maximum likelihood refinement with stereochemically unrestrained updating of the model, proved to be of crucial importance and should be of general relevance.EIAV dUTPase is a homotrimer where each subunit folds into a twisted antiparallel beta-barrel with the N and C-terminal portions interacting with adjacent subunits . The C-terminal 14 and 17 amino acid residues are disordered in the crystal structure of the unliganded and complexed enzyme, respectively . Interactions along the 3-fold axis include a water-containing volume (size 207 A3) which has no contact with bulk solvent.It has earlier been shown that a divalent metal ion is essential for catalysis . For the first time, a putative binding site for such a metal ion, in this case Sr2+, is established . The positions of the inhibitor (the non-hydrolysable substrate analogue dUDP) and the metal ion in the complex are consistent with the location of the active centre established for trimeric dUTPase structures, in which subunit interfaces form three surface clefts lined with evolutionary conserved residues . However, a detailed comparison of the active sites of the EIAV and E . coli enzymes reveals some structural differences . The viral enzyme undergoes a small conformational change in the uracil-binding beta-hairpin structure upon dUDP binding not observed in the other known dUTPase structures . J Mol Biol, 1999 Jan 15, 285(2), 567 - 80 Characterization of in vitro and in vivo mutations in non-conserved nucleotides in the ribosomal RNA recognition domain for the ribotoxins ricin and sarcin and the translation elongation factors; Macbeth MR et al.; The sarcin/ricin domain in 23 S/28 S rRNA is crucial for ribosome function, since it constitutes at least part of the binding site for the elongation factors and hence is essential for binding aminoacyl-tRNA and for translocation . The domain is also the site of action of ricin and sarcin and analysis of the effect of mutations in the RNA on recognition by the cytotoxins has helped to define the structure and to understand the function of the region . We have constructed deletions, separately, of pairs of non-conserved, juxtaposed but non-hydrogen-bonded nucleotides that correspond to C4317 and C4331, and to U4316 and C4332, in an oligoribonucleotide that mimics the sarcin/ricin domain in rat 28 S rRNA . The deletions had no effect on the depurination of A4324 by ricin nor on the cleavage of the phosphodiester bond on the 3' side of G4325 by sarcin . However, simultaneous deletion of the four nucleotides decreased cleavage by sarcin but did not affect depurination by ricin . Removal of the non-canonical A4318.A4330 pair abolished recognition by both toxins . Deletion from oligoribonucleotides, that reproduce the sarcin/ricin domain of Escherichia coli 23 S rRNA, of U2653 and C2667 (equivalent to U4316, C4317 and C4331, C4332 in 28 S rRNA), or substitution of guanosine for U2653 (designed to form a Watson-Crick G2653.C2667 pair), reduced cleavage by sarcin whereas depurination by ricin was slightly increased . An increase in the stability of the mutant oligoribonucleotides may be the basis of the impairment in sarcin action . The tm for the wild-type RNA is 60 degreesC; for the double-deletion mutant U2653Delta/C2667Delta it is 65 degreesC; and for the U2653G transversion it is 69 degreesC . Expression of a mutant 23 S rRNA gene lacking U2653 and C2667 is lethal and a U2653G transversion mutation impairs growth . The mutant ribosomes are less active in protein synthesis than the wild-type and ribosomes with the U2653G mutation are resistant to sarcin . The binding of EF-G to oligoribonucleotides with a U2653/C2667 double deletion is reduced and an effect on the affinity of the factor for the sarcin/ricin domain may account in part for the decrease in ribosome efficiency . The results stress the potential importance in rRNA structure and function of non-conserved nucleotides, and suggest that the sarcin/ricin domain in ribosomes requires a region of structural flexibility for optimal efficiency . J Mol Biol, 1999 Jan 15, 285(2), 555 - 66 Orientation of OmpR monomers within an OmpR:DNA complex determined by DNA affinity cleaving; Harrison-McMonagle P et al.; Escherichia coli OmpR is a transcription factor that regulates the differential expression of the porin genes ompF and ompC . Phosphorylated OmpR binds as a dimer to a 20-bp region of DNA consisting of two tandemly arranged 10-bp half-sites . Expression of the ompF gene is achieved by the hierarchical occupation of three adjacent 20-bp binding sites, designated F1, F2, and F3 and a distally located site, F4 . Despite genetic, biochemical, and structural studies, specific details of the interaction between phosphorylated OmpR and the DNA remain unknown . We have linked the DNA cleaving moiety o-phenanthroline-copper to eight different sites within the DNA binding domain of OmpR in order to determine the orientation of the two OmpR monomers in the OmpR:F1 complex . Five of the resulting conjugates exhibited DNA cleaving activity, and four of these yielded patterns that could be used to construct a model of the OmpR:F1 complex . We propose that OmpR binds asymmetrically to the F1 site as a tandemly arranged dimer with each monomer having its recognition helix in the major groove . The N-terminal end of the recognition helix is promoter-proximal and flanked by "wings" W1 and W2 positioned proximally and distally, respectively, to the transcription start site of ompF . We further propose that the C-terminal end of the recognition helix makes the most extensive contacts with DNA and predict bases within the F1 site that are sufficiently close to be contacted by the recognition helix . J Mol Biol, 1999 Jan 15, 285(2), 469 - 83 In vivo and in vitro activities of the Escherichia coli sigma54 transcription activator, PspF, and its DNA-binding mutant, PspFDeltaHTH; Jovanovic G et al.; Transcription of the phage-shock protein (psp) operon in Escherichia coli is driven by a sigma54 promoter, stimulated by integration host factor and dependent on an upstream, cis-acting sequence and an activator protein, PspF . PspF belongs to the enhancer binding protein family but lacks an N-terminal regulatory domain . Purified PspF is not modified and has an ATPase activity that is increased twofold in the presence of DNA carrying the psp cis-acting sequence . Purified mutant His-tagged PspF that lacks the C-terminal DNA-binding motif has a DNA-independent ATPase activity when present at 30-fold the concentration of the wild-type protein . Both proteins oligomerize in solution in an ATP and DNA-independent manner . The wild-type activator protein, but not the DNA-binding mutant, binds specifically to the cis-acting sequence . Analysis of the sequence protected by PspF demonstrates the presence of two upstream binding sites within the sequence, UAS I and UAS II, which together constitute the psp enhancer . Protection at low protein concentrations is more pronounced and more extensive on a supercoiled DNA than on a linear template . Full expression of the psp operon upon hyperosmotic shock depends on wild-type PspF, but only partially requires the presence of the psp enhancer . J Mol Biol, 1999 Jan 8, 285(1), 107 - 13 Nucleotides in 23S rRNA protected by the association of 30S and 50S ribosomal subunits; Merryman C et al.; We have studied the effect of subunit association on the accessibility of nucleotides in 23S and 5S rRNA . Escherichia coli 50S subunits and 70S ribosomes were subjected to a combination of chemical probes and the sites of attack identified by primer extension . Since the ribose groups and all of the bases were probed, the present study provides a comprehensive map of the nucleotides that are likely to be involved in subunit-subunit interactions . Upon subunit association, the bases of 22 nucleotides and the ribose groups of more than 60 nucleotides are protected in 23S rRNA; no changes are seen in 5S rRNA . Interestingly, the bases of nucleotides A1866, A1891 and A1896, and G2505 become more reactive to chemical probes, indicating localized rearrangement of the structure of the 50S subunit upon association with the 30S subunit . Most of the protected nucleotides are located in four stem-loop structures around positions 715, 890, 1700, and 1920 . In free 50S subunits, virtually all of the ribose groups in these four regions are strongly cleaved by hydroxyl radicals, suggesting that these stems protrude from the 50S subunit . When the 30S subunit is bound, most of the ribose groups in the 715, 890, 1700 and 1920 stem-loops are protected, as are many bases in and around the corresponding apical loops . Intriguingly, three of the protected regions of 23S rRNA are known to be linked via tertiary interactions to features of the peptidyl transferase center . Together with the juxtaposition of the subunit-protected regions of 16S rRNA with the small subunit tRNA binding sites, our findings suggest the existence of a communication pathway between the codon-anticodon binding sites of the 30S subunit with the peptidyl transferase center of the 50S subunit via rRNA-rRNA interactions . J Mol Biol, 1998 Dec 18, 284(5), 1717 - 25 Mutations of Gly to Ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione; Lo Bello M et al.; Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding . The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis . Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies . All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the {S}0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant . This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding . Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication . Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results . J Mol Biol, 1998 Dec 18, 284(5), 1529 - 46 The X-ray structure of Escherichia coli enoyl reductase with bound NAD+ at 2.1 A resolution; Baldock C et al.; Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor . The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16 . The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules . There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding . Each monomer is composed of a seven-stranded parallel beta-sheet flanked on each side by three alpha-helices with a further helix lying at the C terminus of the beta-sheet . This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes . Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity . Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate . J Mol Biol, 1998 Dec 18, 284(5), 1379 - 90 The C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase is required for efficient rho-dependent transcription termination; Kainz M et al.; We screened a collection of single alanine residue substitution mutants spanning the entire C-terminal domain of the alpha subunit (alphaCTD) of Escherichia coli RNA polymerase (RNAP) for defects in rho-dependent transcription termination at lambdatR1 in vivo and in vitro, and thereby identified a patch of amino acid residues in the alphaCTD required for efficient rho-dependent termination . NusA addition led to the stimulation of rho-dependent termination under our conditions in vitro . The termination defects of a few mutant RNAPs could be attributed to altered interactions with the NusA protein, but rho-dependent termination by most of the defective RNAPs was still stimulated normally by NusA . The NusA-enhanced transcription pausing behaviors of the mutant RNAPs did not always correlate with their rho-dependent termination phenotypes . We conclude that the alphaCTD is a target for interactions with NusA that influence both termination and pausing, but in addition it participates in rho-dependent transcription termination in a NusA-independent manner . J Mol Biol, 1998 Dec 18, 284(5), 1367 - 78 Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure; Lieberman KR et al.; L15, a 15 kDa protein of the large ribosomal subunit, interacts with over ten other proteins during 50 S assembly in vitro . We have probed the interaction L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting, and have used localized hydroxyl radical probing, generated from Fe(II) tethered to unique sites of L15, to characterize the three-dimensional 23 S rRNA environment of L15 . Footprinting of L15 was done by reconstituting purified, recombinant L15 with core particles derived from Escherichia coli 50 S subunits by treatment with 2 M LiCl . The cores migrate as compact 50 S-like particles in sucrose gradients, contain 23 S and 5 S rRNA, and lack a subset of the 50 S proteins, including L15 . Using both Fe(II).EDTA and dimethyl sulfate, we have identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23 S rRNA . This footprint cannot be detected when L15 is incubated with "naked" 23 S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle.Protein-tethered hydroxyl radical probing was done using mutants of L15 containing single cysteine residues at amino acid positions 68, 71 and 115 . The mutant proteins were derivatized with 1-{p-(bromo-acetamido)benzyl}-EDTA . Fe(II), bound to core particles, and hydroxyl radical cleavage was initiated . Distinct but overlapping sets of cleavages were obtained in the footprinted region of domain II, and in specific regions of domains I, IV and V of 23 S rRNA . These data locate L15 in proximity to several 23 S rRNA elements that are dispersed in the secondary structure, consistent with its central role in the latter stages of 50 S subunit assembly . Furthermore, these results indicate the proximity of these rRNA regions to one another, providing constraints on the tertiary folding of 23 S rRNA . J Mol Biol, 1998 Dec 18, 284(5), 1353 - 65 Identification of a contact site for different transcription activators in region 4 of the Escherichia coli RNA polymerase sigma70 subunit; Lonetto MA et al.; The sigma subunit of RNA polymerase orchestrates basal transcription by first binding to core RNA polymerase and then recognizing promoters . Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli sigma70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC . Homology modeling of region 4 of sigma70 to the closely related NarL or 434 Cro proteins, suggests that the five basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the -35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators . The only substitution that has a significant effect on activator-independent transcription is at R603, indicating that this residue of sigma70 may play a distinct role in transcription initiation . J Mol Biol, 1998 Dec 18, 284(5), 1243 - 6 Functional interaction between tRNA2Gly2 at the ribosomal P-site and RF1 during termination at UAG; Zhang S et al.; The glycine codons GGA or GGG, on the 5' side of stop codons UAG and UGA, are associated with a uniquely low termination efficiency in Escherichia coli, as compared to other codons, including the two glycine codons GGU and GGC . In contrast to the wild-type strain, all four glycine codons have a similar effect on termination at UAG in a strain with a mutant release factor 1 (RF1) . Thus, these two glycine codon pairs, when present at the ribosomal P-site, affect termination efficiency of mutant or wild-type RF1 at UAG differently . If reading of GGA/G by tRNAGly2 is eliminated in the RF1 wild-type strain and replaced by a mutant form of tRNAGly3, termination efficiency is increased to the same level as for GGU/C, normally read by tRNAGly3 . The results suggest an unusual interaction between the P-site tRNAGly2 and wild-type RF1 at the ribosomal A-site that is not present with mutant RF1 . J Surg Res, 1998 Dec, 80(2), 309 - 14 Escherichia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle; Scott WS et al.; The soluble isoform of guanylate cyclase (sGC) is activated by nitric oxide (NO) to form guanosine 3':5'-cyclic monophosphate (cGMP) . Cyclic GMP levels cause smooth muscle relaxation and regulate vascular tone to various vascular beds, including the lung . Under conditions of cytokine excess the inducible synthesis of NO may result in cGMP overproduction, generalized vasodilation, and septic shock . In the pulmonary bed the opposite response may occur, pulmonary hypertension . We hypothesized that sGC activity becomes downregulated in the face of Escherichia coli lipopolysaccharide (LPS) . We tested the effects of LPS on alpha1-subunit sGC mRNA abundance, Western analysis, and enzyme activity in cultured rat pulmonary artery smooth muscle cells . LPS increased extracellular cGMP production by pulmonary artery smooth muscle cells, with increased levels being first detectable at 3-6 h (10 microg/ml LPS) and exceeding 140 pmol/ml by 24 h (P < 0.05) . The response was inhibited by 0.05 mM l-NG-monomethyl-l-arginine (l-NMA) and, in turn, restored by 1 mM l-arginine, indicating a NO synthase-dependent response . Pretreating cells with LPS for >/= 3 h inhibited subsequent cGMP synthesis in response to 10(-4) M SNAP for 60 min . Coincubating cells with 0.05 mM l-NMA also reversed this effect . Soluble GC enzyme activity in cells exposed to basal medium alone measured 0.74 pmol cGMP/ml per minute; activity in cells exposed to 10 microg/ml LPS for 24 h decreased to 0.04 pmol cGMP/ml per minute (P < 0.05) . LPS pretreatment decreased sGC mRNA abundance and protein mass, but did not totally eliminate them . It is concluded that LPS affects cGMP synthesis at the level of enzyme activity, enzyme mass, and mRNA abundance . Over the short term (<24 h) LPS causes the synthesis of large amounts of cGMP . As the duration of exposure progresses (>/=3 h), mechanisms come into play that decrease cGMP production significantly and include decreases in mRNA abundance, enzyme mass, and enzyme activity . J Surg Res, 1998 Dec, 80(2), 259 - 65 Early effect of low-dose endotoxin on rat cecal mucosa ex vivo; Mayer JM et al.; It has been suggested that endotoxin triggers translocation of intestinal bacteria in vivo, either by directly damaging intestinal mucosa or by inducing a systemic inflammatory reaction that leads to mucosal disruption . To address this issue, we examined the immediate effect of extraluminal endotoxin on structure and function of isolated rat cecal mucosa without other inflammatory cells in vitro . The cecal mucosa of 12 male Wistar rats was mounted in modified Ussing chambers filled with Dulbecco's modified Eagle's medium and the ampicillin-resistant Escherichia coli HB101:K12 incubated on the mucosal side . Endotoxin was added to the submucosal side at concentrations of 1 and 10 EU/ml, respectively . Under gassing with carbogene at 37 degreesC, the potential difference across the mucosa was measured continuously . Samples of the mucosal and submucosal solutions were removed at 60, 120, and 180 min and plated out on McConkey ampicillin-agar . After 180 min, the mucosal specimens were retrieved and examined by light and scanning electron microscopy . No significant change in potential difference was observed in control or endotoxin-incubated mucosa within the observation period . Neither light nor scanning electron microscopy showed a significant change in the structure of the epithelium, mucosa, or submucosa . No significant translocation of the E . coli across the mucosa was seen . We concluded that endotoxin alone does not induce immediate structural and functional damage to rat cecal mucosa in vitro . Therefore, it seems unlikely that a short endotoxemia alone directly triggers bacterial translocation by disrupting intestinal mucosa, but rather, entotoxin induces a local and systemic inflammatory reaction that leads to mucosal disruption . Genomics, 1998 Dec 15, 54(3), 443 - 52 Cloning of two new human helicase genes of the RecQ family: biological significance of multiple species in higher eukaryotes; Kitao S et al.; Two new human DNA helicase genes, RecQ4 and RecQ5, that belong to the RecQ helicase family were cloned and characterized . The addition of these genes increases the total to five helicase genes in the human RecQ family, which includes helicases involved in Bloom and Werner syndromes, the genetic diseases manifesting the distinctive but overlapping clinical phenotypes of immunodeficiency, premature aging, and an enhanced risk of cancer . The RecQ4 helicase is as large as the Bloom (BLM) and Werner (WRN) helicases, and its gene expression profile is organ-specific, resembling that of BLM helicase . In contrast, the RecQ5 helicase has a low molecular weight, similar to the human progenitor RecQ1 helicase, and is expressed in all the organs examined . All five human helicase genes are expressed in cultured K562 leukemia and fibroblast cells . Synchronized K562 cell cultures showed that the genes RecQ4 and BLM, and RecQ1 and WRN, seem to be upregulated at the G1/S and G2/M phases, respectively, of the cell cycle . The biological significance of multiple species of human RecQ helicases, which are apparently nonessential for life but may be related to distinct diseases, is discussed in light of the fact that unicellular organisms, like Escherichia coli and yeast, contain only one species of helicase of this particular family . J Infect Dis, 1999 Feb, 179(2), 503 - 7 Shiga toxin induces superoxide production in polymorphonuclear cells with subsequent impairment of phagocytosis and responsiveness to phorbol esters; King AJ et al.; The role of inflammatory cells in the pathogenesis of hemolytic-uremic syndrome induced by Shiga toxin (Stx)-producing Escherichia coli remains unclear . The hypothesis that Stx has direct effects on polymorphonuclear cell (PMN) viability and function was examined by measuring apoptosis, necrosis, phagocytosis, and spontaneous and phorbol myristate acetate (PMA)-stimulated production of reactive oxygen intermediates . PMN from 6 healthy persons were exposed to medium, Stx1 (0.01-100 ng/mL), or heat-inactivated Stx1 or Stx1 B subunit (100 ng/mL) . Stx1 induced a prominent dose-dependent respiratory burst from PMN at doses as low as 0.01 ng/mL; they were less responsive to PMA stimulation and had reduced ability for phagocytosis . This dysfunction was not due to cell death, as the magnitude of apoptosis and necrosis of PMN treated with Stx1 (100 ng/mL) for 20 h was identical to that of medium control . These results suggest that Stx has direct effects on PMN that could contribute to tissue injury early in the disease. J Infect Dis, 1999 Feb, 179(2), 382 - 9 Epidemiology of enterotoxigenic Escherichia coli diarrhea in a pediatric cohort in a periurban area of lower Egypt; Abu-Elyazeed R et al.; Enterotoxigenic Escherichia coli (ETEC) are diverse pathogens that express heat-labile (LT) and/or heat-stable (ST) enterotoxins, yet little is known about whether epidemiologic patterns of pediatric ETEC diarrhea vary by the expressed ETEC toxin phenotype . In total, 242 Egyptian children aged <3 years were prospectively followed in 1993-1995 . ETEC episodes were detected during twice-weekly home visits, and asymptomatic ETEC excretion was identified from monthly cross-sectional surveys . ETEC episodes were 0.6 per child-year . ST-only ETEC was 2.6 times (P<.001) more common in warmer than cooler months, while LT-only ETEC showed no seasonal variation . Ownership of a household sanitary latrine, but not breast-feeding, was associated with a lower risk of both enterotoxin phenotypes . Coexpression of a colonization factor by LT- or ST-only ETEC strengthened the association with diarrhea . These findings indicate that the epidemiologic patterns of LT-only and ST-only ETEC are not identical and that disease interventions should include improved household sanitation. EMBO J, 1999 Jan 4, 18(1), 249 - 57 In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters; Liere K et al.; We report here the in vitro characterization of PrpoB-345, the tobacco rpoB promoter recognized by NEP, the phage-type plastid RNA polymerase . Transcription extracts were prepared from mutant tobacco plants lacking PEP, the Escherichia coli-like plastid-encoded RNA polymerase . Systematic dissection of a approximately 1 kb fragment determined that the rpoB promoter is contained in a 15-nucleotide segment (-14 to +1) upstream of the transcription initiation site (+1) . Point mutations at every nucleotide reduced transcription, except at the -5 position which was neutral . Critical for rpoB promoter function was a CRT-motif (CAT or CGT) at -8 to -6 (transcription <30%), defining it as the promoter core . The core CAT sequence is also present in the maize rpoB promoter, which is faithfully recognized by tobacco extracts . Alignment of NEP promoters identified a CATA or TATA (=YATA) sequence at the rpoB core position, also present in plant mitochondrial promoters . Furthermore, NEP and the phage T7 RNA polymerase exhibit similar sensitivity to inhibitors of transcription . These data indicate that the nuclear RpoZ gene, identified by sequence conservation with mitochondrial RNA polymerases, encodes the NEP catalytic subunit. EMBO J, 1999 Jan 4, 18(1), 65 - 74 The acidic C-terminal domain of protein disulfide isomerase is not critical for the enzyme subunit function or for the chaperone or disulfide isomerase activities of the polypeptide; Koivunen P et al.; Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4-hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding . We report here that deletion of PDI sequences corresponding to the entire C-terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4-hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI . However, partially overlapping critical regions for all these functions were identified at the C-terminal end of the preceding thioredoxin-like domain a' . Point mutations introduced into this region identified several residues as critical for prolyl 4-hydroxylase assembly . Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes . The critical region identified here corresponds to the C-terminal alpha helix of domain a', but this is not the only critical region for any of these functions. J Pharm Pharmacol, 1998 Nov, 50(11), 1307 - 15 The role of the iminium bond in the inhibition of reverse transcriptase by quaternary benzophenanthridines; Kerry MA et al.; The quaternary benzo{c}phenanthridines fagaronine, nitidine and O-methylfagaronine have been reviewed as potential antitumour and antiviral agents . Their mode of action has not been established, but their ability to bind with DNA by intercalation is believed to be involved . Of the three synthetic analogues of O-methylfagaronine which we have synthesized, methoxidine and ethoxidine are active against HIV-1 reverse transcriptase (IC50 values 2.8 microM and 2.4 microM respectively) whereas hydroxidine is inactive . One of the prerequisites for the enzyme inhibitory activity of this class of molecule is the presence of an iminium group--it is well known that a positive charge on a polyaromatic nucleus facilitates intercalative binding with DNA . Through UV spectrophotometric and modelling studies, we have shown that the iminium bond plays a more fundamental role in enzyme inhibition through its susceptibility to nucleophilic attack--the inactive analogue hydroxidine has a non-electrophilic iminium bond . Consequently, we have demonstrated that iminium bond electrophilicity is a parameter which needs to be considered in ternary complex formation with reverse transcriptase. FEBS Lett, 1998 Dec 11, 441(1), 53 - 8 Production and characterization of monoclonal antibodies directed against native epitopes of NhaA, the Na+/H+ antiporter of Escherichia coli; Padan E et al.; Monoclonal antibodies (mAbs) recognizing native epitopes are an important tool for functional and structural studies of proteins, yet they have rarely been used with transport proteins . In an attempt to raise monoclonal antibodies against the NhaA Na+/H+ antiporter of Escherichia coli we encountered difficulties in the screening procedure, which is based on the standard enzyme-linked immunosorbent assay (ELISA) . Here we report a rapid and efficient method of screening for anti-NhaA mAbs which recognize native epitopes of the antiporter . The method is based on the use of His-tagged protein, Ni2+-nitrilotriacetic acid coated plates and non-denaturing conditions in the assay . With this procedure four mAbs were obtained, three of which recognize the NhaA in its native conformation and one preferentially recognizes the denatured form . The latter mAb is Western blot positive, the others are Western blot negative and bind the detergent solubilized NhaA as assayed by gel filtration chromatography . Competition experiments show that the native epitopes are common to both the His-tagged and the wild-type protein . We suggest that in the standard ELISA the NhaA protein is not presented to the antibody in the native conformation whereas the His tag based protocol favors this presentation . Indeed, we could remarkably improve the low reactivity of the standard ELISA by coating the plates with anti-NhaA mAb and use it to present NhaA ('sandwich' ELISA or two antibodies assay) . Remarkably, two of the mAbs (5H4, 2C5) which bind native NhaA inhibit drastically the deltapH driven 22Na uptake mediated by His-tagged NhaA reconstituted in proteoliposomes . Hence, these mAbs afford a new tool to study the structure/function relationship of the antiporter. Int J Clin Pharmacol Ther, 1998 Dec, 36(12), 642 - 51 Autoactivation and activation of the cytochrome P450s; Ekins S et al.; OBJECTIVE: In order to reliably predict in vivo pharmacokinetic parameters from in vitro data, we must thoroughly understand the systems we currently use to determine enzyme kinetic parameters . There have been a number of reports of atypical Michaelis-Menten kinetics for cytochrome- (CYP) P4503A mediated metabolism in vitro but little discussion of its clinical relevance . In this manuscript, we examined the scope of CYP autoactivation and confirmed that CYP1A2 demonstrates atypical Michaelis-Menten kinetics in vitro . MATERIALS: Human liver microsomes, baculovirus-expressed CYP1A2, CYP1A2 in the RECO format, and E . coli expressed CYP1A2 were utilized . METHODS: Enzyme kinetics were performed using the various human CYP1A2 sources and ethoxyresorufin O-deethylation as a prototypical biotransformation . The data were fit to various models of enzyme kinetics . In some cases the data best fit the Hill equation, which was used to empirically model allosteric-type autoactivation kinetics . RESULTS: RECO CYP1A2 and E . coli expressed CYPIA2 both demonstrated autoactivation kinetics for ethoxyresorufin O-deethylation . When the data were fit to the Hill equation, n (the slope factor) was found to be 1.4 and 1.8 for RECO and E . coli expressed CYP1A2, respectively . Human liver microsomal and insect expressed sources of CYP1A2 illustrated classical Michaelis-Menten kinetics for the O-deethylation of ethoxyresorufin . CONCLUSION: Data generated in the current study and previous work suggest many CYPs, not only CYP3A, appear to behave as allosteric enzymes . We would argue that this is not necessarily a classical allosteric mechanism because n is frequently a non-integer . This autoactivation appears to be a function of several factors including substrate physicochemical characteristics, specific interactions of the substrates (activators) with the enzyme active site, and presence of other enzyme modulators . These factors interact to increase the catalytic activity of CYP and thus the complexity of predicting enzyme kinetic parameters or drug interactions. Intensive Care Med, 1998 Nov, 24(11), 1181 - 6 Effect of plasma and LPS on respiratory burst of neutrophils in septic patients; Pascual C et al.; OBJECTIVE: To compare the respiratory burst of neutrophils in sepsis and control patients using lipopolysaccharide (LPS), autologous plasma, and a combination of the two . DESIGN: Prospective, consecutive case study . SETTING: A 16-bed intensive care unit (ICU) in a university teaching hospital . INTERVENTIONS: None . PATIENTS: Plasma was obtained from 23 healthy patients scheduled for minor surgery immediately prior to induction of anesthesia (controls) and from 23 ICU patients within 24 h of diagnosis of sepsis or septic shock . MEASUREMENTS AND MAIN RESULTS: Respiratory burst was determined by lucigenin chemiluminescence expressed as mean +/- SEM of peak values of relative light units per neutrophil . There were no significant differences between neutrophils of septic patients and controls for the stimuli saline, phorbol myristate acetate, formyl-methionyl-leucyl-phenylalanine, and LPS alone . Septic patients showed a lower respiratory burst than controls (p < 0.05) under the following stimuli: plasma alone (5911 +/- 803 vs 15,397 +/- 3038) and LPS and plasma combined (13,857 +/- 1537 vs 23,026 +/- 2640) . However, when stimulated with plasma after priming with LPS, septic patients elicited a higher value than control subjects (11,373 +/- 1758 vs 5987 +/- 1234, p < 0.05) . CONCLUSIONS: (1) Some components of the plasma of septic patients may have a profound effect on neutrophil response; (2) plasma as a respiratory burst stimulus differentiates between sepsis and non-sepsis samples better than other common stimuli; (3) precautions must be taken when using plasma together with LPS because of the different response depending on whether LPS-priming precedes the plasma stimulus or both are introduced simultaneously and whether septic or nonseptic plasma is used. Protein Eng, 1998 Nov, 11(11), 1103 - 9 Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins; Feil IK et al.; Members of the cholera toxin family, including Escherichia coli heat-labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein . The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity . The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft . Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I . Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I . Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT-II . Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site . The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I . Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I . The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I . Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity . The most striking result of this study is that the active site loop of LT-I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'. Protein Eng, 1998 Nov, 11(11), 1083 - 8 Use of fortuitous in vitro mutations in a synthetic Cu-Zn superoxide dismutase gene to illuminate protein structure-function relationships; Kanan JH et al.; A completely synthetic bovine copper-zinc superoxide dismutase gene (Cu-ZnSOD), designed using the most favoured codons for expression in yeast, was constructed . Fortuitous mutations introduced while cloning the synthetic gene permitted the additional construction of four altered-polypeptide products representing two single (Pro121-->Leu and Gly128-->Asp), one double (Pro100-->Leu, Arg113-->Lys) and one triple (Pro100-->Leu, Arg113-->Lys, Pro121-->Leu) mutant . All five versions of the gene were expressed in a SOD-deficient Escherichia coli strain . The 'wild-type' version of the gene and the two single-mutants were expressed to equal extents (approximately 8% of total soluble protein) . However, compared with the 'wild-type' enzyme, one single-mutant (Gly128-->Asp) showed almost twice as much dismutase activity whilst the other (Pro121-->Leu) exhibited only 70% of the 'wild-type' level . In contrast, the double and triple mutants showed diminished expression of the gene (approximately 1 and 3% of total soluble protein, respectively) and almost no detectable SOD activity . Polyclonal antibovine SOD antibody bound all the recombinant proteins, although some of the products showed decreased size and probably altered conformations . The 'wild-type' superoxide dismutase recombinant was correctly dimerized and possessed dismutase activity, as did the Gly128-->Asp mutant despite the change in charge . Mutations in the other three versions affected enzyme folding and activity . The effect of the different mutations appeared to be additive, with the Pro121-->Leu substitution leading to the apparent proteolytic degradation of the enzyme in vivo. Protein Eng, 1998 Nov, 11(11), 1057 - 64 Effect of replacing a conserved proline residue on the function and stability of bovine adrenodoxin; Grinberg AV et al.; A proline residue in the C-terminal part of the polypeptide chain is highly conserved among many {2Fe-2S} ferredoxins . To investigate the requirement for proline at this position, we constructed steric (4-108W), charged (4-108K), polar (4-108S) and non-polar (4-108A) truncated mutants of adrenodoxin and studied them for biological function and stability . Although the variants were expressed in Escherichia coli with a significantly lower yield compared with wild-type adrenodoxin, successful incorporation of the iron-sulfur cluster suggested their proper folding . Similar absorption, CD and EPR spectra indicated that the cluster environment was not affected by the mutations . No evidence for an essential role of Pro108 in determining the redox potential of adrenodoxin or its interactions with the redox partners was found . However, replacement of this residue results in a dramatic decrease in the overall protein stability . The differences in the Gibbs energy of unfolding at 37 degrees C, delta{delta(d)G(37 degrees C)}, are -5.0, -7.8, -10.1 and -10.7 kJ/mol for 4-108A, 4-108S, 4-108W and 4-108K mutants, respectively, compared with 4-108P as a control . We conclude that the principle function of Pro108 is to stabilize adrenodoxin threefold: (i) through limitation of the conformation of the polypeptide chain in this region, (ii) through a hydrogen bond to Arg14 and (iii) favorable hydrophobic contacts. Avian Dis, 1998 Oct-Dec, 42(4), 711 - 20 Alterations in the lymphocytic and mononuclear phagocytic systems of turkey poults associated with exposure to poult enteritis and mortality syndrome; Heggen CL et al.; In vivo and in vitro mononuclear phagocytic system functions, expression of lymphocyte subset cell surface markers in the thymus and bursa of Fabricius, and lymphocyte subset dynamics during the course of poult enteritis and mortality syndrome (PEMS) were examined . PEMS is an acute, transmissible, infectious intestinal disease accompanied by high mortality and morbidity . The etiology of this multifactorial disease remains to be elucidated; however, turkey coronavirus was initially assumed to be one of the primary agents involved . Further investigation demonstrated that turkey coronavirus was not always detectable in poults exhibiting PEMS symptoms, and, thus, PEMS poults began to be identified as positive or negative for turkey coronavirus . In each trial, uninfected hatchmate controls were compared with turkey poults that were contact exposed to PEMS poults at 7 days of age . Following intravenous inoculation, control poults cleared Escherichia coli from their circulation by 60 min, whereas viable E . coli were still present in the circulation of PEMS poults at 60 min postinoculation . Inflammatory response measured by Sephadex-elicited abdominal exudate cell recruitment and the adherence potential of abdominal exudate cells was not significantly different between uninfected and PEMS poults . The percentage of glass-adherent abdominal exudate macrophages was higher in PEMS poults . However, the ability of these macrophages to phagocytize sheep red blood cells and the average number of sheep red blood cells per phagocytic macrophage were both lower compared with uninfected controls . CD4+ expression in thymic tissue of PEMS poults at 9 days postinfection was significantly lower . The CD4+:CD8+ lymphocyte ratio in peripheral blood leukocytes from coronavirus-negative PEMS poults was lower than that from both uninfected and coronavirus-positive PEMS poults at 14 days postinfection . In the spleen, the CD4+:CD8+ lymphocyte ratio was higher in coronavirus-positive PEMS poults as compared with the other treatments . In conclusion, immune system dysfunction in PEMS is associated with impaired mononuclear phagocytic system function and alterations in lymphocyte populations. JSLS, 1998 Jul-Sep, 2(3), 285 - 8 Tandem subdiaphragmatic and pleural sequelae due to lost gallstones following cholecystectomy; Paramesh A et al.; We report two similar thoracoabdominal complications we encountered due to retained gallstones after cholecystectomy . These patients had had an open cholecystectomy after a failed laparoscopic attempt, with spillage of gallbladder debris intraoperatively . They were admitted more than 12 months later with subdiaphragmatic abscesses . Attempted computerized axial tomography (CT) guided drainage of these abscesses resulted in these patients developing pleural fluid collections, which required surgical drainage . The patients underwent exploratory laparotomies, and drainage of the subdiaphragmatic abscesses had revealed gallstones within the abscess cavity . A detailed presentation of these cases, with review of current literature and clinicopathologic issues for discussion are described. JSLS, 1998 Jul-Sep, 2(3), 263 - 8 The fate of retained gallstones following laparoscopic cholecystectomy in a prairie dog model; Bonar JP et al.; BACKGROUND AND OBJECTIVES: Reported complications of retained gallstones following laparoscopic cholecystectomy (LC) are increasing . This study was undertaken to evaluate the effects of retained gallstones following LC in a prairie dog model . METHODS: Twenty-seven prairie dogs with diet-induced gallstones were divided into three groups of nine . Group I (control) had LC with removal of stones . Group II had LC followed by return of native stones intra-abdominally . Group III had LC followed by return of infected stones (stones dipped in Escherichia coli) intra-abdominally . Animals were euthanized at two months and the character and extent of intra-abdominal adhesions were scored . RESULTS: Adhesions were present in 56% of animals in Group I, 89% in Group II, and 100% in Group III . The character and extent of adhesions in groups II & III were significantly greater than the control group (p < 0.03) . Group III exhibited the highest degree of adhesions when compared to control (p < 0.007) . Histopathology revealed evidence of micro-abscess formation, foreign body giant cell reaction, and fat necrosis adjacent to retained stones . CONCLUSION: Retained intra-abdominal gallstones, especially if infected, are associated with increased adhesions and inflammatory response in this LC model . Further investigation into the long-term consequences of this entity is warranted. JSLS, 1998 Apr-Jun, 2(2), 189 - 90 Laparoscopically assisted surgery for colonic perforation with peritonitis--a case report; Fine A; Elective laparoscopic colonic surgery is increasingly recognized as feasible and perhaps preferential . A case of laparoscopically assisted surgery for trauma to the rectum with bacterial peritonitis is presented . It presents an example of the application of this modality to the treatment of iatrogenic colon perforations and perhaps selected diverticulitis. Braz J Med Biol Res, 1998 Oct, 31(10), 1319 - 27 Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin; Brito GA et al.; In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration . The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 +/- 0.19 and Dex = 0.35 +/- 0.13 vs saline (S) = 2.85 +/- 0.59; fMLP: Ptx = 0.43 +/- 0.09 and Dex 0.01 +/- 0.01 vs S = 1.08 +/- 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 +/- 1.4 and Dex = 3.06 +/- 0.76 vs S = 15.94 +/- 3.97; fMLP: Ptx = 3.85 +/- 0.56 and Dex = 0.40 +/- 0.16 vs S = 7.15 +/- 1.17 neutrophils/field) induced by the two agonists in the rat mesentery . The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 microns), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient . The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 +/- 0.38 vs S = 4.20 +/- 1.01; fMLP: Dex = 0.25 +/- 0.11 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue . In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 +/- 2.25 vs S = 4.20 +/- 1.01; fMLP: Ptx = 4.66 +/- 1.24 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field) . This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space . In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances . The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration. Crit Care Med, 1998 Dec, 26(12), 2021 - 8 Treatment of septic shock in rats with nitric oxide synthase inhibitors and inhaled nitric oxide; Pedoto A et al.; OBJECTIVE: To evaluate the effect of treatment with a combination of nitric oxide synthase inhibitors and inhaled nitric oxide on systemic hypotension during sepsis . DESIGN: Prospective, randomized, controlled study on anesthetized animals . SETTING: A cardiopulmonary research laboratory . SUBJECTS: Forty-seven male adult Sprague-Dawley rats . INTERVENTIONS: Animals were anesthetized, mechanically ventilated with room air, and randomized into six groups: a) the control group (C, n=6) received normal saline infusion; b) the endotoxin-treated group received 100 mg/kg i.v . of Escherichia coli lipopolysaccharide (LPS, n=9); c) the third group received LPS, and 1 hr later the animals were treated with 100 mg/kg i.v . Nw-nitro-L-arginine (LNA, n=9); d) the fourth group received LPS, and after 1 hr, the animals were treated with 100 mg/kg i.v . aminoguanidine (AG, n=9); e) the fifth group received LPS and 1 hr later was treated with LNA plus 1 ppm inhaled nitric oxide (LNA+NO, n=7); f) the sixth group received LPS and 1 hr later was treated with aminoguanidine plus inhaled NO (AG+NO, n=7) . Inhaled NO was administered continuously until the end of the experiment . MEASUREMENTS AND MAIN RESULTS: Systemic mean blood pressure (MAP) was monitored through a catheter in the carotid artery . Mean exhaled NO (ENO) was measured before LPS (T0) and every 30 mins thereafter for 5 hrs . Arterial blood gases and pH were measured every 30 mins for the first 2 hrs and then every hour . No attempt was made to regulate the animal body temperature . All the rats became equally hypothermic (28.9+/-1.2 degrees C {SEM}) at the end of the experiment . In the control group, blood pressure and pH remained stable for the duration of the experiment, however, ENO increased gradually from 1.3+/-0.7 to 17.6+/-3.1 ppb after 5 hrs (p< .05) . In the LPS treated rats, MAP decreased in the first 30 mins and then remained stable for 5 hrs . The decrease in MAP was associated with a gradual increase in ENO, which was significant after 180 mins (58.9+/-16.6 ppb) and reached 95.3+/-27.5 ppb after 5 hrs (p< .05) . LNA and AG prevented the increase in ENO after LPS to the level in the control group . AG caused a partial reversal of systemic hypotension, which lasted for the duration of the experiment . LNA reversed systemic hypotension almost completely but only transiently for 1 hr, and caused severe metabolic acidosis in all animals . The co-administration of NO with AG had no added benefits on MAP and pH . In contrast, NO inhalation increased the duration of the reversal in MAP after LNA, alleviated the degree of acidosis, and decreased the mortality rate (from 55% to 29%) . CONCLUSIONS: In this animal model, LPS-induced hypotension was alleviated slightly and durably after AG, but only transiently after LNA . Furthermore, co-administration of NO with AG had no added benefits but alleviated the severity of metabolic acidosis and mortality after LNA . We conclude that nitric oxide synthase (NOS) inhibitors, given as a single large bolus in the early phase of sepsis, can exhibit some beneficial effects . Administration of inhaled NO with NOS inhibitors provided more benefits in some conditions and therefore may be a useful therapeutic combination in sepsis . NO production in sepsis does not seem to be a primary cause of systemic hypotension . Other factors are likely to have a major role. Cell, 1998 Dec 23, 95(7), 1027 - 36 frizzled and frizzled 2 play a partially redundant role in wingless signaling and have similar requirements to wingless in neurogenesis; Bhat KM; The Drosophila Frizzled (Fz) and Frizzled2 (DFz2) proteins function as receptors for Wingless (Wg) in tissue culture cells . While previous results indicate that loss of function for fz has tissue polarity defects, loss-of-function effects of Dfz2 are not known . Here, we have examined the requirements of fz and Dfz2 during neurogenesis . Our results indicate that both Fz and DFz2 function in Wg signaling, and loss of either of the two affects the same subset of neuroblasts as those affected by loss of wg . While these defects are partially penetrant in embryos lacking either fz or Dfz2, the penetrance is significantly enhanced in embryos lacking both . Since the penetrance of the CNS phenotypes is not complete in double mutants, additional components that allow some degree of Wg signaling must exist in vivo. Cell, 1998 Dec 23, 95(7), 1017 - 26 Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway; Kennerdell JR et al.; We investigated the potential of double-stranded RNA to interfere with the function of genes in Drosophila . Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested . In contrast, single-stranded RNA weakly interfered with gene activity . The method was used to determine the reception mechanism of the morphogen Wingless . Interference of the frizzled and Drosophila frizzled 2 genes together produced defects in embryonic patterning that mimic loss of wingless function . Interference of either gene alone had no effect on patterning . Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway . Our results demonstrate that dsRNA interference can be used to analyze many aspects of gene function. Cell, 1998 Dec 23, 95(7), 975 - 9 Reconstitution of an SOS response pathway: derepression of transcription in response to DNA breaks; Anderson DG et al.; E . coli responds to DNA damage by derepressing the transcription of about 20 genes that make up the SOS pathway . Genetic analyses have shown that SOS induction in response to double-stranded DNA (dsDNA) breaks requires LexA repressor, and the RecA and RecBCD enzymes--proteins best known for their role as initiators of dsDNA break repair and homologous recombination . Here we demonstrate that purified RecA protein, RecBCD enzyme, single-stranded DNA-binding (SSB) protein, and LexA repressor respond to dsDNA breaks in vitro by derepressing transcription from an SOS promoter . Interestingly, derepression is more rapid if the DNA containing the dsDNA break has a chi recombination hot spot (5'-GCTGGTGG-3'), suggesting a novel regulatory role for one of the most overrepresented octamers in the E . coli genome. J Hepatol, 1998 Dec, 29(6), 910 - 4 HVJ-liposome mediated gene transfer into hepatocytes in vivo; Hirano T et al.; BACKGROUND/AIMS: The efficient transduction of appropriate target cells will be critical for gene therapy . We evaluated the suitability of hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer for gene therapy of liver diseases . METHODS: The Escherichia coli beta-galactosidase (beta-gal) gene was introduced into rat liver by HVJ-liposome to examine gene transfer efficacy and persistence of expression with or without partial hepatectomy prior to transfection . RESULTS: About 30% of hepatocytes were transduced after portal vein injection . Gene expression was transient, with only 2% of hepatocytes expressing beta-gal after 4 weeks . However, partial hepatectomy performed 24 h prior to injection resulted in persistently high levels of beta-gal for 4 weeks after injection . A 247-bp beta-gal polymerase chain reaction fragment transcript was detected in livers of transfected rats, but not in livers of control rats . The rat livers following gene transfer were histologically normal, and serum glutamic-pyruvic transaminase was not found to be elevated in rats . CONCLUSIONS: Our results demonstrate that HVJ-liposome-mediated gene transfer produced high gene transduction and persistent gene expression in the liver. Arch Pharm Res, 1998 Jun, 21(3), 291 - 7 Purification and characterization of Cop, a protein involved in the copy number control of plasmid pE194; Kwak JH et al.; Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system . Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system . The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl . Cop protein was calculated to contain 39.1% alpha-helix, 16.8% beta-sheet, 17.4% turn, and 26.8% random structure . The DNA binding property of Cop protein expressed in E . coli was preserved during the expression and purification process . The isoelectric point of Cop was determined to be 9.0 . The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E . coli. Mutat Res, 1998 Dec 14, 409(3), 107 - 21 Eukaryotic mismatch repair: an update; Jiricny J; The discovery that mutations in mismatch repair genes segregate with hereditary nonpolyposis colon cancer has awakened a great deal of interest in the study of the process of postreplicative mismatch repair . The characterisation of the principal players involved in this important metabolic pathway has been greatly facilitated by the amino acid sequence conservation among functional homologues of bacteria, yeast and mammals . The phenotypes of mismatch repair deficient mutants are also similar in many ways . In humans, mismatch repair malfunction demonstrates itself in the form of a mutator phenotype of the affected cells, an instability of microsatellite sequences and increased levels of somatic recombination . Moreover, mismatch repair deficient cells display also varying levels of tolerance to DNA damaging agents and are thought to be involved in the cell killing mediated by these agents . This article discusses some recent developments in this fast-moving field. Biochem Biophys Res Commun, 1998 Dec 9, 253(1), 53 - 8 Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi; Kaplan D et al.; Cruzipain, the major proteinase of Trypanosoma cruzi, plays an important role in the biology of this parasite . This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag163B6 hybridoma against cruzipain . This procedure involves the use of cDNAs obtained with the aid of a specific set of primers complementary to the complete light kappa chain (L kappa) and the first two domains of the IgG1 heavy chain (VH/CH1) . These products were subsequently cloned in the pComb3 system, from which the gIII gene had been removed, and expressed in Escherichia coli cells . The recombinant Fab molecule recognized cruzipain by ELISA, in a fashion similar to the original mAb anti-Ag163B6 . Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicated the immunoglobulin nature of the recombinant product . Moreover, both the mAb anti-Ag163B6 and the soluble Fab fragment described here react similarly with the intact parasite surface, as observed in an indirect immunofluorescence assay . In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals. Acta Anaesthesiol Sin, 1998 Sep, 36(3), 113 - 26 Effects of dobutamine, norepinephrine and epinephrine on intramucosal pH and hemodynamics of dogs during endotoxic shock; Hayes JK et al.; This study assessed the effects of dobutamine (DOB), epinephrine (EPI) and norepinephrine (NE) on gastric tissue oxygenation indicated by gastric intramucosal pH (pHi) and hemodynamics in dogs subjected to endotoxic shock . Twenty-four dogs were assigned to four groups of 6 dogs each: endotoxin without catecholamine and endotoxin with DOB, or EPI or NE . Endotoxic shock was induced by intravenous injection of 3 mg/kg of E . coli over 1 min, with an additional 3 mg/kg over the next 2 hrs . Dogs were resuscitated with normal saline to maintain pulmonary capillary wedge pressure (PCWP) near baseline levels . Catecholamines were infused at 0.1, 0.4 and 1.6 micrograms/kg/min (EPI and NE) and 2.5, 5.0 and 10.0 micrograms/kg/min (DOB) for 30 min at each rate . After 2 hrs of endotoxemia, mean arterial pressure (MAP) and cardiac index (CI) and oxygenation delivery index (DO2I) for all dogs decreased by 46.5%, 43.9% and 15.1% respectively, while pHi decreased from 7.47 to 7.10 . Endotoxemia increased blood lactate by 142% . Following fluid resuscitation, EPI (1.6 micrograms/kg/min) further increased lactate by 178% (1.22 to 3.4 mmol/L) . No correlation was found between tonometry pHi and lactate (R2 = 0.003), pHi and pHa (R2 = 0.231), pHi and DO2I (R2 = 0.056) nor between intramucosal PCO2 and PaCO2 (R2 = 0.005) . pHi did not reflect the improvements in cardiovascular hemodynamics observed following administration of catecholamines . NE improved MAP, CI and DO2I whereas DOB produced similar effects as NE but further reduced SVR . EPI produced similar effects as NE . DOB, NE and EPI further decreased pHi . EPI significantly (P < 0.05) increased blood lactate levels more than DOB and NE. Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 151 - 6 A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins; Xu Y et al.; We describe a method for assaying protein interactions that offers some attractive advantages over previous assays . This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest . Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission . By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers . BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles. Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 35 - 7 Proton exit from the heme-copper oxidase of Escherichia coli; Puustinen A et al.; Pathways of proton entry have been identified in the proton-translocating heme-copper oxidases, but the proton exit pathway is unknown . Here we report experiments with cytochrome bo3 in Escherichia coli cells that may identify the beginning of the exit pathway . Systematic mutations of arginines 438 and 439 (R481 and R482 in the E . coli enzyme), numbering as in cytochrome aa3 from bovine heart mitochondria, which interact with the ring D propionates of the two heme groups, reveal that the D propionate of the oxygen-binding heme is involved in proton pumping; its anionic form must be stabilized in order for proton translocation to occur . This may locate the beginning of the pathway by which pumped protons exit from the enzyme structure. J Gen Physiol, 1999 Jan, 113(1), 81 - 96 Antisense knock out of the inositol 1,3,4,5-tetrakisphosphate receptor GAP1(IP4BP) in the human erythroleukemia cell line leads to the appearance of intermediate conductance K(Ca) channels that hyperpolarize the membrane and enhance calcium influx; Lu X et al.; To study the role of the inositol 1,3,4,5-trisphosphate-binding protein GAP1(IP4BP) in store-operated Ca2+ entry, we established a human erythroleukemia (HEL) cell line in which the expression of GAP1(IP4BP) was substantially reduced by transfection with a vector containing antisense DNA under control of a Rous Sarcoma virus promoter and the Escherichia coli LacI repressor (AS-HEL cells) . Control cells were transfected with vector lacking antisense DNA (V-HEL cells) . GAP1(IP4BP) protein, which is a member of the GTPase-activating protein (GAP1) family, was reduced by 85% in AS-HEL cells and was further reduced by 96% by treatment with isopropylthio-beta-D- galactoside to relieve LacI repression . The loss of GAP1(IP4BP) was associated with both a membrane hyperpolarization and a substantially increased Ca2+ entry induced by thrombin or thapsigargin . The activation of intermediate conductance Ca2+-activated K+ channels in AS-HEL cells (not seen in V-HEL cells) was responsible for the membrane hyperpolarization and the enhanced Ca2+ entry, and both were blocked by charybdotoxin . Stimulated V-HEL cells did not hyperpolarize and basal Ca2+ influx was unaffected by charybdotoxin . In V-HEL cells hyperpolarized by removal of extracellular K+, the thapsigargin-stimulated Ca2+ influx was increased . Expression of mRNA for the human Ca2+-activated intermediate conductance channel KCa4 was equivalent in both AS-HEL and V-HEL cells, suggesting that the specific appearance of calcium-activated potassium current (IK(Ca)) in AS-HEL cells was possibly due to modulation of preexisting channels . Our results demonstrate that GAP1(IP4BP), likely working through a signaling pathway dependent on a small GTP-binding protein, can regulate the function of K(Ca) channels that produce a hyperpolarizing current that substantially enhances the magnitude and time course of Ca2+ entry subsequent to the release of internal Ca2+ stores. Clin Diagn Lab Immunol, 1999 Jan, 6(1), 30 - 40 Polymeric display of immunogenic epitopes from herpes simplex virus and transmissible gastroenteritis virus surface proteins on an enteroadherent fimbria; Rani DB et al.; The strong immunogenicity of bacterial fimbriae results from their polymeric and proteinaceous nature, and the protective role of these immunogens in experimental or commercial vaccines is associated with their capacity to induce antiadhesive antibodies . Fimbria-mediated intestinal colonization by enteropathogens typically leads to similar antibody responses . The possibility of taking advantage of these properties was investigated by determining whether enteroadhesive fimbriae, like the 987P fimbriae of enterotoxigenic Escherichia coli, can serve as carriers for foreign antigens without losing their adhesive characteristics . Random linker insertion mutagenesis of the fasA gene encoding the major 987P subunit identified five different mutants expressing wild-type levels of fimbriation . The linker insertion sites of these mutants were used to introduce three continuous segments of viral surface glycoproteins known to be accessible to antibodies . These segments encode residues 11 to 19 or 272 to 279 of herpes simplex virus type 1 (HSV-1) glycoprotein D {gD(11-19) and gD(272-279), respectively} or residues 379 to 388 of the transmissible gastroenteritis virus (TGEV) spike protein {S(379-388)} . Studies of bacteria expressing fimbriae incorporating mutated FasA subunits alone or together with wild-type FasA subunits (hybrid fimbriae) indicated that foreign epitopes were best exported and displayed on assembled fimbriae when they were inserted near the amino terminus of FasA . Fimbriated bacteria expressing FasA subunits carrying the HSV gD(11-19) or the TGEV S(379-388) epitope inserted between the second and third residues of mature FasA elicited high levels of foreign epitope antibodies in all rabbits immunized parenterally . Antibodies against the HSV epitope were also shown to recognize the epitope in the context of the whole gD protein . Because the 987P adhesive subunit FasG was shown to be present on mutated fimbriae and to mediate bacterial attachment to porcine intestinal receptors, polymeric display of foreign epitopes on 987P offers new opportunities to test the potential beneficial effect of enteroadhesion for mucosal immunization and protection against various enteric pathogens. Clin Diagn Lab Immunol, 1999 Jan, 6(1), 24 - 9 Evaluation of recombinant dense granule antigen 7 (GRA7) of Toxoplasma gondii for detection of immunoglobulin G antibodies and analysis of a major antigenic domain; Jacobs D et al.; Dense granule protein 7 (GRA7) of Toxoplasma gondii was expressed in Escherichia coli as a fusion protein . The leader peptide contained a 25-amino-acid mouse tumor necrosis factor fragment and six histidyl residues . After purification by metal chelate affinity chromatography, the antigen was evaluated in an enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG) . For two sets of IgG-positive human serum samples, obtained from routine screening, an overall sensitivity of 81% was obtained . For chronic-phase sera, the sensitivity of detection was 79%, but chronic-phase sera with low titers were more difficult to detect (65% sensitivity for sera with immunofluorescence titer of 1/64) . When GRA7 was combined with Tg34AR (rhoptry protein 2 C-terminal fragment), the sensitivity rose to 96% . For a set of acute-phase serum samples tested on GRA7, the sensitivity of detection was 94%, and high-titer IgM-positive sera were detected at an especially high rate . In contrast, when Tg34AR was used, the sensitivity was only 85% for this latter set of serum samples . Three truncated GRA7 fragments containing the same leader peptide as that of recombinant GRA7 were produced . The shortest fragment (97 N-terminal amino acids) was not reactive with human sera or with a specific anti-GRA7 monoclonal antibody, while the two larger fragments were reactive . The most important antigenic domain of GRA7 for human sera was localized between residues 97 and 146 . The epitope for the specific monoclonal antibody could be further narrowed down by the use of synthetic peptides, but this epitope is not recognized by sera from T . gondii-infected humans . These results indicate that GRA7 may be considered as an additional tool for studying the immune response to T . gondii. J Food Prot, 1998 Dec, 61(12), 1674 - 80 Distribution of Norwalk virus within shellfish following bioaccumulation and subsequent depuration by detection using RT-PCR; Schwab KJ et al.; Consumption of raw bivalve mollusks contaminated with pathogens from human feces continues to present a human health risk . The purpose of this study was to monitor the uptake, localization, and removal of Norwalk virus (NV) in shellfish (oyster and clam) tissues by analyzing virus distribution in selected dissected tissues . Live shellfish were allowed to bioaccumulate different input titers of NV for time periods from 4 to 24 h . In some experiments, depuration by shellfish that bioaccumulated NV and Escherichia coli bacteria was allowed to proceed for 24 or 48 hours . Dissected stomach (St), digestive diverticula (DD), adductor muscle (AM), and hemolymph cells (HC) tissues were assayed for NV by the reverse transcription polymerase chain reaction (RT-PCR) method . An internal RNA standard control was added to the RT-PCR to identify the presence of inhibitors to RT-PCR . NV titers in DD tissues before and after depuration were estimated using quantitative RT-PCR end-point dilution . NV was found in the alimentary tract (DD or St) at all concentrations of input virus, but was present more frequently after exposure to higher levels of virus . NV was detected in AM and HC only following exposure to higher levels of virus . In experiments where depuration by oysters was continued for 48 h, depuration of bacteria was efficient (95% reduction of bacteria), but minimal (7%) reduction of NV titers from DD tissues was detected . These findings indicate that NV can localize both within and outside the alimentary tract of shellfish, and NV is poorly depurated using conditions favorable for E . coli depuration. Eur J Biochem, 1998 Dec 1, 258(2), 869 - 78 Structure/function relationship of CYP11B1 associated with Dahl's salt-resistant rats--expression of rat CYP11B1 and CYP11B2 in Escherichia coli; Nonaka Y et al.; Dahl's salt-resistant normotensive rats (DR rats) have been previously reported to express cytochrome P-450 (CYP11B1) containing five missense mutations {Matsukawa, N., Nonaka, Y., Higaki, J., Nagano, M., Mikami H., Ogihara, T . & Okamoto, M . (1993) J . Biol . Chem . 268, 9117-9121} . To investigate structure-function relationships of CYP11B, wild-type rat CYP11B1 and CYP11B2 and DR-CYP11B1 (mutant CYP11B1 in Dahl's salt-resistant rats) have been successfully expressed in Escherichia coli . Steroid 11beta-hydroxylase (11beta-OHase) activity observed with DR-CYP11B1 was similar to that of wild-type CYP11B1, while 18-hydroxylase (18-OHase) activity of DR-CYP11B1 was lower than that of wild-type CYP11B1 . Mutant CYP11B1s containing a single or a double amino acid substitution associated with DR-CYP11B1 have been also expressed in E . coli to investigate effects of the substitutions on enzymatic activity . Each of the single mutant enzymes showed lower 18-OHase activity than wild-type CYP11B1, but not as low as DR-CYP11B1 . A double mutant CYP11B1 with V381L and I384L showed 18-OHase activity at a similar low level to that of DR-CYP11B1 . The 19-hydroxylation (19-OHase) activity of DR-CYP11B1 was about one-third of that of the wild-type enzyme and this low activity appeared due to the V443M mutation . These results suggest that three of five amino acid substitutions present in DR-CYP11B1 account for the decreased 18-OHase and 19-OHase activities . A decrease in these enzyme activities may be responsible for the normotension of the DR rats when fed a high-salt diet. Eur J Biochem, 1998 Dec 1, 258(2), 863 - 8 Purification, cDNA cloning and expression of human NG,NG-dimethylarginine dimethylaminohydrolase; Kimoto M et al.; cDNA encoding N(G),N(G)-dimethylarginine dimethylaminohydrolase from rat kidney had been cloned {Kimoto, M., Sasakawa, T., Tsuji, H., Miyatake, S., Oka, T., Nio, N . & Ogawa, T . (1997) Biochim . Biophys . Acta 1337, 6-10} . The enzyme hydrolyzes N(G),N(G)-dimethyl-L-arginine and N(G)-monomethyl-L-arginine, which are known as endogenous inhibitors for the nitric oxide-generating system . In the present study, human N(G),N(G)-dimethylarginine dimethylaminohydrolase has been purified to homogeneity from liver and characterized . The cDNA clone encoding human N(G),N(G)-dimethylarginine dimethylaminohydrolase was isolated from a human kidney lambda gt10 library using a probe prepared from a plasmid containing the entire coding region of rat N(G),N(G)-dimethylarginine dimethylaminohydrolase . Its open reading frame encoded a protein of 285 amino acids with a molecular mass of 31,121 Da . The deduced amino acid sequence exhibits 93% identity with that of rat . The cDNA was expressed as a fusion protein in Escherichia coli and the recombinant protein exhibited enzyme activity which is the same as that of natural enzyme. Eur J Biochem, 1998 Dec 1, 258(2), 854 - 62 The roles of the N-linked glycans and extension regions of soybean beta-conglycinin in folding, assembly and structural features; Maruyama N et al.; Beta-conglycinin, one of the dominant storage proteins of soybean, is a trimer composed of three subunits, alpha, alpha' and beta . All subunits are N-glycosylated and alpha and alpha' contain extension regions in addition to the core regions common to all subunits . Non-glycosylated individual subunits and deletion mutants (alpha(c) and alpha'(c)) lacking the extension regions of alpha and alpha' were expressed in Escherichia coli . All recombinant proteins were purified to near homogeneity and appeared to have the correct conformation, as judged by CD, density-gradient centrifugation and gel-filtration profiles, indicating that the N-linked glycans and extension regions are not essential for the folding and the assembly into trimers of beta-conglycinin . Density-gradient centrifugation, gel-filtration and differential scanning calorimetry profiles of the recombinant proteins and the native beta-conglycinin indicated that the N-linked glycans and extension regions contribute to the dimension of beta-conglycinin but not to the density and the thermal stability . Comparing the solubilities of the individual subunits with those of deletion mutants, only the alpha and alpha' subunits were soluble at lower ionic strength (mu < 0.25) at around the pH value of the endoplasmic reticulum . This suggests that the extension regions play an important role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N-linked glycans. Eur J Biochem, 1998 Dec 1, 258(2), 794 - 802 Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata); Fujimoto Y et al.; Synthetic urea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities . Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (Ka for 4PU30 = 4x10(10) M(-1)) in the soluble fraction of etiolated mung bean seedlings {Nagata, R., Kawachi, E., Hashimoto, Y . & Shudo, K . (1993) Biochem . Biophys . Res . Commun . 191, 543-549} . In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU . We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT) PCR . The cDNA encoded a protein with a calculated molecular mass of 17 kDa . A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family . Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins. Eur J Biochem, 1998 Dec 1, 258(2), 729 - 35 Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore; Crompton M et al.; A cyclophilin-D affinity matrix was employed to isolate components of the mitochondrial permeability transition pore . A cDNA encoding cyclophilin-D was cloned from a rat liver library and ligated into pGEX to allow expression of a glutathione S-transferase/cyclophilin-D fusion protein in Escherichia coli XL1 cells . The cyclophilin-D in the fusion was functionally normal as judged by its peptidylprolyl cis-trans-isomerase activity and its inhibition by cyclosporin A . The fusion protein was bound to glutathione-agarose to form the cyclophilin-D affinity matrix . The matrix selectively bound 32-kDa proteins of mitochondrial membrane extracts, but no H2O-soluble proteins were bound . The 32-kDa band on SDS/PAGE resolved into a doublet and reacted with antibodies against the voltage-dependent anion channel (porin) and the adenine nucleotide translocase . These two proteins were also selectively retained by the affinity matrix in the presence of cyclosporin A . The thus-purified voltage-dependent anion channel, adenine nucleotide translocase and the fusion protein were incorporated into phosphatidylcholine liposomes containing fluorescein sulphonate . The proteoliposomes were permeabilized by Ca2+ plus phosphate, and this was blocked completely by cyclosporin A . These properties are identical to those of the permeability transition pore in mitochondria . It is concluded that the basic permeability transition pore structure comprises the voltage-dependent anion channel (outer membrane), adenine nucleotide translocase (inner membrane) and cyclophilin-D, and forms at contact sites between the two membranes. Eur J Biochem, 1998 Dec 1, 258(2), 579 - 85 Inactivation of isocitrate dehydrogenase kinase/phosphatase by 5'-{p-(fluorosulfonyl)benzoyl}adenosine is not due to the labeling of the invariant lysine residue found in the protein kinase family; Oudot C et al.; The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5'-{p-(fluorosulfonyl)benzoyl}adenosine (FSBA) . This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process . About 2 mol {3H}FSBA/mol IDHK/P were bound during the time course of the inactivation . However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol . This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond . Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523 . The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue . However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480. Eur J Biochem, 1998 Dec 1, 258(2), 540 - 5 The deoxyribonuclease activity attributed to ribosome-inactivating proteins is due to contamination; Day PJ et al.; The mode of action of ribosome-inactivating proteins (RIPs) has, for many years, been considered to be depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis . Recently, this view has been challenged by the observation that many RIP preparations have significant DNase activity in addition to their N-glycosidase activity . In this study, we have investigated the putative DNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases, the DNase activity is due to the presence of contaminating nucleases . The N-glycosidase and DNase activities of PAP were separately and specifically inactivated by chemical modification and heat . Gel filtration of ricin allowed physical separation of the two activities . Furthermore, neither recombinant PAP nor recombinant ricin A-chain purified from Escherichia coli displayed DNase activity. Eur J Biochem, 1998 Dec 1, 258(2), 454 - 9 Molecular cloning and expression of active Ole e 3, a major allergen from olive-tree pollen and member of a novel family of Ca2+-binding proteins (polcalcins) involved in allergy; Ledesma A et al.; A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced . A strategy based on two-step PCR amplification towards the 5' end and 3' end, with an internal specific primer, has been used . The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type . The cDNA was inserted into the pET-11b expression vector and over-expressed in Escherichia coli . The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3 . The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens . RNA transcripts of Ole e 3 were only detected in pollen tissue . Northern-blot and Western-blot analyses of poly(A)+ RNA and protein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologous proteins . This indicates a sequence conservation and widespread distribution for this family of Ca2+-binding proteins that can be responsible for allergenic cross-reactivity . We suggest the tentative generic name of polcalcins for the members of this family of Ca2+-binding proteins from pollen. Eur J Biochem, 1998 Dec 1, 258(2), 445 - 53 Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein; Gauthier SY et al.; The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10-30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition . Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol . Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP . The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA . All eight cysteine residues appear to be involved in disulfide bond formation . Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh . NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation . Both NMR and CD spectra indicate the presence of extensive beta structure (70-80%) with little or no alpha helix . The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0 degrees C to 20 degrees C), and the presence of 50% trifluoroethanol. Eur J Biochem, 1998 Dec 1, 258(2), 437 - 44 Structure/function correlation of spermine-analogue-induced modulation of peptidyltransferase activity; Karahalios P et al.; In a cell-free system derived from Escherichia coli, various analogues of spermine were used to study their effect on the binding of AcPhe-tRNA to poly (U)-programmed ribosomes and on the puromycin reaction carried out at 6 mM Mg2+ (Ac, acetyl) . In the absence of factors washable from ribosomes (FWR fraction), mono-acylated or di-acylated analogues of spermine stimulate the binding of AcPhe-tRNA to a lesser degree than spermine, in the order: N1-acetylspermine > N1,N12-diacetylspermine approximately = N1,N12-dipivaloylspermine . Also, the above analogues do not show any sparing effect on Mg2+ requirements for AcPhe-tRNA binding to ribosomes, in contrast to spermine . The presence of FWR fraction during the binding or acetylation of the secondary amines of spermine moderates or abolishes the stimulatory effect . In addition, all analogues tested enhance the stability of the ternary complex AcPhe-tRNA-poly(U)-ribosome and the extent of AcPhe-puromycin synthesis, particularly in the absence of the FWR fraction . At the kinetic phase of AcPhe-puromycin synthesis, the analogues display both stimulatory and inhibitory effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with partial noncompetitive inhibition) . Detailed kinetic analysis shows that the analogues tested can mimic the behaviour of spermine, however, the potency to affect the peptidyltransferase activity depends on their degree of acylation, acyl-substituent size, charge distribution and on their chain flexibility. Eur J Biochem, 1998 Dec 1, 258(2), 411 - 8 Refolding of native and recombinant chicken riboflavin carrier (or binding) protein: evidence for the formation of non-native intermediates during the generation of active protein; Pattanaik P et al.; Riboflavin carrier (or binding) protein (RCP) is a phosphoglycoprotein originally purified from the egg white, yolk and serum of laying hens . The 18 cysteine residues present in RCP form nine disulfide bridges, allowing the protein to form a compact structure to generate a hydrophobic pocket in which riboflavin sits . We studied the refolding of totally reduced and denatured egg white RCP and found that the protein initially folded to generate a molecule that did not possess riboflavin-binding activity, despite near-complete oxidation of the cysteine residues . Riboflavin-binding activity was then slowly regained, but the final refolded form of the protein was less compact in structure than the native molecule, due to incomplete oxidation of all the cysteine residues . Denatured and reduced dephosphorylated RCP refolded as efficiently as the native protein, with similar rates of disulfide-bond oxidation and generation of riboflavin binding, showing that the phosphoserine stretch of RCP has little role to play during refolding . In order to study the role of glycosylation in the refolding process, the cDNA for full-length RCP was expressed in Escherichia coli and purified . Recombinant RCP refolded only in the presence of redox buffers, demonstrating that glycosylation of RCP could allow the formation of high yields of productive intermediates in the folding pathway . Using a panel of conformation-specific monoclonal antibodies to RCP, it appeared that the folding intermediates of RCP possessed a structure distinctly different to the native protein, indicating that the correct folding pathway of RCP passed through conformation(s) generated by non-native disulfide bridges. Vet Microbiol, 1998 Nov, 64(1), 75 - 81 A study of relationships among F17 a producing enterotoxigenic and non-enterotoxigenic Escherichia coli strains isolated from diarrheic calves; Contrepois M et al.; We investigated the clonal relationships among 41 enterotoxigenic (ETEC) or non-enterotoxigenic (NETEC) Escherichia coli strains producing the F17 a fimbriae isolated from diarrheic calves in France or Belgium in the early 1980s . Twenty-three of the 26 ETEC strains were highly clonally related, most of them with a O101:K32:H9-serotype . The NETEC strains were also divided in clonal subgroups, most of them with O101:H-serotype . The F17 a positive ETEC strains are no longer isolated from diarrheic calves in these countries . It is postulated that the use of a vaccine including O101, K32 and H9 antigens in addition to K99 (F5) explains the strongly reduced isolation of the O101:K32:H9, K99 (F5) E . coli clone. Bioorg Med Chem Lett, 1998 Sep 22, 8(18), 2545 - 8 A new spacer group derived from arylmalonaldehydes for glucuronylated prodrugs; Papot S et al.; A new glucuronylated prodrug of doxorubicin, potentially useful for ADEPT or PMT cancer chemotherapy, has been prepared from 4-methyl phenyl malonaldehyde . The enol ether spacer, linked via a carbamate to the 3'-amino group of doxorubicin is rapidly cleaved after beta-glucuronidase (E coli) catalyzed hydrolysis at pH 7.2 and 37 degrees C. Bioorg Med Chem Lett, 1998 Sep 22, 8(18), 2419 - 22 A photoactivated prodrug; Wei Y et al.; A photolabile derivative (1) of the anticancer drug, 5-fluorodeoxyuridine (2), was designed and synthesized as a model prodrug . Photolysis of 1 with long-wavelength UV light rapidly released 2 in solution . While compound 1 alone is nontoxic to cells, the presence of both 1 and UV irradiation (lambda = 350 nm) resulted in potent inhibition of cell growth. Bioorg Med Chem Lett, 1998 Jul 7, 8(13), 1629 - 34 A new series of cyclic amino acids as inhibitors of S-adenosyl L-methionine synthetase; Lavrador K et al.; Optically active 3-amino-3-(tetrahydrofuranyl) carboxylic acid, 3-amino-3-(tetrahydrothienyl) carboxylic acid and their corresponding six membered ring analogues have been synthesised and examined as potential inhibitors of the enzyme S-adenosylmethionine (AdoMet) synthetase . The kinetic behaviour of these compounds was studied using recombinant rat liver AdoMet synthetase (alpha-isoform) fractionated from E . coli transformed with the plasmid pSSRL-T7N . All the compounds tested were competitive inhibitors of the enzyme with respect to L-methionine. Mol Vis . 1998 Dec 31;4:33. Structure-function relationships in the four repeats of human interphotoreceptor retinoid-binding protein (IRBP); Nickerson JM et al.; PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) binds hydrophobic ligands in the interphotoreceptor space . Human IRBP consists of 1230 amino acids in four 300 amino acid long repeats . We asked: 1 . Whether each of the four repeats can bind retinoids or fatty acids, 2 . Whether each repeat can prevent retinol degradation in aqueous solutions, 3 . Whether a ligand can stabilize the protein from thermal denaturation, 4 . Whether the four repeats can be further classified into two groups . Our rationale was to make each repeat from the human cDNA and then examine structural and functional characteristics . METHODS: Individual repeats were produced in E . coli and the whole protein was expressed in baculovirus . Binding properties with all-trans-retinol were characterized by ligand fluorescence enhancement . The quenching of protein fluorescence by retinol, 9-cis-retinal, all-trans-retinoic acid, beta-ionine, alpha-ionine, trans-parinaric acid, and DHA was also examined . Binding curves were analyzed by nonlinear regression . Prevention of retinol decomposition was measured by absorption spectroscopy . Circular dichroism was examined in the far UV range to study protein secondary structure and the near UV range to study ligand binding effects on the tryptophan environment . RESULTS: Temperature dependent denaturation suggests that EcR1 is the most stable of the four repeats . Each repeat possesses the capability of binding 9-cis-retinal, all-trans-retinol, all-trans retinoic acid, docosahexaenoic acid, alpha- and beta-ionine, and trans-parinaric acid . Protein fluorescence quenching by retinol and retinol fluorescence enhancement assays yielded similar binding parameters for each repeat . Each expressed repeat prevents the degradation of retinol in aqueous solutions . CONCLUSIONS: The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand . Each repeat binds both retinoids and analogs, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands . Together, the results suggest that each repeat retains all functions of the whole protein . However, there are distinguishing characteristics among the repeats in their ligand binding properties, though the four repeats cannot be classified into just two distinctive groups . Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein. Mol Vis . 1998 Dec 30;4:30. Arginine to glutamine substitutions in the fourth module of Xenopus interphotoreceptor retinoid-binding protein; Baer CA et al.; PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) is unusual for a lipid-binding protein in that its gene is expressed uniquely by cells of photoreceptor origin and consists of four homologous repeats, each coding for a module of approximately 300 amino acid residues . All-trans retinol binding domains, which appear to be present in each module, are composed of conserved hydrophobic regions {Baer et al, Exp Eye Res 1998; 66:249-262} . Here we investigate the role of highly conserved arginines contained in these regions . METHODS: To study the arginines in an individual module without the interference of ligand-binding activity elsewhere in the protein, we expressed in E . coli the fourth module of Xenopus IRBP by itself as a soluble thioredoxin fusion protein (X4IRBP) . Arginines 1005, 1041, 1073 and 1122 were separately replaced by glutamine using PCR overlap extension mutagenesis . The glutamine substitutions were confirmed by liquid chromatography-tandem mass spectrometry . The binding of all-trans retinol and 9-(9-anthroyloxy)stearic acid (9-AS) to X4IRBP and each of the mutants was evaluated by fluorescence spectroscopy . Binding was followed by monitoring the enhancement of ligand fluorescence and the quenching of protein endogenous fluorescence . The ability of the recombinant proteins to protect all-trans retinol from oxidative degradation was evaluated by monitoring absorbance at 325 nm over time . RESULTS: The substitution of Gln for Arg1005 about doubled the amount of ligand necessary to attain saturation and about doubled the level of fluorescence enhancement obtained at saturation for both all-trans retinol and 9-AS . Although there was not a significant change in the Kd, the substitution increased the calculated number of binding sites (N) from approximately 2 to approximately 4 per polypeptide . The other Arg->Gln mutants did not significantly change the Kd or N . None of the mutations compromised the ability of the module to protect all-trans retinol from degradation . CONCLUSIONS: Our data suggest that the function of the conserved arginines in IRBP is fundamentally different from that of other retinoid-binding proteins . These residues do not appear to play a role in defining the specificity of the ligand-binding domain . Rather, Arg1005 appears to play a role in defining the capacity of the domain . Our data suggest that the binding site consists of a single hydrophobic cavity promiscuous for fatty acids and all-trans retinol. J Biol Chem, 1999 Jan 8, 274(2), 881 - 8 Extremely thermostable serine-type protease from Aquifex pyrophilus . Molecular cloning, expression, and characterization; Choi IG et al.; A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe . Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases . The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation . When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms . The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride . The half-life of the protein is 6 h at 105 degreesC, suggesting that it is one of the most heat-stable proteases . The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent. J Biol Chem, 1999 Jan 8, 274(2), 762 - 9 Expression, purification, and spectroscopic characterization of human thromboxane synthase; Hsu PY et al.; Thromboxane A2 (TXA2) is a potent inducer of vasoconstriction and platelet aggregation . Large scale expression of TXA2 synthase (TXAS) is very useful for studies of the reaction mechanism, structural/functional relationships, and drug interactions . We report here a heterologous system for overexpression of human TXAS . The TXAS cDNA was modified by replacing the sequence encoding the first 28 amino acid residues with a CYP17 amino-terminal sequence and by adding a polyhistidine tag sequence prior to the stop codon; the cDNA was inserted into the pCW vector and co-expressed with chaperonins groES and groEL in Escherichia coli . The resulting recombinant protein was purified to electrophoretic homogeneity by affinity, ion exchange, and hydrophobic chromatography . UV-visible absorbance (UV-Vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectra indicate that TXAS has a typical low spin cytochrome P450 heme with an oxygen-based distal ligand . The UV-Vis and EPR spectra of recombinant TXAS were essentially identical to those of TXAS isolated from human platelets, except that a more homogenous EPR spectrum was observed for the recombinant TXAS . The recombinant protein had a heme:protein molar ratio of 0.7:1 and a specific activity of 12 micromol of TXA2/min/mg of protein at 23 degreesC . Furthermore, it catalyzed formation of TXA2, 12-hydroxy-5,8,10-heptadecatrienoic acid, and malondialdehyde in a molar ratio of 0.94:1.0:0.93 . Spectral binding titrations showed that bulky heme ligands such as clotrimazole bound strongly to TXAS (Kd approximately 0.5 microM), indicating ample space at the distal face of the heme iron . Analysis of MCD and EPR spectra showed that TXAS was a typical low spin hemoprotein with a proximal thiolate ligand and had a very hydrophobic distal ligand binding domain. J Biol Chem, 1999 Jan 8, 274(2), 681 - 6 Interaction of the sarcin/ricin domain of 23 S ribosomal RNA with proteins L3 and L6; Uchiumi T et al.; We investigated interaction of an RNA domain covering the target site of alpha-sarcin and ricin (sarcin/ricin domain) of Escherichia coli 23 S rRNA with ribosomal proteins . RNA fragments comprising residues 2630-2788 (Tox-1) and residues 2640-2774 (Tox-2) of 23 S rRNA were transcribed in vitro and used to analyze the binding proteins by gel shift and filter binding . Protein L6 bound to both Tox-1 (Kd: 0.31 microM) and Tox-2 (Kd: 0.18 microM), and L3 bound only to Tox-1 (Kd: 0.069 microM) in a solution containing 10 mM MgCl2 and 175 mM KCl at 0 degreesC . Footprinting studies were performed using the chemical probe dimethyl sulfate on full-length 23 S rRNA . Binding of L6 protected a single base, A-2757, and strongly enhanced reactivity of C-2752 . A direct role of A-2757 in the L6 binding was verified by site-directed mutagenesis; replacements of A-2757 with G and C impaired the L6 binding . On the other hand, binding of L3 protected A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750 . Interestingly, binding of L6 and L3 together protected additional bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the other stem-loop . This appears to be due to cooperative interaction of L3 and L6 with the RNA . The results are discussed with respect to conformational modulation of the sarcin/ricin domain by the protein binding. J Biol Chem, 1999 Jan 8, 274(2), 666 - 72 Quantitative assessment of EF-1alpha.GTP binding to aminoacyl-tRNAs, aminoacyl-viral RNA, and tRNA shows close correspondence to the RNA binding properties of EF-Tu; Dreher TW et al.; A ribonuclease protection assay was used to determine the equilibrium dissociation constants (Kd) for the binding of various RNAs by wheat germ EF-1alpha.GTP . Aminoacylated fully modified tRNAs and unmodified tRNA transcripts of four specificities (valyl, methionyl, alanyl, and phenylalanyl) from higher plants or Escherichia coli were bound with Kd values between 0.8 and 10 nM . A valylated 3'-fragment of turnip yellow mosaic virus RNA, which has a pseudoknotted amino acid acceptor stem, was bound with affinity similar to that of Val-tRNAVal . Uncharged tRNA and initiator Met-tRNAMet from wheat germ, RNAs that are normally excluded from the ribosomal A site in vivo, bound weakly . The discrimination against wheat germ initiator Met-tRNAMet was almost entirely due to the 2'-phosphoribosyl modification at nucleotide G64, since removal resulted in tight binding by EF-1alpha.GTP . A 44-nucleotide RNA representing a kinked acceptor/T arm obtained by in vitro selection to bacterial EF-Tu formed an Ala-RNA.EF-1alpha.GTP complex with a Kd of 29 nM, indicating that much of the binding affinity for aminoacylated tRNA is derived from interaction with the acceptor/T half of the molecule . The pattern of tRNA interaction observed for EF-1alpha (eEF1A) therefore closely resembles that of bacterial EF-Tu (EF1A). Appl Environ Microbiol, 1999 Jan, 65(1), 294 - 6 Organic solvent tolerance of Escherichia coli is independent of OmpF levels in the membrane; Asako H et al.; The organic solvent tolerance of Escherichia coli was measured under conditions in which OmpF levels were controlled by various means as follows: alteration of NaCl concentration in the medium, transformation with a stress-responsive gene (marA, robA, or soxS), or disruption of the ompF gene . It was shown that solvent tolerance of E . coli did not depend upon OmpF levels in the membrane. Nature, 1998 Dec 17, 396(6712), 679 - 82 Heterodimerization is required for the formation of a functional GABA(B) receptor; White JH et al.; GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABA(A/C)) receptors to produce fast synaptic inhibition and metabotropic (GABA(B)) receptors to produce slow, prolonged inhibitory signals . The gene encoding a GABA(B) receptor (GABA(B)R1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes . Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABA(B)R2), which we identified by mining expressed-sequence-tag databases . Yeast two-hybrid screening showed that this new GABA(B)R2-receptor subtype forms heterodimers with GABA(B)R1 through an interaction at their intracellular carboxy-terminal tails . Upon expression with GABA(B)R2 in HEK293T cells, GABA(B)R1 is terminally glycosylated and expressed at the cell surface . Co-expression of the two receptors produces a fully functional GABA(B) receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor . These results indicate that, in vivo, functional brain GABA(B) receptors may be heterodimers composed of GABA(B)R1 and GABA(B)R2. J Basic Microbiol, 1998, 38(5-6), 395 - 404 The gluconate high affinity transport of GntI in Escherichia coli involves a multicomponent complex system; Porco A et al.; Within the main system for gluconate utilization in E . coli, the gntT gene (located at the minute 76.4) that encodes a permease, is currently the only element involved in the high affinity transport . In this paper, the nucleotide sequence of the upstream region of this locus was determined . Two open reading frames of 729 bp (gntX) and 573 bp (gntY) were identified as additional gnt genes by complementation studies . Our observations suggest that these loci might conform an operon distinct of gntT under the control of the gntR gene product . Such operon encodes a gluconate periplasmic binding protein (GntX) and a putative membrane-bound protein (GntY) . These products and the permease encoded by the gntT gene seem to conform a high-affinity complex transport system for gluconate . We suggest that this novel system could belong to the TRAP transporters. Shock, 1998 Dec, 10(6), 436 - 41 Acetazolamide treatment prevents in vitro endotoxin-stimulated tumor necrosis factor release in mouse macrophages; West MA et al.; We previously showed that incubation in carbon dioxide (CO2), but not air or helium (He), markedly decreased macrophage intracellular pH (pHi) and resulted in reversible inhibition of lipopolysaccharide- (LPS) stimulated tumor necrosis factor (TNF) and interleukin-1 release . We sought to determine whether carbonic anhydrase inhibition with acetazolamide would prevent CO2-mediated inhibition of LPS-stimulated TNF release . Murine peritoneal macrophages were treated with acetazolamide for 1 h under control atmosphere (95% air/5% CO2) and then switched to incubator modules containing: 1) 80% CO2/20% O2, 2) 80% He/20% O2, or 3) 100% air . Before transfer to experimental atmospheric conditions the macrophages were stimulated with 0 or 1 microg/mL of LPS (Escherichia coli 0111 B4) . Supernatant TNF was measured 4 h later by bioassay . In parallel experiments LPS-stimulated cytokine mRNA was estimated using reverse transcriptase polymerase chain reaction (RT-PCR) 2 h after LPS stimulation . Viability was determined using dye uptake . Incubation in CO2 or helium had no effect on TNF production in the absence of LPS . In the absence of acetazolamide CO2 produced marked inhibition of LPS-stimulated TNF release, but this was not blocked by the presence of acetazolamide . This CO2-mediated inhibition of TNF was associated with normal levels of TNF mRNA . In acetazolamide-treated macrophages, LPS resulted in a dose-dependent inhibition of TNF release when the cells were incubated in air or helium . Maintenance of normal intracellular pH is required for TNF release, but not TNF mRNA induction by LPS . Factors that alter intracellular pH regulation may modulate LPS-stimulated cytokine production. Poult Sci, 1998 Dec, 77(12), 1893 - 8 Lipopolysaccharide-induced reductions in body weight gain and feed intake do not reduce the efficiency of arginine utilization for whole-body protein accretion in the chick; Webel DM et al.; The effects of repeated injections of 400 microg Escherichia coli lipopolysaccharide (LPS) on chick performance from 11 to 22 d posthatching were examined in chicks fed casein-based diets containing graded levels of arginine . Administration of LPS reduced (P < 0.05) weight gain, feed intake, and protein accretion, and there was a tendency (P=0.07) for LPS administration to be more growth-depressing at the higher than at the lower levels of supplemental arginine . Regression analysis of protein accretion for the first three doses of arginine indicated that protein accretion was a linear (P < 0.01) function of supplemental arginine intake for both saline-injected (r2=0.94) and LPS-injected (r2=0.93) chicks . Slopes of the best-fit regression lines for both treatment groups were equal, indicating that arginine utilization for protein accretion was not affected by LPS administration . The dietary arginine concentration required to maximize weight gain and feed efficiency was unaffected by LPS administration, with both saline- and LPS-injected chicks reaching plateaus in weight gain and feed efficiency at 0.90 and 0.98% digestible arginine, respectively. FEBS Lett, 1998 Dec 4, 440(3), 273 - 6 Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides; Kanda T et al.; Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis . We demonstrate here that virtually a single tRNA species in a crude E . coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide . One out of five oligomers complementary to tRNA(Asp) blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested . This 'knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons. Hear Res, 1998 Dec, 126(1-2), 37 - 46 OCP2 exists as a dimer in the organ of Corti; Henzl MT et al.; OCP2 is one of the most abundant proteins in the organ of Corti (OC), comprising approximately 5% of the total protein in the supporting cell population . Although the very close homolog, Skp1p, has been implicated in regulating cell-cycle progression, the function of OCP2 in the terminally differentiated cochlea is presently unknown . We have purified recombinant OCP2 from Escherichia coli and examined the protein by analytical ultracentrifugation . Interestingly, sedimentation equilibrium data collected at 20 degrees C unequivocally indicate that, at the concentrations present in the OC, free OCP2 would exist as a dimeric species . The apparent sedimentation coefficient is independent of concentration at loading concentrations between 10 and 100 microM, indicating the absence of a significant monomer-dimer equilibrium in this concentration range . The functional significance of this finding is discussed. Microbios, 1998, 95(381), 71 - 7 The use of cryopreserved apical protoplasts from Curvularia lunata for electrotransformation; Dlugonski J et al.; An electroporation method, utilizing cryopreserved protoplasts, has been developed for the steroid 11-hydroxylating fungus Curvularia lunata strain IM 2901 . Protoplasts released from the apical parts of 24- and 48-h-old mycelia were suspended in cryopreservation buffer and stored at -75 degrees C for several weeks . The thawed and freshly prepared (control) protoplasts were electroporated with pAN 7-1 plasmid carrying the Escherichia coli hygromycin B resistance gene (hph) under the control of Aspergillus nidulans sequences . The electroporation efficiency of the control protoplasts with plasmid pAN 7-1 was 7.5 and 12.0 transformants per microgram DNA (protoplasts liberated from 24- and 48-h-old mycelia, respectively) . Protoplasts released from the younger mycelium were more stable according to their reversion ability to mycelial form and transformation efficiency . After 16 weeks of cryopreservation the yield of electroporation was 61.3% of the control value . All isolated electrotransformants proved to be stable for a period of > 4 months even without selective pressure. Bioorg Med Chem Lett, 1998 Jan 6, 8(1), 97 - 100 DNA gyrase inhibitory activity of ellagic acid derivatives; Weinder-Wells MA et al.; Ellagic acid was found to inhibit E . coli DNA gyrase supercoiling with approximately the same potency as nalidixic acid . Tricyclic analogs of ellagic acid, which vary in the number and position of the hydroxy groups as well as their replacement with halogens, have been synthesized . The biological activity of these analogs is discussed. Bioorg Med Chem Lett, 1998 Apr 7, 8(7), 875 - 80 Effects of metal ions on the rates and enantioselectivities of reactions catalyzed by a series of semisynthetic transaminases created by site directed mutagenesis; Qi D et al.; Fatty acid binding proteins are a class of small 15 kDa proteins with a simple architecture that forms a large solvent sequestered cavity . In previous work, we demonstrated that reductive amination reactions could be performed in this cavity by covalent attachment of a pyridoxamine cofactor and that the rate, enantioselectivity and substrate specificity of these reactions could be altered by site directed mutagenesis . Herein, we show that the chemistry performed by these conjugates can be extended to include catalytic transamination and describe the effects of added metal ions on reaction rate and enantioselectivity . We conclude that metal ions can be used to increase the rate of reactions catalyzed by semisynthetic transaminases; however, the addition of metal ions can also retard the reaction rate . Furthermore, it appears that the presence of metal ions almost always results in an erosion of reaction enantioselectivity . This limits their utility as a practical means of increasing reaction rate . The results reported here, for four independent systems, should be considered in future designs of artificial transaminases. Plant Cell Physiol, 1998 Oct, 39(10), 1045 - 53 Structural studies of the vacuolar H(+)-pyrophosphatase: sequence analysis and identification of the residues modified by fluorescent cyclohexylcarbodiimide and maleimide; Maruyama C et al.; We determined the amino acid residues of the H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) of pumpkin which are covalently labeled by two fluorescent labeling reagents; N-cyclohexyl-N'-{4-(dimethyl amino)-alpha-naphthyl} carbodiimide (NCD) and N-pyrenylmaleimide (NPM) . NCD and NPM are fluorescent analogues of N,N-dicycrohexylcarbodiimide and N-ethylmaleimide, respectively, and inactivate H(+)-PPase activity . Excess Mg2+ protected the H(+)-PPase from the inactivation by these reagents . Furthermore, we identified the cDNA clone encoding the pumpkin H(+)-PPase in order to determine the position of labeled residues . The nucleotide sequence of the cDNA clone contains a 2,304 bp open reading frame encoding a polypeptide with 768 amino acids . Chemical sequence analysis of fluorescent peptide fragments revealed that Glu749 located in the C-terminal putative transmembrane alpha-helix was a NCD-labeled residue, and Cys632 was a NPM-labeled residue located in a putative cytosolic domain . The amino acid sequence of the region that includes Glu749 is highly conserved in H(+)-PPases from other plants and it also shows some sequence similarity with the region of the carbodiimide-reactive Glu (or Asp) of F0F1-ATPase c-subunit . The reactive glutamic acids in these proteins are located at the last C-terminal transmembrane alpha-helix . We also found that the H(+)-PPase shows significant amino acid sequence similarity to Kdp-ATPase A chain of E . coli . This similarity between the two different proteins suggest that they have evolved from a common ancestor and may utilize a common basic mechanism for ion transport. Curr Microbiol, 1999 Feb, 38(2), 113 - 21 Regulation of the Escherichia coli secA gene is mediated by two distinct RNA structural conformations; Kiser KB et al.; Expression from the secA gene, encoding a key component of the general secretory pathway of Escherichia coli, is influenced by the secretion status of the cell, autogenous translational repression, and translational coupling to the upstream gene, X . SecA binds to its mRNA in a region overlapping its ribosome binding site, thus competing with ribosomes that would initiate secA translation . Mapping of the geneX-secA mRNA secondary structure has demonstrated that the RNA can adopt two distinct conformations in solution . The first conformation arises from the base-pairing of the secA Shine-Dalgarno (SD) sequence with the geneX terminus . The second conformation, in which the secA SD sequence is no longer paired with the geneX terminus, contains a GC-rich stem upstream of the secA SD sequence . The presence of this GC-rich stem is supported by structure mapping of a mutant RNA containing a deletion in the geneX terminus . The former structure appears to be involved in translational coupling by directly linking the geneX and secA sequences, where geneX translation activates secA translational initiation through the unpairing and unmasking of the secA SD sequence . As indicated by SecA-RNA binding assays, the latter structure is probably involved in SecA binding and translational repression of the secA gene . The stabilizing effect of magnesium ions toward occlusion of the secA SD sequence supports the presence of RNA tertiary structure in this regulatory domain. Curr Microbiol, 1999 Feb, 38(2), 80 - 5 The integration host factor (IHF) affects the expression of the phosphate-binding protein and of alkaline phosphatase in Escherichia coli; Spira B et al.; The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon . Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst . IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA . It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA. Mol Biol Rep, 1998 Nov, 25(4), 211 - 6 The dual identities of mammalian tRNA(Sec) for SerRS and selenocysteine synthase; Mizutani T et al.; Se is an essential trace element and is found as a selenocysteine in the active site of Se-enzymes, such as glutathione peroxidase . tRNASec is first aminoacylated with serine by Ser RS and further is converted to selenocysteyl-tRNA by selenocysteine synthase . Mammalian selenocysteine tRNA has dual identities with Ser RS and selenocysteine synthase . Key identity elements for selenocysteine synthase are the long 9 bp AA- and long 6 bp D-stems . Major serine tRNA was converted to a mutant with a 9 bp AA-stem and 6 bp D-stem, instead of a 7 bp AA-stem and 3 bp D-stem . This mutant was active for selenylation as well as serylation . The relative kinetic parameter (Vmax/Km) of the mutant was 0.052 of the value (1.00) of wild-type Sec tRNA . This low value suggests that there is an unknown fine base specific for selenocysteine synthase . For serylation, mutant having 12 bp and wild type tRNASec having 13 bp of the total length of AA- + T-stems were active but the mutants having 11 or 14 bp were inactive . This shows that SerRS measures the distance between the discrimination base and long extra arm for recognition of tRNASer. Virus Res, 1998 Oct, 57(2), 163 - 70 Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus; Jimenez-Clavero MA et al.; Swine vesicular disease virus (SVDV) is the aetiological agent of a highly contagious viral disease of pigs, whose symptoms are indistinguishable from those caused by foot-and-mouth disease virus (FMDV) . The gene coding for the capsid protein precursor of SVDV (P1) from a recent spanish isolate (SPA/1/'93) was cloned and expressed in bacteria, and the antigenicity and immunogenicity of the recombinant product were evaluated . The recombinant P1 was recognised by antibodies against SVDV induced in pigs infected experimentally with different SVDV strains . Immunisation of swine with recombinant P1-induced SVDV-specific cellular and humoral immune responses . The implications of these results in SVD diagnostic as well as in vaccine development are discussed. Genes Dev, 1998 Dec 15, 12(24), 3910 - 22 Combinatorial signaling codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm; Carmena A et al.; Mesodermal progenitors arise in the Drosophila embryo from discrete clusters of lethal of scute (l'sc)-expressing cells . Using both genetic loss-of-function and targeted ectopic expression approaches, we demonstrate here that individual progenitors are specified by the sequential deployment of unique combinations of intercellular signals . Initially, the intersection between the Wingless (Wg) and Decapentaplegic (Dpp) expression domains demarcate an ectodermal prepattern that is imprinted on the adjacent mesoderm in the form of a L'sc precluster . All mesodermal cells within this precluster are competent to respond to a subsequent instructive signal mediated by two receptor tyrosine kinases (RTKs), the Drosophila epidermal growth factor receptor (DER) and the Heartless (Htl) fibroblast growth factor receptor . By monitoring the expression of the diphosphorylated form of mitogen-associated protein kinase (MAPK), we found that these RTKs are activated in small clusters of cells within the original competence domain . Each cluster represents an equivalence group because all members initially resemble progenitors in their expression of both L'sc and mesodermal identity genes . Thus, localized RTK activity induces the formation of mesodermal equivalence groups . The RTKs remain active in the single progenitor that emerges from each cluster under the subsequent inhibitory influence of the neurogenic genes . Moreover, DER and Htl are differentially involved in the specification of particular progenitors . We conclude that distinct cellular identity codes are generated by the combinatorial activities of Wg, Dpp, EGF, and FGF signals in the progressive determination of embryonic mesodermal cells. Genes Dev, 1998 Dec 15, 12(24), 3889 - 99 Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis; Gonzalez M et al.; Most SOS mutagenesis in Escherichia coli is dependent on the UmuD and UmuC proteins . Perhaps as a consequence, the activity of these proteins is exquisitely regulated . The intracellular level of UmuD and UmuC is normally quite low but increases dramatically in lon- strains, suggesting that both proteins are substrates of the Lon protease . We report here that the highly purified UmuD protein is specifically degraded in vitro by Lon in an ATP-dependent manner . To identify the regions of UmuD necessary for Lon-mediated proteolysis, we performed 'alanine-stretch' mutagenesis on umuD and followed the stability of the mutant protein in vivo . Such an approach allowed us to localize the site(s) within UmuD responsible for Lon-mediated proteolysis . The primary signal is located between residues 15 and 18 (FPLF), with an auxiliary site between residues 26 and 29 (FPSP), of the amino terminus of UmuD . Transfer of the amino terminus of UmuD (residues 1-40) to an otherwise stable protein imparts Lon-mediated proteolysis, thereby indicating that the amino terminus of UmuD is sufficient for Lon recognition and the ensuing degradation of the protein. Genes Dev, 1998 Dec 15, 12(24), 3882 - 8 Complete inhibition of Cdk/cyclin by one molecule of p21(Cip1); Hengst L et al.; Cell-cycle phase transitions are controlled by cyclin-dependent kinases (Cdks) . Key to the regulation of these kinase activities are Cdk inhibitors, proteins that are induced in response to various antiproliferative signals but that can also oscillate during cell-cycle progression, leading to Cdk inactivation . A current dogma is that kinase complexes containing the prototype Cdk inhibitor p21 transit between active and inactive states, in that Cdk complexes associated with one p21 molecule remain active until they associate with additional p21 molecules . However, using a number of different techniques including analytical ultracentrifugation of purified p21/cyclin A/Cdk2 complexes we demonstrate unambiguously that a single p21 molecule is sufficient for kinase inhibition and that p21-saturated complexes contain only one stably bound inhibitor molecule . Even phosphorylated forms of p21 remain efficient inhibitors of Cdk activities . Therefore the level of Cdk inactivation by p21 is determined by the fraction of kinase complexed with the inhibitor and not by the stoichiometry of inhibitor bound to the kinase or the phosphorylation state of the Cdk inhibitor. Mol Carcinog, 1998 Dec, 23(4), 201 - 6 Arrest of replication by mammalian DNA polymerases alpha and beta caused by chromium-DNA lesions; Bridgewater LC et al.; We have previously shown that trivalent chromium, and hexavalent chromium in the presence of one of its primary in vivo reductants, ascorbate, can bind to DNA and form interstrand crosslinks capable of obstructing replication . This effect was demonstrated in vitro by using Sequenase Version 2.0 T7 DNA polymerase; its parent enzyme, the unmodified T7 DNA polymerase; and Escherichia coli polymerase I large (Klenow) fragment; and it was demonstrated ex vivo by using Taq polymerase and DNA from chromium-treated human lung cells as template . This study was performed to determine whether DNA-bound chromium affects mammalian DNA polymerases in the same manner . Two mammalian enzymes, DNA polymerase alpha and DNA polymerase beta, were used . DNA polymerase alpha is a processive enzyme believed to be the primary lagging-stand synthetase, whereas DNA polymerase beta is a non-processive enzyme believed to function in DNA repair by filling single stranded gaps one base at a time . DNA polymerase arrest assays were performed with each of these enzymes to replicate DNA with toxicologically relevant levels of chromium adducts produced by either trivalent chromium or hexavalent chromium and ascorbate . Both enzymes responded to chromium-DNA damage by arresting replication, and the arrests increased in a dose-dependent manner . Furthermore, the guanine-specific pattern of arrests produced when an exonuclease-free preparation of DNA polymerase beta was used corresponded exactly to the arrest patterns produced in vitro by the exonuclease-free enzyme Sequenase and ex vivo by Taq polymerase . These results suggest that replication arrest may be a common response of polymerases to DNA-chromium lesions and provide a plausible mechanism for the inhibition of DNA synthesis and S-phase cell-cycle delay that occurs in mammalian cells treated with genotoxic chromium compounds. Plant Mol Biol, 1998 Dec, 38(6), 1235 - 42 A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests; Pernas M et al.; Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized . Its full-length cDNA clone was isolated from an immature chestnut cotyledon library . The inhibitor was expressed in Escherichia coli and purified from bacterial extracts . Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing . CsC has a molecular mass of 11,275 Da and pI of 6.9 . Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%) . Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM) . By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM) . CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests . Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin. Plant Mol Biol, 1998 Dec, 38(6), 1179 - 90 Characterization of a germin-like protein gene expressed in somatic and zygotic embryos of pine (Pinus caribaea Morelet); Neutelings G et al.; Germin-like proteins (GLPs) ionically bound to the walls of preglobular somatic embryos of Pinus caribaea Morelet are markers of this early developmental stage . In order to reveal the physiological implications of such markers during early embryo development, we isolated a cDNA clone from somatic embryos predicted to encode a protein with sequence similarity to GLPs . PcGER1 has an open reading frame corresponding to a 220 amino acid polypeptide with a putative N-glycosylation site on Asn-69 . The presence of a 24 amino acid putative signal peptide supports the hypothesis of an apoplastic location . The N-terminal 20 amino acid sequence of the predicted mature protein is identical to the amino terminal sequence of GP111, one of the extracellular pine GLPs previously identified . Southern blot hybridizations indicate that PcGER1 is probably unique in the pine genome . Transcripts homologous to PcGER1 are abundant in all embryogenic lines, absent from nonembryogenic lines, and present in quiescent zygotic embryos but not in the female gametophyte, the haploid storage tissue of conifers . Their abundance sharply decreases during germination . Isolation of gf-0.8, a genomic fragment identical to PcGER1 cDNA sequence, confirms that no introns disrupt the coding region as it has been already described for wheat gf-2.8 and gf-3.8 genomic clones . Recombinant PcGER1, produced in Escherichia coli, is recognized by antibodies raised against the GP111 N-terminal nonapeptide and the unglycosylated wheat germin monomer . The implications of GLPs in pine embryogenesis are discussed. Plant Mol Biol, 1998 Dec, 38(6), 1123 - 35 Alfin1, a novel zinc-finger protein in alfalfa roots that binds to promoter elements in the salt-inducible MsPRP2 gene; Bastola DR et al.; Alfin1 cDNA, obtained by differential screening of a poly(A)+ library from salt-tolerant alfalfa cells, encodes a novel protein with a Cys4 and His/Cys3 putative zinc-binding domain that suggests a possible role for this protein in transcriptional regulation . We have expressed the cDNA in Escherichia coli and show that the recombinant Alfin1 protein binds DNA in a sequence-specific manner . The DNA recognition sequence was determined from individual clones isolated after four rounds of random oligonucleotide selection in gel retardation assays, coupled with PCR amplification of the selected sequences . The consensus binding site for Alfin1 is shown to contain two to five G-rich triplets with the conserved core of GNGGTG or GTGGNG in clones showing high-efficiency binding . DNA binding of the recombinant Alfin1 was inhibited by EDTA . Alfin1 mRNA was found predominantly in alfalfa roots . Growth of salt-sensitive Medicago sativa L on 171 mM NaCl led to a slight decrease in Alfin1 mRNA, while the salt-tolerant plants showed no decrease in Alfin1 mRNA levels . Interestingly, recombinant Alfin1 binds efficiently to three fragments of the MsPRP2 promoter, each containing consensus sequences identified by the random oligonucleotide selection . Since MsPRP2 transcripts were shown to be root-specific and accumulated in alfalfa roots in a salt-inducible manner, Alfin1 may play a role in the regulated expression of MsPRP2 in alfalfa roots and contribute to salt tolerance in these plants. Liver, 1998 Dec, 18(6), 398 - 404 Mature hepatocytes actively divide and express gamma-glutamyl transpeptidase after D-galactosamine liver injury; Kitten O et al.; AIMS/BACKGROUND: We studied the fate of hepatocytes in the rat liver after D-galactosamine injury by genetic labeling using recombinant retroviruses carrying the Escherichia coli lacZ gene coupled to a nuclear localization signal . METHODS: Hepatocytes were either labeled by direct injection of 2.5 ml high-titer retrovirus-containing medium in the regenerating liver parenchyma after administration of a single dose of D-galactosamine . Alternatively hepatocytes were pre-labeled, 24 h after a two-thirds hepatectomy, by injecting the same volume of retroviral solution in the portal vein and D-galactosamine was administered 15 days later . Gamma-glutamyl transpeptidase and beta-galactosidase activities were assessed on cryostat sections, along with localization of the hepatocyte-specific HES6 antigen . RESULTS: Morphological observations, as well as beta-galactosidase activity detection, showed that hepatocytes actively divide as early as 1 day after D-galactosamine injection . Gamma-glutamyl transpeptidase activity was detected in biliary cells, but also in mature hepatocytes, pre-labeled with beta-galactosidase before D-galactosamine administration . CONCLUSIONS: These experiments demonstrate that hepatocytes can divide to restore the liver mass after D-galactosamine liver injury . Furthermore, we also show that gamma-glutamyl transpeptidase, which has been reported to be expressed only by fetal or preneoplastic hepatocytes, can be re-expressed by mature hepatocytes during the recovery process. J Photochem Photobiol B, 1998 Sep, 45(2-3), 75 - 81 A non-excision uvr-dependent DNA repair pathway of Escherichia coli (involvement of stress proteins); Sedliakova M; In UV-irradiated excision-proficient (uvr+) Escherichia coli, pre-induced by simultaneous pre-starvation for thymine (T) and amino acids (AAs), and/or a low UV pre-dose applied after prestarvation for AAs, pyrimidine dimer excision (PDE) is reduced without an adequate increase of UV sensitivity and UV mutagenesis . The unexcised lesions are tolerated by a putative repair pathway that is uvr dependent but does not involve excision . The process consists of PDE inhibition, which requires outer membrane protease OmpT, and subsequent pyrimidine dimer (PD) toleration, which may be mediated by interaction with a sister duplex using a number of SOS and stress-inducible proteins. FEMS Microbiol Lett, 1998 Dec 15, 169(2), 403 - 8 Co-regulation of lipoamide dehydrogenase and 2-oxoglutarate dehydrogenase synthesis in Escherichia coli: characterisation of an ArcA binding site in the lpd promoter; Cunningham L et al.; The lipoamide dehydrogenase gene (lpdA) encoding the E3 subunits of both the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes of Escherichia coli, is expressed from the upstream pdh and internal lpd promoters of the pdh operon (pdhR-aceEF-lpdA) . Under aerobic conditions, the specific components of the 2-oxoglutarate dehydrogenase complex encoded by the sucAB genes in the sdhCDAB-sucABCD operon are expressed from the sdh promoter . The provision of lipoamide dehydrogenase subunits for assembly into the 2-oxoglutarate dehydrogenase complex could thus be controlled by co-regulation of the lpd promoter with the sdh promoter . Here, the transcription start point of the lpd promoter was defined by primer extension analysis, and an ArcA binding site, TGTTAACAAT, overlapping the lpd promoter and matching the consensus at 8 out of 10 positions, was identified by in vitro footprint analysis . PdhR was not bound to the lpd promoter nor was ArcA bound specifically to the pdh promoter . These results support the view that co-regulation of the lpd and sdh promoters is mediated primarily by ArcA. Trends Biochem Sci, 1998 Dec, 23(12), 457 - 60 Patch engineering: a general approach for creating proteins that have new binding activities; Smith G; Patch engineering is a technique for creating folded proteins that have new binding activities . Different protein scaffolds are used to present a patch of discontinuous residues on a folded-protein surface . By varying simultaneously the residues in these patches and displaying these mutant proteins on phage, one can select proteins that have new binding activities . Patch engineering is applicable to any protein fold . Novel proteins derived by this approach might replace antibodies in certain applications or provide lead molecules for the design of non-peptide analogues. J Biol Chem, 1999 Jan 1, 274(1), 397 - 402 Regulation of serine biosynthesis in Arabidopsis . Crucial role of plastidic 3-phosphoglycerate dehydrogenase in non-photosynthetic tissues; Ho CL et al.; In plants, Ser is synthesized through a couple of pathways . 3-Phosphoglycerate dehydrogenase (PGDH), the first enzyme that is involved in the phosphorylated pathway of Ser biosynthesis, is responsible for the oxidation of 3-phosphoglycerate to phosphohydroxypyruvate . Here we report the first molecular cloning and characterization of PGDH from Arabidopsis thaliana . Sequence analysis of cDNA and a genomic clone revealed that the PGDH gene is composed of three exons, encoding a 623-amino acid polypeptide (66, 453 Da) . The deduced protein, containing three of the most conserved regions in the NAD-dependent 2-hydroxyacid dehydrogenase family, has 38-39% identity to its animal and bacterial counterparts . The presence of an N-terminal signal sequence for translocation into plastids was confirmed by particle-gun bombardment experiments using green fluorescence protein as a reporter protein for subcellular localization . Southern hybridization analysis and restriction fragment length polymorphism mapping indicated that PGDH is a single-copy gene that is mapped to the upper arm of chromosome 1 . Northern hybridization analysis indicated preferential expression of PGDH mRNA in root tissues of light-grown plants, suggesting that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying Ser to non-photosynthetic tissues . The recombinant enzyme overproduced in Escherichia coli displayed hyperbolic kinetics with respect to 3-phosphoglycerate and NAD+. J Biol Chem, 1999 Jan 1, 274(1), 328 - 34 Cooperative formation of the ligand-binding site of the inositol 1,4, 5-trisphosphate receptor by two separable domains; Yoshikawa F et al.; Limited trypsin digestion of mouse cerebellar membrane fractions leads to fragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K . (1999) J . Biol . Chem . 274, 316-327) . Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity . This is despite the fact that the IP3-binding core (residues 226-578), which is close to the minimum for high affinity binding, is completely split into two tryptic fragments at the Arg-343 and/or Arg-345, around the center of the core . Furthermore, we have examined whether binding activity could be complemented in vitro by mixing two distinct glutathione S-transferase (GST) fusion proteins, which were respectively composed of residues 1-343 and 341-604, almost corresponding to two split binding components, and separately expressed in Escherichia coli . The GST-fused residues 1-343 (GN) showed no binding affinity for IP3, whereas the GST-fused residues 341-604 (GC) displayed weak but definite activity with an affinity >100-fold lower than that of the native receptor . Upon mixing of both GN and GC, a high affinity site comparable to the native site appeared . We suggest that the IP3-binding pocket consists of two non-covalently but tightly associated structural domains each of which has a discrete function: the C-terminal domain alone has low affinity for IP3, whereas the N-terminal one alone is incapable of binding but is capable of potentiating binding affinity. J Biol Chem, 1999 Jan 1, 274(1), 249 - 56 A new heat shock protein that binds nucleic acids; Korber P et al.; We describe the isolation of Hsp15, a new, very abundant heat shock protein that binds to DNA and RNA . Hsp15 is well conserved and related to a number of RNA-binding proteins, including ribosomal protein S4, RNA pseudouridine synthase, and tyrosyl-tRNA synthetase . The region shared between these proteins appears to represent a common, but previously unrecognized, RNA binding motif . Filter binding studies showed that Hsp15 binds to a 17-mer single-stranded RNA with a dissociation constant of 9 microM in 22.5 mM Hepes, pH 7 . 0, 5 mM MgCl2 . A role of Hsp15 in binding nucleic acids puts this protein into a different functional category from that of many other heat shock proteins that act as molecular chaperones or proteases on protein substrates. J Biol Chem, 1999 Jan 1, 274(1), 189 - 95 Evidence for cysteine persulfide as reaction product of L-Cyst(e)ine C-S-lyase (C-DES) from Synechocystis . Analyses using cystine analogues and recombinant C-DES; Lang T et al.; The pyridoxal phosphate-dependent monomeric L-cysteine/cystine C-S-lyase (C-DES), previously isolated from Synechocystis PCC 6714 by its capacity to direct {2Fe-2S} cluster assembly of ferredoxin in vitro (Leibrecht, I., and Kessler, D . (1997) J . Biol . Chem . 272, 10442-10447), has now been cloned, sequenced, and overexpressed in Escherichia coli . The amino acid sequence of C-DES was found to be nearly identical (92% identity) to the open reading frame slr2143 of Synechocystis PCC 6803 and showed a more distant relationship to the NifS family of proteins (about 27% identity) . Recombinant C-DES displayed activities equal to the isolate from Synechocystis in terms of the cyst(e)ine lyase reaction and holoferredoxin formation which recommended its use for functional and mechanistic studies . Investigation of the substrate spectrum for beta-elimination found L-cysteine to be a poor substrate (kcat approximately 0.15 s-1) in contrast to L-cystine (kcat = 36 s-1) and several related compounds . Of these compounds, desaminocystine (S-(carboxyethylthio)-L-cysteine) was used for C-DES-mediated persulfide generation . Stabilization of the linear persulfide 3-(disulfanyl)-propionic acid was achieved by cyclization as a novel intramolecular trapping reaction; this yielded 1,2-dithiolan-3-one which was isolated and identified by chemical analyses. J Biotechnol, 1998 Dec 11, 66(2-3), 203 - 10 Expression of the second epidermal growth factor-like domain of human factor VII in Escherichia coli; Hellebust H et al.; The second epidermal growth factor (EGF)-like domain of human coagulation factor VII is a potent inhibitor of the FVIIa/tissue factor complex, the predominant initiator of coagulation in vivo . This domain has now for the first time been cloned and expressed in Escherichia coli as an affinity fusion protein . The fusion protein was secreted into the periplasm of E . coli and purified by affinity chromatography . The purified protein consisted of a fusion protein with the expected molecular weight, and in addition, a significant fraction of oligomers cross-linked by intermolecular disulfide bonds . Despite the presence of oligomers, the purified protein was a potent inhibitor of the extrinsic blood coagulation pathway with an IC50 value of about 20 microM . The biological activity was retained after liberation of the EGF domain by proteolytic cleavage. Anal Biochem, 1998 Nov 15, 264(2), 263 - 70 Measurement of photosystem I activity with photoreduction of recombinant flavodoxin; Zhao J et al.; Flavodoxin can function as an alternative electron acceptor for photosystem I (PSI) in place of ferredoxin under iron-limiting conditions . The isiB gene, encoding the flavodoxin in Synechococcus sp . PCC 7002, was overexpressed in Escherichia coli . Under the conditions employed, most recombinant flavodoxin (rFlvd) was in soluble form with cofactor correctly inserted . The absorption spectrum of rFlvd was identical to that of the native flavodoxin of the cyanobacteria . Photoreduction of rFlvd by PSI particles and thylakoid membranes was determined directly by monitoring the absorption change at 467 nm . The optimal conditions for rFlvd photoreduction were determined . Compared to other methods currently employed to measure PSI activity such as oxygen uptake in the presence of methyl viologen and NADP+ photoreduction in the presence of ferredoxin and ferredoxin:NADP+ oxidoreductase, measurement of PSI activity with flavodoxin as an electron acceptor has several advantages . It measures the full-chain electron transfer chain of PSI since flavodoxin accepts electrons from FA/FB and it is much simpler than the method with NADP+ photoreduction . With this method, we found that the affinity of wild-type PSI for rFlvd was 35% higher than that of the PsaE-less PSI, showing that this method is sensitive to structural changes of PSI . Our results demonstrate that rFlvd photoreduction is an effective and simple method for PSI activity measurement. Protein Sci, 1998 Dec, 7(12), 2602 - 12 Localization of basic residues required for receptor binding to the single alpha-helix of the receptor binding domain of human alpha2-macroglobulin; Huang W et al.; To better understand the structural basis for the binding of proteinase-transformed human alpha2-macroglobulin (alpha2M) to its receptor, we have used three-dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human alpha2M . Assignment of the backbone NMR resonances of RBD was made using 13C/15-N and 15N-enriched RBD expressed in Escherichia coli . The secondary structure of RBD was determined using 1H and 13C chemical shift indices and inter- and intrachain nuclear Overhauser enhancements . The secondary structure consists of eight strands in beta-conformation and one alpha-helix, which together comprise 44% of the protein . The beta-strands form three regions of antiparallel beta-sheet . The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the alpha-helix, which also contains an (Arg1378) . Secondary structure predictions of other alpha-macroglobulins show the conservation of this alpha-helix and suggest an important role for this helix and for basic residues within it for receptor binding. Protein Sci, 1998 Dec, 7(12), 2541 - 9 The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii; Sridhar Prasad G et al.; NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii . The crystal structure of AvFPR has been determined at 2.0 A resolution . The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR) . AvFPR is overall most homologous to EcFldR . The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold . The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed . The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes . The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue . The structure of AvFPR displays three unique features in the environment of the bound FAD . Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin . Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR . An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI) . This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR . The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD . Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif . The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258. Protein Sci, 1998 Dec, 7(12), 2511 - 21 Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose; Hars U et al.; The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution . The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family . This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices . The effector binding pocket is at the interface of these subdomains . In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules . According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate . The binding affinity for the former is lower than for the latter . The noninducer trehalose thus binds competitively to the repressor . Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA . The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source. Clin Cancer Res, 1998 Dec, 4(12), 2999 - 3004 Identification of novel mutations in the dihydropyrimidine dehydrogenase gene in a Japanese patient with 5-fluorouracil toxicity; Kouwaki M et al.; 5-Fluorouracil (5-FU) is used widely in the treatment of several common neoplasms . Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-FU . Several recent studies have described a pharmacogenetic disorder in which cancer patients with decreased DPD activity develop life-threatening toxicity following exposure to 5-FU . We reported recently the first Japanese case of decreased DPD activity accompanied by severe 5-FU toxicity . The present study describes the results of molecular analysis of this patient and her family, in which three novel mutations (Arg21Gln, Val335Leu, and Glu386Ter) of the gene coding for DPD were identified . We also revealed that Arg21Gln and Glu386Ter are on the same allele and that Val335Leu is on the other allele, on the basis of analysis of the family genome . Expression analysis in Escherichia coli showed that Val335Leu and Glu386Ter led to mutant DPD protein with significant loss of enzymatic activity and no activity, respectively . The Arg21Gln mutation, however, resulted in no decrease in enzymatic activity compared with the wild type . The present data represent the first molecular genetic analysis of DPD deficiency accompanied by severe 5-FU toxicity in a Japanese patient. Cell, 1998 Dec 11, 95(6), 771 - 8 Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes; Locher KP et al.; FhuA protein facilitates ligand-gated transport of ferrichrome-bound iron across Escherichia coli outer membranes . X-ray analysis at 2.7 A resolution reveals two distinct conformations in the presence and absence of ferrichrome . The monomeric protein consists of a hollow, 22-stranded, antiparallel beta barrel (residues 160-714), which is obstructed by a plug (residues 19-159) . The binding site of ferrichrome, an aromatic pocket near the cell surface, undergoes minor changes upon association with the ligand . These are propagated and amplified across the plug, eventually resulting in substantially different protein conformations at the periplasmic face . Our findings reveal the mechanism of signal transmission and suggest how the energy-transducing TonB complex senses ligand binding. Biochimie, 1998 Aug-Sep, 80(8-9), 729 - 37 Interferon-gamma is a target for binding and folding by both Escherichia coli chaperone model systems GroEL/GroES and DnaK/DnaJ/GrpE; Vandenbroeck K et al.; IFN-gamma can be physicochemically distinguished from interferons-alpha, -beta or -omega through the loss of its tertiary structure and biological activity upon exposure to acid or heat . This loss is due to the irreversible aggregation of an unfolded or partially folded state . The conformational instability of IFN-gamma is reflected by its impairment to fold properly when overexpressed in Escherichia coli, resulting in its accumulation in cytoplasmic inclusion bodies . Chaperones were originally identified as a heterogeneous group of proteins that mediate the folding and correct assembly of various polypeptide substrates, and protect thermolabile proteins against inactivation . In either of both cases, chaperones prevent irreversible misfolding by assisting the substrate protein along its pathway to a stable tertiary conformation . Among the best characterized chaperones are the Escherichia coli Hsp60 and Hsp70 heat shock protein complexes, i.e., GroEL/GroES and DnaK/DnaJ/GrpE . They exhibit entirely different reaction mechanisms, which, however, both depend on hydrolysis of ATP . The unfolding of recombinant IFN-gamma by acid or heat can be used as a tool to assess its in vitro interaction with each of both chaperone systems at physiological temperature (35 degrees C) . Using such an experimental set-up, both the DnaK and GroEL chaperone systems appeared to form complexes with IFN-gamma from which correctly folded protein was released in an ATP-dependent manner . In addition to the biotechnological implication of these observations, the relevance to de novo folding of IFN-gamma is discussed. Toxicology, 1998 Sep 15, 130(2-3), 107 - 13 Penetration of cisplatin into mouse brain by lipopolysaccharide; Minami T et al.; We investigated the penetration of cisplatin into the mouse cerebral cortex-rich region (CCR) induced by lipopolysaccharide (LPS) . With the injection of cisplatin into mice 3 h after the LPS treatment, platinum was detected in the CCR during the 7 days after the injection, while platinum was not detected in the CCR of cisplatin-injected mice without LPS pretreatment and of mice simultaneous treated with cisplatin and LPS . The N(G)-nitro-L-arginine methyl ester dose-dependently lowered the platinum level . A dose of 5 mg/kg of aminoguanidine reduced the increase in the platinum level of the LPS-treated mouse, and platinum was no longer detected at doses of 20 mg/kg in the aminoguanidine-injected group . At doses of 500 mg/kg aminoguanidine, however, no effect was seen on the platinum level of the CCR induced by LPS . Regarding indomethacin, the injection of 5 mg/kg resulted in a decrease in the platinum content of the CCR, but not undetectable level . These results suggest that LPS increases the penetration of cisplatin into the mouse brain, and platinum may be accumulated in the CCR . Nitric oxide and prostaglandins contribute to the penetration of platinum into the cerebral cortex. J Hered, 1998 Nov-Dec, 89(6), 525 - 30 Comparison of acute endotoxin-induced lesions in A/J and C57BL/6J mice; O'Malley J et al.; Resistance to the action of endotoxin varies among inbred strains of mice, indicating that a component of this resistance has a genetic basis . Different responses to endotoxin that are characteristic of individual inbred strains represent phenotypes that can be used to genetically map the response modifier genes . This study compares the acute histologic lesions in 8-week-old male A/J and C57BL/6J (B6) mice injected intraperitoneally with endotoxin of E . coli O265:B6 (15 mg/kg) . Animals of both strains exhibited splenitis, splenic lymphoid hyperplasia, splenic lymphoid necrosis, and sequestration of neutrophils in the pulmonary alveoli . The B6 mice showed increased margination of white blood cells to the pulmonary vascular endothelium relative to A/J mice . A large number of degenerating neutrophils was observed in the liver sinusoids of most B6 animals, while this lesion was much less severe in A/J mice . This difference was quantified, demonstrating a highly significant difference in neutrophil infiltration in B6 mice relative to A/J mice . Analysis of this phenotype in F1 mice demonstrates that major genes encoding the trait are not X-linked, imprinted, or maternally inherited and do not show the codominant inheritance expected if Lps(d) were primarily responsible . The distinctive, quantitative nature of these differences provides a useful assay for mapping genes that modify endotoxin responsiveness using the AXB and BXA recombinant inbred (RI) strains derived from A/J and B6 mice. Oncol Rep, 1999 Jan-Feb, 6(1), 107 - 11 A polyclonal antibody against synthetic peptide conserved in N-Myc protein reacts with water-soluble recombinant N-Myc protein; Yang HW et al.; The importance of determining the N-Myc protein has been emphasized in neuroblastoma . We attempted to obtain a water-soluble N-Myc protein, and an antibody highly specific for the N-Myc protein . A plasmid was constructed from partial exons 2 and 3 of the N-myc gene, and expressed in Escherichia coli by isopropyl-beta,-D-thiogalactopyranoside . Newly obtained N-Myc (rN-Myc) was water-soluble with a M.W . of 38 kDa . An N-Myc-specific peptide, GVAPPRPGG RQTSGGDH, was used to raise an antibody . Specificity of the obtained antibody was confirmed and we found the rN-Myc protein reacts positively with the anti-N-Myc IgG raised here . The rN-Myc protein and the anti-N-Myc IgG obtained are expected to be used in an ELISA for N-Myc. J Bacteriol, 1999 Jan, 181(1), 331 - 3 (R)-citramalate synthase in methanogenic archaea; Howell DM et al.; The Methanococcus jannaschii gene MJ1392 was cloned, and its protein product was hyperexpressed in Escherichia coli . The resulting protein was purified and shown to catalyze the condensation of pyruvate and acetyl coenzyme A, with the formation of (R)-citramalate . Thus, this gene (cimA) encodes an (R)-citramalate synthase (CimA) . This is the first identification of this enzyme, which is likely involved in the biosynthesis of isoleucine. J Bacteriol, 1999 Jan, 181(1), 284 - 90 An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes beta-1,4 bonds in mixed-linkage (1-->3),(1-->4)-beta-D-glucans and cellulose; Bauer MW et al.; The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli . The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa . The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain . The P . furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12 . The purified recombinant enzyme hydrolyzed beta-1,4 but not beta-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization {DP} = 5) and cellohexaose (DP = 6) oligosaccharides . To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only beta-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter . The highest specific activity toward polysaccharides occurred with mixed-linkage beta-glucans such as barley beta-glucan and lichenan . Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (-I, -II, and -III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage . The enzyme had temperature and pH optima of 100 degreesC and 6.0, respectively; a half-life of 40 h at 95 degreesC; and a denaturing temperature of 112 degreesC as determined by differential scanning calorimetry . The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments. J Bacteriol, 1999 Jan, 181(1), 197 - 203 Protein mobility in the cytoplasm of Escherichia coli; Elowitz MB et al.; The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo . In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli . These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M . B . Elowitz, M . G . Surette, P . E . Wolf, J . Stock, and S . Leibler, Curr . Biol . 7:809-812, 1997) . The apparent diffusion coefficient, Da, of GFP in E . coli DH5alpha was found to be 7.7 +/- 2.5 microm2/s . A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with Da of 2.5 +/- 0.6 microm2/s . In addition, GFP mobility can depend strongly on at least two factors: first, Da is reduced to 3.6 +/- 0.7 microm2/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces Da to 4.0 +/- 2.0 microm2/s . Thus, a single effective cytoplasmic viscosity cannot explain all values of Da reported here . These measurements have implications for the understanding of intracellular biochemical networks. J Bacteriol, 1999 Jan, 181(1), 177 - 85 Mutations affecting the ability of the Escherichia coli UmuD' protein to participate in SOS mutagenesis; Ohta T et al.; The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism . The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids . A previous genetic screen for nonmutable umuD mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J . R . Battista, T . Ohta, T . Nohmi, W . Sun, and G . C . Walker, Proc . Natl . Acad . Sci . USA 87:7190-7194, 1990) . To identify elements of the UmuD' protein necessary for its role in translesion synthesis, we began with umuD', a modified form of the umuD gene that directly encodes the UmuD' protein, and obtained missense umuD' mutants deficient in UV and methyl methanesulfonate mutagenesis . The D39G, L40R, and T51I mutations affect residues located at the UmuD'2 homodimer interface and interfere with homodimer formation in vivo . The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded beta-sheet and appears to interfere with UmuD'2 homodimer formation indirectly by affecting the structure of the UmuD' monomer . When expressed from a multicopy plasmid, the L40R umuD' mutant gene exhibited a dominant negative effect on a chromosomal umuD+ gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD' function that goes beyond its impairment of homodimer formation . The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal beta-strand and results in a mutant UmuD' protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD+ strain . The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD'2 homodimer, which are mobile in solution. J Bacteriol, 1999 Jan, 181(1), 68 - 77 Cloning and expression of three new Aazotobacter vinelandii genes closely related to a previously described gene family encoding mannuronan C-5-epimerases; Svanem BI et al.; The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously been reported . The corresponding proteins catalyze the Ca2+-dependent polymer-level epimerization of beta-D-mannuronic acid to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate . Here we report the identification of three additional structurally similar genes, designated algE6, algE7, and algY . All three genes were sequenced and expressed in Escherichia coli . AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase . The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks . AlgY did not display epimerase activity, but a hybrid gene in which the 5'-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase . Southern blot analysis of genomic A . vinelandii DNA, using the 5' part of algE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here. J Bacteriol, 1999 Jan, 181(1), 47 - 54 Convergent pathways for utilization of the amino sugars N-acetylglucosamine, N-acetylmannosamine, and N-acetylneuraminic acid by Escherichia coli; Plumbridge J et al.; N-Acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NANA) are good carbon sources for Escherichia coli K-12, whereas N-acetylmannosamine (ManNAc) is metabolized very slowly . The isolation of regulatory mutations which enhanced utilization of ManNAc allowed us to elucidate the pathway of its degradation . ManNAc is transported by the manXYZ-encoded phosphoenolpyruvate-dependent phosphotransferase system (PTS) transporter producing intracellular ManNAc-6-P . This phosphorylated hexosamine is subsequently converted to GlcNAc-6-P, which is further metabolized by the nagBA-encoded deacetylase and deaminase of the GlcNAc-6-P degradation pathway . Two independent mutations are necessary for good growth on ManNAc . One mutation maps to mlc, and mutations in this gene are known to enhance the expression of manXYZ . The second regulatory mutation was mapped to the nanAT operon, which encodes the NANA transporter and NANA lyase . The combined action of the nanAT gene products converts extracellular NANA to intracellular ManNAc . The second regulatory mutation defines an open reading frame (ORF), called yhcK, as the gene for the repressor of the nan operon (nanR) . Mutations in the repressor enhance expression of the nanAT genes and, presumably, three distal, previously unidentified genes, yhcJIH . Expression of just one of these downstream ORFs, yhcJ, is necessary for growth on ManNAc in the presence of an mlc mutation . The yhcJ gene appears to encode a ManNAc-6-P-to-GlcNAc-6-P epimerase (nanE) . Another putative gene in the nan operon, yhcI, likely encodes ManNAc kinase (nanK), which should phosphorylate the ManNAc liberated from NANA by the NanA protein . Use of NANA as carbon source by E . coli also requires the nagBA gene products . The existence of a ManNAc kinase and epimerase within the nan operon allows us to propose that the pathways for dissimilation of the three amino sugars GlcNAc, ManNAc, and NANA, all converge at the step of GlcNAc-6-P. J Bacteriol, 1999 Jan, 181(1), 15 - 23 Pkg2, a novel transmembrane protein Ser/Thr kinase of Streptomyces granaticolor; Nadvornik R et al.; A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases . It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases . The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure . The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues . The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm . Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes . Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae. Infect Immun, 1999 Jan, 67(1), 403 - 9 Detection of live Trypanosoma cruzi in tissues of infected mice by using histochemical stain for beta-galactosidase; Buckner FS et al.; The pathogenesis of tissue damage in chronic Trypanosoma cruzi infection has been a subject of long-standing debate . Conventional staining methods reveal a paucity of parasites in tissues from chronically infected individuals, which has led to the theory that the pathologic findings may be primarily autoimmune in origin . Immunostaining for T . cruzi antigens or in situ PCR methods show evidence for parasite components in chronic tissues; however, these methods do not address whether the stained material represents parasite debris or live organisms . An improved method for detecting intact T . cruzi in tissues was developed by making a genetically engineered strain that expresses Escherichia coli beta-galactosidase . The expression of this enzyme allows the detection of T . cruzi in tissues by using the histochemical stain 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) . The technique was used to monitor tissue parasitism and its relation to pathologic findings in the mouse model of Chagas' disease . Parasites were easily visible as bright blue structures in skeletal muscle, heart, bladder, peripheral nerve, liver, spleen, adrenal gland, brain, and adipose tissue in acutely infected mice . The number of viable parasites diminished >100-fold when tissues from 3-week-infected mice were compared with those from 10-month-infected mice . However, even at the lower level, parasites were clearly recognizable in sections of skeletal muscle and bladder at the 10-month time point . Inflammation remained robust in skeletal muscle, bladder, and sciatic nerve despite the near disappearance of parasites, suggesting three possibilities: exuberant host reactions to the few remaining parasites, autoimmune inflammation, or reactions to retained parasite antigens in the tissues. Infect Immun, 1999 Jan, 67(1), 259 - 65 Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors; Stevens LA et al.; Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase . LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation . All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins . Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins . S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively . The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation) . All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K) . Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose . V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase . Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. Infect Immun, 1999 Jan, 67(1), 165 - 72 Identification of an intestinal neutral glycosphingolipid as a phenotype-specific receptor for the K88ad fimbrial adhesin of Escherichia coli; Grange PA et al.; In this study, we identified a receptor for the K88ad fimbrial adhesin of Escherichia coli in neutral glycosphingolipid preparations from intestinal epithelial cells of K88ad-adhesive pigs, which was absent in preparations from K88ad-nonadhesive pigs . Neither K88ab nor K88ac adhesin variants bound to this neutral glycosphingolipid . Because this receptor is an intestinal glycosphingolipid that binds K88ad adhesin, it has been designated IGLad . Carbohydrate compositional analysis of a partially purified preparation of IGLad identified galactose, glucose, and N-acetylglucosamine in a ratio of 1.5:1.0:0.5 as the major monosaccharides . Preliminary characterization experiments using lectins showed that IGLad contains the terminal glycanic structure Galbeta1-4GlcNAc . Removal of terminal beta-linked galactose residues from IGLad decreased the recognition of IGLad by the K88ad adhesin, indicating that terminal beta-linked galactose is an essential component of the K88ad adhesin recognition site on IGLad . Studies with purified glycosphingolipid standards demonstrated that K88ad adhesin binds to neolactotetraosylceramide (nLc4Cer) (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) , lactotriosylceramide (GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) and lactotetraosylceramide (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) . Based on these studies, IGLad appears to be nLc4Cer. Blood, 1999 Jan 1, 93(1), 363 - 9 Protein replacement by receptor-mediated endocytosis corrects the sensitivity of Fanconi anemia group C cells to mitomycin C; Youssoufian H et al.; Current methods for direct gene transfer into hematopoietic cells are inefficient . Here we show that functional complementation of Fanconi anemia (FA) group C cells by protein replacement can be as efficacious as by transfection with wild-type FAC cDNA . We expressed a chimeric protein (called His-ILFAC) consisting of the mature coding portion of gibbon interleukin-3 (IL-3) and full-length FAC in Escherichia coli . The purified bacterial protein is internalized by hematopoietic cells via IL-3 receptors . The intracellular half-life of His-ILFAC is approximately 60 minutes, which is comparable to that of the transgene-encoded FAC protein . In this cell-culture model His-ILFAC completely corrects the sensitivity of FA group C cells to mitomycin C, but it has no effect on FA cells that belong to complementation groups A and B . We suggest that receptor-mediated endocytosis of cytokine-fusion proteins may be of general use to deliver macromolecules into hematopoietic progenitor cells. Wei Sheng Wu Xue Bao, 1997 Jun, 37(3), 228 - 31 {Conditions for the overexpressions of copper--resistant proteins from Escherichia coli}; Liu Z et al.; There were five copper--resistant proteins encoded in the copper resistant plasmid pRJ1004 from Escherichia coli . The conditions for the overexpressions of two of the five, PcoC and PcoE, were reported in this paper . For the overexpression of PcoC using IPTG (Isopropyl-beta-D-thiogalactoside) as inducer, the best conditions were: medium, LB; IPTG concentration, 0.1 mmol/L; inducing time, one and half hours . For the overexpression of PcoE using CuSO4 as inducer, the best conditions were: medium, LB; CuSO4 concentration, 2.0 mmol/L; inducing time, one and halfhours. Wei Sheng Wu Xue Bao, 1997 Aug, 37(4), 304 - 6 {Identification and expression of ribulose 1,5-bisphosphate carboxylase/oxygenase gene from Thiobacillus versutus}; Liu Z et al.; The chromosomal DNA of Thiobacillus versutus was hybridized with various heterologous ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene as probes . Only Rhodobacter sphaeroides form I RubisCO gene showed homology with T . versutus . The RubisCO gene fragment of T . versutus was isolated using the RubisCO gene of R . sphaeroides as a probe . And the RubisCO gene of T . versutus can express in E . coli cell. Wei Sheng Wu Xue Bao, 1997 Apr, 37(2), 87 - 94 {Nucleotide sequence of gltB gene encoding the large subunit of Rhodobacter sphaeroides glutamate synthase}; Lu T et al.; The complete nucleotide sequence of a 5.4-kb chromosomal EcoRI-SalI fragment was determined, which contains the structural gene (gltB) for the large subunit of Rhodobacter sphaeroides glutamate synthase, as well as the 5'- and 3'- flanking regions . A open reading frame of 4636 base pairs was identified as R . sphaeroides gltB gene . The MW of the large subunit, as deduced from the nucleotide sequence, was estimated as 164kD . Comparision of the nucleotide sequences revealed a high similarity among gltB genes of R . sphaeroides, Azospirillum brasilense and Escherichia coli . The deduced amino acid sequence of R . sphaeroides GltB showed a high similarity with that of A . brasilense GltB. Wei Sheng Wu Xue Bao, 1997 Feb, 37(1), 21 - 5 {Studies on selectivity of recognizing factor for rmf promoter}; Ding Q et al.; The truncted DNA template carrying rmf gene promoter was transcripted by E . coli RNA polymorase holoenzyme (E sigma) reconstituted with core enzyme (alpha 2 beta beta') and sigma 70 or sigma 38 in vitro . The initional site of the transcription of rmf was confirmed with different restriction endonuclease . rmf promoter can be recognized by E sigma 70 but not E sigma 38 . The suitable temperature for in vitro transcription was 37 degrees C, NaCl concentration was 50 mmol/L. Nucleic Acids Res, 1999 Jan 15, 27(2), 637 - 42 5S rRNA gene deletions cause an unexpectedly high fitness loss in Escherichia coli; Ammons D et al.; In Escherichia coli, ribosomal RNAs (16S, 23S and 5S) are co-transcribed in a highly regulated manner from seven genomically dispersed operons . Previous studies on the cellular effects of altered levels of two of these rRNAs (16S and 23S) have been useful in better understanding the regulation of rRNA expression . Furthering these studies, we have investigated the effect of 5S rRNA deficiencies on cell fitness through the sequential deletion of 5S rRNA genes . Our findings indicate that the loss of 5S rDNA from multiple genes decreases cell fitness more rapidly than loss of a similar number of 16S and 23S rRNA genes . These results suggest that the cell's innate ability to up-regulate rRNA operons does not compensate for 5S rRNA deficiencies, as was previously shown for 16S and 23S rRNAs . A plasmid-borne 5S rRNA gene is able to compensate for the deleted 5S rRNA genes. Nucleic Acids Res, 1999 Jan 15, 27(2), 596 - 600 Conserved sequence preference in DNA binding among recombination proteins: an effect of ssDNA secondary structure; Biet E et al.; Repetitive sequences have been proposed to be recombinogenic elements in eukaryotic chromosomes . We tested whether dinucleotide repeats sequences are preferential sites for recombination because of their high affinity for recombination enzymes . We compared the kinetics of the binding of the scRad51, hsRad51 and ecRecA proteins to oligonucleotides with repeats of dinucleotides GT, CA, CT, GA, GC or AT . Since secondary structures in single-stranded DNA (ssDNA) act as a barrier to complete binding we measured whether these oligonucleotides are able to form stable secondary structures . We show that the preferential binding of recombination proteins is conserved among the three proteins and is influenced mainly by secondary structures in ssDNA. Nucleic Acids Res, 1999 Jan 15, 27(2), 455 - 61 Pogo transposase contains a putative helix-turn-helix DNA binding domain that recognises a 12 bp sequence within the terminal inverted repeats; Wang H et al.; Pogo is a transposable element with short terminal inverted repeats . It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B . We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this . The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region . The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA . The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long . The inner copies occur as direct repeats, also with a single mismatch. Nucleic Acids Res, 1999 Jan 15, 27(2), 426 - 8 A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells; Piechaczek C et al.; We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions . Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells . It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure. Structure, 1998 Dec 15, 6(12), 1601 - 12 GDP-fucose synthetase from Escherichia coli: structure of a unique member of the short-chain dehydrogenase/reductase family that catalyzes two distinct reactions at the same active site; Somers WS et al.; Background: . In all species examined, GDP-fucose is synthesized from GDP-mannose in a three-step reaction catalyzed by two enzymes, GDP-mannose 4,6 dehydratase and a dual function 3, 5-epimerase-4-reductase named GDP-fucose synthetase . In this latter aspect fucose biosynthesis differs from that of other deoxy and dideoxy sugars, in which the epimerase and reductase activities are present as separate enzymes . Defects in GDP-fucose biosynthesis have been shown to affect nodulation in bacteria, stem development in plants, and are associated with the immune defect leukocyte adhesion deficiency type II in humans . Results: . We have determined the structure of GDP-fucose synthetase from Escherichia coli at 2.2 A resolution . The structure of GDP-fucose synthetase is closely related to that of UDP-galactose 4-epimerase and more distantly to other members of the short-chain dehydrogenase/reductase family . We have also determined the structures of the binary complexes of GDP-fucose synthetase with its substrate NADPH and its product NADP+ . The nicotinamide cofactors bind in the syn and anti conformations, respectively . Conclusions: . GDP-fucose synthetase binds its substrate, NADPH, in the proper orientation (syn) for transferring the 4-pro-S hydride of the nicotinamide . We have observed a single binding site in GDP-fucose synthetase for the second substrate, GDP-4-keto,6-deoxy-mannose . This implies that both the epimerization and reduction reactions occur at the same site in the enzyme . As is the case for all members of the short-chain family of dehydrogenase/reductases, GDP-fucose synthetase retains the Ser-Tyr-Lys catalytic triad . We propose that this catalytic triad functions in a mechanistically equivalent manner in both the epimerization and reduction reactions . Additionally, the X-ray structure has allowed us to identify other residues that are potentially required for substrate binding and catalysis. Structure, 1998 Dec 15, 6(12), 1517 - 27 Structures of escherichia coli CMP kinase alone and in complex with CDP: a new fold of the nucleoside monophosphate binding domain and insights into cytosine nucleotide specificity; Briozzo P et al.; Background: . Nucleoside monophosphate kinases (NMP kinases) catalyze the reversible transfer of a phosphoryl group from a nucleoside triphosphate to a nucleoside monophosphate . Among them, cytidine monophosphate kinase from Escherichia coli has a striking particularity: it is specific for CMP, whereas in eukaryotes a unique UMP/CMP kinase phosphorylates both CMP and UMP with similar efficiency . Results: . The crystal structure of the CMP kinase apoenzyme from E . coli was solved by single isomorphous replacement and refined at 1.75 A resolution . The structure of the enzyme in complex with CDP was determined at 2.0 A resolution . Like other NMP kinases, the protein contains a central parallel beta sheet, the strands of which are connected by alpha helices . The enzyme differs from other NMP kinases in the presence of a 40-residue insert situated in the NMP-binding (NMPbind) domain . This insert contains two domains: one comprising a three-stranded antiparallel beta sheet, the other comprising two alpha helices . Conclusions: . Two features of the CMP kinase from E . coli have no equivalent in other NMP kinases of known structure . Firstly, the large NMPbind insert undergoes a CDP-induced rearrangement: its beta-sheet domain moves away from the substrate, whereas its helical domain comes closer to it in a motion likely to improve the protection of the active site . Secondly, residues involved in CDP recognition are conserved in CMP kinases and have no counterpart in other NMP kinases . The structures presented here are the first of a new family of NMP kinases specific for CMP. Chem Biol, 1998 Dec, 5(12), 689 - 97 Strategies for protein-based nanofabrication: Ni2+-NTA as a chemical mask to control biologically imposed symmetry; Dabrowski MJ et al.; BACKGROUND: Technologies that improve control of protein orientation on surfaces or in solution, through designed molecular recognition, will expand the range of proteins that are useful for biosensors, molecular devices and biomaterials . A limitation of some proteins is their biologically imposed symmetry, which results in indistinguishable recognition surfaces . Here, we have explored methods for modifying the symmetry of an oligomeric protein that exhibits useful self-assembly properties . RESULTS: Escherichia coli glutamine synthetase (GS) contains 24 solvent-exposed histidines on two symmetry-related surfaces . These histidines drive a metal-dependent self-assembly of GS tubes . Immobilization of GS on the affinity resin Ni2+-NTA followed by on-column modification with diethyl pyrocarbonate affords asymmetrically modified GS that self-assembles only to the extent of 'short' dimeric GS tubes, as demonstrated by electron microscopy, dynamic light scattering and atomic force microscopy . The utility of Ni2+-NTA as a chemical mask was also demonstrated for asymmetric modification of engineered cysteines adjacent to the natural histidines . CONCLUSIONS: Current genetic methods do not provide distinguishable recognition elements on symmetry-related surfaces of biologically assembled proteins . Ni2+-NTA serves as a mask to control chemical modification in vitro of residues within symmetry-related pairs, on proteins containing functional His-tags . This strategy may be extended to modification of a wide range of amino acids with a myriad of reagents. Chem Biol, 1998 Dec, 5(12), 729 - 41 In vitro evolution of molecular cooperation in CATCH, a cooperatively coupled amplification system; Ellinger T et al.; BACKGROUND: One of the key issues in the investigation of evolution is how complex systems evolved from simple chemical replicators . Theoretical work proposed several models in which complex replicating systems are kinetically stabilized . The development of powerful isothermal amplification technique allows complex nucleic acid based evolving in vitro systems to be set up, which may then serve to verify experimentally current theories of evolution . Recently such a system based on the 3SR (self-sustained sequence replication) reaction has been established to investigate the evolution of cooperation: the trans-cooperatively coupled CATCH (cooperative amplification by cross hybridization) . RESULTS: Over four rounds of serial transfer, the cooperatively coupled two species CATCH system evolved into a more complex cooperative four species system, which then was overgrown by CATCH-derived RNA-Z-like hairpin species . In contrast to the classical RNA-Z species, these molecules have complementary loop sequences and self-amplify using a dual mechanism that includes concentration-dependent phases of noncooperative and cooperative amplification . CONCLUSIONS: The evolution of a cooperative system, under conditions that were alternately unfavorable and favorable for cooperative amplification, led to a system showing facultative cooperation . This principle of facultative cooperation preserves the complexity of the system investigated and could have general implications for the evolution and stabilization of cooperation under oscillating reaction conditions. Chem Biol, 1998 Dec, 5(12), 699 - 711 MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor; Matharu AL et al.; BACKGROUND: It has been proposed that Streptomyces malonyl CoA: holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis . Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs) . In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self-malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis . RESULTS: We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro . When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed . When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT . The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar AC:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex . CONCLUSIONS: The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration . There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question . MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP. J Immunol, 1998 Dec 15, 161(12), 7031 - 9 Molecular and immunologic characterization of a highly cross-reactive two EF-hand calcium-binding alder pollen allergen, Aln g 4: structural basis for calcium-modulated IgE recognition; Hayek B et al.; Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a lambda gt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen . rAln g 4 was overexpressed in Escherichia coli and purified to homogeneity . It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens . Exposure of two E . coli-expressed rAln g 4 fragments comprising amino acids 1-41 and 42-85 to patients' IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein . IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium . Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of alpha helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion . Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation . Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients . It may hence be useful for allergy diagnosis and for specific immunotherapy. J Natl Cancer Inst, 1998 Dec 16, 90(24), 1881 - 7 Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen; Carroll MW et al.; BACKGROUND: Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved . The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes . Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen . METHODS: We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination . A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E . coli lacZ (encodes beta-galactosidase, the model antigen), and E . coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene) . The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation . RESULTS: Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro . Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given . Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12 . CONCLUSION: This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer. Plant Mol Biol, 1998 Nov, 38(5), 889 - 97 Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms; Liu Z et al.; cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized . The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids . Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development . Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm . The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development . The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns . The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation . Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK . The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants. Plant Mol Biol, 1998 Nov, 38(5), 827 - 38 Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain; Chye ML; Acyl-CoA binding proteins (ACBPs) are small (ca . 10 kDa) highly-conserved cytosolic proteins that bind long-chain acyl-CoAs . A novel cDNA encoding ACBP1, a predicted membrane protein of 24.1 kDa with an acyl-CoA binding protein domain at its carboxy terminus, was cloned from Arabidopsis thaliana . At this domain, ACBP1 showed 47% amino acid identity to Brassica ACBP and 35% to 40% amino acid identity to yeast, Drosophila, bovine and human ACBPs . Recombinant (His)6-ACBP1 fusion protein was expressed in Escherichia coli and was shown to bind 14{C}oleoyl-CoA . A hydrophobic domain, absent in the 10 kDa ACBPs, was located at the amino terminus of ACBP1 . Using antipeptide polyclonal antibodies in western blot analysis, ACBP1 was shown to be a membrane-associated glycosylated protein with an apparent molecular mass of 33 kDa . The ACBP1 protein was also shown to accumulate predominantly in siliques and was localized to the seed within the silique . These results suggest that the biological role of ACBP1 is related to lipid metabolism in the seed, presumably in which acyl-CoA esters are involved . Northern blot analysis showed that the 1.4 kb ACBP1 mRNA was expressed in silique, root, stem, leaf and flower . Results from Southern blot analysis of genomic DNA suggest the presence of at least two genes encoding ACBPs in Arabidopsis. Plant Mol Biol, 1998 Nov, 38(5), 775 - 83 A novel protein associated with citrus blight has sequence similarities to expansin; Ceccardi TL et al.; A protein associated with citrus blight (CB), a disease of unknown cause, was partially characterized . The 12 kDa protein, designated p12, is diagnostic of CB and is present in leaves and xylem fluid from roots and stems of CB-affected trees . The protein, and up to six other CB-specific proteins, are readily detected by SDS-PAGE of xylem fluid from CB-affected trees . The partial N-terminal amino acid sequence of p12 was found to be unique based on database searches . A cDNA library from CB-affected root cambium was screened with a 60 bp fragment, obtained by PCR amplification of cDNA with degenerate primers designed using the amino acid sequence of p12, and two clones were selected . These clones were sequenced revealing a 674 nucleotide cDNA with a 393 nt ORF which included sequence predicted by the N-terminal amino acid sequence of p12 . The amino acid sequence based on the p12 ORF was found to be up to 49% similar and 31% identical to expansins . Bacterial expression of the cloned ORF, which encodes an 11.8 kDa protein plus an N-terminal hydrophobic signal peptide, produced an immunoreactive protein of the expected size . By northern blot analysis, it was determined that p12 transcripts are present in root and stem cambium, but not in leaves of CB-affected trees, suggesting transport of the protein to leaves . Southern hybridization analysis of citrus genomic DNA indicated that p12 is a citrus encoded protein. Plant Mol Biol, 1998 Nov, 38(5), 725 - 34 The presence of CYP79 homologues in glucosinolate-producing plants shows evolutionary conservation of the enzymes in the conversion of amino acid to aldoxime in the biosynthesis of cyanogenic glucosides and glucosinolates; Bak S et al.; A cDNA encoding CYP79B1 has been isolated from Sinapis alba . CYP79B1 from S . alba shows 54% sequence identity and 73% similarity to sorghum CYP79A1 and 95% sequence identity to the Arabidopsis T42902, assigned CYP79B2 . The high identity and similarity to sorghum CYP79A1, which catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, suggests that CYP79B1 similarly catalyses the conversion of amino acid(s) to aldoxime(s) in the biosynthesis of glucosinolates . Within the highly conserved 'PERF' and the heme-binding region of A-type cytochromes, the CYP79 family has unique substitutions that define the family-specific consensus sequences of FXP(E/D)RH and SFSTG(K/R)RGC(A/I)A, respectively . Sequence analysis of PCR products generated with CYP79B subfamily-specific primers identified CYP79B homologues in Tropaeolum majus, Carica papaya, Arabidopsis, Brassica napus and S . alba . The five glucosinolate-producing plants identified a CYP79B amino acid consensus sequence KPERHLNECSEVTLTENDLRFISFSTGKRGC . The unique substitutions in the 'PERF' and the heme-binding domain and the high sequence identity and similarity of CYP79B1, CYP79B2 and CYP79A1, together with the isolation of CYP79B homologues in the distantly related Tropaeolaceae, Caricaceae and Brassicaceae within the Capparales order, show that the initial part of the biosynthetic pathway of glucosinolates and cyanogenic glucosides is catalysed by evolutionarily conserved cytochromes P450 . This confirms that the appearance of glucosinolates in Capparales is based on a cyanogen 'predisposition' . Identification of CYP79 homologues in glucosinolate-producing plants provides an important tool for tissue-specific regulation of the level of glucosinolates to improve nutritional value and pest resistance. Mol Gen Genet, 1998 Nov, 260(2-3), 251 - 60 Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides; Velayos A et al.; Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway . These mutants were found to be deficient either in orotidine-5'-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity . Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation . To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M . circinelloides was isolated by direct complementation of E . coli . The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants . We therefore concluded that they were all altered at the same locus, the pyrF locus . The corresponding alleles were cloned and sequenced . Comparisons of the amino acid sequence of M . circinelloides OPRTase with those of E . coli and S . typhimurium revealed a high degree of similarity in secondary and tertiary structure . As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M . circinelloides mature protein can be assumed . This would also explain the interallelic complementation between some pyrF mutants . The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Mol Gen Genet, 1998 Nov, 260(2-3), 226 - 31 Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli; Xu SY et al.; The genes encoding the ApaLI (5'-GTGCAC-3'), NspI (5'-RCATGY-3'), NspHI (5'-RCATGY-3'), SacI (5'-GAGCTC-3'), SapI (5'-GCTCTTCN1-3', 5'-N4GAAGAGC-3') and ScaI (5'-AGTACT-3') restriction-modification systems have been cloned in E . coli . Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases . NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence . The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonucleases share significant similarity . 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion . External 5mC modification of a SacI site has no effect on SacI digestion . N4mC modification of the second base in the sequence 5'-GCTCTTC-3' blocks SapI digestion . N4mC modification of the other cytosines in the SapI site does not affect SapI digestion . N4mC modification of ScaI site blocks ScaI digetion . A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system . A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene. Mol Gen Genet, 1998 Nov, 260(2-3), 199 - 206 DnaK-dependent ribosome biogenesis in Escherichia coli: competition for dominance between the alleles dnaK756 and dnaK+; Sbai M et al.; The Escherichia coli chaperone DnaK is vital for many cellular functions, including ribosome biogenesis at high temperature . Thus, the dnaK756-ts (lambdaR) mutant, at the non-permissive temperature, is inhibited at a late stage of ribosome assembly, yielding 21S, 32S and 45S precursor particles . This defect, unlike the lambda resistance and thermosensitivity phenotypes, is not complemented by lysogenisation with a transducing phage lambda dnaK+ bearing the wild-type dnaK gene . However this dominant phenotype becomes recessive when dnaK+ is expressed from a medium-copy-number plasmid . On the other hand, an excess of DnaK causes an unexpected dominant-lethal effect of the dnaK756 allele near non-permissive temperatures . This interplay between the dnaK+ and dnaK756 alleles supports the idea of that DnaK oligomers form in the cell. Mol Gen Genet, 1998 Nov, 260(2-3), 131 - 43 The sugarless mutation affects the expression of the white eye color gene in Drosophila melanogaster; Benevolenskaya EV et al.; Investigation of a modifier locus displaying a darker eye phenotype in white-apricot flies led to the isolation of the gene encoding UDP-glucose dehydrogenase (UDPGDH) . The P-element insertion l(3)05007 occurs upstream of the transcription start site of the sugarless (sgl) gene and greatly reduces its transcription at various developmental stages . A single abundant sgl transcript shows a ubiquitous distribution and encodes a 53-kDa protein which is 64% identical in sequence to bovine UDP-glucose dehydrogenase . Overexpression of sgl in E . coli resulted in synthesis of a protein with high levels of UDPGDH activity . Expression of three genes that participate in pigment deposition, white, scarlet and brown, was significantly affected in populations segregating for sgl, suggesting that it is the decrease in UDPGDH level that produces the modifying effect observed . In addition, genetic effects on white-apricot were observed in sgl-wingless and sgl-hedgehog double mutants . Recent data have indicated an effect of UDPGDH on cell surface glycosaminoglycans (GAGs) which modulate the activity of growth factors, and in particular wingless signaling . Our results suggest that the levels of GAGs are rate limiting for cell-cell signaling pathways which mediate changes in gene expression. FEBS Lett, 1998 Nov 27, 440(1-2), 231 - 4 Expression of an oncogenic mutant G alpha i2 activates Ras in Rat-1 fibroblast cells; Edamatsu H et al.; It has been reported that expression of the active mutant of heterotrimeric GTP-binding protein alpha subunit G alpha i2 transforms Rat-1 cells . However, the G alpha i2-mediated mitogenic signaling pathways remain to be elucidated . Here, we demonstrate that inducible expression of the active mutant of G alpha i2 (G alpha i2(Q205L)) activates Ras and c-Jun N-terminal kinase (JNK) in addition to extracellular signal-regulated kinase (ERK) in Rat-1 cells . Our findings suggest that Ras may play a critical role in the G alpha i2-induced transformation and G alpha i2 can transduce signals from the Gi-coupled receptor to JNK and ERK in certain types of mammalian cells.
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