J Pathol, 2000 Dec, 192(4), 526 - 32 Changes in gastrointestinal morphology associated with obstructive jaundice; Parks RW et al.; Bacterial translocation has been consistently demonstrated in experimental models of obstructive jaundice . An important factor which promotes this phenomenon is physical injury of the intestinal mucosa . Some previous studies have presented suggestive evidence of this, following bile duct ligation . The aims of this study were to analyse objectively intestinal mucosal morphometric characteristics, to examine for evidence of bacterial translocation, and to assess enterocytes for ultrastructural abnormalities . Adult female Wistar rats were assigned to one of three groups: control (n=8), bile duct ligation (BDL; n=11), or sham operation (n=10) . One week later, portal blood, mesenteric lymph nodes, liver, and spleen were harvested and cultured aerobically and anaerobically for evidence of bacterial translocation . Segments of jejunum, ileum, caecum, and large bowel were examined histologically, using light microscopy and morphometrically, using an image analysis system . Electron microscopy was performed on regions of the gastrointestinal tract where significant morphometric alterations had been identified . Significant bacterial translocation was identified following BDL (63 . 6% BDL vs . 0% sham vs . 0% control, p<0.01, Fisher's exact test) . There was a significant reduction in total mucosal thickness (standard error) {650 microm (23) BDL vs . 731 microm (27) sham vs . 744 microm (95) control} and villous height {451 microm (20) BDL vs . 515 microm (18) sham vs . 559 microm (79) control} in jaundiced animals, compared with sham-operated and control animals (p<0.02, Mann-Whitney U-test) . Electron microscopy revealed oedematous change associated with mild inflammation, disruption of desmosomes, and the formation of lateral spaces between enterocytes . In addition, enterocytes showed vacuolation of their cytoplasm and mitochondrial swelling . Increased numbers of bacteria appeared to be attached to the mucosa . These data provide evidence of physical disruption of intestinal mucosa in jaundiced animals, most marked in the distal ileum . Significant bacterial translocation occurs following bile duct ligation and this supports the hypothesis of gut barrier dysfunction with obstructive jaundice . Biochim Biophys Acta, 2000 Dec 15, 1524(2-3), 238 - 46 Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4; Shaw DM et al.; The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface . It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule . Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome . Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies . The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents . However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules . The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA . A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli . The addition of a eukaryotic leader sequence allowed production in mammalian cells . The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS . Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay . MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M . The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs. Biochim Biophys Acta, 2000 Dec 15, 1524(2-3), 162 - 70 Enhanced oxidative damage by the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants; Kang JH et al.; Some cases of familial amyotrophic lateral sclerosis (FALS), a degenerative disorder of motor neurons, is associated with mutation in the Cu,Zn-superoxide dismutase (SOD) gene SOD1 . The purified FALS mutant and wild-type Cu,Zn-SODs expressed in Escherichia coli cells have identical dismutation activity whereas the hydroxyl radical formation of FALS mutants was enhanced relative to that of the wild-type enzyme . These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules . The reaction of the mutants with hydrogen peroxide enhanced DNA strand breaks and lipid peroxidation . The results suggested that the enhanced oxidative damage of macromolecules is mediated in the Cu,Zn-SOD mutants and hydrogen peroxide system via the generation of hydroxyl radicals by a combination of the higher free radical-generating activities of mutants and a Fenton-like reaction of copper ions released from oxidatively damaged Cu,Zn-SODs . Carnosine has been proposed to act as antioxidant in vivo . We investigated whether carnosine could protect the oxidative damage induced by FALS mutants . Carnosine effectively inhibited the DNA cleavage and lipid peroxidation . These results suggest that the higher free radical-generating function of FALS mutants can lead to increased oxidative damage of macromolecules which further implicates free radical-mediated motor neuronal injury in the pathogenesis of FALS and carnosine may be explored as potential therapeutic agents for FALS patients. Biochim Biophys Acta, 2000 Dec 15, 1524(2-3), 87 - 93 Expression, purification and characterization of beta domain and beta domain dimer of metallothionein; Zhou Y et al.; In order to examine the independent self-assembly of the beta fragment of metallothionein and the interaction between two domains with the linker sequence, Lys-Lys-Ser, the chemically synthesized genes of the beta domain and its dimer (beta-KKS-beta) were cloned into vector pGEX-4T-1 and expressed as carboxyl terminal extension of glutathione-S-transferase (GST) . After the GST fusion proteins had been digested with thrombin on a glutathione-Sepharose 4B affinity chromatography column, the beta domain and its dimer were purified with gel filtration and analyzed for their biochemical and spectroscopic properties . Amino acid composition and molecular mass are determined to be consistent with the expected value . The analysis of metal content shows that the beta domain and its dimer can bind with about 3eq and 6eq divalent metals, respectively . The characteristic peak presented around 254 nm in the UV and CD spectrum indicated that both the beta domain and its dimer are able to form the cadmium-thiolate clusters without the aid of the alpha domain . Furthermore, the absorption peak of the beta domain dimer is much higher than that of the beta domain, which suggested that there is an interaction between two beta domains . Finally, the metal-binding ability was determined by DTNB competitive reaction and the value of half dissociation pH, the results reveal that the beta domain dimer has stronger metal-binding ability than the single beta domain, which provides further evidence of the interaction between the two domains. Mutat Res, 2000 Nov 20, 455(1-2), 71 - 80 The Escherichia coli lacZ reversion mutagenicity assay; Josephy PD; The Escherichia coli lacZ reversion assay, based on the set of episomal lacZ alleles engineered by Miller et al., provides an attractive system for studies of mutagenesis and mutational specificity . Each strain in the lacZ set reverts by a specific base substitution or frameshift event . Revertants are selected by growth on lactose minimal medium . In this review, I describe the development of the assay and its subsequent modifications and improvements . Examples of its application are presented and detailed protocols for the implementation of the assay are given. FEBS Lett, 2000 Dec 8, 486(2), 178 - 82 The asp-rich region at the carboxyl-terminus of calsequestrin binds to Ca(2+) and interacts with triadin; Shin DW et al.; Calsequestrin (CSQ) is a high capacity Ca(2+) binding protein in the junctional sarcoplasmic reticulum of striated muscles, and has been shown to regulate the ryanodine receptor (RyR) through triadin and junctin . In order to identify the functional roles of specific regions on CSQ, several CSQ deletion mutants were prepared by molecular cloning and Escherichia coli expression . 45Ca(2+) overlay assay using a native gel system revealed that the major Ca(2+) binding motif of CSQ resides in the asp-rich region (amino acids 354-367) . In an in vitro binding assay using a glutathione-S-transferase affinity column, the interaction between CSQ and triadin was found to be Ca(2+)-dependent, and the site of interaction was confined to the asp-rich region of CSQ . Our results suggest that the asp-rich region of CSQ could participate in the RyR-mediated Ca(2+) release process by offering a direct binding site to luminal Ca(2+) as well as triadin. FEBS Lett, 2000 Dec 8, 486(2), 126 - 30 Inhibition of Mycobacterium smegmatis topoisomerase I by specific oligonucleotides; Bhaduri T et al.; DNA topoisomerase I from Mycobacterium smegmatis unlike many other type I topoisomerases is a site specific DNA binding protein . We have investigated the sequence specific DNA binding characteristics of the enzyme using specific oligonucleotides of varied length . DNA binding, oligonucleotide competition and covalent complex assays show that the substrate length requirement for interaction is much longer ( approximately 20 nucleotides) in contrast to short length substrates (eight nucleotides) reported for Escherichia coli topoisomerase I and III . P1 nuclease and KMnO(4) footprinting experiments indicate a large protected region spanning about 20 nucleotides upstream and 2-3 nucleotides downstream of the cleavage site . Binding characteristics indicate that the enzyme interacts efficiently with both single-stranded and double-stranded substrates containing strong topoisomerase I sites (STS), a unique property not shared by any other type I topoisomerase . The oligonucleotides containing STS effectively inhibit the M . smegmatis topoisomerase I DNA relaxation activity. Bone, 2000 Dec, 27(6), 795 - 802 Functional analysis of bone sialoprotein: identification of the hydroxyapatite-nucleating and cell-binding domains by recombinant peptide expression and site-directed mutagenesis; Harris NL et al.; Mammalian bone sialoprotein (BSP) is a mineralized tissue-specific protein containing an RGD (arginine-glycine-aspartic acid) cell-attachment sequence and two distinct glutamic acid (glu)-rich regions, with each containing one contiguous glu sequence . These regions have been proposed to contribute to the attachment of bone cells to the extracellular matrix and to the nucleation of hydroxyapatite (HA), respectively . To further delineate the domains responsible for these activities, porcine BSP cDNA was used to construct expression vectors coding for two partial-length recombinant BSP peptides: P2S (residues 42-87), containing the first glutamic acid-rich domain; and P1L (residues 69-300), containing the second glutamic acid-rich region and the RGD sequence . These peptides were expressed in Escherichia coli as his-tag fusion proteins and purified by nickel affinity columns and FPLC chromatography . Digestion with trypsin released the his-tag fusion peptide, which generated P2S-TY (residues 42-87) and P1L-TY (residues 132-239) . Using a steady-state agarose gel system, P2S-TY promoted HA nucleation, whereas P2S, P1L, and P1L-TY did not . This implies that the minimum requirement for nucleation of HA resides within the amino acid sequence of the first glutamic acid-rich domain, whereas the second glutamic acid-rich domain may require posttranslational modifications for activity . P1L, but not P2S, promoted RGD-mediated attachment of human gingival fibroblasts in a manner similar to that of native BSP . Deletion of the RGD domain or conversion of it to RGE (arginine-glycine-glutamic acid) abolished the cell-attachment activity of P1L . This suggests that, at least for human gingival fibroblasts, the major cell-attachment activity in the recombinant BSP peptides studied (residues 42-87 and 69-300) requires the RGD sequence located at the C-terminal domain. Mol Cell Biol, 2001 Jan, 21(1), 100 - 8 Sequence-specific recognition and cleavage of telomeric repeat (TTAGG)(n) by endonuclease of non-long terminal repeat retrotransposon TRAS1; Anzai T et al.; The telomere of the silkworm Bombyx mori consists of (TTAGG/CCTAA)(n) repeats and harbors a large number of telomeric repeat-specific non-long terminal repeat retrotransposons, such as TRAS1 and SART1 . To understand how these retrotransposons recognize and integrate into the telomeric repeat in a sequence-specific manner, we expressed the apurinic-apryrimidinic endonuclease-like endonuclease domain of TRAS1 (TRAS1 EN), which is supposed to digest the target DNA, and characterized its enzymatic properties . Purified TRAS1 EN could generate specific nicks on both strands of the telomeric repeat sequence between T and A of the (TTAGG)(n) strand (bottom strand) and between C and T of the (CCTAA)(n) strand (top strand) . These sites are consistent with insertion sites expected from the genomic structure of boundary regions of TRAS1 . Time course studies of nicking activities on both strands revealed that the cleavages on the bottom strand preceded those on the top strand, supporting the target-primed reverse transcription model . TRAS1 EN could cleave the telomeric repeats specifically even if it was flanked by longer tracts of nontelomeric sequence, indicating that the target site specificity of the TRAS1 element was mainly determined by its EN domain . Based on mutation analyses, TRAS1 EN recognizes less than 10 bp around the initial cleavage site (upstream 7 bp and downstream 3 bp), and the GTTAG sequence especially is essential for the cleavage reaction on the bottom strand (5' . TTAGGTT downward arrow AGG . 3') . TRAS1 EN, the first identified endonuclease digesting telomeric repeats, may be used as a genetic tool to shorten the telomere in insects and some other organisms. J Biol Chem, 2001 Mar 16, 276(11), 8500 - 6 Epub 2000 Dec 11. The regulation of type 7 adenylyl cyclase by its C1b region and Escherichia coli peptidylprolyl isomerase, SlyD; Yan SZ et al.; Mammalian membrane-bound adenylyl cyclase consists of two highly conserved cytoplasmic domains (C1a and C2a) separated by a less conserved connecting region, C1b, and one of two transmembrane domains, M2 . The C1a and C2a domains form a catalytic core that can be stimulated by forskolin and the stimulatory G protein subunit alpha (Galpha(s)) . In this study, we analyzed the regulation of type 7 adenylyl cyclase (AC7) by C1b . The C1a, C1b, and C2a domains of AC7 were purified separately . Escherichia coli SlyD protein, a cis-trans peptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b) . SlyD protein can inhibit the Galpha(s)- and/or forskolin-activated activity of both soluble and membrane-bound AC7 . Mutant forms of SlyD with reduced PPIase activity are less potent in the inhibition of AC7 activity . Interestingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein, raising the possibility that mammalian PPIase may regulate enzymatic activity of mammalian adenylyl cyclase . Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than SlyD alone . This inhibition by 7C1b is abolished in a 7C1b mutant in which a conserved glutamic acid (amino acid residue 582) is changed to alanine . Inhibition of adenylyl cyclase activity by 7C1b is further confirmed by using 7C1b purified from an E . coli slyD-deficient strain . This inhibitory activity of AC7 is also observed with the 28-mer peptides derived from a region of C1b conserved in AC7 and AC2 but is not observed with a peptide derived from the corresponding region of AC6 . This inhibitory activity exhibited by the C1b domain may result from the interaction of 7C1b with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated state. J Biol Chem, 2001 Mar 9, 276(10), 7681 - 8 Epub 2000 Dec 11. Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1; Wan L et al.; The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain . The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined . However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA . We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA . The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity . We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding . We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding . These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein . The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates. J Biol Chem, 2001 Mar 16, 276(11), 7791 - 6 Epub 2000 Dec 11. Biologically active recombinant human progastrin(6-80) contains a tightly bound calcium ion; Baldwin GS et al.; Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo . The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro . Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein . After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry . Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion . Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC . The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor . We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion . With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone. Biochemistry, 2000 Dec 19, 39(50), 15620 - 5 Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo; Kobayashi K et al.; To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity quinone binding site (Q(H)), using N-methylnicotinamide (NMA) as an electron mediator . With the ubiquinone bound enzyme, the reduction of the oxidase occurred in two phases as judged from kinetic difference spectra . In the faster phase, the transient species with an absorption maximum at 440 nm, a characteristic of the formation of ubisemiquinone anion radical, appeared within 10 micros after pulse radiolysis . In the slower phase, a decrease of absorption at 440 nm was accompanied by an increase of absorption at 428 and 561 nm, characteristic of the reduced form . In contrast, with the bound ubiquinone-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, though the reduction yield was low . These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at the Q(H) site to heme b exists in cytochrome bo . The first-order rate constant of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1 . The rate constant for the intramolecular electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected . The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo {Ingledew, W . J., Ohnishi, T., and Salerno, J . C . (1995) Eur . J . Biochem . 227, 903-908}, indicating that electron transfer from the bound ubisemiquinone at the Q(H) site to the hemes slows down at the alkaline pH where the bound ubisemiquinone can be stabilized . These findings are consistent with our previous proposal that the bound ubiquinone at the Q(H) site mediates electron transfer from the low-affinity quinol oxidation site in subunit II to low-spin heme b in subunit I. Biochemistry, 2000 Dec 19, 39(50), 15592 - 602 Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the "pseudomature" recombinant enzyme; Peters RJ et al.; The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid . A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products . Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies . Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties . Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir . Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process . The structural implications of these findings are discussed in the context of primary sequence elements considered to be responsible for binding the substrate and intermediate and for initiating the respective cyclization steps. Biochemistry, 2000 Dec 19, 39(50), 15540 - 7 Evolutionary coadaptation of the motif 2--acceptor stem interaction in the class II prolyl-tRNA synthetase system; Burke B et al.; Known crystal structures of class II aminoacyl-tRNA synthetases complexed to their cognate tRNAs reveal that critical acceptor stem contacts are made by the variable loop connecting the beta-strands of motif 2 located within the catalytic core of class II synthetases . To identify potential acceptor stem contacts made by Escherichia coli prolyl-tRNA synthetase (ProRS), an enzyme of unknown structure, we performed cysteine-scanning mutagenesis in the motif 2 loop . We identified an arginine residue (R144) that was essential for tRNA aminoacylation but played no role in amino acid activation . Cross-linking experiments confirmed that the end of the tRNA(Pro) acceptor stem is proximal to this motif 2 loop residue . Previous work had shown that the tRNA(Pro) acceptor stem elements A73 and G72 (both strictly conserved among bacteria) are important recognition elements for E . coli ProRS . We carried out atomic group "mutagenesis" studies at these two positions of E . coli tRNA(Pro) and determined that major groove functional groups at A73 and G72 are critical for recognition by ProRS . Human tRNA(Pro), which lacks these elements, is not aminoacylated by the bacterial enzyme . An analysis of chimeric tRNA(Pro) constructs showed that, in addition to A73 and G72, transplantation of the E . coli tRNA(Pro) D-domain was necessary and sufficient to convert the human tRNA into a substrate for the bacterial synthetase . In contrast to the bacterial system, base-specific acceptor stem recognition does not appear to be used by human ProRS . Alanine-scanning mutagenesis revealed that motif 2 loop residues are not critical for tRNA aminoacylation activity of the human enzyme . Taken together, our results illustrate how synthetases and tRNAs have coadapted to changes in protein-acceptor stem recognition through evolution. Biochemistry, 2000 Dec 19, 39(50), 15418 - 28 The substrate activation process in the catalytic reaction of Escherichia coli aromatic amino acid aminotransferase; Islam MM et al.; Aromatic amino acid aminotransferase is active toward both aromatic and dicarboxylic amino acids, and the mechanism for this dual substrate recognition has been an issue in the enzymology of this enzyme . Here we show that, in the reactions with aromatic and dicarboxylic ligands, the pK(a) of the Schiff base formed between the coenzyme pyridoxal 5'-phosphate and Lys258 or the substrate increases successively from 6.6 in the unliganded enzyme to approximately 8.8 in the Michaelis complex and to >10.5 in the external Schiff base complex . Mutations of Arg292 and Arg386 to Leu, which mimic neutralization of the positive charges of the two arginine residues by the ligand carboxylate groups, increased the Schiff base pK(a) by 0.1 and 0.7 unit, respectively . In contrast to these moderate effects of the Arg mutations, the cleavage of the Lys258 side chain of the Schiff base, which was brought about by preparing a mutant enzyme in which Lys258 was changed to Ala and the Schiff base was reconstituted with methylamine, produced the Schiff base pK(a) value of 10.2, that being 3.6 units higher than that of the wild-type enzyme . The observation indicates that the Schiff base pK(a) in the enzyme is lowered by the torsion around the C4-C4' axis of the Schiff base and suggests that the pK(a) is mainly controlled by changing the torsion angle during the course of catalysis . This mechanism, first observed for the reaction of aspartate aminotransferase with aspartate {Hayashi, H., Mizuguchi, H., and Kagamiyama, H . (1998) Biochemistry 37, 15076-15085}, does not require the electrostatic contribution from the omega-carboxylate group of the substrate, and can explain why in aromatic amino acid aminotransferase the aromatic substrates can increase the Schiff base pK(a) during catalysis to the same extent as the dicarboxylic substrates . This is the first example in which the torsion pK(a) coupling of the pyridoxal 5'-phosphate Schiff base has been demonstrated in pyridoxal enzymes other than aspartate aminotransferase, and suggests the generality of the mechanism in the catalysis of aminotransferases related to aspartate aminotransferase. Biochemistry, 2000 Dec 19, 39(50), 15410 - 7 Identification of tyrosine 204 as the photo-cross-linking site in the DNA-EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry; Wong DL et al.; We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition . The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC) . UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield . SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex . The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry . Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography . A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA . Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment . Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer . Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition . Our method provides an accurate characterization of picomole quantities of DNA-protein complexes. Biochemistry, 2000 Dec 19, 39(50), 15399 - 409 High-level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain; Feng L et al.; Phosphatidylcholine transfer protein (PC-TP) is a 214-amino acid cytosolic protein that promotes intermembrane transfer of phosphatidylcholines, but no other phospholipid class . To probe mechanisms for membrane interactions and phosphatidylcholine binding, we expressed recombinant human PC-TP in Escherichia coli using a synthetic gene . Optimization of codon usage for bacterial protein translation increased expression of PC-TP from trace levels to >10% of the E . coli cytosolic protein mass . On the basis of secondary structure predictions of an amphipathic alpha-helix (residues 198-212) in proximity to a hydrophobic alpha-helix (residues 184-193), we explored whether the C-terminus might interact with membranes and promote binding of phosphatidylcholines . Consistent with this possibility, truncation of five residues from the C-terminus shortened the predicted amphipathic alpha-helix and decreased PC-TP activity by 50%, whereas removal of 10 residues eliminated the alpha-helix, abolished activity, and markedly decreased the level of membrane binding . Circular dichroic spectra of synthetic peptides containing one ((196-214)PC-TP) or both ((183-214)PC-TP) predicted C-terminal alpha-helices in aqueous buffer were most consistent with random coil structures . However, both peptides adopted alpha-helical configurations in the presence of trifluoroethanol or phosphatidylcholine/phosphatidylserine small unilamellar vesicles . The helical content of (196-214)PC-TP increased in proportion to vesicle phosphatidylserine content, consistent with stabilization of the alpha-helix at the membrane surface . In contrast, the helical content of (183-214)PC-TP was not influenced by vesicle composition, implying that the more hydrophobic of the alpha-helices penetrated into the membrane bilayer . These studies suggest that tandem alpha-helices located near the C-terminus of PC-TP facilitate membrane binding and extraction of phosphatidylcholines. Biochemistry, 2000 Dec 19, 39(50), 15316 - 21 Farnesyl diphosphate synthase . Altering the catalytic site to select for geranyl diphosphate activity; Stanley Fernandez SM et al.; Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C(5) substrate dimethylallyl diphosphate (DMAPP) to the C(15) product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate (IPP) . The synthesis of FPP proceeds in two steps, where the C(10) product of the first addition, geranyl diphosphate (GPP), is the substrate for the second addition . The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the binding pocket for the hydrocarbon residue of the allylic substrate . Amino acid substitutions that reduced the size of the binding pocket were identified by molecular modeling . FPPase mutants containing seven promising modifications were constructed . Initial screens using DMAPP and GPP as substrates indicated that two of the substitutions, A116W and N144'W, strongly discriminated against binding of GPP to the allylic site . These observations were confirmed by an analysis of the products from reactions with DMAPP in the presence of excess IPP and by comparing the steady-state kinetic constants for the wild-type enzyme and the A116W and N114W mutants. J Biol Chem, 2001 Apr 13, 276(15), 11895 - 901 Epub 2000 Nov 28. Identification of beta-glucosidase aggregating factor (BGAF) and mapping of BGAF binding regions on Maize beta -glucosidase; Blanchard DJ et al.; In certain maize genotypes (nulls), beta-glucosidase does not enter the gel and therefore cannot be detected on zymograms . Such genotypes were initially thought to be homozygous for a null allele at the glu1 gene . We have shown that a beta-glucosidase aggregating factor (BGAF) is responsible for the null phenotype, and it specifically interacts with maize beta-glucosidases and forms large insoluble aggregates . To understand the mechanism of the beta-glucosidase-BGAF interaction, we constructed chimeric enzymes by domain swapping between the maize beta-glucosidase isozymes Glu1 and Gu2, to which BGAF binds, and the sorghum beta-glucosidase (dhurrinase) isozyme Dhr1, to which BGAF does not bind . The results of binding assays with 12 different chimeric enzymes showed that an N-terminal region (Glu(50)-Val(145)) and an extreme C-terminal region (Phe(466)-Ala(512)) together form the BGAF binding site on the enzyme surface . In addition, we purified BGAF, determined its N-terminal sequence, amplified the BGAF cDNA by reverse transcriptase-polymerase chain reaction, expressed it in Escherichia coli, and showed that it encodes a protein whose binding and immunological properties are identical to the native BGAF isolated from maize tissues . A data base search revealed that BGAF is a member of the jasmonite-induced protein family . Interestingly, the deduced BGAF sequence contained an octapeptide sequence (G(P/R)WGGSGG) repeated twice . Each of these repeat units is postulated to be involved in forming a site for binding to maize beta-glucosidases and thus provides a plausible explanation for the divalent function of BGAF predicted from binding assays. Protein Eng, 2000 Oct, 13(10), 725 - 34 Expression of a bispecific dsFv-dsFv' antibody fragment in Escherichia coli; Schmiedl A et al.; A bispecific disulfide-stabilized Fv antibody fragment (dsFv-dsFv') consisting of two different disulfide-stabilized Fv antibody fragments connected by flexible linker peptides was produced by secretion of three polypeptide chains into the periplasm of Escherichia coli . The dsFv-dsFv' molecules were enriched by immobilized metal affinity chromatography and further purified by anion-exchange chromatography . The recombinant antibody constructs retained the two parental antigen binding specificities and were able to cross-link the two different antigens . The described dsFv-dsFv' design might be of particular value for therapeutic in vivo applications since improved stability is expected to be combined with minimal immunogenicity. Biochem Biophys Res Commun, 2000 Dec 9, 279(1), 223 - 8 Cloning and recombinant expression of human group IIF-secreted phospholipase A(2); Valentin E et al.; Mammalian-secreted phospholipases A(2) (sPLA(2)) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids . We report here the cloning and characterization of human group IIF sPLA(2) (hGIIF sPLA(2)) . The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA(2)s . hGIIF sPLA(2) gene is located on chromosome 1 and lies within a sPLA(2) gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA(2)s . In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney . Finally, recombinant expression of hGIIF sPLA(2) in Escherichia coli shows that the enzyme is Ca(2+)-dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference . Am J Respir Crit Care Med, 2000 Dec, 162(6), 2278 - 86 Halothane reduces the early lipopolysaccharide-induced lung inflammation in mechanically ventilated rats; Giraud O et al.; Several studies suggest that anesthetics modulate the immune response . The aim of this study was to investigate the effect of halothane and thiopental on the lung inflammatory response . Rats submitted or not to intratracheal (IT) instillation of lipopolysaccharides (LPS) were anesthetized with either halothane (0 . 5, 1, or 1.5%) or thiopental (60 mg . kg(-1)) and mechanically ventilated for 4 h . Control rats were treated or not by LPS without anesthesia . Lung inflammation was assessed by total and differential cell counts in bronchoalveolar lavage fluids (BALF) and by cytokine measurements (tumor necrosis factor-alpha {TNF-alpha}, interleukin-6 {IL-6}, macrophage inflammatory protein-2 {MIP-2}, and monocyte chemoattractant protein-1 {MCP-1}) in BALF and lung homogenates . In the absence of LPS treatment, neither halothane nor thiopental modified the moderate inflammatory response induced by tracheotomy or mechanical ventilation . Cell recruitment and cytokine concentrations were increased in all groups receiving IT LPS . However, in halothane-anesthetized rats (halothane > or = 1%), but not in thiopental-anesthetized rats, the LPS-induced lung inflammation was altered in a dose-dependent manner . Indeed, when using 1% halothane, polymorphonuclear leukocyte (PMN) recruitment was decreased by 55% (p < 0.001) and TNF-alpha, IL-6, and MIP-2 concentrations in BALF and lung homogenates were decreased by more than 60% (p < 0.001) whereas total protein and MCP-1 concentrations remained unchanged . The decrease of MIP-2 (observed at the protein and messenger RNA {mRNA} level) was strongly correlated to the decrease of PMN recruitment (r = 0.73, p < 0.05) . This halothane-reduced lung inflammatory response was transient and was reversed 20 h after the end of the anesthesia . Our study shows that halothane > or = 1%, delivered during 4 h by mechanical ventilation, but not mechanical ventilation per se, alters the early LPS-induced lung inflammation in the rat, suggesting a specific effect of halothane on this response. Transpl Int, 2000, 13 Suppl 1, S329 - 32 Adenovirus-mediated gene transfer of CTLA4Ig gene results in prolonged survival of heart allograft; Hayashi S et al.; It has been demonstrated that the administration of CTLA4Ig protein can induce the suppression of allograft and xenograft rejection . The purpose of this study is to determine the effect of adenovirus-mediated gene transfer with CTLA4Ig gene on the transgene expression and suppression of alloimmune response in allogeneic cardiac transplantation of rats . Adenoviral vectors with beta galactosidase or human CTLA4Ig cDNA (Adex/LacZ, Adex/hCTLA4Ig) were constructed . These vectors were transduced to the liver of Lewis (LEW) rats by intravenous injection . Then LEW rats received heterotopic cardiac transplantation from Dark Agouti rats . Experiments were performed using both CTLA4Ig gene transduction and/or immunosuppressant FK506 . The transgene expression after adenovirus-mediated transfer with CTLA4Ig cDNA lasted for several months, compared with several weeks after that in controls, which was proportional to the blood concentration of CTLA4Ig protein . By the administration of 1 x 10(9) PFU of Adex/hCTLA4Ig, the survival of cardiac grafts was significantly prolonged, compared with controls or the use of 1 x 10(8) PFU of Adex/hCTLA4Ig . In the rats with beating grafts over 100 days, the blood concentrations of CTLA4Ig were undetectable . The combination therapy using a low titer Adex/hCTLA4Ig and low-dose of FK506 was synergistically effective on this cardiac transplantation model . In conclusion, adenovirus-mediated gene transfer with CTLA4Ig gene was efficient for the prolongation of both transgene expression and allograft survival. Int J Med Microbiol, 2000 Oct, 290(4-5), 491 - 6 Enterotoxins and the enteric nervous system--a fatal attraction; Farthing MJ; Although there has been extensive investigation of the biochemical consequences of the interactions between bacterial enterotoxins and intestinal epithelial cells and the mechanisms by which they induce intestinal secretion, relatively little attention has been given to other aspects of the host response to these enterotoxins . There is now compelling evidence that the enteric nervous system has a major role in enhancing the secretory state induced by cholera toxin, the E . coli enterotoxins and possibly C . difficile toxin A . Cholera toxin for example is thought to activate a neural reflex via the release of 5-hydroxytryptamine from enterochromaffin cells . Neurotransmitters involved in the reflex include substance P and vasoactive intestinal polypeptide . Delineation of these neural pathways may offer new possibilities for the pharmacological control of enterotoxin-mediated secretion. Int J Med Microbiol, 2000 Oct, 290(4-5), 317 - 23 Structure-function relationships in the Bvg and Evg two-component phosphorelay systems; Bantscheff M et al.; The unorthodox two-component phosphorelay systems BvgAS and EvgAS of Bordetella pertussis and E . coli, respectively, are suitable model systems to investigate the molecular basis of signalling specificity, because, despite their high relatedness on the sequence level, they do not cross-talk to each other . We could show that the two systems belong to the obligate type of phosphorelay systems and that signalling specificity is mediated by the HPt modules of the histidine kinases and the receiver domains of the effector proteins . To gain more insight into signalling specificity on the molecular level, we started a detailed structural analysis of the respective proteins using a combination of genetic and biochemical methods including limited proteolysis and chemical modification of purified proteins and their mass spectrometrical analysis. Gene, 2000 Nov 27, 258(1-2), 173 - 81 Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein; Jones HM et al.; A synthetic gene coding for the Chlamydia trachomatis serovar L2 major outer membrane protein (MOMP) was designed, constructed and expressed in Escherichia coli . The native amino acid sequence was reverse translated and the resulting nucleotide combinations manipulated in order to evenly distribute 25 unique restriction sites along the length of the gene while retaining the native amino acid sequence . The synthetic gene was cloned into a T7 promoter-controlled plasmid (pET-3a) and the expressed product was analyzed to assess antigenicity, cellular localization and function . Monoclonal antibodies specific for native MOMP reacted to the expressed product by immunoblot . Outer membrane fractionation confirmed that the processed protein was located in the outer membrane . MOMP expressed in E . coli and present in the outer membrane was shown to function as a general diffusion porin . This system provides the means to produce readily modifiable MOMP either in purified form or as a membrane-associated protein, and so facilitate the investigation of its functional, structural and antigenic properties. Gene, 2000 Nov 27, 258(1-2), 95 - 108 Stability of DNA repeats in Escherichia coli dam mutant strains indicates a Dam methylation-dependent DNA deletion process; Troester H et al.; In this study we report on the stabilization of short direct repetitive DNA elements . We arranged a 20 bp SK-primer element in a direct repeat manner within the cloning vector pBluescript KS (+) . This resulted in an array of 27 direct repeats consisting of 24 bp units . We show that this plasmid could only be propagated without deletion of repeats in dam mutant Escherichia coli hosts, whereas all efforts to use strains that were defective in the methylation-dependent restriction system and the recA- or the mismatch repair-dependent deletion system failed . The deletions always affected whole repeat units and not parts of them, leading to an unpredictable reduction of the unit number down to a range of between 12 and two during propagation . We conclude that a Dam methylation-dependent, but recA- and mismatch repair-independent, deletion mechanism caused the DNA rearrangements without an obvious involvement of the known methylated-DNA restriction systems. FEMS Microbiol Lett, 2000 Dec 15, 193(2), 217 - 21 Cloning and heterologous expression of a sulfur oxygenase/reductase gene from the thermoacidophilic archaeon Acidianus sp . S5 in Escherichia coli; He Z et al.; A thermoacidophilic, obligately chemolithotrophic, facultatively aerobic archaebacterium, Acidianus sp . S5, was isolated from acidothermal springs in southwest China . The sulfur oxygenase/reductase (SOR) gene of Acidianus sp . S5 was cloned and expressed in Escherichia coli . Several primers were designed and successfully applied for detection and cloning of the sor gene . A 3.7-kb EcoRI fragment containing the sor gene and three neighboring open reading frames was sequenced . Sequence analysis indicated that the sor gene of Acidianus sp . S5 showed 81% identity to the sor gene of Acidianus ambivalens . E . coli cells carrying the sor gene on pBV220SOR were able to overproduce SOR upon a temperature shift from 30 to 42 degrees C . SOR produced in E . coli catalyzes the oxidation of elemental sulfur and concomitant production of sulfite, thiosulfate and hydrogen sulfide . The recombinant enzyme exhibits the same catalytic properties as the one from Acidianus S5. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2000 Dec, 17(6), 404 - 8 {Study on a downstream signal molecule of human CASK/LIN-2}; Qi J et al.; OBJECTIVE: To elucidate the function of human CASK/LIN-2, a novel member of membrane-associated guanylate kinase homologs family (MAGUK), by using the yeast two-hybrid system(LexA) to screen the protein interacting with the guanylate kinase-like domain(GK) of hCASK and identify possible protein that might involve downstream signal transduction of hCASK . METHODS: PCR strategy was used to amplify GK domain cDNA fragment of hCASK . The DNA was subcloned into pLexA to construct bait vector pLexA-GK . The pLexA-GK was transformed into yeast host strain EGY48; through testing the bait protein and a repression assay, it was demonstrated that the bait protein did not have transcription activation for leu and lacZ reporter genes, however it could enter nucleus, bind LexA operator . Human fetus brain cDNA library plasmids were transformed into EGY48 containing pLexA-GK and p8op-lacZ, and screened on plates of Gal/Raf his- trp- ura- leu- and Gal/Raf his- trp- ura-X-gal . The clones with galactose dependent leu+ and lacZ+ were isolated; their library plasmids were rescued by means of transforming E.coli KC8 . These library plasmids were transformed into the yeast again to re-screen . The remaining positive clones were analyzed by PCR and restriction endonuclease . Finally two kinds of target DNA fragments were obtained . RESULTS: The specificity testing indicated that the two target proteins could specially bind GK domain of hCASK . After DNA sequencing and BLASTn analysis on NCBI, it was shown that the two target DNA fragments, 732 and 683 bp, had high sequence similarity with human inhibitors of differentiation 1(Id1) mRNA, with identities of 97%(630/645) and 98%(656/666), respectively . CONCLUSION: GK domain of hCASK could specially interact with Id1 in yeast . Id1 might be a downstream signal molecule of hCASK, which might involve in regulation of cell differentiation. J Infect Dis, 2001 Jan 15, 183(2), 347 - 350 Epub 2000 Dec 08. Intervention with Shiga toxin (Stx) antibody after infection by Stx-producing Escherichia coli; Matise I et al.; Shiga toxins (Stxs) produced by Escherichia coli (STEC) cause systemic vascular damage, manifested as hemolytic uremic syndrome in humans and as edema disease in pigs . Edema disease, a naturally occurring disease of pigs, was used to determine whether Stx antibodies, administered after infection and after the onset of Stx production, could prevent the systemic vascular damage and clinical disease caused by Stxs . A total of 119 STEC-infected pigs were treated with low, medium, or high doses of Stx antibody or with placebo . After inoculation with STEC, antibodies or placebo was injected intraperitoneally at 2 days postinoculation (DPI; low dose) or 4 DPI (medium and high doses) . Edema disease was prevented with the low- and high-dose Stx antibody treatments administered at 2 and 4 DPI, respectively . High-dose antibody treatment also reduced the incidence and extent of vascular lesions . The degree of protection depended on the dose of antibody and the time of administration. J Vet Pharmacol Ther, 2000 Jun, 23(3), 153 - 8 The impact of acute phase response on the plasma clearance of antipyrine, theophylline, phenytoin and nifedipine in rabbits; Saitoh T et al.; The impact of acute phase response (APR) on the plasma clearances of antipyrine, theophylline, phenytoin and nifedipine was studied using 50 female rabbits . APR was induced by a bolus intramuscular injection of Escherichia coli lipopolysaccharide (LPS, 50 microg/kg) . No abnormal findings, other than an increase in rectal body temperature and the plasma concentration of interleukin-6 (IL-6), were observed in the LPS-treated animals . Twenty-four hours after LPS injection, the pharmacokinetic parameters of the four drugs were obtained following intravenous administrations of antipyrine (7 mg/kg), theophylline (5 mg/kg), phenytoin (10 mg/kg) and nifedipine (1 mg/kg) . Total body clearances of antipyrine, theophylline, phenytoin and nifedipine in LPS-treated rabbits decreased, and terminal elimination half-life and the mean residence time of these drugs increased compared with those in the control rabbits . The apparent volume of distribution for phenytoin and nifedipine increased after the LPS injection, although the binding percentage of the drugs with plasma protein did not change . These results suggested that APR appears to decrease the plasma clearances of these drugs in rabbits, which may be due to the suppression of the activity of cytochrome P450 enzymes. Int J Hyg Environ Health, 2000 Oct, 203(2), 159 - 64 A multiplex-PCR method to detect enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli in artificially contaminated foods; Cocolin L et al.; In view of the considerable public health risk of the enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, a multiplex-PCR based method was used for the amplification of the slt genes and of the eaeA gene . Three pairs of primers, different from the oligonucleotides previously used by other authors, were exploited for the amplification . Different E . coli serotypes were tested with the optimized protocol . Fifty three artificially contaminated samples and sixty naturally contaminated samples were processed with the multiplex-PCR . All the artificially contaminated samples gave positive results independently of the number of cells inoculated . On the contrary, the naturally contaminated samples were all negative . The results obtained from this experiment demonstrated that this protocol could be used for monitoring the spread of these organisms in food. Exp Anim, 2000 Jul, 49(3), 229 - 33 Generation of transgenic rats with YACs and BACs: preparation procedures and integrity of microinjected DNA; Takahashi R et al.; The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats from the standpoint of transgenic production efficiency and integrity of the introduced gene . In yeast artificial chromosome (YAC) transgenesis, three methods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the amplification element (AMP/RE), and no amplification of the YAC in yeast (AMP-) . Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP- . Based on these results, we favor the AMP method in spite of the thymidine kinase gene-induced male sterility . In bacterial artificial chromosome (BAC) transgenesis, linear DNA fragments for microinjection prepared by three kinds of purification procedures were compared: Not I digestion and CsCl gradient ultra-centrifugation (Prep . 1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and beta-agarase digestion (Prep . 2), and CsCl gradient ultra-centrifugation, Not I digestion, pulse field gel electrophoresis, and beta-agarase digestion (Prep . 3) . Although the efficiency of producing transgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep . 3 procedure . We therefore favor the Prep . 3 method for preparing BAC DNA fragments . These results indicate that the method used to prepare a large DNA fragment such as YAC and BAC DNAs is important in order to produce transgenic rats with an intact transgene. Can J Microbiol, 2000 Nov, 46(11), 1087 - 90 Use of lactose to induce expression of soluble NifA protein domains of Herbaspirillum seropedicae in Escherichia coli; Monteiro RA et al.; Overexpression and purification are procedures used to allow functional and structural characterization of proteins . Many overexpressed proteins are partially or completely insoluble, and can not be easily purified . The NifA protein is an enhancer-binding protein involved in activating the expression of nif and some fix genes . The NifA protein from many organisms is usually insoluble when over-expressed, and therefore difficult to work with in vitro . In this work we have overexpressed the central + C-terminal and the central domains of the Herbaspirrilum seropedicae NifA protein in an Escherichia coli background . Expression was induced with either IPTG or lactose . The data showed that induction with lactose promoted a significantly higher percentage of these proteins in the soluble fraction than with IPTG . This probably reflects a slower kinetics of induction by lactose. Adv Exp Med Biol, 2000, 485, 143 - 50 Examination of regulatory cross-talk between the decay accelerating factor-binding fimbrial/afimbrial adhesins and type I fimbriae; Holden N et al.; The revised sequence of DaaA means that the protein is 98% identical to the afimbrial adhesin regulator AFAA-III . While PapB repressed FimB OFF to ON switching in strain AAEC370A, this was not the case for DaaA . PapB, but not DaaA, reduced the level of expression of type 1 fimbriae, in static LB media . Attention is now focused on the amino terminus of the PapB protein as the possible domain involved in cross talk with type 1 fimbriae. Biochim Biophys Acta, 2000 Dec 20, 1498(2-3), 233 - 51 Ca(2+)-binding proteins in the retina: from discovery to etiology of human disease(1); Sokal I et al.; Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states . In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy . Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells . Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1 . In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage . Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina . Several members of this subfamily are also present in other tissues . In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood . CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase . These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed. FEBS Lett, 2000 Dec 1, 486(1), 57 - 62 The high affinity ATP binding site modulates the SecA-precursor interaction; van Voorst F et al.; SecA is the central component of the protein-translocation machinery of Escherichia coli . It is able to interact with the precursor protein, the chaperone SecB, the integral membrane protein complex SecYEG, acidic phospholipids and its own mRNA . We studied the interaction between prePhoE and SecA by using a site-specific photocrosslinking strategy . We found that SecA is able to interact with both the signal sequence and the mature domain of prePhoE . Furthermore, this interaction was dependent on the type of nucleotide bound . SecA in the ADP-bound conformation was unable to crosslink with the precursor, whereas the ATP-bound conformation was active in precursor crosslinking . The SecA-precursor interaction was maintained in the presence of E . coli phospholipids but was loosened by the presence of phosphatidylglycerol bilayers . Examining SecA ATP binding site mutants demonstrated that ATP hydrolysis at the N-terminal high affinity binding site is responsible for the changed interaction with the preprotein. J Lipid Res, 2000 Dec, 41(12), 1947 - 51 A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification; Saulnier-Blache JS et al.; The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA) . For that, a recombinant rat LPA acid acyltransferase (LPAAT) produced in Escherichia coli was used . In the presence of {(14)C}oleoyl-CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into {(14)C}phosphatidic acid . Acylation of LPA was complete and linear from 0 to 200 pmol with a minimal detection of 0.2 pmol . This method was used to quantify LPA in butanol-extracted lipids from bovine sera, as well as from human and mouse plasma.This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids. Int Microbiol, 1999 Mar, 2(1), 33 - 8 Inactive and temperature-sensitive folding mutants of aldehyde dehydrogenase of Escherichia coli; Limon A et al.; Aldehyde dehydrogenase, encoded by the aldA gene in Escherichia coli, is inactive in nitrosoguanidine induced mutant strain ECL40 and temperature-sensitive in spontaneous mutant strain JA104 . Both mutants were proven, by complementation experiments, to have a functional aldA regulator and promoter . In spite of no immunodetection of the aldA product, its specific transcript was present in the mutant extracts . It was subsequently proven that the immunodetection of aldehyde dehydrogenase in these mutants required denaturation, revealing that cells lacking the enzyme activity had the inactive protein in their extracts . Thus, the mutations seemed to affect the protein conformation . The temperature-sensitive aldehyde dehydrogenase did not show, neither in vivo nor in vitro, a different thermal stability compared to the wild type enzyme . In this temperature-sensitive strain, the recovery of active aldehyde dehydrogenase, in the presence of rifampicin but not of chloramphenicol, when cells grown at 37 degrees C were shifted to 30 degrees C indicated that this mutation affected the folding process of the protein at the restrictive temperature . Sequencing of the two mutant aldA corresponding genes determined a single amino acid change of Pro to Leu at position 182 for strain ECL40, and of Val to Met at position 145 for strain JA104 . These mutations were thought to possibly promote changes in the local flexibility in the first case, and to perturb the packing of residues by steric hindrance in the second case. J Biol Chem, 2001 Mar 16, 276(11), 8213 - 8 Epub 2000 Dec 06. 7-Methylxanthine methyltransferase of coffee plants . Gene isolation and enzymatic properties; Ogawa M et al.; Caffeine is synthesized through sequential three-step methylation of xanthine derivatives at positions 7-N, 3-N, and 1-N . However, controversy exists as to the number and properties of the methyltransferases involved . Using primers designed on the basis of conserved amino acid regions of tea caffeine synthase and Arabidopsis hypothetical proteins, a particular DNA fragment was amplified from an mRNA population of coffee plants . Subsequently, this fragment was used as a probe, and four independent clones were isolated from a cDNA library derived from coffee young leaves . Upon expression in Escherichia coli, one of them was found to encode a protein possessing 7-methylxanthine methyltransferase activity and was designated as CaMXMT . It consists of 378 amino acids with a relative molecular mass of 42.7 kDa and shows similarity to tea caffeine synthase (35.8%) and salicylic acid methyltransferase (34.1%) . The bacterially expressed protein exhibited an optimal pH for activity ranging between 7 and 9 and methylated almost exclusively 7-methylxanthine with low activity toward paraxanthine, indicating a strict substrate specificity regarding the 3-N position of the purine ring . K(m) values were estimated to be 50 and 12 microM for 7-methylxanthine and S-adenosyl-l-methionine, respectively . Transcripts of CaMXMT could be shown to accumulate in young leaves and stems containing buds, and green fluorescent protein fusion protein assays indicated localization in cytoplasmic fractions . The results suggest that, in coffee plants, caffeine is synthesized through three independent methylation steps from xanthosine, in which CaMXMT catalyzes the second step to produce theobromine. J Biol Chem, 2001 Mar 16, 276(11), 7876 - 83 Epub 2000 Dec 06. Dimer formation of octaprenyl-diphosphate synthase (IspB) is essential for chain length determination of ubiquinone; Kainou T et al.; Ubiquinone (Q), composed of a quinone core and an isoprenoid side chain, is a key component of the respiratory chain and is an important antioxidant . In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-diphosphate synthase, which is encoded by an essential gene, ispB . To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants and expressed them in E . coli and Saccharomyces cerevisiae . The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length . E . coli cells (ispB::cat) harboring an Arg-321 mutant were temperature-sensitive for growth, which indicates that Arg-321 is important for thermostability of IspB . Intriguingly, E . coli cells harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished . When a heterodimer of His-tagged wild-type IspB and glutathione S-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid . These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length. J Vasc Surg, 2000 Dec, 32(6), 1215 - 8 Repair of a saccular aortic aneurysm with superficial femoral-popliteal vein in the presence of a pancreatic abscess; Rosen SF et al.; When one is faced with impending rupture, repair of an aortic aneurysm cannot be delayed . In the presence of coexisting intra-abdominal sepsis, traditional therapy would call for aneurysm exclusion and axillofemoral bypass grafting . Consequences of this choice of treatment include limited long-term graft patency and recurrent prosthetic infection . Autogenous deep veins from the lower extremities have demonstrated exceptional patency and resilience to infection when used to replace infected aortic grafts . We now report a case of concomitant open drainage of a pancreatic abscess and repair of a saccular abdominal aortic aneurysm using the superficial femoral-popliteal vein as a conduit. Mutat Res, 2000 Dec 20, 457(1-2), 1 - 13 Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1; Koch WH et al.; Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins . The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation . Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids . We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay . To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an approximately 7kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB((R394)) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively . Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein . While R394 was unable to restore mutagenesis functions to a DeltaumuDC E . coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog. Biophys J, 2000 Dec, 79(6), 3217 - 25 Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation; Moraczewska J et al.; Mutations in the human TPM3 gene encoding gamma-tropomyosin (alpha-tropomyosin-slow) expressed in slow skeletal muscle fibers cause nemaline myopathy . Nemaline myopathy is a rare, clinically heterogeneous congenital skeletal muscle disease with associated muscle weakness, characterized by the presence of nemaline rods in muscle fibers . In one missense mutation the codon corresponding to Met-8, a highly conserved residue, is changed to Arg . Here, a rat fast alpha-tropomyosin cDNA with the Met8Arg mutation was expressed in Escherichia coli to investigate the effect of the mutation on in vitro function . The Met8Arg mutation reduces tropomyosin affinity for regulated actin 30- to 100-fold . Ca(2+)-sensitive regulatory function is retained, although activation of the actomyosin S1 ATPase in the presence of Ca(2+) is reduced . The poor activation may reflect weakened actin affinity or reduced effectiveness in switching the thin filament to the open, force-producing state . The presence of the Met8Arg mutation severely, but locally, destabilizes the tropomyosin coiled coil in a model peptide, and would be expected to impair end-to-end association between TMs on the thin filament . In muscle, the mutation may alter thin filament assembly consequent to lower actin affinity and altered binding of the N-terminus to tropomodulin at the pointed end of the filament . The mutation may also reduce force generation during activation. Am J Pathol, 2000 Dec, 157(6), 2045 - 53 Immunohistochemical analysis of endothelial-monocyte-activating polypeptide-II expression in vivo; Murray JC et al.; Endothelial-monocyte activating polypeptide (EMAP)-II is a novel molecule with cytokine-like pro-inflammatory properties, inducing procoagulant activity on the surface of endothelial cells and monocyte/macrophages in vitro, as well as up-regulating E- and P-selectin expression . EMAP-II is chemotactic for monocytes/macrophages and neutrophils, and stimulates myeloperoxidase release from neutrophils . Injection of EMAP-II into the mouse footpad induces an acute inflammatory response, although some regression occurs in response to direct injection of EMAP-II into murine tumors . Very little is known about the expression of EMAP-II in normal tissues of mice or humans, or about its function in vivo . We developed polyclonal antibodies against EMAP-II using recombinant protein produced in Escherichia coli, and used these antibodies to carry out an immunohistochemical study of the occurrence and distribution of EMAP-II in human tissues . The distribution of EMAP-II protein is relatively restricted, occurring primarily in endocrine organs, in cells of neuroendocrine origin, but also in tissues with high turnover . EMAP-II is strongly expressed in secretory epithelial cells of the thyroid, pancreas, adrenal and salivary glands, among others, as well as in neurons and subsets of monocytes/macrophages . It is also found in the epithelium of the small and large intestines . We conclude that EMAP-II expression is usually, but not always, associated with tissues that display high turnover and high levels of protein synthesis. J Infect Dis, 2001 Jan 1, 183(1), 154 - 9 Epub 2000 Nov 28. Molecular comparison of extraintestinal Escherichia coli isolates of the same electrophoretic lineages from humans and domestic animals; Johnson JR et al.; Molecular typing methods were used to characterize 38 Escherichia coli strains that originally were isolated from extraintestinal infections and represented 5 multilocus enzyme electrophoretic types (ETs) recovered from both humans and animals . Within each ET, the human and animal isolates did not consistently segregate by host group, according to individual virulence factors (VFs), composite VF-serotype profiles, or pulsed-field gel electrophoresis profiles . Several close matches with respect to VF-serotype profiles were identified between human and canine isolates from different locales . One canine and 2 human isolates of serogroup O6 closely resembled archetypal human pyelonephritis isolate 536 (O6:K15:H31), according to papA sequence and VF-serotype profile . These findings support the hypothesis that certain pathogenic lineages of E . coli cause disease in both humans and animals and that humans may acquire pathogenic E . coli from domestic pets. J Infect Dis, 2001 Jan 1, 183(1), 78 - 88 Epub 2000 Nov 22. Phylogenetic distribution of extraintestinal virulence-associated traits in Escherichia coli; Johnson JR et al.; The 72 member strains of the Escherichia coli Reference collection were assessed as to genotype for 31 putative extraintestinal virulence factor (VF) genes and DNA sequence for papA, the P fimbrial structural subunit gene . Although most VFs were concentrated in phylogenetic group B2 or jointly in groups B2 and D, others were concentrated primarily in group D, were broadly distributed (without group-specific associations), and/or occurred only outside of group B2 . Statistical correlations among VFs suggested linkage on pathogenicity-associated islands or plasmids . Isolates from humans and nonhuman primates had more VFs than did isolates from other animals . Sequence diversity was minimal within each F type-specific papA allele group but was substantial among different papA allele groups . The distribution patterns of papA variants and other VFs suggested multiple horizontal transfer events . These findings provide new insights into the phylogenetic origins of extraintestinal VFs in E . coli. Biochemistry, 2000 Dec 12, 39(49), 15166 - 78 Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein; Miller JR et al.; The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis {Vanden Boom, T . J., Reed, K . E., and Cronan, J . E., Jr . (1991) J . Bacteriol . 173, 6411-6420}, although in vitro lipoate biosynthesis with LipA has never been observed . In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E . coli as soluble proteins . The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide . Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of {3Fe-4S} and {4Fe-4S} cluster states . Reduction with sodium dithionite results in small quantities of an S = 1/2 {4Fe-4S}(1+) cluster with the majority of the protein containing a species consistent with an S = 0 {4Fe-4S}(2+) cluster . LipA was assayed for lipoate or lipoyl-ACP formation using E . coli lipoate-protein ligase A (LplA) or lipoyl-{acyl-carrier-protein}-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) {Jordan, S . W., and Cronan, J . E . (1997) Methods Enzymol . 279, 176-183} . When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed . As shown by this assay, octanoic acid is not a substrate for LipA . Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction . These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry. Biochemistry, 2000 Dec 12, 39(49), 15156 - 65 Engineered eglin c variants inhibit yeast and human proprotein processing proteases, Kex2 and furin; Komiyama T et al.; We engineered eglin c, a potent subtilisin inhibitor, to create inhibitors for enzymes of the Kex2/furin family of proprotein processing proteases . A structural gene was synthesized that encoded "R(1)-eglin", having Arg at P(1) in the reactive site loop in place of Leu(45) . Ten additional variants were created by cassette mutagenesis of R(1)-eglin . These polypeptides were expressed in Escherichia coli, purified to homogeneity, and their interactions with secreted, soluble Kex2 and furin were examined . R(1)-eglin itself was a modest inhibitor of Kex2, with a K(a) of approximately 10(7) M(-)(1) . Substituting Arg (in R(4)R(1)-eglin) or Met (in M(4)R(1)-eglin) for Pro(42) at P(4) created potent Kex2 inhibitors exhibiting K(a) values of approximately 10(9) M(-)(1) . R(4)R(1)-eglin inhibited furin with a K(a) of 4.0 x 10(8) M(-)(1) . Introduction of Lys at P(1), in place of Arg in R(4)R(1)-eglin reduced affinity only approximately 3-fold for Kex2 but 15-fold for furin . The stabilities of enzyme-inhibitor complexes were characterized by association and dissociation rate constants and visualized by polyacrylamide gel electrophoresis . R(4)R(1)-eglin formed stable 1:1 complexes with both Kex2 and furin . However, substitution of Lys at P(2) in place of Thr(44) resulted in eglin variants that inhibited both Kex2 and furin but which were eventually cleaved (temporary inhibition) . Surprisingly, R(6)R(4)R(1)-eglin, in which Arg was substituted for Gly(40) in R(4)R(1)-eglin, exhibited stable, high-affinity complex formation with Kex2 (K(a) of 3.5 x 10(9) M(-)(1)) but temporary inhibition of furin . This suggests that enzyme-specific interactions can alter the conformation of the reactive site loop, converting a permanent inhibitor into a substrate . Eglin variants offer possible avenues for affinity purification, crystallization, and regulation of proprotein processing proteases. Biochemistry, 2000 Dec 12, 39(49), 15150 - 5 Regulation of the successive reaction catalyzed by rat neuronal nitric oxide synthase; Iwanaga T et al.; The rat neuronal nitric oxide synthase (nNOS) catalyzes two monooxygenase reactions successively from L-arginine (L-Arg) to L-citrulline (L-Cit) via N(omega)-hydroxy-L-arginine (OH-Arg) without most of OH-Arg leaving the substrate-binding site . In the steady-state reaction conditions, the amount of OH-Arg produced is about 1/30-1/50 that of L-Cit . We found in this study using nNOS purified from an Escherichia coli expression system that the ratio of the amount of OH-Arg to L-Cit (OH-Arg/L-Cit) increased to about 1 at low concentration of NADPH . In one cycle of the nNOS reaction, the decrease in NADPH concentration was found to reduce the rates of two monooxygenase reactions but had little effect on the rate constant of OH-Arg dissociation from the enzyme . The addition of NADP(+), the competitive inhibitor for NADPH, caused the decrease in the rates of monooxygenase reactions in a single cycle of the reaction and the increase in the ratio of OH-Arg/L-Cit in the steady state . At low CaM concentrations, the ratio of OH-Arg/L-Cit was about the same as that at high CaM . In a single cycle of the nNOS reaction, the rate of monooxygenation was not altered by the CaM concentration but the amount of metabolized L-Arg decreased with the decrease in CaM concentration, showing that the amount of active nNOS was regulated by complex formation between nNOS and CaM . It becomes clear that there are two regulatory mechanisms for the successive reaction of nNOS . One controls the rates of monooxygenations and the other controls the amount of active species of nNOS. Biochemistry, 2000 Dec 12, 39(49), 15136 - 43 Evidence for two distinct effector-binding sites in threonine deaminase by site-directed mutagenesis, kinetic, and binding experiments; Wessel PM et al.; A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase {Schuller, D . J., Grant, G . A., and Banaszak, L . J . (1995) Nat . Struct . Biol . 2, 69-76} and the regulatory domain of E . coli threonine deaminase {Gallagher, D . T., Gilliland, G . L., Xiao, G., Zondlo, J., Fisher, K . E., Chinchilla, D . , and Eisenstein, E . (1998) Structure 6, 465-475} led us to make the hypothesis that threonine deaminase could have two binding sites per monomer . To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase . Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A . thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543 . Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543 . Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme . Y449 interacts with both regulators, isoleucine and valine . However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition. Biochemistry, 2000 Dec 12, 39(49), 15129 - 35 Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P; Cottrell GS et al.; The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4 . 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases . To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases . Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells . Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed . Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive . Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E . coli AP-P, are likely to play a role in shuttling protons during catalysis . These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site . The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase. Biochemistry, 2000 Dec 12, 39(49), 15121 - 8 Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme; Cottrell GS et al.; The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E . C . 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG . AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products . A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE) . The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da . The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa . The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro . Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin . Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E . coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc . The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione . The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory . Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline . The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II) . Neither Co(II) nor Zn(II) reactivated the metal-free enzyme . On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo. J Biol Chem, 2001 Mar 2, 276(9), 6516 - 23 Epub 2000 Dec 05. Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi . Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma; Nozaki T et al.; Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis . We cloned and characterized cDNA encoding cystathionine beta-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi . T . cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine . T . cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes . The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has serine sulfhydrylase and cysteine synthase (CS) activities in vitro . Expression of the T . cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS and CS activities are functional in vivo . Enzymatic studies on T . cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression . We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway . Dissimilar to bacterial and plant SAT, a recombinant T . cruzi SAT showed allosteric inhibition by l-cysteine, l-cystine, and, to a lesser extent, glutathione . Together, these studies demonstrate the T . cruzi is a unique protist in possessing both transsulfuration and sulfur assimilatory pathways. J Biol Chem, 2001 Mar 2, 276(9), 6739 - 46 Epub 2000 Dec 05. Physiochemical properties of rat liver mitochondrial ribosomes; Patel VB et al.; In the present study, the physiochemical properties of rat liver mitochondrial ribosomes were examined and compared with Escherichia coli ribosomes . The sedimentation and translational diffusion coefficients as well as the molecular weight and buoyant density of rat mitochondrial ribosomes were determined . Sedimentation coefficients were established using the time-derivative algorithm (Philo, J . S . (2000) Anal . Biochem . 279, 151-163) . The sedimentation coefficients of the intact monosome, large subunit, and small subunit were 55, 39, and 28 S, respectively . Mitochondrial ribosomes had a particle composition of 75% protein and 25% RNA . The partial specific volume was 0.688 ml/g, as determined from the protein and RNA composition . The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.41 g/cm(3) . The molecular masses of mitochondrial and E . coli ribosomes determined by static light-scattering experiments were 3.57 +/- 0.14 MDa and 2.49 +/- 0.06 MDa, respectively . The diffusion coefficient obtained from dynamic light-scattering measurements was 1.10 +/- 0.01 x 10(-7) cm(2) s(-1) for mitochondrial ribosomes and 1.72 +/- 0.03 x 10(-7) cm(2) s(-1) for the 70 S E . coli monosome . The hydration factor determined from these hydrodynamic parameters were 4.6 g of water/g of ribosome and 1.3 g/g for mitochondrial and E . coli ribosomes, respectively . A calculated hydration factor of 3.3 g/g for mitochondrial ribosomes was also obtained utilizing a calculated molecular mass and the Svedberg equation . These measurements of solvation suggest that ribosomes are highly hydrated structures . They are also in agreement with current models depicting ribosomes as porous structures containing numerous gaps and tunnels. J Biol Chem, 2001 Mar 16, 276(11), 7906 - 12 Epub 2000 Dec 05. DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction; Genevaux P et al.; DjlA is a 30-kDa type III membrane protein of Escherichia coli with the majority, including an extreme C-terminal putative J-domain, oriented toward the cytoplasm . No other regions of sequence similarity aside from the J-domain exist between DjlA and the known DnaK (Hsp70) co-chaperones DnaJ (Hsp40) and CbpA . In this study, we explored whether and to what extent DjlA possesses DnaK co-chaperone activity and under what conditions a DjlA-DnaK interaction could be important to the cell . We found that the DjlA J-domain can substitute fully for the J-domain of DnaJ using various in vivo functional complementation assays . In addition, the purified cytoplasmic fragment of DjlA was shown to be capable of stimulating DnaK ATPase in a manner indistinguishable from DnaJ, and, furthermore, DjlA could act as a DnaK co-chaperone in the reactivation of chemically denatured luciferase in vitro . DjlA expression in the cell is tightly controlled, and even its mild overexpression leads to induction of mucoid capsule . Previous analysis showed that DjlA-mediated induction of the wca capsule operon required the RcsC/RcsB two-component signaling system and that wca induction by DjlA was lost when cells contained mutations in either the dnaK or grpE gene . We now show using allele-specific genetic suppression analysis that DjlA must interact with DnaK for DjlA-mediated stimulation of capsule synthesis . Collectively, these results demonstrate that DjlA is a co-chaperone for DnaK and that this chaperone-co-chaperone pair is implicated directly, or indirectly, in the regulation of colanic acid capsule. Eur J Biochem, 2000 Dec, 267(24), 7109 - 17 Cloning and expression of a distinct subclass of plant thioredoxins; Juttner J et al.; mRNAs encoding a novel thioredoxin were isolated from pollen RNA of Lolium perenne (LpTrx), Hordeum bulbosum (HbTrx), Phalaris coerulescens (PTrx) and Secale cereale (ScTrx) . The cDNAs contain a single ORF of 393 bp encoding a protein of 131 amino acids . The predicted proteins showed highest homology to plant thioredoxins of the h class yet form a distinct subgroup that is characterized by a high level of sequence conservation (95.4-97.7% identity) . GenBank searches revealed additional members of this subclass in tomato, soybean, rice and pine . LpTrx and PTrx were expressed as recombinant proteins in Escherichia coli and tested for thioredoxin activity . Both proteins displayed typical thioredoxin activity in the nonspecific insulin reduction assay, however, were not reduced by E . coli NADPH-dependant thioredoxin reductase. Eur J Biochem, 2000 Dec, 267(24), 7075 - 81 Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli; Patra AK et al.; An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli . Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded . Use of a low concentration of urea (2 M) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding . Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot . Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation . CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both alpha-helical and beta-sheet conformations in the refolded r-bmZPC . Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC . The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization. Eur J Biochem, 2000 Dec, 267(24), 7006 - 14 Hev b 9, an enolase and a new cross-reactive allergen from hevea latex and molds . Purification, characterization, cloning and expression; Wagner S et al.; Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products . A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho-D-glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR . The PCR primers were designed according to conserved regions of enolases from plants . The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa . Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens . In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved . Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography . The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture . The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments . The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography . The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity . Patients' IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules . Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9 . Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials. Protein Sci, 2000 Oct, 9(10), 2026 - 33 Dimer formation by a "monomeric" protein; Park C et al.; Dimeric proteins can arise by the swapping of structural domains between monomers . The prevalence of this occurrence is unknown . Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions . Here, this hypothesis is tested and found to be imprecise . The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein . The H12A and H119A variants have 10(5)-fold less ribonucleolytic activity than does the wild-type enzyme . Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 degrees C results in a 10(3)-fold increase in ribonucleolytic activity . A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid . At pH 6.5 and 65 degrees C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer-dimer equilibrium . The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 degrees C . This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo . The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins . Dimers of "monomeric" proteins could be more prevalent than is usually appreciated. Protein Sci, 2000 Oct, 9(10), 2018 - 25 Purification and refolding of vascular endothelial growth factor-B; Scrofani SD et al.; Vascular endothelial growth factor (VEGF)-A interacts with the receptor tyrosine kinases VEGF-R1 and R2, and the importance of this interaction in endothelial cell (EC) function and blood vessel development has been well documented . Other ligands that interact differentially with these receptors and that are structurally related to VEGF-A include VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PLGF) . Compared with VEGF-A, relatively little is known about the biological role of the VEGF-R1 specific ligand, VEGF-B . Two splice variant isoforms that differ at the COOH-terminus and which retain unique solubility characteristics are widely expressed throughout embryonic and postnatal development . Recent analysis of mice with a targeted deletion of the VEGF-B gene has revealed a defect in heart development and function consistent with an important role in vascularization of the myocardium (Bellomo D et al., 2000, Circ Res 86:E29-E35) . To facilitate further characterization of VEGF-B, we have developed a protocol for expression and purification of refolded recombinant protein from Escherichia coli inclusion bodies (IBs) . The approach developed resolves a number of significant issues associated with VEGF-B, including the ability to heterodimerize with endogenous VEGF-A when co-expressed in mammalian cells, a complex secondary structure incorporating inter- and intrachain disulfide bonds and hydrophobic characteristics that preclude the use of standard chromatographic resins . The resulting purified disulfide-linked homodimer was demonstrated to bind to VEGF-R1 and to compete with VEGF-A for binding to this receptor. Protein Sci, 2000 Oct, 9(10), 2009 - 17 Engineering the substrate specificity of Escherichia coli asparaginase . II . Selective reduction of glutaminase activity by amino acid replacements at position 248; Derst C et al.; The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme . To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties . We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis . In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate . In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine . Smaller differences were seen with other N248 variants . Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules. Protein Sci, 2000 Oct, 9(10), 1993 - 2000 Role of a solvent-exposed aromatic cluster in the folding of Escherichia coli CspA; Rodriguez HM et al.; Escherichia coli CspA is a member of the cold shock protein family . All cold shock proteins studied to date fold rapidly by an apparent two-state mechanism . CspA contains an unusual cluster of aromatic amino acids on its surface that is necessary for nucleic acid binding and also provides stability to CspA (Hillier et al., 1998) . To elucidate the role this aromatic cluster plays in the determining the folding rate and pathway of CspA, we have studied the folding kinetics of mutants containing either leucine or serine substituted for Phe 18, Phe20, and/or Phe31 . The leucine substitutions are found to accelerate folding and the serine substitutions to decelerate folding . Because these residues exert effects on the free energy of the folding transition state, they may be necessary for nucleating folding . They are not responsible, however, for the very compact, native-like transition state ensemble seen in the cold shock proteins, as the refolding rates of the mutants all show a similar, weak dependence of unfolding rate on denaturant concentration . Using mutant cycle analysis, we show that there is energetic coupling among the three residues between the unfolded and transition states, suggesting that the cooperative nature of these interactions helps to determine the unfolding rate . Overall, our results suggest that separate evolutionary pressures can act simultaneously on the same group of residues to maintain function, stability, and folding rate. Protein Sci, 2000 Oct, 9(10), 1922 - 9 Lipoylating and biotinylating enzymes contain a homologous catalytic module; Reche PA; Biotin and lipoic acid moieties are the covalently attached coenzyme cofactors of several multicomponent enzyme complexes that catalyze key metabolic reactions . Attachment of these moieties to the biotinyl- and lipoyl-dependent enzymes is post-translationally catalyzed by specific biotinylating and lipoylating protein enzymes . In Escherichia coli, two different enzymes, LplA and LipB, catalyze independent pathways for the lipoylation of the relevant enzymes, whereas only one enzyme, the BirA protein, is responsible for all the biotinylation . Counterparts of the E . coli BirA, LplA, and LipB enzymes have been previously identified in many organisms, but homology among the three families has never been reported . Computational analysis based on PSI-BLAST profiles and secondary structure predictions indicates, however, that lipoylating and biotinylating enzymes are evolutionarily related protein families containing a homologous catalytic module . Sequence conservation among the three families is very poor, but a single lysine residue is strictly conserved in all of them, which, according to the available X-ray crystal structure of the E . coli BirA protein, is expected to contribute to the binding of lipoic acid in the LplA and LipB enzymes. Protein Sci, 2000 Oct, 9(10), 1914 - 21 Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI; Goedken ER et al.; Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands . Using circular dichroism spectroscopy . we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI . This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies . Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate . As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics . Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions . Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate . Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10) . A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal. Protein Sci, 2000 Oct, 9(10), 1866 - 72 Exchanging the active site between phytases for altering the functional properties of the enzyme; Lehmann M et al.; By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase-1, that was 15-26 degrees C more thermostable than the parent fungal phytases used in its design (Lehmann et al., 2000) . We now sought to use the backbone of consensus phytase-1 and to modify its catalytic properties . This was done by replacing a considerable part of the active site (i.e., all the divergent residues) with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH-activity profile . For the new protein termed consensus phytase-7, a major - although not complete - shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach . Although the exchange of the active site was associated with a 7.6 degrees C decrease in unfolding temperature (Tm) as measured by differential scanning calorimetry, consensus phytase-7 still was >7 degrees C more thermostable than all wild-type ascomycete phytases known to date . Thus, combination of the consensus approach with the selection of a "preferred" active site allows the design of a thermostabilized variant of an enzyme family of interest that (most closely) matches the most favorable catalytic properties found among its family members. RNA, 2000 Nov, 6(11), 1610 - 24 RBP45 and RBP47, two oligouridylate-specific hnRNP-like proteins interacting with poly(A)+ RNA in nuclei of plant cells; Lorkovic ZJ et al.; Introns in plant nuclear pre-mRNAs are highly enriched in U or U + A residues and this property is essential for efficient splicing . Moreover, 3'-untranslated regions (3'-UTRs) in plant pre-mRNAs are generally UA-rich and contain sequences that are important for the polyadenylation reaction . Here, we characterize two structurally related RNA-binding proteins (RBPs) from Nicotiana plumbaginifolia, referred to as RBP45 and RBP47, having specificity for oligouridylates . Both proteins contain three RBD-type RNA-binding domains and a glutamine-rich N-terminus, and share similarity with Nam8p, a protein associated with U1 snRNP in the yeast Saccharomyces cerevisiae . Deletion analysis of RBP45 and RBP47 indicated that the presence of at least two RBD are required for interaction with RNA and that domains other than RBD do not significantly contribute to binding . mRNAs for RBP45 and RBP47 and mRNAs encoding six related proteins in Arabidopsis thaliana are constitutively expressed in different plant organs . Indirect immunofluorescence and fractionation of cell extracts showed that RBP45 and RBP47 are localized in the nucleus . In vivo UV crosslinking experiments demonstrated their association with the nuclear poly(A)+ RNA . In contrast to UBP1, another oligouridylate-binding nuclear three-RBD protein of N . plumbaginifolia (Lambermon et al., EMBO J, 2000, 19:1638-1649), RBP45 and RBP47 do not stimulate mRNA splicing and accumulation when transiently overexpressed in protoplasts . Properties of RBP45 and RBP47 suggest they represent hnRNP-proteins participating in still undefined steps of pre-mRNA maturation in plant cell nuclei. RNA, 2000 Nov, 6(11), 1585 - 96 Alternative designs for construction of the class II transfer RNA tertiary core; Nissan TA et al.; The structural requirements for assembly of functional class II transfer RNA core regions have been examined by sequence analysis and tested by reconstruction of alternative folds into the tertiary domain of Escherichia coli tRNA(2)Gln . At least four distinct designs have been identified that permit stable folding and efficient synthetase recognition, as assessed by thermal melting profiles and glutaminylation kinetics . Although most large variable-arm tRNAs found in nature possess an enlarged D-loop, lack of this feature can be compensated for by insertion of nucleotides either 3' to the variable loop or within the short acceptor/D-stem connector region . Rare pyrimidines at nt 9 in the core region can be accommodated in the class II framework, but only if specific nucleotides are present either in the D-loop or 3' to the variable arm . Glutaminyl-tRNA synthetase requires one or two unpaired uridines 3' to the variable arm to efficiently aminoacylate several of the class II frameworks . Because there are no specific enzyme contacts in the tRNAGln core region, these data suggest that tRNA discrimination by GlnRS depends in part on indirect readout of RNA sequence information. RNA, 2000 Nov, 6(11), 1483 - 91 Coupled nucleotide covariations reveal dynamic RNA interaction patterns; Gultyaev AP et al.; Evolutionarily conserved structures in related RNA molecules contain coordinated variations (covariations) of paired nucleotides . Analysis of covariations is a very powerful approach to deduce phylogenetically conserved (i.e., functional) conformations, including tertiary interactions . Here we discuss conserved RNA folding pathways that are revealed by covariation patterns . In such pathways, structural requirements for alternative pairings cause some nucleotides to covary with two different partners . Such "coupled" covariations between three or more nucleotides were found in various types of RNAs . The analysis of coupled covariations can unravel important features of RNA folding dynamics and improve phylogeny reconstruction in some cases . Importantly, it is necessary to distinguish between multiple covariations determined by mutually exclusive structures and those determined by tertiary contacts. Biochem J, 2000 Dec 15, 352 Pt 3, 841 - 9 Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulness of in vitro selective infective phage technology; Cebe R et al.; The selective infective phage (SIP) technology allows a rapid positive selection of interacting pairs of biological molecules that restore to non-infectious phages their ability to infect the bacterial host . After a successful infection, the phage is amplified and the DNA encoding the interacting ligand is isolated from the phage genome and sequenced . In our studies we have evaluated the usefulness of SIP for the identification and cloning of proteins interacting with a biotinylated target binding to a newly designed adapter molecule consisting of streptavidin fused to the C-terminus of the extracellular domain of the phage minor coat protein III . The new adapter was expressed in Escherichia coli and refolded from inclusion bodies . The two different domains joined within the chimaera were found to be biologically functional . We also demonstrated that non-covalent interactions between a non-infectious phage displaying a short peptide, which specifically binds the streptavidin, and the adapter molecule restore phage infectivity . To evaluate the potential of SIP as a general and generic tool for the screening of cDNA libraries that encode the ligands displayed at the surface of the phage and binding to biotinylated targets, we have increased both the size of the displayed ligand on the phage and the size of the biotinylated target bound to the streptavidin domain of the adapter molecule . In our model systems we show that the size of either the ligand or the target is a limiting factor for the technology. Biochem J, 2000 Dec 15, 352 Pt 3, 755 - 61 Biochemical characterization of a trypanosome enzyme with glutathione-dependent peroxidase activity; Wilkinson SR et al.; In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides . Numerous studies have reported that trypanosomatids lack this activity . Here we show that this is not the case, at least for the American trypanosome Trypanosoma cruzi . We have isolated a single-copy gene from T . cruzi with the potential to encode an 18 kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases fro