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