Escherichia coli

J Biol Chem, 1999 May 7, 274(19), 13403 - 9
Yeast methionine aminopeptidase I . Alteration of substrate specificity by site-directed mutagenesis; Walker KW et al.; In eukaryotes, two isozymes (I and II) of methionine aminopeptidase (MetAP) catalyze the removal of the initiator methionine if the penultimate residue has a small radius of gyration (glycine, alanine, serine, threonine, proline, valine, and cysteine) . Using site-directed mutagenesis, recombinant yeast MetAP I derivatives that are able to cleave N-terminal methionine from substrates that have larger penultimate residues have been expressed . A Met to Ala change at 329 (Met206 in Escherichia coli enzyme) produces an average catalytic efficiency 1.5-fold higher than the native enzyme on normal substrates and cleaves substrates containing penultimate asparagine, glutamine, isoleucine, leucine, methionine, and phenylalanine . Interestingly, the native enzyme also has significant activity with the asparagine peptide not previously identified as a substrate . Mutation of Gln356 (Gln233 in E . coli MetAP) to alanine results in a catalytic efficiency about one-third that of native with normal substrates but which can cleave methionine from substrates with penultimate histidine, asparagine, glutamine, leucine, methionine, phenylalanine, and tryptophan . Mutation of Ser195 to alanine had no effect on substrate specificity . None of the altered enzymes produced cleaved substrates with a fully charged residue (lysine, arginine, aspartic acid, or glutamic acid) or tyrosine in the penultimate position.

J Biol Chem, 1999 May 7, 274(19), 13229 - 34
Unusual sites of arginine methylation in Poly(A)-binding protein II and in vitro methylation by protein arginine methyltransferases PRMT1 and PRMT3; Smith JJ et al.; Arginine methylation is a post-translational modification found mostly in RNA-binding proteins . Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to contain NG, NG-dimethylarginine at 13 positions in its amino acid sequence . Two additional arginine residues were partially methylated . Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein . These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation . Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli were in vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, with S-adenosyl-L-methionine as the methyl group donor . Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites.

J Biol Chem, 1999 May 7, 274(19), 13002 - 9
Interactions between the molybdenum cofactor and iron-sulfur clusters of Escherichia coli dimethylsulfoxide reductase; Rothery RA et al.; We have used site-directed mutagenesis to study the interactions between the molybdo-bis(molybdopterin guanine dinucleotide) cofactor (Mo-bisMGD) and the other prosthetic groups of Escherichia coli Me2SO reductase (DmsABC) . In redox-poised preparations, there is a significant spin-spin interaction between the reduced Em,7 = -120 mV {4Fe-4S} cluster of DmsB and the Mo(V) of the Mo-bisMGD of DmsA . This interaction is significantly modified in a DmsA-C38S mutant that contains a {3Fe-4S} cluster in DmsA, suggesting that the {3Fe-4S} cluster is in close juxtaposition to the vector connecting the Mo(V) and the Em,7 = -120 mV cluster of DmsB . In a DmsA-R77S mutant, the interaction is eliminated, indicating the importance of this residue in defining the interaction pathway . In ferricyanide-oxidized glycerol-inhibited DmsAC38SBC, there is no detectable interaction between the oxidized {3Fe-4S} cluster and the Mo-bisMGD, except for a minor broadening of the Mo(V) spectrum . In a double mutant, DmsAS176ABC102SC, which contains an engineered {3Fe-4S} cluster in DmsB, no significant paramagnetic interaction is detected between the oxidized {3Fe-4S} cluster and the Mo(V) . These results have important implications for (i) understanding the magnetic interactions between the Mo(V) and other paramagnetic centers and (ii) delineating the electron transfer pathway from the {4Fe-4S} clusters of DmsB to the Mo-bisMGD of DmsA.

Hum Gene Ther, 1999 Apr 10, 10(6), 889 - 98
Augmentation of melanoma-specific gene expression using a tandem melanocyte-specific enhancer results in increased cytotoxicity of the purine nucleoside phosphorylase gene in melanoma; Park BJ et al.; The lineage-specific human tyrosinase promoter has been used to successfully target gene expression at the transcriptional level to melanoma cells . The tyrosinase promoter, alone and in combination with a single, or a dual, tandem melanocyte-specific enhancer, was used to regulate expression of the firefly luciferase reporter gene . Transient transfections of these tissue-specific luciferase constructs in human and murine melanoma (Pmel, B16mel) and colon carcinoma (WiDr, MC38) cell lines resulted in melanoma-specific luciferase expression that was amplified 5- and 500-fold with the addition of a single or double enhancer, respectively, to the tyrosinase promoter . When the double enhancer-promoter construct expressed the highly toxic Escherichia coli purine nucleoside phosphorylase (PNP) gene, transfection of the same cell lines followed by administration of the prodrug 6-methyl purine deoxyriboside (6-MPDR) at a concentration of 50 microM caused melanoma-specific in vitro cell killing . Within 5 days after prodrug administration methylthiazol-tetrazolium (MTT) cytotoxicity assays showed that only 15 and 9% of Pmel and B16mel cells, respectively, remained viable compared with controls . This effect was highly specific, as 90 and 96% of WiDr and MC38 colon carcinoma cells remained viable 5 days after identical treatment . This effect was a direct result of increased tissue-specific conversion of 6-MPDR to the toxic metabolite 6-methylpurine (6-MP), as documented by HPLC analysis of culture supernatants . These results show that the dual tandem melanocyte-specific enhancer provides powerful amplification of the transcriptional targeting of gene expression afforded by use of the tyrosinase promoter . This amplification translates into increased, highly specific cytotoxicity to melanoma by the PNP/6-MPDR enzyme/prodrug system and, therefore, has potential efficacy in the use of gene therapy for the treatment of metastatic melanoma.

Biol Chem, 1999 Mar, 380(3), 397 - 401
Site-specific fluorescence labelling of recombinant polyomavirus-like particles; Schmidt U et al.; For the development of gene therapy protocols based on polyomavirus-like particles, we describe a method for fluorescence labelling of virions in order to study virus-cell interactions preceding gene delivery . Site-specific fluorescence labelling of polyomavirus-like particles is achieved via a single cysteine residue and maleimide conjugates of fluorescence dyes (fluorescein, Texas Red) . Polyomavirus-like particles can be assembled in vitro from recombinant capsomers produced in E . coli . Since the assembly process is independent of disulfide bond formation, all cysteine residues of the wild-type protein were replaced by serines, and a new unique cysteine residue was introduced for the attachment of the fluorescence marker.

Biol Chem, 1999 Mar, 380(3), 381 - 6
Yeast cells allow high-level expression and formation of polyomavirus-like particles; Sasnauskas K et al.; Polyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy . Although VLPs can be generated in E . coli or insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans . We selected the yeast Saccharomyces cerevisiae for expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV) . Two entire HaPV VP1-coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels in Saccharomyces cerevisiae . The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid . Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.

Biol Chem, 1999 Mar, 380(3), 277 - 83
The core antigen of hepatitis B virus as a carrier for immunogenic peptides; Murray K et al.; The core antigen of hepatitis B virus (HBcAg) made in Escherichia coli yields particles that closely resemble the viral nucleocapsid . Extensive modifications can be made to the primary structure of HBcAg without impairing particle assembly . This enables other peptide sequences, including very long sequences, to be added, substituted, or inserted into the nucleocapsid subunit while retaining the ability to form highly immunogenic particles . These also retain the T cell epitopes of HBcAg and constitute powerful delivery systems for a diverse range of immunogenic epitopes and have significant potential for development of multicomponent vaccines.

Vet Microbiol, 1999 Mar 31, 66(1), 15 - 27
Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function; Oleksiewicz MB et al.; Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged as an important cause of reproductive disorders and pneumonia in domestic pigs throughout the world . Acute cytocidal replication of PRRSV in alveolar lung macrophages causes the acute pneumonia; however, it remains largely unresolved whether there may also be a predisposition to longer-term local immunodeficiency in the PRRSV-convalescent lung . We applied various flow cytometric techniques to study the interplay between PRRSV replication and macrophage viability/function in pure cultures of porcine alveolar lung macrophages . Monitored by flow cytometric detection of intracellular PRRSV nucleocapsid protein, acute (24 h post infection) PRRSV replication did not impede the ability of alveolar macrophages to ingest fluorescently labelled Escherichia coli . At 48 h post infection, PRRSV-induced cytotoxicity (quantitated by flow analysis of cell size and membrane integrity) led to 40% reduction in the total number of phagocytozing cells . However, viable/uninfected macrophages in PRRSV-infected cultures exhibited normal phagocytic ability at 48 h, indicating that no soluble phagocytosis-suppressive mediators were induced by PRRSV infection in this system . In short, in our minimal system containing only a single cell type, phagocytosis-suppressive effects of PRRSV infection were detected, that acted at the culture level by reducing the total number of alveolar lung macrophages.

J Mol Biol, 1999 Apr 16, 287(5), 845 - 51
The 5 A projection structure of the transmembrane domain of the mannitol transporter enzyme II; Koning RI et al.; The uptake of mannitol in Escherichia coli is controlled by the phosphoenolpyruvate dependent phosphotransferase system . Enzyme II mannitol (EIIMtl) is part of the phosphotransferase system and consists of three covalently bound domains . IICMtl, the integral membrane domain of EIIMtl, is responsible for mannitol transport across the cytoplasmic membrane . In order to understand this molecular process, two-dimensional crystals of IICMtl were grown by reconstitution into lipid bilayers and their structure was investigated by cryo-electron crystallography . The IICMtl crystals obey p22121 symmetry and have a unit cell of 125 Ax65 A, gamma=90 degrees . A projection structure was determined at 5 A resolution using both electron images and electron diffractograms . The unit cell contains two IICMtl dimers with a size of about 40 Ax90 A, which are oriented up and down in the crystal . Each monomer exhibits six domains of high density which most likely correspond to transmembrane alpha-helices and cytoplasmic loops .

Anal Biochem, 1999 May 1, 269(2), 403 - 9
Simultaneous determination of pyrimidine or purine deoxyribonucleoside triphosphates using a polymerase assay; Roy B et al.; In this paper, we describe an improved enzymatic assay for the determination of deoxyribonucleoside triphosphates (dNTPs) . This is based on the elongation of 32P 5'-end-labeled oligonucleotide primers annealed to complementary oligonucleotide templates . Incorporation within the primer/template (p/t) was catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I under conditions where the concentration of the dNTP to be analyzed is limiting . Using a combination of two different sized p/t pairs, dCTP and dTTP (or dATP and dGTP) were assayed together . Since the elongated products were clearly separated after electrophoresis on a denaturing 10% polyacrylamide gel, the two dNTPs could be quantified in a single lane . This method allows for the first time the simultaneous determination of two pyrimidine or two purine deoxyribonucleoside triphosphates . Consequently, a large number of biological samples can be tested in a single experiment . The high sensitivity of this method enables the quantification of low concentrations of dNTPs, such as those found in resting nondividing cells . Furthermore, this new protocol is well suited for the determination of dNTPs in cells treated with the antiretroviral ddI, since the Klenow fragment has a low affinity for ddATP, the active form of ddI .

Allergy, 1999 Feb, 54(2), 119 - 27
Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen; Wang NM et al.; BACKGROUND: Previously, we have identified several Per a 1 (Cr-PII) allergens from a deltagt22A cDNA library of Periplaneta americana . This study aimed to sequence clone C42 and determine its molecular and antigenic properties . METHODS: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21 . The recombinant proteins were purified by ion-exchange and affinity chromatographies . Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE . RESULTS: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50kDa) determined . The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients . Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen . Interestingly, these allergens all contain internal repeats, and the crude B . germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies . In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion . Overlapping peptides were then generated by expression of restriction enzyme fragments in E . coli . The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42 . CONCLUSIONS: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat . The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies.

Biofactors, 1999, 9(1), 27 - 36
Selenophosphate as a substrate for mammalian selenocysteine synthase, its stability and toxicity; Mizutani T et al.; The mechanism of selenocysteine synthesis on tRNASec in mammals was previously studied by means of HSe- as a Se donor to synthesize selenocysteine . It has been recently established that HSe- in E . coli is activated by ATP to become selenophosphate (SeP) . In this study, we provide evidence that {75Se}selenocysteine is produced by bovine selenocysteine synthase from Ser-tRNASec and {75Se}Sep, synthesized from elemental 75Se and Tris(trimethylsilyl)phosphite . We also studied the stability of SeP by NMR measurement . SeP was stable during storage under nitrogen at -80 degrees C for 3 months in 0.2 M Hepes buffer at pH 6.8 . However, SeP decomposed at 0 degree C in air (half life 32 hrs) or at 22 degrees C under nitrogen (half life 30 hrs) at pH 6.8 . The half lives of SeP at -19 degrees C in air and at 0 degree C under nitrogen at pH 6.8 were 740 and 840 hrs, respectively . At pH 4 under nitrogen at 22 degrees C, the half life was 240 hrs . The half life was only 9.2 hrs at pH 9 under nitrogen at 0 degree C . Thus, SeP was proved to be stable at low temperature, under acidic and anaerobic conditions, but labile under neutral and alkaline conditions . The LD50 of SeP administered i.p . to mice was 37.5 mg/kg body weight.

Anal Chem, 1999 Apr 15, 71(8), 1485 - 90
An integrated microfabricated device for dual microdialysis and on-line ESI-ion trap mass spectrometry for analysis of complex biological samples; Xiang F et al.; A microfabricated dual-microdialysis device in a single integrated microfabricated platform was constructed using laser micromachining techniques for the rapid fractionation and cleanup of complex biological samples . On-line dual microdialysis and ESI-MS of biological samples was demonstrated using an ion trap mass spectrometer . The mass spectra obtained demonstrated the efficiency of dual microdialysis for removing both high-molecular-weight and low-molecular-weight species that interfere with effective ESI-MS analysis of target biopolymers . Signal-to-noise ratios were also greatly improved compared to direct sample infusion . In addition to its compactness, negligible dead volume, and robustness, the device can be used at a flow rate of only 200 nL/min, an order of magnitude lower than that obtained previously . This reduced sample consumption and improved sensitivity with ESI-MS . The results suggest the potential for integration of such microfabricated devices with other sample manipulations for the rapid ESI-MS analysis of complex biological samples.

Pediatr Int, 1999 Apr, 41(2), 209 - 12
Factors influencing the development of hemolytic uremic syndrome caused by enterohemorrhagic Escherichia coli infection: from a questionnaire survey to in vitro experiment; Honda T; Enterohemorrhagic Escherichia coli (EHEC) is known as an etiological agent of not only hemorrhagic colitis, but also hemolytic uremic syndrome (HUS) and other serious complications . In this paper, a summary of the results, especially in relation to possible factors involving HUS complication, of a questionnaire survey conducted on the occasion of the massive EHEC outbreak in Sakai city in 1996, is presented . A brief review of the virulence factors and pathogenic mechanism involved in the development of HUS in EHEC infection is also described.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 211 - 5
Mutational analysis of the feedback sites of lysine-sensitive aspartokinase of Escherichia coli; Kikuchi Y et al.; In Escherichia coli, thrA, metLM, and lysC encode aspartokinase isozymes that show feedback inhibition by threonine, methionine, and lysine, respectively . In vitro chemical mutagenesis of the cloned lysC gene was used to identify residues and regions of the polypeptide essential for feedback inhibition by lysine . The isolated lysine-insensitive mutants were demonstrated to have missense mutations in amino acid residues 323-352, and at position 250 of aspartokinase III.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 175 - 82
Functional analysis of the hemK gene product involvement in protoporphyrinogen oxidase activity in yeast; Le Guen L et al.; The Escherichia coli hemK gene has been described as being involved in protoporphyrinogen oxidase activity; however, there is no biochemical evidence for this . In the context of characterizing the mechanisms of protoporphyrinogen oxidation in the yeast Saccharomyces cerevisiae, we investigated the yeast homolog of HemK, which is encoded by the ORF YNL063w, to find out whether it has any protoporphyrinogen oxidase activity and/or whether it modulates protoporphyrinogen oxidase activity . Phenotype analysis and enzyme activity measurements indicated that the yeast HemK homolog is not involved in protoporphyrinogen oxidase activity . Complementation assays in which the yeast HemK homolog is overproduced do not restore wild-type phenotypes in a yeast strain with deficient protoporphyrinogen oxidase activity . Protein sequence analysis of HemK-related proteins revealed consensus motif for S-adenosyl-methionine-dependent methyltransferase.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 111 - 6
Transformation of the nematode-trapping fungus Arthrobotrys oligospora; Tunlid A et al.; The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters . Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl2 treatment . Transformation frequencies varied between 1-6 transformants per microgram DNA . Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations . The addition of restriction enzymes during transformations increased the frequency of single copy integrations . Co-transformation, using the E . coli uidA gene encoding the beta-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 55 - 61
Characterization of enteroaggregative Escherichia coli isolates; Rich C et al.; Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes . Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction . None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon . Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E . coli 17.2 . No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E . coli 17.2.

Mol Pharmacol, 1999 May, 55(5), 873 - 82
Functional roles of aromatic residues in the ligand-binding domain of cyclic nucleotide-gated channels; Li J et al.; The ligand-binding domains of cyclic nucleotide-gated (CNG) channels show sequence homology to corresponding region(s) of the Escherichia coli catabolite gene-activator protein (CAP) and to the regulatory subunit of cAMP-dependent or cGMP-dependent protein kinases . The structure of CAP and that of a cAMP-dependent protein kinases regulatory subunit have been solved, prompting efforts to generate structural models for the binding domains in CNG channel . These models explicitly predicted that an aromatic residue in the CNG channel aligning with leucine 61 of CAP forms an interaction with the bound cyclic nucleotide . We tested this hypothesis by site-directed mutagenesis in a rat olfactory channel (rOCNC1) and a bovine rod photoreceptor channel (Brcng) . We found that mutations at this site had only weak effects that were not specific to the aromatic or the hydrophobic nature of the substituted residue . This result weakens the hypothesis of a strong or specific interaction at this site . We also separately mutated most of the other aromatic residues in the binding domain to alanine; most of these mutations resulted in channels that either did not function or had only minor changes in sensitivity . However, replacing tyrosine 565 with alanine (Y565A) in rOCNC1 increased agonist sensitivity by approximately 10-fold and resulted in prominent spontaneous activities . Y565 presumably lies between two alpha helices in the binding domain; one of these, the C helix, probably rotates during channel activation . The position of Y565 at the "hinge" between the C helix and another portion of the binding domain, and the consequences of Y565 mutations, strongly suggest that this portion of the binding domain is involved in channel gating processes.

Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 5227 - 32
Disruption of gene mg218 of Mycoplasma genitalium through homologous recombination leads to an adherence-deficient phenotype; Dhandayuthapani S et al.; Although the complete genome of Mycoplasma genitalium has been sequenced, the functional identification of various genes, including those involved in virulence, has not been accomplished . Further compounding these difficulties has been the failure to develop genetic tools in mycoplasmas that permit the assessment of gene and operon function and regulation . To determine whether homologous recombination could be developed as a tool to analyze the function of genes in M . genitalium, a plasmid that replicates in Escherichia coli but not in M . genitalium was constructed to disrupt the cytadherence-related gene mg218 of M . genitalium . The electroporation of this disruption plasmid into wild-type hemadsorption-positive (HA+) M . genitalium cells permitted the isolation of HA- (strain JB1) and partial HA+ (strains JB2 and JB20) transformants . Analysis of the transformants by Southern hybridization indicated that homologous recombination occurred at the mg218 locus by single-crossover events in JB1 and JB2 and by a double-crossover event in JB20 . While integration of the disruption construct abolished the expression MG218 in JB1, strains JB2 and JB20 exhibited a truncated MG218 protein (160 kDa), possibly because of in-frame fusion of the disrupted mg218 gene with sequences downstream of the gentamycin-resistance gene present in the disruption construct . Strain JB1, which lacked MG218, displayed a post-translational defect, being unable to maintain the structural integrity of the major adhesin P140 and its operon-related protein P110, in contrast to JB2 and JB20 . It appears that MG218 influences the stability of other cytadherence-related proteins in vivo . Thus, targeted gene disruption through homologous recombination will be a powerful and promising tool for investigating the biology and pathogenesis of M . genitalium.

Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 5221 - 6
Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin; Loregian A et al.; We report an intracellular peptide delivery system capable of targeting specific cellular compartments . In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1 . Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus . The peptide, designated Pol, is able to dissociate this interaction . The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex . When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication . Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments . The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus . The system we describe is suitable for delivery of peptides that specifically disrupt protein-protein interactions and may be developed to target specific cellular compartments.

Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 5089 - 94
Hypermutation in derepressed operons of Escherichia coli K12; Wright BE et al.; This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation . Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response) . A quantitative correlation was established between leuB mRNA abundance and leuB- reversion rates . To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter . When the leu operon was induced by isopropyl-D-thiogalactoside, both leuB mRNA abundance and leuB- reversion rates increased . These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon.

Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 4971 - 6
Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli; Raskin DM et al.; Accurate placement of the division septum at the midpoint of Escherichia coli cells requires the combined action of a general division inhibitor (MinC), a site-specific suppressor of division inhibition (MinE), and an ATPase (MinD) that is required for proper functioning of both MinC and MinE . We previously showed that a functional MinE-Gfp fusion accumulates in a ring structure at/near the middle of cells . Here we show that functional Gfp-MinD accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle imposed by MinE . The results indicate that MinD represents a novel type of dynamic cellular element in bacteria, with multiple roles in directing the division apparatus to the middle of the cell.

Biochemistry, 1999 Apr 27, 38(17), 5620 - 32
Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A; Gallinari P et al.; The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion . The serine protease activity is required for proteolytic processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B polyprotein cleavage sites . NS3 forms a complex with NS4A, a 54-residue polypeptide that was shown to act as an essential cofactor of the NS3 protease . We have expressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4A occurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complex with a sub-nanomolar dissociation constant . We have assessed the minimal ionic strength and detergent and glycerol concentrations required for maximal proteolytic activity and stability of the purified NS3-NS4A complex . Using a peptide substrate derived from the NS5A-NS5B junction, the catalytic efficiency (kcat/Km) of NS3-NS4A-associated protease under optimized conditions was 55 000 s-1 M-1, very similar to that measured with a recombinant complex purified from eukaryotic cells . Dissociation of the NS3-NS4A complex was found to be fully reversible . No helicase activity was exhibited by the purified NS3-NS4A complex, but NS3 was fully active as a helicase upon dissociation of NS4A . On the other hand, both basal and poly(U)-induced NTPase activity and ssRNA binding activity associated with the NS3-NS4A complex were very similar to those exhibited by NS3 alone . Therefore, NS4A appears to uncouple the ATPase/ssRNA binding and RNA unwinding activities associated with NS3.

Biochemistry, 1999 Apr 27, 38(17), 5563 - 71
Mutagenesis studies of the FeSII protein of Azotobacter vinelandii: roles of histidine and lysine residues in the protection of nitrogenase from oxygen damage; Lou J et al.; The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen . One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions . Herein we report the construction and characterization of five site-directed mutants of the FeSII protein (H12Q, H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after being individually overexpressed in Escherichia coli . These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein . The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the mixture to oxygen . In these assays, the H12Q mutant functioned as well as the wild type protein . However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much less efficient protection of nitrogenase . These results are consistent with pH titrations in both oxidation states, which show that His12 is insensitive to 2Fe-2S cluster oxidation state . His55's pK is weakly responsive to oxidation state, and the pK increase of 0 . 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner . Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability . The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions . These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.

Biochemistry, 1999 Apr 27, 38(17), 5521 - 7
Active sites of diacylglycerol kinase from Escherichia coli are shared between subunits; Lau FW et al.; We show that residues from different subunits participate in forming the active site of the trimeric membrane protein diacylglycerol kinase (DGK) from Escherichia coli . Five likely active-site mutants were identified: A14Q, N72S, E76L, K94L, and D95N . All five of these mutants possessed significantly impaired catalytic function, without evidence of gross structural alterations as judged by their similar near-UV and far-UV circular dichroism spectra . We found that mixtures of either A14Q or E76L with N72S or K94L possessed much greater activity than the mutant proteins by themselves, suggesting that Ala14 and Glu76 may be on one half-site while Asn72 and Lys94 are on another half-site . Consistent with the shared site model, we also found that (1) peak activity of A14Q and N72S subunit mixtures occur at equimolar concentrations; (2) the maximum activity of the A14Q and N72S mixture was 20% of the wild-type enzyme, in good agreement with the theoretical maximum of 25%; (3) the activity of mutant subunits could not be recovered by mixing with the wild-type subunits; (4) a double mutant, A14Q/N72S, bearing mutations in both putative half-sites was found to inactivate wild-type subunits; (5) the concentration dependence of inactivation by the A14Q/N72S mutant could be well described by a shared site model for a trimeric protein . Unexpectedly, we found that the single mutant D95N behaved in a manner similar to the double mutant, A14Q/N72S, inactivating wild-type subunits . We propose that Asp95 plays a role in more than one active site.

Biochemistry, 1999 Apr 27, 38(17), 5401 - 11
Assembly of the type 1 procollagen molecule: selectivity of the interactions between the alpha 1(I)- and alpha 2(I)-carboxyl propeptides; Alvares K et al.; Assembly of the heterotrimeric procollagen I molecule is initiated by interactions between the carboxyl propeptide domains of the completed nascent pro alpha chains . The {pro alpha 1(I)}2{pro alpha 2(I)} heterotrimer is the predominant molecule, with much smaller amounts of stable {pro alpha 1(I)}3 homotrimer also being formed . However, the {pro alpha 2(1)}3 homotrimer has not been detected, raising questions as to the mechanism of chain assembly and why {pro alpha2(1)}3 homotrimers are not formed . These questions have been examined here by expressing the intact and amino- or carboxyl-terminal truncated C-propeptides of the pro alpha chains recombinantly in bacteria and in a coupled transcription/translation reticulocyte lysate system . Their interactions were studied in vitro by binding analyses and in vivo by using the yeast two-hybrid system . The C-pro alpha 1(I) interacted with itself, and with C-pro alpha 2(I), as expected . Surprisingly, the C-pro alpha 2(I) also interacted with itself, both in vitro and in vivo . While the interaction of C-pro alpha 2(I) with itself and C-pro alpha 1(I) in vitro was strong, these interactions were weaker in vivo . Deletion of 36 amino acids from the C-terminal domain of C-pro alpha 1 had no effect on its binding to intact self or intact C-pro alpha 2, but the same deletion in C-pro alpha 2 completely abolished its binding to intact C-pro alpha 2 and to C-pro alpha 1 . Comparable N-terminal deletions in C-pro alpha 1 or C-pro alpha 2 diminished, but did not abolish, their binding to intact C-pro alpha 1 and C-pro alpha 2 . In the yeast two-hybrid system, C-pro alpha 2 interacted with itself more weakly than with C-pro alpha 1 . Molecular modeling and circular dichroism analyses showed that C-pro alpha 1 and C-pro alpha 2 have different folded structures and stability . Studies with antibodies specific to the C-pro alpha1 and alpha2 peptides showed them to precipitate different, specific, and distinct cell proteins from fibroblast lysates . The C-pro alpha 2(I) antibody complexed with more cell proteins . We hypothesize that the lack of pro alpha 2(I) homotrimers is not due to the inability of the C-pro alpha 2(I) to interact with itself, but rather to the competing presence of other cell proteins . The specificity of these interactions may reside in conformational differences in N- and C-terminal sequences of the two propeptides or in their different folding patterns.

Biochemistry, 1999 Apr 27, 38(17), 5337 - 45
The crystal structure of the Escherichia coli maltodextrin phosphorylase-acarbose complex; O'Reilly M et al.; Acarbose is a naturally occurring pseudo-tetrasaccharide . It has been used in conjunction with other drugs in the treatment of diabetes where it acts as an inhibitor of intestinal glucosidases . To probe the interactions of acarbose with other carbohydrate recognition enzymes, the crystal structure of E . coli maltodextrin phosphorylase (MalP) complexed with acarbose has been determined at 2.95 A resolution and refined to crystallographic R-values of R (Rfree) = 0.241 (0.293), respectively . Acarbose adopts a conformation that is close to its major minimum free energy conformation in the MalP-acarbose structure . The acarviosine moiety of acarbose occupies sub-sites +1 and +2 and the disaccharide sub-sites +3 and +4 . (The site of phosphorolysis is between sub-sites -1 and +1.) This is the first identification of sub-sites +3 and +4 of MalP . Interactions of the glucosyl residues in sub-sites +2 and +4 are dominated by carbohydrate stacking interactions with tyrosine residues . These tyrosines (Tyr280 and Tyr613, respectively, in the rabbit muscle phosphorylase numbering scheme) are conserved in all species of phosphorylase . A glycerol molecule from the cryoprotectant occupies sub-site -1 . The identification of four oligosaccharide sub-sites, that extend from the interior of the phosphorylase close to the catalytic site to the exterior surface of MalP, provides a structural rationalization of the substrate selectivity of MalP for a pentasaccharide substrate . Crystallographic binding studies of acarbose with amylases, glucoamylases, and glycosyltranferases and NMR studies of acarbose in solution have shown that acarbose can adopt two different conformations . This flexibility allows acarbose to target a number of different enzymes . The two alternative conformations of acarbose when bound to different carbohydrate enzymes are discussed.

Biochemistry, 1999 Apr 27, 38(17), 5290 - 5
The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins; Moenne-Loccoz P et al.; Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core . A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site . Recent stopped-flow absorption and rapid freeze-quench Mossbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation {Pereira, S . A., Small, W., Krebs, C., Tavares, P., Edmondson, D . E., Theil, E . C., and Huynh, B . H . (1998) Biochemistry 37, 9871-9876} . To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation . Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mossbauer conclusions . The frequency at 851 cm-1 is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm-1 is attributed, respectively, to nus and nuas of Fe-O2-Fe . Identification of the chromophore as a micro-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands . Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta9 stearoyl-acyl carrier protein desaturase (Delta9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein . Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.

Biol Pharm Bull, 1999 Mar, 22(3), 234 - 9
Molecular cloning, expression and characterization of a phenol sulfotransferase cDNA from mouse intestine; Tamura H et al.; A sulfotransferase (ST) cDNA was isolated from a mouse intestinal cDNA library using a probe which was generated by reverse transcription (RT)-PCR based on the conserved amino acid sequences of the ST molecules . The isolated cDNA (1.1 kb) contained an 858 bp open reading frame encoding a 286 amino acid polypeptide with molecular weight of 33439 . The deduced amino acid sequence shares 55.1% and 40.2% identity with mouse liver aryl/phenol (mSTp1) and alcohol (mSTa1 or mSTa2) STs, respectively, and it is highly similar to those of rat and human liver phenol ST (P-ST) isozymes, ST1B1 (87.8%) and ST1B2 (71.0%), respectively . RT-PCR analyses showed abundant expression of the P-ST mRNA in the intestine as well as in the liver in the mouse tissues examined (brain, heart, intestine, kidney, liver and lung) of both sexes . E . coli-expressed enzyme is capable of catalyzing the sulfation of 2-naphthol at Km = 3.3 microM and Vmax = 3.33 nmol/min/mg protein and also showed sulfation activity for L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine . Among food constituents tested, tannic acid and epigallocatechin gallate strongly inhibited the P-ST activity in vitro.

Scand J Immunol, 1999 Apr, 49(4), 431 - 40
Different Plasmodium falciparum recombinant MSP1(19) antigens differ in their capacities to stimulate in vitro peripheral blood T lymphocytes in individuals from various endemic areas; Garraud O et al.; This study reports on T-cell proliferative responses to the 19-kDa C-terminal domain of the Plasmodium falciparum merozoite surface protein (MSP1(19)) . Three different recombinant proteins were used: an Escherichia coli product expressing the first EGF-like domain and Saccharomyces cerevisiae and baculovirus/insect-cell-produced proteins containing both EGF-like domains, the latter protein being produced with or without N-glycosylation . Cell donors were P . falciparum-immune adults with no recent history of clinical malaria and recruited from three Senegalese settings with different epidemiological parasite transmission . Each mononuclear-blood-cell preparation was stimulated with a range of concentrations of the three proteins . Most subjects' mononuclear cells were reactive to at least one protein, but significant differences in lymphoproliferation were seen between the settings and within individual cultures depending on the protein source and concentration . Importantly, lymphoproliferation indices correlated inversely with the intensity of P . falciparum malaria transmission . When purified T lymphocytes were cultured in the presence of MSP1(19) plus autologous monocytes, B lymphocytes or a proposed CD1+ dendritic-cell population as costimulatory cells, significant differences were observed depending on the individual's previous exposure to parasites . This study shows that the stimulation of lymphocyte proliferation in vitro with MSP1(19) depends on several factors, including epidemiological conditions and protein preparations.

FEMS Immunol Med Microbiol, 1999 Mar, 23(3), 181 - 8
Phenotypic and genetic features of Escherichia coli strains showing simultaneous expression of localized and diffuse adherence; Scaletsky IC et al.; We have previously shown that some Escherichia coli strains isolated from children with diarrhea present the so-called 'localized and diffused adherence (LA/DA) pattern' in which both localized adherence (LA) and diffused adherence (DA) are expressed simultaneously . In the present study, we show that the LA adherence of these strains is genetically and phenotypically similar to that so far described for enteropathogenic E . coli (EPEC) as determined by DNA hybridization and electron microscopy . On the other hand, the DA is encoded by genes not homologous to the DAEC or AIDA-I DNA probes . In addition, the LA/DA strains are able to invade eukaryotic cells both in vitro and in vivo . In the rabbit ileal loop assay their invasion capacity goes beyond the enterocyte and reaches the muscularis mucosae as determined by transmission electron microscopy . These findings suggest that the LA/DA adherence pattern may be linked to a new E . coli virulence category which in the case of the strains studied may be associated to other virulence traits that enable them to more deeply invade the intestinal mucosa.

Clin Chem Lab Med, 1999 Feb, 37(2), 121 - 5
An ELISA for the H-subunit of human ferritin which employs a combination of rabbit poly- and mice monoclonal antibodies and an enzyme labeled anti-mouse-IgG; Vaisman B et al.; We describe a sensitive ELISA for measuring the H-type subunit of human ferritin . A high detection sensitivity was attained by the use of antibodies from different species and an enzyme-conjugated secondary antibody . It consisted of a sandwich assay using a solid phase coated with a rabbit polyclonal antibody for human ferritin from term placenta and a soluble monoclonal antibody for human H-ferritin, followed by a secondary anti-mouse immunoglobulin (Ig)G conjugated to beta-galactosidase . The assay was calibrated with purified recombinant human H-ferritin from E . coli . The colorigenic chlorophenol red beta-D-galactopyranoside and the fluorogenic 4-methyl-umbelliferyl-beta-D-galactopyranoside substrates were used with similar outcome . The described method permits the measurement of human H-ferritin at a concentration ranging from 0.1 to 100 micrograms/l (or 20-20,000 pg per 200 microliters sample) and is accurate at a concentration as low as 0.3 microgram/l . The coefficient of variation of the assay was 6.05-10.3% and the recovery of H-ferritin added to cell lysates was 105.8 +/- 7.52% . Depending on the H-ferritin content of the cell line tested, only 600 to 60,000 cells of different human cell lines were needed to measure their H-ferritin content.

Nucleic Acids Res, 1999 May 15, 27(10), 2227 - 34
Tetrahymena telomerase ribonucleoprotein RNA-protein interactions; Autexier C et al.; Telomerase is an enzyme that is essential for the replication and maintenance of chromosomal termini . It is a ribonucleoprotein consisting of a catalytic subunit, one or more associated proteins, and an integral RNA subunit that serves as a template for the synthesisof telomeric repeats . We identified a Tetrahymena telomerase RNA-protein complex by an electrophoretic mobility shift assay, using telomerase partially purified from whole cell extracts and radiolabeled, in vitro transcribed wild-type Tetrahymena telomerase RNA . Complex formation was specific as unlabeled Tetra-hymena telomerase RNA, but not Escherichia coli ribo-somal RNAs, competitively inhibited complex formation . Binding required concentrations of MgCl2of at least 10 mM and occurred over a wide range of potassium glutamate concentrations (20-220 mM) . The RNA-protein complex was optimally reconstituted with a 30 degrees C preincubation for </=5 min, prior to electrophoresis . Certain Tetrahymena telomerase RNAs containing deletions of structures and sequences previously predicted to be involved in protein binding were unable to competitively and specifically inhibit complex formation, suggesting a role in protein binding for the deleted residues or structures.

Bioorg Med Chem, 1999 Feb, 7(2), 401 - 9
Selective inhibition of beta-1,4- and alpha-1,3-galactosyltransferases: donor sugar-nucleotide based approach; Takayama S et al.; A combined rational and library approach was used to identify bisphosphonates (IC50 = 20 microM) and galactose type 1-N-iminosugar (IC50=45 microM) as novel motifs for selective inhibition of beta-1,4-galactosyltransferase (beta-1,4-GalT) and alpha-1,3-galactosyltransferase (alpha-1,3-GalT), respectively . Our results demonstrate that, though these two galactosyltransferases both utilize the same donor sugar-nucleotide (UDP-Gal), the difference in their mechanisms can be utilized to design donor sugar or nucleotide analogues with inhibitory activities selective for only one of the galactosyltransferases . Investigation of beta-1,4-GalT inhibition using UDP-2-deoxy-2-fluorogalactose (UDP-2-F-Gal), UDP, and bisphosphonates, also led to the observation of metal dependent inhibition of beta-1,4-GalT . These observations and the novel inhibitor motifs identified in this study pave the way for the design and identification of even more potent and selective galactosyltransferase inhibitors.

FEBS Lett, 1999 Mar 19, 447(1), 1 - 4
A single uridine modification at the wobble position of an artificial tRNA enhances wobbling in an Escherichia coli cell-free translation system; Takai K et al.; 5-Methoxyuridine was introduced into the first position of the anticodon of the unmodified form of tRNA(1Ser) from Escherichia coli . The codon reading efficiencies of this tRNA (tRNA(5-methoxyuridine UGA)) relative to those of the unmodified counterpart (tRNA(UGA)) were measured in a cell-free translation system . tRNA(5-methoxyuridine UGA) was more efficient than tRNA(UGA) in the reading of the UCU and UCG codons and was less efficient in the reading of the UCA codon . Thus, the single modification of U to 5-methoxyuridine can enhance the wobble readings.

FEBS Lett, 1999 Apr 9, 448(2-3), 257 - 60
A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases; Yamamoto Y et al.; A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced . Active inhibitor proteins were expressed in Escherichia coli using the cDNA . The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence . The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences . The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.

J Pediatr Orthop B, 1999 Apr, 8(2), 100 - 2
Complications of Küntscher intramedullary nailing in a child: a case report; Hosny GA et al.; A fracture of the femur in a 7-year-old boy who was treated with retrograde Kuntscher nailing is described . The follow-up period was 8 1/2 years . Deep infection, physeal injury of the femoral head, and trochanteric epiphysiodesis were serious consequences of the surgery.

Adv Enzymol Relat Areas Mol Biol, 1999, 73, 183 - 207
Solution structure and mechanism of the MutT pyrophosphohydrolase; Mildvan AS et al.; The MutT enzyme prevents errors in DNA replication by hydrolyzing mutagenic nucleotide substrates such as 8-oxo-dGTP . It does so by catalyzing nucleophilic attack at the electron rich P beta of nucleoside triphosphates . Members of this small mechanistic class of enzymes require two divalent cations per active site for activity--one coordinated by the enzyme and the other by the enzyme-bound NTP--and show low catalytic powers of 10(7)- to 10(9)-fold . The first structure of an enzyme of this class, obtained by NMR methods in solution, shows MutT to be a compact globular protein with an alpha + beta-fold . The binding of the essential divalent cation activator Mg2+ and the substrate analog Mg(2+)-AMPCPP to the MutT enzyme to form the quaternary E-Mg(2+)-AMPCPP-Mg2+ complex does not alter the global fold of the enzyme but produces localized small conformational changes at or near the metal and substrate binding sites . The adenine-ribose moiety binds in a hydrophobic cleft near 3-strands of a mixed beta-sheet, with the 6-NH2 group of the purine ring approaching the -NH2 side chain of Asn-119 . With a 6-keto group, GTP would interact more favorably with Asn-119, consistent with the substrate preference of MutT for guanine over adenine nucleotides . The enzyme-bound metal is coordinated by three conserved Glu residues (Glu-56, Glu-57, and Glu-98), the backbone carbonyl of a conserved Gly residue (Gly-38), and by two water ligands . The metal-triphosphate moiety of the metal-AMPCPP complex binds in the second coordination sphere of the enzyme-bound divalent cation . One of the water ligands of the enzyme-bound metal ion is well positioned to attack P beta with inversion and to be deprotonated or oriented by Glu-53, which in turn may be oriented by Arg-52 . Lys-39 is positioned to interact electrostatically with the alpha-phosphoryl group and thereby to facilitate the departure of the NMP-leaving group . Quantitatively, the 10(9)-fold rate acceleration produced by the MutT enzyme may be ascribed to catalysis by approximation and polarization of the attacking water by the enzyme-bound metal ion (> or = 10(5)-fold), activation of the NMP leaving group by Lys-39 (10-fold), charge neutralization at P beta by the nucleotide-bound divalent cation (> or = 10-fold), and orientation and/or deprotonation of the attacking water by Glu-53 (> or = 10(2)-fold) . This reaction mechanism, derived from the solution structure of the quaternary MutT complex, is both qualitatively and quantitatively consistent with the results of mutagenesis studies and may well be applicable to other enzymes that catalyze nucleophilic substitution at the electron-rich P beta of NTP substrates.

J Bacteriol, 1999 May, 181(9), 2973 - 8
The MobA-linked primase is the only replication protein of R1162 required for conjugal mobilization; Henderson D et al.; Cells newly transformed with plasmid R1162 DNA were used as donors in conjugal matings to determine if the plasmid replication genes are necessary for transfer . An intact system for vegetative replication is not required for transfer at normal frequency, but the plasmid primase, in the form linked to the nickase, must be present in donor cells.

J Bacteriol, 1999 May, 181(9), 2963 - 5
The C-terminal domain of dnaQ contains the polymerase binding site; Taft-Benz SA et al.; The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III) . Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit . We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain . Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core . The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit . In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit . A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant . The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.

J Bacteriol, 1999 May, 181(9), 2958 - 62
Expression of two glutathione S-transferase genes in the yeast Issatchenkia orientalis is induced by o-dinitrobenzene during cell growth arrest; Tamaki H et al.; Glutathione S-transferases (GSTs) Y-1 and Y-2 from the yeast Issatchenkia orientalis were purified by passage through a glutathione-agarose column, and the cDNA for GST Y-1 was cloned and sequenced . The deduced amino acid sequence consisted of 188 residues with a total calculated molecular mass of 21,001 Da and showed 36.7% identity to that of GST Y-2, another GST isoenzyme expressed in this strain . Escherichia coli DH5alpha transformed with pUC119 harboring the GST Y-1 gene under the control of the lac promoter exhibited 29-fold-higher GST activity than the same strain with pUC119 . Northern blot analysis revealed that both genes were highly expressed in cells cultured in the presence of 200 microM o-dinitrobenzene (DNB), one of the substrates of GST, while only the GST Y-1 gene was expressed, and only slightly, under normal (DNB-free) culture conditions . The DNB in the medium arrested cell growth until it was reduced by conjugation with reduced glutathione . Kinetic analysis of GST gene expression during detoxification of DNB revealed that the levels of expression of both genes were elevated within 3 h after the addition of DNB and that they further increased until 12 h postaddition . The levels of expression of both genes were decreased markedly when the DNB concentration in the culture medium was lowered . These results suggest that I . orientalis cells sense xenobiotics and arrest cell growth as a mechanism for preventing the induction of mutations by these compounds, while the levels of expression of the GST genes are up-regulated for detoxification.

J Bacteriol, 1999 May, 181(9), 2953 - 7
Adenosylcobalamin-mediated methyl transfer by toluate cis-dihydrodiol dehydrogenase of the TOL plasmid pWW0; Lee JY et al.; We identified and characterized a methyl transfer activity of the toluate cis-dihydrodiol (4-methyl-3,5-cyclohexadiene-cis-1, 2-diol-1-carboxylic acid) dehydrogenase of the TOL plasmid pWW0 towards toluene cis-dihydrodiol (3-methyl-4,5-cyclohexadiene-cis-1, 2-diol) . When the purified enzyme from the recombinant Escherichia coli containing the xylL gene was incubated with toluene cis-dihydrodiol in the presence of NAD+, the end products differed depending on the presence of adenosylcobalamin (coenzyme B12) . The enzyme yielded catechol in the presence of adenosylcobalamin, while it gave 3-methylcatechol in the absence of the cofactor . Adenosylcobalamin was transformed to methylcobalamin as a result of the enzyme reaction, which indicates that the methyl group of the substrate was transferred to adenosylcobalamin . Other derivatives of the cobalamin such as aquo (hydroxy)- and cyanocobalamin did not mediate the methyl transfer reaction . The dehydrogenation and methyl transfer reactions were assumed to occur concomitantly, and the methyl transfer reaction seemed to depend on the dehydrogenation . To our knowledge, the enzyme is the first dehydrogenase that shows a methyl transfer activity as well.

J Bacteriol, 1999 May, 181(9), 2922 - 9
The C-terminal sequence of the lambda holin constitutes a cytoplasmic regulatory domain; Blasi U et al.; The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic and acidic residues, with the overall net charge predicted to be positive . The C-terminal domain of lambda S was found to be cytoplasmic, as defined by protease accessibility in spheroplasts and inverted membrane vesicles . C-terminal nonsense mutations were constructed in S and found to be lysis proficient, as long as at least one basic residue is retained at the C terminus . In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis . However, the capacity of each truncated lytic allele for inhibition by the S107 inhibitor product of S is retained . The K97am allele, when incorporated into the phage context, confers a plaque-forming defect because its early lysis significantly reduces the burst size . Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis defect of the mutant SA52G, which causes lysis at or before the time when the first phage particle is assembled in the cell . This mutation scrambles the C-terminal sequence of S, resulting in a predicted net charge increase of +4, and retards lysis by about 30 min, thus permitting a viable quantity of progeny to accumulate . Thus, the C-terminal domain is not involved in the formation of the lethal membrane lesion nor in the "dual-start" regulation conserved in lambdoid holins . Instead, the C-terminal sequence defines a cytoplasmic regulatory domain which affects the timing of lysis . Comparison of the C-terminal sequences of within holin families suggests that these domains have little or no structure but act as reservoirs of charged residues that interact with the membrane to effect proper lysis timing.

J Bacteriol, 1999 May, 181(9), 2878 - 82
DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli; Berardini M et al.; DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents . A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = {number of transformants from inter-HN2-pTZSV28}/{number of transformants from control}) was determined to be approximately 0.6 . Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination . (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB+ ( approximately 0 . 6) than in a DeltapolB (approximately 0.1) strain . Complementation with a polB+-containing plasmid restores RE to wild-type levels, which corroborates this conclusion . In separate experiments, E . coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA approximately uvrA . Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing, E . coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway . Furthermore, these results show that some cross-links are uniquely repaired by each pathway . This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E . coli . It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.

J Bacteriol, 1999 May, 181(9), 2872 - 7
Isoaspartate in ribosomal protein S11 of Escherichia coli; David CL et al.; Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its beta-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age . The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal alpha-linked aspartyl bonds and is thought to serve an important repair function in cells . Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E . coli proteins, a result predicted by the repair hypothesis . The present study demonstrates that this is indeed the case; E . coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase . Moreover, the distribution of isoaspartate-containing proteins in E . coli differed dramatically between logarithmic- and stationary-phase cultures . In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11 . The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.

J Bacteriol, 1999 May, 181(9), 2807 - 15
IncC of broad-host-range plasmid RK2 modulates KorB transcriptional repressor activity In vivo and operator binding in vitro; Jagura-Burdzy G et al.; The korAB operon of broad-host-range plasmid RK2 encodes five genes, two of which, incC and korB, belong to the parA and parB families, respectively, of genome partitioning functions . Both korB and a third gene, korA, are responsible for coordinate regulation of operons encoding replication, transfer, and stable inheritance functions . Overexpression of incC alone caused rapid displacement of RK2 . Using two different reporter systems, we show that incC modulates the action of KorB . Using promoter fusions to the reporter gene xylE, we show that incC potentiates the repression of transcription by korB . This modulation of korB activity was only observed with incC1, which encodes the full-length IncC (364 amino acids {aa}), whereas no effect was observed with incC2, which encodes a polypeptide of 259 aa that lacks the N-terminal 105 aa . Using bacterial extracts with IncC1 and IncC2 or IncC1 purified through the use of a His6 tail and Ni-agarose chromatography, we showed that IncC1 potentiates the binding of KorB to DNA at representative KorB operators . The ability of IncC to stabilize KorB-DNA complexes suggests that these two proteins work together in the global regulation of many operons on the IncP-1 genomes, as well in plasmid partitioning.

J Bacteriol, 1999 May, 181(9), 2765 - 72
Role of CIS in replication of an IncB plasmid; Praszkier J et al.; Replication of the IncB plasmid pMU720 requires the synthesis of the cis-acting RepA protein and the presence of two DNA elements, ori and CIS . CIS is the 166-bp sequence separating the RepA coding sequence from ori . To investigate how this organization of the pMU720 replicon contributes to the mechanism of initiation of replication, mutations in the sequence and/or the length of CIS were introduced into the CIS region and their effects on the efficiency of replication of the pMU720 replicon in vivo was determined . The CIS region was found to be composed of two domains . The repA-proximal domain, which showed strong transcription termination activity, could be replaced by equivalent sequences from I-complex and IncL/M plasmids, whose replicons are organized in the same fashion as pMU720 . Replacement by a trpA transcription terminator afforded only partial replication activity . The repA-distal domain was shown to be a spacer whose role was to position sequence(s) within ori on the correct face of the DNA helix vis-a-vis the repA-proximal portion of CIS . A model for the loading of RepA protein onto ori is discussed.

J Bacteriol, 1999 May, 181(9), 2759 - 64
In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress; Michan C et al.; Simultaneous expression of seven genes in Escherichia coli was measured by a reverse transcription-multiplex PCR fluorescence procedure . Genes studied were (i) oxyR (transcriptional regulator); (ii) katG, dps, gorA, and ahpCF (controlled by OxyR); (iii) sodA (controlled by SoxRS); and (iv) trxA (not related to OxyR or SoxRS) . Except for trxA, transcription of all genes was activated during the course of growth of wild-type bacteria, though notable variations were observed with respect to both the time and extent of activation . Whereas oxyR, katG, dps, and gorA were activated during exponential growth, ahpCF and sodA were stimulated in stationary phase . Maximal induction ranged from 4.6- to 86.5-fold, for gorA and dps, respectively . Treatment with H2O2 stimulated expression of the genes (katG, dps, ahpCF, and gorA) previously identified as members of the OxyR regulon, except for oxyR itself . Induction by H2O2 was a remarkably rapid and reversible process that took place in an OxyR-dependent and sigmaS-independent manner . NaCl induced expression of the genes controlled by OxyR, including the oxyR locus . This transcriptional up-regulation was preserved in a strain with the DeltaoxyR::kan mutation, but it was abolished (ahpCF) or significantly reduced (oxyR and dps) in a strain with the rpoS::Tn10 mutation, potentially reflecting positive transcriptional regulation of the oxyR regulon by sigmaS . Expression of trxA was not increased either by H2O2 stress or by a shift to high-osmolarity conditions.

J Bacteriol, 1999 May, 181(9), 2703 - 9
Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli; Anton AI et al.; The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection . The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study . The size of the gene was highly conserved (2902 to 2905 nucleotides) . Most polymorphic sites were clustered in five secondary-structure helices . Those regions in a large number of operons were sequenced, and several variations were found . Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed . Intercistronic variability was detected, although it seemed to be lower than for the rrs gene . The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected . The mode of variation of the rrl gene seems to be quite similar to that of the rrs gene . Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation . The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3' end.

J Bacteriol, 1999 May, 181(9), 2697 - 702
The global nitrogen regulator NtcA regulates transcription of the signal transducer PII (GlnB) and influences its phosphorylation level in response to nitrogen and carbon supplies in the Cyanobacterium synechococcus sp . strain PCC 7942; Lee HM et al.; The PII protein is encoded by a unique glnB gene in Synechococcus sp . strain PCC 7942 . Its expression has been analyzed in the wild type and in NtcA-null mutant cells grown under different conditions of nitrogen and carbon supply . RNA-DNA hybridization experiments revealed the presence of one transcript species 680 nucleotides long, whatever the nutrient conditions tested . A second transcript species, 620 nucleotides long, absent in the NtcA null mutant, was observed in wild-type cells that were nitrogen starved for 2 h under both high and low CO2 and in the presence of nitrate under a high CO2 concentration . Primer extension analysis indicated that the two transcript species are generated from two tandem promoters, a sigma70 Escherichia coli-type promoter and an NtcA-dependent promoter, located 120 and 53 nucleotides, respectively, from the glnB initiation codon . The NtcA-dependent promoter is up-regulated under the conditions mentioned above, while the sigma70 E . coli-type promoter displays constitutive levels of transcripts in the NtcA null mutant and slightly different levels in the wild-type cells, depending on the nitrogen and carbon supplies . In general, a good correlation between the amounts of the two transcript species and that of the PII protein was observed, as revealed by immunodetection with specific antibodies . The phosphorylation level of PII in the wild type is inversely correlated with nitrogen availability and directly correlated with higher CO2 concentration . This regulation is correspondingly less stringent in the NtcA null mutant cells . In contrast, the dephosphorylation of PII is NtcA independent.

J Bacteriol, 1999 May, 181(9), 2683 - 8
DnaA boxes are important elements in setting the initiation mass of Escherichia coli; Christensen BB et al.; The binding of DnaA protein to its DNA binding sites-DnaA boxes-in the chromosomal oriC region is essential for initiation of chromosome replication . In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass . The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the oriC region into Escherichia coli and by growing the strains at different generation times to obtain different plasmid copy numbers . In fast-growing cells, where the DnaA box plasmid copy number per oriC locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass . In slowly growing cells, where the DnaA box plasmid copy number per oriC locus was higher, we observed more pronounced increases in the initiation mass . Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in E . coli cells.

Nature, 1999 Apr 15, 398(6728), 579 - 85
Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein; Handa N et al.; The Sex-lethal (Sxl) protein of Drosophila melanogaster regulates alternative splicing of the transformer (tra) messenger RNA precursor by binding to the tra polypyrimidine tract during the sex-determination process . The crystal structure has now been determined at 2.6 A resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNA derived from the tra polypyrimidine tract . The two RNA-binding domains have their beta-sheet platforms facing each other to form a V-shaped cleft . The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognized by the protein . This structure offers the first insight, to our knowledge, into how a protein binds specifically to a cognate RNA without any intramolecular base-pairing.

Acta Crystallogr D Biol Crystallogr . 1999 May;55(5):1108.
Initiating a crystallographic study of UDP-galactopyranose mutase from escherichia coli . erratum
McMahon SA, Leonard GA, Buchanan LV, Giraud MF, Naismith JH.
In the paper by McMahon, Leonard, Buchanan, Giraud & Naismith {Acta Cryst . (1999) . D55, 399-402} an author's error has resulted in the fifth sentence of the Abstract being incorrect . The sentence should read 'They are monoclinic, space group P21, with unit-cell dimensions a = 71.12, b = 58.42, c = 96.38 A, beta = 96.38 degrees . 92% (native) and 94% (selenomethionine) complete data sets have been recorded to 2.9 A (Rmerge = 5.0%) and 3.0 A (Rmerge = 6.9%), respectively.'

Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1101 - 4
Expression, purification and crystallization of recombinant human TRAIL; Cha SS et al.; TRAIL (also known as Apo-2L) belongs to the tumour necrosis factor (TNF) cytokine family and induces rapid apoptosis in a wide variety of tumour cell lines upon binding to the death-signalling receptors on the cell membrane . Normal cells are resistant to TRAIL, owing to the expression of decoy receptors which lack functional death domains and antagonize TRAIL-induced apoptosis . Soluble and functional human TRAIL, expressed in Escherichia coli and refolded into a functional form, has been crystallized . The crystals belong to space group P63 with unit-cell dimensions a = b = 65.61, c = 131 . 70 A . The asymmetric unit contains two molecules of TRAIL, with a crystal volume per protein mass (Vm) of 2.41 A3 Da-1 and a solvent content of about 42% by volume . A native and a platinum-derivative data set to 2.8 and 3.5 A resolution, respectively, were obtained from frozen crystals . Structure determination by a combined molecular replacement and isomorphous replacement method is in progress.

Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1091 - 2
Crystallization and preliminary X-ray crystallographic analysis of the protease inhibitor ecotin in complex with chymotrypsin; Lee CS et al.; Ecotin, a homodimeric protein composed of 142-residue subunits, is a novel protease inhibitor present in the periplasm of Escherichia coli . It shows a broad inhibitory specificity towards a group of serine proteases and binds two molecules of protease to form a tetrameric complex in a distinct chelation mechanism . The ecotin-chymotrypsin complex has been crystallized in the triclinic space group P1 with unit-cell parameters a = 57.29, b = 57.39, c = 79.75 A, alpha = 91.49, beta = 88.63 and gamma = 112.45 degrees . The asymmetric unit contains the whole tetrameric complex, consisting of two molecules of chymotrypsin bound to the ecotin dimer, with a corresponding crystal volume per protein mass (VM) of 2.58 A3 Da-1 and a solvent fraction of 48.9% . The crystals diffract beyond 2.0 A with Cu Kalpha X-rays and are very stable in the X-ray beam . Native X-ray data have been collected from a crystal to approximately 2.0 A Bragg spacing.

Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1069 - 73
Purification, crystallization and preliminary crystallographic studies of a two fibronectin type-III domain segment from chicken tenascin encompassing the heparin- and contactin-binding regions; Bisig D et al.; A fragment of chicken tenascin consisting of fibronectin type-III domains 5 and 6 has been expressed in Escherichia coli . After modifying a previously reported purification protocol, an electrophoretically homogeneous recombinant protein was obtained from which various crystal forms could be grown under identical conditions . Only one form was suitable for structure determination . These crystals belong to space group P21, with unit-cell parameters a = 45.2, b = 57.9, c = 72.2 A, beta = 91.4 degrees, and diffract to at least 2.6 A resolution using synchrotron radiation . From density measurements of the crystals, it was found that there are two molecules in the asymmetric unit . Diffraction data of native, two platinum-derivative and one palladium-derivative crystals were collected.

Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1046 - 7
Crystallization and preliminary diffraction analysis of Escherichia coli cysteinyl-tRNA synthetase; Newberry KJ et al.; Crystals of the 52 kDa monomeric Escherichia coli cysteinyl-tRNA synthetase complexed with ATP and cysteine have been grown by hanging-drop vapor diffusion from solutions containing ammonium sulfate as the precipitating agent . The crystals form long hexagonal rods in the space group P321 with unit-cell dimensions a = b = 82.3, c = 168.9 A . There is one enzyme molecule in the asymmetric unit . A complete native data set has been collected from a rotating-anode source to a resolution of 2.7 A at 103 K, with an Rmerge of 6.7%.

Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1086 - 8
Cubic crystals of chloramphenicol phosphotransferase from Streptomyces venezuelae in complex with chloramphenicol; Ellis J et al.; Chloramphenicol 3-O-phosphotransferase (CPT) from Streptomyces venezuelae ISP5230, a novel chloramphenicol-inactivating kinase, has been overexpressed and purified using Escherichia coli as the heterologous host . Crystals of CPT in complex with its substrate chloramphenicol (Cm) were obtained which were suitable for X-ray diffraction . The crystals belong to the cubic space group I4132 with unit-cell dimension a = 200.0 A . The initial CPT crystals diffracted to 3.5 A and the diffraction was improved significantly upon adding acetonitrile and Cm to the crystallization drop . The CPT-Cm crystals diffract to at least 2.8 A resolution.

Vaccine, 1999 Apr 9, 17(15-16), 2089 - 95
DNA inoculation with a plasmid vector carrying the faeG adhesin gene of Escherichia coli K88ab induced immune responses in mice and pigs; Turnes CG et al.; pCB01, an eukaryotic expression vector, was constructed by cloning faeG, the gene that encodes the fimbrial adhesin of Escherichia coli K88ab, in pcDNA3 . Mice and swine were inoculated by the intramuscular route with different quantities of plasmid DNA to evaluate its capacity to induce an immune response . The immune response was monitored by ELISA and immunoblotting, using purified fimbriae and whole suspensions of fimbriated bacteria . Mice showed seroconversion 21 days after the inoculation of 8.9 microg and swine after the administration of 1100 microg of plasmid DNA . Seroconversions increased after successive reinoculations . Immunoblotting showed that sera collected 73 days after the first inoculation recognized exclusively a protein of 27 kDa present in purified fimbrial suspensions and in whole suspensions of E . coli K88ab . The immune response elicited by pCB01 was mainly due to IgG2a, while that elicited by a bacterin was due to IgG2b and IgG3 . Antibodies were still detected 14 months after the initiation of the immunization.

Vaccine, 1999 Apr 9, 17(15-16), 2020 - 9
Induction of immune responses in pigs following oral administration of purified F4 fimbriae; Van den Broeck W et al.; An effective way of stimulating the mucosal immune system was examined in piglets, using F4 fimbriae of enterotoxigenic Escherichia coli (ETEC) . It was demonstrated that purified F4 fimbriae, as opposed to ovalbumin (OVA), are powerful oral immunogens . Indeed, oral administration of purified F4 induced antigen-specific antibody-secreting cells (ASC) in the Peyer's patches, mesenteric lymph nodes (LN), blood and lamina propria 4, 7, 9 and 11 days postimmunization, respectively, indicating a stimulation of the mucosal immune system, whereas upon oral administration of OVA, no immune response was observed . Moreover, the induced F4-specific IgA and IgG antibody responses were comparable with those obtained upon oral infection with viable E . coli and intramuscular (i.m.) F4 injection, respectively . Furthermore, a priming of the mucosal immune system is better obtained by oral infection (ASC localized in mesenteric LN) than by i.m . F4 injection (ASC localized in spleen and retropharyngeal LN) since an oral boost with purified F4 induced a secondary response in the orally infected animal (mainly IgA and IgG ASC, rapid increase of IgA antibodies) while in the i.m . primed animal a secondary (more circulating antigen-specific ASC than in the unprimed animal) as well as a primary IgM and IgA response (mainly IgM ASC, slow increase of IgA antibodies), suggesting a primary mucosal response, were seen . An oral challenge of the naive control displayed a primary response (mainly IgM ASC, slow increase of IgA and IgG antibodies) . The capacity of purified F4 to activate the mucosal immune system on oral administration, is of importance for the development of oral vaccines against ETEC infections.

Microbiology, 1999 Mar, 145 ( Pt 3), 681 - 8
Multiple haem-utilization loci in Helicobacter pylori; Worst DJ et al.; To identify genes responsible for the utilization of haem as an iron source in Helicobacter pylori, a siderophore synthesis mutant of Escherichia coli was transformed with an ordered cosmid library of H . pylori NCTC 11638 . Four independent cosmids were found that were able to complement this mutant on iron-restrictive solid media containing different haem compounds as the sole source of iron . Hybridization experiments revealed that the four cosmids contained unrelated DNA fragments . No major differences were observed in the growth of the four transformants on iron-restrictive solid media to which different haem compounds had been added . None of the cosmids could confer the ability to use haem as an iron source to an E . coli aroB tonB mutant, which means that transport of iron and/or haem across the outer membrane requires a functional TonB protein . Further characterization of the cosmids revealed that one of them was also able to complement E . coli aroB hemA, indicating that the haem molecule is taken up as a whole by this haem-biosynthesis mutant . Expression of this haem-uptake system could not be repressed by excess iron . Another cosmid expressed two polypeptides in E . coli which were specifically immunoreactive with a polyclonal antiserum raised against whole cells of H . pylori . The production of these proteins appeared to be iron repressible . One of these proteins has the same molecular mass as a previously described 77 kDa haem-binding iron-repressible outer-membrane protein (IROMP) of H . pylori.

Microbiology, 1999 Mar, 145 ( Pt 3), 663 - 71
Conversion of dTDP-4-keto-6-deoxyglucose to free dTDP-4-keto-rhamnose by the rmIC gene products of Escherichia coli and Mycobacterium tuberculosis; Stern RJ et al.; dTDP-rhamnose is made from glucose-1-phosphate and dTTP by four enzymes encoded by rmIA-D . An Escherichia coli rmIC mutant was constructed and a crude enzyme extract prepared from it did not produce dTDP-4-keto-rhamnose, in contrast to a crude enzyme extract prepared from a wild-type E . coli strain where small amounts of this intermediate were found after incubation with dTDP-glucose in the absence of NADPH . These results showed that dTDP-4-keto-rhamnose, the product of RmIC, exists as a free intermediate . Further, the Mycobacterium tuberculosis rmIC gene was expressed and incubation of the resulting purified M . tuberculosis RmIC enzyme with dTDP-4-keto-6-deoxyglucose resulted in the conversion of approximately 7% of dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-rhamnose . The enzyme also allowed for the incorporation of two deuterium atoms from deuterium oxide solvent into dTDP-4-keto-glucose . Thus the rmIC gene encodes dTDP-4-keto-6-deoxyglucose epimerase capable of epimerizing at both C-3' and C-5'; this enzyme produces free dTDP-4-keto-rhamnose but the equilibrium of the 4-keto sugar nucleotides lies strongly on the side of the gluco configuration.

Microbiology, 1999 Mar, 145 ( Pt 3), 655 - 61
A theoretical and empirical investigation of the invasion dynamics of colicinogeny; Gordon DM et al.; A mathematical model describing the dynamics of a colicinogenic and a colicin-sensitive population propagated under serial transfer culture conditions was formulated . In addition, a series of in vitro invasion experiments using six representatives of the E colicin group was undertaken, together with the estimation of the growth rates and colicinogenic characteristics of the strains . Growth rates among the strains varied by up to 44% . There were 14-fold differences among strains in their lysis rates and there were up to 10-fold differences in the amount of colicin produced per lysed cell . The in vitro serial transfer invasion experiments revealed that regardless of initial frequency all colicinogenic strains succeeded in displacing the sensitive cell populations . The amount of time required for the colicin-sensitive cell population to be displaced declined as the initial frequency of the colicinogenic population increased and strains producing higher titres of colicin tended to displace the sensitive strain more rapidly . Overall, the observed dynamics of the invasion of colicinogenic strains was adequately described by the theoretical model . However, despite there being substantial differences among the strains in their growth rates and colicinogenic characteristics there were relatively few differences, observed or predicted, in the invasion dynamics of the six colicinogenic strains . These results suggest that the characteristics of different colicinogenic strains cannot be used to explain the extensive variation in the relative abundance of different colicins in natural populations of bacteria.

Microbiology, 1999 Mar, 145 ( Pt 3), 569 - 76
Role for the leucine-responsive regulatory protein (Lrp) as a structural protein in regulating the Escherichia coli gcvTHP operon; Stauffer LT et al.; The Escherichia coli glycine-cleavage enzyme system (gcvTHP and lpd gene products) provides C1 units for cellular methylation reactions . Both the GcvA and leucine-responsive regulatory (Lrp) proteins are required for regulation of the gcv operon . One model proposed for gcv regulation is that Lrp plays a structural role, bending the DNA to allow GcvA to function as either an activator or a repressor in response to environmental signals . This hypothesis was tested by replacing all but the upstream 22 bp of the Lrp-binding region in a gcvT::lacZ fusion with the I1A site from phage lambda . Integration host factor (IHF) binds the I1A site and bends the DNA about 140 degrees . Shifting the I1A site by increments of 1 base around the DNA helix resulted in IHF-dependent activation and repression of gcvT::lacZ expression that were face-of-the-helix dependent . Activation was also dependent on the GcvA protein, and repression was dependent on both the GcvA and GcvR proteins, demonstrating that the roles for these proteins were not altered . The results are consistent with Lrp playing primarily a structural role in gcv regulation, although they do not completely rule out the possibility that Lrp also interacts with another gcv-regulatory protein or with RNA polymerase.

FEBS Lett, 1999 Apr 1, 448(1), 19 - 22
RNA helicase activity of Semliki Forest virus replicase protein NSP2; Gomez de Cedron M et al.; Semliki Forest virus replicase protein nsP2 shares sequence homology with several putative NTPases and RNA helicases . NsP2 has RNA-dependent NTPase activity . Here we expressed polyhistidine-tagged nsP2 in Escherichia coli, purified it by metal-affinity chromatography, and used it in RNA helicase assays . RNA helicase CI of plum pox potyvirus was used as a positive control . Unwinding of alpha-32P-labelled partially double-stranded RNA required nsP2, Mg2+ and NTPs . NsP2 with a mutation, K192N, in the NTP-binding sequence GVPGSGK192SA could not unwind dsRNA and had no NTPase activity . This is the first demonstration of RNA helicase activity within the large alphavirus superfamily.

J Neurochem, 1999 May, 72(5), 2145 - 53
Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme; Nakashima A et al.; Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine . To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography . The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form . Although maximum velocities of all N-terminus-deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(L-erythro-1',2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahy dropteridine (6RBPH4) among the four enzymes . The iron contents incorporated into the three N-terminus-deleted mutants were significantly lower than that of wild type . However, there was no substantial difference in incorporated iron contents among the three mutants . The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form . Dopamine bound to the del-38 more than to the del-35 TH form . However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del-38 than from the del-35 TH form . These observations suggest that the amino acid sequence Gly36-Arg37-Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.

J Physiol Biochem, 1998 Sep, 54(3), 155 - 60
Functional expression of the rabbit intestinal Na+/L-proline cotransporter (IMINO system) in Xenopus laevis oocytes; Urdaneta E et al.; Proline absorption across small intestine takes place mainly through a Na+-dependent cotransporter localized at the brush border membrane of the enterocyte named IMINO system . It transports L-proline and 4-OH-proline but not L-alanine, neither cationic nor anionic amino acids . The present work demonstrates the functional expression of this transporter in Xenopus laevis oocytes by mRNA microinjection and radiotracer uptake techniques . Poly (A)+-RNA was isolated from rabbit jejunal mucosa and injected into oocytes . Five days after the injection, results showed 1.5 fold stimulation of 50 microM 3H-proline uptake by the injected oocytes when compared to the non injected oocytes uptake . Poly (A)+-RNA was sized fractionated and fractions were injected again . Increase on Na+-dependent L-proline uptake was obtained with a mRNA fraction between 2,4 and 4,4 kb, which was used to construct a cDNA library . The library was sequentially divided and cRNAs injected into oocytes in order to screen for an increment on the signal . A subdivision containing around 2,000 colonies was found to augment L-proline uptake 25 fold over the non injected oocytes uptake . This cRNA pool was used to further characterize the transporter . Results showed that in the absence of Na+ there was no L-proline uptake, 2 mM 4-OH-L-proline completely inhibited 50 microM proline uptake and there was no 50 microM alanine uptake . In summary, these results demonstrate the expression of the rabbit small intestine IMINO transporter in Xenopus laevis oocytes and support the next steps in the isolation of the clone.

Environ Mol Mutagen, 1999, 33(2), 132 - 43
Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice; Harbach PR et al.; Big Blue mice harbor a recoverable transgene in a lambda/LIZ shuttle vector . In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor-intensive color plaque assay . Applying a simpler assay {Jakubczak et al . (1996): Proc Natl Acad Sci USA 93:9073-9078}, we measured mutations in the lambda cII gene portion of the transgene . Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice . Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations . Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion . G:C --> A:T transitions were the predominant base substitution (78% of these at CpG sites) . The major mutation hotspot, a six G run and its 3' flanking T at bases 179 to 185, comprised 18.7% of the independent mutations . Other hotspots were positions 103, 196, and 212 . The in vivo cII spectrum had a significantly higher proportion of G --> A and G --> T mutations and fewer frameshifts than reported in vitro . The cII and published lacI spectra are similar, though G --> A transitions and deletions were fewer in the cII gene . The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts . We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations.

Int J Biochem Cell Biol, 1999 Jan, 31(1), 243 - 54
Conformational variability in Escherichia coli 70S ribosome as revealed by 3D cryo-electron microscopy; Agrawal RK et al.; During protein biosynthesis, ribosomes are believed to go through a cycle of conformational transitions . We have identified some of the most variable regions of the E . coli 70S ribosome and its subunits, by means of cryo-electron microscopy and three-dimensional (3D) reconstruction . Conformational changes in the smaller 30S subunit are mainly associated with the functionally important domains of the subunit, such as the neck and the platform, as seen by comparison of heat-activated, non-activated and 50S-bound states . In the larger 50S subunit the most variable regions are the L7/L12 stalk, central protuberance and the L1-protein, as observed in various tRNA-70S ribosome complexes . Difference maps calculated between 3D maps of ribosomes help pinpoint the location of ribosomal regions that are most strongly affected by conformational transitions . These results throw direct light on the dynamic behavior of the ribosome and help in understanding the role of these flexible domains in the translation process.

Mol Microbiol, 1999 Apr, 32(1), 193 - 202
The interdomain linker of Escherichia coli initiation factor IF3: a possible trigger of translation initiation specificity; de Cock E et al.; Initiation factor IF3 is responsible for the accuracy of translation initiation in bacteria, by destabilizing complexes involving non-initiator tRNA and/or nonstart codons . This proofreading is performed on the 30S subunit to which IF3 binds selectively . IF3 has an unusual architecture, with two globular domains connected by a mobile, positively charged linker . Here, we have investigated the function of this flexible tether by probing its conformation when IF3 is bound to the ribosomal RNA . Using site-directed mutagenesis of the linker region, we have also selectively modified its length, its flexibility and its chemical composition . The function of the mutant genes was assayed in vivo, and the structural and biochemical properties of some of the corresponding variant proteins were characterized in vitro . The two isolated domains of IF3 were also co-expressed in order to test the requirement for their covalent attachment . The results indicate that the physical link between the two domains of IF3 is essential for the function of this protein, but that the exact length and chemical composition of the linker can be varied to a large extent . A model is presented in which the extended linker would act as a 'strap', triggering a conformational change in the 30S subunit, which would then ensure initiator tRNA selection.

Mol Microbiol, 1999 Apr, 32(1), 159 - 68
Enzymatic and physiological properties of the tungsten-substituted molybdenum TMAO reductase from Escherichia coli; Buc J et al.; The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of +130 mV . We have successfully substituted the molybdenum with tungsten and obtained an active tungsto-TMAO reductase . Kinetic studies revealed that the catalytic efficiency of the tungsto-substituted TMAO reductase (W-TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo-TMAO reductase (Mo-TorA) . W-TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo-TorA . Most importantly, the W-TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates . The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.

Mol Microbiol, 1999 Apr, 32(1), 151 - 8
Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells; Hartland EL et al.; Enteropathogenic Escherichia coli (EPEC) induce characteristic attaching and effacing (A/E) lesions on epithelial cells . This event is mediated, in part, by binding of the bacterial outer membrane protein, intimin, to a second EPEC protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane . In this study, we have localized the intimin-binding domain of Tir to a central 107-amino-acid region, designated Tir-M . We provide evidence that both the amino- and carboxy-termini of Tir are located within the host cell . In addition, using immunogold labelling electron microscopy, we have confirmed that intimin can bind independently to host cells even in the absence of Tir . This Tir-independent interaction and the ability of EPEC to induce A/E lesions requires an intact lectin-like module residing at the carboxy-terminus of the intimin polypeptide . Using the yeast two-hybrid system and gel overlays, we show that intimin can bind both Tir and Tir-M even when the lectin-like domain is disrupted . These data provide strong evidence that intimin interacts not only with Tir but also in a lectin-like manner with a host cell intimin receptor.

Mol Microbiol, 1999 Apr, 32(1), 29 - 40
Lrp binds to two regions in the dadAX promoter region of Escherichia coli to repress and activate transcription directly; Zhi J et al.; The dadAX operon is expressed by multiple promoters that are repressed by leucine-responsive regulatory protein (Lrp) and activated by cyclic AMP-CRP . In previous work, we found that alanine or leucine acted as inducers to antagonize Lrp repression of the three major promoters directly . Here, we identify 11 Lrp binding sites located within 350 bp of dad DNA . A mutational analysis, coupled with in vivo and in vitro transcription experiments, indicated that Lrp sites that overlap the dad promoters were involved in repression . In contrast, sites upstream of the promoters did not appear to be necessary for repression, but were required for activation by Lrp plus alanine or leucine of one of the major dad promoters, P2 . This activation by alanine or leucine was not simply relief of repression, as P2 transcription from a constitutive template was increased fivefold compared with the basal level of transcription found in the absence of Lrp and the co-activator cyclic AMP-CRP . Alanine or leucine decreased the affinity of Lrp to repressor sites, while having little or no effect on the binding of Lrp to activator sites . This differential effect of alanine and leucine on Lrp binding helps to explain how these modifiers influence both repression and activation of the dad operon.

Surgery, 1999 Apr, 125(4), 421 - 30
Effect of lazaroid U-74389G and methylprednisolone on endotoxin-induced shock in mice; Fukuma K et al.; BACKGROUND: Lazaroids are nonglucocorticoid analogs of methylprednisolone with multiple actions . We investigated whether lazaroid U-74389G could attenuate endotoxin-induced liver injury . We hypothesized that U-74389G treatment may protect against hepatic injury by suppressing proinflammatory gene up-regulation through inhibition of activation of nuclear factor kappa B (NF-kappa B) . We also compared the efficacy of U-74389G with methylprednisolone in endotoxin-induced liver injury . METHODS: Lipopolysaccharide (Escherichia coli, 30 mg/kg given intraperitoneally) was administered to male ICR mice, and U-74389G (3 mg/kg intraperitoneally) or methylprednisolone (30 mg/kg intravenously) was administered simultaneously . Phosphate-buffered saline solution (0.15 mL intravenously) was administered to mice that served as a control group . RESULTS: U-74389G and methylprednisolone treatment significantly increased survival rates 48 hours after lipopolysaccharide injection and protected against lipopolysaccharide-induced liver injury in vivo, as indicated by the decreased hepatic lipid peroxidation, tumor necrosis factor-alpha, and inducible nitric oxide synthase messenger RNA formation, hepatic enzyme release, and neutrophil infiltration in the liver . U-74389G and methylprednisolone also showed inhibitory effects on NF-kappa B activation in the liver . CONCLUSIONS: These findings suggest that U-74389G can suppress proinflammatory gene up-regulation through inhibition of NF-kappa B activation and that it is a promising new antioxidant drug for the treatment of endotoxin shock.

Biochim Biophys Acta, 1999 Apr 19, 1438(1), 131 - 9
Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli; Qiao N et al.; Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter . When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies . The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid . Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively . In contrast, linoleic acid was a poor substrate for this enzyme . The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min . The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme . The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography . The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells . When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography . Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid . Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation . In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.

Gene, 1999 Apr 16, 230(2), 163 - 70
Interaction of DnaK with native proteins and membrane proteins correlates with their accessible hydrophobicity; de Crouy-Chanel A et al.; Molecular chaperones are involved in protein folding, protein targeting to membranes, and protein renaturation after stress . They interact specifically with hydrophobic sequences that are exposed in unfolded proteins, and buried in native proteins . We have studied the interaction of DnaK with native water-soluble proteins and membrane proteins . DnaK-native protein interactions are characterized by dissociation constants between 1 and 50 microM (compared with 0.01-1 microM for unfolded proteins) . This affinity is within the range of most intracellular protein concentrations, suggesting that DnaK interacts with a greater number of native proteins than previously suspected . We found a correlation between the affinity of native proteins for DnaK and their affinity for hydrophobic-interaction chromatography adsorbents, suggesting that DnaK interacts with exposed hydrophobic groups in native proteins . The interaction between DnaK and membrane proteins is characterized by DnaK's high affinity for detergent-solubilized membrane proteins, and its lower affinity for membrane proteins inserted in lipid bilayers, suggesting that the chaperone can interact with the hydrophobic sequences of the former, while it cannot penetrate the hydrophobic core of lipid bilayers . Thus, the specificity of DnaK for hydrophobic sequences is involved in its interaction with not only unfolded proteins, but also native water-soluble proteins and membrane proteins . All proteins interact with DnaK according to their exposed hydrophobicity.

Biochim Biophys Acta, 1999 Apr 21, 1411(1), 159 - 69
A catalytically active complex formed from the recombinant dI protein of Rhodospirillum rubrum transhydrogenase, and the recombinant dIII protein of the human enzyme; Peake SJ et al.; Transhydrogenase is a proton pump . It has three components: dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively, and dII spans the membrane . We have expressed dIII from Homo sapiens transhydrogenase (hsdIII) in Escherichia coli . The purified protein was associated with stoichiometric amounts of NADP(H) bound to the catalytic site . The NADP+ and NADPH were released only slowly from the protein, supporting the suggestion that nucleotide-binding by dIII is regulated by the membrane-spanning dII . HsdIII formed a catalytically active complex with recombinant dI from Rhodospirillum rubrum (rrdI), even in the absence of dII . The rates of forward and reverse transhydrogenation catalysed by this complex are probably limited by slow release from dIII of NADPH and NADP+, respectively . The hybrid complex also catalysed high rates of 'cyclic' transhydrogenation, indicating that hydride transfer, and exchange of nucleotides with dI, are rapid . Stopped-flow experiments revealed a rapid, monoexponential, single-turnover burst of reverse transhydrogenation in pre-steady-state . The apparent first-order rate constant of the burst increased with the concentration of rrdI . A deuterium isotope effect (kH/kD approximately 2 at 27 degrees C) was observed when {4B-1H}NADPH was replaced with {4B-2H}NADPH . The characteristics of the burst of transhydrogenation with rrdI:hsdIII differed from those previously reported for rrdI:rrdIII (J.D . Venning et al., Eur . J . Biochem . 257 (1998) 202-209), but the differences are readily explained by a greater dissociation constant of the hybrid complex . The steady-state rate of reverse transhydrogenation by the rrdI:hsdIII complex was almost independent of pH, but there was a single apparent pKa ( approximately 9.1) associated with the cyclic reaction . The reactions of the dI:dIII complex probably proceed independently of those protonation/deprotonation reactions which, in the complete enzyme, are associated with H+ translocation.

Eur J Biochem, 1999 May, 261(3), 689 - 97
Human high-Km 5'-nucleotidase effects of overexpression of the cloned cDNA in cultured human cells; Rampazzo C et al.; 5'-Nucleotidases participate, together with nucleoside kinases, in substrate cycles involved in the regulation of deoxyribonucleotide metabolism . Three major classes of nucleotidases are known, one on the plasma membrane and two in the cytosol . The two cytosolic classes have been named high-Km nucleotidases and 5'(3')-nucleotidases . Starting from two plasmids with partial sequences (Oka, J., Matsumoto, A., Hosokawa, Y . & Inoue, S . (1994) Biochem . Biophys . Res . Commun . 205, 917-922) we cloned the complete cDNA of the human high-Km nucleotidase into vectors suitable for transfection of Escherichia coli or mammalian cells . After transfection, E . coli overproduced large amounts of the enzyme . Most of the enzyme was present in inclusion bodies that also contained many partially degraded products of the protein . Part of the enzyme, corresponding to approximately 2% of the soluble proteins, was in a soluble active form . Stably transfected human 293 cells were obtained with a vector where the 3'-end of the nucleotidase coding sequence is linked to the 5'-end of the green fluorescent protein coding sequence . Several green clones overproduced both mRNA and fusion protein . Two clones with 10-fold higher enzyme activity were analyzed further . The nucleotidase activity of cell extracts showed the same substrate specificity and allosteric regulation as the high-Km enzyme . The growth rate of the two clones did not differ from the controls . The cells were not resistant to deoxyguanosine or deoxyadenosine, and did not show an increased ability to phosphorylate dideoxyinosine . Both ribonucleoside and deoxyribonucleoside triphosphate pools were decreased slightly, suggesting participation of the enzyme in their regulation.

Eur J Biochem, 1999 May, 261(3), 629 - 39
The structures of the carbohydrate backbones of the lipopolysaccharides from Escherichia coli rough mutants F470 (R1 core type) and F576 (R2 core type); Vinogradov EV et al.; The lipopolysaccharides (LPS) from Escherichia coli rough mutant strains F470 (R1 core type) and F576 (R2 core type) were deacylated yielding in each case a mixture of oligosaccharides with one predominant product which was isolated using high-performance anion-exchange chromatography . In addition, one oligosaccharide present in minor quantities was isolated from LPS of E . coli strain F576 (R2 core type) . The structures of the oligosaccharides were determined by chemical analyses and NMR spectroscopic experiments . Furthermore, de-O-acylated and dephosphorylated LPS preparations were investigated by fast-atom bombardment and collision induced dissociation tandem mass spectrometry . The combined data allow us to deduce the following carbohydrate backbones of the E . coli R1 and R2 core types which share the following structure (Scheme 1): but differ in the substituents R1 and R2 which for the R1 core type are predominantly: and to a minor extent: and for the R2 core type predominantly: and to a minor extent: in which all sugars are d-pyranoses (l,d-Hep, lglycerodmanno-heptopyranose; P, phosphate).

Eur J Biochem, 1999 May, 261(3), 610 - 7
Linear free-energy model description of the conformational stability of uracil-DNA glycosylase inhibitor A thermodynamic characterization of interaction with denaturant and cold denaturation; Reddy GB et al.; The equilibrium unfolding of uracil DNA glycosylase inhibitor (Ugi), a small acidic protein of molecular mass 9474 Da, has been studied by a combination of thermal-induced and guanidine hydrochloride (GdnCl)-induced denaturation . The analysis of the denaturation data provides a measure of the changes in conformational free energy, enthalpy, entropy and heat capacity DeltaCp that accompany the equilibrium unfolding of Ugi over a wide range of temperature and GdnCl concentration . The unfolding of Ugi is a simple two-state, reversible process . The protein undergoes both low-temperature and high-temperature unfolding even in the absence of GdnCl but more so in the presence of denaturant . The data are consistent with the linear free-energy model and with a temperature independent DeltaCp over the large temperature range of unfolding . The small DeltaCp (6.52 kJ.mol-1.K-1) for the unfolding of Ugi, is perhaps a reflection of a relatively small, buried hydrophobic core in the folded form of this small monomeric protein . Despite a relatively low value of DeltaG(H2O), 7.40 kJ.mol-1 at pH 8.3, Ugi displays considerable stability with the temperature of maximum stability being 301.6 K.

Eur J Biochem, 1999 Apr, 261(2), 569 - 76
Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family; Timanova A et al.; A fatty acid-binding protein from the nematode Ascaridia galli was characterized . The gene was isolated and recombinantly expressed in Escherichia coli . According to the deduced amino acid sequence A . galli fatty acid-binding protein (AgFABP) belongs to the family of nematode polyprotein allergens, as shown by Western blotting and PCR analysis with genomic DNA and cDNA . Both native and recombinant proteins bind fatty acids and retinoids with high affinity . The fluorescent fatty acid analogue 11-{(5-dimethylaminonaphthalene-1-sulfonyl)amino} undecanoic acid (DAUDA) shows substantial changes in its emission spectrum when bound to AgFABP; this binding is reversed by fatty acids such as oleate . Moreover, changes of the intrinsic fluorescence of retinol and retinoic acid confirm retinoid binding activity of AgFABP . Fluorescence titration experiments with DAUDA indicate stoichiometric binding to a single binding site per monomer unit with affinities (Kd) of 1.6 and 1.8 x 10(-7) m for native and the recombinant protein, respectively . The apparent binding affinities of the nonfluorescent ligands were calculated in displacement experiments with DAUDA and values in the same range were obtained for myristic, palmitic, oleic, linoleic, arachidonic and retinoic acid . Additionally, the binding affinity of AgFABP for oleate and palmitate was determined by direct and indirect radiochemical analysis and the values obtained were similar to those from the fluorescent experiments . Both proteins show a preference for the binding of long-chain saturated and unsaturated fatty acids, but not for short chain (C3-C12) and branched fatty acids, cholesterol and tryptophan.

Eur J Biochem, 1999 Apr, 261(2), 509 - 17
Identification of a novel human adenylate kinase . cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein; Van Rompay AR et al.; Adenylate kinases have an important role in the synthesis of adenine nucleotides that are required for cellular metabolism . We report the cDNA cloning of a novel 22-kDa human enzyme that is sequence related to the human adenylate kinases and to UMP/CMP kinase of several species . The enzyme was expressed in Escherichia coli and shown to catalyse phosphorylation of AMP and dAMP with ATP as phosphate donor . When GTP was used as phosphate donor, the enzyme phosphorylated AMP, CMP, and to a small extent dCMP . Expression as a fusion protein with the green fluorescent protein showed that the enzyme is located in the cytosol . Northern blot analysis with mRNA from eight different human tissues demonstrated that the enzyme was expressed exclusively in brain, with two mRNA isoforms of 2.4 and 4.0 kb . The gene that encoded the enzyme was localized to chromosome 1p31 . Based on the substrate specificity and the sequence similarity with the previously identified human adenylate kinases, we have named this novel enzyme adenylate kinase 5.

Eur J Biochem, 1999 Apr, 261(2), 430 - 7
31P nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase; Evrard A et al.; SiR-FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has been analysed by 31P-NMR spectroscopy . This protein was reported previously as a reliable simplified model for native SiR-FP {Zeghouf, M., Fontecave, M., Macherel, D., & Coves, J . (1998) Biochemistry 37, 6117-6123} . SiR-FP60 was examined in its native form, as a complex with NADP+ and after monoelectronic reduction either with NADPH or dithionite . In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe . The paramagnetic effect of added manganese was also studied . In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase . Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR-FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface . This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle.

Eur J Biochem, 1999 Apr, 261(2), 354 - 60
The identity determinants required for the discrimination between tRNAGlu and tRNAAsp by glutamyl-tRNA synthetase from Escherichia coli; Sekine S et al.; We previously elucidated the major determinant set for Escherichia coli tRNAGlu identity (U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) and showed that the set is sufficient to switch the identity of tRNAGln to Glu {Sekine, S., Nureki, O., Sakamoto, K., Niimi, T., Tateno, M., Go, M., Kohno, T., Brisson, A., Lapointe, J . & Yokoyama, S . (1996) J . Mol . Biol . 256, 685-700} . In the present study, we attempted to switch the identity of tRNAAsp, which has a sequence similar to that of tRNAGlu, and consequently possesses many nucleotide residues corresponding to the Glu identity determinants (U35, C36, A37, G1*C72, and U11*A24) . A simple transplantation of the rest of the major determinants (U34, U2*A71, U13*G22**Alpha46, and Delta47) to the framework of tRNAAsp did not result in a sufficient switch of the tRNAAsp identity to Glu . To confer an optimal glutamate accepting activity to tRNAAsp, two other elements, C4*G69 in the middle of the acceptor stem and C12*G23**C9 in the augmented D helix, were required . Consistently, the two base pairs, C4*G69 and C12*G23, in tRNAGlu had been shown to exist in the interface with glutamyl-tRNA synthetase (GluRS) by phosphate-group footprinting . We also found the two elements in the framework of tRNAGln, and determined that their contributions successfully changed the identity of tRNAGln to Glu in the previous study . By the identity-determinant set (C4*G69 and C12*G23**C9 in addition to U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) the activity of GluRS was optimized and efficient discrimination from the noncognate tRNAs was achieved.

Br J Surg, 1999 Apr, 86(4), 514 - 7
Comparison of two closure techniques for the repair of experimental colonic perforations; Edwards DP et al.; BACKGROUND: Primary repair of penetrating wounds of the colon is gaining increasing acceptance in surgical practice . This study compared two techniques for the repair of experimental colonic perforations in the presence of peritonitis . METHODS: Multiple colonic perforations were created in the colon of 24 pigs . Following a 6-h delay the perforations were closed either by local excision and suture or by skin staples applied to the seromuscular layers of the colon . The repairs were assessed biomechanically and histologically for up to 14 days after surgery . RESULTS: All animals had diffuse peritonitis at the time of colonic repair . Stapled repairs were completed significantly faster than sutured repairs (mean(s.d.) 4.8(1.6) versus 30.7(4.0) min, P < 0.001) . Bio- mechanical evaluation of repairs revealed no significant differences between the two techniques . Histological examination of repairs closed by staples demonstrated more advanced healing compared with suture closure, on the basis of tissue apposition and inflammatory changes . CONCLUSION: Experimental colonic injuries may be treated successfully by primary repair in the presence of peritonitis . The use of skin staples for repair does not appear to prejudice colonic wound healing.

J Pharmacol Exp Ther, 1999 May, 289(2), 807 - 15
Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates; Reimer DL et al.; The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes) . A murine i.p . B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes . DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid) . The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency . Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors . Plasmid DNA delivery was estimated using {3H}-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis . Liposomal lipid delivery was estimated using {14C}-dioleoylphosphatidylethanolamine as a liposomal lipid marker . Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor . There was a tendency to observe enhanced transfection in small (<250 mg) tumors . Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p . administration . Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration . DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.

J Pharmacol Exp Ther, 1999 May, 289(2), 661 - 7
Competition between cytochrome P-450 isozymes for NADPH-cytochrome P-450 oxidoreductase affects drug metabolism; Li DN et al.; NADPH-cytochrome P-450 oxidoreductase (CPR) is essential for the catalytic activity of cytochrome P-450 (P-450) . On a molar basis, the amount of P-450 exceeds that of CPR in human liver . In this study, we investigated whether drug-drug interactions can occur as a result of competition between P-450 isozymes for this ancillary protein . For this purpose, combinations of P-450 isozymes were coexpressed together with P-450 reductase in Escherichia coli . We show that testosterone inhibited the CYP2D6-mediated bufuralol 1'-hydroxylase activity in bacterial membranes containing both CYP2D6 and CYP3A4 but not in membranes containing CYP2D6 alone . Conversely, bufuralol inhibited the CYP3A4-mediated testosterone 6beta-hydroxylase activity in bacterial membranes containing both CYP3A4 and CYP2D6 but not in membranes containing only CYP3A4 . In each case, inhibition was seen even at a P-450 to P-450 reductase ratio of 1.9:1, which is more favorable than the ratio of 4 reported for human liver . The physiological significance of this mechanism was demonstrated by the observation that testosterone inhibited several prototypical P-450 enzyme activities, such as bufuralol 1'-hydroxylase, coumarin 7-hydroxylase, and 7-ethoxyresorufin O-dealkylase, in human liver microsomes, but not if tested against a panel of bacterial membranes containing the human P-450 isozymes that mainly catalyze these reactions.

Biochem J, 1999 May 1, 339 ( Pt 3), 721 - 7
Thermodynamic and kinetic analysis of the Escherichia coli thioredoxin-C' fragment complementation system; Ghoshal AK et al.; Escherichia coli thioredoxin was cleaved with CNBr at its single Met residue at position 37, which lies in the middle of a long alpha-helix . The two fragments, 1-37 and 38-108, were purified and characterized by using CD and fluorescence spectroscopy . Both fragments lack structure at neutral pH and room temperature . The secondary and tertiary structural contents of the non-covalent complex formed on the mixing of the two peptide fragments are 47% and 35% of the intact protein respectively . The thermodynamics and kinetics of fragment association were characterized by titration calorimetry and stopped-flow fluorescence spectroscopy . Single phases were observed for both association and dissociation, with rate constants at 298 K of kon=4971+/-160 M-1.s -1 and koff=0 . 063+/-0.009 s-1 respectively . The ratio kon/koff was very similar to the binding constant determined by titration calorimetry, suggesting that binding is a two-state process . The values for DeltaCp, DeltaH0 and DeltaG0 at 298 K for dissociation of the complex were 5.7 kJ . mol-1.K-1, 45.3 kJ.mol-1 and 29.8 kJ.mol-1 respectively . The value for DeltaH0 was linearly dependent on temperature from 8-40 degrees C, suggesting that DeltaCp is independent of temperature . The values for DeltaCp and DeltaG0 are very similar to the corresponding values for the unfolding of intact thioredoxin at 25 degrees C . However, both DeltaH0 and DeltaS are significantly more positive for dissociation of the complex, suggesting a decreased hydrophobic stabilization of the complex relative to the situation for intact thioredoxin.

Biochem J, 1999 May 1, 339 ( Pt 3), 649 - 55
Identification of the amine-polyamine-choline transporter superfamily 'consensus amphipathic region' as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents . Role of Cys-300 in restoring thiol sensitivity to Gabp lacking Cys; Hu LA et al.; The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily . Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E . coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents . The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged inverted question mark2-sulphonatoethyl methanethiosulphonate or {2-(trimethylammonium)ethyl} methanethiosulphonate inverted question mark thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS) . The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate . These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally {Closs, Lyons, Kelly and Cunningham (1993) J . Biol . Chem . 268, 20796-20800} and the gab permease particularly {Hu and King (1998) Biochem . J . 300, 771-776}.

Biochem J, 1999 May 1, 339 ( Pt 3), 497 - 502
Aminopeptidase B is structurally related to leukotriene-A4 hydrolase but is not a bifunctional enzyme with epoxide hydrolase activity; Fukasawa KM et al.; Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contains the consensus sequence HEXXHX18E (324-347), conserved among the M1 family of metallopeptidases . To determine if these putative zinc-binding residues (His324, His328 and Glu347) and the active-site Glu325 are essential for the enzyme activity, we replaced the histidines with tyrosines and the glutamic acid residues with alanines using site-directed mutagenesis . The cDNAs were expressed in Escherichia coli, and the resulting recombinant proteins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogeneity . None of the expressed mutated proteins showed aminopeptidase activity . The E325A enzyme contained 1 mol of zinc per mol of protein, and the other three mutants, H324Y, H328Y and E347A, did not contain significant amounts of zinc, as determined by atomic absorption spectrometry . From sequence-homology searches, Ap B is known to be closely related to leukotriene (LT)-A4 hydrolase (EC 3.3.2.6) . We examined human placental Ap B and recombinant rat Ap B, both of which had been purified previously {Fukasawa, Fukasawa, Kanai, Fujii and Harada (1996) J . Biol . Chem . 271, 30731-30735}, to determine whether or not they had epoxide hydrolase activities . However, neither enzyme hydrolysed LTA4 into LTB4 . We then replaced some amino acids in the domain of the rat enzyme similar to the LTA4-binding site of LTA4 hydrolase . However, these mutants, Y408F, N409S and NE409-410SS also did not possess any epoxide hydrolase activity . We concluded that Ap B is an M1-family zinc metallopeptidase without epoxide hydrolase activity.

FEBS Lett, 1999 Mar 26, 447(2-3), 329 - 33
Involvement of a chloroplast homologue of the signal recognition particle receptor protein, FtsY, in protein targeting to thylakoids; Kogata N et al.; We isolated an Arabidopsis thaliana cDNA whose translated product shows sequence similarity to the FtsY, a bacterial homologue of SRP receptor protein . The Arabidopsis FtsY homologue contains a typical chloroplast transit peptide . The in vitro-synthesized 37 kDa FtsY homologue was imported into chloroplasts, and the processed 32 kDa polypeptide bound peripherally on the outer surface of thylakoids . Antibodies raised against the FtsY homologue also reacted with a thylakoid-bound 32 kDa protein . The antibodies inhibited the cpSRP-dependent insertion of the light-harvesting chlorophyll alb-binding protein into thylakoid membranes suggesting that the chloroplast FtsY homologue is involved in the cpSRP-dependent protein targeting to the thylakoid membranes.

FEBS Lett, 1999 Mar 26, 447(2-3), 269 - 73
Thioredoxin peroxidase in the Cyanobacterium Synechocystis sp . PCC 6803; Yamamoto H et al.; The amino acid sequence deduced from the open reading frame designated sll0755 in Synechocystis sp . PCC 6803 is similar to the amino acid sequences of thioredoxin peroxidases from other organisms . In the present study, we found that a recombinant SLL0755 protein that was expressed in Escherichia coli was able to reduce H2O2 and tertiary butyl hydroperoxide with thioredoxin from E . coli as the electron donor . Targeted disruption of open reading frame sll0755 in Synechocystis sp . PCC 6803 cells completely eliminated the H2O2-dependent and tertiary butyl hydroperoxide-dependent photosynthetic evolution of oxygen and the electron flow in photosystem II . These results indicate that the product of open reading frame sll0755 is a thioredoxin peroxidase whose activities are coupled to the photosynthetic electron transport system in Synechocystis sp . PCC 6803.

FEBS Lett, 1999 Mar 26, 447(2-3), 241 - 6
Biochemical analysis of interleukin-2 receptor beta chain phosphorylation by p56(lck); Delespine-Carmagnat M et al.; Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL-2 signaling and leads to recruitment of SH2 or PTB domain-containing proteins to the receptor . In this study, we have used subdomains of the IL-2 receptor beta chain (IL-2Rbeta) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56(lck), one of the critical tyrosine kinases activated by IL-2 . We report that recombinant p56(lck) phosphorylates in vitro tyrosine residues within the IL-2Rbeta chain but not those within the IL-2Rgamma chain . p56(lck) phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2Rbeta chain acidic subdomain . Interestingly, phosphorylation of Tyr-358 appears to require the presence of either Tyr-355 or Tyr-361 . p56(lck) also phosphorylates very efficiently the two tyrosines present in the IL-2Rbeta chain C-terminal region, Tyr-392 and Tyr-510 . We also investigated the association of p56(lck) with the IL-2Rbeta chain which was found to depend on a short stretch of the IL-2Rbeta chain acidic subdomain, and to be independent of the presence of its tyrosine residues.

FEBS Lett, 1999 Mar 26, 447(2-3), 144 - 8
Kup is the major K+ uptake system in Escherichia coli upon hyper-osmotic stress at a low pH; Trchounian A et al.; The K+ uptake was observed in washed cells of Escherichia coli, wild-type, upon hyper-osmotic stress at pH 5.5 when glucose was supplemented . This uptake had apparent a Km of 0.58 mM and Vmax of 0.10 micromol K+/min/mg protein . Such a K+ uptake was investigated using a mutant defective in Kdp and TrkA but with a functional Kup and a mutant defective in Kdp and Kup but having an active TrkA . The K+ uptake to reach the steady state level as well as the initial K+ influx rate in the first mutant were at least 3.5-fold greater than these values with the second mutant and similar to those of the wild-type . Such differences in the K+ uptake activity were correlated with K+ requirements for growth of these mutants . Moreover, the K+ uptake in the wild-type was blocked by a protonophore (carbonyl cyanide m-chlorophenylhydrazone) . Valinomycin, arsenate and N,N'-dicyclohexylcarbodiimide were not effective in changing the K+ uptake . It is suggested that Kup is the major K+ uptake system in E . coli upon hyper-osmotic stress at a low pH.

Biochimie, 1999 Jan-Feb, 81(1-2), 59 - 67
Base excision repair of 8-hydroxyguanine protects DNA from endogenous oxidative stress; Boiteux S et al.; A particularly important stress for all cells is the one produced by reactive oxygen species (ROS) that are formed as a byproduct of endogenous metabolism or the exposure to environmental oxidizing agents . An oxidatively damaged guanine, 8-hydroxyguanine (8-OH-G), is abundantly produced in DNA exposed to ROS . The biological relevance of this kind of DNA damage has been unveiled by the study of two mutator genes in E . coli, fpg and mutY . Both genes code for DNA glycosylases that cooperate to prevent the mutagenic effects of 8-OH-G . Inactivation of any of those two genes leads to a spontaneous mutator phenotype characterized by the exclusive increase in G:C to T:A transversions . In the simple eukaryote Saccharomyces cerevisiae, the OGG1 gene encodes an 8-OH-G DNA glycosylase which is the functional homolog of the bacterial fpg gene product . Moreover, the inactivation of OGG1 in yeast creates a mutator phenotype that is also specific for the generation of G:C to T:A transversions . The presence of such system in mammals has been confirmed by the cloning of the OGG1 gene coding for a human homolog of the yeast enzyme . Human cells also possess a MutY homolog encoded by the MYH gene . Analysis of the human OGG1 gene and its transcripts in normal and tumoral tissues reveals alternative splicing, polymorphisms and somatic mutations . The aim of this review is to summarize recent findings dealing with the biochemical properties and the biological functions of 8-OH-G DNA glycosylases in bacterial, yeast, insect and mammalian cells . These results point to 8-OH-G as an endogenous source of mutations and to its likely involvement in the process of carcinogenesis.

Mater Med Pol, 1997 Jan-Dec, 29(1-4), 14 - 6
Release of human monocytes protein C and the cofactor protein S in vitro; Giedrojc J et al.; Monocytes may contribute to a coagulation process by expression of the tissue factor and by synthesizing factors V, VII, X and XIII . Cultured blood monocytes also express both the tissue plasminogen activator and the plasminogen activator inhibitor I . The present study assesses the ability of human monocytes to secrete protein C and its cofactor protein S, which are potent inhibitors of the clotting cascade . Monocytes derived from the blood of healthy volunteers and prepared according to Boyum were cultured for up to 36 hours with or without lipopolisaccharide from Escherichia coli . After different times of incubation, the concentrations of proteins C and S in the supernatants were measured in order to determine synthesis of proteins, monocytes were cultured in the presence or absence of cycloheximide, the protein synthesis inhibitor . The concentration of protein C was estimated by means of the ELISA Protein C test (Boehringer Mannheim) . Protein S concentrations were measured by rocket immunoelectrophoresis according to Laurell, using monospecific antisera (American Diagnostica Inc., N.Y.) . The study showed that human monocytes, when stimulated by lipopolisaccharide, release proteins C and S in vitro . The concentration of these proteins in the culture supernatants markedly increased with time during the 36-hour observation . The supernatants obtained from the culture of unstimulated monocytes did not contain detectable quantities of the investigated proteins . The exposure of the cells to cycloheximide did not suppress the release of proteins C and S . In conclusion, our results suggest that monocytes are not able to synthesize proteins C and S . They can only release these factors . Furthermore, monocytes may be responsible for coagulation due to the inactivation of factors Va and VIIIa by protein C.

Acta Vet Hung, 1999, 47(1), 137 - 50
Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals; Morenkov OS et al.; Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed . The rec-gE-ELISA is based on the E . coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum . The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography . The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity . The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E . coli . The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions . Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%) . This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2) . We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.

Science, 1999 Apr 23, 284(5414), 611 - 5
Mechanism of intrinsic transcription termination and antitermination; Yarnell WS et al.; Gene expression is modulated by regulatory elements that influence transcription elongation by RNA polymerase: terminators that disrupt the elongation complex and release RNA, and regulators that overcome termination signals . RNA release from Escherichia coli RNA polymerase can be induced by a complementary oligonucleotide that replaces the upstream half of the RNA hairpin stem of intrinsic terminator transcripts, implying that RNA hairpins act by extracting RNA from the transcription complex . A transcription antiterminator inhibits this activity of oligonucleotides and therefore protects the elongation complex from destabilizing attacks on the emerging transcript . These effects illuminate the structure of the complex and the mechanism of transcription termination.

Biochemistry, 1999 Apr 20, 38(16), 5045 - 53
Solution structures of apo and holo biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy; Roberts EL et al.; A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro . The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy . That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP . In agreement with X-ray crystallographic studies {Athappilly, F . K., and Hendrickson, W . A . (1995) Structure 3, 1407-1419}, the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues . The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101 . However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1' . The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms . The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.

Philos Trans R Soc Lond B Biol Sci, 1999 Mar 29, 354(1383), 569 - 82
Historical overview of research on the tobacco mosaic virus genome: genome organization, infectivity and gene manipulation; Okada Y; Early in the development of molecular biology, TMV RNA was widely used as a mRNA {corrected} that could be purified easily, and it contributed much to research on protein synthesis . Also, in the early stages of elucidation of the genetic code, artificially produced TMV mutants were widely used and provided the first proof that the genetic code was non-overlapping . In 1982, Goelet et al . determined the complete TMV RNA base sequence of 6395 nucleotides . The four genes (130K, 180K, 30K and coat protein) could then be mapped at precise locations in the TMV genome . Furthermore it had become clear, a little earlier, that genes located internally in the genome were expressed via subgenomic mRNAs . The initiation site for assembly of TMV particles was also determined . However, although TMV contributed so much at the beginning of the development of molecular biology, its influence was replaced by that of Escherichia coli and its phages in the next phase . As recombinant DNA technology developed in the 1980s, RNA virus research became more detached from the frontier of molecular biology . To recover from this setback, a gene-manipulation system was needed for RNA viruses . In 1986, two such systems were developed for TMV, using full-length cDNA clones, by Dawson's group and by Okada's group . Thus, reverse genetics could be used to elucidate the basic functions of all proteins encoded by the TMV genome . Identification of the function of the 30K protein was especially important because it was the first evidence that a plant virus possesses a cell-to-cell movement function . Many other plant viruses have since been found to encode comparable 'movement proteins' . TMV thus became the first plant virus for which structures and functions were known for all its genes . At the birth of molecular plant pathology, TMV became a leader again . TMV has also played pioneering roles in many other fields . TMV was the first virus for which the amino acid sequence of the coat protein was determined and first virus for which cotranslational disassembly was demonstrated both in vivo and in vitro . It was the first virus for which activation of a resistance gene in a host plant was related to the molecular specificity of a product of a viral gene . Also, in the field of plant biotechnology, TMV vectors are among the most promising . Thus, for the 100 years since Beijerinck's work, TMV research has consistently played a leading role in opening up new areas of study, not only in plant pathology, but also in virology, biochemistry, molecular biology, RNA genetics and biotechnology.

Biochemistry, 1999 Apr 20, 38(16), 5222 - 31
Expression, purification, characterization, and reconstitution of the large and small subunits of yeast acetohydroxyacid synthase; Pang SS et al.; Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyzes the first step in the biosynthesis of the branched-chain amino acids . In bacteria, the enzyme has a large subunit containing the catalytic machinery and a small subunit with a regulatory role . In eucaryotes, the evidence for a regulatory subunit is largely indirect and circumstantial . We investigated the possibility that the yeast open reading frame YCL009c is an AHAS small subunit . Analysis of the DNA sequence shows that it contains all the appropriate transcription, translation and regulatory signals . YCL009c was shown to be expressed in yeast and the protein localized in mitochondria where it undergoes removal of a transit peptide targeting sequence . This putative small subunit protein (ilv6) and the catalytic subunit of yeast AHAS (ilv2) were each overexpressed in Escherichia coli and purified to near homogeneity . Reconstitution studies showed that the ilv6 protein stimulates the catalytic activity of the ilv2 protein by up to 7-fold (from 6.8 +/- 0.7 to 49.0 +/- 1.8 U/mg) and confers upon it sensitivity to inhibition by valine (Ki = 0.16 +/- 0.02 mM) . Valine inhibition is partially reversed by ATP . The reconstitution is favored by high concentrations of potassium phosphate ( approximately 1 M) and at neutral pH . Under optimal conditions for reconstitution, a dissociation constant for the subunits of 70 +/- 7 nM was determined . Valine inhibition is partial, resulting in a specific activity that is similar to that of the ilv2 protein alone . However, measurements of the Km for substrate rule out the possibility that valine inhibition is accomplished by dissociation of the subunits.

Biochemistry, 1999 Apr 20, 38(16), 5111 - 6
Zinc stabilizes the SecB binding site of SecA; Fekkes P et al.; The molecular chaperone SecB targets preproteins to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli . SecA recognizes SecB via its carboxyl-terminal 22 aminoacyl residues, a highly conserved domain that contains 3 cysteines and 1 histidine residue that could potentially be involved in the coordination of a metal ion . Treatment of SecA with a zinc chelator resulted in a loss of the stimulatory effect of SecB on the SecA translocation ATPase activity, while the activity could be restored by the addition of ZnCl2 . Interaction of SecB with the SecB binding domain of SecA is disrupted by chelators of divalent cations, and could be restored by the addition of Cu2+ or Zn2+ . Atomic absorption and electrospray mass spectrometry revealed the presence of one zinc atom per monomeric carboxyl terminus of SecA . It is concluded that the SecB binding domain of SecA is stabilized by a zinc ion that promotes the functional binding of SecB to SecA.

Biochemistry, 1999 Apr 20, 38(16), 5096 - 102
Replacements of basic and hydroxyl amino acids identify structurally and functionally sensitive regions of the mitochondrial phosphate transport protein; Briggs C et al.; The mitochondrial phosphate transport protein (PTP) from the yeast Saccharomyces cerevisiae has been expressed in Escherichia coli, purified, and reconstituted . Basic and hydroxyl residues were replaced to identify structurally and functionally important regions in the protein . Physiologically relevant unidirectional transport from extraliposomal (cytosol) pH 6.8 to intraliposomal (matrix) pH 8.0 was assayed . Replacements that affect transport most dramatically are at Lys42 (matrix end of helix A), Thr79 (helix B), Lys90 (cytosol end of helix B), Arg140 and Arg142 (matrix end of helix C), Lys179 and Lys187 (helix D), Ser232 (helix E), and Arg276 (helix F) . The deleterious nature of these mutations was confirmed by the observation that the yeast PTP null mutant transformed with any one of these mutant genes cannot grow or has difficulties growing with glycerol as the primary carbon source . More than 90% of transport activity can be blocked by various mutations without affecting growth on glycerol . Alterations in the structure of the transport protein caused by the mutations were characterized by determining the fraction of PTP incorporated into liposomes during reconstitution . The incorporation of all PTPs (wild type and mutant) into liposomes is 15.5 +/- 8.4 ng of PTP/25 microL and fairly independent of the amount of PTP in the initial reconstitution mix (49-212 ng of PTP/25 microL) . Arg159Ala and Lys295Gln show the smallest incorporation of 2.3 +/- 1.6 ng of PTP/25 microL and 2.6 +/- 0.2 ng of PTP/25 microL, respectively . Ser145Ala shows the largest incorporation of 37.0 ng of PTP/25 microL . These three mutants show near wild-type reconstituted transport activity . Two of these three mutations are located in the loop connecting the matrix ends of helices C and D, Ser145 at its N-terminal (the matrix end of helix C) and Arg159 near its center . Lys295 is located at the C-terminal of PTP beyond helix F . These results, together with those from other mutations, suggest that like helix A, the protein segment consisting of the loop connecting helices C and D and helix D as well as the C-terminal of PTP beyond helix F faces the subunit interface of this homodimer . The role of the replacement-sensitive residues in the phosphate or in the coupled proton transport path is discussed.

Biochemistry, 1999 Apr 20, 38(16), 5006 - 16
Outer membrane protein A of Escherichia coli inserts and folds into lipid bilayers by a concerted mechanism; Kleinschmidt JH et al.; Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea . In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time {Kleinschmidt, J . H., and Tamm, L . K . (1999) Biochemistry 38, 4996-5005} . Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilayers via three structurally distinct membrane-bound folding intermediates . To take full advantage of the TDFQ technique and to further dissect the folding pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein . All mutants refolded in vivo and in vitro and, as judged by SDS-PAGE, trypsin fragmentation, and Trp fluorescence, their refolded state was indistinguishable from the native state of OmpA . TDFQ analysis of the translocation across the lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30 degrees C, all Trps started from a far distance from the bilayer center and then gradually approached a distance of approximately 10 A from the bilayer center . In a narrow temperature range between 30 and 35 degrees C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center . When monitored at 40 degrees C, which resolved the last steps of OmpA refolding, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 A from the center after refolding was complete . In contrast Trp-7 approached the 10 A distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer . The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four beta-hairpins of the eight-stranded beta-barrel of OmpA were very similar to one another . This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate provide strong evidence for a synchronous translocation of all four beta-hairpins of OmpA across the lipid bilayer and suggest that OmpA inserts and folds into lipid bilayers by a concerted mechanism.

Biochemistry, 1999 Apr 20, 38(16), 4996 - 5005
Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding; Kleinschmidt JH et al.; The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli . This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process . Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers . Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center . The first folding intermediate is stable at 2 degrees C for at least 1 h . A second intermediate has been isolated at temperatures between 7 and 20 degrees C . The Trps move 4-5 A closer to the center of the bilayer at this stage . Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer . The final (native) structure is observed at higher temperatures of refolding . In this structure, the Trps are located on average about 9-10 A from the bilayer center . Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.

Biochemistry, 1999 Apr 20, 38(16), 4965 - 71
Different adaptations of the same peptide motif for tRNA functional contacts by closely homologous tRNA synthetases; Steer BA et al.; The N73 nucleotide at the end of the tRNA acceptor stem is commonly used by tRNA synthetases for discrimination . Because only a few synthetase-tRNA cocrystal structures have been determined, understanding of the molecular basis for N73 discrimination is limited . Here we investigated the possibility that, for at least some synthetases, the capacity to recognize different N73 nucleotides resides in the variable sequence of the loop of motif 2, a motif found in all class II enzymes . In the cocrystal of the class II yeast aspartyl-tRNA synthetase, atomic groups of the G73 discriminator of tRNAAsp interact with three side chains of the enzyme . We examined lysyl-tRNA synthetase, a close structural homologue of the aspartyl enzyme . Different substitutions were introduced into the Escherichia coli enzyme (A73 discriminator) to make its loop more like that of the human enzyme (G73 discriminator) . Our data show that the loop of motif 2 of the lysine enzyme makes tRNA functional contacts, as predicted from the structural comparison . And yet, the E . coli enzyme with the "humanized" loop sequence had the same quantitative kinetic preference for A73 versus G as the wild-type enzyme . We conclude that discriminator base selectivity in the lysine enzyme requires residues in addition to or other than those in the loop of motif 2 . Thus, even tRNA synthetases that are close structural homologues may use the same RNA binding element to make functional contacts with places (in the acceptor stem) that are idiosyncratic to each synthetase-tRNA pair.

Biochemistry, 1999 Apr 20, 38(16), 4948 - 57
Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases; Place C et al.; We have used UV-laser mediated cross-linking, DNase I footprinting and KMnO4 reactivity to probe the interaction between T7 RNA polymerase (RNAP) and a consensus promoter during the early stages of transcription . In a binary complex formed in the absence of substrate on a supercoiled plasmid, direct contacts were observed on the template (T) strand at positions -17, -5, and +3 and on the nontemplate (NT) strand at position -8 . These contacts lie within the DNase I cleavage footprint from positions -21 to +11 on the T strand and from positions -17 to +16 on the NT strand and straddle sites of enhanced reactivity of thymines to KMnO4 at position -3 on the T strand and position -2 on the NT strand . Use of supercoiled plasmid templates has allowed the mapping of contacts in the initiation region of the promoter in the binary complex for the first time . Upon addition of GTP, T7 RNAP enters a reiterative mode of synthesis, producing a ladder of poly(G) products . Under these conditions the downstream contact on the T strand switched from position +3 to +4 and +5 while the contact at position -17 was maintained . Under conditions in which the synthesis of transcription products is limited to 6-7 nucleotides, only the contact at position -17 on the T strand was preserved . A comparison of these results with the interaction of Escherichia coli RNA polymerase at the lac promoter reveals strong similarities in the manner in which these polymerases recognize their promoters.

Inflammation, 1999 Apr, 23(2), 131 - 9
Increased levels of transforming growth factor beta 1 and basic fibroblast growth factor in patients on CAPD: a study during non-infected steady state and peritonitis; Mlambo NC et al.; Long-term influence of continuous ambulatory peritoneal dialysis (CAPD) on concentrations of transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) in the peritoneal effluent, and the effect of peritonitis on these cytokines were investigated . TGF-beta1 and bFGF were assayed in effluent samples from dialysate bags collected during the initial week of treatment with CAPD and at 5 months . To determine the effect of peritonitis, dialysate bags were collected on admission to the hospital and on days 3 and 10 and also during non-infected steady state . Serum was drawn prior to infection and on days 1 and 10 . TGF-beta1 increased more than threefold during the longitudinal follow-up period, median concentrations of 35 pg/ml to 106 pg/ml (P<0.05) . No change in bFGF was seen during this initial 5 months . TGF-beta1 was increased on the first day of peritonitis (median concentration 169 pg/ml) and reached its maximum on day 3 of infection, (median concentration 216 pg/ml) (P<0.05 vs non-infected state, median concentration 39 pg/ml) . Basic FGF reached a maximum on day three of infection (median concentration 7.7 pg/ml; P=0.01 vs non-infected state) and then slowly declined . In conclusion, TGF-beta1 is influenced by CAPD treatment per se, and together with bFGF is increased during peritonitis, indicating its importance in the peritoneum and its potential involvement in the development of tissue fibrosis and eventually ultrafiltration failure.

J Inorg Biochem, 1999 Jan-Feb, 73(1-2), 1 - 5
Preparation, 195Pt NMR spectra and biological activity of platinum (IV) complexes with dipeptides; Watabe M et al.; Three dipeptide complexes of the form K{Pt(IV) (dipep) Cl(OH)2} and four dipeptide complexes of the form K{Pt(IV)-(Hdipep)Cl2(OH)2} were newly prepared . The 195 Pt NMR peak of the K{Pt(IV) (dipep)Cl(OH)2} complexes appeared at about 1200 ppm and these chemical shifts were about 3150 ppm downfield compared with those of the K{Pt(II) (dipep) Cl} complexes . The chemical shifts of the K{Pt(IV) (Hdipep) Cl2 (OH)2} complexes were at about 900 ppm, i.e., about 3050 ppm downfield compared with those of the K{Pt(II) (Hdipep)Cl} complexes . The H{Pt(IV) (Hdigly) Cl2(OH)2} and K{Pt(IV) (Hdigly) Cl2(OH)2} complexes inhibited the growth of C . albicans at a more diluted concentration than cisplatin at 1 microgram/ml, but the platinum complexes only weakly inhibited the growth of these cells compared with the cisplatin-inhibited growth of Meth-A and Hep-2 cells at 10 micrograms/ml . These results suggested that the platinum complexes selectively inhibited the growth of fungal cells.

J Biol Chem, 1999 Apr 30, 274(18), 12730 - 7
DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes; Boiziau C et al.; In vitro selection was performed in a DNA library, made of oligonucleotides with a 30-nucleotide random sequence, to identify ligands of the human immunodeficiency virus type-1 trans-activation-responsive (TAR) RNA element . Aptamers, extracted after 15 rounds of selection-amplification, either from a classical library of sequences or from virtual combinatorial libraries, displayed an imperfect stem-loop structure and presented a consensus motif 5'ACTCCCAT in the apical loop . The six central bases of the consensus were complementary to the TAR apical region, giving rise to the formation of RNA-DNA kissing complexes, without disrupting the secondary structure of TAR . The RNA-DNA kissing complex was a poor substrate for Escherichia coli RNase H, likely due to steric and conformational constraints of the DNA/RNA heteroduplex . 2'-O-Methyl derivatives of a selected aptamer were binders of lower efficiency than the parent aptamer in contrast to regular sense/antisense hybrids, indicating that the RNA/DNA loop-loop region adopted a non-canonical heteroduplex structure . These results, which allowed the identification of a new type of complex, DNA-RNA kissing complex, demonstrate the interest of in vitro selection for identifying non-antisense oligonucleotide ligands of RNA structures that are of potential value for artificially modulating gene expression.

J Biol Chem, 1999 Apr 30, 274(18), 12675 - 84
Kinetics and inhibition of recombinant human cystathionine gamma-lyase . Toward the rational control of transsulfuration; Steegborn C et al.; The gene encoding human cystathionine gamma-lyase was cloned from total cellular Hep G2 RNA . Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian cystathionine gamma-lyase overproduced in a bacterial system . About 90% of the heterologous gene product was insoluble, and renaturation experiments from purified inclusion bodies met with limited success . About 5 mg/liter culture of human cystathionine gamma-lyase could also be extracted from the soluble lysis fraction, employing a three-step native procedure . While the enzyme showed high gamma-lyase activity toward L-cystathionine (Km = 0.5 mM, Vmax = 2.5 units/mg) with an optimum pH of 8.2, no residual cystathionine beta-lyase behavior and only marginal reactivity toward L-cystine and L-cysteine were detected . Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and aminoethoxyvinylglycine . Propargylglycine inactivated human cystathionine gamma-lyase much more strongly than trifluoroalanine, in agreement with the enzyme's preference for C-gamma-S bonds . Aminoethoxyvinylglycine showed slow and tight binding characteristics with a Ki of 10.5 microM, comparable with its effect on cystathionine beta-lyase . The results have important implications for the design of specific inhibitors for transsulfuration components.

J Biol Chem, 1999 Apr 30, 274(18), 12488 - 98
Escherichia coli DNA helicase II is active as a monomer; Mechanic LE et al.; Helicases are thought to function as oligomers (generally dimers or hexamers) . Here we demonstrate that although Escherichia coli DNA helicase II (UvrD) is capable of dimerization as evidenced by a positive interaction in the yeast two-hybrid system, gel filtration chromatography, and equilibrium sedimentation ultracentrifugation (Kd = 3.4 microM), the protein is active in vivo and in vitro as a monomer . A mutant lacking the C-terminal 40 amino acids (UvrDDelta40C) failed to dimerize and yet was as active as the wild-type protein in ATP hydrolysis and helicase assays . In addition, the uvrDDelta40C allele fully complemented the loss of helicase II in both methyl-directed mismatch repair and excision repair of pyrimidine dimers . Biochemical inhibition experiments using wild-type UvrD and inactive UvrD point mutants provided further evidence for a functional monomer . This investigation provides the first direct demonstration of an active monomeric helicase, and a model for DNA unwinding by a monomer is presented.

J Biol Chem, 1999 Apr 30, 274(18), 12414 - 22
Regulation of tissue plasminogen activator activity by cells . Domains responsible for binding and mechanism of stimulation; Sinniger V et al.; A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics . We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays . To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of full-length glycosylated and nonglycosylated tPA (F-G-K1-K2-P), DeltaFtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P) . Deletion variants were made in Escherichia coli and were nonglycosylated . Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA . Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value . Loss of N-terminal domains as in DeltaFtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value . The protease domain alone was stimulated at very low levels of up to 2-fold . Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin . A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants . Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.

J Biol Chem, 1999 Apr 30, 274(18), 12339 - 45
Phospholipid-assisted refolding of an integral membrane protein . Minimum structural features for phosphatidylethanolamine to act as a molecular chaperone; Bogdanov M et al.; Escherichia coli-derived phosphatidylethanolamine (PE) or PE with fully saturated fatty acids was able to correct in vitro a defect in folding in the lipid-dependent epitope 4B1 of lactose permease (LacY) resulting from in vivo assembly in the absence of PE . PE plasmalogen, PE with two unsaturated fatty acids, and lyso-PE, which all do not favor bilayer organization, did not support proper refolding . Proper refolding occurred when these latter lipids were mixed with a bilayer-forming lipid (phosphatidylglycerol), which alone could not support refolding . L-Phosphatidylserine (PS; natural diastereomer) did support proper refolding . PE derivatives of increasing degrees of methylation were progressively less effective in supporting refolding, with phosphatidylcholine being completely ineffective . Therefore, the properties of nonmethylated aminophospholipids capable of organization into a bilayer configuration are essential for the recovery of the native state of epitope 4B1 after misassembly in vivo in the absence of PE . Neither D-PS (sn-glycero-1-phosphate backbone) nor P-D-S (D-serine in the head group) is competent in supporting proper refolding unless used in binary mixtures with phosphatidylglycerol . The detailed characterization of phospholipid-assisted refolding reported here further supports a specific rather than nonspecific role for PE in structural maturation of lactose permease in vivo (Bogdanov, M., and Dowhan, W . (1998) EMBO J . 17, 5255-5264).

J Biol Chem, 1999 Apr 30, 274(18), 12316 - 22
The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism; Hoidal HK et al.; The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate . The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks) . AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C . Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity . During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks . These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.

Protein Sci, 1999 Apr, 8(4), 921 - 9
A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation; Beckett D et al.; The Escherichia coli biotin holoenzyme synthetase, BirA, catalyzes transfer of biotin to the epsilon amino group of a specific lysine residue of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase . Sequences of naturally biotinylated substrates are highly conserved across evolutionary boundaries, and cross-species biotinylation has been demonstrated in several systems . To define the minimal substrate requirements in BirA-catalyzed biotinylation, we have measured the kinetics of modification of a 23-residue peptide previously identified by combinatorial methods . Although the sequence of the peptide bears little resemblance to the biotinylated sequence in BCCP, it is enzymatically biotinylated in vivo . Rates of biotin transfer to the 23-residue peptide are similar to those determined for BCCP . To further elucidate the sequence requirements for biotinylation, transient kinetic measurements were performed on a series of amino- and carboxy-terminal truncations of the 23-mer . The results, determined by stopped-flow fluorescence, allowed identification of a 14-residue peptide as the minimum required sequence . Additional support was obtained using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides that had been incubated with an excess of biotinyl-5'-adenylate intermediate and catalytic amounts of BirA . Results of these measurements indicate that while kinetically inactive truncations showed no significant shift in molecular mass to the values expected for biotinylated species, kinetically active truncations exhibited 100% biotinylation . The specificity constant (k(cat)/Km) governing BirA-catalyzed biotinylation of the 14-mer minimal substrate is similar to that determined for the natural substrate, BCCP . We conclude that the 14-mer peptide efficiently mimics the biotin acceptor function of the much larger protein domain normally recognized by BirA.

Protein Sci, 1999 Apr, 8(4), 731 - 40
In vitro evolution of thermostable p53 variants; Matsumura I et al.; The tumor suppressor p53 is conformationally unstable at physiological temperature . Even the activated p53delta30 variant, which lacks the self-inhibiting carboxy terminal domain, has a half-life of only 8 min at 37 degrees C in vitro . We have developed a genetic approach to identify p53 variants that stabilize the active conformation . The human p53delta30 gene was randomly mutated, and the resulting library was expressed in Escherichia coli under conditions that apparently denatured the parental protein . Stable p53 variants were identified based on their ability to specifically bind a p53 consensus site . The initial thermostable variants were randomly recombined by DNA shuffling, and substitutions that were functionally additive or synergistic were identified in a second more stringent round of screening . The DNA binding activity of N239Y/N268D/E336V p53delta30 variant has a half-life of 100 min at 37 degrees C, 12 times longer than that of the parental protein . The thermostable variants should be more amenable to crystallographic studies and more effective in gene therapies than the wild-type protein.

Br J Radiol, 1998 Oct, 71(850), 1083 - 5
Renal malacoplakia: an important consideration in the differential diagnosis of renal masses in the presence of Escherichia coli infection; Evans NL et al.; Renal malacoplakia is an uncommon condition with a variety of radiological characteristics which may initially suggest an alternative diagnosis . Three cases of renal malacoplakia were diagnosed in our hospital during a 2 year period . This apparent cluster of cases probably reflects the increased use of imaging and biopsy in the investigation of elderly hospitalized patients . It is important to make a definitive diagnosis as correct management may result in cure.

BJU Int, 1999 Mar, 83(4), 476 - 82
Monocyte tissue factor levels in patients with urological tumours: an association between tumour presence and progression; Lwaleed BA et al.; OBJECTIVE: To examine the hypothesis that increased monocyte tissue factor (mTF) levels may reflect urological tumour presence and progression . PATIENTS, SUBJECTS AND METHODS: Using a two-stage kinetic chromogenic assay, mTF levels were measured in 60 controls (normal subjects {60} and patients awaiting hernia repair or cholecystectomy {60}), patients with benign and malignant disease of the bladder (73), or prostate (81), and in patients with and without recurrent malignant disease of the bladder (30) . The levels were assessed under fresh resting conditions (baseline) and after incubation for 6 h without (unstimulated) and with (stimulated) Escherichia coli endotoxin . Each benign disease group was subdivided into inflammatory and non-inflammatory categories . RESULTS: Patients with bladder and prostate malignancy showed significantly higher mTF levels than did each control for baseline and stimulated cells . The benign inflammatory groups for both organs had significantly higher mTF levels than had each control for baseline cells . There was no difference between malignant and benign inflammatory groups . Stimulated mTF levels showed better discrimination between the study groups . The mTF levels were associated with histological tumour progression, serum prostate specific antigen level and static bone scan images . Levels were also higher in patients with bladder cancer recurrence than in those with a normal check cystoscopy . CONCLUSION: Stimulated mTF levels are raised in malignant and inflammatory disease compared with controls and patients with non-inflammatory conditions, and give maximal discrimination between these groups . mTF levels showed an association with tumour grade and other markers of tumour progression.

Protein Sci, 1999 Jan, 8(1), 122 - 36
Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between beta-galactosidase and other glycohydrolases; Juers DH et al.; Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer . Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues . A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank . Many structures include an alpha/beta barrel . Those that are most similar to the alpha/beta barrel of E . coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases . The structure comparison suggests that beta-amylase should also be included in this family . Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures . Domains 1, 2, and 4 of E . coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains . This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases . The fold of domain 1 of E . coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs . This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts . It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft . The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.

Protein Sci, 1999 Jan, 8(1), 65 - 74
Interaction of thioredoxins with target proteins: role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2-oxoacid dehydrogenase complexes; Bunik V et al.; The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated . The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes . Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide . Site-directed mutagenesis of D26, W31, F/W12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes . Sequences of 11 thioredoxin species tested biochemically are aligned . The thioredoxin residues at the contact between the alpha3/3(10) and alpha1 helices, the length of the alpha1 helix and the charges in the alpha2-beta3 and beta4-beta5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28) . The distribution of the charges on the surface of the thioredoxin molecules is analyzed . The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems . The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule . Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action . The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.

Brain Res Bull, 1999 Jan 1, 48(1), 89 - 92
Rapid expression of polymorphic ovine prion proteins and studies on their protease sensitivity; Dear DV et al.; We have used coupled and uncoupled in vitro transcription/translation to express rapidly aglycosyl ovine prion proteins from ovine genomic DNA genotyped for scrapie susceptible and nonsusceptible polymorphisms . Unlike previous in vitro studies of prion proteins, this method does not require cloning or laborious extractions . To our knowledge, this is the first report of ovine PrP expression at low (ng) levels under the control of an Escherichia coli promoter and ribosome binding site both coded for in the polymerase chain reaction primer . The rapidity of this approach could form the basis of a high throughput screening assay for PrP interactions, as proteins were expressed in a matter of hours from genomic DNA as the starting material . There was no difference observed in proteinase K sensitivity between prion translation products containing either scrapie susceptible or nonsusceptible polymorphisms.

Hum Gene Ther, 1999 Mar 20, 10(5), 787 - 800
Modulation of cell-mediated immunity prolongs adenovirus-mediated transgene expression in sciatic nerve; Jani A et al.; In a previous report, we demonstrated that a first-generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E . coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1 . Adenovirus-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral transgene expression over time . In our current work we have optimized adenoviral vector-mediated transgene expression after intraneural injection into sciatic nerve . Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old rats, decreases thereafter, and there is minimal associated tissue injury . In contrast, few lacZ-expressing Schwann cells are found in nerve of adult animals 10 days after injection, probably owing to immune clearance of virus-infected cells . Consistent with this notion, high levels of LacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult beta2-microglobulin-deficient mice (beta2M4-/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506 . In addition, adenovirus-infected Schwann cells cocultured with axons in vitro, in the absence of a host immune response, ensheathe axons and express lacZ for at least 8 weeks . These data thus demonstrate that lacZ transgene expression of first-generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity . Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Mol Microbiol, 1999 Mar, 31(6), 1853 - 61
Recruitment of ZipA to the division site by interaction with FtsZ; Liu Z et al.; ZipA is an essential cell division protein in Escherichia coli that is recruited to the division site early in the division cycle . As it is anchored to the membrane and interacts with FtsZ, it is a candidate for tethering FtsZ filaments to the membrane during the formation of the Z ring . In this study, we have investigated the requirements for ZipA localization to the division site . ZipA requires FtsZ, but not FtsA or FtsI, to be localized, indicating that it is recruited by FtsZ . Consistent with this, apparently normal Z rings are formed in the absence of ZipA . The interaction between FtsZ and ZipA occurs through their carboxy-terminal domains . Although a MalE-ZipA fusion binds to FtsZ filaments, it does not affect the GTPase activity or dynamics of the filaments . These results are consistent with ZipA acting after Z ring formation, possibly to link the membrane to FtsZ filaments during invagination of the septum.

Mol Microbiol, 1999 Mar, 31(6), 1825 - 33
Autoregulation of lactose uptake through the LacY permease by enzyme IIAGlc of the PTS in Escherichia coli K-12; Hogema BM et al.; Bacterial growth on one or more carbon sources requires careful control of the uptake and metabolism of these carbon sources . In Escherichia coli, the phosphorylation state of enzyme IIAGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in this control in two ways . The unphosphorylated form of IIAGlc causes 'inducer exclusion', the inhibition of uptake of a number of non-PTS carbon sources, including lactose uptake by the lactose permease . The phosphorylated form of enzyme IIAGlc probably activates adenylate cyclase . In cells growing on lactose, enzyme IIAGlc was approximately 50% dephosphorylated, suggesting that lactose could inhibit its own uptake . This inhibition could be demonstrated by comparing lactose uptake rates in the wild-type strain and in a mutant in which the lactose carrier was insensitive to inducer exclusion . In this deregulated mutant strain, lactose was consumed much faster, and large amounts of glucose were excreted . It was shown that enzyme IIAGlc was dephosphorylated more strongly and that the cAMP level was lower in the mutant, most probably causing the observed decrease in lac expression level . When the lac expression level in the mutant strain was increased to that of the parent strain by adding exogenous cAMP, growth on lactose was slower, suggesting that enzyme IIAGlc-mediated inhibition of lactose uptake and downregulation of the lac expression level protected the cells against excessive lactose influx . An even stronger increase in the lac expression level in a mutant lacking enzyme IIAGlc caused complete growth arrest . We conclude that the autoregulatory mechanism that controls lactose uptake is an important mechanism for the cells in adjusting the uptake rate to their metabolic capacity.

Mol Microbiol, 1999 Mar, 31(6), 1809 - 24
Protonmotive force, ExbB and ligand-bound FepA drive conformational changes in TonB; Larsen RA et al.; TonB couples the cytoplasmic membrane protonmotive force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes . Previous studies of a TonB transmembrane domain mutant (TonB-delta V17) and its phenotypical suppressor (ExbB-A39E) suggested that TonB is conformationally sensitive . Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB-S16L and -H20Y, as were two new suppressors, ExbB-V35E and -V36D . Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB-delta V17, for which both the conserved motif and the register of the predicted transmembrane domain alpha-helix are affected, was the most refractory . As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present . Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant-suppressor pairs . The phenotype of TonB-delta V17 was more complex than the -S16L and -H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable . This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB-delta V17 . Importantly, this degradation was dependent upon the presence of a TonB-dependent ligand, suggesting that TonB conformation also changes following the energy transduction event . Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.

Mol Microbiol, 1999 Mar, 31(6), 1783 - 93
Escherichia coli nusG mutations that block transcription termination by coliphage HK022 Nun protein; Burova E et al.; The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the lambda nutR site in vivo . We show that it is also essential for Nun termination at lambda nutL . Three recessive mis-sense nusG mutations have been isolated that inhibit termination by Nun at lambda nutR . The mutations are ineffective in a lambda pL nutL fusion, even when lambda nutR replaces lambda nutL . The mutant strains support lambda growth, indicating that lambda N antitermination activity is not impaired . Transcription arrest by Nun in vitro is stimulated by NusG protein at both lambda nutR and lambda nutL . Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site . The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination . These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.

Mol Microbiol, 1999 Mar, 31(6), 1775 - 82
The assembly and migration of SeqA-Gfp fusion in living cells of Escherichia coli; Onogi T et al.; SeqA protein, which binds to hemi-methylated GATC sequences of DNA, is localized to discrete fluorescent foci in wild-type Escherichia coli cells . In this work, we observed cellular localization of the SeqA-Gfp fusion in living cells . SeqA-Gfp was localized to a discrete focus or foci in wild-type and seqA null mutant cells, but the fusion was dispersed in the whole cell in dam null mutant cells lacking Dam methyltransferase . These results were consistent with the previous description of the localization of SeqA by immunofluorescence microscopy . Time-lapse experiments revealed that duplicated SeqA-Gfp foci migrated rapidly in opposite directions . Flow cytometry demonstrated that the fusion restored synchronous replication of chromosomal DNA from multiple origins in seqA null mutant cells, indicating that SeqA-Gfp is biologically active . Immunoprecipitation of the fusion from cell extracts using anti-Gfp antibody indicated that the fusion was assembled with the wild-type SeqA protein.

Mol Microbiol, 1999 Mar, 31(6), 1747 - 57
Amino acid residue Ala-62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis-associated Escherichia coli to collagens; Pouttu R et al.; Adhesion of meningitis-associated Escherichia coli O18acK1H7 to collagens was characterized . The E . coli strain IHE 3034 adhered to type IV and type I collagens but not to type III collagen immobilized on glass . Collagens lack terminal mannosyl units, yet the bacterial adhesion was completely abolished in the presence of alpha-methyl-D-mannoside . A cat cassette was introduced into the filmA gene of IHE 3034, and the resulting mutant strain IHE 3034-2 failed to adhere to collagens . In contrast, insertion of a Gm cassette into the sfaA gene of IHE 3034, encoding the S-fimbrillin, had no significant effect on the adhesiveness . The fim cluster from IHE 3034 was cloned and expressed in trans in the fimA::cat mutant strain IHE 3034-2 . The complemented strain IHE 3034-2(pRPO-1) exhibited adhesiveness to type IV and type I collagens, confirming the function of the type 1 fimbria in the adhesion . We have previously shown that the type 1 fimbria from E . coli K-12 strain PC31 does not confer bacterial adhesiveness to collagens . The fimH genes from E . coli IHE 3034 as well as from PC31 were expressed in the fimH-null strain MS4 . The FimH from IHE 3034 potentiated collagen adherence, whereas the FimH from PC31 was inactive . Sequence comparison of fimH from IHE 3034 and PC31 revealed five amino-acid differences in the predicted mature FimH proteins: at residues 27, 62, 70, 78 and 201 . Each of these residues in the IHE 3034-FimH were individually substituted to the corresponding amino acid in the PC31-FimH . The substitution S62-->A completely abolished collagen adhesiveness . The reverse substitution A62-->S in the PC31-FimH as well as in the FimH from another E . coli strain induced collagen adhesiveness to the level seen with IHE 3034-FimH . Out of nine fimH genes analysed from isolates of E . coli, collagen adhesiveness as well as alanine at position 62 in FimH were found only in two O18acK1H7 isolates with the isoenzyme profile ET type 1 . Our results demonstrate that the amino-acid residue Ala-62 in the FimH lectin is critical for the adhesion to collagens by a highly virulent clonal group of E . coli.

Mol Microbiol, 1999 Mar, 31(6), 1643 - 52
IHF protein inhibits cleavage but not assembly of plasmid R388 relaxosomes; Moncalian G et al.; Relaxosomes are specific nucleoprotein structures involved in DNA-processing reactions during bacterial conjugation . In this work, we present evidence indicating that plasmid R388 relaxosomes are composed of origin of transfer (oriT) DNA plus three proteins TrwC relaxase, TrwA nic-cleavage accessory protein and integration host factor (IHF), which acts as a regulatory protein . Protein IHF bound to two sites (ihfA and ihfB) in R388 oriT, as shown by gel retardation and DNase I footprinting analysis . IHF binding in vitro was found to inhibit nic-cleavage, but not TrwC binding to supercoiled DNA . However, no differences in the frequency of R388 conjugation were found between IHF- and IHF+ donor strains . In contrast, examination of plasmid DNA obtained from IHF- strains revealed that R388 was obtained mostly in relaxed form from these strains, whereas it was mostly supercoiled in IHF+ strains . Thus, IHF could have an inhibitory role in the nic-cleavage reaction in vivo . It can be speculated that triggering of conjugative DNA processing during R388 conjugation can be mediated by IHF release from oriT.

Biochim Biophys Acta, 1999 Apr 12, 1431(1), 212 - 22
Purification of beef kidney D-aspartate oxidase overexpressed in Escherichia coli and characterization of its redox potentials and oxidative activity towards agonists and antagonists of excitatory amino acid receptors; Negri A et al.; The flavoenzyme d-aspartate oxidase from beef kidney (DASPO, EC 1.4 . 3.1) has been overexpressed in Escherichia coli . A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E . coli paste . rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9-20% depending on the preparation) . This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution . Redox potentials of -97 mV and -157 mV were determined for free and l-(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25 degrees C . The large positive shift in the redox potential of the coenzyme compared to free FAD (-207 mV) is in agreement with similar results obtained with other flavooxidases . rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d-aspartatic acid, d-glutamic acid, N-methyl-d-aspartic acid, d,l-cysteic acid, d-homocysteic acid, d, l-2-amino-3-phosphonopropanoic acid, d-alpha-aminoadipic acid, d-aspartic acid-beta-hydroxamate, glycyl-d-aspartic acid and cis-2, 3-piperidine dicarboxylic acid . Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25 degrees C are reported.

Biochim Biophys Acta, 1999 Apr 12, 1431(1), 166 - 78
Crotonobetaine reductase from Escherichia coli consists of two proteins; Preusser A et al.; Crotonobetaine reductase from Escherichia coli is composed of two proteins (component I (CI) and component II (CII)) . CI has been purified to electrophoretic homogeneity from a cell-free extract of E . coli O44 K74 . The purified protein shows l(-)-carnitine dehydratase activity and its N-terminal amino acid sequence is identical to the caiB gene product from E . coli O44 K74 . The relative molecular mass of CI has been determined to be 86100 . It is composed of two identical subunits with a molecular mass of 42600 . The isoelectric point of CI was found to be 4.3 . CII was purified from an overexpression strain in one step by ion exchange chromatography on Fractogel EMD TMAE 650(S) . The N-terminal amino acid sequence of CII shows absolute identity with the N-terminal sequence of the caiA gene product, i.e . of the postulated crotonobetaine reductase . The relative molecular mass of the protein is 164400 and it is composed of four identical subunits of molecular mass 41500 . The isoelectric point of CII is 5.6 . CII contains non-covalently bound FAD in a molar ratio of 1:1 . In the crotonobetaine reductase reaction one dimer of CI associates with one tetramer of CII . A still unknown low-molecular-mass effector described for the l(-)-carnitine dehydratase is also necessary for crotonobetaine reductase activity . Monoclonal antibodies were raised against the two components of crotonobetaine reductase.

Biochim Biophys Acta, 1999 Apr 12, 1431(1), 157 - 65
Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas; Liu X et al.; A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1 . The protein was produced as insoluble inclusion bodies . After partial purification by washing, the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12 . The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2 . The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2 . To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done . The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

Biochim Biophys Acta, 1999 Apr 12, 1431(1), 1 - 13
Multiple folding pathways for heterologously expressed human prion protein; Jackson GS et al.; Human PrP (residues 91-231) expressed in Escherichia coli can adopt several conformations in solution depending on pH, redox conditions and denaturant concentration . Oxidised PrP at neutral pH, with the disulphide bond intact, is a soluble monomer which contains 47% alpha-helix and corresponds to PrPC . Denaturation studies show that this structure has a relatively small, solvent-excluded core and unfolds to an unstructured state in a single, co-operative transition with a DeltaG for folding of -5.6 kcal mol-1 . The unfolding behaviour is sensitive to pH and at 4.0 or below the molecule unfolds via a stable folding intermediate . This equilibrium intermediate has a reduced helical content and aggregates over several hours . When the disulphide bond is reduced the protein adopts different conformations depending upon pH . At neutral pH or above, the reduced protein has an alpha-helical fold, which is identical to that observed for the oxidised protein . At pH 4 or below, the conformation rearranges to a fold that contains a high proportion of beta-sheet structure . In the reduced state the alpha- and beta-forms are slowly inter-convertible whereas when oxidised the protein can only adopt an alpha-conformation in free solution . The data we present here shows that the human prion protein can exist in multiple conformations some of which are known to be capable of forming fibrils . The precise conformation that human PrP adopts and the pathways for unfolding are dependent upon solvent conditions . The conditions we examined are within the range that a protein may encounter in sub-cellular compartments and may have implications for the mechanism of conversion of PrPC to PrPSc in vivo . Since the conversion of PrPC to PrPSc is accompanied by a switch in secondary structure from alpha to beta, this system provides a useful model for studying major structural rearrangements in the prion protein.

Curr Biol, 1999 Apr 8, 9(7), R254 - 7
Membrane proteins: A tale of barrels and corks; Sansom MS; Crystal structures have been solved for two bacterial outer membrane proteins, FhuA and FepA, which mediate active transport of chelated iron . Analysis of ligand-induced changes in the structure of FhuA has provided our first structural insights into an active transport mechanism for a complex solute.

Nat Biotechnol, 1999 Apr, 17(4), 360 - 4
Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis; Brune W et al.; Herpesviruses are important pathogens in animals and humans . The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive . Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines . For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli . Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system . Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes . Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments . We also constructed revertant genomes by allelic exchange in bacteria . These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.

J Cell Biol, 1999 Apr 19, 145(2), 279 - 89
Overexpression of CALNUC (nucleobindin) increases agonist and thapsigargin releasable Ca2+ storage in the Golgi; Lin P et al.; We previously demonstrated that CALNUC, a Ca2+-binding protein with two EF-hands, is the major Ca2+-binding protein in the Golgi by 45Ca2+ overlay (Lin, P., H . Le-Niculescu, R . Hofmeister, J.M . McCaffery, M . Jin, H . Henneman, T . McQuistan, L . De Vries, and M . Farquhar . 1998 . J . Cell Biol . 141:1515-1527) . In this study we investigated CALNUC's properties and the Golgi Ca2+ storage pool in vivo . CALNUC was found to be a highly abundant Golgi protein (3.8 microg CALNUC/mg Golgi protein, 2.5 x 10(5) CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 microM, binding capacity = 1.1 micromol Ca2+/micromol CALNUC) . 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC . Deletion of the first EF-hand alpha helix from CALNUC completely abolished its Ca2+-binding capability . CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, alpha-mannosidase II (Man II) . Approximately 70% of the 45Ca2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca2+ pump) blocker . Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from Golgi stores . By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi . These results provide direct evidence that CALNUC binds Ca2+ in vivo and together with SERCA and IP3R is involved in establishment of the agonist-mobilizable Golgi Ca2+ store.

J Cell Biol, 1999 Apr 19, 145(2), 255 - 64
CRM1-mediated recycling of snurportin 1 to the cytoplasm; Paraskeva E et al.; Importin beta is a major mediator of import into the cell nucleus . Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs . Both adapters have an NH2-terminal importin beta-binding domain for binding to, and import by, importin beta, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus . We have shown previously that CAS mediates export of importin alpha . Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs) . However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs . First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain . Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity . Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free . This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.

Biol Reprod, 1999 May, 60(5), 1231 - 8
Utilization of endoscopic inoculation in a mouse model of intrauterine infection-induced preterm birth: role of interleukin 1beta; Reznikov LL et al.; A novel murine model of intrauterine infection/inflammation-induced preterm birth based on direct endoscopic intracervical inoculation is described . Using this model, we investigated infection-induced premature pregnancy loss in normal and interleukin (IL) 1beta-deficient mice . Seventy-four CD-1, HS, C57BL/6J wild type (IL-1beta+/+), and C57BL/6J IL-1beta-deficient (IL-1beta-/-) mice were inoculated intracervically using a micro-endoscope, at a time corresponding to 70% of average gestation . Intracervical injection of lipopolysaccharide (LPS) or Escherichia coli reliably induced premature birth: 100% of mice intracervically injected with LPS and 92% of mice with a positive endometrial E . coli culture delivered prematurely within 36 h after inoculation . No losses were observed in mice inoculated with saline . Pregnancy loss was associated with increased uterine tissue cyclooxygenase-2 gene expression and uterine content of IL-1beta, tumor necrosis factor alpha, macrophage inflammatory protein-1alpha, and IL-6, as well as elevation of nuclear factor-kappaB activity in uterine tissues . Although IL-1beta-/- mice exhibited decreased uterine cytokine production in response to bacteria and LPS, IL-1beta deficiency did not affect the rate of pregnancy loss . This model using direct intracervical bacterial or LPS inoculation is useful for studying preterm pregnancy loss in genetically altered mice in order to develop novel interventions for infection-associated preterm labor.

Res Vet Sci, 1999 Apr, 66(2), 85 - 91
Studies of endotoxin-dependent fever in pre-pubertal pigs following acute activation of the pituitary-adrenocortical axis: towards a new hypothesis of fever regulation; Parrott RF et al.; The possibility that adrenocortical activation might alter the pyretic effects of bacterial lipopolysaccharide endotoxin in growing pigs was investigated . In a series of four experiments, animals received increasing doses of porcine adrenocorticotrophic hormone ACTH (1.5, 4.5, 13.5 IU kg-1) or CRH (7 microg kg-1), all of which markedly affected cortisol release . Unexpectedly, these treatments tended to increase body temperature during the early and middle stages of the febrile response, although they did appear to induce an earlier deferscence . These results suggest that acute stress may not modify fever induced by immunological challenge, although a different situation could obtain during chronic stress . Furthermore, a hypothesis of fever regulation is proposed which attempts to reconcile the present findings with those from previous studies in swine .

Biochem Biophys Res Commun, 1999 Apr 21, 257(3), 792 - 7
High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli; Yoshikawa F et al.; Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an inositol 1, 4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region . Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins . We have found that the structures of IP3 binding constructs expressed in E . coli significantly affect their production as soluble protein . Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction . As a result, soluble active T604 would be 19 mg per liter of culture . The affinity for IP3 of T604 (Kd = 45 nM) is comparable to that of the native IP3R1, whereas that of an R441Q mutant is much higher (8.1 nM) . This system should provide an invaluable and powerful means to unveil the molecular recognition of IP3R1 for IP3 .

Biochem Biophys Res Commun, 1999 Apr 21, 257(3), 753 - 8
Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth; Polo L et al.; Irradiation of selected Escherichia coli defective strains with red-light induces a stimulation of the cell growth rate . Such effect is wavelength-dependent and is accompanied by a transient increase of the cell volume and some enzymic activities . The presence of oxygen appears to be essential for the occurrence of a significant photostimulatory effect . The results obtained upon irradiation in the presence of quenchers (tryptophan, histidine, azide) or enhancers (deuterium oxide) of singlet oxygen (1O2) strongly suggest that this activated oxygen derivative is generated by excitation of endocellular chromophores (possibly cytochromes) . The reaction of 1O2 with nearby cellular targets could induce a sublethal cell damage which in turn promotes an accelerated cell metabolism .

Biochem Biophys Res Commun, 1999 Apr 21, 257(3), 731 - 7
The 37-amino-acid interdomain of dengue virus NS5 protein contains a functional NLS and inhibitory CK2 site; Forwood JK et al.; The dengue virus NS5 RNA-dependent RNA polymerase has been detected in the nucleus of virus-infected mammalian cells . We demonstrate here for the first time using in vitro and in vivo assay systems that the 37-amino-acid linker interdomain of NS5 (residues 369 to 405) contains a nuclear localization sequence (NLS) which is capable of targeting b-galactosidase to the nucleus . Further, we show that the linker is recognized by subunits of the NLS-binding importin complex with an affinity similar to that of the bipartite NLS of the retinoblastoma protein and, in analogous fashion to proteins such as the SV40 large tumor antigen, contains a functional protein kinase CK2 phosphorylation site (threonine 395) . Interestingly, this site appears to inhibit NS5 nuclear targeting, probably through a cytoplasmic retention mechanism . The linker may have an important role in targeting NS5 to the nucleus in a regulated manner during the dengue virus infectious cycle .

Biochem Biophys Res Commun, 1999 Apr 21, 257(3), 662 - 7
In vitro codon-reading specificities of unmodified tRNA molecules with different anticodons on the sequence background of Escherichia coli tRNASer; Takai K et al.; The codon-reading properties of wobble-position variants of the unmodified form of Escherichia coli tRNASer1 (the UGA anticodon) were measured in a cell-free translation system . Two variants, with the AGA and CGA anticodons, each exclusively read a single codon, UCU and UCG, respectively . The only case of efficient wobbling occurred with the variant with the GGA anticodon, which reads the UCU codon in addition to the UCC codon . Surprisingly, this wobble reading is more efficient than the Watson-Crick reading by the variant with the AGA anticodon . Furthermore, we prepared tRNA variants with AA, UC, and CU, instead of GA, in the second and third positions and measured their relative efficiencies in the reading of codons starting with UU, GA, and AG, respectively . The specificity concerning the wobble position is essentially the same as that in the case of the codons starting with UC .

Atherosclerosis, 1999 Mar, 143(1), 75 - 80
Lipopolysaccharide enhances oxidative modification of low density lipoprotein by copper ions, endothelial and smooth muscle cells; Maziere C et al.; The effect of lipopolysaccharide (LPS, endotoxin) on low density lipoprotein (LDL) oxidative modification by copper ions, endothelial and smooth muscle cells was studied by determination of the level of lipid peroxidation products (thiobarbituric acid reactive substances or TBARS), the diene level and the electrophoretic mobility of the LDL particle . LPS 25-75 microg/ml induced a dose-dependent increase in LDL oxidation by copper ions, endothelial and smooth muscle cells . At 75 microg LPS/ml, the TBARS content was 1.9, 1.6, and 1.8-fold increased, respectively . The LDL degradation by J774 macrophage-like cells was concomitantly stimulated . Preincubation of the LDL particle with LPS induced a marked increase in the subsequent LDL oxidative modification either by copper ions or by endothelial and smooth muscle cells . In addition, pretreatment of endothelial and smooth muscle cells with LPS also induced an enhancement of LDL oxidative modification performed in the absence of LPS . This effect was accompanied by a parallel increase in superoxide anion release by the cells . These results point at one of the mechanisms involved in the described association between bacterial infection and acute myocardial infarction as well as coronary heart disease.

Novartis Found Symp, 1999, 221, 183 - 96; discussion 196-9
The molecular mechanism of regulation of the NhaA Na+/H+ antiporter of Escherichia coli, a key transporter in the adaptation to Na+ and H+; Padan E et al.; The NhaA Na+/H+ antiporter is the main system responsible for adaptation to Na+ and alkaline pH (in the presence of Na+) in Escherichia coli and many other enteric bacteria . It is under intricate control . At the protein level it is regulated directly by pH, one of its regulatory signals . A pH shift from 7 to 8.5 activates the antiporter and, in a fashion correlated with the activity change, confers a conformation change that, in isolated membrane vesicles, is reflected in the exposure of trypsin-cleavable sites . H225 and G338 are essential for the pH response of NhaA . nhaA transcription is dependent on NhaR, a positive regulator of the LysR family, and is regulated by Na+, the other environmental signal . Na+ affects the NhaR/nhaA interaction directly by changing the footprint of NhaR on nhaA in a pH-dependent fashion . The expression of nhaA is also under global regulation of H-NS . We suggest that the pattern of regulation of nhaA found in E . coli is a paradigm for the response of proteins and genes to H+ and Na+, the most common ions that challenge every cell.

Novartis Found Symp, 1999, 221, 93 - 106; discussion 106-11
Acid tolerance induced by metabolites and secreted proteins, and how tolerance can be counteracted; Rowbury RJ; Several metabolites and salts including glucose, L-glutamate, L-aspartate, FeCl3, KCl and L-proline induce acid tolerance at neutral external pH (pHo) in log phase Escherichia coli . For induction by glucose and L-glutamate, the processes are independent of integration host factor (IHF), H-NS, CysB, ferric uptake regulator (Fur) and RelA . For most of the above, tolerance does not appear if induction occurs and NaCl, sucrose, SDS or DOC are present . For several responses, cAMP inhibits induction . For many established acid tolerance and sensitization processes, including those tolerance responses switched on at pH 5.0 and by glucose, glutamate or aspartate, induction is associated with secretion of extracellular induction proteins . These proteins bring about the response if added to organisms under normally non-inducing conditions . Secreted components also influence inherent acid tolerances and sensitivities . Analysis of some established tolerance responses indicates that induction is a two-stage process, secreted extracellular proteins playing an obligate role in induction . For example, the functioning of the acid-induced medium protein(s) is essential for acid habituation at pHo 5.0 . It seems likely that such two-stage mechanisms are essential for many inducible processes in bacteria.

Novartis Found Symp, 1999, 221, 75 - 83; discussions 83-92
Acid and base regulation in the proteome of Escherichia coli; Slonczewski JL et al.; Acid and base conditions have many significant effects on the growth of Escherichia coli . External and internal pH perturbations induce different classes of genes . pH-dependent regulation of genes intersects with other regulatory responses, e.g . oxygen level or osmolarity . 2D electrophoretic gels were used to compare global patterns of protein induction in Escherichia coli grown in complex media buffered at the acid or alkaline ends of the pH range for growth (pH 4.4 vs . pH 9.1) . Preliminary results indicate new classes of acid- and base-dependent regulation, in some cases highly dependent on oxygen level . Other proteins are induced strongly at both extremes of pH, compared to pH 7 . Current work continues to dissect the relationship between effects of pH, oxygen level and osmolarity.

Genome Res, 1999 Apr, 9(4), 325 - 33
A cluster of ABA-regulated genes on Arabidopsis thaliana BAC T07M07; Wang ML et al.; Arabidopsis thaliana BAC T07M07 encoding the abscisic acid-insensitive 4 (ABI4) locus has been sequenced completely . It contains a 95,713-bp insert and 24 predicted genes . Most putative genes were confirmed by gel-based RNA profiling and a cluster of ABA-regulated genes was identified . One of the 24 genes, designated PP2C5, encodes a putative protein phosphatase 2C . The encoded protein was expressed in Escherichia coli, and its enzyme activity in vitro was confirmed.

Nature, 1999 Apr 8, 398(6727), 533 - 8
Structural basis for self-association and receptor recognition of human TRAF2; Park YC et al.; Tumour necrosis factor (TNF)-receptor-associated factors (TRAFs) form a family of cytoplasmic adapter proteins that mediate signal transduction from many members of the TNF-receptor superfamily and the interleukin-1 receptor . They are important in the regulation of cell survival and cell death . The carboxy-terminal region of TRAFs (the TRAF domain) is required for self-association and interaction with receptors . The domain contains a predicted coiled-coil region that is followed by a highly conserved TRAF-C domain . Here we report the crystal structure of the TRAF domain of human TRAF2, both alone and in complex with a peptide from TNF receptor-2 (TNF-R2) . The structures reveal a trimeric self-association of the TRAF domain, which we confirm by studies in solution . The TRAF-C domain forms a new, eight-stranded antiparallel beta-sandwich structure . The TNF-R2 peptide binds to a conserved shallow surface depression on one TRAF-C domain and does not contact the other protomers of the trimer . The nature of the interaction indicates that an SXXE motif may be a TRAF2-binding consensus sequence . The trimeric structure of the TRAF domain provides an avidity-based explanation for the dependence of TRAF recruitment on the oligomerization of the receptors by their trimeric extracellular ligands.

Mol Cell Biol, 1999 May, 19(5), 3727 - 35
Distinct mechanisms of activation of Stat1 and Stat3 by platelet-derived growth factor receptor in a cell-free system; Vignais ML et al.; Ligand-dependent activation of the platelet-derived growth factor receptor (PDGFR) in fibroblasts in culture leads to the activation of the JAK family of protein-tyrosine kinases and of the transcription factors Stat1 and Stat3 . To determine the biochemical mechanism of STAT activation by PDGFR, we devised a cell-free system composed of a membrane fraction from cells overexpressing PDGFR . When supplemented with crude cytosol, the membrane fraction supported PDGF- and ATP-dependent activation of both Stat1 and Stat3 . However, the extent of Stat3 activation differed depending on the source of the cytosolic fraction . Using purified recombinant STAT proteins produced in Escherichia coli, we found that Stat1 could be activated by immunopurified PDGFR and showed no additional requirement for membrane- or cytosol-derived proteins . In contrast, activation of Stat3 exhibited a strong requirement for the cytosolic fraction . The activity present in the cytosolic fraction could be depleted with antibodies to JAK proteins . We conclude that the mechanisms of activation of Stat1 and Stat3 by PDGFR are distinct . Stat1 activation appears to result from a direct interaction with the receptor, whereas Stat3 activation additionally requires JAK proteins.

J Biol Chem, 1999 Apr 23, 274(17), 12157 - 62
Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin; Sipma H et al.; A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E . coli . The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1 . Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM . This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 microM Ca2+ . The maximal effect of Ca2+ on Lbs-1 (5 microM Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%) . Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R . (1997) J . Biol . Chem . 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1 . Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 microM . Maximal inhibition (10 microM calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%) . Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 microM Ca2+ (together 74.5 +/- 5.1%) . These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.

J Biol Chem, 1999 Apr 23, 274(17), 11848 - 53
Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22 . Direct involvement of protein myristoylation in calmodulin-target protein interaction; Takasaki A et al.; Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated . Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown . Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain . In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin . Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined . Only the former bound to calmodulin . Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated . The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain . Phosphorylation of a single serine residue in the N-terminal domain (Ser5) by protein kinase C abolished the binding . Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.

J Biol Chem, 1999 Apr 23, 274(17), 11679 - 86
The glucocorticoid response element II is functionally homologous in rat and human insulin-like growth factor-binding protein-1 promoters; Schweizer-Groyer G et al.; In vivo, insulin-like growth factor-binding protein-1 (IGFBP-1) modulates the IGFs' bioavailability and may contribute to their delivery to peripheral tissues . In rat and human hepatocytes, glucocorticoids stimulate IGFBP-1 gene transcription through homologous glucocorticoid response units (GRU) . Transfection experiments have shown that one of these, GRU2 (nucleotide (nt) -121 to -85 and nt -111 to -74 in human and rat promoters, respectively), was on its own able to mediate the glucocorticoid response in rat but not in human species (Suwanichkul, A., Allander, S., Morris, S . L . & Powell, D . R . (1994) J . Biol . Chem . 269, 30835-30841, Goswami, R., Lacson, R., Yang, E., Sam, R . & Unterman, T . (1994) Endocrinology 134, 736-743, and Suh, D . S., Ooi, G . T . & Rechler, M . M . (1994) Mol . Endocrinol . 8, 794-805) . A close comparison of GRU2 sequences has pointed out a C to A transition in the underlying GREII, which creates a GATC tetranucleotide in rat species . This tetranucleotide is submitted to adenosyl methylation (dam methylation) in most Escherichia coli bacterial strains, but not in eucaryotic cells . We showed (i) that on its own, the unmethylated rat GRU2 (propagated in dam E . coli strains) was inactive, as is the case for its human counterpart (nonsignificant glucocorticoid inductions, 1.48 +/- 0.23 and 1.7 +/- 0.35-fold in Chinese hamster ovary cells, respectively) and (ii) that its adenosyl methylation in standard dam+ bacterial strains yielded a functional GRU (6.5 +/- 1 . 1 and 13.1 +/- 3.9-fold glucocorticoid inductions in Chinese hamster ovary and HepG2 cells, respectively) . Transient transfection in HepG2 hepatoma cells clearly showed that the interaction of liver-enriched trans-acting factor(s) with the 5'-overlapping insulin response element does not enable the unmethylated rat GRU2 or the human GRU2 to become responsive to glucocorticoids (nonsignificant 2.21 +/- 0.48 and 1.20 +/- 0.06-fold induction, respectively) . Furthermore, we have correlated these functional data with in vitro DNA-protein interaction studies: the dam methylated rat GREII displayed a 2.8-fold higher affinity for the glucocorticoid receptor than its unmethylated counterpart.

J Biol Chem, 1999 Apr 23, 274(17), 11623 - 8
Transcription termination factor Rho contains three noncatalytic nucleotide binding sites; Kim DE et al.; The active form of transcription termination factor rho from Escherichia coli is a homohexamer, but several studies suggest that the six subunits of the hexamer are not functionally identical . Rho has three tight and three weak ATP binding sites . Based on our findings, we propose that the tight nucleotide binding sites are noncatalytic and the weak sites are catalytic . In the presence of RNA, the rho-catalyzed ATPase rate is fast, close to 30 s-1 . However, under these conditions the three tightly bound nucleotides dissociate from the rho hexamer at a slow rate of 0.02 s-1, indicating that the three tight nucleotide binding sites of rho do not participate in the fast ATPase turnover . These slowly exchanging nucleotide binding sites of rho are capable of hydrolyzing ATP, but the resulting products (ADP and Pi) bind tightly and dissociate from rho about 1500 times slower than the fast ATPase turnover . Both RNA and excess ATP in solution are necessary for stabilizing nucleotide binding at these sites . In the absence of RNA, or when solution ATP is hydrolyzed to ADP, a faster dissociation of nucleotides was observed . Based on these results, we propose that the rho hexamer is similar to the F1-ATPase and T7 DNA helicase-containing noncatalytic sites that do not participate in the fast ATPase turnover . We propose that the three tight sites on rho are the noncatalytic sites and the three weak sites are the catalytic sites.

J Biol Chem, 1999 Apr 23, 274(17), 11463 - 8
Interaction of SeqA and Dam methylase on the hemimethylated origin of Escherichia coli chromosomal DNA replication; Kang S et al.; Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase . Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end of oriC were examined . We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers . Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R . On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences . The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication . Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding . Also, SeqA protein delayed the in vitro methylation of hemimethylated 13-mers by Dam methylase . These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.

Microbiology, 1999 Jan, 145 ( Pt 1), 41 - 55
Transcriptional regulation of molybdoenzyme synthesis in Escherichia coli in response to molybdenum: ModE-molybdate, a repressor of the modABCD (molybdate transport) operon is a secondary transcriptional activator for the hyc and nar operons; Self WT et al.; Escherichia coli growing under anaerobic conditions produces several molybdoenzymes, such as formate hydrogenlyase (formate to H2 and CO2; hyc and fdhF genes) and nitrate reductase (narGHJI genes) . Synthesis of these molybdoenzymes, even in the presence of the cognate transcriptional activators and effectors, requires molybdate in the medium . Besides the need for molybdopterin cofactor synthesis, molybdate is also required for transcription of the genes encoding these molybdoenzymes . In E . coli, ModE was previously identified as a repressor controlling transcription of the operon encoding molybdate transport components (modABCD) . In this work, the ModE protein was also found to be a required component in the activation of hyc-lacZ to an optimum level, but only in the presence of molybdate . Mutant ModE proteins which are molybdate-independent for repression of modA-lacZ also restored hyc-lacZ expression to the wild-type level even in the absence of molybdate . Nitrate-dependent enhancement of transcription of narX-lacZ was completely abolished in a modE mutant . Nitrate-response by narG-lacZ and narK-lacZ was reduced by about 50% in a modE mutant . DNase I footprinting experiments revealed that the ModE protein binds the hyc promoter DNA in the presence of molybdate . ModE-molybdate also protected DNA in the intergenic region between narXL and narK from DNase I hydrolysis . DNA sequences (5' TAYAT 3' and 5' GTTA 3') found in ModE-molybdate-protected modABCD operator DNA were also found in the ModE-molybdate-protected region of hyc promoter DNA (5' GTTA-7 bp-CATAT 3') and narX-narK intergenic region (5' GTTA-7 bp-TACAT 3') . Based on these results, a working model is proposed in which ModE-molybdate serves as a secondary transcriptional activator of both the hyc and narXL operons which are activated primarily by the transcriptional activators, FhlA and NarL, respectively.

Microbiology, 1999 Jan, 145 ( Pt 1), 33 - 40
The TATA-box binding protein of Entamoeba histolytica: cloning of the gene and location of the protein by immunofluorescence and confocal microscopy; Luna-Arias JP et al.; A 309 bp DNA fragment from Entamoeba histolytica was amplified by PCR using primers derived from the Acanthamoeba castellanii consensus TATA-box binding protein amino acid sequence . The amplified fragment was used to isolate cDNA and genomic DNA clones containing an ORF encoding the complete E . histolytica TATA-box binding protein (Ehtbp, 702 bp, 234 aa, molecular mass 26 kDa) . The EhTBP functional domain showed 55% sequence identity to that of Homo sapiens, 54% to A . castellanii and 37% to Plasmodium falciparum TBPs . In Southern blot experiments we detected a single Ehtbp band, which was transcribed as a 1.3 kb mRNA containing a 420 nt 5' untranslated region . However, the probe hybridized with the 0.8 and 1.5 Mb chromosomes, suggesting that this sequence is diploid . In situ PCR assays showed two signals in 95% of trophozoites, one located in the nucleus and another in EhkO, the novel DNA-containing organelle recently reported . The recombinant E . histolytica TATA-box binding protein was expressed in Escherichia coli . Antibodies against it recognized two proteins of 26 and 29 kDa in E . histolytica nuclear extracts . Confocal microscopy immunofluorescence analysis located the protein in both the nucleus and EhkO.

J Pediatr Hematol Oncol, 1999 Mar-Apr, 21(2), 123 - 8
Hemolytic uremic syndrome in children: platelet aggregation and membrane glycoproteins; Sassetti B et al.; PURPOSE: Several mechanisms have been proposed to explain the fibrin-platelet thrombosis at the microcirculation level in the different clinical conditions of hemolytic uremic syndrome (HUS) . The relationships between platelet structure and function during the first 4 weeks of evolution of the disease were studied to understand the mechanism of platelet alteration . PATIENTS AND METHODS: Coagulation parameters, platelet counts, and aggregation were studied in 49 children, and membrane glycoproteins (GPs) in 20 of the 49 children (mean age, 17 months) with HUS (Group 2) were studied during the first 4 weeks of evolution of the disease . RESULTS: No disseminated intravascular coagulation was found in patients with recurrent or persistent thrombocytopenia . Platelet aggregation was sequentially performed during the first weeks of evolution . All patients had a functional decrease in the acute period of HUS . Platelet GPs GPIb, GPIIbIIIa, GPIIb, and GPIIIa were evaluated . GPIIbIIIa complex presented low level and never reached normal values during the first 4 weeks of disease . CONCLUSIONS: Platelet alterations are probably caused by multiple mechanisms: "exhausted" platelets, structural membrane alterations caused by arginine-glycine-aspartic peptide blockade, or diminished or nonfunctional membrane GPIb and GPIIbIIIa complexes.

Vet Immunol Immunopathol, 1999 Mar 1, 67(4), 359 - 72
Molecular cloning and physiological effects of brushtail possum interleukin-1beta; Wedlock DN et al.; Interleukin-1beta (IL-1beta) was isolated from LPS-stimulated brushtail possum alveolar macrophages using PCR primers based on conserved regions of mammalian IL-1beta . The complete cDNA was cloned by 5' and 3' rapid amplification of cDNA ends (RACE) . The predicted protein of 269 amino acids shared 4346% identity with several mammalian IL-1beta proteins . Constructs were made to express the mature IL-1beta in Escherichia coli and two recombinant IL-1beta proteins, rpIL-1beta1 and rpIL-1beta2, which differed in length by four amino acids at the N-terminus, were produced . Both proteins induced a weak proliferative response in a possum thymocyte assay . Possums injected intravenously with 100 microg of rpIL-1beta1 or rpIL-1beta2 showed profound changes in body temperature and numbers of circulating leukocytes . A sharp decrease in temperature occurred within 2 h of administration followed by an elevation of temperature peaking at 24 h . The smaller rpIL-1beta1 protein had a greater effect on temperature than rpIL-1beta2 . Both rpIL-1beta proteins caused a marked decrease in number of neutrophils and lymphocytes at 2-6 h after injection . At 24 h after injection, neutrophil and lymphocyte numbers were elevated 6.0-fold and 2.6-fold, respectively in the possums injected with rpIL-1beta1 and 3.9-fold and 1.5-fold, respectively in the possums injected with rpIL-1beta2 . Fibrinogen levels were elevated at 24 and 72 h after injection with both proteins . In comparison, neither recombinant bovine IL-1beta (rbIL-1beta) nor PBS had significant effects on body temperature or blood haematology . The studies have shown that the two recombinant forms of IL-1beta were biologically active in possums and that the IL-1beta with four fewer amino acids at the N-terminus was the more active.

Plant J, 1999 Mar, 17(5), 467 - 77
Chloroplast Cpn20 forms a tetrameric structure in Arabidopsis thaliana; Koumoto Y et al.; Chloroplast chaperonin 20 (Cpn20) in higher plants is a functional homologue of the Escherichia coli GroES, which is a critical regulator of chaperonin-mediated protein folding . The cDNA for a Cpn20 homologue of Arabidopsis thaliana was isolated . It was 958 bp long, encoding a protein of 253 amino acids . The protein was composed of an N-terminal chloroplast transit peptide, and the predicted mature region comprised two distinct GroES domains that showed 42% amino acid identity to each other . The isolated cDNA was constitutively expressed in transgenic tobacco . Immunogold labelling showed that Cpn20 is accumulated in chloroplasts of transgenic tobacco . A Northern blot analysis revealed that mRNA for the chloroplast Cpn20 is abundant in leaves and is increased by heat treatment . To examine the oligomeric structure of Cpn20, a histidine-tagged construct lacking the transit peptide was expressed in E . coli and purified by affinity chromatography . Gel-filtration and cross-linking analyses showed that the expressed products formed a tetramer . The expressed products could substitute for GroES to assist the refolding of citrate synthase under non-permissive conditions . The analysis on the subunit stoichiometry of the GroEL-Cpn20 complex also revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.

Plant J, 1999 Feb, 17(4), 385 - 95
Glyoxalase I from Brassica juncea: molecular cloning, regulation and its over-expression confer tolerance in transgenic tobacco under stress; Veena et al.; Despite its ubiquitous presence, the role of glyoxalase I has not been well investigated in plants . In order to find out its physiological functions, we have cloned and characterized a cDNA from Brassica juncea encoding glyoxalase I (Gly I) and made transgenic tobacco plants harbouring Gly I in both sense and antisense orientation . The transgenic nature of the plants was confirmed by Southern blotting, and the estimated number of genes inserted ranged from one to six . The transcript and protein levels of glyoxalase I were also monitored in transgenic plants . The expression of glyoxalase I in B . juncea was upregulated in response to salt, water and heavy metal stresses . In response to a high concentration of salt, the transcript level averaged threefold higher in 72 h, and an increase in the protein was also seen by immunoblotting . The transgenic plants over-expressing glyoxalase I showed significant tolerance to methylglyoxal and high salt, as tested in detached leaf disc senescence assay . A comparison of plants expressing high and low levels of glyoxalase I showed that the tolerance to different salt concentrations was correlated with the degree of glyoxalase I expression . Our results suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions.

Plant J, 1999 Feb, 17(4), 341 - 51
Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant Delta; Ronen G et al.; The red colour of tomato (Lycopersicon esculentum) fruits is provided by the carotenoid pigment lycopene whose concentration increases dramatically during the ripening process . A single dominant gene, Del, in the tomato mutant Delta changes the fruit colour to orange as a result of accumulation of delta-carotene at the expense of lycopene . The cDNA for lycopene epsilon-cyclase (CrtL-e), which converts lycopene to delta-carotene, was cloned from tomato . The primary structure of CRTL-E is 71% identical to the homologous polypeptide from Arabidopsis and 36% identical to the tomato lycopene beta-cyclase, CRTL-B . The CrtL-e gene was mapped to a single locus on chromosome 12 of the tomato linkage map . This locus co-segregated with the Del gene . In the wild-type tomato, the transcript level of CrtL-e decreases at the 'breaker' stage of ripening to a non-detectable level in the ripe fruit . In contrast, it increases approximately 30-fold during fruit ripening in the Delta plants . The Delta mutation does not affect carotenoid composition nor the mRNA level of CrtL-e in leaves and flowers . These results strongly suggest that the mutation Del is an allele of the gene for epsilon-cyclase . Together with previous data, our results indicate that the primary mechanism that controls lycopene accumulation in tomato fruits is based on the differential regulation of expression of carotenoid biosynthesis genes . During fruit development, the mRNA levels for the lycopene-producing enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) increase, while the mRNA levels of the genes for the lycopene beta- and epsilon-cyclases, which convert lycopene to either beta- or delta-carotene, respectively, decline and completely disappear.

Parasitology, 1999 Mar, 118 ( Pt 3), 297 - 304
Cloning and characterization of glutamate dehydrogenase (GDH) from the gut of Haemonchus contortus; Skuce PJ et al.; Vaccination of lambs with the membrane-bound (S3) thiol-Sepharose binding protein (TSBP) fraction derived from the gut of Haemonchus contortus confers significant protection against homologous challenge . The S3 TSBP peptide profile is dominated by a major protein of ca . 60 kDa which is strongly recognized by antisera from sheep demonstrably protected following immunization with S3 TSBP . In an attempt to identify this protein, sera from protected lambs were employed to screen a lambda gt11 cDNA library of the adult parasite and resulted in the isolation of numerous clones encoding a homologue of the mitochondrial enzyme, glutamate dehydrogenase (GDH) . GDH enzyme activity was readily demonstrable in S3 TSBP material and immunolocalization studies showed that the enzyme was localized to the cytoplasm of the parasite's gut . Furthermore, the enzyme appeared to be developmentally regulated, with both GDH mRNA and protein expressed almost exclusively during the blood-feeding parasitic stages.

Biochem Mol Biol Int, 1999 Feb, 47(2), 301 - 8
Hepatitis B virus: DNA polymerase activity of deletion mutants; Kim Y et al.; The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities . Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made . Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E . coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared . Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively . However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region . The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity . In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain.

Biochem Mol Biol Int, 1999 Feb, 47(2), 267 - 73
High level expression of soluble angiogenin in Escherichia coli; Yoon JM et al.; Human angiogenin was genetically engineered and contained the E . coli Omp A signal sequence for secreting soluble angiogenin to the periplasm under tac promoter control . The angiogenin sequence was encoded in a single gene and expressed as a 14.4 kilodalton soluble protein in E . coli . It was purified by CM-Sepharose ion-exchange chromatography and by a heparin-Sepharose affinity chromatography procedure . The biological activity of angiogenin was established by its ability to inhibit mRNA-dependent rabbit reticulocyte cell-free translation.

Gut, 1999 May, 44(5), 615 - 9
Vagotomy inhibits the jejunal fluid secretion activated by luminal ileal Escherichia coli STa in the rat in vivo; Rolfe VE et al.; BACKGROUND: Escherichia coli heat stable enterotoxin (STa) is a major cause of secretory diarrhoea in humans . AIMS: To assess the effects of instilling STa into the ileum on remote fluid secretion in the jejunum and colon in rats in vivo by a gravimetric technique . METHODS AND RESULTS: Ileal STa (55 ng/ml) stimulated fluid secretion in both ileal and jejunal loops but not in the colon . The fluid secretion induced by ileal STa was inhibited by L-NAME (Nomega-nitro-L-arginine methyl ester, 40 mg/kg intraperitoneally) but not by D-NAME (Nomega-nitro-D-arginine methyl ester) . Ileal carbachol (183 mg/ml) instilled into the lumen stimulated ileal secretion but not jejunal secretion, and was unaffected by L-NAME . Capsaicin (10 microM), instilled luminally with STa in the ileum, blocked both the ileal and jejunal fluid secretion . Acute bilateral vagotomy prevented luminal ileal STa from inducing jejunal fluid secretion but not from activating ileal fluid secretion . CONCLUSION: Ileal E coli STa stimulates remote secretion in the rat jejunum but not in the colon, probably by a nitrinergic, vagal reflex mediated by C fibres . This neural pathway will amplify the action of the toxin in its generation of secretory diarrhoea.

EMBO J, 1999 Apr 15, 18(8), 2304 - 10
Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences; Brendler T et al.; The SeqA protein binds to the post-replicative forms of the origins of replication of the Escherichia coli chromosome (oriC) and the P1 plasmid (P1oriR) at hemimethylated GATC adenine methylation sites . It appears to regulate replication by preventing premature reinitiation . However, SeqA binding is not exclusive to replication origins: different fragments with hemimethylated GATC sites can bind SeqA in vitro when certain rules apply . Most notably, more than one such site must be present on a bound fragment . The protein appears to recognize individual hemimethylated sites, but must undergo an obligate cooperative interaction with a nearby bound protein for stable binding . SeqA contacts both DNA strands in a discrete patch at each hemimethylated GATC sequence . All four GATC bases are contacted and are essential for binding . Although the recognized sequence is symmetrical, the footprint on the methylated strand is always broader, suggesting that the bound protein is positioned asymmetrically with its orientation dictated by the position of the unique methyl group . Studies of alternative spacings and relative orientations of adjacent sites suggest that each site may be recognized by a symmetrical dimer with an induced asymmetry in one of the subunits similar to that seen with certain type II restriction endonucleases.

Science, 1999 Apr 16, 284(5413), 482 - 5
Dissecting and exploiting intermodular communication in polyketide synthases; Gokhale RS et al.; Modular polyketide synthases catalyze the biosynthesis of medicinally important natural products through an assembly-line mechanism . Although these megasynthases display very precise overall selectivity, we show that their constituent modules are remarkably tolerant toward diverse incoming acyl chains . By appropriate engineering of linkers, which exist within and between polypeptides, it is possible to exploit this tolerance to facilitate the transfer of biosynthetic intermediates between unnaturally linked modules . This protein engineering strategy also provides insights into the evolution of modular polyketide synthases.

Ross Fiziol Zh Im I M Sechenova, 1998 Dec, 84(12), 1432 - 7
{Urokinase induces adhesion of monocytes to fibrinogen}; Arefi'eva TI et al.; Urokinase activates adhesion of monocytic U937 cells to fibrinogen-coated surface . This effect is due to urokinase proteolytic activity and does not depend on the urokinase binding to its receptor.

Biochem Mol Biol Int, 1999 Mar, 47(3), 417 - 25
Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease; Jeon OH et al.; A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library . Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain . The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E . coli . The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity . Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion . The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.

J Clin Microbiol, 1999 May, 37(5), 1642 - 5
Genetic diversity among Escherichia coli isolates carrying f18 genes from pigs with porcine postweaning diarrhea and edema disease; Nagy B et al.; Multilocus enzyme electrophoresis was applied to detect allelic variation and multilocus genotypes (electrophoretic types {ETs}) among 43 Escherichia coli isolates from weaned pigs suffering from edema disease or from diarrhea . ETs were analyzed in relation to O serogroups and virulence genes (sta, stb, lt, stx2, and f18) by DNA hybridization . Genomic diversity was the lowest in serogroup O138, while virulence genes (stx2 and f18) were the most uniform in serogroup O139 . In general, the serogroups or toxin and F18 fimbria types were not related to selected ETs, suggesting that the toxin and f18 fimbria genes in E . coli isolates from pigs with postweaning diarrhea or edema disease occur in a variety of chromosomal backgrounds.

J Clin Microbiol, 1999 May, 37(5), 1575 - 8
Molecular cloning and sequencing of the aroA gene from Actinobacillus pleuropneumoniae and its use in a PCR assay for rapid identification; Hernanz Moral C et al.; The gene (aroA) of Actinobacillus pleuropneumoniae, serotype 2, encoding 5-enolpyruvylshikimate-3-phosphate synthase was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined . A pair of primers from the 5' and 3' termini were selected to be the basis for development of a specific PCR assay . A DNA fragment of 1,025 bp was amplified from lysed A . pleuropneumoniae serotypes 1 to 12 of biovar 1 or from isolated DNA . No PCR products were detected when chromosomal DNAs from other genera were used as target DNAs; however, a 1,025-bp DNA fragment was amplified when Actinobacillus equuli chromosomal DNA was used as a target, which could be easily differentiated by its NAD independence . The PCR assay developed was very sensitive, with lower detection limits of 12 CFU with A . pleuropneumoniae cells and 0.8 pg with extracted DNA . Specificity and sensitivity make this PCR assay a useful method for the rapid identification and diagnosis of A . pleuropneumoniae infections.

J Clin Microbiol, 1999 May, 37(5), 1554 - 60
Evaluation of recombinant antigens for serodiagnosis of Chagas' disease in South and Central America; Umezawa ES et al.; The commercially available diagnostic tests for Chagas' disease employ whole extracts or semipurified fractions of Trypanosoma cruzi epimastigotes . Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents . The use of recombinant antigens for the serodiagnosis of Chagas' disease is recommended to increase the sensitivity and specificity of serological tests . Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T . cruzi recombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas' disease . The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela) . The optimal concentration of each recombinant antigen for coating of plates was determined with the help of 125I-labelled recombinant proteins . While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6% . Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients . In this way, it is proposed that a mixture of a few T . cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas' disease.

Ann Surg, 1999 Apr, 229(4), 478 - 86
Intestinal cytokine response after gut ischemia: role of gut barrier failure; Grotz MR et al.; OBJECTIVE: To investigate the effect of intestinal ischemia with and without a reperfusion injury on intestinal cytokine production and gut permeability . SUMMARY BACKGROUND DATA: In humans and in animal models, the gut has been implicated as a cytokine-producing organ after ischemia/reperfusion (I/R)-type injuries . Because of the limitations of in vivo models, it has been difficult to demonstrate directly that the gut releases cytokines after an I/R injury or whether there is a relation between the magnitude of the ischemic process and the cytokine response . METHODS: Ileal mucosal membranes from rats subjected to sham or 45 or 75 min of superior mesenteric occlusion (SMAO) or 45 minutes of SMAO and 30 minutes of reperfusion (SMAO 45/30) were mounted in the Ussing chamber system . Levels of tumor necrosis factor-alpha and interleukin-6 were serially measured in the mucosal and serosal reservoirs of the Ussing system, as was mucosal permeability as reflected by the passage of bacteria or phenol red across the ileal membrane . In a second group of experiments, Escherichia coli C25 was added to the mucosal reservoir to determine if the cytokine response would be increased . RESULTS: Mucosal and serosal levels of tumor necrosis factor-alpha were equally increased after SMAO, with the highest levels in the 75-minute SMAO group . The highest levels of interleukin-6 were found in rats subjected to 75 minutes of SMAO or SMAO 45/30; the serosal levels of interleukin-6 were four to sixfold higher than the mucosal levels . The addition of E . coli C25 resulted in a significant increase in the amount of interleukin-6 or tumor necrosis factor-alpha recovered from the mucosal reservoir . Increased ileal membrane permeability was observed only in rats subjected to 75 minutes of SMAO or SMAO 45/30 . CONCLUSION: These results directly document that the levels of tumor necrosis factor-alpha and interleukin-6 released from the gut increase after an ischemic or I/R injury, such as SMAO, and that there is a relation between the magnitude of the gut ischemic or I/R insult and the cytokine response.

Plant Cell Physiol, 1999 Feb, 40(2), 149 - 54
Chloroplast NADH dehydrogenase from Pisum sativum: characterization of its activity and cloning of ndhK gene; Elortza F et al.; The pea chloroplast ndhK gene coding for a component of a NADH-plastoquinone oxidoreductase has been cloned and sequenced . This gene codes for a polypeptide of 227 amino acids and a predicted molecular mass of 25,495 Da which belongs to the family of the 20 kDa PSST subunit of the bovine mitochondrial complex I . A fragment of this gene has been overexpressed in Escherichia coli, and antibodies against the expressed polypeptide recognize a protein of the predicted molecular mass from pea thylakoid membranes . This polypeptide is a component of a protein complex with NADH dehydrogenase activity and is not associated with ferredoxin-NADP+ reductase.

Biol Psychiatry, 1999 Apr 1, 45(7), 853 - 62
Heightened transcription for enzymes involved in norepinephrine biosynthesis in the rat locus coeruleus by immobilization stress; Serova LI et al.; BACKGROUND: The locus coeruleus (LC), a target for CRH neurons, is critically involved in responses to stress . Various physiological stresses increase norepinephrine turnover, tyrosine hydroxylase (TH) enzymatic activity, protein and mRNA levels in LC cell bodies and terminals; however, the effect of stress on other enzymes involved in norepinephrine biosynthesis in the LC is unknown . METHODS: Rats were exposed to single (2 hour) or repeated (2 hour daily) immobilization stress (IMO) . Recombinant rat dopamine b-hydroxylase (DBH) cDNA was expressed in E . coli and used to generate antisera for immunohistochemistry and immunoblots in LC . Northern blots were used to assess changes in mRNA levels for TH, DBH, and GTP cyclohydrolase I (GTPCH) in the LC in response to the stress . Conditions were found to isolate nuclei from LC and to use them for run-on assays of transcription . RESULTS: Repeated stress elevated the DBH immunoreactive protein levels in LC . Parallel increases in TH, DBH and GTPCH mRNA levels of about 300% to 400% over control levels were observed with single IMO, and remained at similar levels after repeated IMO . This effect was transcriptionally mediated, and even 30 min of a single IMO significantly increased the relative rate of transcription . CONCLUSIONS: This study is the first to reveal transcriptional activation of the genes encoding catecholamine biosynthetic enzymes in the LC by stress . In addition to TH, changes in DBH and GTPCH gene expression may also contribute to the development of stress-triggered affective disorders.

EMBO J, 1999 Apr 1, 18(7), 1730 - 7
Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity; Levina N et al.; Mechanosensitive channels are ubiquitous amongst bacterial cells and have been proposed to have major roles in the adaptation to osmotic stress, in particular in the management of transitions from high to low osmolarity environments . Electrophysiological measurements have identified multiple channels in Escherichia coli cells . One gene, mscL, encoding a large conductance channel has previously been described, but null mutants were without well-defined phenotypes . Here, we report the characterization of a new gene family required for MscS function, YggB and KefA, which has enabled a rigorous test of the role of the channels . The channel determined by KefA does not appear to have a major role in managing the transition from high to low osmolarity . In contrast, analysis of mutants of E.coli lacking YggB and MscL shows that mechanosensitive channels are designed to open at a pressure change just below that which would cause cell disruption leading to death.

Nat Struct Biol, 1999 Apr, 6(4), 374 - 9
Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy; Opella SJ et al.; The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples . Both M2 segments form straight transmembrane alpha-helices with no kinks . The AChR M2 peptide inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular . A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.

Nat Struct Biol, 1999 Apr, 6(4), 359 - 65
The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia; Guenther BD et al.; Elevated plasma homocysteine levels are associated with increased risk for cardiovascular disease and neural tube defects in humans . Folate treatment decreases homocysteine levels and dramatically reduces the incidence of neural tube defects . The flavoprotein methylenetetrahydrofolate reductase (MTHFR) is a likely target for these actions of folate . The most common genetic cause of mildly elevated plasma homocysteine in humans is the MTHFR polymorphism A222V (base change C677-->T) . The X-ray analysis of E . coli MTHFR, reported here, provides a model for the catalytic domain that is shared by all MTHFRs . This domain is a beta8alpha8 barrel that binds FAD in a novel fashion . Ala 177, corresponding to Ala 222 in human MTHFR, is near the bottom of the barrel and distant from the FAD . The mutation A177V does not affect Km or k(cat) but instead increases the propensity for bacterial MTHFR to lose its essential flavin cofactor . Folate derivatives protect wild-type and mutant E . coli enzymes against flavin loss, and protect human MTHFR and the A222V mutant against thermal inactivation, suggesting a mechanism by which folate treatment reduces homocysteine levels.

Nat Struct Biol, 1999 Apr, 6(4), 336 - 9
Pilus chaperone FimC-adhesin FimH interactions mapped by TROSY-NMR; Pellecchia M et al.; The 23 kDa two-domain periplasmic chaperone FimC from Escherichia coli is required for the assembly of type-1 pili, which are filamentous, highly oligomeric protein complexes anchored to the outer bacterial membrane that mediate adhesion of pathogenic E . coli strains to host cell surfaces . Here we identified the contact sites on the surface of the NMR structure of FimC that are responsible for the binding of the 28 kDa mannose-binding type-1 pilus subunit FimH by 15N and 1H NMR chemical shift mapping, using transverse relaxation-optimized spectroscopy (TROSY) . The FimH-binding surface of FimC is formed nearly entirely by the N-terminal domain, and its extent and shape indicate that FimC binds a folded form of the pilus subunits.

Nature, 1999 Apr 1, 398(6726), 427 - 31
Local inhibition and long-range enhancement of Dpp signal transduction by Sog; Ashe HL et al.; Extracellular gradients of signalling molecules can specify different thresholds of gene activity in development . A gradient of Decapentaplegic (Dpp) activity subdivides the dorsal ectoderm of the Drosophila embryo into amnioserosa and dorsal epidermis . The proteins Short gastrulation (Sog) and Tolloid (Tld) are required to shape this gradient . Sog has been proposed to form an inhibitory complex with either Dpp or the related ligand Screw, and is subsequently processed by the protease Tld . Paradoxically, Sog appears to be required for amnioserosa formation, which is specified by peak Dpp signalling activity . Here we show that the misexpression of sog using the even-skipped stripe-2 enhancer redistributes Dpp signalling in a mutant background in which dpp is expressed throughout the embryo . Dpp activity is diminished near the Sog stripe and peak Dpp signalling is detected far from this stripe . However, a tethered form of Sog suppresses local Dpp activity without augmenting Dpp activity at a distance, indicating that diffusion of Sog may be required for enhanced Dpp activity and consequent amnioserosa formation . The long-distance stimulation of Dpp activity by Sog requires Tld, whereas Sog-mediated inhibition of Dpp does not . The heterologous Dpp inhibitor Noggin inhibits Dpp signalling but fails to augment Dpp activity . These results suggest an unusual strategy for generating a gradient threshold of growth-factor activity, whereby Sog and its protease specify peak Dpp signalling far from a localized source of Sog.

Mol Microbiol, 1999 Mar, 31(5), 1321 - 32
Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30); Drummelsmith J et al.; The group 1 K30 antigen from Escherichia coli (O9a:K30) is present on the cell surface as both a capsular structure composed of high-molecular-weight K30 polysaccharide and as short K30 oligosaccharides linked to lipid A-core in a lipopolysaccharide molecule (K30LPS) . To determine the molecular processes that are responsible for the two forms of K antigen, the 16 kb chromosomal cps region has been characterized . This region encodes 12 gene products required for the synthesis, polymerization and translocation of the K30 antigen . The gene products include four glycosyltransferases responsible for synthesis of the K30 repeat unit; a PST (1) exporter (Wzx), required to transfer lipid-linked K30 units across the plasma membrane to the periplasmic space; and a K30-antigen polymerase (Wzy) . These gene products are typical of those seen in O-antigen biosynthesis gene clusters and they interact with the lipopolysaccharide translocation pathway to express K30LPS on the cell surface . The same gene products also provide the biosynthetic intermediates for the capsule assembly pathway, although they are not in themselves sufficient for synthesis of the K30 capsule . Three additional genes, wza, wzb and wzc, encode homologues to proteins that are encoded by gene clusters involved in expression of a variety of bacterial exopolysaccharides . Mutant analysis indicates that Wza and Wzc are required for wild-type surface expression of the capsular structure but are not essential for polymerization and play no role in the translocation of K30LPS . These surface expression components provide the key feature that distinguishes the assembly systems for O antigens and capsules.

J Immunol, 1999 Apr 1, 162(7), 4342 - 8
Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen; Asturias JA et al.; Inhalation of allergens produced by the American cockroach (Periplaneta americana) induces IgE Ab production and the development of asthma in genetically predisposed individuals . The cloning and expression in Escherichia coli of P . americana tropomyosin allergen have been achieved . The protein shares high homology with other arthropod tropomyosins (80% identity) but less homology with vertebrate ones (50% identity) . The recombinant allergen was produced in E . coli as a nonfusion protein with a yield of 9 mg/l of bacterial culture . Both natural and recombinant tropomyosins were purified by isoelectric precipitation . P . americana allergen 1 (Per a 1) and Per a 7 (tropomyosin) are to date the only cross-reacting allergens found in cockroaches . ELISA and Western blot inhibition experiments, using natural and recombinant purified tropomyosins from shrimp and cockroach, showed that tropomyosin induced cross-reactivity of IgE from patients allergic to these allergens, suggesting that this molecule could be a common allergen among invertebrates.

J Biotechnol, 1999 Mar 26, 69(1), 63 - 7
N-terminal methionine in recombinant proteins expressed in two different Escherichia coli strains; Vassileva-Atanassova A et al.; Two genes coding for chloramphenicol acetyltransferase and human interferon gamma, respectively, were overexpressed constitutively in two different strains of Escherichia coli (E . coli LE392 and E . coli XL1) . The N-terminal amino acid analysis of the purified proteins showed that: (a) the N-terminal methionine is processed more efficiently in E . coli LE392 rather than in E . coli XL1 cells; (b) the N-terminal methionine is removed better from the heterologous human interferon gamma in comparison with the homologous chloramphenicol acetyltransferase protein: and (c) there is no strong correlation between the efficiency of N-terminal procession and the yield of recombinant protein.

J Biotechnol, 1999 Mar 26, 69(1), 1 - 7
High performance flow injection analysis of recombinant protein G; Hagedorn J et al.; Chromatographic discs were investigated for their potential to substitute for the hitherto used cartridges in heterogeneous flow injection analysis . Originally designed for fast high performance liquid chromatography (HPLC) of biopolymers, the discs combine reliability with speed and resolution . This together with their price and their long-standing time made them attractive for use in flow injection analysis . The base material of the discs is a glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) co-polymer . The epoxy groups inherent to this base structure can be used for immobilization purposes . In this first demonstration, antibodies were immobilized and the resulting affinity discs used for the fast analysis (< 5 min) of protein G from cell lysate of recombinant Escherichia coli . A linear calibration curve over several orders of magnitude as well as excellent reproducibility and correlation with data produced by conventional protein assay were obtained.

Kidney Int, 1999 Apr, 55(4), 1367 - 74
A laboratory model of toxin-induced hemolytic uremic syndrome; Taylor CM et al.; BACKGROUND: Verocytotoxin-producing (Shiga-like toxin-producing) Escherichia coli infection is the principal cause of hemolytic uremic syndrome (HUS) . The pathogenesis is unclear, and there is a need for animal models . These are impeded by the different distribution of verocytotoxin receptors between species . We have circumvented this restriction using ricin, which gains entry into cells via various galactose receptors . Like verocytotoxin, ricin specifically cleaves a single adenine from ribosomal RNA . METHODS: Rats were given ricin at a dose of 6.7 micrograms/100 g body wt, with or without lipopolysaccharide at 10 micrograms/100 g body wt . Lipopolysaccharide alone or saline were used as controls . Changes in glomerular filtration rate, hematological parameters, histology, and plasma cytokine concentrations were measured . RESULTS: Extensive glomerular thrombosis, pyknotic nuclei, and an infiltration of ED1-positive cells into glomeruli were observed eight hours after an injection of ricin . Other vascular beds were unaffected . Histologic changes were preceded by oliguric renal failure, hemolysis, and thrombocytopenia . Ricin produced a rise in plasma concentrations of monocyte chemotactic protein-1, > tumor necrosis factor-alpha, > interleukin-1 beta, > interleukin-6 . Interferon-gamma showed a small increase at the end of the experiment . CONCLUSIONS: Ricin induces glomerular thrombotic microangiopathy, closely resembling that which occurs in verocytotoxin-producing E . coli-induced HUS . As in HUS, high concentrations of proinflammatory cytokines are present, which are probably a result of cytokine superinduction by the toxin.

Kidney Int, 1999 Apr, 55(4), 1234 - 40
Gene therapy for renal anemia in mice with polycystic kidney using an adenovirus vector encoding the human erythropoietin gene; Osada S et al.; BACKGROUND: Recombinant human erythropoietin (rHuEPO) is primarily used for patients with anemia associated with end-stage renal disease . We evaluated the efficacy of EPO gene therapy using adenovirus vector for chronic renal failure mice expressing severe renal anemia . METHODS: Recombinant HuEPO gene transfer to mesothelial cells was performed in vitro and in vivo . Recombinant replication-deficient adenoviruses containing rHuEPO cDNA (AdCMVEPO), E . coli lacZ gene (AdCMVlacZ), or an nonexogenous gene (AdNull as control vector) driven by the cytomegalovirus promotor/enhancer were constructed . The oligosaccharides associated with the rHuEPO from AdCMVEPO-treated mesothelial cells were analyzed . For in vivo study, the DBA/2FG-pcy mouse, a model for human autosomal recessive polycystic kidney disease resulting in chronic renal failure with progressive anemia, was used . RESULTS: The sialylated oligosaccharides associated with the rHuEPO produced in AdCMVEPO-treated mesothelial cells occupied 78 +/- 0.7% of the total oligosaccharide pool . A single intraperitoneal administration of AdCMVEPO induced rHuEPO synthesis in the peritoneal cells and a marked increase in erythrocyte production . The maximal increase in hematocrit (43 +/- 4%) was observed on day 28, and it remained elevated for 40 days . CONCLUSION: These results indicate that intraperitoneal administration of AdCMVEPO improves renal anemia in mice with chronic renal failure and that the mesothelial cell is an appropriate target cell for gene transfer.

Mol Microbiol, 1999 Mar, 31(5), 1537 - 48
Genetic and functional characterization of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric tissue; Odenbreit S et al.; In this study, we isolated and characterized a chromosomal locus of Helicobacter pylori previously identified by transposon shuttle mutagenesis as being involved in the adhesion of the pathogen to gastric epithelial cells . Two closely homologous genes were identified, designated as alpA and alpB, encoding outer membrane (OM) proteins of 518 amino acids each . They are members of the outer membrane protein supergene family identified in the H . pylori 26695 complete genome sequence . AlpA carries a functional lipoprotein signal sequence . AlpB carries a putative standard N-terminal signal sequence and shows a strong amino-acid sequence identity to AlpA . Transposon insertion mutagenesis, immunoblotting and primer extension studies indicate that both genes are organized in an operon, but no obvious consensus promoter sequence was found upstream of the transcriptional start site . The C-terminal portion of both proteins is predicted to form a porin-like beta-barrel in the outer membrane, consisting of 14 transmembrane amphipathic beta-strands . Adhesion experiments with defined isogenic mutants indicate that both proteins are necessary for specific adherence of H . pylori to human gastric tissue . The pattern of AlpAB-dependent adherence of H . pylori to the gastric epithelial surface shows a clear difference to the BabA2-mediated adherence to Lewis, suggesting that a different receptor is involved.

Mol Microbiol, 1999 Mar, 31(5), 1429 - 41
Characterization of Escherichia coli cspE, whose product negatively regulates transcription of cspA, the gene for the major cold shock protein; Bae W et al.; Escherichia coli contains nine members of the CspA protein family from CspA to Cspl . To elucidate the cellular function of CspE, we constructed a delta cspE strain . CspE is highly produced at 37 degrees C . The synthesis level of CspE transiently increased during the growth lag period after dilution of stationary-phase cells into the fresh medium at 37 degrees C . This is consistent with the delta cspE phenotype of the longer growth lag period after dilution . The protein synthesis patterns of the delta cspE strain and the wild-type strain were compared using two-dimensional gel electrophoresis . In the delta cspE strain, the synthesis of a number of proteins at 37 degrees C was found to be altered and cspA was derepressed . The derepression of cspA in the delta cspE strain was at the level of transcription in a promoter-independent fashion but was not caused by stabilization of the cspA mRNA, which was shown to be a major cause of CspA induction after cold shock . In vitro transcription assays demonstrated that both CspE and CspA enhanced transcription pause at the region immediately downstream of the cold box, a putative repressor binding site on the cspA mRNA . In a cell-free protein synthesis system using S-30 cell extracts, CspA production was specifically inhibited by the addition of CspE . These results indicate that CspE functions as a negative regulator for cspA expression at 37 degrees C, probably by interacting with the transcription elongation complex at the cspA cold box region.

Mol Microbiol, 1999 Mar, 31(5), 1359 - 72
Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice; Chevalier C et al.; Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H . pylori isolates . Helicobacter pylori GGT was purified from sonicated extracts of H . pylori strain 85P by anion exchange chromatography . The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H . pylori library . The H . pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa . Escherichia coli clones harbouring the H . pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E . coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C . GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae . Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H . pylori GGT is synthesized in both H . pylori and E . coli as a pro-GGT that is processed into a large and a small subunit . Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H . pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro . These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain . These results demonstrate that H . pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4698 - 703
Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation; Thomas SG et al.; A major catabolic pathway for the gibberellins (GAs) is initiated by 2beta-hydroxylation, a reaction catalyzed by 2-oxoglutarate-dependent dioxygenases . To isolate a GA 2beta-hydroxylase cDNA clone we used functional screening of a cDNA library from developing cotyledons of runner bean (Phaseolus coccineus L.) with a highly sensitive tritium-release assay for enzyme activity . The encoded protein, obtained by heterologous expression in Escherichia coli, converted GA9 to GA51 (2beta-hydroxyGA9) and GA51-catabolite, the latter produced from GA51 by further oxidation at C-2 . The enzyme thus is multifunctional and is best described as a GA 2-oxidase . The recombinant enzyme also 2beta-hydroxylated other C19-GAs, although only GA9 and GA4 were converted to the corresponding catabolites . Three related cDNAs, corresponding to gene sequences present in Arabidopsis thaliana databases, also encoded functional GA 2-oxidases . Transcripts for two of the Arabidopsis genes were abundant in upper stems, flowers, and siliques, but the third transcript was not detected by Northern analysis . Transcript abundance for the two most highly expressed genes was lower in apices of the GA-deficient ga1-2 mutant of Arabidopsis than in wild-type plants and increased after treatment of the mutant with GA3 . This up-regulation of GA 2-oxidase gene expression by GA contrasts GA-induced down-regulation of genes encoding the biosynthetic enzymes GA 20-oxidase and GA 3beta-hydroxylase . These mechanisms would serve to maintain the concentrations of biologically active GAs in plant tissues.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4610 - 4
Escherichia coli genes regulated by cell-to-cell signaling; Baca-DeLancey RR et al.; Utilizing the bicistronic reporter transposon mini-Tn5 lacZ-tet/1, we have identified lacZ fusions to four Escherichia coli genes/operons that are strongly activated by the accumulation of self-produced extracellular signals . These fusions were designated cma9, cma48, cma113, and cma114 for conditioned medium activated . Each of the cma fusions was expressed in a growth phase-dependent manner, and the presence of conditioned medium from a stationary phase E . coli culture resulted in the premature activation of these fusions in cells at early to mid-logarithmic phase . The cma48 and cma114 fusions were dependent on RpoS for growth phase expression and response to extracellular factors . The extracellular factors that activated the cma9, cma48, and cma114 fusions were produced in both rich complex and defined minimal media . The cma fusions were shown to be within the cysK (cma9), astD (cma48), tnaB (cma113), and gabT (cma114) genes . These genes function in the uptake, synthesis, or degradation of amino acids that yield pyruvate and succinate.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4604 - 9
Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology; Scherzinger E et al.; Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine {poly(Q)} repeat expansion in the first exon of the huntingtin protein . Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease . Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time . Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils . Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4366 - 71
A molecular trigger of lipid binding-induced opening of a helix bundle exchangeable apolipoprotein; Narayanaswami V et al.; Apolipophorin III (apoLp-III) from the sphinx moth, Manduca sexta, is a helix bundle protein that interacts reversibly with lipoproteins . Its five elongated amphipathic alpha-helices are organized in an antiparallel fashion, with helices 3 and 4 connected by a short 6-residue (PDVEKE) linker helix, termed helix 3' . Upon interaction with lipoproteins, apoLp-III opens to expose a continuous hydrophobic interior . It was postulated that helix bundle opening is preceded by an initiation step wherein helix 3' serves to recognize available lipoprotein surface binding sites . To test this hypothesis, helix 3' was replaced by residues that have a propensity to form a type I beta-turn, NPNG . This mutant apoLp-III was defective in lipoprotein binding assays . To define a more precise mode of interaction, the relevance of the presence of the hydrophobic Val-97 flanked by Asp-96 and Glu-98 was investigated by site-directed mutagenesis . V97N and D96N/V97N/E98Q apoLp-III were unable to compete with wild-type apoLp-III to initiate an interaction with lipoproteins, whereas D96N/E98Q apoLp-III was as competent as wild-type apoLp-III . The results suggest that Val-97 is critical, whereas Asp-96 and Glu-98 are irrelevant for initiating binding to lipoproteins . A model of binding is presented wherein apoLp-III is oriented with the helix 3' end of the molecule juxtaposed to the lipoprotein surface . Recognition of lipoprotein surface hydrophobic defects by Val-97 triggers opening of the helix bundle and facilitates formation of a stable binding interaction.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4285 - 8
Assigning protein functions by comparative genome analysis: protein phylogenetic profiles; Pellegrini M et al.; Determining protein functions from genomic sequences is a central goal of bioinformatics . We present a method based on the assumption that proteins that function together in a pathway or structural complex are likely to evolve in a correlated fashion . During evolution, all such functionally linked proteins tend to be either preserved or eliminated in a new species . We describe this property of correlated evolution by characterizing each protein by its phylogenetic profile, a string that encodes the presence or absence of a protein in every known genome . We show that proteins having matching or similar profiles strongly tend to be functionally linked . This method of phylogenetic profiling allows us to predict the function of uncharacterized proteins.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4268 - 72
A mutation in the heterotrimeric stimulatory guanine nucleotide binding protein alpha-subunit with impaired receptor-mediated activation because of elevated GTPase activity; Warner DR et al.; It has been reported that substitution of Arg258, a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) alpha-subunit (alphas), to alanine (alphas-R258A) results in decreased activation by receptor or aluminum fluoride (AlF4-) and increased basal GDP release . Arg258 interacts with Gln170 in the helical domain, and, presumably, loss of this interaction between the GTPase and helical domain leads to more rapid GDP release, resulting in decreased activation by AlF4- and increased thermolability . In this study, we mutate Gln170 to alanine (alphas-Q170A) and demonstrate that this mutant, like alphas-R258A, has decreased activation by AlF4-, increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release . However, unlike alphas-R258A, alphas-Q170A does not have impaired receptor-mediated activation . Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by AlF4-) but is not important for receptor-mediated activation . In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of alphas-R258A was 14-fold higher than normal whereas that of alphas-Q170A was unaffected . Examination of the alphas crystal structure suggests that Arg258, through interactions with Glu50, might constrain the position of Arg201, a residue critical for catalyzing the GTPase reaction . This is an example of a mutation in a heterotrimeric G protein that results in an increased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction.

Biochemistry, 1999 Apr 13, 38(15), 4834 - 45
Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis; Shroyer MJ et al.; The role of the conserved histidine-187 located in the leucine intercalation loop of Escherichia coli uracil-DNA glycosylase (Ung) was investigated . Using site-directed mutagenesis, an Ung H187D mutant protein was created, overproduced, purified to apparent homogeneity, and characterized in comparison to wild-type Ung . The properties of Ung H187D differed from Ung with respect to specific activity, substrate specificity, DNA binding, pH optimum, and inhibition by uracil analogues . Ung H187D exhibited a 55000-fold lower specific activity and a shift in pH optimum from pH 8.0 to 7.0 . Under reaction conditions optimal for wild-type Ung (pH 8.0), the substrate preference of Ung H187D on defined single- and double-stranded oligonucleotides (25-mers) containing a site-specific uracil target was U/G-25-mer > U-25-mer > U/A-25-mer . However, Ung H187D processed these same DNA substrates at comparable rates at pH 7.0 and the activity was stimulated approximately 3-fold relative to the U-25-mer substrate . Ung H187D was less susceptible than Ung to inhibition by uracil, 6-amino uracil, and 5-fluorouracil . Using UV-catalyzed protein/DNA cross-linking to measure DNA binding affinity, the efficiency of Ung H187D binding to thymine-, uracil-, and apyrimidinic-site-containing DNA was (dT20) = (dT19-U) >/= (dT19-AP) . Comparative analysis of the biochemical properties and the X-ray crystallographic structures of Ung and Ung H187D {Putnam, C . D., Shroyer, M . J . N., Lundquist, A . J., Mol, C . D., Arvai, A . S., Mosbaugh, D . W., and Tainer, J . A . (1999) J . Mol . Biol . 287, 331-346} provided insight regarding the role of His-187 in the catalytic mechanism of glycosylic bond cleavage . A novel mechanism is proposed wherein the developing negative charge on the uracil ring and concomitant polarization of the N1-C1' bond is sustained by resonance effects and hydrogen bonding involving the imidazole side chain of His-187.

Biochemistry, 1999 Apr 13, 38(15), 4794 - 9
DnrD cyclase involved in the biosynthesis of doxorubicin: purification and characterization of the recombinant enzyme; Kendrew SG et al.; Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester (AAME) to aklaviketone (AK), an intermediate in the biosynthetic pathway to daunorubicin (DNR) and doxorubicin . To investigate the role of DnrD in this transformation, its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized . The enzyme was shown to catalyze the conversion of AAME to AK presumably via an intramolecular aldol condensation mechanism . In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin (TCM) C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the AAME precursor . Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring . As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters that are important for aromatic ring formation . In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.

Biochemistry, 1999 Apr 13, 38(15), 4782 - 93
Identifying groups involved in the binding of prephenate to prephenate dehydrogenase from Escherichia coli; Christendat D et al.; Site-directed mutagenesis was used to investigate the importance of Lys178, Arg286, and Arg294 in the binding of prephenate to the bifunctional enzyme chorismate mutase-prephenate dehydrogenase . From comparison of the kinetic parameters of wild-type enzyme and selected mutants, we conclude that only Arg294 interacts specifically with prephenate . The R294Q substitution reduces the enzyme's affinity for prephenate without affecting V/Et of the dehydrogenase reaction or the kinetic parameters of the mutase reaction . Arg294 likely interacts with the ring carboxylate at C-1 of prephenate since the dissociation constants for a series of inhibitors missing the ring carboxyl group were similar for wild-type and R294Q enzymes . The pH dependencies of log (V/KprephenateEt) and of pKi for hydroxyphenyllactate show that the wild-type dehydrogenase possesses a group with a pK of 8.8 that must be protonated for binding prephenate to the enzyme . None of the three conserved residues is this group since its titration is observed in the V/KprephenateEt profiles for the mutants K178Q, R286A, and R294Q . This group is also seen in the pH-rate profiles of the binding of two substrate analogues, hydroxyphenyllactate and deoxoprephenate . Their only common structural feature at C-1 is the side chain carboxylate, indicating that the protonated residue (pK 8.8) must interact with prephenate's side chain carboxylate . Gdn-HCl-induced denaturation was conducted on wild-type and selected mutant proteins . Unfolding of the wild-type enzyme proceeds through a partially unfolded dimer which dissociates into unfolded monomers . The order of stability is wild-type = R294Q > K178Q > R286A > K178R . The least unstable mutants have reduced mutase and dehydrogenase activities.

Biochemistry, 1999 Apr 13, 38(15), 4736 - 42
The folding and structural integrity of the first LIN-12 module of human Notch1 are calcium-dependent; Aster JC et al.; Notch1 is a member of a conserved family of large modular type 1 transmembrane receptors that control differentiation in multicellular animals . Notch function is mediated through a novel signal transduction pathway involving successive ligand-induced proteolytic cleavages that serve to release the intracellular domain of Notch, which then translocates to the nucleus and activates downstream transcription factors . The extracellular domain of all Notch receptors have three iterated LIN-12 modules that appear to act as negative regulatory domains, possibly by limiting proteolysis . Each LIN-12 module contains three disulfide bonds and three conserved aspartate (D) or asparagine (N) residues . To begin to understand the structural basis for LIN-12 function, the first LIN-12 module of human Notch1 (rLIN-12.1) has been expressed recombinantly in Escherichia coli and purified in a reduced form . In redox buffers, rLIN-12.1 forms only one disulfide isomer in the presence of millimolar Ca2+ concentrations, whereas multiple disulfide isomers are observed in the presence of Mg2+ and EDTA . Further, mutation of conserved residues N1460, D1475, and D1478 to alanine abolishes Ca2+-dependent folding of this module . Mass spectrometric analysis of partially reduced rLIN-12.1 has been used to deduce that disulfide bonds are formed between the first and fifth (C1449-C1472), second and fourth (C1454-C1467), and third and sixth (C1463-C1479) cysteines of this prototype module . This arrangement is distinct from that observed in other modules, such as EGF and LDL-A, that also contain three disulfide bonds . One-dimensional proton nuclear magnetic resonance shows that Ca2+ induces a dramatic increase in the extent of chemical shift dispersion of the native rLIN-12.1 amide protons, as seen for the Ca2+-binding LDL-A modules . We conclude that Ca2+ is required both for proper folding and for the maintenance of the structural integrity of Notch/LIN-12 modules.

Am J Physiol, 1999 Apr, 276(4 Pt 2), H1207 - 14
Hypoxic contraction of small pulmonary arteries from normal and endotoxemic rats: fundamental role of NO; Terraz S et al.; The present study was aimed at examining the role of nitric oxide (NO) in the hypoxic contraction of isolated small pulmonary arteries (SPA) in the rat . Animals were treated with either saline (sham experiments) or Escherichia coli lipolysaccharide {LPS, to obtain expression of the inducible NO synthase (iNOS) in the lung} and killed 4 h later . SPA (300- to 600-micrometer outer diameter) were mounted as rings in organ chambers for the recording of isometric tension, precontracted with PGF2alpha, and exposed to either severe (bath PO2 8 +/- 3 mmHg) or milder (21 +/- 3 mmHg) hypoxia . In SPA from sham-treated rats, contractions elicited by severe hypoxia were completely suppressed by either endothelium removal or preincubation with an NOS inhibitor {NG-nitro-L-arginine methyl ester (L-NAME), 10(-3) M} . In SPA from LPS-treated rats, contractions elicited by severe hypoxia occurred irrespective of the presence or absence of endothelium and were largely suppressed by L-NAME . The milder hypoxia elicited no increase in vascular tone . These results indicate an essential role of NO in the hypoxic contractions of precontracted rat SPA . The endothelium independence of HPV in arteries from LPS-treated animals appears related to the extraendothelial expression of iNOS . The severe degree of hypoxia required to elicit any contraction is consistent with a mechanism of reduced NO production caused by a limited availability of O2 as a substrate for NOS.

Transplantation, 1999 Mar 27, 67(6), 775 - 83
Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation; Bilbao G et al.; BACKGROUND: Liver function after transplantation is determined by the quality of the donor organ and the influences of preservation, flush, and reperfusion injury . In this regard, cell death (apoptosis) plays an important role in organ preservation and rejection . Therefore, we examined the possibility of genetic modification of the liver graft with a recombinant adenovirus vector encoding the Bcl-2 gene to reduce apoptosis during the preservation time . METHODS: Liver grafts from C57B1/6 mice were procured and preserved using standard techniques . A replication defective adenovirus vector (deltaE1) containing the human Bcl-2 gene (AdCMVhBcl-2) was developed in our laboratory . An adenovirus vector encoding an irrelevant gene (Escherichia coli beta-galactosidase) was used as a control . Each mouse received 1 x 10(9) plaque forming units administered i.v . 48 hr before the liver procurement . Analyses of liver enzyme activities were determined in the preservation solution . Apoptosis in liver biopsies was determined by DNA fragmentation with an in situ histochemical assay . RESULTS: Immunohistochemical analysis and RT-PCR confirmed the expression of hBcl-2 in the grafts . Grafts from livers expressing hBcl-2 showed significant reduction of the aspartame amino transferase (AST) and lactate dehydrogenase (LDH) release compared with grafts from the control groups . After rewarming, significant cytoprotection was also observed in grafts from animals treated with AdCMVhBcl-2 . Histological analysis correlated with the hepatocellular injury determined with transaminases and LDH in the preservation solution . Significant reduction in the number of apoptotic cells was observed in grafts expressing hBcl-2 . CONCLUSIONS: We have demonstrated a novel approach to reducing the preservation injury to liver grafts with the human Bcl-2 gene . This approach may allow a longer preservation time, potentially reduce the incidence of primary nonfunction, decrease the immunogenicity of the cold injured organ, and increase the safer use of "marginal" liver grafts.

Xenobiotica, 1999 Feb, 29(2), 187 - 93
Contribution of human hepatic cytochrome P450s and steroidogenic CYP17 to the N-demethylation of aminopyrine; Niwa T et al.; 1 . Aminopyrine N-demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli . 2 . Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible . The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by Lineweaver-Burk plots . CYP2C19 exhibited the highest affinity and a high capacity for the aminopyrine N-demethylation . CYP2C8 showed the highest Vmax, followed by CYP2C19, 2D6 and 1A2, whereas the Km for CYP2C8, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19 . Accordingly, the Vmax/Km for CYP2C19 was more than nine times higher than that of other P450s . 3 . Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver . The activity was increased 1.5-fold by the addition of cytochrome b5, whereas the activity was not affected by the addition of Mg2+ . 4 . These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.

RNA, 1999 Apr, 5(4), 495 - 502
The peculiar architectural framework of tRNASec is fully recognized by yeast AspRS; Rudinger-Thirion J et al.; The wild-type transcript of Escherichia coli tRNASec, characterized by a peculiar core architecture and a large variable region, was shown to be aspartylatable by yeast AspRS . Similar activities were found for tRNASec mutants with methionine, leucine, and tryptophan anticodons . The charging efficiency of these molecules was found comparable to that of a minihelix derived from tRNAAsp and is accounted for by the presence of the discriminator residue G73, which is a major aspartate identity determinant . Introducing the aspartate identity elements from the anticodon loop (G34, U35, C36, C38) into tRNASec transforms this molecule into an aspartate acceptor with kinetic properties identical to tRNAAsp . Expression of the aspartate identity set in tRNASec is independent of the size of its variable region . The functional study was completed by footprinting experiments with four different nucleases as structural probes . Protection patterns by AspRS of transplanted tRNASec and tRNAAsp were found similar . They are modified, particularly in the anticodon loop, upon changing the aspartate anticodon into that of methionine . Altogether, it appears that recognition of a tRNA by AspRS is more governed by the presence of the aspartate identity set than by the structural framework that carries this set.

Crit Care Med, 1999 Mar, 27(3), 597 - 604
Combined antithrombin III and C1-esterase inhibitor treatment decreases intravascular fibrin deposition and attenuates cardiorespiratory impairment in rabbits exposed to Escherichia coli endotoxin; Giebler R et al.; OBJECTIVE: To assess the effect of a combined antithrombin III and C1-esterase inhibitor treatment on intravascular organ fibrin deposition and cardiorespiratory changes following intravenous Escherichia coli endotoxin (lipopolysaccharide {LPS} 80 microg/kg i.v.) exposure . DESIGN: Prospective, randomized trial . SETTING: Research laboratory of a university medical center . SUBJECTS: Anesthetized, instrumented and mechanically ventilated rabbits ({Chbb:CH); n = 40) . INTERVENTIONS: Endotoxin was given to 30 animals . Ten animals received no inhibitor (endotoxin control group) . The other animals were either treated by high-dose (300 units/kg; n = 10) or low-dose (100 units/kg; n = 10) combined antithrombin III and C1-esterase inhibitor administration . Ten rabbits (time control group) were given placebo (sodium chloride 0.9%) . Cardiorespiratory variables were assessed at baseline, 120 mins, and 240 mins after endotoxin or placebo administration . Four hours after endotoxin injection, liver, lung, and kidney tissue samples were examined for intravascular fibrin deposition by light microscopy . MEASUREMENTS AND MAIN RESULTS: Inhibitor treatment significantly decreased clot formation in lungs and livers without, however, demonstrating a clear dose-dependent effect . Combined antithrombin III/C1-esterase treatment attenuated the decrease of mean arterial pressure and cardiac output observed following endotoxin injection . Blood pressure improvement was significantly dependent on dosage administered . CONCLUSION: Combination of antithrombin III and C1-esterase inhibitor treatment during early endotoxin shock decreased organ fibrin deposition and improved cardiovascular stability.

Crit Care Med, 1999 Mar, 27(3), 588 - 96
Large-pore hemodialysis in acute endotoxin shock; Kline JA et al.; OBJECTIVE: This study was undertaken to test the hypothesis that hemodialysis with a large-pore membrane would improve heart function during acute endotoxin shock . SETTING: Large animal laboratory . DESIGN: Eighteen mongrel dogs were instrumented to measure left ventricular maximum end-systolic elastance (left ventricular maximum elastance at end systole), cardiac output, circumflex artery blood flow, and myocardial mechanical efficiency (CO x MAP/MVO2, where CO is cardiac output, MAP is mean arterial pressure, and MVO2 is myocardial oxygen consumption) . Plasma catecholamine concentrations were determined by high-performance liquid chromatography . Endotoxin shock was induced by infusing 5.0 microg/kg/min of Escherichia col 0127:B8 endotoxin in the portal vein for 60 mins, followed by 2.0 microg/kg/min of constant infusion . Control dogs (n = 6) received 4.0 mL/kg/min of saline; hemodialysis dogs (n = 6) underwent venovenous hemodialysis in 50-min intervals using a polysulfone filter (1.2 m2; mean pore size, 0.50 nm; blood flow rate, 400 mL/min; ultrafiltrate, "zero-balanced"); shams (n = 5) were treated identically to hemodialysis dogs, except that no convective dialysis was performed . A fourth group (n = 6) was treated with dopamine (5.0-7.0 microg/kg/min, optimal dose for contractile increase based on dose-response studies) . MEASUREMENTS AND MAIN RESULTS: After 2 hrs of treatment, left ventricular maximum elastance at end systole increased and was unchanged in controls (30 +/- 5 mm Hg/mm) and shams (24 +/- 6 mm Hg/mm) compared with basal control . Hemodialysis treatment increased contractility (53 +/- 4 mm Hg/mm), as did dopamine treatment (54 +/- 7 mm Hg/mm) . Endotoxin shock reduced mechanical efficiency to 45% of basal control; with hemodialysis treatment, left ventricular efficiency returned to 64% of basal control measurement, compared with 49% with dopamine treatment . During treatment, myocardial glucose uptake was increased with hemodialysis compared with other groups . No difference was observed among groups for left ventricular end-diastolic pressures or dimensions, or catecholamine concentrations . CONCLUSIONS: Large-pore hemodialysis increased left ventricular contractility to a similar degree as dopamine and provided a marginal improvement in myocardial glucose uptake and mechanical efficiency.

Cell, 1999 Apr 2, 97(1), 85 - 97
Transformation of MutL by ATP binding and hydrolysis: a switch in DNA mismatch repair; Ban C et al.; The MutL DNA mismatch repair protein has recently been shown to be an ATPase and to belong to an emerging ATPase superfamily that includes DNA topoisomerase II and Hsp90 . We report here the crystal structures of a 40 kDa ATPase fragment of E . coli MutL (LN40) complexed with a substrate analog, ADPnP, and with product ADP . More than 60 residues that are disordered in the apoprotein structure become ordered and contribute to both ADPnP binding and dimerization of LN40 . Hydrolysis of ATP, signified by subsequent release of the gamma-phosphate, releases two key loops and leads to dissociation of the LN40 dimer . Dimerization of the LN40 region is required for and is the rate-limiting step in ATP hydrolysis by MutL . The ATPase activity of MutL is stimulated by DNA and likely acts as a switch to coordinate DNA mismatch repair.

Nucleic Acids Res, 1999 May 1, 27(9), 2051 - 6
Open complex formation during transcription initiation at the Escherichia coli galP1 promoter: the role of the RNA polymerase alpha subunit at promoters lacking an UP-element; Burns HD et al.; We have studied the role of the C-terminal domain of the alpha subunit (alphaCTD) of Escherichia coli RNA polymerase during transcription initiation at promoters lacking an UP-element . The temperature requirement for open complex formation was used as an indication of the kinetics of this process . We have previously shown that alphaCTD is required for transcription initiation at low temperature at the galP1 promoter, a promoter containing an UP-element . DNase I footprinting has been used to reveal the structure of open promoter complexes and the temperature requirement for open complex formation has been determined using potassium permanganate as a probe . In this work we show that, although alphaCTD is not absolutely required for transcription initiation at promoters lacking an UP-element, it does play a role during transcription initiation . This role is independent of the sequence of the promoter upstream from the -35 region and does not require stable alphaCTD-DNA interactions as determined by DNase I footprinting . The role of alphaCTD at promoters lacking an UP-element is discussed.

Am J Physiol, 1999 Apr, 276(4 Pt 1), G1016 - 26
Mechanisms underlying the anti-inflammatory actions of central corticotropin-releasing factor; Casadevall M et al.; Immune activation of hypothalamic corticotropin-releasing factor (CRF) provides a negative feedback mechanism to modulate peripheral inflammatory responses . We investigated whether central CRF attenuates endothelial expression of intercellular adhesion molecule 1 (ICAM-1) and leukocyte recruitment during endotoxemia in rats and determined its mechanisms of action . As measured by intravital microscopy, lipopolysaccharide (LPS) induced a dose-dependent increase in leukocyte rolling, adhesion, and emigration in mesenteric venules, which was associated with upregulation of endothelial ICAM-1 expression . Intracisternal injection of CRF abrogated both the increased expression of ICAM-1 and leukocyte recruitment . Intravenous injection of the specific CRF receptor antagonist astressin did not modify leukocyte-endothelial cell interactions induced by a high dose of LPS but enhanced leukocyte adhesion induced by a low dose . Blockade of endogenous glucocorticoids but not alpha-melanocyte-stimulating hormone (alpha-MSH) receptors reversed the inhibitory action of CRF on leukocyte-endothelial cell interactions during endotoxemia . In conclusion, cerebral CRF blunts endothelial upregulation of ICAM-1 and attenuates the recruitment of leukocytes during endotoxemia . The anti-inflammatory effects of CRF are mediated by adrenocortical activation and additional mechanisms independent of alpha-MSH.

Am J Physiol, 1999 Apr, 276(4 Pt 1), E635 - 41
Mechanism of adipose tissue iNOS induction in endotoxemia; Kapur S et al.; The aim of the present study was to investigate the mechanism of adipose tissue inducible nitric oxide synthase (iNOS) induction in endotoxemia . Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) to rats for </=8 h markedly increased iNOS mRNA and protein levels in white and brown adipose tissues . This effect was comparable to or greater than the induction of iNOS in liver, kidney, or skeletal muscle . iNOS activity was also found to be greatly enhanced in both white and brown adipose tissues of LPS-treated rats (an approximately 12- to 20-fold increase) . Treatment of cultured 3T3-L1 adipocytes with LPS, tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone failed to induce iNOS activity . However, when used in combination, TNF-alpha, IFN-gamma, and LPS markedly and synergistically increased iNOS activity in these cells . In conclusion, these results suggest that adipose tissue is a major site of iNOS expression in endotoxemia . Our data further indicate that iNOS induction can be reproduced in vitro in cultured adipocytes and that a concerted action of cytokines and endotoxin is needed for maximal activation of the enzyme.

Biochem Biophys Res Commun, 1999 Apr 13, 257(2), 414 - 7
Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase; Zhou H et al.; NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins . It is a member of the short-chain dehydrogenase family of enzymes . A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+ . Site-directed mutagenesis was used to examine the important role of this residue . Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E . coli . Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein . Mutants T188A and T188Y were found to be inactive . Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold . These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH .

Biochem Biophys Res Commun, 1999 Apr 13, 257(2), 400 - 4
Physical interaction between interleukin-12 receptor beta 2 subunit and Jak2 tyrosine kinase: Jak2 associates with cytoplasmic membrane-proximal region of interleukin-12 receptor beta 2 via amino-terminus; Yamamoto K et al.; IL-12 is a heterodimeric cytokine, composed of p40 and p35 subunits, that exerts its biological effects by binding to specific cell surface receptors . Two human IL-12 receptor proteins, designated IL-12R beta 1 and IL-12R beta 2, have been previously identified . IL-12R beta 2 has box 1 motif, box 2 motif, and three tyrosine residues in its cytoplasmic domain . In response to IL-12, Jak2 and Tyk2, family members of Janus family protein tyrosine kinases, are phosphorylated in PHA-activated T lymphocytes . The present study demonstrates that Jak2 binds to the cytoplasmic membrane-proximal region of IL-12R beta 2, and box 2 motif and tyrosine residues in the cytoplasmic domain were not required for binding . The amino-terminus of Jak2 is necessary for association with IL-12R beta 2 .

Biochem Biophys Res Commun, 1999 Apr 13, 257(2), 348 - 50
Determination of mean and standard deviation of dihedral angles; Doker R et al.; Backbone torsional angles are a characteristic and useful parameter for the description and characterisation of protein structures determined by x-ray crystallography or NMR spectroscopy . For the comparison of an ensemble of three-dimensional structures the calculation of the statistical parameters mean and standard deviation would be very useful . However, they are not defined unambiguously for periodic quantities such as the dihedral angles . In this paper a plausible and unique definition of these parameters is introduced and a straightforward method for their calculation is given .

Biochem Biophys Res Commun, 1999 Apr 13, 257(2), 327 - 32
S-form lipopolysaccharide (LPS), but not lipid A or R-chemo-type LPS, induces interleukin-6 production in vitamin D3-differentiated THP-1 cells; Suda Y et al.; Bacterial lipopolysaccharide (LPS) induces the production of various inflammatory cytokines and the inducibility is considered attributable to the glycolipid part of LPS called lipid A . We report an in vitro model in which lipid A is not necessarily a minimal structure for the LPS activity . Vitamin D3-differentiated THP-1 cells, cultured human monocytic leukemia cells, produced a high level of interleukin-6 (IL-6) by stimulating LPS from Escherichia coli O111:B4, but not by stimulating synthetic E . coli-type lipid A (compound 506), E . coli Re mutant LPS (ReLPS), or alkali-treated LPS . The induction by LPS was inhibited by the anti-CD14 antibodies or by the synthetic lipid A precursor (compound 406) . An alkali-treated LPS or compound 506 partially inhibited the LPS-induced IL-6 production . These facts suggest that lipid A alone is not sufficient for the IL-6-inducing activity, but the polysaccharide part in LPS contributes or acts as a co-factor for activation of differentiated THP-1 cells .

J Mol Biol, 1999 Mar 12, 286(5), 1581 - 96
Foldability of barnase mutants obtained by permutation of modules or secondary structure units; Tsuji T et al.; Modules, defined as stable, compact structure units in a globular protein, are good candidates for the construction of novel foldable proteins by permutation . Here we decomposed barnase into six modules (M1-M6) and constructed 23 barnase mutants containing permutations of the internal four (M2-M5) out of six modules . Globular proteins can also be subdivided into secondary structure units based on the extended structures that control the mutual relationships of the modules . We also decomposed barnase into six secondary structure units (S1-S6) and constructed 21 barnase mutants containing permutations of the internal four (S2-S5) out of six secondary structure units . Foldability of these two types of mutants was assessed by means of circular dichroism, fluorescence, and 1H-NMR measurements . A total of 15 of 23 module mutants and 15 of 21 secondary structure unit mutants formed definite secondary structures, such as alpha-helix and beta-sheet, at 20 microM owing to intermolecular interactions, but most of them converted to random coil structures at a lower concentration (1 microM) . Of the 44 mutants, only two, M3245 and S2543, gave distinct near-UV CD spectra . S2543 especially showed definite signal dispersion in the amide and methyl regions of the 1H-NMR spectrum, though M3245 did not . Furthermore, urea-induced unfolding of S2543 monitored by far-UV CD and fluorescence measurements showed a distinct cooperative transition . These results strongly suggest that S2543 takes partially folded conformations in aqueous solution . Our results also suggest that building blocks such as secondary structure units capable of taking different stable conformations by adapting themselves to the surrounding environment, rather than building blocks such as modules having a specified stable conformation, are required for the formation of foldable proteins . Therefore, the use of secondary structure units for the construction of novel globular proteins is likely to be an effective approach .

Electrophoresis, 1999 Feb, 20(2), 344 - 8
Interferon gamma regulates a unique set of proteins in fresh human bladder transitional cell carcinomas; Aboagye-Mathiesen G et al.; Poly(A) mRNA was isolated from human placental trophoblast cells stimulated with 100 U/mL of interleukin-2 and 5 microg/mL of phytohemagglutinin and reverse-transcribed . The cDNA coding for the mature interferon-gamma (IFN-gamma) protein was amplified using specific primers, cloned into the pGEX-4T2 vector, and expressed in Escherichia coli . Treatment of four fresh bladder transitional cell carcinoma (TCC) biopsies (TCCs 845-1, grade II, Ta; TCC 925-1, grade II, Ta; TCC 919-1, grade III, T1; TCC 950-1, grade III, T1) with the purified recombinant trophoblast IFN-gamma (50 U/mL, 20 h), followed by proteome analysis using two-dimensional gel electrophoresis, revealed several major proteins whose level of expression were affected by this cytokine . Of these, five (tryptophanyl-tRNA synthetase, the interferon gamma-inducible protein gamma3, mangase superoxide dismutase, and two unknown proteins of apparent molecular masses of 35.8 and 11.2 kDa, respectively) were upregulated in at least 75% of the tumors analyzed while one was downregulated (aldose reductase) . Proteins were identified using a combination of techniques that included microsequencing, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) immunoblotting and comparison with the transitional cell carcinoma isoelectric focusing (IEF) database . Proteome profile analysis of primary cultures from a low-grade lesion (TCC 846-1, Grade II, Ta) labeled in the presence and absence of IFN-gamma showed that all of the proteins disregulated in vivo were also affected in the cultures . The cultured cells, on the other hand, exhibited additional changes that were not detected in vivo and that may reflect adaptation to the culturing conditions . Taken together, the results provide a first glance at the effect of IFN-gamma on the protein expression profiles of TCCs, and in due course may form the basis for more comprehensive studies aimed at evaluating the usefulness of this cytokine in bladder cancer management.

Mol Biol Cell, 1999 Apr, 10(4), 1133 - 46
Elucidation of a PTS-carbohydrate chemotactic signal pathway in Escherichia coli using a time-resolved behavioral assay; Lux R et al.; Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)-carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA . Responses initiated by flash photorelease of a PTS substrates D-glucose and its nonmetabolizable analog methyl alpha-D-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis . This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway . The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity . Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 +/- 3.1 s-1 . The response threshold was <10 nM for glucose . Responses to methyl alpha-D-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable . These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP-CheW-CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation . The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.

Mol Biol Cell, 1999 Apr, 10(4), 1119 - 31
beta-catenin can be transported into the nucleus in a Ran-unassisted manner; Yokoya F et al.; The nuclear accumulation of beta-catenin plays an important role in the Wingless/Wnt signaling pathway . This study describes an examination of the nuclear import of beta-catenin in living mammalian cells and in vitro semi-intact cells . When injected into the cell cytoplasm, beta-catenin rapidly migrated into the nucleus in a temperature-dependent and wheat germ agglutinin-sensitive manner . In the cell-free import assay, beta-catenin rapidly migrates into the nucleus without the exogenous addition of cytosol, Ran, or ATP/GTP . Cytoplasmic injection of mutant Ran defective in its GTP hydrolysis did not prevent beta-catenin import . Studies using tsBN2, a temperature-sensitive mutant cell line that possesses a point mutation in the RCC1 gene, showed that the import of beta-catenin is insensitive to nuclear Ran-GTP depletion . These results show that beta-catenin possesses the ability to constitutively translocate through the nuclear pores in a manner similar to importin beta in a Ran-unassisted manner . We further showed that beta-catenin also rapidly exits the nucleus in homokaryons, suggesting that the regulation of nuclear levels of beta-catenin involves both nuclear import and export of this molecule.

J Bacteriol, 1999 Apr, 181(8), 2648 - 51
Bordetella pertussis waaA encodes a monofunctional 2-keto-3-deoxy-D-manno-octulosonic acid transferase that can complement an Escherichia coli waaA mutation; Isobe T et al.; Bordetella pertussis lipopolysaccharide (LPS) contains a single 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residue, whereas LPS from Escherichia coli contains at least two . Here we report that B . pertussis waaA encodes an enzyme capable of transferring only a single Kdo during the biosynthesis of LPS and that this activity is sufficient to complement an E . coli waaA mutation.

J Bacteriol, 1999 Apr, 181(8), 2612 - 9
Acarbose, a pseudooligosaccharide, is transported but not metabolized by the maltose-maltodextrin system of Escherichia coli; Brunkhorst C et al.; The pseudooligosaccharide acarbose is a potent inhibitor of amylases, glucosidases, and cyclodextrin glycosyltransferase and is clinically used for the treatment of so-called type II or insulin-independent diabetes . The compound consists of an unsaturated aminocyclitol, a deoxyhexose, and a maltose . The unsaturated aminocyclitol moiety (also called valienamine) is primarily responsible for the inhibition of glucosidases . Due to its structural similarity to maltotetraose, we have investigated whether acarbose is recognized as a substrate by the maltose/maltodextrin system of Escherichia coli . Acarbose at millimolar concentrations specifically affected the growth of E . coli K-12 on maltose as the sole source of carbon and energy . Uptake of radiolabeled maltose was competitively inhibited by acarbose, with a Ki of 1.1 microM . Maltose-grown cells transported radiolabeled acarbose, indicating that the compound is recognized as a substrate . Studying the interaction of acarbose with purified maltoporin in black lipid membranes revealed that the kinetics of acarbose binding to LamB is asymmetric . The on-rate of acarbose is approximately 30 times lower when the molecule enters the pore from the extracellular side than when it enters from the periplasmic side . Acarbose could not be utilized as a carbon source since the compound alone was not a substrate of amylomaltase (MalQ) and was only poorly attacked by maltodextrin glucosidase (MalZ).

J Bacteriol, 1999 Apr, 181(8), 2572 - 83
Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1; Paterson ES et al.; The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions . Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins . This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT) . Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups . All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation . The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins . Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required . The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability . Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.

J Bacteriol, 1999 Apr, 181(8), 2519 - 26
A region near the C-terminal end of Escherichia coli DNA helicase II is required for single-stranded DNA binding; Mechanic LE et al.; The role of the C terminus of Escherichia coli DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDDelta107C (deletion of the last 107 C-terminal amino acids), UvrDDelta102C, and UvrDDelta40C . This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles . Genetic complementation assays demonstrated that mutant proteins UvrDDelta107C and UvrDDelta102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair . UvrDDelta40C protein fully complemented the loss of helicase II in both repair pathways . UvrDDelta102C and UvrDDelta40C were purified to apparent homogeneity and characterized biochemically . UvrDDelta102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (kcat = 0.01% of the wild-type level) . UvrDDelta40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured . These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.

J Bacteriol, 1999 Apr, 181(8), 2338 - 45
Specific contacts between residues in the DNA-binding domain of the TyrR protein and bases in the operator of the tyrP gene of Escherichia coli; Hwang JS et al.; In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter . Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J . S . Hwang, J . Yang, and A . J . Pittard, J . Bacteriol . 179:1051-1058, 1997) . In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR . Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein . Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay . HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding . Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the +/-5 positions in the TyrR boxes.

Genes Dev, 1999 Apr 1, 13(7), 901 - 11
The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi, resulting in constitutive recombination activation; Churchill JJ et al.; Double-strand DNA break repair and homologous recombination in Escherichia coli proceed by the RecBCD pathway, which is regulated by cis-acting elements known as chi sites . A crucial feature of this regulation is the RecBCD enzyme-directed loading of RecA protein specifically onto the 3'-terminal, chi-containing DNA strand . Here we show that RecBC enzyme (lacking the RecD subunit) loads RecA protein constitutively onto the 3'-terminal DNA strand, with no requirement for chi . This strand is preferentially utilized in homologous pairing reactions . We propose that RecA protein loading is a latent property of the RecBCD holoenzyme, which is normally blocked by the RecD subunit and is revealed following interaction with chi.

Genes Dev, 1999 Apr 1, 13(7), 890 - 900
Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits; Taylor AF et al.; We report here an unusual mechanism for enzyme regulation: the disassembly of all three subunits of RecBCD enzyme after its interaction with a Chi recombination hot spot . The enzyme, which is essential for the major pathway of recombination in Escherichia coli, acts on linear double-stranded DNA bearing a Chi site to produce single-stranded DNA substrates for strand exchange by RecA protein . We show that after reaction with DNA bearing Chi sites, RecBCD enzyme is inactivated and the three subunits migrate as separate species during glycerol gradient ultracentrifugation or native gel electrophoresis . This Chi-mediated inactivation and disassembly of purified RecBCD enzyme can account for the previously reported Chi-dependent loss of Chi activity in E . coli cells containing broken DNA . Our results support a model of recombination in which Chi regulates one RecBCD enzyme molecule to make a single recombinational exchange ('one enzyme-one exchange' hypothesis).

Cancer Res, 1999 Apr 1, 59(7), 1514 - 9
Alteration of the conserved residue tyrosine-158 to histidine renders human O6-alkylguanine-DNA alkyltransferase insensitive to the inhibitor O6-benzylguanine; Xu-Welliver M et al.; The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from alkylation damage . O6-Benzylguanine (BG) is a potent inactivator of human AGT (ED50 of 0.1 microM) that is currently undergoing clinical trials to enhance chemotherapy by alkylating agents . In a screen of AGT mutants randomly mutated at position glycine-160, we found that the double mutant Y158H/G160A protected Escherichia coli from killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) even in the presence of BG and that the AGT activity of this mutant was strongly resistant to BG (ED50 of 180 microM) . Because the single mutant G160A was not resistant to BG, this suggested that the presence of the charged histidine residue at position 158 was responsible . This hypothesis was confirmed by the construction of the single mutation Y158H . The Y158H-mutant AGT was slightly less active than wild-type AGT for the repair of methylated DNA in vitro, but it protected E . coli from killing by MNNG even in the presence of BG and had an ED50 for the inactivation by BG of 620 microM . In contrast, mutant Y158F had an ED5o of 0.2 microM . Previous studies (M . Xu-Welliver et al., Cancer Res., 58: 1936-1945, 1998) have shown that mutant P140K is highly resistant to BG (ED50 of >1200 microM) . Models of human AGT suggest that the side chain of the lysine inserted into this mutant is close to tyrosine-158 and that the positively charged lysine side-chain may interfere with BG binding . The double mutants P140K/Y158H and P140K/Y158F resembled P140K and Y158H in being highly resistant to BG, but the use of a sensitive assay for reaction of BG with AGT indicated that their abilities to react were in the order P140K/ Y158H < P140K < P140K/Y158F . These results confirm that the presence of a positively charged residue close to the active site of human AGT renders it highly resistant to BG without substantially affecting activity toward methylated DNA substrates . Such mutants may limit the value of BG therapy if they arise in malignant cells during chemotherapy, but the mutant sequences may be useful for gene therapy approaches in which BG-resistant human AGTs are used to prevent hematopoietic toxicity . At least 28 AGT sequences (from 25 species) have now been described . In 25 of these, the position equivalent to 158 in the human AGT is also a tyrosine, and in the other 3, it is a phenylalanine . The importance of an aromatic ring side chain at this position is emphasized by previous studies (S . Edara et al., Carcinogenesis, 16: 1637-1642, 1995), which show that the replacement by alanine renders human AGT inactive . Our results show that histidine can also substitute for tyrosine at this position.

J Radiat Res (Tokyo), 1998 Dec, 39(4), 263 - 70
An improved system for selection of forward mutations in an Escherichia coli supF gene carried by plasmids; Obata F et al.; An improved system to examine forward mutations that occurred in the supF gene of Escherichia coli carried on a multicopy plasmid is described . The system was validated by measuring spontaneous mutations of supF plasmids propagated in wild-type, recA- and mutM- mutY- E . coli strains, the mutation frequencies of which were 1.3 x 10(-7), 6.3 x 10(-7) and 1.5 x 10(-6), respectively . Sequence analysis of the supF mutant plasmids revealed that G:C-->T:A and G:C-->C:G transversions dominated . This improved system allows rapid scoring and sequencing forward mutations in the supF gene, thus permitting its use as a genetic target for repair and mutagenesis studies in bacteria and mammalian cells.

Chin J Biotechnol, 1998, 14(2), 75 - 84
Cloning of 1-aminocyclopropane-1-carboxylate (ACC) synthetase cDNA and the inhibition of fruit ripening by its antisense RNA in transgenic tomato plants; Liu C et al.; A 1.7 kb fragment of ACC synthetase cDNA, one member of the ACC synthetase multigene family, was amplified from total tomato cDNA through a polymerase chain reaction (PCR) and cloned in E . coli . Restriction mapping and sequencing analysis confirmed its fidelity and correctness . The cloned ACC synthetase gene was then inserted into a binary vector pBin437, in an inverted orientation between the CaMV 35S promoter with duplicated enhancers and the Nos 3' transcriptional termination sequence, to construct an expression vector pBACC . Transgenic tomato plants were obtained by A . tumefaciens-mediated transformation of cotyledons . PCR detection and Southern blot analysis confirmed the integration of the antisense ACC synthetase gene in the transformed tomato genome . The results from RT-PCR of RNAs isolated from transgenic tomato leaves confirmed that antisense ACC synthetase RNA was synthesized in these transgenic plants . The amount of ethylene released from transgenic tomato fruits was reduced significantly to about 30% of that released by non-transformed controls . The inhibition effect of antisense RNA on fruit ripening was observed in transgenic plants and their progeny (T1) . The shelf life of transgenic tomato fruits was at least 60 days at room temperature without significant change in hardness and color . After 15-20 days of treatment of the transgenic fruits with ethylene, most of them reached the ripe stage . The antisense ACC synthetase gene was inherited as a single gene in the progenies of transgenic tomatoes determined by T1 progeny analysis, consistent with the results of Southern blot analysis . Transgenic homozygotes expressing antisense ACC synthetase RNA showed prolonged shelf life in the T2 progeny.

Chin J Biotechnol, 1998, 14(2), 67 - 74
Study on the genetic determinants responsible for expression and assembly of CS3 fimbriae; Dong Z et al.; Colonization factor antigens (CFAs) are important virulent factors and protective antigens of enterotoxigenic Escherichia coli (ETEC) . Among the known CFAs of human origin, CS3 is a major fimbriae antigen . To study the genes associated with the expression and assembly of CS3 is a key step to delineate the regulating mechanism of expression and assembly of the fimbriae . In this paper we analyzed the restriction map of the CS3 genetic determinant and successfully cloned the CS3 subunit gene and the region encoding the auxiliary proteins . Expression results of the recombinant plasmids in minicells showed that the genetic determinant of CS3 fimbriae encodes six polypeptides with molecular weights 15, 17, 24, 27, 48, and 90 kDa . Western blotting and complementary expression analysis among the different mutants showed that the 15 kDa/17 kDa proteins compose the CS3 subunit and the 17 kDa protein is the precursor . The others may be necessary for the assembly of CS3 fimbriae . The relative locations of various genes of the CS3 genetic determinant were also determined.

Acta Chir Hung, 1998, 37(1-2), 39 - 44
Serologic examinations in acute appendicitis; Antal A et al.; Authors studied the formation of endotoxic antibody level in healthy adults and in patients with appendicitis with a technique (indirect haemagglutination) not used till now . They found the antibody level against endotoxin to be increased in 91% of their patients in the postoperative period . Decrease in the antibody level against endotoxin was observed in two patients with gangrenous appendicitis and two patients with perforated appendicitis . Summarizing their results, authors consider mixed (aerobic, anaerobic) infection to be of decisive importance in the development of acute appendicitis, contributing to the weakened immune response of the host.

J Biol Chem, 1999 Apr 16, 274(16), 11275 - 82
Interaction between the nucleotide exchange factor Mge1 and the mitochondrial Hsp70 Ssc1; Sakuragi S et al.; Function of Hsp70s such as DnaK of the Escherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively . A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1 . Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed . Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein . K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required . In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins . We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction . The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for function in vivo.

J Biol Chem, 1999 Apr 16, 274(16), 11007 - 12
Plastidic pathway of serine biosynthesis . Molecular cloning and expression of 3-phosphoserine phosphatase from Arabidopsis thaliana; Ho CL et al.; In plants, Ser is biosynthesized by two different pathways: a photorespiratory pathway via Gly and a plastidic pathway via the phosphorylated metabolites from 3-phosphoglycerate . In contrast to the better characterization of the photorespiratory pathway at a molecular level, the molecular regulation and significance of the plastidic pathway are not yet well understood . An Arabidopsis thaliana cDNA encoding 3-phosphoserine phosphatase, the enzyme that is responsible for the conversion of 3-phosphoserine to Ser in the final step of the plastidic pathway of Ser biosynthesis, was cloned by functional complementation of an Escherichia coli serB- mutant . The 1.1-kilobase pair full-length cDNA, encoding 295 amino acids in its open reading frame, contains a putative organelle targeting presequence . Chloroplastic targeting has been demonstrated by particle gun bombardment using an N-terminal 60-amino acid green fluorescence protein fusion protein . Southern hybridization suggested the existence of a single-copy gene that mapped to chromosome 1 . 3-Phosphoserine phosphatase enzyme activity was detected in vitro in the overexpressed protein in E . coli . Northern analysis revealed preferential gene expression in leaf and root tissues of light-grown plants with an approximately 1.5-fold abundance in the root compared with the leaf tissues . This indicates the possible role of the plastidic pathway in supplying Ser to non-photosynthetic tissues, in contrast to the function of the photorespiratory pathway in photosynthetic tissues . This work completes the molecular cloning and characterization of the three genes involved in the plastidic pathway of Ser biosynthesis in higher plants.

J Biol Chem, 1999 Apr 16, 274(16), 10840 - 5
Transcription activation by CooA, the CO-sensing factor from Rhodospirillum rubrum . The interaction between CooA and the C-terminal domain of the alpha subunit of RNA polymerase; He Y et al.; CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator from Rhodospirillum rubrum that binds specific DNA sequences in response to CO . The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are analogous to class II CRP-dependent promoters . In this study, we developed an in vivo CooA reporter system in Escherichia coli and an in vitro transcription assay using RNA polymerases (RNAP) from E . coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP . The ability of CooA to activate CO-dependent transcription in vivo in heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required . This hypothesis was confirmed in vitro with purified CooA and purified RNAP . Use of a mutant form of E . coli RNAP with alpha subunits lacking their C-terminal domain (alpha-CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R . rubrum promoter PcooF in vitro, which indicates that alpha-CTD plays an important role in this activation . DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not alpha-CTD-truncated RNAP, to PcooF . This facilitated binding provides evidence for a direct contact between CooA and alpha-CTD of RNAP during activation of transcription . Mapping the CooA-contact site in alpha-CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the alpha-CTD at class II promoters.

J Biol Chem, 1999 Apr 16, 274(16), 10777 - 83
The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independently of its acylation status; Basar T et al.; The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the epsilon-amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC . However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12 . We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues . Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta9 C16:1) fatty-acyl groups . Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity . This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin . These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status.

J Struct Biol, 1999 Mar, 125(1), 63 - 75
Two-dimensional crystallization of Escherichia coli lactose permease; Zhuang J et al.; A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside . Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-dimensional (2D) crystals as shown by electron microscopy of negatively stained specimens . Single-particle analysis of 16 383 protein particles in densely packed vesicles reveals a 5.4-nm-long trapeziform protein of 4.1 to 5.1 nm width, with a central stain-filled indentation . Depending on reconstitution conditions, trigonal and rectangular crystallographic packing arrangements of these elongated particles assembled into trimers are observed . The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes . Projection maps calculated to a resolution of 2 nm show that these crystals consist of two layers .

Methods, 1999 Apr, 17(4), 313 - 9
Caspases: preparation and characterization; Stennicke HR et al.; Caspases and their involvement in programmed cell death have been an area of significant interest since their initial identification in 1992 . To facilitate the search for new components involved in cell death, and to aid researchers in understanding the interactions between currently known cell death proteins, we describe a number of techniques commonly used in the preparation and characterization of caspases .

J Exp Zool, 1999 May 1, 283(6), 510 - 21
Facilitated geranylgeranylation of shrimp ras-encoded p25 fusion protein by the binding with guanosine diphosphate; Huang CF et al.; A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning . Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains . Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals . As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein . In other words, the GTP bound form of ras-encoded p25 fusion protein prevails . Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively . That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form.

Cancer Gene Ther, 1999 Mar-Apr, 6(2), 155 - 62
Combined radiation and p53 gene therapy of malignant glioma cells; Badie B et al.; More than half of malignant gliomas reportedly have alterations in the p53 tumor suppressor gene . Because p53 plays a key role in the cellular response to DNA-damaging agents, we investigated the role of p53 gene therapy before ionizing radiation in cultured human glioma cells containing normal or mutated p53 . Three established human glioma cell lines expressing the wild-type (U87 MG, p53wt) or mutant (A172 and U373 MG, p53mut) p53 gene were transduced by recombinant adenoviral vectors bearing human p53 (Adp53) and Escherichia coli beta-galactosidase genes (AdLacZ, control virus) before radiation (0-20 Gy) . Changes in p53, p21, and Bax expression were studied by Western immunoblotting, whereas cell cycle alterations and apoptosis were investigated by flow cytometry and nuclear staining . Survival was assessed by clonogenic assays . Within 48 hours of Adp53 exposure, all three cell lines demonstrated p53 expression at a viral multiplicity of infection of 100 . p21, which is a p53-inducible downstream effector gene, was overexpressed, and cells were arrested in the G1 phase . Bax expression, which is thought to play a role in p53-induced apoptosis, did not change with either radiation or Adp53 . Apoptosis and survival after p53 gene therapy varied . U87 MG (p53wt) cells showed minimal apoptosis after Adp53, irradiation, or combined treatments . U373 MG (p53mut) cells underwent massive apoptosis and died within 48 hours of Adp53 treatment, independent of irradiation . Surprisingly, A172 (p53mut) cells demonstrated minimal apoptosis after Adp53 exposure; however, unlike U373 MG cells, apoptosis increased with radiation dose . Survival of all three cell lines was reduced dramatically after >10 Gy . Although Adp53 transduction significantly reduced the survival of U373 MG cells and inhibited A172 growth, it had no effect on the U87 MG cell line . Transduction with AdLacZ did not affect apoptosis or cell cycle progression and only minimally affected survival in all cell lines . We conclude that responses to p53 gene therapy are variable among gliomas and most likely depend upon both cellular p53 status and as yet ill-defined downstream pathways involving activation of cell cycle regulatory and apoptotic genes.

Cancer Gene Ther, 1999 Mar-Apr, 6(2), 99 - 106
Use of L-plastin promoter to develop an adenoviral system that confers transgene expression in ovarian cancer cells but not in normal mesothelial cells; Chung I et al.; The objective of this study was to develop an adenoviral vector system that would generate a pattern of expression of exogenous therapeutic genes appropriate for the treatment of ovarian cancer . For this purpose, we have generated a replication-deficient recombinant adenoviral vector, AdLPLacZ, which contains the human L-plastin (LP) promoter (LP-P) driving the Escherichia coli LacZ gene . LP is constitutively expressed at high levels in malignant epithelial cells but is not expressed in normal tissues, except at low levels in mature hematopoietic cells . Because adenoviral vectors infect early hematopoietic multilineage precursor cells only poorly or not at all, this vector would be of use in the peritoneal cavity and in vitro for marrow purging . We first analyzed the expression of the LacZ reporter gene in ovarian and breast cancer cell lines, normal fibroblasts, and leukemia cell lines using the adenoviral vector in which the LacZ gene is governed by the LP-P promoter (AdLPLacZ) or in which the LacZ gene is governed by the cytomegalovirus (CMV) promoter (AdCMVLacZ) . We found equivalent and high levels of expression of beta-galactosidase (beta-gal) by AdLPLacZ and AdCMVLacZ vectors in the breast or ovarian cancer cell lines as well as in a fibrosarcoma cell line, indicating that the adenoviral vectors infected these cells and expressed their transgenes equally with the LP and CMV promoters . Expression of the LacZ gene with the CMV vector but not with the LP-P vector was observed in experiments with normal fibroblasts, indicating that the vectors infected the cells, but that the LP-P was not active within them . In hematopoietic cells such as U937 cells, no measurable beta-gal activity was detected in cells infected either by AdLPLacZ or by AdCMVLacZ, indicating that the adenoviral vectors were not infecting the cells . Although beta-gal activity was observed in fresh ascitic ovarian cancer cells after infection with adenoviral vectors containing CMV or the LP promoters, beta-gal activity was detected in a portion of a biopsy of normal peritoneum when the tissues were exposed to the AdCMVLacZ vector, but not when tissues were exposed to the AdLPLacZ vector . These results suggest that the transcription of therapeutic genes in cells infected by the AdLP vectors would be restricted to LP expression-positive ovarian carcinoma cells but would not be seen in the normal mesothelial cells of the peritoneal cavity . This possibility implies that adenoviral vectors carrying therapeutic genes driven by the LP-P would be of use for the intracavitary treatment ovarian cancer.

Vaccine, 1999 Mar 17, 17(11-12), 1442 - 53
Novel modifications to the C-terminus of LTB that facilitate site-directed chemical coupling of antigens and the development of LTB as a carrier for mucosal vaccines; O'Dowd AM et al.; To facilitate site-directed chemical coupling of antigens to the heat labile enterotoxin B subunit of Escherichia coli (LTB), a series of genetically modified fusion proteins of LTB were constructed . The LTB fusion proteins had modified versions of a short (14 amino acid) spacer epitope called the Pk tag attached at their C termini . The LTB-Pk.cys mutants differed from each other in the position of a single cysteine residue within the Pk tag . The presence of a cysteine residue at any position within the Pk spacer tag did not prevent the LTB-Pk.cys proteins from forming pentamers or binding to GM1 gangliosides, but the position of the cysteine had variable impact on the yield of the fusion proteins . Following site-directed chemical coupling of antigens to the cysteine residue within the Pk tag, the LTB antigen conjugates retained their ability to bind GM1 on the surface of eukaryotic cells . Intranasal immunisation of mice with an experimental antigen (HRP) chemically linked to LTB-Pk.cys induced high levels of anti-HRP antibodies that could be detected in the serum, saliva and nasal and lung washes . No antibody responses to HRP were detected when HRP was co-administered with, but not linked to, LTB-Pk.cys.

Vaccine, 1999 Mar 17, 17(11-12), 1394 - 403
Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection; Myers GA et al.; Recombinant Helicobacter pylori urease (rUre) has been shown to confer protection against challenge with Helicobacter felis in mice . The purpose of the present study was to examine duration of the immune response and long-term protective efficacy of immunization with rUre . Swiss Webster mice were orally immunized four times at weekly intervals with 100 microg rUre plus 5 microg heat-labile enterotoxin of Escherichia coli (LT) adjuvant, or with LT only . At 4, 10, 20 or 40 weeks post immunization, 25 rUre-immunized mice and control mice were challenged with H . felis and sacrificed at 2 or 10 weeks post-challenge . H . felis infection was assessed by gastric urease assay and by histology . Anti-H . pylori urease specific antibody levels were measured in serum and saliva both pre- and post-challenge . Over the 40 week time period, the infection rates in rUre-immunized mice were significantly lower than those in controls (p < 0.05) as assessed by gastric urease activity . Protection ranged from 79 100% at 2 weeks post-challenge and 63-78% at 10 weeks post-challenge . Gastric bacterial density in rUre-immunized mice was significantly lower than that of controls (p < 0.03) as determined by histologic assessment . Anti-urease antibody levels remained elevated in the serum and mucosal compartments at 39 weeks following immunization . This study shows that immunization with rUre plus LT results in long-lasting protective immunity against challenge with H . felis.

Vaccine, 1999 Mar 5, 17(9-10), 1272 - 81
Construction and murine immunogenicity of recombinant Bacille Calmette Guérin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin; Hayward CM et al.; Three recombinant strains of Mycobacterium bovis Bacille Calmette Guerin (rBCG) were prepared in which the immunogenic B subunit of human Escherichia coli heat labile enterotoxin (LT-Bh) was expressed either as a cytoplasm protein, a cell wall associated lipoprotein or a secreted protein . Intraperitoneal immunisation of mice with these rBCG induced IgG and IgA antibodies to LT-Bh and shifted the serum IgG subclass response to subsequent challenge with purified LT-Bh from IgG1 to an IgG2a . Oral administration of recombinant BCG induced mucosal and serum IgA antibodies to LT-Bh which peaked four months after immunisation . Antibody responses were greater when LT-Bh was expressed as a secreted protein or lipoprotein rather than in the cytoplasm . Oral vaccination with recombinant BCG may be an effective approach, particularly to induce mucosal IgA and prime for a serum TH1 recall response.

Vaccine, 1999 Mar 5, 17(9-10), 1130 - 5
Comparison between targeted and untargeted systemic immunizations with adjuvanted urease to cure Helicobacter pylori infection in mice; Guy B et al.; Outbred OF1 mice infected in a first step with a mouse-adapted Helicobacter pylori strain were immunized in a second step by systemic or mucosal routes: systemic immunizations were performed subcutaneously with adjuvanted urease either in the infra or supra-diaphragmatic region of the body, while mucosal immunization was done with urease in the presence of E . coli heat Labile toxin . Mucosal and subcutaneous immunizations induced in infected mice a significant reduction in bacterial density whatever the site of injection but complete eradication was preferentially observed in mice immunized subcutaneously in the back . Systemic immunization with appropriate schedules and formulations could constitute a valuable approach to cure Helicobacter pylori infection.

J Hum Virol, 1998 May-Jun, 1(4), 251 - 6
Lipopolysaccharide from an Escherichia coli htrB msbB mutant induces high levels of MIP-1 alpha and MIP-1 beta secretion without inducing TNF-alpha and IL-1 beta; Hone DM et al.; OBJECTIVE: To identify a lipopolysaccharide (LPS) that retains the capacity to induce beta-chemokine secretion without the concomitant activation of pyrogenic cytokines . METHODS: LPS was extracted from strain MLK986 (mLPS), an htrB1::Tn10, msbB::ocam mutant of Escherichia coli that is defective for lipid A synthesis, and from wild-type parent E coli strains, W3110 (wtLPS) . The capacity of these LPS preparations to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory proteins 1 alpha (MIP-1 alpha) and MIP-1 beta was assessed using a human peripheral blood mononuclear cell (PBMC) activation assay . RESULTS: Stimulation of PBMCs with mLPS did not induce measurable levels of pyrogenic cytokines TNF-alpha and IL-1 beta, whereas wtLPS induced high levels of these cytokines . Furthermore, mLPS antagonized the induction of TNF-alpha secretion by wtLPS . Nonetheless, mLPS retained a discrete agonist activity that induced MIP-1 alpha and MIP-1 beta secretion by PBMCs . This latter agonist activity appears to be unique to mLPS, since two previously documented LPS antagonists, Rhodobacter sphaeroides diphosphoryl lipid A and synthetic lipid IVA, did not induce MIP-1 alpha and MIP-1 beta secretion . Furthermore, synthetic lipid IVA was an antagonist of MIP-1 alpha and MIP-1 beta induction by mLPS . CONCLUSION: These results show that mLPS exhibits a novel bipartite activity, being an effective antagonist of TNF-alpha induction by wtLPS, while paradoxically being an agonist of MIP-1 alpha and MIP-1 beta secretion.

Math Biosci, 1999 Mar 15, 157(1-2), 269 - 86
Dynamics of the inducing signal for the SOS regulatory system in Escherichia coli after ultraviolet irradiation; Aksenov SV; SOS response in Escherichia coli is induced by various DNA-damaging treatments, for example by ultraviolet irradiation, to help a cell to recover from the damage . During induction of the SOS regulatory system, generation of the inducing signal for the system is the early step . In the present study a model for quantitative description of the signal dynamics is developed . We derive the inducing signal, in terms of concentration of single-stranded DNA, as a function of time since the moment of ultraviolet irradiation . Simulation of the signal level after irradiation with two doses of 5 and 20 J m-2 is presented . This provides quantitative description of the event that controls various cellular physiological reactions induced in the course of the SOS response . The dynamics of the signal level are then used as an input for a dynamical equation description of the SOS regulatory system that we proposed earlier . This allows for a quantitative analysis of the subsequent step in the SOS induction: cleavage of LexA protein, a negative regulator of the SOS system . The model is verified against available experimental data for LexA protein level in ultraviolet radiation-induced Escherichia coli cells.

J Biotechnol, 1999 Feb 19, 68(2-3), 149 - 58
Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography; Batas B et al.; The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding . We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL) . It was found that the most significant purification occurred during the removal of cell debris . Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100 . Centrifugation between each wash step gave a purer product with a higher rHEWL yield . With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane . Significant flux decline was observed for both membranes . Second, we studied the refolding of rHEWL . Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity . Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material . Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.

J Biotechnol, 1999 Feb 19, 68(2-3), 125 - 34
Enhancing recombinant protein yields in Escherichia coli using the T7 system under the control of heat inducible lambda PL promoter; Gupta JC et al.; A recombinant plasmid containing the complete lacZ gene downstream of the T7 promoter was used to transform Escherichia coli containing another plasmid which had the T7 RNA polymerase gene under the control of heat inducible lambda PL promoter . This recombinant E . coli containing the two plasmids was studied in order to enhance beta-galactosidase expression . The heat shock time which effectively regulates the T7 RNA polymerase was optimized and best expression of beta-galactosidase was obtained with 2 min heat shock . Substrate feeding increased the duration of log phase and allowed induction at a higher cell density without affecting the specific activity . A high cell density (7 g l-1) and high specific activity (approximately 20,000 U) were achieved which effectively increased the product concentration 18-fold.

J Clin Invest, 1999 Apr, 103(7), 1047 - 54
Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury; Otterbein LE et al.; Heme oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury . In this study, we examined whether exogenous administration of HO-1 by gene transfer could also confer protection . We first demonstrated the feasibility of overexpressing HO-1 in the lung by gene transfer . A fragment of the rat HO-1 cDNA clone containing the entire coding region was cloned into plasmid pAC-CMVpLpA, and recombinant adenoviruses containing the rat HO-1 cDNA fragment Ad5-HO-1 were generated by homologous recombination . Intratracheal administration of Ad5-HO-1 resulted in a time-dependent increase in expression of HO-1 mRNA and protein in the rat lungs . Increased HO-1 protein expression was detected diffusely in the bronchiolar epithelium of rats receiving Ad5-HO-1, as assessed by immunohistochemical studies . We then examined whether ectopic expression of HO-1 could confer protection against hyperoxia-induced lung injury . Rats receiving Ad5-HO-1, but not AdV-betaGal, a recombinant adenovirus expressing Escherichia coli beta-galactosidase, before exposure to hyperoxia (>99% O2) exhibited marked reduction in lung injury, as assessed by volume of pleural effusion and histological analyses (significant reduction of edema, hemorrhage, and inflammation) . In addition, rats receiving Ad5-HO-1 also exhibited increased survivability against hyperoxic stress when compared with rats receiving AdV-betaGal . Expression of the antioxidant enzymes manganese superoxide dismutase (Mn-SOD) and copper-zinc superoxide dismutase (CuZn-SOD) and of L-ferritin and H-ferritin was not affected by Ad5-HO-1 administration . Furthermore, rats treated with Ad5-HO-1 exhibited attenuation of hyperoxia-induced neutrophil inflammation and apoptosis . Taken together, these data suggest the feasibility of high-level HO-1 expression in the rat lung by gene delivery . To our knowledge, we have demonstrated for the first time that HO-1 can provide protection against hyperoxia-induced lung injury in vivo by modulation of neutrophil inflammation and lung apoptosis.

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