Escherichia coli

J Biol Chem, 1998 Apr 3, 273(14), 8447 - 53
Replication origin of the broad host range plasmid RK2 . Positioning of various motifs is critical for initiation of replication; Doran KS et al.; The 393-base pair minimal origin, oriV, of plasmid RK2 contains three iterated motifs essential for initiation of replication: consensus sequences for binding the bacterial DnaA protein, DnaA boxes, which have recently been shown to bind the DnaA protein; 17-base pair direct repeats, iterons, which bind the plasmid encoded replication protein, TrfA; and A + T-rich repeated sequences, 13-mers, which serve as the initial site of helix destabilization . To investigate how the organization of the RK2 origin contributes to the mechanism of replication initiation, mutations were introduced into the minimal origin which altered the sequence and/or spacing of each particular region relative to the rest of the origin . These altered origins were analyzed for replication activity in vivo and in vitro, for localized strand opening and for DnaB helicase mediated unwinding . Mutations in the region between the iterons and the 13-mers which altered the helical phase or the intrinsic DNA curvature prevented strand opening of the origin and consequently abolished replication activity . Insertions of more or less than one helical turn between the DnaA boxes and the iterons also inactivated the replication origin . In these mutants, however, strand opening appeared normal but the levels of DnaB helicase activity were substantially reduced . These results demonstrate that correct helical phasing and intrinsic DNA curvature are critical for the formation of an open complex and that the DnaA boxes must be on the correct side of the helix to load DnaB helicase.

J Biol Chem, 1998 Apr 3, 273(14), 8419 - 24
The protein translocation apparatus contributes to determining the topology of an integral membrane protein in Escherichia coli; Prinz WA et al.; The assembly of integral membrane proteins is determined by features of these proteins and the protein translocation apparatus . We used alkaline phosphatase fusions to the membrane protein MalF to investigate the role of the protein translocation machinery in the arrangement of proteins in the cytoplasmic membrane of Escherichia coli . In particular, we studied the effects of prlA mutations on membrane protein topology . These mutations lie in the secY gene, which encodes a core component of the protein translocation apparatus . We find that the topology of some of the fusion proteins is changed and, in one case, is completely inverted in prlA mutants . We discuss the mechanism of prlA-mediated export and the role of the protein translocation apparatus in contributing to membrane protein topology.

J Biol Chem, 1998 Apr 3, 273(14), 8294 - 300
Cooperative binding properties of restriction endonuclease EcoRII with DNA recognition sites; Reuter M et al.; EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA . To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers . Maximal restriction was observed at dimer/site ratios of 0.25 and 0 . 5 . The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites . We conclude that one EcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites . A Lac repressor "barrier" bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch . Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance . At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.

J Biol Chem, 1998 Apr 3, 273(14), 8193 - 202
Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro; Sullivan FX et al.; We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose . The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity . The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation . Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E . coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved . We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR . Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro . This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide . Finally, we show that the two homologous enzymes from E . coli are sufficient to carry out the same enzymatic pathway in bacteria.

J Biol Chem, 1998 Apr 3, 273(14), 7949 - 56
Thermodynamic evidence for conformational coupling between the B and C domains of the mannitol transporter of escherichia coli, enzyme IImtl; Meijberg W et al.; The transport across the cytoplasmic membrane and concomitant phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol-specific transport protein from the phosphoenolpyruvate-dependent phosphotransferase system, enzyme IImtl . Interactions between the cytoplasmic B and the membrane embedded C domain play an important role in the catalytic cycle of this enzyme, but the nature of this interaction is largely unknown . We have studied the thermodynamics of binding of (i) mannitol to enzyme IImtl, (ii) the substrate analog perseitol to enzyme IImtl, (iii) perseitol to phosphorylated enzyme IImtl, and (iv) mannitol to enzyme IImtl treated with trypsin to eliminate the cytoplasmic domains . Analysis of the heat capacity increment of these reactions showed that approximately 50-60 residues are involved in the binding of mannitol and perseitol, but far less in the phosphorylated state or after removal of the B domain . A model is proposed in which binding of mannitol leads to the formation of a contact interface between the two domains, either by folding of unstructured parts or by docking of preexisting surfaces, thus positioning the incoming mannitol close to the phosphorylation site on the B domain to facilitate the transfer of the phosphoryl group.

J Biol Chem, 1998 Apr 3, 273(14), 7865 - 72
Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase; Benen JA et al.; The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected from a cDNA library constructed from mRNA isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol by immunochemical screening . The vaoA-cDNA nucleotide sequence revealed an open reading frame of 1680 base pairs encoding a 560-amino acid protein with a deduced mass of 62,915 Da excluding the covalently bound FAD . The deduced primary structure shares 31% sequence identity with the 8alpha-(O-tyrosyl)-FAD containing subunit of the bacterial flavocytochrome p-cresol methyl hydroxylase . The vaoA gene was isolated from a P . simplicissimum genomic library constructed in lambdaEMBL3 using the vaoA-cDNA as a probe . Comparison of the nucleotide sequence of the vaoA gene with the cDNA nucleotide sequence demonstrated that the gene is interrupted by five short introns . Aspergillus niger NW156 prtF pyrA leuA cspA transformed with the pyrA containing plasmid and a plasmid harboring the complete vaoA gene including the promoter and terminator was able to produce vaoA mRNA and active vanillyl-alcohol oxidase when grown on veratryl alcohol and anisyl alcohol . A similar induction of the vaoA gene was found for P . simplicissimum, indicating that similar regulatory systems are involved in the induction of the vaoA gene in these fungi . Introduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA-cDNA resulted in elevated expression levels of active vanillyl-alcohol oxidase from the lac promoter in Escherichia coli TG2 . The catalytic and spectral properties of the purified recombinant enzyme were indistinguishable from the native enzyme.

J Biol Chem, 1998 Apr 3, 273(14), 7818 - 27
Introduction of a tryptophan reporter group into the ATP binding motif of the Escherichia coli UvrB protein for the study of nucleotide binding and conformational dynamics; Hildebrand EL et al.; The DNA-dependent ATPase activity of UvrB is required to support preincision steps in nucleotide excision repair in Escherichia coli . This activity is, however, cryptic . Elicited in nucleotide excision repair by association with the UvrA protein, it may also be unmasked by a specific proteolysis eliminating the C-terminal domain of UvrB (generating UvrB*) . We introduced fluorescent reporter groups (tryptophan replacing Phe47 or Asn51) into the ATP binding motif of UvrB, without significant alteration of behavior, to study both nucleotide binding and those conformational changes expected to be essential to function . The inserted tryptophans occupy moderately hydrophobic, although potentially heterogeneous, environments as evidenced by fluorescence emission and time-resolved decay characteristics, yet are accessible to the diffusible quencher acrylamide . Activation, via specific proteolysis, is accompanied by conformational change at the ATP binding site, with multiple changes in emission spectra and a greater shielding of the tryptophans from diffusible quencher . Titration of tryptophan fluorescence with ATP has revealed that, although catalytically incompetent, UvrB can bind ATP and bind with an affinity equal to that of the active UvrB* form (Kd of approximately 1 mM) . The ATP binding site of UvrB is therefore functional and accessible, suggesting that conformational change either brings amino acid residues into proper alignment for catalysis and/or enables response to effector DNA.

J Biol Chem, 1998 Apr 3, 273(14), 7787 - 90
A single BIR domain of XIAP sufficient for inhibiting caspases; Takahashi R et al.; The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli . Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases . These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain . To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells . The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells . Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases . The apparent inhibition constants (Ki) for BIR2-mediated inhibition of caspases-3 and -7 were 2-5 nM, indicating that this single BIR domain possesses potent anti-caspase activity . Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not . These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases.

Arch Biochem Biophys, 1998 Apr 1, 352(1), 144 - 52
Overexpression, single-step purification, and site-directed mutagenetic analysis of casbene synthase; Huang K et al.; Casbene synthase is a diterpene cyclase isolated from castor bean (Ricinus communis L), which catalyzes the cyclization of geranylgeranyl diphosphate to form the phytoalexin casbene . We here report the overexpression of casbene synthase in Escherichia coli in soluble form using a thioredoxin fusion system . The amplified DNA by PCR carried on pCS7 was inserted into the expression vector pET32b(+) to form pCAS.2 . The resulting transformants of pCAS . 2/BL21(DE3) produced a thioredoxin casbene synthase fusion protein (20-30% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside at 20 degrees C . Recombinant casbene synthase was purified to homogeneity in a single step with a His-binding metal-affinity column . Casbene synthase has a conserved aspartate-rich region {amino acids 355-359 (DDTID)}, one cysteine, and three histidines with several prenyl transferases and terpene cyclases . Seven mutants were constructed by site-directed mutagenesis . The importance of Asp 355 and Asp 356 for catalysis was established by an increase in Km as well as a reduction in kcat in the corresponding glutamate mutants . These results indicate that the first and the second aspartate are involved in catalysis, while the third aspartate and the conserved cysteine and histidine residues selected for mutagenesis appear not to be involved in catalysis .

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3627 - 31
Regulation of the Escherichia coli water channel gene aqpZ; Calamita G et al.; Osmotic movement of water across bacterial cell membranes is postulated to be a homeostatic mechanism for maintaining cell turgor . The molecular water transporter remained elusive until discovery of the Escherichia coli water channel, AqpZ, however the regulation of the aqpZ gene expression and physiological function of the AqpZ protein are unknown . Northern analysis revealed a transcript of 0.7 kb, confirming the monocistronic nature of aqpZ . Regulatory studies performed with an aqpZ::lacZ low copy plasmid demonstrate enhanced expression during mid-logarithmic growth, and expression of the gene is dependent upon the extracellular osmolality, which increased in hypoosmotic environments but strongly reduced in hyperosmolar NaCl or KCl . While disruption of the chromosomal aqpZ is not lethal for E . coli, the colonies of the aqpZ knockout mutant are smaller than those of the parental wild-type strain . When cocultured with parental wild-type E . coli, the aqpZ knockout mutant exhibits markedly reduced colony formation when grown at 39 degrees C . Similarly, the aqpZ knockout mutant also exhibits greatly reduced colony formation when grown at low osmolality, but this phenotype is reversed by overexpression of AqpZ protein . These results implicate AqpZ as a participant in the adaptive response of E . coli to hypoosmotic environments and indicate a requirement for AqpZ by rapidly growing cells.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3578 - 82
Oxidized, deaminated cytosines are a source of C --> T transitions in vivo; Kreutzer DA et al.; The most common base substitution arising from oxidative damage of DNA is a GC --> AT transition . In an effort to determine the oxidized lesion(s) that gives rise to this mutation, the mutagenicity of three oxidized cytosines, 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, were investigated in Escherichia coli . An M13 viral genome was constructed to contain a single oxidized cytosine at a specific site . Replication in vivo of the single-stranded genomes yielded mutation frequencies of 0.05%, 83%, and 80% for 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, respectively . The predominant mutation observed was C --> T . A model for C --> T oxidative mutagenesis is suggested in which initial cytosine oxidation is followed by deamination to a poorly repaired uracil derivative that is strongly miscoding during replication.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3555 - 60
Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant; Vaz AD et al.; P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion . In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs . In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine . Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis- or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation . Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate . Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene . These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO2H)3+ and oxenoid-iron (FeO)3+, can effect olefin epoxidation . The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3531 - 6
The domain organization of NaeI endonuclease: separation of binding and catalysis; Colandene JD et al.; NaeI is a remarkable type II restriction endonuclease . It must bind two recognition sequences to cleave DNA, forms a covalent protein-DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity . The latter activities apparently derive from reactivation of a cryptic DNA ligase active site . Here, we demonstrate that NaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa . The domains were purified by cloning . The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intact NaeI . Analytical ultracentrifugation showed this domain to be a monomer in solution . The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association . DNA greatly inhibited proteolysis of the linker region . The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure for NaeI.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3525 - 30
23S rRNA positions essential for tRNA binding in ribosomal functional sites; Bocchetta M et al.; rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation . Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis . In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit . By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits from Escherichia coli and capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin . These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3478 - 82
Thiolate ligands in metallothionein confer redox activity on zinc clusters; Maret W et al.; We postulate a novel and general mechanism in which the redox-active sulfur donor group of cyst(e)ine confers oxidoreductive characteristics on stable zinc sites in proteins . Thus, the present, an earlier, and accompanying manuscripts {Maret, W., Larsen, K . S . & Vallee, B . L . (1997) Proc . Natl . Acad . Sci . USA 94, 2233-2237; Jiang, L.-J., Maret, W . & Vallee, B . L . (1998) Proc . Natl . Acad . Sci . USA 95, 3483-3488; and Jacob, C., Maret, W . & Vallee, B . L . (1998) Proc . Natl . Acad . Sci . USA 95, 3489-3494} demonstrate that the interactive network featuring multiple zinc/sulfur bonds as found in the clusters of metallothionein (MT) constitutes a coordination unit critical for the concurrent oxidation of cysteine ligands and the ensuing release of zinc . The low position of MT (<-366 mV) on a scale of redox reagents allows its effective oxidation by relatively mild cellular oxidants, in particular disulfides . When MT is exposed to an excess of dithiodipyridine, all of its 20 cysteines are oxidized within 1 hr with the concomitant release of all 7 zinc atoms; similarly, the thiol/disulfide oxidoreductase DsbA reacts stoichiometrically with MT to release zinc . Zinc and sulfur ligands in the clusters are in a spatial arrangement that seemingly favors disulfide bond formation . Jointly, this and the above-mentioned manuscripts conclude that the control of cellular zinc distribution as a function of the energy state of the cell is the long sought role of MT . This specific MT function renders dubious the widely held belief that MT primarily scavenges radicals or detoxifies metals and is consistent with the frequent use of cysteine as a zinc ligand in proteins as a means of both tight and weak zinc binding of thiols and disulfides, respectively . Thus, we relate changes in the reducing power of the cell to the stability of the zinc/sulfur network in MT and the relative mobility of zinc and its control.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3472 - 7
Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli; Wilce MC et al.; The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-A resolution, for a dipeptide-inhibited complex at 2.3-A resolution, and for a low-pH inactive form at 2.7-A resolution . The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions . The monomer folds into two domains . The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro . The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits . The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme) . The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3402 - 7
A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides; Astatke M et al.; Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate . The Klenow fragment of E . coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold . Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2'-OH of an incoming rNTP . A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 "steric gate" can be fully effective . Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides.

J Mol Biol, 1998 Mar 6, 276(4), 819 - 27
A single mutation disrupts the pH-dependent dimerization of glycinamide ribonucleotide transformylase; Mullen CA et al.; Monomeric GART reversibly associates into a dimeric form as a function of decreasing solution pH . The transition is consistent with a three-proton transfer reaction with an apparent pKa near 7 . We now report that a single mutation, which replaces a glutamic acid at position 70 in the dimer interface with alanine (E70A), disrupts the pH-dependent dimerization of GART based on dynamic light scattering and gel filtration studies . A comparison of data obtained from UV-absorbance difference spectroscopy for both the wild-type and mutant forms of GART indicates that a tyrosine residue(s) undergoes a change in solvent exposure over the pH range 6.55 to 8.19 . A conformational change in tertiary structure that accompanies dimerization accounts for 60% of the observed optical difference, while the remaining 40% can be attributed to a pH-dependent process unrelated to dimerization . In addition, fluorescence studies of the mutant protein indicate that a pH-dependent change in tryptophan fluorescence exhibited by the wild-type protein is unrelated to quaternary structural changes and is likely a result of simple fluorescence quenching by nearby protonated histidine side-chains . Taken together, our results indicate that a single amino acid change at the dimer interface is sufficient to interrupt the highly specific, pH-dependent assembly reaction of GART, although pH-dependent conformational changes present in the wild-type protein also occur in E70A GART . This work is a first application of structure-based site-directed mutagenesis to the analysis of this pH-dependent assembly reaction .

J Mol Biol, 1998 Mar 6, 276(4), 689 - 703
Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis; Sun W et al.; Escherichia coli primase/SSB/single-stranded phage G4oric is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication . By a strategy of deletion analysis and antisense oligonucleotide protection on small single-stranded G4oric fragments, we have identified the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein . Together with the previous data, we have defined the structure of the primase/SSB/G4oric priming complex . Two SSB tetramers bind to the G4oric secondary structure, which dictates the spacing of 3' and 5' bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure . Two primase molecules then bind separately to specific DNA sequences in the 3' and 5' SSB-free G4oric regions . Binding of the 3' SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming . The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems . Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template .

Development, 1998 Mar, 125(5), 949 - 58
Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change; Ludwig MZ et al.; Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function . Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view . Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them . To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D . melanogaster, D . yakuba, D . erecta and D . pseudoobscura . Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer . Some of the binding sites in D . melanogaster are either absent or modified in other species . One functionally important bicoid-binding site in D . melanogaster appears to be recently evolved . We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation . This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D . melanogaster embryos . Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein . We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe . We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites . This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.

Neurochem Res, 1998 Jun, 23(6), 907 - 11
Mycobacterium leprae binds to a 25-kDa phosphorylated glycoprotein of human peripheral nerve; Suneetha LM et al.; Mycobacterium leprae, the causative agent of leprosy, specifically invades and destroys the peripheral nerve, which results in the main clinical manifestation of the disease . Little is known about the bacteria-nerve protein interaction . We show in the present work that M leprae binds to a 25 kDa glycoprotein from human peripheral nerve . This protein is phosphorylatable and it binds to lectins which have alpha-mannose specificity . This M leprae-protein interaction could be of importance in the pathogenesis of leprosy.

Neurosci Lett, 1998 Mar 13, 244(2), 112 - 4
Are the capsaicin-sensitive structures of ventral medulla involved in the temperature response to endotoxin in rats?
Koulchitsky SV.
In chronic experiments on rats pretreated with bilateral microinjection of 25 nl 1% capsaicin to the caudal ventrolateral medulla under ketamine-xylazine-acepromazine anesthesia, an enhancement of the temperature response to intraperitoneal application of 3 microg/kg E . coli lipopolysaccharide as compared to animals who received vehicle to the caudal ventrolateral medulla was found . This is indicative of the involvement of the capsaicin-sensitive bulbar structures in thermoregulatory processes during endotoxemia.

Neurosci Lett, 1998 Mar 13, 244(2), 81 - 4
Efficient mutagenesis of zebrafish by a DNA cross-linking agent; Ando H et al.; We developed a novel procedure for efficient mutagenesis of zebrafish using a DNA cross-linking agent 4,5',8-trimethylpsoralen (TMP), which is known to frequently induce small deletions in Escherichia coli and Caenorhabditis elegans . A specific-locus test and pilot screenings indicated that the TMP mutagenesis procedure was efficient . To confirm the successful mutagenesis by TMP, we characterized mutants with selective impairments in the nervous system . The no tectal neuron mutation hindered the development of the tectal neurons, while the edawakare mutation resulted in the enhancement of the extension and branching of the peripheral axons of trigeminal ganglion and Rohon-Beard sensory neurons . These results suggest that the TMP mutagenesis will provide an efficient method to isolate and characterize zebrafish mutants at molecular level.

Arch Virol, 1998, 143(3), 467 - 74
Transient marker stabilisation: a general procedure to construct marker-free recombinant vaccinia virus; Scheiflinger F et al.; Recombinant vaccinia viruses based on the highly attenuated Modified Vaccinia Ankara (MVA) strain expressing HIV-1 antigen genes were constructed by a novel procedure involving the transient use of two marker genes . The selectable markers used, the Escherichia coli guanine phosphoribosyltransferase (gpt) and the beta-galactosidase (lacZ) genes, are not retained within the final recombinant virus . The transient marker stabilisation (TMS) procedure allows the generation of marker-free recombinant viruses in a series of simple plaque purification steps . HIV-1 gag pol genes were inserted into two loci of vaccinia MVA demonstrating the efficiency of the procedure.

J Neurochem, 1998 May, 70(5), 1793 - 8
Molecular cloning and expression of a mouse brain cDNA encoding a novel protein target of calcyclin; Filipek A et al.; A protein target of mouse calcyclin, p30, which we call calcyclin-binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells . The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library . A 1.4-kb positive clone was detected, isolated, and sequenced . The analyzed clone contains an open reading frame encoding a protein of a molecular mass of approximately 26 kDa . The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein . The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells . Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed . The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca2+ in solution during affinity chromatography and on blots . Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.

J Biotechnol, 1998 Feb 5, 60(1-2), 55 - 66
Estimation of biomass and specific growth rate in a recombinant Escherichia coli batch cultivation process using a chemical multisensor array; Bachinger T et al.; A chemical multisensor array is used in combination with an artificial neural network to estimate the biomass concentration and specific growth rate in a recombination Escherichia coli batch cultivation . It is shown that by providing sufficient information to the artificial neural network, an accuracy comparable to that of an established dry weight method can be achieved . The obtained prediction error (1 sigma) of 0.043 g l-1 for biomass compares well with the error of the dry weight method in this low biomass concentration range (0.1-3 g l-1) . The prediction for the specific growth rate is accurate during important parts of the cell growth (1 sigma = 0.025 h-1) . The results show that this non-invasive method is potentially useful for estimating biomass and specific growth rate on-line in bioprocesses.

J Biotechnol, 1998 Feb 5, 60(1-2), 23 - 35
Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xynl from Rhodothermus marinus; Karlsson EN et al.; The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli . The catalytic domain belongs to glycosyl hydrolase family 10 . The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase . The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9 . The pH and temperature optima for activity were determined to be 7.5 and 80 degrees C, respectively . At that temperature the enzyme had a half-life of 1 h 40 min . An addition of 1 mM calcium stabilized the activity of the enzyme at 80 degrees C . The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans) . No exo- or endo-cellulase activity was observed . Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose . The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan . The R . marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase.

Biosci Biotechnol Biochem, 1998 Mar, 62(3), 564 - 6
cDNA cloning, expression, and mutagenesis of scytalone dehydratase needed for pathogenicity of the rice blast fungus, Pyricularia oryzae; Motoyama T et al.; We purified scytalone dehydratase from the rice blast fungus, Pyricularia oryzae, and cloned its cDNA on the basis of its amino acid sequence . The deduced amino acid sequence was 62% identical to the scytalone dehydratase from Colletotrichum lagenarium . The expression of this gene was induced transcriptionally in the stationary phase when melanin synthesis occurs . We constructed a heterologous expression system for the enzyme in Escherichia coli, did deletion analysis with this system, and found that a C-terminal region is essential for the enzyme function.

Biosci Biotechnol Biochem, 1998 Mar, 62(3), 448 - 52
Catalase catalyzes of peroxynitrite-mediated phenolic nitration; Kono Y et al.; Catalase catalyzed the peroxynitrite-mediated nitration of 4-hydroxyphenylacetic acid . The curve for the pH dependence of nitration was similar to that for the reaction between peroxynitrite and phenol . Cyanide, azide, and 3-amino-1,2,4-triazole inhibited the nitration in a dose-dependent way . When catalase was mixed with peroxynitrite, Compound I was detected as an intermediate . Because azide was an electron donor for the peroxidatic action of catalase, and because 3-amino-1,2,4-triazole inhibited catalase activity by binding with Compound I, peroxynitrite-mediated phenolic nitration was probably accompanied by Compound I formation . Both catalase and superoxide dismutase protected Escherichia coli from peroxynitrite toxicity.

Mol Cells, 1998 Feb 28, 8(1), 96 - 100
Expression of C5 protein, the protein component of Escherichia coli RNase P, from the tac promoter; Park BH et al.; The accurate function of C5 protein, the protein component of Escherichia coli RNase P, is uncertain in vivo . A controllable expression system for C5 protein was constructed which can be used to investigate effects of C5 protein on various cellular functions including biosynthesis of RNase P in vivo . The semisynthetic rnpA gene encoding C5 protein was fused to the tac promoter of the pKK223-3 expression vector . This tac promoter expression system produced a high level of C5 protein upon induction with isopropyl-beta-D-thiogalacto-pyranoside . When the overexpressed C5 protein was purified and used for reconstitution of RNase P, the reconstituted enzyme was active . The N-terminal amino acid of the overexpressed C5 protein was leucine specified by the second codon of the rnpA gene . The more controllable expression system was constructed by introducing the lacIq gene into the vector sequence itself.

Mol Cells, 1998 Feb 28, 8(1), 43 - 8
Characterization of protein interaction among subunits of protein kinase CKII in vivo and in vitro; Kim MS et al.; Protein kinase CKII (CKII) is a ubiquitous protein serine/threonine kinase . CKII usually exists in tetrameric complexes composed of two catalytic (CKII alpha and/or CKII alpha') and two regulatory (CKII beta) subunits . In the present study, using a combined in vivo and in vitro approach, we have investigated the role of CKII subunits in the formation of the tetrameric structure of CKII and the formation of the polymeric structure of CKII holoenzyme . Our in vivo experiments show that CKII beta interacts with either another CKII beta or CKII alpha and that CKII alpha does not interact with another CKII alpha (or CKII alpha') . Our in vitro experiments also show that CKII beta is able to associate with both CKII alpha and another CKII beta and that CKII alpha exists as a monomeric form in solution . These data indicate that CKII beta mediates the formation of a tetramer by both the dimerization of CKII beta and the interaction of CKII beta with CKII alpha . The results of this study also suggest that CKII beta may be involved in the formation of the polymeric structure of the CKII holoenzyme.

Biochem Biophys Res Commun, 1998 Apr 17, 245(2), 478 - 82
Expression of a hepatitis C virus NS3 protease-NS4A fusion protein in Escherichia coli; Inoue H et al.; Both the NS3 protease and the NS4A protein are required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein . The NS3 protease domain was fused at its C-terminal end with full length NS4A and expressed in Escherichia coli . This protein (NS3 delta-NS4A) was purified to apparent homogeneity after refolding from extracts recovered from inclusion bodies . During the expression and purification process, NS3 delta-NS4A was not auto-processed in either a cis or trans manner at NS3/NS4A junction site . When the kcat/K(m) values and thermostability of NS3 delta-NS4A were compared with those for maltose binding protein-NS3 fusion protein (MBP-NS3), which contains only NS3 region, the single-chain NS3 delta-NS4A showed enhanced proteolytic activities and thermostability.

Biochem Biophys Res Commun, 1998 Apr 17, 245(2), 319 - 24
In vitro and in vivo potentiation of radiosensitivity of malignant gliomas by antisense inhibition of the RAD51 gene; Ohnishi T et al.; The mammalian RAD51 gene is a homologue of the yeast RAD51 and E . coli RecA genes, which are related to the repair of DNA double-strand breaks and are also involved in recombination repair and various SOS responses to DNA damage by gamma-irradiation and alkylating reagents . In this study, we investigated both in vitro and in vivo whether inhibition of the RAD51 gene by antisense oligonucleotides (ODNs) enhances the radiosensitivity of mouse malignant gliomas . A volume of 100 nM of RAD51 antisense ODNs inhibited the level of mRNA by more than 95% and reduced the protein expression by about 70% . Treatment of mouse 203G glioma cells with 100 nM of RAD51 antisense ODNs significantly enhanced the radiation-induced cell kill compared to control cells, and cells treated with sense or scrambled ODNs . When the glioma cells were implanted in the cisterna magna of mice followed by treatment with RAD51 antisense ODNs, the survival time of the mice was markedly prolonged compared to that of the untreated group (p < 0.001, logrank test) . In addition, the combination of antisense ODNs and irradiation extended the survival time of the glioma-bearing mice much longer than could be achieved with radiation alone (p < 0.0001, logrank test) . These results suggest that inhibition of RAD51 can be expected to serve as a novel potentiator for radiation therapy in malignant gliomas by inhibiting DNA double-strand break repair.

Gen Comp Endocrinol, 1998 May, 110(2), 201 - 11
Rainbow trout glucocorticoid receptor overexpression in Escherichia coli: production of antibodies for western blotting and immunohistochemistry; Tujague M et al.; Fragments of cDNA that encode the N-terminal and DNA-binding domains (DBD) of the rainbow trout glucocorticoid receptor (rtGR) were expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) . The fusion proteins induced by IPTG could readily be detected as 45- and 40-kDa bands, respectively, in crude extracts, as well as in proteins purified on glutathione-agarose . These purified hybrid proteins were used to immunize rabbits . The antisera produced were tested for specificity by Western blot analysis using extracts from COS-1 cells transfected with an rtGR expression vector and from trout liver cells . The antisera raised against the DBD domain did not detect any bands on Western blots, even at low antiserum dilution . However, the purified DBD fusion protein specifically bound GRE-containing DNA fragments in gel-shift assays, and the retarded complexes were supershifted by these antibodies . The antisera raised against the N-terminal domain consistently detected two protein bands at 104 and 100 kDa in the two cell extracts and allowed specific immunohistochemical staining in fish brain and pituitary . For the first time in fish, these antibodies will allow analysis of GR expression in different cortisol target tissues.

Anal Biochem, 1998 May 1, 258(2), 305 - 10
Detection of lectins using ligand blotting and polyacrylamide-type glycoconjugate probes; Kamemura K et al.; A sensitive and convenient method for detection of the carbohydrate-binding activity of lectins was established using the combination of blotting of lectins on polyvinylidene difluoride membranes, carbohydrate-conjugated biotinylated polyacrylamide-type probes (carbohydrate-bp probes), horseradish peroxidase-streptavidin, and detection by enhanced chemiluminescence of the enzyme reaction . This method was tested for detection of four plant lectins blotted on the membrane: concanavalin A was detectable down to 100 ng by mannose-bp probe, Ricinus communis agglutinin 120 to as low as 5 ng by N-acetyllactosamine-bp probe, soybean agglutinin to 1 microgram by beta-N-acetyl-D-galactosamine-bp probe, and wheat germ agglutinin to 5 ng by beta-N-acetyl-D-glucosamine-bp probe . All four lectins were detectable on an electroblotted membrane after SDS-polyacrylamide gel electrophoresis . This method was used to detect recombinant human galectin-3 in Escherichia coli cell lysates and mannan-binding protein in human serum . These results indicate that this method is widely applicable to convenient detection and characterization of lectins in crude samples.

Mol Microbiol, 1998 Mar, 27(6), 1247 - 60
EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli; Wainwright LA et al.; Enteropathogenic Escherichia coli uses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium . Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export . Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion . CesD shows sequence homology to chaperone proteins from other type III secretion pathways . Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC . A mutation in cesD abolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA . The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis . CesD was shown to interact with EspD but not EspB or EspA . CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane . Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family.

Mol Microbiol, 1998 Mar, 27(6), 1213 - 21
Synthesis of Escherichia coli O9a polysaccharide requires the participation of two domains of WbdA, a mannosyltransferase encoded within the wb* gene cluster; Kido N et al.; WbdA (previously MtfA) is one of the mannosyltransferases encoded within the Escherichia coli O9a wb* gene cluster . It is composed of two domains of similar size, connected by an alpha-helix chain . Elimination of the C-terminal half by transposon insertion or gene deletion caused synthesis of an altered structural O-polysaccharide consisting only of alpha-1,2-linked mannose . O9a polysaccharide synthesis was restored by the C-terminal half of WbdA in trans . No membrane incorporation of mannose from GDP mannose was observed in a strain carrying only the gene for truncated WbdA . For mannose incorporation, it was necessary to introduce both wbdB and wbdC genes into the strain . Therefore, it is likely that the N-terminal half of truncated WbdA synthesizes the altered O-polysaccharide together with other mannosyltransferases which participate in the initial reactions of the O9a polysaccharide synthesis . Both N- and C-terminal domains of WbdA are required for the synthesis of the complete E . coli O9a polysaccharide . The chi sequence location between the two domains and homology plot analyses of the wbdA and the WbdA protein suggested that the wbdA gene might have arisen by fusion of two independent genes.

Mol Microbiol, 1998 Mar, 27(6), 1141 - 56
Influence of FIS on the transcription from closely spaced and non-overlapping divergent promoters for an aminoacyl-tRNA synthetase gene (gltX) and a tRNA operon (valU) in Escherichia coli; Champagne N et al.; The gltX gene, encoding the glutamyl-tRNA synthetase (GluRS), and the valU operon, whose transcripts contain three tRNAVal/UAC and one tRNALys/UUU, are adjacent and divergently transcribed . It is the only known case of adjacent genes encoding an aminoacyl-tRNA synthetase and a tRNA precursor in Escherichia coli . The gltX promoters (P1, P2 and P3) direct the synthesis of transcripts non-overlapping with and divergent from the one initiated at the valU promoter . We report that their promoter region (250 bp) contains three binding sites for the factor for inversion stimulation (FIS), centred at positions -71, -91 and -112 from the valU transcription initiation site, and that the destruction of any of these sites does not prevent the binding of FIS to the others . As FIS is one of the major positive regulators of stable RNA operons, we have studied its role on gltX and valU transcription . FIS stimulates valU transcription in vitro and about twofold in vivo during steady-state exponential growth . In contrast, gltX transcription is repressed by the presence of FIS in vitro and about twofold in vivo during growth acceleration when a decrease in GluRS concentration was observed . Under all conditions tested, most of the gltX transcripts start at the P3 promoter . Nested deletions of this regulatory region reveal that the FIS-dependent repression of the gltX-P3 promoter is abolished after the removal of the valU promoter, and is not altered by the additional removal of the FIS binding sites; moreover, in vivo transcription from gltX-P1 and/or gltX-P2 present on some of these regulatory region variants is modulated by the nature of the upstream region by FIS and is sometimes stronger than that from gltX-P3 . These results show that the strength and the site of gltX transcription initiation are influenced by the upstream region up to and including the valU promoter; furthermore, they indicate that although these adjacent genes are involved in the first step of protein biosynthesis and share cis and trans regulatory elements, their transcription is non-co-ordinate.

Mol Microbiol, 1998 Mar, 27(6), 1119 - 27
Mutational analysis of the NH2-terminal arms of the trp repressor indicates a multifunctional domain; Mackintosh SG et al.; The NH2-terminal arms of the Escherichia coli trp repressor have been implicated in three functions: formation of repressor-operator complexes via association with non-operator DNA; stabilization of repressor oligomers bound to DNA; and oligomerization of the aporepressor in the absence of DNA . To begin to examine the structural aspects of the arms that are responsible for these varied activities, we generated an extensive set of deletion and substitution mutants and measured the activities of these mutants in vivo using reporter gene fusions . Deletion of any part of the arms resulted in a significant decrease in repressor activity at both the trp and the trpR operons . Positions 4, 5 and 6 were the most sensitive to missense changes . Most substitutions at these positions resulted in repressors with less than 5% of the activity of the wild-type trp repressor . A large percentage of the missense mutants were more active than the wild-type repressor in medium containing tryptophan and less active in medium without tryptophan . This phenotype can be explained in terms of altered oligomerization of both the repressor and the aporepressor . Also, nine super-repressor mutants, resulting from substitutions clustered at both ends of the arms, were found . Our results support the hypothesis that the NH2-terminal arm of the trp repressor is a multifunctional domain and reveal structural components likely to be involved in the various functions.

RNA, 1998 Feb, 4(2), 231 - 8
Construction of an in vivo-regulated U6 snRNA transcription unit as a tool to study U6 function; Luukkonen BG et al.; U6 snRNA is the only spliceosomal snRNA transcribed by RNA polymerase III in yeast . We have constructed a regulated U6 snRNA transcription unit by introducing the binding site for the Escherichia coli lacI repressor protein in the U6 snRNA promoter . GAL-induced expression of lacI protein led to a decrease in U6 snRNA levels and blocked cell growth . lacI dissociation from the promoter, and consequent U6 snRNA transcription, could be induced by addition of IPTG and repression of lacI transcription . To test the usefulness of this system in studying spliceosomal U6 snRNA function, we conditionally expressed U6 snRNAs with a single base substitution in position A51 . We demonstrate that expression of the U6-A51 mutations confers a strong dominant negative phenotype as shown by severe reductions in growth rate . In these strains, splicing of endogenous pre-mRNAs was blocked before the second step.

RNA, 1998 Feb, 4(2), 189 - 94
Mutational analysis of the donor substrate binding site of the ribosomal peptidyltransferase center; Saarma U et al.; Previous experiments have shown that the top of helix 90 of 23S rRNA is highly important for the ribosomal peptidyltransferase activity and might be part of the donor (P) site . Developing on these studies, mutations in the 23S rRNA at the highly conserved positions G2505, G2582, and G2583 were investigated . None of the mutations affected assembly, subunit association, or the capacity of tRNA binding to A and P sites . A "selective transpeptidation assay" revealed that the mutations specifically impaired peptide bond formation . Results with a modified "fragment" assay using the minimal donor substrate pA-fMet are consistent with a model where the nucleotides psiGG2582 form a binding pocket for C75 of the tRNA.

Cancer Gene Ther, 1998 Mar-Apr, 5(2), 119 - 26
Transfection of primary tumor cells and tumor cell lines with plasmid DNA/lipid complexes; Stopeck AT et al.; Cancer vaccines that utilize genetically modified tumor cells require gene transfer methods capable of producing immunostimulatory doses of transgenes from fresh or short-term cultures of human tumor cells . Our studies optimize in vitro transfection of primary tumor cells using cationic lipids and a plasmid encoding the gene for human interleukin-2 (IL-2) . Established tumor cell lines produced 10- to 100-fold more IL-2 than did fresh or short-term tumor cultures as measured by enzyme-linked immunoabsorbent analysis . Importantly, transfection of primary tumor cells produced immunostimulatory levels of IL-2 as determined by increased thymidine incorporation by autologous peripheral blood mononuclear cells and lymphokine-activated killer cell activity . IL-2 secretion by tumor cells persisted for at least 30 days post-transfection and was unaffected by freeze thawing or irradiation to 8000 rads . Multiple solid tumor types were successfully transfected, but normal blood mononuclear cells and leukemic blasts were resistant to transfection . Enzyme-linked immunoabsorbent analysis of the amount of IL-2 secreted into the medium by transfected tumor cells correlated with the percentage of tumor cells expressing intracellular IL-2 as measured by flow cytometry . Plasmids utilizing a cytomegalovirus promoter yielded superior transfection efficiencies compared with plasmids containing a Rous sarcoma virus promoter . These results suggest that a clinical vaccine trial using autologous tumor cells genetically modified to secrete IL-2 is feasible in patients with solid tumors.

Cancer Gene Ther, 1998 Mar-Apr, 5(2), 83 - 91
Phosphorylation and cytotoxicity of therapeutic nucleoside analogues: a comparison of alpha and gamma herpesvirus thymidine kinase suicide genes; Cazaux C et al.; Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain . In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity . When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR) . The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK . TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs.

Microbiol Immunol, 1998, 42(3), 195 - 202
Construction of the chimeric reverse transcriptase of simian immunodeficiency virus sensitive to nonnucleoside reverse transcriptase inhibitor; Isaka Y et al.; A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) . The compounds can be grouped into two broad classes; nucleoside analogs and nonnucleoside RT inhibitors . The nonnucleoside RT inhibitors are quite specific for HIV-1 RT but not human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) RT . We have investigated the property of SIV/HIV-1 chimeric viruses in which portions of SIV(MAC) RT were exchanged with the corresponding domain of HIV-1 RT; amino acids 176-190, 176-383 and 176-495 of HIV-1 RT . The chimeric virus, which was substituted amino acids 176-190 of RT, had detectable RT activity, and this chimeric RT was sensitive to three nonnucleoside RT inhibitors {nevirapine, HEPT derivative (E-EBU-dM) and TIBO derivative (R82913)} . To further study this chimeric virus, we purified the chimeric RT enzyme expressed in Escherichia coli and determined its kinetic properties; the Km, and Vmax values, and the Ki value of HEPT derivative calculated for the DNA polymerase activity . This study reveals that amino acids 176-190 of SIV(MAC) RT were important for the enzymatic activity and the SIV/HIV-1 chimeric RT, which had amino acids 176-190 of HIV-1, was sensitive to the nonnucleoside RT inhibitor.

Mol Hum Reprod, 1998 Mar, 4(3), 235 - 42
Expression of extracellular superoxide dismutase in the human male reproductive tract, detected using antisera raised against a recombinant protein; Williams K et al.; Mammalian spermatozoa are particularly susceptible to the deleterious effects of reactive oxygen species and lipid peroxidation, which ultimately lead to impaired fertility . A number of enzymes are present in the male reproductive tract which may play a role in preventing oxidative damage; in particular, the epididymis is the site of synthesis and secretion of large amounts of extracellular superoxide dismutase (eSOD) . In order to study the distribution of eSOD in the male reproductive tract, and distinguish it from other related superoxide dismutase isoenzymes (e.g . cytosolic SOD), polyclonal antisera have been raised against a recombinant human eSOD fusion protein, expressed in bacterial cells . This protein was expressed from a synthetic gene fragment, using preferred Escherichia coli codons, designed to overcome the problems associated with the high guanine+cytosine content of the natural human eSOD transcript . Using this antiserum, eSOD can be readily detected in a range of human reproductive tissues as well as in human seminal plasma . However, the presence of similar levels of eSOD in the seminal plasma of vasectomized men (probably of prostatic origin) precludes its use as a simple diagnostic indicator of eSOD activity levels in the epididymis.

Int J Biochem Cell Biol, 1997 Dec, 29(12), 1475 - 83
The effect of a nested set of C-terminal substituted deletions on the function of the alpha subunit of Escherichia coli RNA polymerase; Thomas MS et al.; A genetic screen was devised to obtain plasmid-borne rpoA alleles exhibiting partial or no complementation of the chromosomal Escherichia coli rpoA341 allele responsible for a pleiotropic phenotype . Nine of the ten mutants obtained carried single base pair deletions within the 3' end of rpoA resulting in frameshifting into a 72 codon +1 orf extending from within codon L262 and terminating 16 bp downstream of the rpoA reading frame . These frameshifts give rise to a set of substituted alpha deletions that are all of the same size (334 aa) and carry segments of the Orf sequence replacing the alpha region from the C-terminus (residue 329) to various points between 272 > 319 . The in vivo properties of this nested set of nine C-terminal-substituted derivatives of the alpha subunit of RNA polymerase have been assessed in terms of their assembly and transcriptional proficiency . The results indicate: (i) replacement of as much as 42 C-terminal residues of the alpha subunit does not prevent formation of a transcriptionally proficient holoenzyme form of RNA polymerase capable of complementing rpoA112(Ts); (ii) the extreme C-terminal Orf region, like that of alpha itself, is exposed in holoenzyme; (iii) these substituted deletions are not commonly functional at class I activated promoters.

FEMS Microbiol Lett, 1998 Apr 15, 161(2), 337 - 43
Isolation and heterologous expression of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from the wheat leaf-spot pathogen, Mycosphaerella graminicola; Keon J et al.; We describe the isolation and sequence of a gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD) (EC 1.13.11.27)) from the wheat leaf-spot fungal pathogen Mycosphaerella graminicola (Septoria tritici), that directs the synthesis of 2,5-dihydroxyphenylacetate (homogentisic acid, HGA) . The sequence of the deduced peptide showed homology to HPPDs from other organisms; the greatest identity was to a T-cell reactive protein, also identified as HPPD, from the human fungal pathogen Coccidioides immitis . As observed for HPPD from other sources, expression of the M . graminicola HPPD gene in Escherichia coli cells could be detected by the gradual development of a brown pigment in cultures as a result of the spontaneous oxidation and polymerisation of HGA . Pigment development in these cultures was prevented by the HPPD inhibitor sulcotrione.

FEMS Microbiol Lett, 1998 Apr 15, 161(2), 325 - 30
Expression of membrane-associated proteins by strains of enteroaggregative Escherichia coli; Spencer J et al.; Certain strains of enteroaggregative Escherichia coli express an outer membrane-associated protein, involved with the adhesion of these bacteria to HEp-2 cells . Strains of enteroaggregative E . coli hybridising with DNA probes for aggregative adhesion, diffuse adhesion and aggregative adhesion fimbriac II expressed an outer membrane-associated protein of 18 kDa regulated by magnesium ions . Strains hybridising with the aggregative adhesion probe only expressed a 20-kDa outer membrane-associated protein regulated by calcium and magnesium . The present study describes two populations of enteroaggregative E . coli which appear to adhere to HEp-2 cells by expressing antigenically distinct, negatively charged membrane-associated proteins.

Growth Factors, 1998, 15(3), 215 - 29
Purification and characterization of a recombinant human cripto-1 protein; Seno M et al.; Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors . To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E . coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein . We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites . Expression of the modified rhCR-1 protein in E . coli was limited to the inclusion bodies . The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence . Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation . By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethylammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent . This modification facilitates solubilization of the protein when rhCR-1 is denatured . The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer . The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit beta-casein expression, to stimulate the tyrosine phosphorylation of Shc and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.

J Med Microbiol, 1998 Apr, 47(4), 283 - 93
The 18th C.L . Oakley Lecture . Pathogenicity of enteropathogenic Escherichia coli; Baldwin TJ; Enteropathogenic Escherichia coli (EPEC) remain an important world-wide cause of diarrhoeal disease and mortality of infants and young children . Research programmes around the world have, in recent times, made enormous strides towards a better understanding of EPEC pathogenesis, yielding unique insights into the molecular intercourse between host and pathogen . Recombinant DNA and cell biology techniques have provided powerful tools, giving the first intriguing glimpses of a wealth of bacterial products mediating complex host:pathogen interactions involving the subversion of normal host signalling processes . Much has been discovered since 1945, when E . coli was first implicated as a cause of diarrhoea . However, many questions remain unanswered and many more remain unasked . Much remains to be discovered, especially in the area of molecular interactions between host and pathogen and how they relate to the manifestation of disease in the patient.

Protein Sci, 1998 Apr, 7(4), 1057 - 60
Crystallization and preliminary X-ray analysis of a 1:1 complex between a designed monomeric interferon-gamma and its soluble receptor; Randal M et al.; A variant of human interferon-gamma (IFN-gamma) has been created in which the two chains of the homodimeric cytokine were linked N- to C-terminus by an eight residue polypeptide linker . The sequence of this linker was derived from a loop in bira bifunctional protein, and was determined from a structural database search . This "single-chain" variant was used to create an IFN-gamma molecule that binds only a single copy of the alpha-chain receptor, rather than the 2 alpha-chain receptor: 1 IFN-gamma binding stoichiometry observed for the native hormone . Crystals have been grown of a 1:1 complex between this single-chain molecule and the extracellular domain of its alpha-chain receptor . These crystals diffract beyond 2.0 A, significantly better than the 2.9 A observed for the native 2:1 complex . Density calculations suggest these crystals contain two complexes in the asymmetric unit; a self-rotation function confirms this conclusion.

Protein Sci, 1998 Apr, 7(4), 961 - 5
A differential scanning calorimetric study of the thermal unfolding of apo- and holo-cytochrome b562; Robinson CR et al.; Cytochrome b562 is a four-helix-bundle protein containing a non-covalently bound b-type heme prosthetic group . In the absence of heme, cytochrome b562 remains highly structured under native conditions . Here we report thermodynamic data for the thermal denaturation of the holo- and apoproteins as determined by differential scanning calorimetry . Thermal denaturation of holocytochrome b562 is a highly reversible process, and unexpectedly does not involve dissociation of the heme prosthetic group . Thermal denaturation of the corresponding apoprotein, with the heme group chemically removed, remains a cooperative, reversible process . Apocytochrome b562 is substantially destabilized relative to the holoprotein: the t1/2 is more than ten degrees lower, and enthalpy and heat capacity changes are about one-half of the holoprotein values . However, the energetic parameters of apocytochrome b562 denaturation are within the range of observed values for small proteins.

Mutagenesis, 1998 Mar, 13(2), 127 - 32
Mutation frequency decline in MMS-treated Escherichia coli K-12 mutS strains; Grzesiuk E et al.; It has been shown that the decline in mutant frequency (MFD) (argE3(ochre)-->Arg+) which occurs in MMS-treated and then transiently starved AB1157 Escherichia coli K-12 cells concerns revertants which arose by supL suppressor formation in a process which is umuDC dependent . Here we have examined whether MMS-induced Arg+ revertants are susceptible to decline when bacteria are deficient in mismatch repair . We show that there is an absence of MFD in MMS-treated M1 (mutS) and in EC2416 (mutS delta umuDC) cells defective in mismatch repair which is associated with a change in the spectrum of MMS-induced mutations formed . In contrast to AB1157, transformation of M1 (mutS) bacteria with plasmids harbouring various combinations of umuD(D')C genes does not enhance the level of MMS-induced mutations but may influence the proportion of supL mutations . These supL mutations show MFD . Repair processes under MFD conditions were confirmed by analysis of plasmid DNA isolated from MMS-treated bacteria at different stages of their starvation and digestion with Fpg protein.

Mutagenesis, 1998 Mar, 13(2), 109 - 14
Spontaneous mutation in lacI transgenic mice: a comparison of tissues; de Boer JG et al.; The nature of spontaneous mutations in the lacI transgene of Big Blue mice was determined in selected tissues . The mutant frequencies ranged from 2.5 x 10(-5) to 7.1 x 10(-5) for liver, spleen, bladder, stomach, kidney, bone marrow, lung and skin . We also determined the DNA sequence alterations in the mutants recovered from these tissues . In all tissues the predominant class of mutations was G:C-->A:T transitions, most of which occurred at 5'-CpG-3' dinucleotide sequences . Bladder, kidney and skin display the highest contribution of G:C-->A:T transitions . The second most common class of mutations was G:C-->T:A transversions . All other base substitution classes contributed < 10% each . Of the non-substitution events, the loss of a single base pair was the most frequently occurring event (< 10%) . The similarity of mutational spectra (in terms of kinds of mutations detected by the lacI transgenic system) in all tissues examined supports the idea that similar mutational pathways function in these tissues in the absence of chemical or physical stimulus.

Br Poult Sci, 1998 Mar, 39(1), 152 - 5
Changes in plasma alpha 1-acid glycoprotein concentration and selected immune response in broiler chickens injected with Escherichia coli lipopolysaccharide; Takahashi K et al.; 1 . Changes in plasma alpha 1-acid glycoprotein (AGP) concentration and immune responses following Escherichia coli lipopolysaccharide (LPS) injection were studied in broiler chickens . 2 . Higher plasma AGP concentrations were observed from 12 to 48 h after a single injection of LPS . 3 . The highest concentration of plasma AGP was observed on day 2 followed by a gradual decrease in chicks injected with 150 micrograms/kg body weight of LPS every day for 13 d . 4 . Plasma AGP concentration in chicks injected daily with LPS at 900 micrograms/kg body weight for 13 d increased on day 2, and decreased on day 4 to the concentration found before the injection . The concentration increased again on day 10 . 5 . Changes in plasma interleukin-1 (IL-1) like activity were similar to those in plasma AGP concentration when LPS was injected daily at 900 micrograms/kg body weight for 3 d . 6 . Responses of blood mononuclear cell (MNC) proliferation to mitogen or concanavalin A, (Con A), and pokeweed mitogen (PWM) were positively correlated with changes in plasma AGP concentration . 7 . The results suggest that plasma AGP concentration could be used as a positive indicator of changes in blood MNC proliferation to a mitogen and in plasma IL-1 like activity.

Virology, 1998 Apr 10, 243(2), 482 - 91
DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1; Zhao KN et al.; Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells . Plasmids carrying both an SV40 origin of replication (ori) and an E . coli ori were introduced into Cos-1 cells by DNA transfection PV capsid proteins were supplied in trans by recombinant vaccinia viruses . Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E . coli as an indication of packaging efficacy . VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1 + L2 packaged plasmid DNA at least 50 times more effectively . BPV-1 L1 + L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BPV sequences . Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BPV VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences . The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb.

Virology, 1998 Apr 10, 243(2), 423 - 31
Expression of human papillomavirus type 16 L1 protein in Escherichia coli: denaturation, renaturation, and self-assembly of virus-like particles in vitro; Zhang W et al.; Major capsid protein L1 of HPV16 was produced in a fused form in Escherichia coli using an inducible expression system . The protein formed insoluble aggregations (inclusion bodies) and the yield was more than 10% of total cell proteins . The inclusion bodies were isolated and solubilised with 8 M urea and the L1 proteins were purified by chromatographic separation . Following removal of the urea by gradual dialysis, the denatured L1 proteins spontaneously renatured and subsequently assembled into polymorphologic aggregations in vitro . Electron microscopy showed that the assembled material included structures resembling native empty capsids as well as incompletely formed capsids . After separation from the pool of polymorphologic structures by sucrose gradient sedimentation, the correctly formed virus-like particles (VLE . coliPs) were recognised by a HPV16 type-specific, conformational-dependent monoclonal antibody in an ELISA . This system offers not only a model for investigation of the intrinsic interactions that occur during L1 assembly, but also a potential route for convenient manufacture of highly purified VLP vaccines.

Virology, 1998 Apr 10, 243(2), 275 - 82
Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies; Kulski JK et al.; Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coli with the expression plasmid pTrcL1 . Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16 . One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype . A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype . The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens.

Eur Surg Res, 1998, 30(2), 79 - 94
Treatment with growth hormone and insulin-like growth factor-1 in septicemia: effects on carbohydrate metabolism; Balteskard L et al.; Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) may be beneficial against the protein catabolism seen in injury and septicemia . Further understanding of their effects on carbohydrate metabolism is needed . In a septic porcine model receiving total parenteral nutrition, pretreatment with GH or IGF-1 (or no treatment in controls) was followed by an infusion of live Escherichia coli bacteria . Endogenous glucose production, carbohydrate oxidation, glucose and lactate fluxes over the liver, gastrointestinal organs, kidney, and hindleg were determined . Endogenous glucose production increased during septicemia in the GH group . The metabolic acidosis induced by septicemia was augmented by GH, but attenuated by IGF-1 . The alanine and lactate levels were significantly higher in the GH- than in the IGF-1 treated animals during septicemia . IGF-1 pretreatment appeared to induce favorable effects while GH pretreatment might produce unfavorable effects on carbohydrate metabolism in septic piglets.

Mol Cell Biol, 1998 May, 18(5), 3081 - 8
Selective disruption of genes transiently induced in differentiating mouse embryonic stem cells by using gene trap mutagenesis and site-specific recombination; Thorey IS et al.; A strategy employing gene trap mutagenesis and site-specific recombination (Cre/loxP) has been used to identify genes that are transiently expressed during early mouse development . Embryonic stem cells expressing a reporter plasmid that codes for neomycin phosphotransferase and Escherichia coli LacZ were infected with a retroviral gene trap vector (U3Cre) carrying coding sequences for Cre recombinase (Cre) in the U3 region . Activation of Cre expression from integrations into active genes resulted in a permanent switching between the two selectable marker genes and consequently the expression of beta-galactosidase (beta-Gal) . As a result, clones in which U3Cre had disrupted genes that were only transiently expressed could be selected . Moreover, U3Cre-activating cells acquired a cell autonomous marker that could be traced to cells and tissues of the developing embryo . Thus, when two of the clones with inducible U3Cre integrations were passaged in the germ line, they generated spatial patterns of beta-Gal expression.

Mol Cell Biol, 1998 May, 18(5), 2721 - 8
Fission yeast rad12+ regulates cell cycle checkpoint control and is homologous to the Bloom's syndrome disease gene; Davey S et al.; The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes . Defects in BLM are responsible for the human disease Bloom's syndrome, which is characterized in part by genomic instability and a high incidence of cancer . Here we describe the cloning of rad12+, which is the fission yeast homolog of BLM and is identical to the recently reported rhq1+ gene . We showed that rad12 null cells are sensitive to DNA damage induced by UV light and gamma radiation, as well as to the DNA synthesis inhibitor hydroxyurea . Overexpression of the wild-type rad12+ gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G2-phase checkpoint control . We showed genetically and biochemically that rad12+ acts upstream from rad9+, one of the fission yeast G2 checkpoint control genes, in regulating exit from the S-phase checkpoint . The physical chromosome segregation defects seen in rad12 null cells combined with the checkpoint regulation defect seen in the rad12+ overproducer implicate rad12+ as a key coupler of chromosomal integrity with cell cycle progression.

Clin Exp Immunol, 1998 Apr, 112(1), 84 - 91
Recombinant human IL-16 inhibits HIV-1 replication and protects against activation-induced cell death (AICD); Idziorek T et al.; The chemoattractant cytokine IL- 16 has been reported to suppress lymphocyte activation and to inhibit HIV-1 replication in acutely infected T cells . We have cloned and expressed human IL-16 in Escherichia coli and investigated whether the recombinant protein could regulate the level of lymphocyte apoptosis from HIV-1-infected subjects . After purification and refolding, only 2-10% of the recombinant cytokine was present in a biologically active homotetrameric form . This could explain the need for high concentrations of the bacterially derived IL- 16 to induce significant inhibition of HIV-1 replication . Addition of IL-16 to unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-1-infected subjects did not modify the observed level of spontaneous lymphocyte apoptosis . In contrast, IL-16 added to PBMC cultures stimulated with anti-CD3, anti-CD95 or dexamethasone reduced significantly the percentage of lymphocytes undergoing AICD . This effect was found to correlate with the ability of the cytokine to decrease CD95 expression on activated CD4+ T cells . Comparative studies on PBMC from healthy individuals indicated that the regulation of apoptosis levels by IL-16 is a complex phenomenon and could depend on the nature of the activator used and/or the immune status of lymphocytes tested . The outcome of CD4 cross-linking on T cells by various ligands is discussed in the context of the observed beneficial activities of IL- 16 and its potential role in the treatment of HIV disease.

Chem Biol Interact, 1998 Feb 20, 109(1-3), 153 - 67
Redefining reductive sulfate assimilation in higher plants: a role for APS reductase, a new member of the thioredoxin superfamily?
Wray JL, Campbell EI, Roberts MA, Gutierrez-Marcos JF.
The reaction steps leading from the intermediate adenosine 5'-phosphosulfate (APS) to sulfide within the higher plant reductive sulfate assimilation pathway are the subject of controversy . Two pathways have been proposed: a 'bound intermediate' pathway in which the sulfo group of APS is first transferred by APS sulfotransferase to a carrier molecule to form a bound sulfite intermediate and is then further reduced by thiosulfonate reductase to bound sulfide; and a 'free intermediate' pathway in which APS is further activated to 3'-phosphoadenosine 5'-phosphosulfate (PAPS) by APS kinase followed by reduction of the sulfo group to free sulfite by PAPS reductase . Sulfite is then reduced to free sulfide by sulfite reductase . Sulfide, either free or bound, is then incorporated into organic form (as cysteine) by the enzyme O-acetylserine (thiol) lyase . In order to better characterize the pathway we attempted to clone PAPS reductase cDNAs by functional complementation of an Escherichia coli cysH mutant to prototrophy . We found no evidence for PAPS reductase cDNAs but did identify cDNAs that encode a small family of novel, chloroplast-localized proteins with APS reductase activity that are new members of the thioredoxin superfamily . We show here that the thioredoxin domain of these proteins is functional . We speculate that rather than proceeding via either of the pathways proposed above, reductive sulfate assimilation proceeds via the reduction of APS to sulfite by APS reductase and the subsequent reduction of sulfite to sulfide by sulfite reductase . In this scheme the product of the APS kinase reaction, PAPS, is not a direct intermediate in the pathway but rather acts as a substrate for sulfotransferase action and perhaps as a store of activated sulfate that can be returned to the pathway as APS via phosphohydrolase action on PAPS . Interactions between enzyme isoforms within the chloroplast stroma may bring about substrate channeling of APS and contribute to the partitioning of APS between sulfotransferase reactions on the one hand and the synthesis of cysteine and related metabolites via the reductive sulfate assimilation pathway on the other.

Chem Biol Interact, 1998 Feb 20, 109(1-3), 129 - 35
Effects of 3'-phosphoadenosine 5'-phosphate on the activity and folding of phenol sulfotransferase; Yang YS et al.; Known spectroscopic and kinetic data are used to formulate pathways of the physiological and transfer reactions and the substrate inhibition of phenol sulfotransferase . Kinetic mechanisms indicate that release of PAP from enzyme complex is required for the physiological reaction but not for the transfer reaction . The pathways explain rate difference between the physiological and transfer reactions since the release of PAP is the rate-limiting step of the former reaction . Two enzyme species of phenol sulfotransferase which distinguish the physiological and transfer reaction were found to involve the binding of PAP . Differences between two forms of phenol sulfotransferase, alpha and beta, indicate that they assemble through different folding process . It is demonstrated that only alpha enzyme renatures in the presence of PAP and beta enzyme renatures only in the absence of PAP in vitro . In the over-expressed system, formation of alpha and beta phenol sulfotransferase is also dependent on the availability of PAP in Escherichia coli . It is concluded that folding of phenol sulfotransferase is assisted by PAP to form alpha enzyme . In the absence of PAP, beta form of phenol sulfotransferase is produced.

Chem Biol Interact, 1998 Feb 20, 109(1-3), 43 - 52
Novel sulfotransferases cloned by RT-PCR: real proteins or PCR artifacts?
Gaedigk A, Lekas P, Berchuk M, Grant DM.
During studies designed to subclone human phenol sulfotransferase (STP and STM) sequences for use in heterologous E . coli-based expression systems, we designed two oligonucleotide primers that would allow for the simultaneous PCR amplification of expression cassettes containing the coding regions of the STP1, STP2 and STM cDNAs . Following total RNA isolation from human liver, reverse transcription of cDNA, PCR amplification under standard conditions, plasmid subcloning and restriction analysis to select for suitable ST recombinants, we recovered plasmids containing inserts corresponding to STP1, STP2 and STM . However, ten additional, closely related but apparently novel ST sequences were also isolated . Alignments of the three known ST sequences (and one published allelic variant) with these new clones revealed that each one appears to be a PCR-generated modular chimera possessing a combination of DNA segments derived from STP1, STP2 and STM . This observation should serve as an alert to the potential pitfalls of using PCR techniques for the cloning of highly related genes and their cDNA products, especially when PCR primer design allows for the amplification of multiple products in a single reaction.

Mol Biochem Parasitol, 1998 Mar 15, 91(2), 237 - 49
Identification and heterologous expression of a new dense granule protein (GRA7) from Toxoplasma gondii; Jacobs D et al.; Immunoscreening of an expression library constructed with Toxoplasma gondii tachyzoite mRNA with sera from toxoplasmosis-positive humans has led to the identification of a new parasite antigen . Sequence analysis of the gene encoding this antigen allowed the calculation of the theoretical molecular mass (25,857 Da) and showed that the protein contains a putative signal sequence . The C-terminal region contains two hydrophobic regions, the last of which has the characteristics of a membrane-spanning domain . When the protein was heterologously expressed in E . coli and tested by Western blot, it reacted with the human sera originally used for screening . The new antigen also reacted with a monoclonal antibody raised against the entire parasite . Ultrastructural analysis showed that the protein is localized in the dense granules . After host cell invasion, the protein is secreted into the vacuolar network, the parasitophorous vacuole membrane, and into extensions protruding in the cytoplasm . Therefore, it is suggested to designate this new dense granule protein GRA7, following the established nomenclature for this protein family.

Mol Biochem Parasitol, 1998 Mar 15, 91(2), 221 - 35
Thioredoxin peroxidase from Onchocerca volvulus: a major hydrogen peroxide detoxifying enzyme in filarial parasites; Lu W et al.; Random screening of an Onchocerca volvulus third-stage (L3) cDNA library identified a highly abundant cDNA encoding a newly discovered antioxidant enzyme, thioredoxin peroxidase (TPx), a member of the peroxidoxin superfamily . This TPx cDNA (Ov-tpx-2) encodes a polypeptide of 199 amino acid residues with a calculated molecular weight of 21,890 Da . The Ov-tpx-2 cDNA represents roughly 2.5% of the total cDNAs from the L3 cDNA library . The gene was expressed in Escherichia coli and the protein product was shown to have antioxidant activity . Antiserum raised against Ov-TPX-2 recognized a native protein from extracts of both the L3 and adult-stages with a molecular weight of 22 kD . The localization and stage-specificity of Ov-TPX-2 protein was analyzed by immunocytochemistry and immunoelectron microscopy using monospecific antibodies . Expression was detected in late first-stage larvae during development in the vector and increased in intensity during differentiation to the infective L3-stage . The antigen was also detected in post-infective larvae and adult worms . In larvae, Ov-TPX-2 protein was predominantly localized to the hypodermis and cuticle, with additional sites in the hypodermal chords and multivesicular bodies . In adult worms, the primary sites of expression were the uterine epithelium and intestine, with additional labeling of the body wall and cuticle . Developing embryos and microfilariae in utero were bathed in Ov-TPX-2 protein discharged from epithelial cells . These results suggest that Ov-TPX-2 may protect the parasites from being damaged by host-generated oxidative stress and that Ov-TPX-2 protein provides the H2O2-detoxifying activity predicted but not previously identified in filarial parasites . Its highly upregulated expression in infective larvae may aid in parasite establishment following transmission to the definitive host.

J Biomol NMR, 1998 Jan, 11(1), 97 - 102
An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli; Cai M et al.; A cost-effective protocol for uniform 15N and/or 13C isotope labeling of bacterially expressed proteins is presented . Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression . This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients . The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients . Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.

J Biomol NMR, 1998 Jan, 11(1), 17 - 29
A NOESY-HSQC simulation program, SPIRIT; Zhu L et al.; A program SPIRIT (Simulation Program considering Incomplete Recovery of z magnetization and INEPT Transfer efficiency) has been developed to simulate three-dimensional NOESY-HSQC spectra . This program takes into account (1) different transfer efficiency during INEPT and reverse INEPT durations due to differential relaxation rates and 1J coupling constants; (2) the different effect of the sensitivity-enhancement scheme on CH, CH2 and CH3 systems; and (3) incomplete recovery of longitudinal magnetization between scans . The simulation program incorporates anisotropic tumbling mode for symmetric tops, and allows for differential external relaxation rates for protons . Some well-defined internal motions, such as the fast rotation of methyl groups, are taken into account . The simulation program also allows for input of multiple conformations and their relative populations to calculate the average relaxation matrix to account for slow internal motions . With the SPIRIT program, the sensitivity-enhanced NOESY-HSQC experiment can be used directly in the evaluation of the accuracy of structures, which can potentially be improved by direct refinement against the primary data.

Prikl Biokhim Mikrobiol, 1998 Jan-Feb, 34(1), 120 - 6
{Preparation of lymphotoxin and properties of it}; Lebedev LR et al.; The process of isolation of highly purified human lymphotoxin was studied and optimized . A set of methods providing an increase in the content of the target product in the biomass (plasmid DNA amplification and selection of clones of transformed cells) was applied at the stage of cultivation . A two-step purification scheme (ion-exchange chromatography on DEAE-Biotone A1 and hydroxylapatite) was developed . A number of physical characteristics were studied.

Mem Inst Oswaldo Cruz, 1997 Sep-Oct, 92(5), 637 - 41
Expression of recombinant antigens in Escherichia coli: application on immunochemical studies of Schistosoma mansoni tegumental antigens; Abath FG et al.; Sm15 and Sm13 are recognized by antibodies from mice protectively vaccinated with tegumental membranes, suggesting a potential role in protective immunity . In order to raise antibodies for immunochemical investigations, the genes for these antigens were expressed in pGEX and pMal vectors so that comparisons could be made among different expression systems and different genes . The fusion proteins corresponding to several parts of the gene for the precursor of Sm15 failed in producing antibodies recognizing the parasite counterpart . On the other hand, antibodies raised against Sm13 MBP-fusion proteins recognized the 13 kDa tegumental protein . Thus the peculiarities of the gene of interest are important and the choice of the expression system must sometimes be decided on an empirical basis.

J Mol Biol, 1998 Mar 13, 276(5), 913 - 25
Molecular genetic analysis of transposase-end DNA sequence recognition: cooperativity of three adjacent base-pairs in specific interaction with a mutant Tn5 transposase; Zhou M et al.; Transposition of Tn5 and IS50 requires the specific binding of transposase (Tnp) to the end inverted repeats, the outside end (OE) and the inside end (IE) . OE and IE have 12 identical base-pairs and seven non-identical base-pairs . Previously we described the isolation of a Tnp mutant, EK54, that shows an altered preference for OE versus IE compared to wild-type (wt) Tnp . EK54 enhances OE recognition and decreases IE recognition both in DNA binding and in overall transposition . Here we report that base-pairs 10, 11 and 12 of the OE are critical for the specific recognition by EK54 Tnp . These three adjacent base-pairs act cooperatively; all three must be present in order for EK54 Tnp to interact very favorably with the end DNA . The existence of only one or two of these three base-pairs decreases binding of EK54 Tnp . The combined use of EK54 Tnp and a new OE/IE mosaic end sequence containing the OE base-pairs 10, 11 and 12 gives rise to an extraordinarily high transposition frequency . Just as the Tnp is a multifunctional protein, the nucleotides in the 19 bp Tn5 ends also affect other functions besides Tnp binding . Furthermore, the fact that we were able to isolate end sequence variants that transpose at a higher frequency than the natural ends (OE and IE) with wt Tnp reveals yet another way in which the wt transposition frequency is depressed, i.e . by keeping the end sequences suboptimal.

J Mol Biol, 1998 Mar 13, 276(5), 861 - 75
On the mechanism of inhibition of phage T7 RNA polymerase by lac repressor; Lopez PJ et al.; We study here the effect on phage T7 RNA polymerase activity of lac repressor bound downstream of the T7 promoter . When repressor binds in vitro at an operator centered at +13 or +15 with respect to transcription start, it does not prevent initiation, though the transcript yield is reduced . However, the processivity of the polymerase is depressed and transcript extension is blocked at positions +4 and +6, respectively . These results indicate that repressor and polymerase do not simply exclude each other from the promoter . Rather, they would come into steric conflict and compete for establishment or retention of interactions with the same segment of DNA, without this leading to the immediate displacement of either polymerase or repressor . The resulting destabilization of the transcription complex would depress both initiation rate and enzyme processivity . In contrast to the above results, little reduction in runoff transcription is observed when operator is centered at +47 . The decreased sensitivity of polymerase to repressor bound at +47 versus +13 or +15 is likely to be due to the higher stability of the elongation complex during the transcription of downstream regions in comparison with the first transcribed nucleotides . We also show that under conditions of leaky repression and with operator centered at +13, a mutant T7 RNA polymerase showing normal promoter affinity but a slower elongation rate is more sensitive to repression than the wild-type enzyme, both in vitro and in vivo . In vitro, this higher sensitivity is largely due to a reduced ability of the mutant to overcome the elongation block at position +4 . The parallel between the in vitro and in vivo data suggests that in vivo the repressor also does not prevent polymerase from binding to promoter, but interferes with subsequent steps in initiation and transcript extension, in this case presumably largely extension beyond +4.

Fold Des, 1998, 3(2), 87 - 93
Coupling protein stability and protein function in Escherichia coli CspA; Hillier BJ et al.; BACKGROUND: CspA is a small protein that binds single-stranded RNA and DNA . The binding site of CspA consists of a cluster of aromatic amino acids, which form an unusually large nonpolar patch on the surface of the protein . Because nonpolar residues are generally found in the interiors of proteins, this cluster may have evolved to bind nucleic acids at the expense of protein stability . RESULTS: Three neighboring phenylalanines have been mutated singly and in combination to leucine and to serine . All mutations adversely affect DNA binding . Surprisingly, all mutations, and especially those to serine, are destabilizing . CONCLUSIONS: The aromatic cluster in CspA is required not only for protein function but also for protein stability . This result is pertinent to the design of beta-sheet proteins and single-stranded nucleic acid binding proteins, whose binding mode is proposed to be of aromatic-aromatic intercalation.

J Chromatogr A, 1998 Mar 6, 798(1-2), 117 - 23
High-performance liquid chromatographic peak identification of 2,4-dinitrophenylhydrazine derivatives of lipid peroxidation aldehydes by photodiode array detection; Cordis GA et al.; Malonaldehyde (MDA), a product of lipid peroxidation, is a presumptive marker for the development of oxidative stress in tissues and plasmas . In this study we report the photodiode array detection of the 2,4-dinitrophenylhydrazine (DNPH) derivatives of MDA using HPLC . Oxidative stress was produced by injecting (i.p.) bacterial lipopolysaccharide (LPS) into rats at a dose of 100 micrograms/kg, or i.v . into rabbits (1 microgram/kg), or added to freshly drawn human blood (200 ng/ml) . Blood was collected at several time points up to 5 h, centrifuged, and equal volumes of 20% TCA were used to precipitate proteins from the plasma . The supernatants were derivatized with DNPH, and the aldehyde-DNPHs were extracted with pentane . After evaporation, aliquots of 10 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column, chromatographed with an acetonitrile-water-acetic acid gradient mobile phase and scanned using Waters 996 photodiode array detector . Peak identification and homogeneity was determined by comparing the experimental peaks and UV scans with those of authentic standards . A significant increase in the DNPH derivative of malonaldehyde (MDA-DNPH), but not of the other aldehyde-DNPH derivatives of formaldehyde (FDA), acetaldehyde (ADA), acetone and propionaldehyde (PDA) was seen over the first hour after LPS administration in anesthetized rats, while in conscious rabbits this trend lasted up to 3 h . The retention times as well as the UV scans of the derivatized aldehydes matched the authentic standards . Thus, photodiode array detection has proved valuable in establishing this HPLC method for estimating oxidative stress . This technique could accurately measure pmol amounts of MDA-DNPH indicating the usefulness of photodiode array detection method for estimating small changes in the oxidative stress.

J Chromatogr A, 1998 Mar 6, 798(1-2), 73 - 82
Accelerated recombinant protein purification process development automated, robotics-based integration of chromatographic purification and analysis; Londo T et al.; Recovery of recombinant proteins from endogenous, host molecules can be an experimentally intensive and time-consuming task . Often the time to analyze material during development of recovery procedures is the rate-limiting step . Nowadays, modern techniques and equipment are being specifically engineered to make this effort much more efficient . We present a case study to illustrate how a new, automation tool, designed for easy, systematic methods development, can be used for very rapid process and analytical optimization . This tool uses robotics to integrate process development with rapid LC-based analysis requiring no user intervention . The methods and procedures described can be generalized to any recombinant protein recovery campaign.

Science, 1998 Apr 10, 280(5361), 286 - 9
Ribosome-catalyzed peptide-bond formation with an A-site substrate covalently linked to 23S ribosomal RNA; Green R et al.; In the ribosome, the aminoacyl-transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks photochemically with G2553 of 23S ribosomal RNA (rRNA) . This covalently linked substrate reacts with a peptidyl-tRNA analog to form a peptide bond in a peptidyl transferase-catalyzed reaction . This result places the conserved 2555 loop of 23S rRNA at the peptidyl transferase A site and suggests that peptide bond formation can occur uncoupled from movement of the A-site tRNA . Crosslink formation depends on occupancy of the P site by a tRNA carrying an intact CCA acceptor end, indicating that peptidyl-tRNA, directly or indirectly, helps to create the peptidyl transferase A site.

Biochemistry, 1998 Mar 24, 37(12), 4160 - 8
Reaction of Escherichia coli cytochrome bo3 with substoichiometric ubiquinol-2: a freeze-quench electron paramagnetic resonance investigation; Schultz BE et al.; The reaction of the quinol oxidase cytochrome bo3 from Escherichia coli with ubiquinol-2 (UQ2H2) was carried out using substoichiometric (0.5 equiv) amounts of substrate . Reactions were monitored through the use of freeze-quench EPR spectroscopy . Under 1 atm of argon, semiquinone was formed at the QB site of the enzyme with a formation rate constant of 140 s-1; the QB semiquinone EPR signal decayed with a rate constant of about 5 s-1 . Heme b and CuB were reduced within the 10-ms dead time of the freeze-quench experiment and remained at a constant level of reduction over the 1-s time course of the experiment . Quantitation of the reduction levels of QB and heme b during this reaction yielded a reduction potential of 30-60 mV for heme b . Under a dioxygen atmosphere, the rates of semiquinone formation and its subsequent decay were not altered significantly . However, accurate quantitation of the EPR signals for heme b and heme o3 could not be made, due to interference from dioxygen . In the reaction between the QB-depleted enzyme and UQ2H2 under substoichiometric conditions, there was no observable change in the EPR spectra of the enzyme over the time course of the reaction, suggesting an electron transfer from heme b to the binuclear site in the absence of QB which occurs within the dead time of the freeze-quench apparatus . Analysis of the thermodynamics and kinetics of electron transfers in this enzyme suggests that a Q-cycle mechanism for proton translocation is more likely than a cytochrome c oxidase-type ion-pump mechanism.

Biochemistry, 1998 Mar 24, 37(12), 4148 - 59
Localization of histidine residues responsible for heme axial ligation in cytochrome b556 of complex II (succinate:ubiquinone oxidoreductase) in Escherichia coli; Vibat CR et al.; Complex II (succinate:ubiquinone oxidoreductase) from Escherichia coli contains four different subunits . Two of the subunits (SDHC and SDHD) are hydrophobic and anchor the two more hydrophilic (flavin and iron-sulfur) subunits (SDHA and SDHB) to the cytoplasmic membrane . Previous studies have shown that the complex of SDHC/SDHD is required to maintain the heme B component of the enzyme and that the heme B is ligated to the protein by two histidine ligands . In the current work, the histidines within SDHC and SDHD have been systematically mutated . SDHC-His91 and SDHD-His14 were eliminated as potential ligands by these studies . SDHC-His84 and SDHD-His71 have been identified as the most likely heme axial ligands in the E . coli enzyme, suggesting that the heme bridges these two subunits in the membrane . Furthermore, the results show that the four-subunit Complex II assembles and retains function despite the absence of the heme B prosthetic group in the membrane . The results do not rule out completely SDHC-His30 as a candidate for heme ligation, but do show that mutation at this position prevents assembly of Complex II in the membrane.

Biochemistry, 1998 Mar 24, 37(12), 4071 - 9
Structural basis for different inhibitory specificities of human cystatins C and D; Hall A et al.; Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S . To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems . In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold . Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity . The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured . By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H . These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively) . Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C . Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency . Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D.

EMBO J, 1998 Mar 16, 17(6), 1838 - 45
Functional interactions between the holliday junction resolvase and the branch migration motor of Escherichia coli; van Gool AJ et al.; Homologous recombination generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks . Two key steps in this process are the branch migration of Holliday junctions followed by their resolution into mature recombination products . In E.coli, branch migration is catalysed by the RuvB protein, a hexameric DNA helicase that is loaded onto the junction by RuvA, whereas resolution is promoted by the RuvC endonuclease . Here we provide direct evidence for functional interactions between RuvB and RuvC that link these biochemically distinct processes . Using synthetic Holliday junctions, RuvB was found to stabilize the binding of RuvC to a junction and to stimulate its resolvase activity . Conversely, RuvC facilitated interactions between RuvB and the junction such that RuvBC complexes catalysed branch migration . The observed synergy between RuvB and RuvC provides new insight into the structure and function of a RuvABC complex that is capable of facilitating branch migration and resolution of Holliday junctions via a concerted enzymatic mechanism.

EMBO J, 1998 Mar 16, 17(6), 1829 - 37
Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair; Villarroya M et al.; The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity . The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively . In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters . However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes . As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down . Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters . These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction . Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region . These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.

EMBO J, 1998 Mar 16, 17(6), 1759 - 67
A common role of CRP in transcription activation: CRP acts transiently to stimulate events leading to open complex formation at a diverse set of promoters; Tagami H et al.; We have shown previously that the cyclic AMP receptor protein (CRP) is not required after the formation of the open complex at the lac promoter (Tagami and Aiba, 1995, Nucleic Acids Res., 19, 6705-6712) . In this paper, we investigate the role of CRP in transcription activation at the malT and gal promoters . At the malT promoter, RNA polymerase (RNAP) forms a nonproductive RNAP-promoter binary complex in the absence of CRP and a productive CRP-RNAP-promoter ternary complex in the presence of CRP . CRP can be removed from the malT ternary complex by a moderate concentration of heparin . The resulting binary complex is functionally identical to the ternary complex . At the gal promoter, RNAP predominantly forms a binary complex at the P2 promoter in the absence of CRP and a ternary complex at the P1 promoter in the presence of CRP . A very high concentration of heparin is able to dissociate CRP from the galP1 ternary complex without changing the properties of the complex . These data indicate that CRP is not required for the maintenance of the ternary complex and plays no role in the subsequent steps, irrespective of the promoter . We conclude that the common role of CRP in the activation of transcription is to stimulate events leading to the formation of a productive open complex at a diverse set of CRP-dependent promoters . We suggest that the interaction between CRP and RNAP is needed only transiently for the activation of transcription.

Biochim Biophys Acta, 1998 Feb 23, 1390(3), 323 - 32
Biochemical characterization of a delta12 acyl-lipid desaturase after overexpression of the enzyme in Escherichia coli; Panpoom S et al.; The Delta12 acyl-lipid desaturase of Synechocystis sp . PCC 6803 was overexpressed in Escherichia coli as an active enzyme . The overexpressed protein was associated with cell membranes; it represented about 10% of the total cellular protein and 25% of the total membrane protein . The enzyme in the membrane fraction exhibited strong fatty-acid desaturase activity . The desaturase in salt-washed membranes was stabilized by the presence of sorbitol . Storage of salt-washed membranes in 2 M sorbitol at 4 degrees C and at pH 7-8 for six days resulted in the loss of less than 10% of the desaturase activity . The desaturase activity had a positive temperature coefficient, a result that suggests that the increase in the desaturation of fatty acids at low temperature might not be caused by the activation of desaturases at low temperature but, rather, by the increased synthesis of desaturases de novo .

Biochim Biophys Acta, 1998 Feb 5, 1390(1), 8 - 20
Cucumber root lipoxygenase can act on acyl groups in phosphatidylcholine; Matsui K et al.; A cDNA encoding cucumber root lipoxygenase was isolated and expressed in E . coli . The enzyme showed highest activity at pH 5.5 when alpha-linolenic acid dispersed with Tween 20 was used as a substrate but showed little activity at above pH 8.0 . On the contrary, it showed the highest activity at pH 9.0 with trilinolenin emulsified with gum arabic . When the assay was performed with linolenic acid dispersed with different concentrations of Tween 20, little activity which could be seen up to the reaction solution became turbid as the linolenic acid/Tween 20 ratio increased, while the activity rapidly emerged afterward . The enzyme could also act on phosphatidylcholine, although the activity was strongly modified by freeze-thaw and sonication treatment on the lipid vesicles . Addition of deoxycholic acid to the phospholipid vesicles drastically enhanced the activity . Addition of free fatty acid was also revealed to be effective to enhance the activity . In the latter case, myristic acid exerted highest activity . Oleic acid enhanced the activity more highly than palmitic acid did . These lines of evidence suggested that the lipoxygenase strictly recognized a specific physical state of the phospholipid substrate in the reaction mixture . The enzyme was irreversibly inactivated as the reaction proceeded, however, the rate of the inactivation was much influenced by the additives . Furthermore, stoichiometry between consumed oxygen and formed conjugated diene could not be observed . (c) 1998 Elsevier Science B.V.

Biochim Biophys Acta, 1998 Feb 5, 1390(1), 1 - 7
Complementary DNA sequence for a 42 kDa rat kidney choline/ethanolamine kinase; Aoyama C et al.; By means of peptide sequence information, several cDNA clones encoding a 42 kDa choline/ethanolamine kinase were isolated from a rat kidney cDNA library . Eight clones were sequenced with all of them resulting in identical overlapping nucleotide sequences . Four of them possessed entire open reading frame which could encode 394 amino acids with a calculated molecular size of 45 100 . The predicted amino acid sequence contained all of the internal peptide fragment sequences derived from the purified 42 kDa enzyme . When the open reading frame was introduced into pGEX-2T vector and transfected into E . coli cells, a significant choline/ethanolamine kinase activity did appear in the cell lysate . A homology comparison with the previously reported choline kinase cDNAs (CKR1 and CKR2) from rat liver showed 66%-68% in entire nucleotide sequences and 57%-59% in amino acid sequences, indicating that the cloned cDNA here must be a novel CK/EK gene product . (c) 1998 Elsevier Science B . V.

Acta Anaesthesiol Scand, 1998 Apr, 42(4), 406 - 13
Retinyl palmitate injections reduce serum levels and effects of endotoxin on systemic haemodynamics and oxygen transport in the pig; Eriksson M et al.; BACKGROUND: Retinyl palmitate {(RP) 230 IU.kg(-1)} modulates the circulatory and respiratory responses of a subsequent infusion of endotoxin in the pig . The aims of this study were: I . To determine if RP (2300 IU.kg(-1)) affects the serum endotoxin levels in this model . II . To evaluate the effect of this dose of RP on circulatory and respiratory variables in our porcine model . III . To investigate the levels of RP and neutrophil count in porcine endotoxaemia . METHODS: Ten anaesthetized pigs were randomly given 2300 IU.kg(-1) of RP or the solvent i.m . prior to the continuous i.v . infusion of E . coli endotoxin (10 microg.kg(-1).h(-1)) . Another 4 sham animals were given either i.m . RP (n=2) or i.m . solvent (n=2) followed by an infusion of saline . Haemodynamics and oxygen extraction were monitored and samples taken for analysis of endotoxin, RP and blood cells . RESULTS: I . Endotoxin levels in serum were lower (P<0.001) in the RP-pretreated pigs . II . These animals had higher cardiac index (P<0.05), mean arterial pressure and left ventricular stroke work index (both P<0.001), and lower oxygen extraction (P<0.01) . III . RP-Pre=pretreatment caused a paradoxical decrease in serum retinyl (P<0.001) and a more rapid restitution of neutrophil count (P<0.05) . CONCLUSION: Pretreatment with RP (2300 IU.kg(-1)) counteracts the progressive increase in serum endotoxin levels in porcine endotoxaemia.

Mol Gen Genet, 1998 Mar, 257(5), 519 - 28
A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation; Heuermann D et al.; A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin . The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H . pylori-specific replicon originally identified on a small cryptic H . pylori plasmid, an oriT sequence and a multiple cloning site . Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (catGC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E . coli and H . pylori . The shuttle plasmids were introduced into the H . pylori strain P1 by natural transformation . A efficiency of 7.0 x 10(-7) and 4.7 x 10(-7) transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H . pylori . An approximately 100-fold higher H . pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H . pylori strain, rather than E . coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation . Interestingly, both shuttle vectors could also be mobilized efficiently from E . coli into different H . pylori recipients, with pHel2 showing an efficiency of 2.0 x 10(-5) transconjugants per viable H . pylori P1 recipient . Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer . The functional complementation of a recA-deficient H . pylori mutant by the cloned H . pylori recA+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H . pylori demonstrate the general usefulness of this system, which will significantly facilitate the molecular analysis of H . pylori virulence factors in the future.

Biol Chem, 1998 Mar, 379(3), 289 - 94
The preprotein translocase of the mitochondrial outer membrane; Ryan MT et al.; The translocase components of the mitochondrial outer membrane (Tom) are essential for the transfer of preproteins from the cytosol to the mitochondria . At least nine Tom components have now been identified and they fall into two categories; receptor components and components of the General Import Pore (GIP) . The receptor components most likely diffuse laterally in the membrane and deposit preproteins atthe GIP where they are then inserted into the mitochondria . This review focuses on these components and their known interactions in the process of preprotein translocation.

Am J Respir Crit Care Med, 1998 Apr, 157(4 Pt 1), 1269 - 76
Allergenic activity of a major grass pollen allergen is elevated in the presence of nasal secretion; Bufe A et al.; Phl p5 is a major allergen of timothy grass and causes rhinitis and bronchial asthma in nearly all patients allergic to grass pollen . The biochemical processing of this molecule by the nasal mucosa at its first encounter and possible changes of its biologic activity are unknown . Two isoforms of the allergen were expressed in Escherichia coli and subsequently purified . Conversion of these preparations to various forms with molecular size between 10 and 20 kD in the presence of nasal secretion was observed . Surprisingly, in skin prick test assays with allergic patients the mixture of converted peptides caused significantly higher allergic response when compared with the parent protein . Allergenic activity of the recombinant N-terminal Phl p5a and the C-terminal Phl p5b as measured by skin prick test and histamine release assays was significantly higher than that of the respective parent molecules . Using pancreatic rather than nasal secretion, Phl p5b was completely degraded and its allergenicity was almost completely reduced . Proteolytic degradation converts Phl p5 to defined fragments with increased allergenicity . Complete degradation of Phl p5 on the mucosa could be a preventive strategy to destroy its potency for the induction of an allergic response.

Am J Vet Res, 1998 Apr, 59(4), 452 - 7
Effect of infusion of hypertonic saline solution of conscious heifers with hypoxemia caused by endotoxin infusion; Suzuki K et al.; OBJECTIVE: To compare effects of i.v . infusion of a small volume (5 ml/kg of body weight) of hypertonic saline (7.2% NaCl) solution (HSS) and i.v . infusion of an equivalent volume of isotonic saline (0.9% NaCl) solution (ISS) on arterial blood gases in conscious heifers with hypoxemia caused by endotoxin infusion . ANIMALS: 9 Holstein heifers . PROCEDURE: All heifers received 2.0 microg of endotoxin/kg, i.v., during a 5-minute period . Twenty-five minutes after endotoxin infusion, 3 heifers received ISS, 3 received HSS, and the remaining 3 did not receive fluids (control) . Heifers were monitored for 150 minutes after initiation of fluid replacement . Arterial blood gases, blood pressure, and serum electrolyte concentrations were measured . RESULTS: Endotoxin administration had a profound effect on pulmonary function, causing severe hypoxemia accompanied by a significant decrease in the partial pressure of oxygen (PaO2) and a significant increase in the arterial-alveolar O2 gradient (P{A-a}O2) . Cattle in the HSS group had progressive and significant increases in PaO2 and O2 saturation, compared with the other groups . The P(A-a)O2 values for the control and ISS groups were slightly decreased until 150 minutes after initiation of fluid replacement; however, HSS infusion induced a progressive and significant decrease in P(A-a)O2 for the remainder of the experimental period . CONCLUSIONS AND CLINICAL RELEVANCE: Rapid infusion of HSS can successfully resuscitate conscious cattle with induced pulmonary dysfunction . Infusion of HSS may be beneficial for initial resuscitation of cattle with naturally developing bovine respiratory disease complex.

FEBS Lett, 1998 Apr 3, 425(3), 382 - 4
Enzymes as chaperones and chaperones as enzymes; Wang CC et al.; Chaperones and foldases are two groups of accessory proteins which assist maturation of nascent peptides into functional proteins in cells . Protein disulfide isomerase, a foldase, and ATP-dependent proteases, responsible for degradation of misfolded proteins in cells, both have intrinsic chaperone activities . Trigger factor and DnaJ, well known Escherichia coli chaperones, show peptidyl prolyl isomerase and protein disulfide isomerase activities respectively . It is suggested that the combination of chaperone and enzyme activities in one molecule is the result of evolution to increase molecular efficiency.

FEBS Lett, 1998 Apr 3, 425(3), 377 - 81
Rewiring a receptor: negative output from positive input; Taylor BL et al.; A point mutation or covalent modification in bacterial chemotaxis receptors causes bacteria to be repelled by attractants, and attracted to repellents . The variety of conditions causing inverse responses suggest that the signal transduction mechanism in receptors can be readily rewired to elicit inverse responses . A model is presented in which the orientation of a critical residue with respect to an active site determines whether the receptor produces normal or inverted signals . The model is consistent with observed responses and can be generalized to include receptors in other signal transduction systems.

Cancer Res, 1998 Apr 15, 58(8), 1624 - 30
Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcinomas; Santamaria I et al.; We have identified and cloned a new member of the papain family of cysteine proteinases from a human brain cDNA library . The isolated cDNA codes for a polypeptide of 334 amino acids that exhibits all of the structural features characteristic of cysteine proteinases, including the active site cysteine residue essential for peptide hydrolysis . Pairwise comparisons of this amino acid sequence with the remaining human cysteine proteinases identified to date showed a high percentage of identity (78%) with cathepsin L; the percentage of identity with all other members of the family was much lower (<40%) . On the basis of these structural characteristics, we have tentatively called this novel protein cathepsin L2 . The cDNA encoding the mature cathepsin L2 was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, which is commonly used as a substrate for cysteine proteinases . Cathepsin L2 proteolytic activity on this substrate was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases, thus providing additional evidence that the isolated cDNA encodes a functional cysteine proteinase . Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues demonstrated that cathepsin L2 is predominantly expressed in the thymus and testis . This finding is in marked contrast with the wide tissue distribution of most cysteine proteinases characterized to date, including cathepsin L, and suggests that cathepsin L2 may play a specialized role in the thymus and testis . Expression analysis of cathepsin L2 in human tumors revealed a widespread expression in colorectal and breast carcinomas but not in normal colon or mammary gland or in peritumoral tissues . Cathepsin L2 was also expressed by colorectal and breast cancer cell lines as well as by some tumors of diverse origin, including ovarian and renal carcinomas . These results open the possibility that this novel enzyme may be involved in tumor processes, as already reported for other cysteine proteinases, including cathepsin L.

Histochem Cell Biol, 1998 Apr, 109(4), 339 - 47
Differential expression of IFN-gamma, IL-4, IL-10, and IL-1beta mRNAs in decalcified tissue sections of mouse lipopolysaccharide-induced periodontitis mandibles assessed by in situ hybridization; Iwasaki Y et al.; To examine the role of T cell subgroups, Th1 and Th2, in the development of periodontitis, the expression of various cytokines was investigated in a mouse model of alveolar bone resorption using in situ hybridization (ISH) with digoxigenin-labeled oligonucleotides . When mice received repetitive injections with Escherichia coli lipopolysaccharide into the gingiva every 48 h, alveolar bone resorption was detectable after the fourth injection, reaching a maximum after the 13th injection . For the best performance of ISH, we first had to choose a decalcification protocol . Among various decalcification protocols, 10% EDTA (4 degrees C, 5-6 days) was the best for 28S rRNA staining . Positive cells for transcripts of interferon-gamma (Th1 product) were detected after the fourth injection, reaching a maximum after the tenth injection . A similar pattern was obtained for interleukin (IL)-10 mRNA (Th2 product) and IL-1beta, while the positive cell number reached a maximum after the 13th and 10th injections, respectively . The number of IL-4 mRNA (Th2 product)-positive cells remained low till the tenth injection, but increased thereafter . Consequently, we found that the population change from Th1 to Th2 in the inflammation site correlated with the transition from gingivitis to periodontitis, indicating differential roles of T cell subgroups in the development of periodontitis.

J Eukaryot Microbiol, 1998 Mar-Apr, 45(2), 13S - 16S
Molecular analysis of the Gal/GalNAc adhesin of Entamoeba histolytica; Mann BJ et al.; Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactose/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin . Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence . Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence . The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit . The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E . coli and also with denatured native adhesin . These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes . The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E.coli, retained at least some of its native conformation.

FEMS Microbiol Lett, 1998 Apr 1, 161(1), 47 - 52
The Bradyrhizobium japonicum phoB gene is required for phosphate-limited growth but not for symbiotic nitrogen fixation; Minder AC et al.; We identified by cloning and DNA sequence analysis the phosphate regulatory gene phoB of Bradyrhizobium japonicum . The deduced gene product displayed pronounced similarity to the PhoB protein of Sinorhizobium meliloti (71.4% identical amino acids) . Escherichia coli (50.2%) and other bacterial species . Insertion of a kanamycin resistance cassette into phoB led to impaired growth of the B . japonicum mutant in media containing approximately 25 microM phosphate or less . A standard plant infection test using wild-type and phoB-defective B . japonicum strains showed that the phoB mutation had no effect on the symbiotic properties of B . japonicum with its soybean host plant.

Lipids, 1998 Mar, 33(3), 327 - 32
Fatty acid composition of the cellular slime mold Polysphondylium pallidum; Saito T et al.; The cellular slime mold Polysphondylium pallidum was grown upon Escherichia coli B/r, and the fatty acid compositions of total lipids obtained from vegetative amebae and aggregation-competent cells were compared . Fatty acids isolated from vegetative cells included C-17 and C-19 cyclopropane fatty acids and also straight-chain, saturated fatty acids . The cyclopropane fatty acids were derived from the ingested bacteria . Development of amebae to aggregation-competent cells was accompanied by a substantial decrease in saturated cyclopropane fatty acids and a concomitant increase in unsaturated fatty acids and unsaturated cyclopropane fatty acids, mostly as 18:3 (5,9,12) . We report here the fatty acid composition and identify the occurrence of delta 5 desaturation of cyclopropane fatty acids, namely, 9,10-methylene 5-hexadecenoic acid and 11,12-methylene 5-octadecenoic acid . These fatty acids have not been reported previously in the related species Dictyostelium discoideum, which also feeds on E . coli B/r and has delta 5-desaturation activity.

Genetics, 1998 Apr, 148(4), 1627 - 35
Reversion of the tyrosine ochre strain Escherichia coli WU3610 under starvation conditions depends on a new gene tas; Timms AR et al.; When 3 x 10(8) bacteria of the Escherichia coli tyrA14(oc) leu308(am) strain WU3610 are plated on glucose salts agar supplemented with leucine only, colonies of slow-growing Tyr+ suppressor mutants begin to appear after about a week and increase in numbers roughly linearly with time thereafter (stationary phase or starvation-associated mutation) . From a library constructed from two of these mutants, a clone was obtained that suppressed the tyrosine requirement of WU3610 when present on a multicopy plasmid . The activity was identified to an open reading frame we call tas, the sequence for which has homology with a variety of known genes with aldo-keto reductase activity . The activity of tas complements the prephenate dehydrogenase dysfunction of tyrA14 (the chorismate mutase activity of tyrA possibly being still functional) . A strain deleted for tas showed no spontaneous mutation under starvation conditions . Whereas neither tas+ nor tas bacteria showed any increase in viable or total count when plated under conditions of tyrosine starvation at 3 x 10(8) cells per plate, at lower density (approximately 10(7) per plate) tas+ but not tas bacteria showed considerable residual growth . We suggest that the single copy of tas present in WU3610 allows cryptic cell or DNA turnover under conditions of tyrosine starvation and that this is an essential prerequisite for starvation-associated mutation in this system . The target gene for mutation is not tas, although an increase in the expression of this gene, for example, resulting from a suppressor mutation affecting supercoiling, could be responsible for the slow-growing Tyr+ phenotype.

Genetics, 1998 Apr, 148(4), 1599 - 610
Mutagenesis and more: umuDC and the Escherichia coli SOS response; Smith BT et al.; The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli . Analyses of the SOS response have led to new insights into the transcriptional and post-translational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis . SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions . Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E . coli cell cycle after DNA damage has occurred, are discussed.

Genetics, 1998 Apr, 148(4), 1559 - 66
Transient and heritable mutators in adaptive evolution in the lab and in nature; Rosenberg SM et al.; Major advances in understanding the molecular mechanism of recombination-dependent stationary-phase mutation in Escherichia coli occurred this past year . These advances are reviewed here, and we also present new evidence that the mutagenic state responsible is transient . We find that most stationary-phase mutants do not possess a heritable stationary-phase mutator phenotype, although a small proportion of heritable mutators was found previously . We outline similarities between this well-studied system and several recent examples of adaptive evolution associated with heritable mutator phenotype in a similarly small proportion of survivors of selection in nature and in the lab . We suggest the following: (1) Transient mutator states may also be a predominant source of adaptive mutations in these latter systems, the heritable mutators being a minority (Rosenberg 1997); (2) heritable mutators may sometimes be a product of, rather than the cause of, hypermutation that gives rise to adaptive mutations.

Genetics, 1998 Apr, 148(4), 1453 - 9
Adaptive mutation: has the unicorn landed?
Foster PL.
Reversion of an episomal Lac- allele during lactose selection has been studied as a model for adaptive mutation . Although recent results show that the mutations that arise during selection are not "adaptive" in the original sense, the mutagenic mechanism that produces these mutations may nonetheless be of evolutionary significance . In addition, a transient mutational state induced in a subpopulation of starving cells could provide a species with a mechanism for adaptive evolution.

Genetics, 1998 Apr, 148(4), 1441 - 51
The lacI gene as a target for mutation in transgenic rodents and Escherichia coli; de Boer JG et al.; The lacI gene has been used extensively for the recovery and analysis of mutations in bacteria with various DNA repair backgrounds and after exposure to a wide variety of mutagens . This has resulted in a large database of information on mutational mechanisms and specificity of many mutagens, as well as the effect of DNA repair background on mutagenicity . Most importantly, knowledge about the mutational sensitivity of the lacI gene is now available, yielding information about mutable nucleotides . This popularity and available knowledge resulted in the use of the lacI gene in transgenic rodents for the study of mutagenesis in mammals, where it resides in approximately 40 repeated copies . As the number of sequenced mutations recovered from these animals increases, we are able to analyze the sites at which mutations have been recovered in great detail and to compare the recovered sites between bacteria and transgenic animals . The nucleotides that code for the DNA-binding domain are nearly saturated with base substitutions . Even after determining the sequences of approximately 10,000 mutations recovered from the animals, however, new sites and new changes are still being recovered . In addition, we compare the nature of deletion mutations between bacteria and animals . Based on the nature of deletions in the animals, we conclude that each deletion occurs in a single copy of the gene.

Biochem J, 1998 May 1, 331 ( Pt 3), 953 - 8
Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase; Tamura HO et al.; Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg . In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum . The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide . Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1 . The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme . These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT-PCR).

Biochem J, 1998 May 1, 331 ( Pt 3), 937 - 45
cDNA sequence and heterologous expression of monomeric spinach pullulanase: multiple isomeric forms arise from the same polypeptide; Renz A et al.; The spinach pullulanase gene was cloned and sequenced using peptide sequences of the purified enzyme as a starting point and employing PCR techniques and cDNA library screening . Its open reading frame codes for a protein of 964 amino acids which represents a precursor of the pullulanase . The N-terminal transit peptide consists of 65 amino acids, and the mature protein, comprising 899 amino acids, has a calculated molecular mass of 99kDa . Pullulanase is a member of the alpha-amylase family . In addition to a characteristic catalytic (beta/alpha)8-barrel domain, it contains a domain, F, that is specific for branching and debranching enzymes . Pullulanase cDNA was expressed in Escherichia coli, and the purified protein was compared with the enzyme from spinach leaves . Identity of the two proteins was confirmed in terms of catalytic properties, N-terminal amino acid sequences and molecular masses . The pullulanase produced by E . coli showed the same microheterogeneity as the spinach leaf enzyme: it could be resolved into two substrate-induced forms by electrophoresis in amylopectin-containing polyacrylamide gels, and, in the absence of substrate, into several free forms (charge isomers) by isoelectric focusing or chromatofocusing . Rechromatofocusing of single free forms resulted in the originally observed pattern of molecular forms . However, heterogeneity of the protein disappeared on isoelectric focusing under completely denaturing conditions when only one protein band was observed . Post-translational modifications such as glycosylation and phosphorylation could be excluded as potential explanations for the protein heterogeneity . Therefore the microheterogeneity of spinach leaf pullulanase results from neither genetic variation nor post-translational modifications, but is a property of the single unmodified gene product . The different interconvertible forms of the pullulanase represent protein populations of different tertiary structure of the same polypeptide.

Biochem J, 1998 May 1, 331 ( Pt 3), 783 - 92
Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity; Smith G et al.; Cytochrome P-450 CYP2D6, human debrisoquine hydroxylase, metabolizes more than 30 prescribed drugs, the vast majority of which are small molecules containing a basic nitrogen atom . In contrast, the similar mouse protein Cyp2d-9 was first characterized as a testosterone 16alpha-hydroxylase . No common substrates have been reported for the two enzymes . Here we investigate the structural basis of this difference in substrate specificity . We have earlier used a combination of NMR data and homology modelling to generate a three-dimensional model of CYP2D6 {Modi, Paine, Sutcliffe, Lian, Primrose, Wolf, C.R . and Roberts (1996) Biochemistry 35, 4541-4550} . We have now generated a homology model of Cyp2d-9 and compared the two models to identify specific amino acid residues that we believe form the substrate-binding site in each protein and therefore influence catalytic selectivity . Although there are many similarities in active site structure, the most notable difference is a phenylalanine residue (Phe-483) in CYP2D6, which in the model is located such that the bulky phenyl ring is positioned across the channel mouth, thus limiting the size of substrate that can access the active site . In Cyp2d-9, the corresponding position is occupied by an isoleucine residue, which imposes fewer steric restraints on the size of substrate that can access the active site . To investigate whether the amino acid residue at this position does indeed influence the catalytic selectivity of these enzymes, site-directed mutagenesis was used to change Phe-483 in CYP2D6 to isoleucine and also to tryptophan . CYP2D6, Cyp2d-9 and both mutant CYP2D6 proteins were co-expressed with NADPH cytochrome P-450 reductase as a functional mono-oxygenase system in Escherichia coli and their relative catalytic activities towards bufuralol and testosterone were determined . All four proteins exhibited catalytic activity towards bufuralol but only Cyp2d-9 catalysed the formation of 16alpha-hydroxytesterone . Uniquely, the CYP2D6F483I mutant acquired the ability to metabolize testosterone to a novel product, which was identified by MS and proton NMR spectroscopy as 15alpha-hydroxytestosterone . NMR spin relaxation experiments were used to measure distances between the haem iron and protons of testosterone bound to the CYP2D6F483I mutant . These experiments demonstrate that very minor modifications to the active site structure of CYP2D6 can have a profound influence on the substrate specificity of the enzyme.

Hum Mutat, 1998, 11(4), 259 - 63
In vitro mutations in dihydrofolate reductase that confer resistance to methotrexate: potential for clinical application; Blakley RL et al.; Mammalian cells cultured in the presence of the chemotherapeutic agent, methotrexate, develop resistance to this drug . Sometimes this is due to mutations in the gene for dihydrofolate reductase, the primary target of methotrexate . However, it has not been possible to link such polymorphism to resistance of neoplastic disease to therapy with methotrexate . Nevertheless, interest in this possibility lead to the introduction of many mutations into the cDNA for human DHFR by mutagenesis . Most of the corresponding enzyme variants have been expressed in Escherichia coli and characterized . Many mutations in codons for hydrophobic residues at the active site greatly decrease inhibition by methotrexate, and by the related substrate analogue, trimetrexate, while allowing the retention of considerable catalytic efficiency . Introduction of some of these mutants into mammalian cells by retroviral transfer provides substantial protection from toxic effects of the inhibitors, and has promise for the myeloprotection of patients receiving therapy with methotrexate or trimetrexate . Another potential use is in therapy for inherited disorders of hematopoiesis, where genetic modification of enough cells is a perennial problem . After transplantation of bone marrow that has been transduced with a bicistronic vector encoding both the mutant DHFR and a therapeutic gene, subsequent administration of methotrexate or trimetrexate should permit selection and enrichment of genetically modified hematopoietic cells.

Rapid Commun Mass Spectrom, 1998, 12(7), 339 - 44
Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry; Ayed A et al.; A mass spectrometer coupling electrospray ionization with time-of-flight mass spectrometry (ESI-TOFMS) has been used to investigate the oligomeric species of Escherichia coli citrate synthase, and to determine the effect of nicotinamide adenine dinucleotide (NADH), an allosteric inhibitor of this enzyme, on the equilibrium between the oligomeric forms . An equilibrium mixture of dimers (M = 95,770 Da) and hexamers (M = 287,310 Da) was found under the conditions used (KA = 6.9 x 10(10) M-2), and NADH was observed to bind selectively to the hexamer (KD = 1.1 microM), shifting the equilibrium to the latter form . The power of ESI-TOFMS to measure ions of very large mass-to-charge ratio (up to m/z approximately 10,000 in this case) is shown to be a valuable tool for obtaining accurate information about compositions of noncovalent complexes and equilibrium constants . The measured constants agree with those determined by conventional means.

Proteins, 1998 Apr 1, 31(1), 21 - 32
Crystal structure of calcium-independent subtilisin BPN' with restored thermal stability folded without the prodomain; Almog O et al.; The three-dimensional structure of a subtilisin BPN' construct that was produced and folded without its prodomain shows the tertiary structure is nearly identical to the wild-type enzyme and not a folding intermediate . The subtilisin BPN' variant, Sbt70, was cloned and expressed in Escherichia coli without the prodomain, the 77-residue N-terminal domain that catalyzes the folding of the enzyme into its native tertiary structure . Sbt70 has the high-affinity calcium-binding loop, residues 75 to 83, deleted . Such calcium-independent forms of subtilisin BPN' refold independently while retaining high levels of activity {Bryan et al., Biochemistry, 31:4937-4945, 1992} . Sbt70 has, in addition, seven stabilizing mutations, K43N, M50F, A73L, Q206V, Y217K, N218S, Q271E, and the active site serine has been replaced with alanine to prevent autolysis . The purified Sbt70 folded spontaneously without the prodomain and crystallized at room temperature . Crystals of Sbt70 belong to space group P2(1)2(1)2(1) with unit cell parameters a = 53.5 A, b = 60.3 A, and c = 83.4 A . Comparison of the refined structure with other high-resolution structures of subtilisin BPN' establishes that the conformation of Sbt70 is essentially the same as that previously determined for other calcium-independent forms and that of other wild-type subtilisin BPN' structures, all folded in the presence of the prodomain . These findings confirm the results of previous solution studies that showed subtilisin BPN' can be refolded into a native conformation without the presence of the prodomain {Bryan et al., Biochemistry 31:4937-4945, 1992} . The structure analysis also provides the first descriptions of four stabilizing mutations, K43N, A73L, Q206V, and Q271E, and provides details of the interaction between the enzyme and the Ala-Leu-Ala-Leu tetrapeptide found in the active-site cleft.

J Immunol, 1998 Jan 1, 160(1), 475 - 84
Effect of C1 inhibitor on inflammatory and physiologic response patterns in primates suffering from lethal septic shock; Jansen PM et al.; We evaluated the effect of C1 inhibitor (C1-inh), an inhibitor of the classical pathway of complement and the contact system, on the physiologic and inflammatory response in baboons suffering from lethal Escherichia coli sepsis . Five animals pretreated with 500 U/kg C1-inh (treatment group; n = 5), followed by a 9-h continuous infusion of 200 U/kg C1-inh subsequent to bacterial challenge, were compared with five controls receiving E . coli alone . Of the treatment group, one animal survived and another lived beyond 48 h, whereas all control animals died within 27 h . In four of five treated animals, less severe pathology was observed in various target organs . C1-inh administration did not prevent the hemodynamic or hematologic changes observed upon E . coli infusion . The activation of fibrinolysis and the development of disseminated intravascular coagulation were essentially unaffected by C1-inh . However, C1-inh supplementation significantly reduced decreases in plasma levels of factor XII and prekallikrein and abrogated the systemic appearance of C4b/c, indicating substantial inhibition of activation of the contact system and the classical complement pathway, respectively . Furthermore, treated animals displayed a reduced elaboration of various cytokines including TNF, IL-10, IL-6, and IL-8 . Thus, the administration of C1-inh may have a beneficial but modest effect on the clinical course and outcome of severe sepsis in nonhuman primates . We suggest that activated complement and/or contact system proteases may, at least in part, contribute to the attendant manifestations of septic shock through an augmentation of the cytokine response.

Curr Microbiol, 1998 May, 36(5), 253 - 8
Unusual gene arrangement of the bidirectional hydrogenase and functional analysis of its diaphorase subunit HoxU in respiration of the unicellular cyanobacterium anacystis nidulans
Boison G, Schmitz O, Schmitz B, Bothe H.
The bidirectional, NAD+-dependent hydrogenase from cyanobacteria is encoded by the structural genes hoxFUYH, which have been found to be clustered, though interspersed with different open reading frames (ORFs), in the heterocystous, N2-fixing Anabaena variabilis and in the unicellular Synechocystis PCC 6803 . In another unicellular, non N2-fixing cyanobacterium, Anacystis nidulans, hoxF has now been identified as being separated by at least 16 kb from the residual structural genes hoxUYH . An ORF (termed hoxE gene) is located immediately upstream of hoxF in A . nidulans and in Synechocystis . Its deduced amino acid sequence shows similarities to the NuoE subunit of NADH dehydrogenase I of E . coli, to the homologous subunit of respiratory complex I in mitochondria, and also to the first 104 amino acids of HoxF in A . nidulans and Synechocystis . The diversity in the arrangement of hydrogenase genes in cyanobacteria is puzzling . The subunits HoxE, HoxF, and HoxU of the diaphorase part of the bidirectional hydrogenase have been discussed to be shared both by respiratory complex I and bidirectional hydrogenase in cyanobacteria . Different hoxU mutants were obtained by inserting a lacZKmR cassette into the gene both in A . nidulans and Anacystis PCC 7942 . Such mutants showed reduced H2-evolution activities catalyzed by the bidirectional hydrogenase, but had nonimpaired respiratory O2-uptake . A common link between respiratory complex I and the diaphorase part of the bidirectional hydrogenase in cyanobacteria may still exist, but this hypothesis could not be verified in the present study by analyzing defined mutants impaired in one of the diaphorase genes.

Free Radic Biol Med, 1998 Mar 1, 24(4), 556 - 62
Different mechanisms of thioredoxin in its reduced and oxidized forms in defense against hydrogen peroxide in Escherichia coli; Takemoto T et al.; The present experiments were done to elucidate the roles of thioredoxin and thioredoxin reductase system in defense against hydrogen peroxide (H2O2) in Escherichia coli . The thioredoxin-deficient mutant (trxA) was more sensitive to H2O2 than was the wild-type strain, when challenged in the stationary and exponentially growing phase . Thioredoxin reductase-deficient mutant (trxB) in the stationary phase also exhibited increased sensitivity, compared with the wild-type strain . These results indicated that reduced form of thioredoxin is required for defense against H2O2, possibly by scavenging radicals generated in the cells . In contrast, the trxB mutant in the growing phase had higher survival after exposure to H2O2 than the wild-type strain . The acquirement of resistance related to increased capacity for removing H2O2 in the trxB mutant and was not observed in a catalase-negative background . Furthermore, enhanced expression of the katG :: lacZ gene occurred in the mutant . Therefore, it was concluded that oxidized form of thioredoxin confers H2O2 resistance on E . coli cells by increasing activity to remove H2O2, which was brought about by enhanced induction of the katG-coded catalase/hydroperoxidase I at the transcriptional level . In addition, this resistance to H2O2 correlated well with reduced amount of DNA damage caused by H2O2, determined by the induction level of the recA :: lacZ fusion gene after treatment with H2O2.

Crit Care Med, 1998 Apr, 26(4), 723 - 9
Prophylaxis against lipopolysaccharide-induced acute lung injury by alpha-tocopherol liposomes; Suntres ZE et al.; OBJECTIVE: To investigate whether intravenously administered liposomal alpha-tocopherol can protect the lung from the injurious action of Escherichia coli lipopolysaccharide (LPS) . DESIGN: Prospective, randomized animal study . SETTING: Government research laboratory . SUBJECTS: Twenty adult male Sprague-Dawley rats . INTERVENTIONS: Animals were intravenously pretreated with alpha-tocopherol liposomes (20 mg alpha-tocopherol/kg body weight), plain liposomes, or saline . Twenty-four hours later, pretreated animals were challenged with an intravenous injection of LPS (E . coli 0111:B4, 1 mg/kg body weight), and killed 2 hrs after LPS challenge . MEASUREMENTS AND MAIN RESULTS: Challenge of saline-pretreated animals with LPS resulted in lung injuries as evidenced by an increase in wet lung weight and a reduction in pulmonary angiotensin converting enzyme (25%) and alkaline phosphatase (28%), injury markers of lung endothelial and epithelial type II cells, respectively . Also, LPS administration resulted in an increase in pulmonary myeloperoxidase and protease activities, indicative of a neutrophilic inflammatory response . Pretreatment of animals with liposomal alpha-tocopherol significantly attenuated the LPS-induced edematous lung weight response, and reduced the extent of injuries to the pulmonary endothelial and epithelial cells, demonstrated by a significantly smaller reduction in the corresponding enzyme marker activities . CONCLUSION: These results suggest that augmentation of the pulmonary antioxidant status can ameliorate LPS-induced lung injuries mediated by oxidative stress mechanisms.

Plant Cell Physiol, 1998 Feb, 39(2), 177 - 85
Cloning of the gene encoding a protochlorophyllide reductase: the physiological significance of the co-existence of light-dependent and -independent protochlorophyllide reduction systems in the cyanobacterium Plectonema boryanum; Fujita Y et al.; Cyanobacteria have two protochlorophyllide (Pchlide) reductases catalyzing the conversion of Pchlide to chlorophyllide, a key step in the biosynthetic pathway of chlorophylls (Chls); a light-dependent (LPOR) and a light-independent (DPOR) reductase . We found an open reading frame (ORF322) in a 2,131-bp EcoRI fragment from the genomic DNA of the cyanobacterium Plectonema boryanum . Because the deduced amino acid sequence showed a high similarity to those of various plant LPORs and the LPOR activity was detected in the soluble fraction of Escherichia coli cells over-expressing the ORF322 protein, ORF322 was defined as the por gene encoding LPOR in P . boryanum . A por-disrupted mutant, YFP12, was isolated by targeted mutagenesis to investigate the physiological importance of LPOR . YFP12 grew as well as wild type under low light conditions (10-25 muE m-2 s-1) . However, its growth was significantly retarded as a result of a significant decrease in its Chl content under higher light conditions (85-130 muE m-2 s-1) . Furthermore, YFP12 stopped growing and suffered from photobleaching under the highest light intensity (170 muE m-2 s-1) . In contrast, a chlL-disrupted (DPOR-less) mutant YFC2 grew as well as wild type irrespective of light intensity . From these phenotypic characteristics, we concluded that, although both LPOR and DPOR contribute to Chl synthesis in the cells growing in the light, the extent of the contribution by LPOR increases with increasing light intensity; without it, the cells are unable to grow under light intensities of more than 130 muE m-2 s-1.

Plant Cell Physiol, 1998 Feb, 39(2), 139 - 43
A protein encoded by din1, a dark-inducible and senescence-associated gene of radish, can be imported by isolated chloroplasts and has sequence similarity to sulfide dehydrogenase and other small stress proteins; Shimada Y et al.; In an attempt to isolate cDNA clones for dark-inducible chloroplast proteins, we screened a cDNA library which was prepared from radish cotyledons by a two-step method . The source plants were grown under continuous light for 14 d and kept in darkness for 24 h . One of the selected clones, S2D12, corresponded to the din1 gene which we previously reported as a dark-inducible, senescence-associated gene {Azumi and Watanabe (1991) Plant Physiol . 95:577} . A 22 kDa polypeptide was produced from the cDNA in an in vitro expression system in the presence of {35S}methionine . This polypeptide was capable of being imported by isolated chloroplasts, processed to a smaller mature form and localized in the stromal fraction . As the amino acid sequence of the putative mature protein has no homology to any known chloroplast protein, din1 was suggested to be the first gene for a chloroplast protein which is negatively controlled by light . The putative mature protein has similarity to sulfide dehydrogenase from Wolinella succinogenes and other small stress proteins; glpE and pspE from Escherichia coli and hsp67B2 from Drosophila melanogaster.

Z Ernahrungswiss, 1998, 37 Suppl 1, 118 - 21
Probing the presumed catalytic triad of a selenium-containing peroxidase by mutational analysis; Maiorino M et al.; Glutathione peroxidases (GPx) are characterized by a catalytically active selenium which forms the center of a strictly conserved triad composed of selenocysteine, glutamine, and tryptophan . In order to check the functional relevance of this structural peculiarity, six molecular mutants of phospholipid hydroperoxide glutathione peroxidase (PHGPx) were designed, isolated, and investigated kinetically . Replacement of the selenocysteine in position 46 by cysteine decreased k + 1, i.e., the reaction rate of reduced enzyme with hydroperoxide, by three orders of magnitude . The rate of regeneration of the reduced enzyme by glutathione (k' + 2) was similarly affected . Additional substitution of Gln81 or Trp136 by acid residues resulted in a further decrease of k + 1 by three orders of magnitude, whereas histidine or neutral residues in these positions proved to be less deleterious . The data support the hypothesis that the typical triad of selenocysteine, glutamine, and tryptophan is indeed a novel catalytic center in which the reactivity of selenium is optimized by hydrogen bonding provided by the adjacent glutamine and tryptophan residues.

Arch Dermatol Res, 1998 Mar, 290(3), 109 - 12
Assessment of microsatellite instability in a cell line from a patient with xeroderma pigmentosum variant; Tzung TY et al.; Cells from patients with xeroderma pigmentosum (XP) variant are thought to be defective in postreplication repair . This DNA repair pathway is not well defined in human cells and the exact genetic defect of XP variant is unknown . In another cancer-prone hereditary disorder, hereditary nonpolyposis colon cancer, tumors are characterized by a DNA mismatch repair defect with microsatellite instability . Since there are some similarities between postreplication repair and mismatch repair, we investigated microsatellite instability, the hallmark of a DNA mismatch repair defect, in a lymphoblastoid cell line from a patient with XP variant . Two normal lines and one nucleotide excision repair-defective XP group A line were used as controls . In a host cell microsatellite instability assay, the recently developed shuttle vector pZCA29 was transfected into these cells and replicated plasmid recovered after 3 days . The plasmid contains two CA repeat tracts that interrupt the reading frame of the lacZ gene . Reversion to active beta-galactosidase, detectable by a color reaction of bacterial transformants, represents the frequency of frameshift mutations in the CA repeat tracts during replication of the plasmid, and thereby the host cells' microsatellite instability . We did not find any significant differences in the mutation frequencies of the plasmids after passage through either cell line . This indicates that there is no microsatellite instability in the examined XP variant cell line.

J Rheumatol, 1998 Apr, 25(4), 748 - 52
Polyarthritis and periostitis induced by Escherichia coli . Lipopolysaccharide injection in young male hamsters; Gruys E et al.; OBJECTIVE: To describe the clinicopathological manifestations of lipopolysaccharide (LPS) induced arthritis in the hamster and to compare its time of onset, duration, and severity with other forms of experimentally induced arthritis . METHODS: A preparation containing 30 microg LPS from Escherichia coli was injected subcutaneously for 5 to 21 days into young male hamsters (Mesocricetus auratus) . Arthritis was quantified by measuring soft tissue swelling of affected joints with calipers . After decalcification, paraffin sections were cut and stained with hematoxylin and eosin, Giemsa, and azan . Acute phase reactant apolipoprotein serum amyloid A (apoSAA) levels were determined by ELISA . RESULTS: Symmetrical polyarthritis developed within 3 days and persisted for 14-21 days, provided the hamsters received daily LPS injections . Most prominent were lesions in the carpal-metacarpal joints of the front legs and in the tarsal-metatarsal joints of the hind legs . Animals in whom LPS injections were discontinued after 4 or 7 days recovered completely . Histological findings of exudative synovitis, periarticular soft tissue swelling, and juxtaarticular periostitis were associated with a sharp rise in serum titers of apoSAA . CONCLUSION: The unusually rapid onset of arthritis and periostitis in this experimental animal model suggests that its systemic manifestations were not mediated by a classical immune response, and may represent an "innate" response of targeted cells within the synovial membrane and periosteum to bacterial cell wall endotoxins.

Glycoconj J, 1998 Feb, 15(2), 147 - 53
Expression, purification, and biochemical characterization of a recombinant lectin of Sarcocystis muris (Apicomplexa) cyst merozoites; Klein H et al.; The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia colias a histidine-tagged fusion protein . The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography . The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits . Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin . To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.

Glycoconj J, 1998 Feb, 15(2), 139 - 45
Combined preparative enzymatic synthesis of dTDP-6-deoxy-4-keto-D-glucose from dTDP and sucrose; Stein A et al.; dTDP-6-deoxy-4-keto-D-glucose (1), the common intermediate in the biosyntheses of the manifold deoxysugars, was synthesized on a gram-scale by the combination of sucrose synthase and dTDP-D-glucose 4,6-dehydratase in a fed batch, starting the reaction with dTDP . This process allowed a dTDP conversion with a 100% rate . An easy and efficient three-step purification with anion-exchange chromatography and gel filtration gave 1.1 g of 1 in an overall yield of 73% . This work realizes a first step for an economic access to activated deoxysugars.

Res Vet Sci, 1998 Jan-Feb, 64(1), 73 - 7
Effect of administration of a novel recombinant bovine interferon on length of oestrous cycle in cattle; Bleach EC et al.; In ruminants, extensive reproductive loss occurs during the process of maternal recognition of pregnancy and it has been suggested that trophoblast interferons may be potential therapeutic agents . This paper reports results from a trial using eight first lactation Holstein-Friesian heifers to test the efficacy of a novel recombinant bovine interferon produced in bacteria in extending the life of the corpus luteum . Oestrus was synchronised in these animals and 0.1 mg of this non-glycosylated interferon was infused into the uterus twice daily for 13 days starting approximately 12 days after oestrus . This treatment resulted in an extension of the lifespan of the corpus luteum by 5.5 days (P=0.028) compared with untreated controls . In these animals the interovulatory period was extended by 6.4 days (P=0.009) . Administration of this protein did not have any adverse effects either on body temperature or on daily milk yields . The results indicate that this novel interferon may have potential therapeutic application for reducing embryo mortality.

J Virol, 1998 May, 72(5), 3751 - 61
The Cys-rich region of hepatitis A virus cellular receptor 1 is required for binding of hepatitis A virus and protective monoclonal antibody 190/4; Thompson P et al.; The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV . The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins . The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region . To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab) . Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells) . Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells . Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1 . To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells . Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable . HAV-infected cr5 and mut2 cells but not mut1, mut3, d1-, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells . These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.

Vet Microbiol, 1998 Jan 31, 59(4), 283 - 94
Phenotypic expression of K88 adhesion alone or simultaneously with K99 and/or F41 adhesins in the bovine enterotoxigenic Escherichia coli strain B41; Bertin A; F41-positive and F41-negative derivatives of bovine enterotoxigenic Escherichia coli strain B41 carrying K88 or K88 and K99 plasmids were investigated for stability and expression of genes for their fimbrial antigens . Either K88 plasmid alone or both K88 and K99 plasmids could be maintained in these strains though stability could depend on culture medium . K99 antigen could be detected in each strain bearing K99 plasmid . Clones that produced K88 antigen or clones that did not produce this antigen could be isolated from each strain, except from the strain that possessed K99 plasmid in the strain that did not possess the ability to produce F41 antigen . Strains possessing K88 plasmid in the strain able to produce F41 antigen produced clones expressing either both K88 and F41 antigens, (also F41 appeared strongly expressed in some clones) or clones that produced only F41 antigen or no antigen at all . Clones that produced only K88 antigen or others that did not produce this antigen could be produced from a strain bearing only K88 plasmid and that did not possess the ability to produce F41 antigen . None of these strains bearing K88 plasmid alone or additionally K99 plasmid produced mannose-resistant hemagglutination of horse or sheep erythrocytes at 20 degrees C as found for K99 and F41 ETEC natural strains, respectively . These results suggested that the structures of pili when several genetic determinants were present simultaneously may not be identical to those of original strains . In this study, clones expressing either one, two or three adhesin bearing antigens could be obtained from the strain B41.

Biochemistry (Mosc), 1998 Apr, 63(4), 485 - 8
Protein sizes and stoichiometry in the chaperone SecB--RBPTI complex estimated by ANS fluorescence; Vekshin NL; Interaction of the SecB E . coli chaperone with model precursor protein, the reduced form of bovine pancreatic trypsin inhibitor (RBPTI), in aqueous solution was studied using ANS fluorescence . Binding of RBPTI by SecB-ANS led to an increase and blue shift in the ANS fluorescence . Two ANS emission centers exist in the proteins and their complex: the short-lived center (lifetime of approximately 1 nsec) where ANS is strongly quenched by polar amino acid residues and the long-lived center (6-12 nsec) where ANS emits from the hydrophobic pocket . Estimation of the volumes and diameters of RBPTI, SecB, and the SecB--RBPTI complex using fluorescence lifetime and polarization of ANS by a modified Levshin--Perrin equation was done . Effective diameter of the SecB equals to 33.4 A . The diameter of the SecB--RBPTI complex is 49.8 A . The volume of the complex equals to sum of volumes of one tetrameric SecB and four RBPTI molecules . The binding of RBPTI molecules into the tetramer SecB is not an insertion to a "pore" without increase of the protein sizes, but "paste" of four polypeptide molecules to four subunits of SecB.

Biochemistry (Mosc), 1998 Apr, 63(4), 382 - 98
GroE chaperonin-assisted folding and assembly of dodecameric glutamine synthetase; Fisher MT; The folding and assembly of Escherichia coli dodecameric glutamine synthetase is facilitated by the E . coli GroE chaperonins, GroEL and GroES . Since endogenous glutamine synthetase monomers are bound to GroEL immediately after cell lysis and are assembly competent, this strongly suggests that glutamine synthetase is an authentic substrate of the GroE chaperonins . At physiological temperatures, the in vitro reactivation of glutamine synthetase increases from 10 to 70-80% of the original activity when the chaperonin GroEL is included . Although nucleotide binding is sufficient to dissociate assembly competent glutamine synthetase monomers from GroEL, the addition of GroES substantially accelerates the dissociation, assembly, and reactivation . The interactions of glutamine synthetase monomers with the activated chaperonin are transient (t1/2 = 10 sec) and these monomers can be released from GroEL at high concentrations without misfolding or inappropriate aggregation . It has been found that the nucleotide-induced conformational change of GroEL is critical for folding success of glutamine synthetase because the simple displacement of glutamine synthetase monomers from the GroEL chaperonin with another protein substrate inhibits reactivation . During glutamine synthetase refolding, the "high affinity" nucleotide-free GroEL is most efficient in preventing initial folding intermediates from partitioning to off-pathway folding routes . Interestingly, the more physiologically relevant "low affinity" nucleotide-bound ((ATP/ADP) GroEL--GroES) complex is not as efficient at capturing the initial folding intermediates of glutamine synthetase . In contrast to glutamine synthetase, non-authentic "model" substrates such as mammalian mitochondrial rhodanese and mitochondrial malate dehydrogenase show no differences in folding efficiencies with either the "low affinity" or "high affinity" complexes . Besides the nature of the chaperonin complex itself, the mechanism of GroE-assisted folding is determined by the folding environment and, most importantly, by initial interactions of chaperonins with folding intermediates . Glutamine synthetase interacts only transiently with chaperonin complexes, while most of the "model" proteins exhibit relatively long interactions times . It may be indicative of a specific evolutionary selected mechanism of chaperonin-assisted folding (optimizing the folding kinetics), different from that observed with non-authentic chaperonin substrates . Since the kinetics of protein folding depends heavily on the solution environment, studies involving in vivo chaperonin substrates under conditions that closely mimic those found in the cell will be required to define and solve the physiologically relevant kinetic mechanism of chaperonin-assisted folding.

Thromb Res, 1997 Dec 1, 88(5), 419 - 26
Production of a recombinant antithrombotic and fibrinolytic protein, PLATSAK, in Escherichia coli; van Zyl WB et al.; The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin . Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets . We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids . The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin . The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene . Gene expression was induced in E . coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography . The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin . The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph . Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside . Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.

Biochem Mol Biol Int, 1998 Mar, 44(3), 507 - 13
Effects of deleting A19 tyrosine from insulin; Du X et al.; A mutant proinsulin gene was constructed through PCR mediated mutagenesis . The code of A19Tyr was deleted . The mutant proinsulin, (deltaY)19-lys-proinsulin {(deltaY)19KPI}, was expressed in E . coli and purified . After treatment with trypsin and carboxypeptidase B, and Resource Q separation, (deltaY)19-human insulin {(deltaY)19HI} was obtained . It retains 63.6% of receptor binding activity but only 2.2% of immune activity, and shows a longer retaining time on reverse-phase FPLC and a slower mobility by native PAGE analysis . These results suggest that the deletion of A19Tyr causes some conformational changes on insulin, which plays a minor role on the affinity of insulin to its receptor, and a major role on immunogenicity of the hormone.

Biol Pharm Bull, 1998 Mar, 21(3), 284 - 8
Hepatoprotective effect of Fe-TPEN on carbon tetrachloride induced liver injury in rats; Moon JO et al.; Fe(II)-tetrakis-N,N,N',N'(2-pyridylmethyl) ethylenediamine (Fe-TPEN) catalyzes the dismutation of superoxide, and blocks the toxic effect of paraquat on Escherichia coli growth and survival . We examined antioxidative effects of Fe-TPEN on lipid peroxidation and t-butyl hydroperoxide induced cell damage . Fe-TPEN inhibited the FeSO4/H2O2 induced lipid peroxidation in the rat liver homogenates with an IC50 value of 30.2 microM, and protected Ac2F cell damage by t-butyl hydroperoxide in a dose-dependent manner (EC50 value is 2.6 microM) . Also, hepatoprotective effect of Fe-TPEN (5 mg/kg, i.p.) was investigated using CCl4 induced liver injury in rats . This complex inhibited the elevation of serum alanine aminotransferase (AST) and aspartate aminotransferase (ALT) levels in CCl4 induced liver injuries, and improved submassive necrosis and fatty degeneration of the hepatocytes . Fe-TPEN also prevented the loss of total and nonprotein SH contents, glutathione peroxidase and glutathione-S-transferase activity in cytosol of rat liver . Although the exact mechanism of action is not clear, antioxidative properties as well as attenuation of hepatocellular defense systems by Fe-TPEN seem to be important on its potent hepatoprotective effect in CCl4-intoxicated rat.

J Bacteriol, 1998 Apr, 180(8), 2237 - 43
The tfdK gene product facilitates uptake of 2,4-dichlorophenoxyacetate by Ralstonia eutropha JMP134(pJP4); Leveau JH et al.; Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Ralstonia eutropha JMP134(pJP4) was studied and shown to be an energy-dependent process . The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 microM, implying the involvement of a protein in the transport process . We identified an open reading frame on plasmid pJP4, which was designated tfdK, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily . An interruption of the tfdK gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake . A tfdA mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D . A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.

J Bacteriol, 1998 Apr, 180(8), 2232 - 6
A novel DNA polymerase family found in Archaea; Ishino Y et al.; One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene . This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical alpha-like DNA polymerase (family B) . Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeon Pyrococcus furiosus, which has also at least one alpha-like DNA polymerase (T . Uemori, Y . Sato, I . Kato, H . Doi, and Y . Ishino, Genes Cells 2:499-512, 1997) . The genes in M . jannaschii encoding the proteins that are homologous to the DNA polymerase II of P . furiosus have been located and cloned . The gene products of M . jannaschii expressed in Escherichia coli had both DNA polymerizing and 3'-->5' exonuclease activities . We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.

J Bacteriol, 1998 Apr, 180(8), 2228 - 31
Oxygen regulation of the ccoN gene encoding a component of the cbb3 oxidase in Rhodobacter sphaeroides 2.4.1T: involvement of the FnrL protein; Mouncey NJ et al.; The ccoNOQP gene cluster of Rhodobacter sphaeroides 2.4.1T encodes a cbb3 cytochrome oxidase which is utilized in oxygen-limited conditions for aerobic respiration . The beta-galactosidase activity of a ccoN::lacZ transcriptional fusion was low under high (30%)-oxygen and anaerobic growth conditions . Maximal ccoN::lacZ expression was observed when the oxygen concentration was lowered to 2% . In an FnrL mutant, ccoN::lacZ expression was significantly lower than in the wild-type strain, suggesting that FnrL is a positive regulator of genes encoding the cbb3 oxidase.

J Bacteriol, 1998 Apr, 180(8), 2212 - 9
Molecular analysis of the gene encoding F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis; Purwantini E et al.; The gene fgd, which codes for F420-dependent glucose-6-phosphate dehydrogenase (FGD), was cloned from Mycobacterium smegmatis, and its sequence was determined and analyzed . A homolog of FGD which has a very high similarity to the M . smegmatis FGD-derived amino acid sequence was identified in Mycobacterium tuberculosis . FGD showed significant homology with F420-dependent N5,N10-methylene-tetrahydromethanopterin reductase (MER) from methanogenic archaea and with several hypothetical proteins from M . tuberculosis and Archaeoglobus fulgidus, but FGD showed no significant homology with NADP-dependent glucose-6-phosphate dehydrogenases . Multiple alignment of FGD and MER proteins revealed four conserved consensus sequences . Multiple alignment of FGD with the hypothetical proteins also revealed portions of the same conserved sequences . Moderately high levels of FGD were expressed in Escherichia coli BL21(DE3) carrying fgd in pBluescript.

J Bacteriol, 1998 Apr, 180(8), 2186 - 93
Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response; Rao NN et al.; Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (Pi), in media deficient in both Pi and amino acids . This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses . Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation . When complemented with a phoB-containing plasmid, phoB mutants regain the ability to accumulate polyP . PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during Pi starvation) . Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp . A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.

J Bacteriol, 1998 Apr, 180(8), 2175 - 85
A membrane-associated protein, FliX, is required for an early step in Caulobacter flagellar assembly; Mohr CD et al.; The ordered assembly of the Caulobacter crescentus flagellum is accomplished in part through the organization of the flagellar structural genes in a regulatory hierarchy of four classes . Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator . In order to identify gene products required for early events in flagellar assembly, we used the known phenotypes of class II mutants to identify new class II flagellar genes . In this report we describe the isolation and characterization of a flagellar gene, fliX . A fliX null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect . Epistasis experiments placed fliX within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly . The fliX gene encodes a 15-kDa protein with a putative N-terminal signal sequence . Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells . Full expression of fliX was found to be dependent on ctrA, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the fliX promoter . The fliX gene is located upstream and is divergently transcribed from the class III flagellar gene flgI, which encodes the basal body P-ring monomer . Analysis of the fliX-flgI intergenic region revealed an arrangement of cis-acting elements similar to that of another set of Caulobacter class II and class III flagellar genes, fliL-flgF, that is also divergently transcribed . In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle.

J Bacteriol, 1998 Apr, 180(8), 2050 - 6
FtsZ dynamics during the division cycle of live Escherichia coli cells; Sun Q et al.; The dynamics and assembly of bacterial cell division protein FtsZ were monitored in individual, growing and dividing Escherichia coli cells in real time by microculture of a merodiploid strain expressing green fluorescent protein (GFP)-tagged FtsZ . Cells expressing FtsZ-GFP at levels less than or equivalent to that of wild-type FtsZ were able to grow and divide over multiple generations, with their FtsZ rings visualized by fluorescence . During the late stages of cytokinesis, which constituted the last one-fourth of the cell cycle, the lumen of the FtsZ ring disappeared as the whole structure condensed . At this time, loops of FtsZ-GFP polymers emanated outward from the condensing ring structure and other unstable fluorescent structures elsewhere in the cell were also observed . Assembly of FtsZ rings at new division sites occurred within 1 min, from what appeared to be single points . Interestingly, this nucleation often took place in the predivisional cell at the same time the central FtsZ ring was in its final contraction phase . This demonstrates directly that, at least when FtsZ-GFP is being expressed, new division sites have the capacity to become fully functional for FtsZ targeting and assembly before cell division of the mother cell is completed . The results suggest that the timing of FtsZ assembly may be normally controlled in part by cellular FtsZ concentration . The use of wide-field optical sectioning microscopy to obtain sharp fluorescence images of FtsZ structures is also discussed.

Nat Biotechnol, 1998 Apr, 16(4), 376 - 80
Selection for a periplasmic factor improving phage display and functional periplasmic expression; Bothmann H et al.; The efficiency of both phage display in Escherichia coli and periplasmic expression of recombinant proteins may be limited by the same periplasmic folding steps . To search for E . coli factors that improve the efficiency of both procedures, a library of E . coli proteins was coexpressed in a phagemid vector that contained a poorly folding single-chain Fv antibody (scFv) fragment fused to g3p . We enriched, by panning for antigen binding, those phagemids in which the amount of displayed scFv is highest . We thus identified the periplasmic protein Skp/OmpH/HlpA as improving phage display of a wide range of scFv fragments . This occurs as a result of an increase in the amount of hybrid protein displayed on the phage . Coexpression of skp also increases the functional yield of scFv fragments when expressed by secretion to the periplasm.

J Am Soc Nephrol, 1998 Apr, 9(4), 632 - 42
Evolution of tubulointerstitial fibrosis in experimental renal infection and scarring; Hewitson TD et al.; Renal tubulointerstitial fibrosis may result from a loss of tubulointerstitial volume, which produces a disproportionate increase in the density of matrix . This study examines the relationship between fibrogenesis and collapse in scar formation after experimental renal infection . Escherichia coli were inoculated into the renal cortex of Sprague Dawley rats, with saline substituted in a control group . Glomerular, tubular, and interstitial profile areas were determined . Density of glomerular profiles was used as a measure of tubulointerstitial collapse . Collagen type I, III, and IV expression was examined by in situ hybridization and immunohistochemistry . Myofibroblasts were identified by alpha smooth muscle actin immunohistochemistry, and matrix metalloproteinase-1 (MMP-1) and MMP-2 were localized with appropriate antisera . Acute interstitial edema was followed by increasing density of glomerular profiles, paralleled by loss of interstitial volume and progressive tubular atrophy . Glomerular profile area remained unchanged . Density of glomerular profiles was not temporally related to myofibroblast accumulation . Procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) transcription was focal, spatially related but temporally ordered . Collagen I, III, and IV immunostaining was increased from days 3, 24, and 100, respectively (P < 0.05 versus day 0 and day 100 saline) . However, when corrected for glomerular density, collagen I immunostaining decreased between days 24 and 100, whereas collagen III and IV no longer differed from day 0 . MMP staining within the lesion was confined to occasional interstitial and epithelial cells throughout . It is concluded that in this model, contraction and collapse of the tubulointerstitial parenchyma has a greater influence than new collagen production on final fibrotic density.

J Cardiovasc Pharmacol, 1998 Apr, 31(4), 479 - 83
Protective effects of ethynylestradiol on the hemodynamic changes induced by lipopolysaccharide in anesthetized rats; Palacios B et al.; Estrogen pretreatment has been reported to protect rats from death induced by endotoxin . We investigated the effects of posttreatment with a synthetic estrogen, ethynylestradiol, on arterial pressure and hemodynamics in thiobutabarbitone-anesthetized rats challenged with Escherichia coli lipopolysaccharide . Rats were i.v . injected with lipopolysaccharide (1 mg/kg) followed by vehicle or a single dose of ethynylestradiol (0.25, 0.5, or 1 mg/kg) 1 h later . Another group (time-matched control) was given the vehicle . In the time-control group, there was a slight decrease in mean arterial pressure (-10 +/- 3 mm Hg) but no significant changes in cardiac output, total peripheral resistance, or heart rate over the 6-h study period . Lipopolysaccharide progressively reduced mean arterial pressure and cardiac output (-27 +/- 8 mm Hg and -52 +/- 6 ml/min, after 6 h) and increased total peripheral resistance and heart rate (+0.33 +/- 0.10 mm Hg/min/ml and +21 +/- 13 beats/min, after 6 h) . None of the time-control rats died, but 36% of the rats treated with lipopolysaccharide died between 3 and 6 h after endotoxin challenge . Ethynylestradiol, at 0.25 and 0.5 completely, and at 1 mg/kg partially, restored mean arterial pressure and cardiac output at 6 h after injection of lipopolysaccharide . Ethynylestradiol at 0.5 and 1 mg/kg, but not 0.25 mg/kg, completely reversed the increase in total peripheral resistance at 6 h after injection of lipopolysaccharide . Mortality was 14% in each of the three groups of rats given ethynylestradiol 1 h after lipopolysaccharide . Therefore posttreatment with ethynylestradiol attenuated hemodynamic changes in endotoxic shock.

Chest, 1998 Apr, 113(4), 1078 - 83
Gastric and esophageal intramucosal PCO2 (PiCO2) during endotoxemia: assessment of raw PiCO2 and PCO2 gradients as indicators of hypoperfusion in a canine model of septic shock; Guzman JA et al.; STUDY OBJECTIVES: To validate capnometric recirculating gas tonometry (CRGT) for continuously monitoring gut intramucosal PCO2 (PiCO2) in a septic shock model, and to compare gastric vs esophageal PCO2 vs intramucosal-arterial PCO2 gradients . INTERVENTIONS: CRTG catheters were placed in the stomach and esophagus of six anesthetized dogs . A saline solution filled balloon tonometry (ST) catheter was also placed in the stomach . After equilibration, 3 mg/kg Escherichia coli lipopolysaccharide (LPS) was administered IV . PiCO2 measurements were made at 0, 45, and 90 min post-LPS by ST and continuously by CRGT . RESULTS: Baseline PiCO2 was 41.5+/-1.9 (+/-SE) in the stomach by CRGT, 38.0+/-1.0 by ST, and 43.0+/-4.4 mm Hg in the esophagus (p=not significant) . Gastric PiCO2 by CRGT increased to 47.0+/-2.4 mm Hg by 25 min post-LPS (p<0.05), whereas gastric (ST) and esophageal PiCO2 increased significantly by 45 min post-LPS . Good agreement was observed between gastric CRGT and ST measurements (mean bias, 1.3 mm Hg) . The PiCO2-PaCO2 gradient increased post-LPS, but was significant only for gastric CRGT measurements 90 min post-LPS infusion . CONCLUSION: CRGT provided continuous gastric PiCO2 measurements that were in close agreement with ST but detected changes earlier than the conventional technique . Continuous esophageal PiCO2 represents a valid alternative for assessing gastric PiCO2.

Appl Biochem Biotechnol, 1998 Mar, 69(3), 147 - 56
Large-scale preparation of the delta10 form of staphylokinase by in vitro processing of recombinant staphylokinase with purified human plasminogen; Chattopadhyay D et al.; The authors have developed a rapid and convenient method for purification of a low molecular weight form (delta 10) of the bacterial plasminogen activator, staphylokinase . Recombinant staphylokinase is expressed in Escherichia coli, with an amino terminal extension that facilitated purification by immobilized metal-affinity chromatography . Purified staphylokinase is treated with human plasminogen, and the resulting truncated form is purified using a combination of immobilized metal affinity chromatography and hydrophobic interaction chromatography . Purified protein is characterized by amino terminal sequencing and in vitro plasminogen activation assay.

Can J Vet Res, 1998 Apr, 62(2), 81 - 6
Localization of potential binding sites for the edema disease verotoxin (VT2e) in pigs; Waddell TE et al.; The purpose of this study was to identify organs and cells to which the edema disease verotoxin (VT2e) could bind in pigs . Frozen 4-5 microns thick sections of organs usually affected in edema disease (colon, spinal cord, cerebellum and eyelid) and organs not usually affected (liver, ileum) from two 5- to 6-week-old weaned pigs were permeabilized with acetone, then exposed to VT2e . Unbound VT2e was removed by washing and bound VT2e was detected by immunohistochemistry . In the eyelid, double-label immunofluorescence was used to identify the cells to which VT2e bound . VT2e was shown to bind to all six organs that were examined . The toxin bound to arteries in all organs, to veins in all organs except the liver, and to enterocytes in the ileal crypts . Double labelling of eyelid with monoclonal antibodies specific for von Willebrand factor or alpha-smooth actin and VT2e showed that the toxin bound to endothelial and vascular smooth muscle cells . The binding of VT2e to endothelium is consistent with findings for other verotoxins but binding to vascular smooth muscle has not been reported for other verotoxins . It is concluded that i) factors other than the presence of receptors for VT2e influence the development of lesions in edema disease, and ii) smooth muscle necrosis, which is characteristic of the vascular lesions in edema disease, may be due to a direct action of toxin on smooth muscle cells.

J Eur Acad Dermatol Venereol, 1998 Jan, 10(1), 28 - 35
Use of recombinant pemphigus vulgaris antigen in development of ELISA and IB assays to detect pemphigus vulgaris autoantibodies; Bhol K et al.; AIM/OBJECTIVE: The objectives of this study are: (1) to measure the titers of pemphigus vulgaris (PV) autoantibody in the sera of patients with active disease, using three different assays: (a) Indirect immunofluorescence (IIF) using monkey esophagus as a substrate, (b) immunoblot (IB) and, (c) enzyme-linked immunosorbent assay (ELISA) using recombinant PV antigen (rPVA) . (2) To compare the sensitivity of these three assays . BACKGROUND: The titer of PV autoantibodies and disease severity and extent do not always correlate . This could be due to the lack of consistency and specificity of the substrate . Different results are obtained using different substrates . A standard substrate with uniformly controlled source of antigen would be more useful and clinically beneficial . METHODS: In this study we studied 25 PV patients, six each with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), mucous membrane pemphigoid (MMP), and herpes gestationis (HG), and sera from 16 normal subjects . IIF was used to determine the PV autoantibody using monkey esophagus . IB assay was used according to standard protocol using normal human epidermis and rPVA as substrates . ELISA was performed using rPVA as antigens expressed in E . coli . RESULTS: Sera of all 25 PV patients showed binding to the rPVA, normal human sera and the sera from the six BP, six OCP, six MMP, and six HG patients did not show any binding . In addition, we used antisera from rabbits immunized with PVA peptides (Bos-1, Bos-6) which also showed binding to rPVA, whereas normal rabbit sera did not show any reactivity . ELISA and IB titers in all the patients were 2.5 to 160 times higher than with the conventionally used IIF assay . The titers of the PV specific autoantibody measured using the rPVA did not show statistically significant differences between the ELISA and IB assays . CONCLUSIONS: IB and ELISA are superior to IIF in evaluating the antibody levels in PV patients . ELISA is more practical and is preferable to IB and is recommended for clinical use.

Bioorg Khim, 1998 Jan, 24(1), 48 - 57
{Expression of synthetic human interleukin-10 gene and its mutant variants in Escherichia coli cells}; Ptitsyn LR et al.; The human interleukin-10 gene was obtained by chemico-enzymatic synthesis, and vectors for cytoplasmic and periplasmic expression of the recombinant IL-10 gene in Escherichia coli cells were constructed . Mutant IL-10 genes bearing substitutions in a region upstream of the ATG codon and in the triplet coding for the second amino acid residue in the protein were obtained by in vitro mutagenesis . High levels of expression were observed for the fusion protein composed of IL-10 and an N-terminal fragment of IL-3 and for the mutant IL-10 containing cysteine as the second amino acid residue.

J Mol Biol, 1998 Feb 27, 276(3), 559 - 69
Slipped misalignment mechanisms of deletion formation: analysis of deletion endpoints; Feschenko VV et al.; To gain insight into the mechanisms of deletion formation between tandem repeats, Escherichia coli plasmids were engineered to carry a 101 bp tandem duplication within the tetA gene such that deletion of one of the repeats restores an intact tetA gene and tetracycline resistance to the cell . Four base-pair changes were introduced into one of the tandem repeats to serve as genetic markers . After selection for deletion, individual plasmid products were sequenced to deduce where within the repeat the deletion had occurred . Our analysis shows most deletions are fusions of the two repeats in a single 20 bp interval . This is consistent with the simple replication slip-pair model for deletion formation and suggests that this interval may have unusual features that promote deletion . Dimer replicon products have experienced a sister-chromosome exchange event in addition to deletion and carry two tetA loci: a deleted locus showing a similar distribution of endpoints as seen-in the monomer products and an unchanged repeat locus . Seemingly reciprocal dimers are occasionally recovered which carry both a deleted and a triplicated tetA locus . These are not truly reciprocal in that the sequence analysis showed that the deletion and triplication had occurred in separate intervals . Sequence analysis of the dimeric products is consistent with predictions from our sister-strand exchange model where slipped alignment of nascent DNA strands induces deletion formation concomitant with sister-chromosome exchange.

J Mol Biol, 1998 Feb 27, 276(3), 547 - 57
Mutations in the leader region of ribosomal RNA operons cause structurally defective 30 S ribosomes as revealed by in vivo structural probing; Balzer M et al.; The biogenesis of functional ribosomes is regulated in a very complex manner, involving different proteins and RNA molecules . RNAs are not only essential components of both ribosomal subunits but also transiently interacting factors during particle formation . In eukaryotes snoRNAs act as molecular chaperones to assist maturation, modification and assembly . In a very similar way highly conserved leader sequences of bacterial rRNA operons are involved in the correct formation of 30 S ribosomal subunits . Certain mutations in the rRNA leader region cause severe growth defects due to malfunction of ribosomes which are assembled from such transcription units . To understand how the leader sequences act to facilitate the formation of the correct 30 S subunits we performed in vivo chemical probing to assess structural differences between ribosomes assembled either from rRNA transcribed from wild-type operons or from operons which contain mutations in the rRNA leader region . Cells transformed with plasmids containing the respective rRNA operons were reacted with dimethylsulphate (DMS) . Ribosomes were isolated by sucrose gradient centrifugation and modified nucleotides within the 16 S rRNA were identified by primer extension reaction . Structural differences between ribosomes from wild-type and mutant rRNA operons occur in several clusters within the 16 S rRNA secondary structure . The most prominent differences are located in the central domain including the universally conserved pseudoknot structure which connects the 5', the central and the 3' domain of 16 S rRNA . Two other clusters with structural differences fall in the 5' domain where the leader had been shown to interact with mature 16 S rRNA and within the ribosomal protein S4 binding site . The other differences in structure are located in sites which are also known as sites for the action of several antibiotics . The data explain the functional defects of ribosomes from rRNA operons with leader mutations and help to understand the altered biogenesis pathway from mutations in an rRNA leader region to the formation of functionally defective ribosomes.

J Mol Biol, 1998 Feb 27, 276(3), 537 - 45
Base-pairing of 23 S rRNA ends is essential for ribosomal large subunit assembly; Liiv A et al.; In ribosomal RNA precursors the spacer sequences bracketing mature 16 S and 23 S rRNA are base-paired to form long helices (processing stems) . In pre-23 S rRNA, the processing stem is continued by eight base-pairs of mature 23 S rRNA known as helix 1 . Recently, we have found that any part of 23 S rRNA between positions 40 and 2773 could be deleted without the loss of ribosome-like particle formation, while both end regions were indispensable . In this paper we have analyzed the role of the 5' and 3' end regions of 23 S rRNA during ribosomal 50 S assembly in vivo by using mutants of the 23 S rRNA gene . Deletions and substitutions in both strands of the helix 1 lead to the loss of plasmid derived 50 S formation . Compensatory mutations restoring helix 1 were assembled into functional 50 S subunits . We conclude that the helix 1 of 23 S rRNA is the main RNA determinant for ribosomal large-subunit assembly . Deletions in both the 5' and 3' strand of the processing stem reduced the ability of the 23 S rRNA to form ribosomal 50 S subunits . However, even the complete removal of either the 5' or the 3' strand of the processing stem did not abolish the 50 S assembly completely . Thus, processing stem facilitates, but is not essential for assembly.

Vet Microbiol, 1998 Jan 16, 59(2-3), 213 - 27
Characterization of an immuno-dominant antigen in Brucella ovis and evaluation of its use in an enzyme-linked immunosorbent assay; Kittelberger R et al.; A panel of 45 Brucella ovis serologically positive sera were tested in immunoblots against B . ovis outer membrane proteins Omp31 and Omp25, purified by preparative SDS-gel electrophoresis . Forty-three sera reacted with Omp31, while only 11 reacted with Omp25, suggesting that Omp31 is identical to the previously reported immuno-dominant 29-kDa protein . Attempts to purify Omp31 on a larger scale by using procedures such as ion exchange-, reversed phase-, affinity- and gel filtration chromatography suggested that the outer membrane proteins were aggregated with rough lipopolysaccharide . Only denaturing SDS-gel filtration chromatography was able to separate proteins of about 29 kDa from rough lipopolysaccharide but did not separate Omp31 from Omp25 in B . ovis preparations . When used in an enzyme-linked immunosorbent assay, this 29-kDa protein preparation was less sensitive and less specific than the routinely used heat-extracted B . ovis antigen . A readily available recombinant E . coli, expressing the gene for Omp31 from Brucella melitensis 16 M, was used to extract and enrich recombinant Omp31 by a temperature-dependent Triton X-114-based technique . When this material was used in immunoblots with the 45 sera from B . ovis-infected sheep and with 10 monoclonal antibodies, raised against B . ovis Omp31, major differences in the antibody reactivity between the recombinant B . melitensis Omp31 and the B . ovis Omp31 were found . Such differences were unexpected because of the known structural and immunological relatedness of outer membrane proteins from various Brucella species . These results indicated that the antibody-response in B . ovis naturally-infected sheep against the immuno-dominant Omp31 was directed against epitopes which were only accessible when the protein was aggregated with rough lipopolysaccharides, or which were formed after aggregation but were not present in the recombinant protein.

Vet Microbiol, 1998 Jan 16, 59(2-3), 203 - 12
Multiple receptors on porcine intestinal epithelial cells for the three variants of Escherichia coli K88 fimbrial adhesin; Billey LO et al.; We evaluated intestinal epithelial membrane preparations from five phenotypes of pigs, distinguished by the variant of K88 fimbrial adhesin (K88ab, K88ac, K88ad) which bind to their intestinal epithelial cells (A-all three variants, B-K88ab and K88ac, C-K88ab and K88ad, D-K88ad, and E-none of the variants), for the presence of K88 adhesin receptors . Intestinal brush border membranes were prepared from 20 animals (four from each phenotype) . Brush border proteins, that had been separated using SDS-PAGE and transferred to nitrocellulose membranes, were overlaid with biotinylated K88 adhesin, 35S-labelled K88+ Escherichia coli, or biotinylated K88+ E . coli . Biotinylated K88ab and K88ac fimbrial adhesins and labelled E . coli expressing K88ab or K88ac adhesin bound to 210- and 240-kDa receptors in phenotype A and B, but not phenotype C, D, or E animals . In contrast, no phenotype-specific receptors were identified for the K88ad adhesin . Previously, purified K88ab and K88ac fimbriae were shown to block K88ad binding, but purified K88ad fimbriae were unable to block K88ab or K88ac binding in phenotype A animals . These results point to the existence of three K88 adhesin receptors to account for the observed phenotypes: (1) Receptor bcd binds all three variants and is found in phenotype A pigs, (2) Receptor bc (210- and 240-kDa receptors) binds K88ab and K88ac and is found in phenotype A and B pigs, and (3) Receptor d binds K88ad and is found in phenotype C and D pigs.

Mutat Res, 1998 Feb 2, 397(2), 247 - 62
Unusual insertion element polymorphisms in the promoter and terminator regions of the mucAB-like genes of R471a and R446b; Kulaeva OI et al.; We have previously identified umu-complementing genes on two incL/M plasmids, R471a and R446b (C . Ho et al., J . Bacteriol., 175 (1993) 5411-5419) . Molecular analysis of these genes revealed that they are more structurally and functionally related to mucAB from the incN plasmid pKM101 than to other members of the previously identified Umu-like family . As a consequence, we have termed these new homologs mucAB(R471a) and mucAB(R446b) respectively . Interestingly, while the location of the mucAB-like genes is essentially the same in both R471a and R446b, the regions immediately flanking the mucAB-like genes are highly polymorphic . For example, 5' to mucAB(R471a) we found an insert that appears to be a novel retroelement encoding a putative reverse transcriptase (RT) . This RT is related to the reverse transcriptases encoded by group II introns but is embedded in a retron-like context . Immediately 3' to the mucAB(R471a) locus is a putative insertion element of a sparsely-dispersed class not previously reported from enteric bacteria . Both the RT and insertion element are absent in R446b . These observations suggest that the mucAB-like genes from R471a and R446b are located within regions of the R-plasmids that perhaps were once (or still are) mobile genetic elements . Such observations might help explain the distribution of umu-like genes on R-plasmids and bacterial chromosomes.

Mutat Res, 1998 Feb 2, 397(2), 209 - 19
Induction of somatic intrachromosomal recombination inversion events by cyclophosphamide in a transgenic mouse model; Sykes PJ et al.; Somatic intrachromosomal recombination (SICR) can result in chromosomal inversion and deletion, mechanisms which are important in carcinogenesis . We have utilised a transgenic mouse model to study SICR inversion events in spleen cells . The transgenic construct is designed so that expression of an Escherichia coli lacZ transgene only occurs in a cell when an SICR inversion event occurs in the region of the transgene . The inversion events can then be detected by histochemical staining of frozen spleen sections for transgene expression and by polymerase chain reaction across the inversion breakpoints . The spontaneous inversion frequency in spleen rose 2-fold from 1.54 +/- 0.24 x 10(-4) (mean +/- SE) in 4-month-old transgenic mice to 3.12 +/- 0.67 x 10(-4) in 22-month-old mice . Four- or 8-month-old mice were treated with a single intraperitoneal injection of cyclophosphamide, with doses ranging from 0.01 to 100 mg/kg . The animals were killed 3 days after treatment . A significant induction of SICR inversions was detected at all doses with a 3.2-fold maximum induction of inversions detected at 10 mg/kg . These results suggest that the transgenic mouse model used here may be a sensitive model for studying the role of SICR in mutation and in studying risk assessment of environmental DNA-damaging agents.

Biochemistry, 1998 Mar 31, 37(13), 4644 - 52
The biochemistry of hemolysin toxin activation: characterization of HlyC, an internal protein acyltransferase; Trent MS et al.; Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins . RTX toxins are products of a gene cluster, CABD . The A gene product, nontoxic hemolysin (proHlyA), is made toxic by posttranslational fatty acylation of two internal lysine residues . HlyC, the C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor . HlyB and HlyD are involved in secretion of the toxin . ProHlyA and HlyC were separately subcloned, expressed, and purified, and acyl-ACPs with diverse radioactive acyl groups were synthesized . With these proteins, the conversion of proHlyA to HlyA by acyl transfer was assayed . Acyl-ACP was the obligate acyl donor . Acyl transfer was catalyzed by HlyC monomer, and an acyl-enzyme intermediate was shown . Reaction was inhibited by ACPSH but not by fatty acid or fatty-acyl CoA . Km and Vmax for HlyA were 0.94 microM and 7.5 pmol of acyl group transferred/min, respectively; Km and Vmax for myristoyl-ACP were 0.48 microM and 6.9 pmol/min . The kinetic parameters of different acyl-ACPs resembled a competitive inhibition as acyl group carbon chain length increased; Km's increased while Vmax's remained unchanged . The different kinetic efficacies in the acyltransferase reaction of the ACPs with different acyl groups contrasted notably with the lytic powers of the corresponding acyl-toxins that they generated.

Biochemistry, 1998 Mar 31, 37(13), 4626 - 34
A triple mutation in the a subunit of the Escherichia coli/Propionigenium modestum F1Fo ATPase hybrid causes a switch from Na+ stimulation to Na+ inhibition; Kaim G et al.; Previously we have shown that the Na+-translocating Escherichia coli (F1-delta)/Propionigenium modestum (Fo+delta) hybrid ATPase acquires a Na+-independent phenotype by the c subunit double mutation F84L, L87V that is reflected by Na+-independent growth of the mutant strain MPC8487 on succinate {Kaim, G., and Dimroth, P . (1995) J . Mol . Biol . 253, 726-738} . Here we describe a new class of mutants that were obtained by random mutagenesis and screening for Na+-independent growth on succinate . All six mutants of the new class contained four mutations in the a subunit (S89P, K220R, V264E, I278N) . Results from site-specific mutagenesis revealed that the substitutions K220R, V264E, and I278N were sufficient to create the new phenotype . The resulting E . coli mutant strain MPA762 could only grow in the absence but not in the presence of Na+ ions on succinate minimal medium . This effect of Na+ ions on growth correlated with a Na+-specific inhibition of the mutant ATPase . The Ki for NaCl was 1 . 5 mM at pH 6.5, similar to the Km for NaCl in activating the parent hybrid ATPase at this pH . On the other hand, activation by Li+ ions was retained in the new mutant ATPase . In the absence of Na+ or Li+, the mutant enzyme had the same pH optimum at pH 6.5 and twice the specific activity as the parent hybrid ATPase . In accordance with the kinetic data, the reconstituted mutant ATPase catalyzed H+ or Li+ transport but no Na+ transport . These results show for the first time that the coupling ion selectivity of F1Fo ATPases is determined by structural elements not only of the c subunit but also of the a subunit.

Biochemistry, 1998 Mar 31, 37(13), 4621 - 5
Involvement of two aspartate residues of Rubisco activase in coordination of the ATP gamma-phosphate and subunit cooperativity; van de Loo FJ et al.; Aspartate residues are involved in coordination of the nucleotide-metal of several nucleotide triphosphatases . To examine interactions between Rubisco activase and ATP, site-directed mutations were made at two species-invariant aspartate residues, D174 and D231 . In the absence of the magnesium cofactor, the mutant proteins D231R, D174Q, and D174A, but not D174E, bound ATP with higher affinity than did wild-type . In the presence of Mg2+, the affinity for ATP of D231R was further increased, but was reduced with mutations at D174 . Although all mutants bound ATP, only D174E aggregated in response to ATP/Mg2+ and retained partial ATPase and Rubisco activation activities . In mixing experiments, the catalytically competent D174E stimulated wild-type ATPase activity, whereas the mutants lacking ATPase activity were inhibitory to wild-type enzyme and prevented aggregation . These results are consistent with a mechanism for activase that involves ATP-binding, subunit aggregation and ATP hydrolysis as sequential steps in the catalytic mechanism . The results also indicated that precise coordination of the gamma-phosphate is required for aggregation and depends on D174 and D231 . To account for the pronounced cooperativity of Rubisco activase subunits, we suggest that coordination of the ATP gamma-phosphate may involve participation of residues from adjacent subunits.

Biochemistry, 1998 Mar 31, 37(13), 4465 - 72
Kinetic analysis of zinc ligand mutants of mammalian protein farnesyltransferase; Fu HW et al.; Protein farnesyltransferase (FTase) is a zinc metalloenzyme that catalyzes the prenylation of several proteins that are important in cellular regulatory events . A specific residue of FTase, Cys299 in the beta subunit previously identified as essential for zinc binding and catalysis, had been tentatively assigned as one of the zinc ligands . This assignment was subsequently confirmed in the X-ray structure of FTase, which also identified two additional residues, Asp297 and His362 in the beta subunit, as the remaining protein-derived metal ligands . To more fully explore the role of zinc in the catalytic mechanism of FTase, site-directed mutagenesis was performed on these two zinc ligands . Although the abilities of all the mutants to bind the farnesyl diphosphate substrate were similar to that of the wild-type enzyme, all the mutants displayed markedly reduced enzymatic activities and zinc affinities . Steady-state and pre-steady-state kinetic analyses of the residual activities indicated that the rate-limiting step changed from product release in the wild-type enzyme to the chemical step of product formation for three of the mutant enzymes . Additionally, single-turnover experiments indicated that the greatest effect of alteration of zinc ligands for all the mutants was on the product formation step, this being reduced 10(3)-10(5)-fold in the mutant forms compared to the wild-type enzyme . These results confirm a critical involvement of the zinc in catalysis by FTase and support a model in which the metal ion is directly involved in the chemical step of the enzymatic reaction.

Biochemistry, 1998 Mar 31, 37(13), 4397 - 406
Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein; Brown KC et al.; The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP) . Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking . The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli . In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation . The cross-linking reaction occurs between two ecotin proteins in a dimer complex . Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin . The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry . The target of the reaction is tyrosine, and the product is bityrosyl cross-links . The yield of the cross-linking is on the order of 15% . However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine . This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.

Biochemistry, 1998 Mar 31, 37(13), 4388 - 96
Crystal structure of fructose-1,6-bisphosphate aldolase from the human malaria parasite Plasmodium falciparum; Kim H et al.; The structure of the glycolytic enzyme class I fructose-1, 6-bisphosphate aldolase from the human malaria parasite Plasmodium falciparum has been determined by X-ray crystallography . Homotetrameric P . falciparum aldolase (PfALDO) crystallizes in space group P3221 with one 80 kDa dimer per asymmetric unit . The final refined PfALDO model has an R-factor of 0.239 and an R-free of 0.329 with respect to data from 8 to 3.0 A resolution . PfALDO is potentially a target for antimalarial drug design as the intraerythrocytic merozoite lifestage of P . falciparum is completely dependent upon glycolysis for its ATP production . Thus, inhibitors directed against the glycolytic enzymes in P . falciparum may be effective in killing the parasite . The structure of PfALDO is compared with the previously determined structure of human aldolase in order to determine possible targets for the structure-based design of selective PfALDO ligands . The salient structural differences include a hydrophobic pocket on the surface of PfALDO, which results from some amino acid changes and a single residue deletion compared with human aldolase, and the overall quaternary structure of the PfALDO tetramer, which buries less surface area than human aldolase.

Biochemistry, 1998 Mar 31, 37(13), 4325 - 35
Preparation and kinetic characterization of a series of betaW37 variants of human hemoglobin A: evidence for high-affinity T quaternary structures; Kwiatkowski LD et al.; Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli . These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules . A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants . The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP . The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations . Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer . The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E . Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state . Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer . Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.

Biochim Biophys Acta, 1998 Mar 25, 1363(3), 231 - 7
pH dependence of the function of sodium ion extrusion systems in Escherichia coli; Sakuma T et al.; Escherichia coli has three systems for sodium ion extrusion, NhaA, NhaB and ChaA . In this study, we examined the effect of pH on the function of these transporters using mutants having one of them, and found that (1) a mutant having NhaB excreted sodium ions at pH 7.5 but not at pH 8.5, (2) the efflux of sodium ions from mutant cells having ChaA was observed at both pH 7.5 and 8.5, but the activity was lower at pH 7.5, and (3) sodium ions were excreted from mutant cells having NhaA at pH 6.5 to 8.5 . The extrusion activity of cells having NhaA was higher than that of cells having NhaB or ChaA . These results indicate that NhaB functions at a pH below 8, and ChaA extrudes sodium ions mainly at an alkaline pH above 8 . It was also suggested that the activity of NhaB and ChaA is not enough to maintain a low level of internal sodium ions when the external concentration of sodium ions is high, and NhaA is induced within a wide range of medium pH under such conditions .

Biochim Biophys Acta, 1998 Mar 25, 1363(3), 217 - 23
Glutamate residues at positions 219 and 252 in the a-subunit of the Escherichia coli ATP synthase are not functionally equivalent; Hatch LP et al.; The role of glutamate-219 in the a-subunit of the Escherichia coli F0F1-ATPase was examined using site-directed mutagenesis . The replacement of Glu-219 by lysine, alanine or glycine resulted in a partially functional F0F1-ATPase . Combining any of these mutations with the substitution of glutamate for Gln-252 did not result in any increase in function . These findings rule out a proposal that glutamate at position 252 can functionally replace glutamate at position 219 {S.B . Vik, B.J . Antonio, J . Biol . Chem . 269 (1994) 30364-30369} . All the single and double mutants grew better at 25 degrees C than at 37 degrees C, suggesting a role for Glu-219 in maintaining the structure of the F0 .

J Biol Chem, 1998 Mar 27, 273(13), 7698 - 702
Fusicoccin effect on the in vitro interaction between plant 14-3-3 proteins and plasma membrane H+-ATPase; Fullone MR et al.; A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced . The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study . The cDNA of GF14-6 was expressed in Escherichia coli as a 32P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H+-ATPase partially purified from maize roots . The results demonstrated that the recombinant protein specifically bound to H+-ATPase . The binding was dependent on Mg2+ and was strongly increased by fusicoccin . Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme . To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments . GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

J Biol Chem, 1998 Mar 27, 273(13), 7529 - 37
Insertions into the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase reveal a role for fingers subdomain in processive polymerization; Kew Y et al.; Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) displays a characteristic poor processivity during DNA polymerization . Structural elements of RT that determine processivity are poorly understood . The three-dimensional structure of HIV-1 RT, which assumes a hand-like structure, shows that the fingers, palm, and thumb subdomains form the template-binding cleft and may be involved in determining the degree of processivity . To assess the influence of fingers subdomain of HIV-1 RT in polymerase processivity, two insertions were engineered in the beta3-beta4 hairpin of HIV-1NL4-3 RT . The recombinant mutant RTs, named FE20 and FE103, displayed wild type or near wild type levels of RNA-dependent DNA polymerase activity on all templates tested and wild type or near wild type-like sensitivities to dideoxy-NTPs . When polymerase activities were measured under conditions that allow a single cycle of DNA polymerization, both of the mutants displayed 25-30% greater processivity than wild type enzyme . Homology modeling the three-dimensional structures of wild type HIV-1NL4-3 RT and its finger insertion mutants revealed that the extended loop between the beta3 and beta4 strands protrudes into the cleft, reducing the distance between the fingers and thumb subdomains to approximately 12 A . Analysis of the models for the mutants suggests an extensive interaction between the protein and template-primer, which may reduce the degree of superstructure in the template-primer . Our data suggest that the beta3-beta4 hairpin of fingers subdomain is an important determinant of processive polymerization by HIV-1 RT.

J Biol Chem, 1998 Mar 27, 273(13), 7462 - 9
The molybdenum cofactor of Escherichia coli nitrate reductase A (NarGHI) . Effect of a mobAB mutation and interactions with {Fe-S} clusters; Rothery RA et al.; We have studied the effect of a mobAB mutation and tungstate on molybdo-molybdopterin-guanine dinucleotide (Mo-MGD) insertion into Escherichia coli nitrate reductase (NarGHI) . Preparation of fluorescent oxidized derivatives of MGD (Form A and Form B) indicates that in a mobAB mutant there is essentially no detectable cofactor present in either the membrane-bound (NarGHI) or purified soluble (NarGH) forms of the enzyme . Electron paramagnetic resonance characterization of membrane-bound cofactor-deficient NarGHI suggests that it has altered electrochemistry with respect to the dithionite reducibility of the {Fe-S} clusters of NarH . Potentiometric titrations of membrane-bound NarGHI indicate that the NarH {Fe-S} clusters have midpoint potentials at pH 8.0 (Em,8.0 values) of +180 mV ({3Fe-4S} cluster), +130, -55, and -420 mV ({4Fe-4S} clusters) in a wild-type background and +180, +80, -35, and -420 mV in a mobAB mutant background . These data support the following conclusions: (i) a model for Mo-MGD biosynthesis and assembly into NarGHI in which both metal chelation and nucleotide addition to molybdopterin precede cofactor insertion; and (ii) the absence of Mo-MGD significantly affects Em,8.0 of the highest potential {4Fe-4S} cluster.

J Biol Chem, 1998 Mar 27, 273(13), 7367 - 74
Tn5 in vitro transposition; Goryshin IY et al.; This communication reports the development of an efficient in vitro transposition system for Tn5 . A key component of this system was the use of hyperactive mutant transposase . The inactivity of wild type transposase is likely to be related to the low frequency of in vivo transposition . The in vitro experiments demonstrate the following: the only required macromolecules for most of the steps in Tn5 transposition are the transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be able to self-dissociate from product DNAs; Tn5 transposes by a conservative "cut and paste" mechanism; and Tn5 release from the donor backbone involves precise cleavage of both 3' and 5' strands at the ends of the specific end sequences.

J Biol Chem, 1998 Mar 27, 273(13), 7351 - 7
Role of the GroEL chaperonin intermediate domain in coupling ATP hydrolysis to polypeptide release; Martin J; Modification of the Escherichia coli chaperonin GroEL with N-ethylmaleimide at residue Cys138 affects the structural and functional integrity of the complex . Nucleotide affinity and ATPase activity of the modified chaperonin are increased, whereas cooperativity of ATP hydrolysis and affinity for GroES are reduced . As a consequence, release and folding of substrate proteins are strongly impaired and uncoupled from ATP hydrolysis in a temperature-dependent manner . Folding of dihydrofolate reductase at 25 degrees C becomes dependent on GroES, whereas folding of typically GroES-dependent proteins is blocked completely . At 37 degrees C, GroES binding is restored to normal levels, and the modified GroEL regains its chaperone activity to some extent . These results assign a central role to the intermediate GroEL domain for transmitting conformational changes between apical and central domains, and for coupling ATP hydrolysis to productive protein release.

Plant Physiol, 1998 Mar, 116(3), 1151 - 61
The chloroplast small heat-shock protein oligomer is not phosphorylated and does not dissociate during heat stress in vivo; Suzuki TC et al.; Plants synthesize several classes of small (15- to 30-kD monomer) heat-shock proteins (sHSPs) in response to heat stress, including a nuclear-encoded, chloroplast-localized sHSP (HSP21) . Cytosolic sHSPs exist as large oligomers (approximately 200-800 kD) composed solely or primarily of sHSPs . Phosphorylation of mammalian sHSPs causes oligomer dissociation, which appears to be important for regulation of sHSP function . We examined the native structure and phosphorylation of chloroplast HSP21 to understand this protein's basic properties and to compare it with cytosolic sHSPs . The apparent size of native HSP21 complexes was > 200 kD and they did not dissociate during heat stress . We found no evidence that HSP21 or the plant cytosolic sHSPs are phosphorylated in vivo . A partial HSP21 complex purified from heat-stressed pea (Pisum sativum L.) leaves contained no proteins other than HSP21 . Mature recombinant pea and Arabidopsis thaliana HSP21 were expressed in Escherichia coli, and purified recombinant Arabidopsis HSP21 assembled into homo-oligomeric complexes with the same apparent molecular mass as HSP21 complexes observed in heat-stressed leaf tissue . We propose that the native, functional form of chloroplast HSP21 is a large, oligomeric complex containing nine or more HSP21 subunits, and that plant sHSPs are not regulated by phosphorylation-induced dissociation.

Plant Physiol, 1998 Mar, 116(3), 1097 - 110
Heat-stress response of maize mitochondria; Lund AA et al.; We have identified maize (Zea mays L . inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots . During heat stress (42 degrees C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly . In contrast, levels of two 22-kD proteins increased dramatically (HSP22) . Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22 . The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species . Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress . Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13 degrees C heat stress . Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery . Under continuous heat-stress conditions, the level of HSP22 remained high . A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database . Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.

Plant Physiol, 1998 Mar, 116(3), 1083 - 9
Herbicide safener-binding protein of maize . Purification, cloning, and expression of an encoding cDNA; Scott-Craig JS et al.; Dichloroacetamide safeners protect maize (Zea mays L.) against injury from chloroacetanilide and thiocarbamate herbicides . Etiolated maize seedlings have a high-affinity cytosolic-binding site for the safener {3H}(R,S)-3-dichloroacetyl-2,2,5-trimethyl-1, 3-oxazol-idine ({3H}Saf), and this safener-binding activity (SafBA) is competitively inhibited by the herbicides . The safener-binding protein (SafBP), purified to homogeneity, has a relative molecular weight of 39,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 5.5 . Antiserum raised against purified SafBP specifically recognizes a 39-kD protein in etiolated maize and sorghum (Sorghum bicolor L.), which have SafBA, but not in etiolated wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), or Arabidopsis, which lack SafBA . SafBP is most abundant in the coleoptile and scarcest in the leaves, consistent with the distribution of SafBA . SBP1, a cDNA encoding SafBP, was cloned using polymerase chain reaction primers based on purified proteolytic peptides . Extracts of Escherichia coli cells expressing SBP1 have strong {3H}Saf binding, which, like binding to the native maize protein, is competitively inhibited by the safener dichlormid and the herbicides S-ethyl dipropylthiocarbamate, alachlor, and metolachlor . SBP1 is predicted to encode a phenolic O-methyltransferase, but SafBP does not O-methylate catechol or caffeic acid . The acquisition of its encoding gene opens experimental approaches for the evaluation of the role of SafBP in response to the relevant safeners and herbicides.

Nucleic Acids Res, 1998 Mar 15, 26(6), 1481 - 6
A liquid chromatography/electrospray mass spectrometric study on the post-transcriptional modification of tRNA; Taniguchi H et al.; Liquid chromatography/electrospray mass spectrometry is one of the rapidly developing techniques with which mass of large hydrophilic polymers such as proteins and nucleic acids can be determined precisely . The technique was applied to studies on the modifications of tRNAs . Various tRNA species purified from Escherichia coli were directly injected into a capillary reversed-phase column and the desalted and concentrated tRNAs were analyzed on-line with an electrospray mass spectrometer . In some cases, small but significant differences were noted between the theoretical and observed molecular masses, suggesting that there exist still unknown modifications . Under high resolution measurements, multiple peaks corresponding to species modified to a varying extent were resolved . To study the structures in detail, the isolated tRNA species were digested with ribonuclease T1, and the resulting mixture of fragments were analyzed by the same liquid chromatography/mass spectrometry . In this way, most of the fragments were easily identified solely from their masses, and the positions where the expected and real structures differ were revealed . The results obtained showed the presence of micro-heterogeneity among tRNAs and demonstrated at the same time the power of the hyphenated technique for the structural analysis on nucleic acids.

Nucleic Acids Res, 1998 Mar 15, 26(6), 1473 - 80
Study of the interaction of DNA with cisplatin and other Pd(II) and Pt(II) complexes by atomic force microscopy; Onoa GB et al.; Modifications in the structure of a 260 bp DNA (hlyM) fragment from Escherichia coli caused by interaction with Pd(II) and Pt(II) complexes were studied . Cisplatin and transplatin {cis- and trans-PtCl2(NH3)2 respectively}, Pt2Cl2(Spym)4 (Spym = 2-mercaptopyrimidine anion), Pd-famotidine and Pt-famotidine were incubated with DNA for 24 h at 37 degrees C and then observed with an atomic force microscope . Atomic force microscopy (AFM) provides the opportunity for nanometer resolution in research on the interaction between nucleic acids and metal complexes . The complexes induced noticeable changes in DNA topography according to their different characteristics and structure . In the case of cisplatin a shortening in DNA strands was observed . Transplatin and Pt2Cl2(Spym)4 caused shortening and compaction, whilst an aggregation of two strands was observed for the Pt-famotidine compound but not for the Pd-famotidine compound or the metal-free famotidine.

Nucleic Acids Res, 1998 Mar 15, 26(6), 1466 - 72
Transcription coupled nucleotide excision repair by isolated Escherichia coli membrane-associated nucleoids; ClGL et al.; One form of nucleotide excision repair (NER) is known to be functionally coupled to transcription, but the nature of this functional link in Escherichia coli is still unclear . Here we have employed the isolated membrane-associated nucleoids from E.coli to examine this issue . We show that the isolated nucleoid fraction is capable of excision of UV-induced pyrimidine dimers when reconstituted with a cytoplasmic fraction resolved by sucrose gradient fractionation . This excision activity by UvrABC is sensitive to rifampicin and is dependent on transcription . By using crosslinking and immunoprecipitation, the damage recognition protein, UvrA, was found to be specifically associated with the RNA polymerase beta subunit on the chromosomal DNA independent of DNA damage . It suggests that at least in one of the NER pathways the search for damage may be directly linked to RNA polymerase . In addition, the role of transcription in the unfolding of the nucleoid structure to allow repair enzymes to gain access to the damaged DNA is described . This study provides insight into the understanding of the transcription-repair coupling in vivo.

Nucleic Acids Res, 1998 Mar 15, 26(6), 1414 - 20
A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli; Smith PA et al.; The high affinity binding interaction of biotin to avidin or streptavidin has been used widely in biochemistry and molecular biology, often in sensitive protein detection or protein capture applications . However, in vitro chemical techniques for protein biotinylation are not always successful, with some common problems being a lack of reaction specificity, inactivation of amino acid residues critical for protein function and low levels of biotin incorporation . This report describes an improved expression system for the highly specific and quantitative in vivo biotinylation of fusion proteins . A short 'biotinylation peptide', described previously by Schatz, is linked to the N-terminus of Escherichia coli thioredoxin (TrxA) to form a new protein, called BIOTRX . The 'biotinylation peptide' serves as an in vivo substrate mimic for E . coli biotin holoenzyme synthetase (BirA), an enzyme which usually performs highly selective biotinylation of E.coli biotin carboxyl carrier protein (BCCP) . A plasmid expression vector carrying the BIOTRX and birA genes arranged as a bacterial operon can be used to obtain high level production of soluble BIOTRX and BirA proteins and, under appropriate culture conditions, BIOTRX protein produced by this system is completely biotinylated . Fusions of BIOTRX to other proteins or peptides, whether these polypeptides are linked to the C-terminus or inserted into the BIOTRX active site loop, are also quantitatively biotinylated . Both types of BIOTRX fusion can be captured efficiently on avidin/streptavidin media for purification purposes or to facilitate interaction assays . We illustrate the utility of the system by measurements of antibody and soluble receptor protein binding to BIOTRX fusions immobilized on streptavidin-conjugated BIAcore chips.

Nucleic Acids Res, 1998 Mar 15, 26(6), 1408 - 13
Single chain dimers of MASH-1 bind DNA with enhanced affinity; Sieber M et al.; By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17 . Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels . MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed . CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding . Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH . On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold . Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.

Nature, 1998 Apr 2, 392(6675), 520 - 3
Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation; Craig AW et al.; In the initiation of translation in eukaryotes, binding of the small ribosomal subunit to the messenger RNA results from recognition of the 5' cap structure (m7GpppX) of the mRNA by the cap-binding complex eIF4F . eIF4F is itself a three-subunit complex comprising the cap-binding protein eIF4E, eIF4A, an ATP-dependent RNA helicase, and eIF4G, which interacts with both eIF4A and eIF4E and enhances cap binding by eIF4E . The mRNA 3' polyadenylate tail and the associated poly(A)-binding protein (PABP) also regulate translational initiation, probably by interacting with the 5' end of the mRNA . In yeast and plants, PABP interacts with eIF4G but no such interaction has been reported in mammalian cells . Here, we describe a new human PABP-interacting protein, PAIP-I, whose sequence is similar to the central portion of eIF4G and which interacts with eIF4A . Overexpression of PAIP-1 in COS-7 cells stimulates translation, perhaps by providing a physical link between the mRNA termini.

Nature, 1998 Apr 2, 392(6675), 512 - 6
Fatty acyl-CoA thioesters are ligands of hepatic nuclear factor-4alpha; Hertz R et al.; Dietary fatty acids specifically modulate the onset and progression of various diseases, including cancer, atherogenesis, hyperlipidaemia, insulin resistances and hypertension, as well as blood coagulability and fibrinolytic defects; their effects depend on their chain length and degree of saturation . Hepatocyte nuclear factor-4alpha (HNF-4alpha) is an orphan transcription factor of the superfamily of nuclear receptors and controls the expression of genes that govern the pathogenesis and course of some of these diseases . Here we show that long-chain fatty acids directly modulate the transcriptional activity of HNF-4alpha by binding as their acyl-CoA thioesters to the ligand-binding domain of HNF-4alpha . This binding may shift the oligomeric-dimeric equilibrium of HNF-4alpha or may modulate the affinity of HNF-4alpha for its cognate promoter element, resulting in either activation or inhibition of HNF-4alpha transcriptional activity as a function of chain length and the degree of saturation of the fatty acyl-CoA ligands . In addition to their roles as substrates to yield energy, as an energy store, or as constituents of membrane phospholipids, dietary fatty acids may affect the course of a disease by modulating the expression of HNF-4alpha-controlled genes.

J Lipid Res, 1998 Mar, 39(3), 633 - 46
Identification of the epitope of a monoclonal antibody that inhibits heparin binding of lipoprotein lipase: new evidence for a carboxyl-terminal heparin-binding domain; Sendak RA et al.; A panel of 13 monoclonal antibodies to avian lipoprotein lipase (LPL) was screened for inhibition of LPL binding to primary avian adipocytes . One monoclonal antibody, designated xCAL (monoclonal antibody to chicken adipose lipoprotein lipase) 3-6a, was found to inhibit the binding of LPL to primary avian adipocytes . In solid phase assays, xCAL 3-6a inhibited the binding of LPL to both heparan sulfate and heparin . XCAL 3-6a did not inhibit the catalytic activity of the avian enzyme . The monoclonal antibody was not found to cross-react significantly with bovine lipoprotein lipase . In order to determine the location of the epitope of xCAL 3-6a on lipoprotein lipase, several avian lipoprotein lipase deletion mutants were constructed and produced as glutathione S-transferase (GST) fusion proteins in E . coli . These mutants were screened for their ability to react with xCAL 3-6a using Western blotting . The minimum continuous fragment of lipoprotein lipase that was required for reactivity contained the amino acids 310 to 450 . Site-directed mutagenesis of basic residues 321, 405, 407, 409, 415, and 416 revealed that Arg 405 is necessary for the interaction of LPL with xCAL 3-6a . Additional deletions of either the amino- or carboxyl-terminal portion of the fragment containing residues 310-450 resulted in loss of antibody binding, suggesting that the epitope is a discontinuous one that is formed when the termini are brought together through protein folding . Heparin-Sepharose chromatography of wild-type LPL and a mutant LPL in which the well-characterized heparin-binding sequence (Arg 281-Lys 282-Arg 284) has been mutated was carried out in the presence and absence of xCAL 3-6a . These experiments indicate that lipoprotein lipase contains a heparin-binding domain, in addition to Arg 281-Arg 284, that can be blocked by xCAL 3-6a.

Bioconjug Chem, 1998 Mar-Apr, 9(2), 201 - 7
Interleukin 7 (IL-7) receptor-specific cell killing by DAB389 IL-7: a novel agent for the elimination of IL-7 receptor positive cells; Sweeney EB et al.; Interleukin 7 (IL-7) induces the proliferation of B cell progenitors in long-term bone marrow cultures, promotes the growth of resting fetal and adult thymocytes, and costimulates mature human T cell proliferation . IL-7 also induces cell growth in hematologic malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, and Sezary syndrome . We have constructed a recombinant fusion protein, DAB389 IL-7, composed of the catalytic and transmembrane domains of diphtheria toxin (DT), fused to IL-7 . We demonstrate that DAB389 IL-7 is selectively cytotoxic for only those cells bearing the IL-7 receptor and that entry into target cells is mediated through the receptor . The nontoxic mutant, DA(E149S)B389 IL-7, was constructed and used to demonstrate that the catalytic domain of DT is responsible for the ADP ribosylation of elongation factor 2 that results in cytotoxicity . Finally, we demonstrate that DA(E149S)B389 IL-7 induces the growth of IL-7-dependent cells, verifying the bioactivity of the IL-7 binding domain of DAB389 IL-7 . We propose that DAB389 IL-7 may be an important reagent in studying the IL-7--IL-7 receptor complex and may possess potential as a therapeutic agent against IL-7 receptor-bearing hematologic malignancies.

J Immunol, 1997 Dec 1, 159(11), 5535 - 44
A cryptic T cell epitope on the apical membrane antigen 1 of Plasmodium chabaudi adami can prime for an anamnestic antibody response: implications for malaria vaccine design; Amante FH et al.; We have investigated the proliferative and Th cell responses to the Plasmodium chabaudi adami DS homologue of the Plasmodium falciparum apical membrane Ag 1 (AMA-1), a leading malaria vaccine candidate . Immunodominant T cell epitopes were defined following immunization of BALB/c mice with Escherichia coli-expressed, refolded P . c . adami DS AMA-1 recombinant protein and testing cells from the draining lymph nodes for responses against a series of overlapping peptides spanning P . c . adami AMA-1 . A limited number of major T cell sites were identified in both conserved and variable regions of the protein . Several cryptic epitopes that evoked T cell responses following immunization with peptides, but not after protein immunization, were also identified . Adoptive transfer of a T cell line specific for a conserved cryptic epitope (corresponding to residues 31-50) provided help for an anti-AMA-1 protein-specific Ab response following in vivo challenge with P . c . adami parasitized RBC, such that AMA-1-specific Abs appeared more rapidly in recipient mice than in controls . Furthermore, T cells specific for cryptic epitopes afforded partial protection against P . c . adami infection in nude mice . The identification of conserved cryptic Th cell epitopes has important implications for malaria vaccine design.

Eur J Pharmacol, 1998 Jan 26, 342(2-3), R1 - R2
Inhibition of ornithine decarboxylase by ifenprodil; Badolo L et al.; Ifenprodil (NMDA receptor antagonist) was tested as an inhibitor of ornithine decarboxylase . It was found that ifenprodil inhibited ornithine decarboxylase activity with the same potency as alpha-difluoromethylornithine, a major inhibitor of ornithine decarboxylase . This result suggests that ifenprodil could target either the polyamine site on the NMDA receptor complex or/and polyamine biosynthesis.

Invest New Drugs, 1997, 15(4), 289 - 93
An in vitro assessment of the antineoplastic potential of 2H-1,3-oxazine-2,6(3H)-dione (3-oxauracil), a novel pyrimidine; Seal L et al.; The pyrimidine (uracil) analogue 3-oxauracil (OU) previously had been shown to completely inhibit the growth of E . coli B and decrease by 96% the replication of herpes simplex virus type 2 when present in the culture fluid at a concentration of 10(2) microM . Limited in vivo studies in mice demonstrated antiviral effects without significant toxicity when given i.p . daily for two weeks at a concentration of 3.23 mg/kg . However, the antineoplastic properties of OU were unknown . We assessed the ability of OU to inhibit the proliferation of various human tumor cell lines (3 pancreatic, 1 colon, 1 neuroendocrine, and 1 lung) in an in vitro radiometric (Bactec) system . In the pancreatic lines (RWP-2, MiaPaCa-2, and PANC-1), the colon line (HT-29), the neuroendocrine line (COLO 320DM), and the lung cancer cell line (SK-MES-1), OU at a concentration of 10(3) microM, produced a dramatic decrease in percent cell survival . When compared with cytotoxic drugs of choice for these tumor cells (gemcitabine, 5-fluorouracil, and adriamycin, respectively) a significantly higher concentration of OU was required usually to achieve comparable results with two exceptions . These were the HT-29 and the COLO 320DM cell lines . These results indicate OU has significant (p < 0.05) cytotoxic activity against pancreatic, colon, neuroendocrine, and nonsmall cell lung cancer lines, when compared to untreated control cultures . Additional in vivo testing of this potential antineoplastic agent is warranted.

Adv Exp Med Biol, 1997, 400A, 365 - 73
PX-52, A novel inhibitor of 14 kDa secretory and 85 kDa cytosolic phospholipases A2; Franson RC et al.; Previously we reported that PGBx, a prostaglandin oligomer with anti-inflammatory activity, inhibited 14 kDa phospholipase A2 (PLA2) activity and blocked arachidonic acid mobilization in prelabeled human neutrophils (Biochim . Biophys . Acta 1006:272-277, 278-286, 1989) This study describes a new inhibitor of phospholipase A2, PX-52, that also blocks agonist induced arachidonic acid mobilization in prelabeled cells . PX-52, a fatty acid polymer, inhibited hydrolysis of 14C-oleate labeled E.coli by a variety of 14 kDa PLA2s including human PMN, sperm, synovial fluid and disc, as well as porcine pancreas, N . naja, and bee venom in a dose-dependent manner with IC50s ranging from 1.0-3.7 uM . Inhibition of activity was comparable at different Ca2+ concentrations, but was relieved by increasing substrate concentration or by methylation of PX-52 . Hydrolysis of {14C}-arachidonyl phosphatidylcholine by 85 kDa, cytosolic PLA2 from U937 cells was similarly inhibited by PX-52, the IC50 = 5 uM . Arachidonic acid mobilization induced by A23187 in prelabeled human PMNs was blocked by PX-52; IC50 = 10-15 uM while concentrations of up to 80 uM oleate had no effect . These results demonstrate that PX-52 inhibits the in vitro activity of secretory and cytosolic PLA2s and agonist-induced arachidonic acid release from human cells . Given its ability to block the arachidonic acid cascade, PX-52 may be useful in the control of inflammation.

Biochim Biophys Acta, 1998 Apr 1, 1397(1), 56 - 64
Design of metal-binding green fluorescent protein variants; Bogdanov A Jr et al.; Diglycylcysteine motifs bind reduced oxo-compounds of technetium-99m, an important isotope in nuclear imaging . We suggested a system for detecting gene expression employing the effect of oxo{99mTc}technetate (Tc(V)O3+) transchelation and coordination with redox amino acid motifs . DNA fragments encoding diglycylcysteine (GGC) binding motifs were prepared by PCR and positioned downstream from the green fluorescent protein (GFP) cDNA insert . Using a Bluescript (+) vector with the fusion protein positioned under the control of a lac promoter, we obtained several E . coli clones expressing the following GFP fusion peptides: (1) GFP-P1 bearing a 'hydrophilic' C-terminal peptide (LEGGGCEGGC) containing two residues of glutamic acid and C-terminal cysteine (2) GFP-P2 carrying a 'hydrophobic' (LGGGGCGGGCGI) peptide (3) a control GFP fusion peptide with deleted C-terminal portion . Bacterial lysates obtained from the corresponding clones were tested for oxo{99mTc} technetate transchelation from a glucoheptonate complex . We found, using a solid phase assay, that radioactivity associated with protein lysates obtained from clones expressing GFP-P2 fusions were 3-4 fold higher than lysates prepared from a clone expressing a truncated GFP fusion protein lacking the C-terminal GGC motifs . High expression of GFP fusions (5-21% of total protein) was demonstrated by electrophoresis and verified by immunoblotting . Specific association of the isotope with GFP-P2 fusion proteins was detected upon incubation of gels in the presence of {99mTc}glucoheptonate, while no binding of oxo{99mTc}technetate to GFP-P1 was revealed . We demonstrated, by using semi-quantitative autoradiography, that there is a 10-fold higher binding of oxotechnetate to GFP-P2 than to a control GFP fusion protein . The implications of the study for in vivo gene expression imaging are discussed.

AIDS Res Hum Retroviruses, 1998 Mar 20, 14(5), 419 - 25
Immunologic cross-reaction between HIV type 1 p17 and Mycoplasma hyorhinis variable lipoprotein; Pincus SH et al.; Monoclonal antibodies directed against the HIV-1 matrix protein p17 that react with a component present on the surface of HIV-1-infected cells have previously been described . In this study we show that one of these monoclonal antibodies binds to persistently HIV-1-infected cell lines that are coinfected with Mycoplasma hyorhinis, but not to cell lines that are uninfected with mycoplasma . Mycoplasma-infected cells secrete HIV-1 at a higher rate, have a slight increase in cell surface expression of gp120 and gp41, and are less sensitive to immunotoxins than uninfected cells . The anti-p17 antibody binds to a protein of M . hyorhinis grown in cell-free culture . The variable expression and size of the protein among strains is typical of the variable lipoprotein (Vlp) system of M . hyorhinis . Confirmation of the reactivity of the antibody with a Vlp was provided by demonstrating its specific binding to recombinant VlpF expressed in E . coli, and to a synthetic peptide representing the carboxy-terminal region of VlpF, but not to other recombinant Vlp products or peptides . This is a true cross-reaction because the antibody also binds to recombinant p17 expressed in E . coli and the binding is inhibited by the VlpF peptide . These analyses highlight the potential of mycoplasma contamination of tissue culture cell lines to cause anomalous results . With regard to HIV-1, mycoplasma infection of cells results in increased rates of virus secretion, and introduces a potential confounding immunologic cross-reaction as well . The existence of a cell surface form of p17 is unlikely.

Eur J Biochem, 1998 Mar 15, 252(3), 467 - 76
An Escherichia coli hydrogenase-3-type hydrogenase in methanogenic archaea; Kunkel A et al.; Methanogenic archaea are known to contain two types of {NiFe} hydrogenases designated F420-reducing hydrogenase and F420-non-reducing hydrogenase . We report here that they additionally contain Escherichia coli hydrogenase-3-type {NiFe} hydrogenases . The evidence is based on biochemical studies and analysis of the subunit primary structure of this hydrogenase (designated Ech) purified from membranes of acetate-grown cells of Methanosarcina barkeri . The subunits EchE and EchC of the EchABCDEF complex showed 34% and 45% sequence identity to the nickel-containing large subunit HycE and to the iron-sulfur cluster containing small subunit HycG, respectively, of the hydrogenase in the formate hydrogen lyase complex from E . coli . Analysis of the totally sequenced genomes of Methanococcus jannaschii and Methanobacterium thermoautotrophicum strain deltaH revealed that these organisms contain similar open reading frames, indicating the presence of an E . coli hydrogenase-3-type hydrogenase also in these methanogenic archaea.

Oncogene, 1998 Mar 12, 16(10), 1241 - 7
Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers; Denissenko MF et al.; Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers . Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues . To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts . We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites . These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.

Cell, 1998 Apr 3, 93(1), 103 - 9
The unusual active site of Gal6/bleomycin hydrolase can act as a carboxypeptidase, aminopeptidase, and peptide ligase; Zheng W et al.; The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin . The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome . In Gal6 the C termini lie in the active site clefts . We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase . The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate . We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin.

Cell, 1998 Apr 3, 93(1), 93 - 101
A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins; Weiner JH et al.; We report the identification of the proteins encoded by the mttABC operon (formerly yigTUW), which mediate a novel Sec-independent membrane targeting and translocation system in Escherichia coli that interacts with cofactor-containing redox proteins having a S/TRRXFLK "twin arginine" leader motif . A pleiotropic-negative mutant in mttA prevents the periplasmic localization of twin arginine redox enzymes, including nitrate reductase (NapA) and trimethylamine N-oxide reductase (TorA) . The mutation also prevents the correct localization of the integral membrane molybdoenzyme dimethylsulfoxide reductase (DmsABC) . The DmsA subunit has a twin arginine leader . Proteins with a Sec-dependent leader or which assemble spontaneously in the membrane are not affected by this mutation . MttA, B, and C are members of a large family of related sequences extending from archaebacteria to higher eukaryotes.

Nat Struct Biol, 1998 Apr, 5(4), 294 - 303
The crystal structure of Dps, a ferritin homolog that binds and protects DNA; Grant RA et al.; The crystal structure of Dps, a DNA-binding protein from starved E . coli that protects DNA from oxidative damage, has been solved at 1.6 A resolution . The Dps monomer has essentially the same fold as ferritin, which forms a 24-mer with 432 symmetry, a hollow core and pores at the three-fold axes . Dps forms a dodecamer with 23 (tetrahedral) point group symmetry which also has a hollow core and pores at the three-folds . The structure suggests a novel DNA-binding motif and a mechanism for DNA protection based on the sequestration of Fe ions.

Nat Struct Biol, 1998 Apr, 5(4), 289 - 93
Crystal structure of aspartate decarboxylase at 2.2 A resolution provides evidence for an ester in protein self-processing; Albert A et al.; The structure of L-aspartate-alpha-decarboxylase from E . coli has been determined at 2.2 A resolution . The enzyme is a tetramer with pseudofour-fold rotational symmetry . The subunits are six-stranded beta-barrels capped by small alpha-helices at each end . The active sites are located between adjacent subunits . The electron density provides evidence for catalytic pyruvoyl groups at three active sites and an ester at the fourth . The ester is an intermediate in the autocatalytic self-processing leading to formation of the pyruvoyl group . This unprecedented structure provides novel insights into the general phenomenon of protein processing.

Biophys Chem, 1998 Mar 9, 70(3), 247 - 57
Irreversible thermal denaturation of uridine phosphorylase from Escherichia coli K-12; Lyubarev AE et al.; Thermal denaturation of uridine phosphorylase from Escherichia coli K-12 has been studied by differential scanning calorimetry . The excess heat capacity vs . temperature profiles were obtained at temperature scanning rates of 0.25, 0.5, and 1 K/min . These profiles were analysed using three models of irreversible denaturation which are approximations to the whole Lumry-Eyring model, namely, the one-step model of irreversible denaturation, the Lumry-Eyring model with the fast equilibrating first step, and the model involving two consecutive irreversible steps . In terms of statistics the latter model describes the kinetics of thermal denaturation of uridine phosphorylase more satisfactorily than the two other models . The values of energy activation for the first and second steps calculated for the model involving two consecutive irreversible steps are the following: Ea,1 = 609.3 +/- 1.8 kJ/mol and Ea,2 = 446.8 +/- 3.2 kJ/mol.

Appl Environ Microbiol, 1998 Apr, 64(4), 1566 - 8
High-pressure inactivation and sublethal injury of pressure-resistant Escherichia coli mutants in fruit juices; Garcia-Graells C et al.; The potential of high-pressure-resistant mutants of Escherichia coli to survive high-pressure pasteurization in fruit juices and in low-pH buffers was investigated . Treatments with up to 500 MPa of pressure caused only a limited direct inactivation of the mutants but resulted in an accelerated low-pH inactivation during subsequent storage.

Appl Environ Microbiol, 1998 Apr, 64(4), 1405 - 11
Structural and kinetic properties of nonglycosylated recombinant Penicillium amagasakiense glucose oxidase expressed in Escherichia coli; Witt S et al.; The gene coding for Penicillium amagasakiense glucose oxidase (GOX; beta-D-glucose; oxygen 1-oxidoreductase {EC 1.1.3.4}) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme . Recombinant Escherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence . When transformed into E . coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E . coli culture containing more than 60% inactive GOX . Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml-1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol . Reactivation followed first-order kinetics and was optimal at 10 degrees C . The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography . Up to 12 mg of active GOX could be purified from a 1-liter E . coli culture . Circular dichroism demonstrated similar conformations for recombinant and native P . amagasakiense GOXs . The purified enzyme has a specific activity of 968 U mg-1 and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P . amagasakiense . In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5 . This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX in E . coli.

Appl Environ Microbiol, 1998 Apr, 64(4), 1313 - 8
Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells; Sheridan GE et al.; The relationship between the detection of mRNA and cellular viability in Escherichia coli was investigated in cells killed by heat or ethanol . Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from rpoH, groEL, and tufA genes . mRNA from all three genes was detected immediately after the cells had been killed by heat or ethanol but gradually disappeared with time when dead cells were held at room temperature . In heat-killed cells, some mRNA targets became undetectable after 2 to 16 h, whereas after ethanol treatment, mRNA was still detected after 16 h . In contrast, 16S rRNA was detected by RT-PCR in all samples containing dead cells and did not disappear during a subsequent incubation of 16 h at room temperature . Of the different types of nucleic acid, mRNA is the most promising candidate for an indicator of viability in bacteria, but its persistence in dead cells depends on the inactivating treatment and subsequent holding conditions.

Helicobacter, 1998 Mar, 3(1), 28 - 38
An investigation of the molecular basis of the spontaneous occurrence of a catalase-negative phenotype in Helicobacter pylori; Manos J et al.; BACKGROUND: The discovery of a highly active catalase in Helicobacter pylori that in some strains may lose its activity has generated strong scientific interest . We have characterized a spontaneous catalase-negative isolate of H . pylori (UNSW-RU1) and sequenced katA in the parent strain and the promoters of both phenotypes as a prelude to understanding the genetic processes leading to the failure to express catalase . MATERIALS AND METHODS: Protein extracts from both phenotypes were examined for catalase on 2D-PAGE and analyzed by Western blot-based immuno-analysis . Presence of catalase mRNA was detected by Northern blot . Hi-Fidelity PCR was used to sequence the katA promoter while katA was sequenced using cycle-sequencing . The transcription start site was located by primer extension . RESULTS: Catalase protein was absent in UNSW-RU1 (KatA-) by 2D-PAGE and Western blot, as was catalase mRNA by Northern blot, indicating that the cause of the KatA- phenotype was at the level of transcription . No mutations were found in the promoter region of the KatA- isolate . The transcription start site was identified 55 bp upstream of the ATG site and putative RNA polymerase binding sites were mapped at "-10" and "-35" . A Fur box was identified 181 bp upstream of the transcription start site . The sequences of an 876 bp ORF and a 366 bp Escherichia coli phnA homologue were identified . CONCLUSIONS: The UNSW-RU1 (KatA-) phenotype does not express KatA or transcribe katA . The absence of defects in its promoter and a large part of its ORF indicates that loss of activity may be due to a mutation in an accessory gene essential for catalase expression, or to the binding of a repressor preventing katA transcription.

Biochim Biophys Acta, 1998 Mar 3, 1383(1), 82 - 92
Interaction of quinones with Arabidopsis thaliana thioredoxin reductase; Bironaite D et al.; In view of the ubiquitous role of the thioredoxin/thioredoxin reductase (TRX/TR) system in living cells, the interaction of Arabidopsis thaliana NADPH-thioredoxin reductase (EC 1.6.4.5) with quinones, an important class of redox cycling and alkylating xenobiotics, was studied . The steady-state reactions of A . thaliana TR with thioredoxin (TRX) and reaction product NADP+ inhibition patterns were in agreement with a proposed model of E . coli enzyme (B.W . Lennon, C.H . Williams, Jr., Biochemistry, vol . 35 (1996), pp . 4704-4712), that involved enzyme cycling between four- and two-electron reduced forms with FAD being reduced . Quinone reduction by TR proceeded via a mixed single- and two-electron transfer, the percentage of single-electron flux being equal to 12-16% . Bimolecular rate constants of quinone reduction (kcat/km) and reaction catalytic constants (kcat) increased upon an increase in quinone single-electron reduction potential . E(1)7 . In several cases, the kcat of quinone reduction exceeded kcat of TRX reduction, suggesting that quinones intercepted electron flux from TR to TRX . Incubation of reduced TR with alkylating quinones resulted in a rapid loss of TRX-reductase activity, while quinone reduction rate was unchanged . In TRX-reductase and quinone reductase reactions of TR, NADP+ exhibited different inhibition patterns . These data point out that FAD and not the catalytic disulfide of TR is responsible for quinone reduction, and that quinones may oxidize FADH2 before it reduces catalytic disulfide . Most probably, quinones may oxidize the two-electron reduced form of TR, and the enzyme may cycle between two-electron reduced and oxidized forms in this reaction . The relatively high rate of quinone reduction by A . thaliana thioredoxin reductase accompanied by their redox cycling, confers pro-oxidant properties to this antioxidant enzyme . These factors make plant TR an attractive target for redox active and alkylating pesticide action.

Biochim Biophys Acta, 1998 Apr 1, 1397(1), 91 - 101
Selection of monoclonal antibodies for probing of functional intermediates in incision of UV-irradiated DNA by Uvr(A)BC endonuclease from Escherichia coli; Kovalsky OI et al.; Monoclonal antibodies (mAbs) were generated that recognize UvrA and UvrB proteins . These proteins are components of the Uvr(A)BC endonuclease, which initiates nucleotide excision repair in Escherichia coli . mAbs, which can be used for probing of structural intermediates of Uvr(A)BC endonuclease functioning, were selected for their ability to: (i) recognize different epitopes; (ii) have a high-affinity for native antigenic protein; (iii) preserve functionality of the Uvr protein in immunocomplex . The adherence of anti-Uvr mAbs with these criteria was verified by additivity and competition tests, and by their influence on the ATPase activities of UvrA and UvrB*, the functionally active proteolytic fragment of UvrB . Two out of twelve anti-UvrA and seven out of thirteen anti-UvrB/anti-UvrB* hybridoma lines were shown to satisfy these criteria . Recognition of UvrA and UvrB deletion mutant proteins by mAbs was used to map their epitopes . Epitopes of A2D1 and A2B1 mAbs were mapped to regions of amino acids 230-281 and 560-680 of UvrA, respectively . Epitopes of anti-UvrB/UvrB* mAbs were assigned to the following amino acid regions of UvrB: B2A1, 8-61; B2C5 and B*2E3, 171-278; B2E2, 631-673; B3C1, 1-7 and/or 62-170; B*2B9, 473-630; B*3E11, 379-472 . The ability of selected mAbs to neutralize the incision function of Uvr(A)BC was analyzed . The results are discussed in terms of the applicability of these mAbs to probe the structures of intermediates in the functioning of Uvr(A)BC.

Gastroenterology, 1998 Apr, 114(4), 791 - 7
Guanylin stimulates regulated secretion from human neuroendocrine pancreatic cells; John M et al.; BACKGROUND & AIMS: Gastroenteropancreatic neuroendocrine cells secrete chemical messengers in a calcium-dependent fashion . So far, other second messenger systems involved in regulated secretion have gained little attention . The aim of this study was to characterize guanosine 3',5'-cyclic monophosphate (cGMP)-mediated vesicular secretion in pancreatic neuroendocrine cells . METHODS: In a human pancreatic cell line, BON, cyclic nucleotide levels and chromogranin A release were monitored with specific immunoassays . Uptake and release of gamma-aminobutyric acid were measured . Intracellular Ca2+ concentration was monitored with fura-2 . Guanylyl cyclase C was analyzed by reverse-transcription polymerase chain reaction . RESULTS: Guanylin increased cGMP concentrations in BON cells via guanylyl cyclase C . Stimulation of the cGMP pathway by guanylin or Escherichia coli heat-stable enterotoxin increased the release of chromogranin A and gamma-aminobutyric acid from BON cells . This effect was mimicked by the cGMP analogue 8-bromo-cGMP . CONCLUSIONS: Guanylin and STa stimulate the regulated secretion from BON cells via guanylyl cyclase C and cGMP . Our study yields novel information about secretory properties of guanylin, mediated via a signal transduction pathway, increasing cGMP and leading to regulated secretion of neuroendocrine cells.

J Biol Chem, 1998 Mar 20, 273(12), 7094 - 8
Identification and characterization of DegP, a serine protease associated with the luminal side of the thylakoid membrane; Itzhaki H et al.; The proteases involved in proteolytic degradation in the thylakoid lumen are largely unknown . Western analysis with an antibody against the Escherichia coli periplasmic serine protease DegP suggested that pea chloroplasts contain a homologue of this protease . This homologue was peripherally bound to the luminal side of the thylakoid membrane and could only be removed by a combination of high salt and non-ionic detergent . Its level increased almost 2-fold in pea seedlings exposed to elevated temperature for 4 h, suggesting this protease's role in the chloroplast's heat response . Isolated thylakoid membranes containing the chloroplastic homologue of DegP degraded beta-casein, an in vitro substrate of the bacterial protease . This activity was partially inhibited by a serine protease inhibitor, suggesting that at least part of the casein-degrading activity in the thylakoid membrane is attributable to DegP . The existence of chloroplastic DegP was further supported by isolating a full-length Arabidopsis cDNA (designated AtDegP) encoding a protein that is 37% identical and 60% similar to the E . coli protease . The amino terminus of the deduced amino acid sequence contained a bipartite transit peptide, typical of proteins targeted to the thylakoid lumen, and the mature portion of the protein contained the highly conserved serine protease catalytic triad His-Asp-Ser . The possible physiological roles of chloroplastic DegP protease are discussed.

J Biol Chem, 1998 Mar 20, 273(12), 6643 - 9
Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE; Pierpaoli EV et al.; The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE . Here, we show that the DnaJ-triggered conversion of DnaK.ATP (T state) to DnaK.ADP.Pi (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously with ATP hydrolysis . This is in contrast with the T-->R conversion in the absence of DnaJ which is biphasic, the first phase occurring simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster, H.-P., Packschies, L., Bukau, B., and Reinstein, J . (1996) J . Mol . Biol . 263, 657-670) . Apparently, DnaJ not only stimulates ATP hydrolysis but also couples it with conformational changes of DnaK . In the absence of GrpE, DnaJ forms a tight ternary complex with peptide.DnaK.ADP.Pi (Kd = 0.14 microM) . However, by monitoring complex formation between DnaK (1 microM) and a fluorophore-labeled peptide in the presence of ATP (1 mM), DnaJ (1 microM), and varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3 microM), substoichiometric concentrations of GrpE were found to shift the equilibrium from the slowly binding and releasing, high-affinity R state of DnaK completely to the fast binding and releasing, low-affinity T state and thus to prevent the formation of a long lived ternary DnaJ . substrate.DnaK.ADP.Pi complex . Under in vivo conditions with an estimated chaperone ratio of DnaK:DnaJ:GrpE = 10:1:3, both DnaJ and GrpE appear to control the chaperone cycle by transient interactions with DnaK.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 3157 - 61
RNA components of Escherichia coli degradosome: evidence for rRNA decay; Bessarab DA et al.; Recently, we found that a multicomponent ribonucleolytic degradosome complex formed around RNase E, a key mRNA-degrading and 9S RNA-processing enzyme, contains RNA in addition to its protein components . Herein we show that the RNA found in the degradosome consists primarily of rRNA fragments that have a range of distinctive sizes . We further show that rRNA degradation is carried out in the degradosome by RNase E cleavage of A+U-rich single-stranded regions of mature 16S and 23S rRNAs . The 5S rRNA, which is known to be generated by RNase E processing of the 9S precursor, was also identified in the degradosome, but tRNAs, which are not cleaved by RNase E in vitro, were absent . Our results, which provide evidence that decay of mature rRNAs occurs in growing Escherichia coli cells in the RNA degradosome, implicate RNase E in degradosome-mediated decay.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2908 - 13
The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli; Zhou YN et al.; In Escherichia coli, stringently controlled genes are highly transcribed during rapid growth, but "turned off" under nutrient limiting conditions, a process called the stringent response . To understand how transcriptional initiation at these promoters is coordinately regulated, we analyzed the interactions between RNA polymerase (RNAP) (both wild type and mutants) and four stringently controlled promoters . Our results show that the interactions between RNAP and stringently controlled promoters are intrinsically unstable and can alternate between relatively stable and metastable states . The mutant RNAPs appear to specifically further weaken interactions with these promoters in vitro and behave like "stringent" RNAPs in the absence of the stringent response in vivo, constituting a novel class of mutant RNAPs . Consistently, these mutant RNAPs also activate the expression of other genes that normally require the response . We propose that the stability of initiation complexes is coupled to the transcription of stringently controlled promoters, and this unique feature coordinates the expression of genes positively and negatively regulated by the stringent response.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2856 - 61
3' exoribonucleolytic trimming is a common feature of the maturation of small, stable RNAs in Escherichia coli; Li Z et al.; In addition to tRNA and 5S RNA, Escherichia coli contains several other small, stable RNA species; these are M1, 10Sa, 6S, and 4.5S RNA . Although these RNAs are initially synthesized as precursor molecules, relatively little is known about their maturation . The data presented here show that 3' exoribonucleolytic trimming is required for the final maturation of each of these molecules . As found previously with tRNA, but not 5S RNA, any one of a number of exoribonucleases can carry out the trimming reaction in vivo, although RNases T and PH are most effective . In their absence, large amounts of immature molecules accumulate for most of the RNAs, and these can be converted to the mature forms in vitro by the purified RNases . A model is proposed that identifies a structural feature present in all the small, stable RNAs of E . coli, and describes how this structure together with the RNases influences the common mechanism for 3' maturation.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2807 - 11
Identification of an activation region in the proteasome activator REGalpha; Zhang Z et al.; Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28 . Three REG subunits, alpha, beta, and gamma, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes . Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REGalpha . Most are monomers that produce functional proteasome activators when mixed with REGbeta subunits . Five REGalpha mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149 . The crystal structure of the REGalpha heptamer shows that this region forms a loop at the base of each REGalpha subunit . One mutation in this loop (N146Y) yields a REGalpha heptamer that binds the proteasome as tightly as wild-type REGalpha but does not activate peptide hydrolysis . Corresponding amino acid substitutions in REGbeta (N135Y) and REGgamma (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts . Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme . Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2784 - 9
Carbon dioxide stimulates peroxynitrite-mediated nitration of tyrosine residues and inhibits oxidation of methionine residues of glutamine synthetase: both modifications mimic effects of adenylylation; Berlett BS et al.; The activity of glutamine synthetase (EC 6.3.1.2) from Escherichia coli is regulated by the cyclic adenylylation and deadenylylation of Tyr-397 in each of the enzyme's 12 identical subunits . The nitration of Tyr-397 or of the nearby Tyr-326 by peroxynitrite can convert the unadenylylated enzyme to a form exhibiting regulatory characteristics similar to the form obtained by adenylylation . The adenylylated conformation can also be elicited by the oxidation of surface-exposed methionine residues to methionine sulfoxide . However, the nitration of tyrosine residues and the oxidation of methionine residues are oppositely directed by the presence and absence of CO2 . At physiological concentrations of CO2, pH 7.4, nitration occurs but oxidation of methionine residues is inhibited . Conversely, in the absence of CO2 methionine oxidation is stimulated and nitration of tyrosine is prevented . It was further established that adenylylation of Tyr-397 precludes its nitration by peroxynitrite . Furthermore, nitration of Tyr-326 together with either nitration or adenylylation of Tyr-397 leads to inactivation of the enzyme . These results demonstrate that CO2 can alter the course of peroxynitrite-dependent reactions and serve notice that (i) the reactions have physiological significance only if they are shown to occur at physiological concentrations of CO2 and physiological pH; and (ii) the peroxynitrite-dependent nitration of tyrosine residues or the oxidation of methionine residues of metabolically regulated proteins can seriously compromise their biological function.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2773 - 7
Overexpression of Escherichia coli oxidoreductases increases recombinant insulin-like growth factor-I accumulation; Joly JC et al.; Transient overexpression of either DsbA or DsbC can double the yield of periplasmic insulin-like growth factor (IGF)-I in Escherichia coli to 8.5 g/liter . Strikingly, most of the overexpressed DsbA or DsbC is found in the reduced form, implying that enhanced disulfide isomerization is responsible for the substantial increase in IGF-I yield . All of the accumulated IGF-I has had the signal sequence removed, underscoring the secretion capacity of this organism as well as its utility for efficient production of polypeptide with the correct amino terminus . The overexpressed IGF-I constitutes approximately 30% of the total cell protein . Overproduction of active site mutants of DsbA instead of the wild-type gene do not produce this increase in yield . With wild-type levels of DsbA and DsbC, most of the secreted IGF-I is found in disulfide-linked aggregates, although 10% is soluble and about 5% is correctly folded . Contrary to expectations, overexpression of the disulfide oxidoreductases decreased the soluble fraction . Because the aggregated protein can be efficiently solubilized and refolded, the increased yield is a significant benefit for the production of IGF-I.

Plant Cell, 1998 Mar, 10(3), 319 - 30
Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2; Muschietti J et al.; We screened for pollen-specific kinase genes, which are potential signal transduction components of pollen-pistil interactions, and isolated two structurally related receptor-like kinases (RLKs) from tomato, LePRK1 and LePRK2 . These kinases are similar to a pollen-expressed RLK from petunia, but they are expressed later during pollen development than is the petunia RLK . The abundance of LePRK2 increases when pollen germinates, but LePRK1 remains constant . Both LePRK1 and LePRK2 are localized to the plasma membrane/cell wall of growing pollen tubes . Both kinase domains have kinase activity when expressed in Escherichia coli . In phosphorylation assays with pollen membrane preparations, LePRK2, but not LePRK1, is phosphorylated, and the addition of tomato style, but not leaf, extracts to these membrane preparations results at least partially in specific dephosphorylation of LePRK2 . Taken together, these results suggest that LePRK1 and LePRK2 play different roles in postpollination events and that at least LePRK2 may mediate some pistil response.

Methods, 1998 Jan, 14(1), 55 - 64
Heterologous expression and purification of recombinant rolipram-sensitive cyclic AMP-specific phosphodiesterases; Salanova M et al.; With the cloning of cDNAs coding for the different phosphodiesterase 4 (PDE4) isoenzymes present in mammals, homogeneous preparations of these forms have become readily available . This strategy has greatly facilitated the understanding of the properties of the myriad of isoforms derived from the four PDE4 genes found in mammals, and has opened a new avenue to develop inhibitors with a different degree of selectivity for each isoform . Here we describe the strategies and methods used to express PDE4 in bacterial, yeast, insect, and mammalian cell heterologous systems, and review the advantages and disadvantages of each of these expression strategies . In addition, procedures to purify the recombinant proteins are described . The recently developed purification of a PDE4 by immunoaffinity chromatography provides a rapid and efficient method to prepare large quantities of PDE4 . This method should be very useful for structural and kinetic studies on the PDE4D isoforms .

EMBO J, 1998 Mar 2, 17(5), 1535 - 41
Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD; Hall MC et al.; UvrD (DNA helicase II) is an essential component of two major DNA repair pathways in Escherichia coli: methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair . In addition, it has an undefined role in the RecF recombination pathway and possibly in replication . In an effort to better understand the role of UvrD in these various aspects of DNA metabolism, a yeast two-hybrid screen was used to search for interacting protein partners . Screening of an E.coli genomic library revealed a potential interaction between UvrD and MutL, a component of the methyl-directed mismatch repair pathway . The interaction was confirmed by affinity chromatography using purified proteins . Deletion analysis demonstrated that the C-terminal 218 amino acids (residues 398-615) of MutL were sufficient to produce the two-hybrid interaction with UvrD . On the other hand, both the N- and C-termini of UvrD were required for interaction with MutL . The implications of this interaction for the mismatch repair mechanism are discussed.

EMBO J, 1998 Mar 2, 17(5), 1507 - 14
Mutations in RNAs of both ribosomal subunits cause defects in translation termination; Arkov AL et al.; Mutations in RNAs of both subunits of the Escherichia coli ribosome caused defects in catalysis of peptidyl-tRNA hydrolysis in a realistic in vitro termination system . Assaying the two codon-dependent cytoplasmic proteins that drive termination, RF1 and RF2, we observed large defects with RF2 but not with RF1, a result consistent with the in vivo properties of the mutants . Our study presents the first direct in vitro evidence demonstrating the involvement of RNAs from both the large and the small ribosomal subunits in catalysis of peptidyl-tRNA hydrolysis during termination of protein biosynthesis . The results and conclusions are of general significance since the rRNA nucleotides studied have been virtually universally conserved throughout evolution . Our findings suggest a novel role for rRNAs of both subunits as molecular transmitters of a signal for termination.

EMBO J, 1998 Mar 2, 17(5), 1497 - 506
The 3' to 5' degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3' to 5' exonucleases of the exosome complex; Anderson JS et al.; One major pathway of mRNA decay in yeast occurs by deadenylation-dependent decapping, which exposes the transcript to 5' to 3' exonucleolytic degradation . We show that a second general pathway of mRNA decay in yeast occurs by 3' to 5' degradation of the transcript . We also show that the SKI2, SKI3, SKI6/RRP41, SKI8 and RRP4 gene products are required for 3' to 5' decay of mRNA . The Ski6p/Rrp41p protein has homology to the Escherichia coli 3' to 5' exoribonuclease RNase PH, and both the Ski6p/Rrp41p and Rrp4p proteins are components of a multiprotein complex, termed the exosome, that contains at least three polypeptides with 3' to 5' exoribonuclease activities . These observations suggest that the exosome may be the nucleolytic activity that degrades the body of the mRNA in a 3' to 5' direction, and the exosome's activity on mRNAs may be modulated by Ski2p, Ski3p and Ski8p . Blocking both 3' to 5' and 5' to 3' decay leads to inviability, and conditional double mutants show extremely long mRNA half-lives . These observations argue that efficient mRNA turnover is required for viability and that we have identified the two major pathways of mRNA decay in yeast.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 967 - 74
An analysis of the role of active site protic residues of cytochrome P-450s: mechanistic and mutational studies on 17alpha-hydroxylase-17,20-lyase (P-45017alpha also CYP17); Lee-Robichaud P et al.; Certain cytochrome P-450s involved in the transformation of steroids catalyse not only the hydroxylation process associated with the group of enzymes, but also an acyl-carbon cleavage reaction . The hydroxylation occurs using an iron-monooxygen species while the acyl-carbon cleavage has been suggested to be promoted by an iron peroxide . In this paper we have studied the role of active site protic residues, Glu305 and Thr306, in modulating the two activities . For this purpose, the kinetic parameters for the hydroxylation reaction (pregnenolone-->17alpha-hydroxypregnenolone) and two different versions of acyl-carbon cleavage (17alpha-hydroxypregnenolone-->dehydroepiandrosterone and 3beta-hydroxyandrost-5-ene-17beta-carbaldehyde-->3beta-hydroxya ndrost -5,16-diene+androst-5-ene-3beta,17alpha-diol) were determined using the wild-type human CYP17 and its eight different single and double mutants . In addition the propensity of the proteins to undergo a subtle rearrangement converting the 450 nm active-form into an inactive counterpart absorbing at 420 nm, was monitored by measuring the t12 of the P-450-->P-420 conversion . The results are interpreted to draw the following conclusions . The functional groups of Glu305 and Thr306 do not directly participate in the two proton delivery steps required for hydroxylation but may be important participants for the provision of a net work of hydrogen bonds for 'activating' water that then acts as a proton donor . The loss of any one of these residues is, therefore, only partially debilitating . That the mutation of Thr306 impairs the hydroxylation reaction more than it does the acyl-carbon cleavage is consistent with the detailed mechanistic scheme considered in this paper . Furthermore attention is drawn to the fact that the mutation of Glu305 and Thr306 subtly perturbed the architecture of the active site, which affects the geometry of this region of the protein and therefore its catalytic properties.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 951 - 8
The NADP+-linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression; Barderi P et al.; NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained . Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced . The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes . Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained . TcGluDH1 encodes an enzymically active protein, since its expression in E . coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 839 - 45
The polypeptide backbone of recombinant human zona pellucida glycoprotein-3 initiates acrosomal exocytosis in human spermatozoa in vitro; Chapman N et al.; Human gamete interaction is of fundamental biological importance, yet the molecular interactions between spermatozoa and the zona pellucida are poorly understood . Surprisingly, the role of the polypeptide backbone of zona pellucida glycoprotein 3 (ZP3), the putative ligand for spermatozoa activation, has been largely overlooked . Purified recombinant human ZP3 was expressed in Escherichia coli as a C-terminal fusion to the dimeric glutathione S-transferase (GST) from Schistosoma japonicum and was shown to induce acrosomal exocytosis in live, capacitated human spermatozoa . The level of exocytosis is comparable with that obtained using purified, glycosylated, recombinant human ZP3 {van Duin, M., Polman, J.E.M., DeBreet, I.T.M., Van Ginneken, K., Bunschoten, H., Grootenhuis, A., Brindle, J . and Aitken, R.J . (1994) . Biol Reprod . 51, 607-617} . These data imply that the polypeptide chain of human ZP3 contributes to recognition of spermatozoa during acrosomal exocytosis in vitro.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 833 - 8
Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases; Mason RW et al.; We used site-directed mutagenesis to alter the specificity of human cystatin C, an inhibitor with a broad reactivity against cysteine proteinases . Nine cystatin C variants containing amino acid substitutions in the N-terminal (L9W, V10W, V10F and V10R) and/or the C-terminal (W106G) enzyme-binding regions were designed and produced in Escherichia coli . It was discovered that the inhibition profile of the cystatin could be altered by changing residues 9 and 10, which are proposed to bind in the S3 and S2 substrate-binding pockets respectively of the enzymes . All of the variants with substitutions in the N-terminal segment displayed decreased binding to cathepsins B and H, indicating that the S3 and S2 pockets of these enzymes cannot easily accommodate large aromatic residues . The introduction of a charged residue into S2 (variant V10R) created a more specific inhibitor to distinguish cathepsin B from cathepsin H . Cathepsin L showed a preference for larger aromatic residues in S2 . In contrast, cathepsin S preferred phenylalanine to valine in S2, but bound less tightly to the V10W cystatin variant . The latter variant proved to be valuable for discriminating between cathepsin L and cathepsin S (Ki 2.4 and 190 pM respectively) . The equilibrium dissociation constant of the complex between cathepsin L and variant L9W/W106G showed little difference in affinity from that of the cathepsin L complex with the singly substituted W106G variant . In contrast, the L9W/W106G variant displayed increased specificity for cathepsin S with a Ki of 10 pM . Our results clearly indicate differences in the specificity of interaction between the N-terminal region of cystatin C and cathepsins B, H, L and S, and that, although cystatin C has evolved to be a good inhibitor of all of the mammalian cysteine proteinases, more specific inhibitors of the individual enzymes can be engineered.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 827 - 31
Identification of cDNAs encoding two human alpha class glutathione transferases (GSTA3 and GSTA4) and the heterologous expression of GSTA4-4; Board PG; The Expressed Sequence Tag database has been searched for examples of previously undescribed human Alpha class glutathione transferases . An incomplete transcript of the previously described GSTA3 gene was identified in a cDNA library derived from 8-9 week placenta . This indicates that the GSTA3 gene is functional and is possibly under specific developmental regulation . A second cDNA, termed GSTA4, was identified in a brain cDNA library . The encoded GSTA4-4 enzyme was expressed in Escherichia coli and was found to be immunologically distinct from GSTA1-1 and to have high activity with alk-2-enals . Although GSTA4-4 appears to be functionally similar to the mouse GST5.7 and rat GST8-8 Alpha class enzymes, sequence comparisons and phylogenetic analysis suggest that GSTA4-4 may be a member of a distinct Alpha class subgroup.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 707 - 12
Intergenic suppression of the gammaM23K uncoupling mutation in F0F1 ATP synthase by betaGlu-381 substitutions: the role of the beta380DELSEED386 segment in energy coupling; Ketchum CJ et al.; We previously demonstrated that the Escherichia coli F0F1-ATP synthase mutation, gammaM23K, caused increased energy of interaction between gamma- and beta-subunits which was correlated to inefficient coupling between catalysis and transport {Al-Shawi, Ketchum and Nakamoto (1997) J . Biol . Chem . 272, 2300-2306} . Based on these results and the X-ray crystallographic structure of bovine F1-ATPase {Abrahams, Leslie, Lutter and Walker (1994) Nature (London) 370, 621-628} gammaM23K is believed to form an ionized hydrogen bond with betaGlu-381 in the conserved beta380DELSEED386 segment . In this report, we further test the role of gamma-beta-subunit interactions by introducing a series of substitutions for betaGlu-381 and gammaArg-242, the residue which forms a hydrogen bond with betaGlu-381 in the wild-type enzyme . betaE381A, D, and Q were able to restore efficient coupling when co-expressed with gammaM23K . All three mutations reversed the increased transition state thermodynamic parameters for steady state ATP hydrolysis caused by gammaM23K . betaE381K by itself caused inefficient coupling, but opposite from the effect of gammaM23K, the transition state thermodynamic parameters were lower than wild-type . These results suggest that the betaE381K mutation perturbs the gamma-beta-subunit interaction and the local conformation of the beta380DELSEED386 segment in a specific way that disrupts the communication of coupling information between transport and catalysis . betaE381A, L, K, and R, and gammaR242L and E mutations perturbed enzyme assembly and stability to varying degrees . These results provide functional evidence that the beta380DELSEED386 segment and its interactions with the gamma-subunit are involved in the mechanism of coupling.

J Theor Biol, 1998 Jan 7, 190(1), 37 - 49
A dynamic model of the Escherichia coli phosphate-starvation response; Van Dien SJ et al.; A mathematical model of the Escherichia coli Pho regulon was developed to study the induction of the phoA gene by starvation for inorganic phosphate . The model includes phosphate transport, detection of the phosphate concentration at the cell surface, and the signal transduction cascade ultimately leading to the induction of various Pho-controlled genes . Four parameters were manipulated to match the dynamic response of a culture growing with phosphate as the growth-limiting substrate to available experimental data for alkaline phosphatase production and internal phosphate concentration . Steady-state analysis demonstrates that the cascade design of this genetic control system gives rise to a harp transition between the uninduced and induced state for a small change in the external phosphate concentration . Parameter sensitivity indicates that the dissociation constant of the repression complex (which holds PhoR in the inactive form when phosphate is in excess), the rate constants for PhoB and PhoR phosphorylation, and the rate constant for induced transcription of Pho genes have the most influence over the expression of Pho-controlled genes . Changes in the repression complex dissociation constant and the PhoB/PhoR phosphorylation rates alter the sensitivity of the phosphate-starvation response to external phosphate concentration, whereas changes in the transcription rate constant affect the gain of the system . The model also predicts that additional Pho promoter (i.e., for the production of a heterologous protein from the phoA promoter on a plasmid) titrate activator protein PhoB A, such that a lower phosphate concentration is required to initiate expression from a high-copy plasmid than from a single-copy plasmid or the chromosome .

Hum Mol Genet, 1998 Mar, 7(3), 371 - 8
Huntingtin interacts with cystathionine beta-synthase; Boutell JM et al.; We have screened a rat brain library to identify proteins which interact with the 5'-end of huntingtin (amino acids 1-171), including the polyglutamine tract, in the yeast two-hybrid system . We detected an interaction with cystathionine beta-synthase (CBS) {L-serine hydrolyase (adding homocysteine), EC 4.2.1.22}, which was confirmed in vitro using His-tagged CBS expressed in Escherichia coli , which was able to specifically bind both rat and human full-length huntingtin . Neither normal nor expanded polyglutamine repeat alone interacted with CBS in the yeast two-hybrid system and nor did constructs containing SBMA or DRPLA with normal or expanded polyglutamine tracts . CBS therefore appears to bind specifically to huntingtin . CBS deficiency is associated with homocystinuria, which is known to affect various physiological systems, including the central nervous system . Homocysteine, one of the substrates of CBS, is known to accumulate in homocystinuria and is metabolized to homocysteate and homocysteine sulphinate, both known to be powerful excitotoxic amino acids . It has been suggested that Huntington's disease involves the action of excitotoxic amino acids and this interaction with CBS may suggest a mechanism for such excitotoxic damage.

Environ Mol Mutagen, 1998, 31(2), 149 - 56
Comparison of the types of mutations induced by 7,12-dimethylbenz{a}anthracene in the lacI and hprt genes of Big Blue rats; Mittelstaedt RA et al.; An important question regarding the use of transgenic reporter genes to detect mutation in rodents is how the types of mutations recovered in transgenes compare with the types of mutations found in endogenous genes . In this study, we examined mutations induced by 7,12-dimethylbenz{a}anthracene in the lacI transgene and the endogenous hprt gene of lymphocytes from Big Blue rats and in the hprt gene of lymphocytes from nontransgenic Fischer 344 rats . The overall mutation profiles found in these genes were remarkably similar: the majority of mutations were base pair substitutions, with the most common mutation being A:T-->T:A transversion . Differences were found for the mutational profiles in the endogenous gene and transgene with respect to the location of the mutations and the orientation of basepair substitutions in the DNA strands . In most cases, these differences could be explained by the nature of the target genes . The results support the use of the lacI transgene for detecting in vivo mutation.

Thyroid, 1998 Mar, 8(3), 229 - 34
Induction of oral tolerance in human autoimmune thyroid disease; Lee S et al.; Our laboratory has reported suppression of experimental autoimmune thyroiditis in mice by oral feeding with antigen . Based on these data, we considered it possible that oral feeding of animal thyroglobulin (TG) might induce tolerance to antigen in human autoimmune thyroid disease (AITD) . Thirteen patients receiving thyroid hormone replacement with synthetic thyroxine (T4) (five patients with Graves' disease, treated with radioiodine 4 to 11 years ago and eight patients with Hashimoto's thyroiditis) were randomly assigned to a test group (switched to replacement with desiccated thyroid from porcine thyroids) and a control group (maintained on synthetic T4) . Humoral and cellular immunologic parameters were evaluated in addition to clinical parameters before and every 3 months after the onset of study for a year . At the onset of study, there was no difference in clinical parameters, or humoral and cellular immunity to thyroid autoantigens, except a finding that one thyroid peroxidase (TPO) peptide (100 approximately 119) appeared to stimulate peripheral blood mononuclear cells (PBMC) during in vitro microproliferation assay more in the test group than control group (p = 0.051 by t test) . Additionally, almost all of TPO and thyrotropin receptor extracellular domain (TSHR) peptides were slightly more stimulatory to PBMC from the test group than the control group, although this was not statistically significant . After treatment, all variables were analyzed at each time point between groups (t test), and also were analyzed over time in each group (analysis of variance, ANOVA) . Among the clinical parameters, thyrotropin (TSH) levels were unchanged and equal . Total serum T4 levels (p < 0.05 at 6 and 12 months after treatment) and free thyroxine indices (FT4I) (p < 0.05 at all time points after treatment) were lower in the test group than the control group . This is an expected result of treatment with desiccated thyroid . We found no change over time nor any difference between groups at time points for titers of antibodies to thyroid autoantigens, ie, human TG, human TPO, and recombinant human TSHR from Escherichia coli . However, cellular immunity, measured by in vitro microproliferation of PBMC to peptides of TPO or TSHR, showed significant differences between groups . At 12 months, stimulatory indices (SI) of PBMC to six peptides, containing the indicated amino acids (764 approximately 95, 100 approximately 119, 110 approximately 129, 261 approximately 275, 441 approximately 448, 708 approximately 727) of 10 TPO peptides, and one peptide (145 approximately 163) of 14 TSHR peptides were lower in the test group than control group (p < 0.05) . SI of PBMC to phytohemagglutinin, purified protein derivative from mycobacteria, and tetanus toxoid were not different between groups nor changed over time in any group . In conclusion, treating patients with AITD with an antigen related to the autoantigen TG did not produce changes in humoral immunity parameters, while cellular immunity to certain peptides were apparently suppressed . While the results are both surprising and intriguing, we need more evidence to justify the use of autoantigen as a form of immunospecific therapy in patients with AITD.

Biophys J, 1998 Apr, 74(4), 2046 - 58
Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase; van den Berg PA et al.; Time-resolved polarized flavin fluorescence was used to study the active site dynamics of Escherichia coli glutathione reductase (GR) . Special consideration was given to the role of Tyr177, which blocks the access to the NADPH binding-site in the crystal structure of the enzyme . By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for approximately 90% of the fluorescence decay could be attributed to quenching by Y177 . Based on the temperature invariance for this lifetime, and the very high quenching rate, electron transfer from Y177 to the light-excited isoalloxazine part of flavin adenine dinucleotide (FAD) is proposed as the mechanism of flavin fluorescence quenching . Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization . This depolarization process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from internal mobility of the flavin, as has previously been proposed . Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the closely related enzyme dihydrolipoyl dehydrogenase . The fluorescence intensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein . Although structural fluctuations are likely to contribute to the nonexponential decay and the probability of quenching by a specific site, the concept of conformational substates need not be invoked to explain the heterogeneous fluorescence dynamics.

Hum Genet, 1998 Mar, 102(3), 305 - 13
Novel and recurrent tyrosine aminotransferase gene mutations in tyrosinemia type II; Huhn R et al.; Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels . The disease results from deficiency in hepatic tyrosine aminotransferase (TAT) . We have previously described one deletion and six different point mutations in four RHS patients . We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States . We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis . We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W) . Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family . Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family . Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG-->TCA) . Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins . In all, twelve different TAT gene mutations have now been identified in tyrosinemia type II.

J Chromatogr B Biomed Sci Appl, 1998 Feb 27, 706(1), 167 - 71
Affinity purification and characterization of recombinant human galectin-1; Fouillit M et al.; Galectin-1, a polypeptidic factor that can have major effects on cell growth and apoptosis, was overexpressed in E . coli . This protein was purified to homogeneity by affinity chromatography on lactose coupled to divinylsulfone-activated agarose . The recombinant galectin-1 (rGAL1) was compared with the homologous protein purified from human brain tissue using two-dimensional electrophoresis on immobilized pH gradient (IPG-DALT) . rGAL1 had a major isoelectric point of 5.4 (major pI of tissular galectin-1, 5.1) and its subunit molecular mass was 14500 . Addition of rGAL1 to Jurkat T-lymphoblastoid cells induced cell death in a concentration-dependent manner.

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