Escherichia coli

Int J Parasitol, 2001 May 15, 31(7), 687 - 96
Differential gene expression in haemocytes of the snail Biomphalaria glabrata: effects of Schistosoma mansoni infection; Miller AN et al.; Parasite encapsulation and destruction in Biomphalaria glabrata has been shown to involve the cellular component of the snail's internal defence system, the haemocytes . To identify genes involved in the immunobiology of these cells, we used the method of differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) to investigate differential gene regulation in haemocytes isolated from Schistosoma mansoni exposed and unexposed snails . RNA isolated from circulating haemocytes from resistant snails (BS-90 stock), previously exposed to S . mansoni, was analysed using 12 different arbitrary primers in conjunction with an anchored Oligo d(T(11)CG) primer . Transcription profiles between haemocytes of parasite exposed and unexposed snails were compared and a total of 87 differentially regulated bands were identified and isolated . Of these, 65 bands were cloned and used as probes in Southern blots to show the presence of corresponding sequences in the snail genome . RT-PCR was performed to verify the regulation of these transcripts . DNA sequence analysis showed that the majority of the cloned sequences were novel, although a few showed a high degree of sequence similarity to other sequences in the DNA and protein databases . One of these included a differentially expressed transcript that showed a significant degree of sequence identity to E . coli transposase Tn5, an enzyme whose activity is normally associated with generating mobility and instability in the genome.

Biochem J, 2001 May 15, 356(Pt 1), 223 - 32
Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis; Bearne SL et al.; Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen . When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis . Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography . Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde . Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis . Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0 . The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate gamma-semialdehyde . Although glutamate gamma-semialdehyde exists in solution primarily in its cyclic form, Delta(1)-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Delta(1)-pyrroline-5-carboxylate (L-proline, L-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition . When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate gamma-semialdehyde is decreased approx . 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.

Virology, 2001 May 10, 283(2), 197 - 206
The long repeat region is dispensable for fowl adenovirus replication in vitro; Ojkic D et al.; Two regions containing tandemly repeated sequences are present in the fowl adenovirus 9 (FAdV-9) genome . The longer repeat region (TR-2) is composed of 13 contiguous 135-bp-long direct repeats, the function of which is unknown . An infectious FAdV-9 genomic clone, constructed by homologous recombination in Escherichia coli, was used for engineering of recombinant viruses . The enhanced green fluorescence protein (EGFP) coding sequence was cloned in both rightward and leftward orientations so as to replace TR-2 . Replication-competent recombinant FAdVs were recovered, demonstrating that TR-2 was dispensable for FAdV-9 propagation in vitro . The expression of EGFP in infected cells was demonstrated by fluorescence microscopy, immunoprecipitation, and RT-PCR .

Can J Anaesth, 2001 Apr, 48(4 Suppl), S32 - 40
Oxygen therapeutics--current concepts; Hill SE; PURPOSE: In an effort to develop agents that enhance the oxygen-delivery capability of blood without the risks associated with allogeneic blood transfusions, several products are undergoing development and clinical trials . These oxygen transport agents can be divided into two main groups, perfluorocarbon (PFC) emulsions and modified hemoglobin solutions . SOURCE: Articles from the literature on the development and clinical trials of oxygen therapeutic agents were reviewed . PRINCIPAL FINDING: PFCs are synthetic fluorinated hydrocarbons that increase dissolved oxygen in the fluid phase of the blood without binding the oxygen molecule . They enhance oxygen delivery significantly and may be used to augment the technique of intraoperative autologous donation . Two PFC products have been tested in Phase III clinical trials . Hemoglobin-based oxygen carriers (HBOCs) are either cross-linked or microencapsulated hemoglobin molecules . Modification of the human hemoglobin molecule with intra- and inter-molecular cross-linking eliminates renal toxicity and improves the oxygen dissociation characteristics of the molecule . These modifications are necessary because stroma-free hemoglobin (Hb) does not release oxygen in the physiologic range and dissociates into dimers which can be rapidly filtered by the kidney, leading to renal toxicity . In addition to human Hb, bovine hemoglobin is another source of raw material for HBOC products . Recombinant human Hb has also been produced, using an E . coli expression system, for HBOC manufacturing . Four cross-linked hemoglobin products have been tested in Phase III clinical trials . CONCLUSION: While no product has yet been approved for clinical use, preliminary studies with oxygen therapeutics show promising results, with effective oxygen carrying capacity and acceptable side effect profiles . In the future, the formation of a hybrid product which combines the best features from several of the products currently undergoing development may yield the ideal oxygen therapeutic agent.

Shock, 2001 May, 15(5), 386 - 91
Endotoxin impairs agonist-stimulated intracellular free calcium (Ca(i)) responses in freshly dispersed aortic endothelial cells; Jones JJ et al.; Impairment in endothelial cell intracellular free calcium (Ca(i)) mobilization mechanisms may contribute to decreased nitric oxide (NO) biosynthesis and impaired vasorelaxation responses of endotoxemic guinea pigs to endothelium-dependent vasodilators . We tested this hypothesis using fura-2 microfluorometry to compare agonist-stimulated Ca(i) responses of aortic endothelial cells freshly dispersed from guinea pigs 16 h after intraperitoneal injection of Escherichia coli endotoxin (lipopolysaccharide, LPS; 4 mg/kg) or saline (CON) . In the presence of normal extracellular Ca2+ (2 mmol/L), basal (non-stimulated) endothelial Ca(i) (340/380 nm fluorescence ratio, R) was not different between CON and LPS cells (1.1 +/- 0.03 and 1.1 +/- 0.03, respectively) . However, exposure to ADP (10 micromol/L) produced a biphasic increase in Ca(i) that was markedly decreased in cells from LPS-treated animals (P < 0.0001) . Peak ADP-stimulated Ca(i) responses averaged 2.2 +/- 0.21 in CON cells and 1.5 +/- 0.11 (P < 0.01) in cells dispersed from LPS-treated animals . Exposure to acetylcholine (ACh; 10 micromol/L) produced sustained increases in Ca(i) (R = 1.4 +/- 0.13) in CON cells; however, LPS abolished Ca(i) responses to ACh . Exposure of endothelial cells to substance P (100 nmol/L) produced a biphasic increase in Ca(i) that was not different between groups . In the absence of extracellular Ca2+ (plus 10 micromol/L EGTA), exposure to ADP (10 micromol/L) produced transient increases in Ca(i) (Ca2+ release) that were decreased in cells from LPS-treated versus CON animals . Exposure to ACh in zero Ca2+ (10 micromol/L) produced smaller increases in Ca(i) (peak R = 1.3 +/- 0.12) in CON cells (when compared to ADP); however, Ca(i) responses to ACh remained absent in cells from LPS-treated animals . Re-exposure to Ca2+ produced sustained ACh-induced Ca(i) responses (Ca2+ influx) in cells from CON, but not LPS-treated animals; LPS markedly impaired (P< 0.05) ADP-induced sustained Ca(i) responses . Our data demonstrate that in vivo LPS exposure elicits decreased agonist-stimulated endothelial Ca(i) responses primarily involving impaired Ca2+ influx mechanisms . Known dependence of endothelial agonist-stimulated NO synthesis on Ca(i) suggests that defects in cell Ca2+ mobilization may contribute to LPS-induced impaired NO biosynthesis and decreased endothelium-dependent relaxation.

Shock, 2001 May, 15(5), 378 - 85
Effects of nucleoside transport inhibition on hepatosplanchnic perfusion, oxygen extraction capabilities, and TNF release during acute endotoxic shock; Zhang H et al.; We explored the effects of the nucleoside transport inhibitor draflazine on regional blood flow, O2 extraction capabilities, and tumor necrosis factor (TNF) release in acute endotoxic shock . Fourteen anesthetized and mechanically ventilated dogs received 2 mg/kg of Escherichia coli endotoxin and were divided into two groups . Seven dogs received 0.1 mg/kg of draflazine 30 min before endotoxin, and 7 dogs served as a control group . Draflazine decreased arterial pressure without influencing cardiac index . Mesenteric and portal blood flow and ileum mucosal perfusion increased, but renal blood flow dramatically decreased . After endotoxemia, the draflazine-treated dogs had a lesser fall in cardiac index, filling pressures, and left ventricular stroke work index, and a lesser increase in pulmonary vascular resistance . After fluid resuscitation, they had a consistently lower renal blood flow and ileum mucosal perfusion, but a higher mixed venous and hepatic oxygen saturation and arterial pH than the control group . When cardiac index was reduced by tamponade to study the O2 extraction capabilities, renal blood flow and ileum mucosal perfusion remained lower in the draflazine group . Draflazine did not influence whole-body O2 extraction capabilities, but it delayed the occurrence of liver O2 supply dependency as indicated by a significantly lower liver DO2crit (27.7 +/- 3.9 vs . 43.3 +/- 10.8 mL/min) and a higher O2ERcrit (62.7 +/- 9.5 vs . 42.5 +/- 7.1%) than controls (both P< 0.05) . On the other hand, draflazine increased intestinal DO2crit (42.4 +/- 15.4 vs . 27.7 +/- 6.5 mL/min, P < 0.05) compared to the control group . TNF levels remained higher in the draflazine group than in the control group, particularly 3 and 4 h after endotoxin administration . We conclude that nucleoside transport inhibition with draflazine does not alter global and hepatosplanchnic hemodynamics but may decrease gut mucosal perfusion and renal blood flow . However, this intervention can improve liver O2 extraction capabilities in acute endotoxic shock.

J Biol Chem, 2001 Jul 20, 276(29), 27555 - 61 Epub 2001 May 02.
The crystallographic structure of the mannitol 2-dehydrogenase NADP+ binary complex from Agaricus bisporus; Horer S et al.; Mannitol, an acyclic six-carbon polyol, is one of the most abundant sugar alcohols occurring in nature . In the button mushroom, Agaricus bisporus, it is synthesized from fructose by the enzyme mannitol 2-dehydrogenase (MtDH; EC ) using NADPH as a cofactor . Mannitol serves as the main storage carbon (up to 50% of the fruit body dry weight) and plays a critical role in growth, fruit body development, osmoregulation, and salt tolerance . Furthermore, mannitol dehydrogenases are being evaluated for commercial mannitol production as alternatives to the less efficient chemical reduction of fructose . Given the importance of mannitol metabolism and mannitol dehydrogenases, MtDH was cloned into the pET28 expression system and overexpressed in Escherichia coli . Kinetic and physicochemical properties of the recombinant enzyme are indistinguishable from the natural enzyme . The crystal structure of its binary complex with NADP was solved at 1.5-A resolution and refined to an R value of 19.3% . It shows MtDH to be a tetramer and a member of the short chain dehydrogenase/reductase family of enzymes . The catalytic residues forming the so-called catalytic triad can be assigned to Ser(149), Tyr(169), and Lys(173).

FEBS Lett, 2001 Apr 27, 495(3), 167 - 71
Modulation of ribosomal recruitment to 5'-terminal start codons by translation initiation factors IF2 and IF3; Grill S et al.; Sequence determinants and structural features of the RNA govern mRNA-ribosome interaction in bacteria . However, ribosomal recruitment to leaderless mRNAs, which start directly with the AUG start codon and do not bear a Shine-Dalgarno sequence like canonical mRNAs, does not appear to rely on 16S rRNA-mRNA interactions . Here, we have studied the effects of translation initiation factors IF2 and IF3 on 30S initiation at a 5'-terminal AUG and at a competing downstream canonical ribosome binding site . We show that IF2 affects the forward kinetics of 30S initiation complex formation at the 5'-terminal AUG as well as the stability of these complexes . Moreover, the IF2:IF3 molar ratio was found to play a decisive role in translation initiation of a leaderless mRNA both in vitro and in vivo indicating that the translational efficiency of an mRNA is not only intrinsically determined but can be altered depending on the availability of components of the translational machinery.

Int Microbiol, 2000 Dec, 3(4), 239 - 45
The purified colicin S8 is a multimeric protein; Concepcion Curbelo JL et al.; Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc . Colicins are commonly inducible and extracellular . Their molecular masses range from 30 to 90 kDa . Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure . In our hands, purified colicin S8 was an aggregation of extremely related polypeptides . Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa . Behavior on molecular filtration indicated a molecular weight higher than 200 kDa . Similar results were obtained when purification was carried out through FPLC . Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide . We conclude that more than one form of colicin S8 exists . The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides . Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components.

Antisense Nucleic Acid Drug Dev, 2001 Apr, 11(2), 77 - 85
Nuclease resistance and RNase H sensitivity of oligonucleotides bridged by oligomethylenediol and oligoethylene glycol linkers; Vorobjev PE et al.; The properties of new chimeric oligodeoxynucleotides made of short sequences (tetramers, pentamers, octamers, and decamers) bridged by hexamethylenediol and hexaethylene glycol linkers have been investigated . These chimeric oligonucleotides showed an improved resistance toward snake venom 3'-phosphodiesterase, with an increased stability when a terminal 3'-3'-internucleotide phosphodiester bond is present . It also has been demonstrated that the hybrid complexes formed by bridged oligonucleotides and a complementary 20-mer RNA are able to elicit the activity of ribonuclease H (RNase H) from Escherichia coli . The substrate properties of chimeric oligonucleotides depend on the length of the oligonucleotide fragments bridged by linkers . Introduction of a nonnucleotide spacer into the native oligonucleotide only slightly hampers the extent of the RNA hydrolysis in the hybrid complexes, whereas a modification of the site of reaction is observed as a possible consequence of the steric disturbance due to the aliphatic linkers . Hence, these new chimeric oligonucleotides, namely, short oligonucleotide fragments bridged by nonnucleotide linkers, demonstrate a favorable combination of exonuclease resistance and high substrate activity toward RNase H . As a consequence, these chimeric oligonucleotides could be proposed as new, promising analogs to be used in the antisense strategy.

Nature, 2001 May 3, 411(6833), 110 - 4
An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes; Fabrega C et al.; Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs . There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each . These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs . Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret . Here we describe a different cysteinyl-tRNA synthetase from M . jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo . This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin . The existence of this protein contrasts with proposals that aminoacylation with cysteine in M . jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase.

J Virol, 2001 Jun, 75(11), 5197 - 204
Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner; Gewurz BE et al.; The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome . We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus . Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules . The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry . These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules . Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2 . The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E . We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM . The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.

J Virol, 2001 Jun, 75(11), 5141 - 50
Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection; Tumpey TM et al.; Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses . We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I . Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone . In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge . Mice that were i.n . administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge . The immune mediators of Het-I were investigated . The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively . beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge . Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice . Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5 . These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.

J Mass Spectrom, 2001 Apr, 36(4), 384 - 91
Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9; Havlicek V et al.; The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp) . The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain . This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP . The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC . However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT) . In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9 . This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323 . Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found . In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence . We also discuss these results in comparison with those from recombinant ACT expressed in E . coli strain K12 XL1-Blue . The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry .

J Neurobiol, 2001 Jun 5, 47(3), 183 - 93
Conditional ablation of neurones in transgenic mice; Isles AR et al.; Conditional targeted ablation of specific cell populations in living transgenic animals is a very powerful strategy to determine cell functions in vivo . This approach would be of particular value to study the functions of distinct neuronal populations; however, the transgene of choice for conditional cell ablation studies in mice, the herpes simplex virus thymidine kinase gene, cannot be used to ablate neurones as its principal mode of action relies on cell proliferation . Here we report that expression of the E.coli nitroreductase gene (Ntr) and metabolism of the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) to its cytotoxic derivative can be used to conditionally and acutely ablate specific neuronal populations in vivo . As proof of principal, we have ablated olfactory and vomeronasal receptor neurones by expressing Ntr under the control of the olfactory marker protein (OMP) gene promoter . We demonstrate that following CB1954 administration, olfactory and vomeronasal receptor neurones expressing the transgene were selectively eliminated from the olfactory epithelium (OE), and projections to the olfactory bulb (OB) were lost . The functional efficacy of cell ablation was demonstrated using a highly sensitive behavioural test to show that ablated mice had lost the olfactory ability to discriminate distinct odors and were consequently rendered anosmic . Targeted expression of Ntr to specific neuronal populations using conventional transgenes, as described here, or by "knock-in" gene targeting using embryonic stem cells may be of significant value to address the functions of distinct neuronal populations in vivo .

Plant Cell Physiol, 2001 Apr, 42(4), 349 - 57
Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds; Mizuno K et al.; cDNA clones encoding an isoform of starch branching enzyme, RBE4, have been identified from a developing rice seed cDNA library, using a synthetic oligonucleotide probe corresponding to the N-terminal amino acid sequence of RBE4 . The cDNA-derived amino acid sequence indicated that RBE4 is initially produced as a precursor protein of 841 amino acids, including a 53-residue transit peptide at the N-terminus . The mature form of RBE4 shared a high degree of sequence identity (80%) with mature RBE3, and possessed an N-terminal extra sequence, as found in RBE3 . Northern blot analysis demonstrated that the RBE4 gene is expressed in both leaves and developing seeds . The RBE4 gene was distinguished from the RBE1 and RBE3 genes by expression at the earlier stages of seed development . To examine enzymatic functions of RBE4, recombinant proteins were produced in Escherichia coli cells, and purified by two chromatographic separations . The branched alpha-glucans produced by the recombinant enzymes from potato amylose revealed the different patterns of oligosaccharide chain transfer . The peak of major branches of the products by RBE3 or RBE4 was 6 glucose units, whereas the peaks of major branches of the products by RBE1 were 6 and 11 glucose units . The similar property between RBE3 and RBE4 is supported by high similarity of the amino acid sequences between them.

J Biol Chem, 2001 Jul 6, 276(27), 24790 - 6 Epub 2001 May 01.
CYP83b1 is the oxime-metabolizing enzyme in the glucosinolate pathway in Arabidopsis; Hansen CH et al.; CYP83B1 from Arabidopsis thaliana has been identified as the oxime-metabolizing enzyme in the biosynthetic pathway of glucosinolates . Biosynthetically active microsomes isolated from Sinapis alba converted p-hydroxyphenylacetaldoxime and cysteine into S-alkylated p-hydroxyphenylacetothiohydroximate, S-(p-hydroxyphenylacetohydroximoyl)-l-cysteine, the next proposed intermediate in the glucosinolate pathway . The production was shown to be dependent on a cytochrome P450 monooxygenase . We searched the genome of A . thaliana for homologues of CYP71E1 (P450ox), the only known oxime-metabolizing enzyme in the biosynthetic pathway of the evolutionarily related cyanogenic glucosides . By a combined use of bioinformatics, published expression data, and knock-out phenotypes, we identified the cytochrome P450 CYP83B1 as the oxime-metabolizing enzyme in the glucosinolate pathway as evidenced by characterization of the recombinant protein expressed in Escherichia coli . The data are consistent with the hypothesis that the oxime-metabolizing enzyme in the cyanogenic pathway (P450ox) was mutated into a "P450mox" that converted oximes into toxic compounds that the plant detoxified into glucosinolates.

J Biol Chem, 2001 Jul 6, 276(27), 25399 - 403 Epub 2001 May 01.
HSP47 binds cooperatively to triple helical type I collagen but has little effect on the thermal stability or rate of refolding; Macdonald JR et al.; HSP47, a collagen-specific molecular chaperone, interacts with unfolded and folded procollagens . Binding of chicken HSP47 to native bovine type I collagen was studied by fluorescence quenching and cooperative binding with a collagen concentration at half saturation (K(half)) of 1.4 x 10(-7) m, and a Hill coefficient of 4.3 was observed . Similar results are observed for the binding of mouse HSP47 recombinantly expressed in Escherichia coli . Chicken HSP47 binds equally well to native type II and type III procollagen without the carboxyl-terminal propeptide (pN type III collagen), but binding to triple helical collagen-like peptides is much weaker . Weak binding occurred to both hydroxylated and nonhydroxylated collagen-like peptides, and a significant chain length dependence was observed . Binding of HSP47 to native type I collagen had no effect on the thermal stability of the triple helix . Refolding of type I collagen in the presence of HSP47 showed minor changes, but these are probably not biologically significant . Binding of HSP47 to bovine pN type III collagen has only minor effects on the thermal stability of the triple helix and does not influence the refolding kinetics of the triple helix.

J Biol Chem, 2001 Jul 20, 276(29), 27345 - 53 Epub 2001 May 01.
Essential amino acids of Escherichia coli DnaC protein in an N-terminal domain interact with DnaB helicase; Ludlam AV et al.; Escherichia coli DnaC protein bound to ATP forms a complex with DnaB protein . To identify the domain of DnaC that interacts with DnaB, a genetic selection was used based on the lethal effect of induced dnaC expression and a model that inviability arises by the binding of DnaC to DnaB to inhibit replication fork movement . The analysis of dnaC alleles that preserved viability under elevated expression revealed an N-terminal domain of DnaC involved in binding to DnaB . Mutant proteins bearing single amino acid substitutions (R10P, L11Q, L29Q, S41P, W32G, and L44P) that reside in regions of predicted secondary structure were inert in DNA replication activity because of their inability to bind to DnaB, but they retained ATP binding activity, as indicated by UV cross-linking to {alpha-(32)P}ATP . These alleles also failed to complement a dnaC28 mutant . Other selected mutations that map to regions carrying Walker A and B boxes are expected to be defective in ATP binding, a required step in DnaB-DnaC complex formation . Lastly, we found that the sixth codon from the N terminus encodes aspartate, resolving a reported discrepancy between the predicted amino acid sequence based on DNA sequencing data and the results from N-terminal amino acid sequencing (Nakayama, N., Bond, M . W., Miyajima, A., Kobori, J., and Arai, K . (1987) J . Biol . Chem . 262, 10475-10480).

J Biol Chem, 2001 Jul 6, 276(27), 24498 - 505 Epub 2001 May 01.
Fused p47phox and p67phox truncations efficiently reconstitute NADPH oxidase with higher activity and stability than the individual components; Ebisu K et al.; Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47(phox), p67(phox), and Rac with the membrane-associated flavocytochrome b(558) . Following cell-free activation, enzymatic activity is highly labile (Tamura, M., Takeshita, M., Curnutte, J . T., Uhlinger, D . J., and Lambeth, J . D . (1992) J . Biol . Chem . 267, 7529-7538) . To try to stabilize the activity and investigate the nature of the complex, fusion proteins between p47N-(1-286) and p67N-(1-210) were constructed . In a cell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficiency and produced a higher activity than the individual proteins, and also resulted in an 8-fold improved efficiency for Rac and a lowered K(m) for NADPH . O(2) generating activity was remarkably stabilized by using p67N-p47N . The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors . In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components . Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system containing cytochrome b(558) relipidated with phosphatidylinositol- or phosphatidylserine-enriched phospholipid mixtures . From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system . The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex . Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b(558) in the active complex.

J Biol Chem, 2001 Jul 6, 276(27), 25014 - 21 Epub 2001 May 01.
The Escherichia coli heat labile toxin binds to Golgi membranes and alters Golgi and cell morphologies using ADP-ribosylation factor-dependent processes; Zhu X et al.; The fate of the catalytic subunit of the Escherichia coli heat labile toxin (LTA(1)) was studied after expression in mammalian cells to assess the requirement for ADP-ribosylation factor (ARF) binding to localization and toxicity and ability to compete with endogenous ARF effectors . A progression in LTA(1) localization from cytosol to binding Golgi stacks to condensation of Golgi membranes was found to correlate with the time and level of LTA(1) expression . At the highest levels of LTA(1) expression the staining of LTA and both extrinsic and lumenal Golgi markers all became diffuse, in a fashion reminiscent of the actions of brefeldin A . Thus, LTA(1) binds to the Golgi and can alter its morphology in two distinct ways . However, point mutants of LTA(1) that are defective in the ability to bind activated ARF were also unable to bind Golgi membranes or modify Golgi morphology . Co-expression of mutants of ARF3 that regained binding to these same mutant LTA(1) proteins restored the localization and activities of the toxin . Thus, binding to ARF is required both for the localization of the toxin to the Golgi and for effects on Golgi membranes . A correlation was also seen between the ability of LTA mutants to bind ARF and the increase in cellular cAMP levels . These results demonstrate the importance of ARF binding to the toxicity and cellular effects of the ADP-ribosylating bacterial toxin and reveal that mutants defective in binding ARF retain basal ADP-ribosylation activity but are the least toxic LTA(1) mutants yet described, making them the best candidates for development as mucosal adjuvants.

Genetics, 2001 May, 158(1), 41 - 64
Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli; Courcelle J et al.; The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor . Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E . coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control . We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E . coli chromosome . We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold . In addition, several transcripts were either downregulated or degraded following UV irradiation . These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.

Genetics, 2001 May, 158(1), 29 - 39
Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli; Onda M et al.; We studied the role of DNA ligase in illegitimate recombination in Escherichia coli . A temperature-sensitive mutation in the lig gene reduced the frequency with which lambdabio-transducing phages were generated to 10-14% of that of wild type under UV irradiation . Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type . Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation . In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp) . However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp) . Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation . These observations support our hypothesis that the illegitimate recombination that generates lambdabio-transducing phages is mediated by the DNA break-and-join mechanism.

Electrophoresis, 2001 Mar, 22(5), 966 - 9
Reliable quantification of in vitro synthesized green fluorescent protein: comparison of fluorescence activity and total protein levels; Nemetz C et al.; At any time in vitro or in vivo expressed unlabeled proteins have to be quantified it is difficult to find a reliable method, especially with nonpurified samples . Quantification via protein activity can result in too low levels if the proteins analyzed tend to aggregate into inactive forms . Here, wild-type green fluorescent protein (GFPwt) was expressed in high amounts in vitro using the Rapid Translation System 500 based on Escherichia coli lysates . Fluorescent activity was determined in dependence of oxygen and compared to total protein levels . In the presence of low amounts of oxygen only 16% of the whole GFPwt amounts were detectable via determination of fluorescence activity . A reliable method to easily quantify whole protein levels even without specific antibodies and without purification steps by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining is described.

Electrophoresis, 2001 Mar, 22(5), 933 - 45
Mass spectrometric imaging of immobilized pH gradient gels and creation of "virtual" two-dimensional gels; Walker AK et al.; We have developed a matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) based technique for the detection of intact proteins directly from immobilized pH gradient gels (IPGs) . The use of this technique to visualize proteins from IPGs was explored in this study . Whole cell Escherichia coli extracts of various loadings were separated on IPGs . These IPGs were processed to remove contaminants and to achieve matrix/analyte cocrystallization on the surface of the gel . Mass spectra were acquired by scanning the surface of the gel and were assimilated into a "virtual" two dimensional (2-D) gel . This virtual 2-D gel is analogous to a "classical" 2-D gel, except that the molecular weight information is acquired by mass spectrometry rather than by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . This mass spectrometry (MS) based technology exemplifies a number of desirable characteristics, some of which are not attainable with classical two-dimensional electrophoresis (2-DE) . These include high sensitivity, high reproducibility, and an inherently higher resolution and mass accuracy than 2-D gels . Furthermore, there is a difference in selectivity exhibited between virtual 2-D gels and classical 2-D gels, as a number of proteins are visible in the virtual gel image that are not present in the stained gels and vice versa . In this report, virtual 2-D gels will be compared to classical 2-D gels to illustrate these features.

Nat Rev Mol Cell Biol, 2001 May, 2(5), 350 - 6
Protein targeting by the twin-arginine translocation pathway; Robinson C et al.; The twin-arginine translocation pathway operates in the thylakoid membrane of chloroplasts and in the plasma membrane of most free-living bacteria . Its main function is to transport fully folded proteins across the membrane . Three important tat genes have been identified and the sequences of the encoded proteins, together with the unusual properties of the pathway, indicate that the Tat system is completely different from other protein translocases.

Proc Natl Acad Sci U S A, 2001 May 8, 98(10), 5521 - 5 Epub 2001 May 01.
The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase; Ribbe MW et al.; It is known that an E146D site-directed variant of the Azotobacter vinelandii iron protein (Fe protein) is specifically defective in its ability to participate in iron-molybdenum cofactor (FeMoco) insertion . Molybdenum-iron protein (MoFe protein) from the strain expressing the E146D Fe protein is partially ( approximately 45%) FeMoco deficient . The "free" FeMoco that is not inserted accumulates in the cell . We were able to insert this "free" FeMoco into the partially pure FeMoco-deficient MoFe protein . This insertion reaction required crude extract of the DeltanifHDK A . vinelandii strain CA12, Fe protein and MgATP . We used this as an assay to purify a required "insertion" protein . The purified protein was identified as GroEL, based on the molecular mass of its subunit (58.8 kDa), crossreaction with commercially available antibodies raised against E . coli GroEL, and its NH(2)-terminal polypeptide sequence . The NH(2)-terminal polypeptide sequence showed identity of up to 84% to GroEL from various organisms . Purified GroEL of A . vinelandii alone or in combination with MgATP and Fe protein did not support the FeMoco insertion into pure FeMoco-deficient MoFe protein, suggesting that there are still other proteins and/or factors missing . By using GroEL-containing extracts from a DeltanifHDK strain of A . vinelandii CA12 along with FeMoco, Fe protein, and MgATP, we were able to supply all required proteins and/or factors and obtained a fully active reconstituted E146D nifH MoFe protein . The involvement of the molecular chaperone GroEL in the insertion of a metal cluster into an apoprotein may have broad implications for the maturation of other metalloenzymes.

J Biol Chem, 2001 Jul 20, 276(29), 27449 - 54 Epub 2001 Apr 30.
Structure of Ala(20) --> Pro/Pro(64) --> Ala substituted subunit c of Escherichia coli ATP synthase in which the essential proline is switched between transmembrane helices; Dmitriev OY et al.; The structure of the A20P/P64A mutated subunit c of Escherichia coli ATP synthase, in which the essential proline has been switched from residue 64 of the second transmembrane helix (TMH) to residue 20 of the first TMH, has been solved by (15)N,(1)H NMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture . The cA20P/P64A mutant grows as well as wild type, and the F(0)F(1) complex is fully functional in ATPase-coupled H(+) pumping . Residues 20 and 64 lie directly opposite to each other in the hairpin-like structure of wild type subunit c, and the prolinyl 64 residue is thought to induce a slight bend in TMH-2 such that it wraps around a more straightened TMH-1 . In solution, the A20P/P64A substituted subunit c also forms a hairpin of two alpha-helices, with residues 41-45 forming a connecting loop as in the case of the wild type protein, but, in this case, Pro(20) induces a bend in TMH-1, which then packs against a more straightened TMH-2 . The essential prolinyl residue, whether at position 64 or 20, lies close to the aspartyl 61 H(+) binding site . The prolinyl residue may introduce structural flexibility in this region of the protein, which may be necessary for the proposed movement of the alpha-helical segments during the course of the H(+) pumping catalytic cycle.

J Biol Chem, 2001 Jun 29, 276(26), 24051 - 8 Epub 2001 Apr 30.
Zinc inhibition of protein trans-splicing and identification of regions essential for splicing and association of a split intein*; Ghosh I et al.; Two important aspects of protein splicing were investigated by employing the trans-splicing intein from the dnaE gene of Synechocystis sp . PCC6803 . First, we demonstrated that both protein splicing and cleavage at the N-terminal splice junction were inhibited in the presence of zinc ion . The trans-splicing reaction was partially blocked at a concentration of 1-10 microm Zn(2+) and completely inhibited at 100 microm Zn(2+); the inhibition by zinc was reversed in the presence of ethylenediaminetetraacetic acid . We propose that inactivation of Cys(160) at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification steps in the splicing pathway . Furthermore, in vivo and in vitro assays were established for the determination of intein residues and regions required for splicing or association between the N- and C-terminal intein halves . N-terminal truncation of the intein C-terminal segment inhibited both splicing and association activities, suggesting this region is crucial for the formation of an interface between the two intein halves . The replacement of conserved residues in blocks B and F with alanine abolished splicing but allowed for association . This is the first evidence showing that the conserved residues in block F are required for protein splicing.

Bioinformatics, 2001 May, 17(5), 445 - 54
The utility of different representations of protein sequence for predicting functional class; King RD et al.; MOTIVATION: Data Mining Prediction (DMP) is a novel approach to predicting protein functional class from sequence . DMP works even in the absence of a homologous protein of known function . We investigate the utility of different ways of representing protein sequence in DMP (residue frequencies, phylogeny, predicted structure) using the Escherichia coli genome as a model . RESULTS: Using the different representations DMP learnt prediction rules that were more accurate than default at every level of function using every type of representation . The most effective way to represent sequence was using phylogeny (75% accuracy and 13% coverage of unassigned ORFs at the most general level of function: 69% accuracy and 7% coverage at the most detailed) . We tested different methods for combining predictions from the different types of representation . These improved both the accuracy and coverage of predictions, e.g . 40% of all unassigned ORFs could be predicted at an estimated accuracy of 60% and 5% of unassigned ORFs could be predicted at an estimated accuracy of 86%.

Bioinformatics, 2001 May, 17(5), 429 - 37
Analysis of genomic sequences by Chaos Game Representation; Almeida JS et al.; MOTIVATION: Chaos Game Representation (CGR) is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to find the coordinates for their position in a continuous space . This distribution of positions has two properties: it is unique, and the source sequence can be recovered from the coordinates such that distance between positions measures similarity between the corresponding sequences . The possibility of using the latter property to identify succession schemes have been entirely overlooked in previous studies which raises the possibility that CGR may be upgraded from a mere representation technique to a sequence modeling tool . RESULTS: The distribution of positions in the CGR plane were shown to be a generalization of Markov chain probability tables that accommodates non-integer orders . Therefore, Markov models are particular cases of CGR models rather than the reverse, as currently accepted . In addition, the CGR generalization has both practical (computational efficiency) and fundamental (scale independence) advantages . These results are illustrated by using Escherichia coli K-12 as a test data-set, in particular, the genes thrA, thrB and thrC of the threonine operon.

Biochemistry, 2001 May 8, 40(18), 5548 - 55
Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase; Rong J et al.; The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain . We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units {Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M . (2000) Biochem . J . 346, 463-468} . In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST . Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho{(35)S}sulfate . Incubations with O-desulfated heparin, predominantly composed of {(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-}(n)(), resulted in 2-O-sulfation of iduronic acid . When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure {(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-}(n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid . Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid . In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).

Biochemistry, 2001 May 8, 40(18), 5506 - 10
Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli; Ding PZ et al.; The melibiose carrier of Escherichia coli is a sugar-cation cotransport system that utilizes Na(+), Li(+), or H(+) . This membrane transport protein consists of 12 transmembrane helices . Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 17-37, which includes all of the residues in membrane helix I . The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloro- mercuribenzenesulfonic acid (PCMBS) . Cysteine substitution caused loss of transport activity in six of the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C) . PCMBS caused greater than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C, Y26C, M27C, Y28C, M30C, and Y31C) . We suggest that the residues whose cysteine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment . Second site revertants were isolated from K18C and Y31C . The revertants were found to have mutations in helices I, IV, and VII.

Biochemistry, 2001 May 8, 40(18), 5488 - 95
Stability and global fold of the mouse prohormone convertase 1 pro-domain; Tangrea MA et al.; We have purified the mouse prohormone convertase 1 (PC1) pro-domain expressed in Escherichia coli cells and demonstrated, using a number of biophysical methods, that this domain is an independent folding unit with a T(m) of 39 degrees C at a protein concentration of 20 microM and pH 7.0 . This differs significantly from similar pro-domains in bacteria and human furin, which are unfolded at 25 degrees C and require the catalytic domain in order to be structured {Bryan et al . (1995) Biochemistry 34, 10310-10318; Bhattacharjya et al . (2000) J . Biomol . NMR 16, 275-276} . Using heteronuclear NMR spectroscopy, we have determined the backbone (1)H, (13)C, and (15)N assignments for the pro-domain of PC1 . On the basis of (1)H/(13)C chemical shift indices, NOE analysis, and hydrogen exchange measurements, the pro-domain is shown to consist of a four-stranded beta-sheet and two alpha-helices . The results presented here show that both the bacterial pro-domain in complex with subtilisin and the uncomplexed mouse PC1 pro-domain have very similar overall folds despite a lack of sequence homology . The structural data help to explain the location of the secondary processing sites in the pro-domains of the PC family, and a consensus sequence for binding to the catalytic domain is proposed.

Biochemistry, 2001 May 8, 40(18), 5376 - 81
A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu; Mursinna RS et al.; Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accurately selecting and activating cognate amino acids for aminoacylation of the correct tRNA . Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection . These two sieves rely on different strategies for amino acid recognition to significantly enhance the accuracy of aminoacylation . We have performed alanine scanning mutagenesis in a conserved threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's CP1 domain that is hypothesized to contain a putative editing active site . Characterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid from being hydrolyzed after aminoacylation of the tRNA . Mutation of this threonine to an alanine eliminates discrimination of the cognate amino acid in the editing active site . This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site.

Appl Microbiol Biotechnol, 2001 Mar, 55(2), 187 - 91
High-level production of heme-containing holoproteins in Escherichia coli; Jung Y et al.; The expression of recombinant protein is essential for the investigation of the functions and properties of heme-containing protein as an electron carrier . For the expression of fully active recombinant protein, conversion of the expressed apoprotein into holoprotein is the most important and difficult problem . In this study, a system was developed for the production of heme-containing protein in a pure, recombinant holoprotein form, using the bovine cytochrome b5 tryptic fragment and Escherichia coli bacterioferritin as heterologous and homologous heme-containing model proteins, respectively . This system is based on the slow synthesis of recombinant apoprotein, which can maintain the balanced consumption of amino acids between protein synthesis and heme synthesis, so that the synthesized apoprotein continues to act as a heme sink . From a 1-1 culture, 15 mg of cytochrome b5 and 40 mg of bacterioferritin were purified as pure holoprotein forms . Our expression system provides a rapid and simple method for obtaining large quantities of the active holo-form of heme-containing proteins.

Biosci Biotechnol Biochem, 2001 Mar, 65(3), 694 - 7
Reactivities of mutants of a major house dust mite allergen Der f 2 to mouse anti-Der f 2 monoclonal antibodies analyzed by immunoblotting; Takai T et al.; A total of sixteen recombinant variants of a major house dust mite allergen Der f 2, the wild-type Der f 2, six cysteine mutants, six proline mutants, and three lysine mutants, were expressed in Escherichia coli . The cells were solubilized and run on SDS-PAGE under reducing conditions . Epitopes for five mouse anti-Der f 2 monoclonal antibodies, 1B2, 7C10, 13A4, 15E11, and 18G8, to the recombinant Der f 2 variants were characterized by immunoblot analysis.

Ultramicroscopy, 2001 Apr, 87(3), 155 - 64
4Pi-confocal microscopy of live cells; Bahlmann K et al.; By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy . So far this improvement was possible only in glycerol-mounted, fixed cells . Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells . Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope . The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria . The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 98 - 100
{Cloning and expression of the genes of glutathione synthetases}; Shen LX et al.; The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively . The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh . E . coli BL21 was transformed by pTrc-gsh for expression of the related enzymes . Analysis of SDS-PAGE showed that the expected products were expressed . E . coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5 . The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG . The expressed products were up to 25% of the total protein of the bacteria . Acetone-treated cells of the engineered strain could synthesize GSH efficiently.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 90 - 3
{A study on the expression of human leptin in the mammary glands of transgenic mice}; Liu JZ et al.; Human leptin expressed by E . coli had been used to treat human obesity in American and scientists had achieved good effects, the researchers here wanted to know whether human leptin can be expressed in the mammary glands of transgenic animas . In this study, human leptin gene about 1.0 kb, the terminator of rabbit whey acid protein gene (rWAP) about 0.2 kb and the promoter including the distal upstream region and part of the first exon of rWAP gene about 6.3 kb were used to construct a expression vector . Before we did the subclonings, the sequences of the human leptin gene were sequenced by ABI377 DNA Sequencer, the results showed that the fragment of human leptin gene included the last nine base pairs of the first exon, the complete sequences of the second exon(172 bp) and parts of the third exon(including part of the encoding sequences and part of the 3' untranslated region) . The final expression vector was digested with NotI and a fragment of 7.5 kb was collected and dissolved in TE(10 mmol/L Tris.Cl, pH7.4; 0.1 mmol/L EDTA) for later microinjection . The concentration of DNA was about 2 micrograms/mL, the copy number in 1 mL was about 2.4 x 10(11), every 1 to 2 pL of the prepared DNA solution was microinjected into the mouse embryos at pronucleus stage . After standard microinjection procedures, 48 live mice were obtained . The tails of the mice were cut(about 0.1 g) at four weeks of age, genomic DNA was extracted and digested completely with EcoRI, two were confirmed to be transgenic mice(both were female) by Southern hybridization using DIG labeled human leptin gene as probe, transgenic rate among the mice born was about 4% (2/48) . The two female transgenic mice(2# and C3) were mated with nontransgenic male mice . The two founder transgenic mice were segregated with their baby mice for at least three hours at the fifth day after parturition and were milked by intraperitoneal injection of 0.3 IU of oxytocin and udder massage . SDS-PAGE was used to analyze whether there were expression of human leptin in the milk of the two founder transgenic mice with the milk of non-transgenic mouse at fifth day after parturition as control . SDS-PAGE results showed that compared with the control there was a new band in both of the founder transgenic mice milk, and its molecular weight was about 16 kD, which was quite similar with that of the human leptin . The researchers estimated that the expression level of this protein in the milk of the transgenic mice was about 1-2 mg/mL.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 78 - 83
{Cloning, expression and preliminary application of a alpha-hydroxynitrile lyase from cassave}; Cheng SH et al.; alpha-Hydroxynitrile lyase (ME-HNLs, E.C . 4.1.2.3.37) from the cyanogenic crop cassava(Manihot esculentz, Crantz) catalyze the condensation of hydrocyanic acid and aldehydes or ketone into (s)-cyanohydrins, which are valuable starting material for various optically active compounds, such as pharmaceuticals and agrochemicals . The cDNA of a ME-HNL were obtained by RT-PCR and cloned . The sequencing result for the cDNA showed that the sequence encoded for the ME-HNL was inconsistent with all those which are published, such as hnl10, hnl24, hnl4 . The full sequence analysis demonstrated that the cloned cDNA was about 75.2%, 79.8%, 99.2% homologous to other three reported HNL genes from cassava, respectively, among which the last was the same to the cloned gene except the five base substitution at the site 142, 337, 476, 634 and 636, respectively . The two base substitutions lead to change the amino acid sequence, i.e., Ser113-->Gly113, Phe158-->Tyr158 . To construct the recombinant plasmid pET30a-hnl, the cDNA was inserted into an expression vector pET30a . After transformation of pET30a-hnl and induction with IPTG, the ME-HNL was efficiently expressed in E . coli . BL21 (DE3) and reached over 2100 units/L of culture with the specific activity 8.5 u/mg protein . By one simple treatment, incubating 10 minutes at 70 degrees C, the recombinant ME-HNL may be used as an catalyst for production of (S)-mandelonitrile with enantiomeric excess of 95.2% and 98.2% yield.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 7 - 10
{Site-directed mutagenesis at disulfide bond Cys206-Cys210 of prochymosin (chymosin)}; Cheng HJ et al.; During the work of site-directed mutagenesis at disulfide bond Cys206-Cys210 of prochymosin, it was found that the corresponding template sequence had the potential to form a loop-stem structure with free energy of -16.1 kcal/mol, which prevent the template from pairing with primer and, in turn, the synthesis of the mutated DNA strand . Rapid annealing can overcome this difficulty . Five expression plasmids of prochymosin muants with deletion of Cys206-Cys210 (C206A, C210A, C206A/C210A, C210S and C206S/C210S) were constructed . Except for C206A they were expressed at high level in E . coli amounting to 50% of the total cellular proteins . Renaturation of the mutant prochymosin indicated that Cys206-Cys210 is dispensable for correct refolding of prochymosin . However, the amino acid residues at Cys206 and/or Cys 210 play a critical role in determining the renaturation . Among the five mutants the reactivation efficiency of C206A/C210A were about 4.5-fold, 20-fold and 30-fold higher than that of C206S/C210S, C210A and C210S respectively . C206A can not correctly refold at all . CD spectra in the far UV region indicate that C206A/C210A and C206S/C210S chymosin analogs have a secondary structure almost identical to that of the wild-type chymosin . Fluorescence spectroscopic analysis revealed that mutant chymosins have the same emission maximum at 333 nm as the wild-type chymosin but their fluorescence intensities at 333 nm are much higher than that of the wild-type chymosin . Considering that the mutants and the wild-type chymosin exhibit almost the same specific activity, it is reasonable to conclude that the mutant proteins assume a native active information with a perturbance around some tryptophan residues.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 68 - 72
{Cloning and expression of urate oxidase and its application in serum uric acid analysis}; Zhu XJ et al.; Anurate oxidase (uricase, EC 1.7.3.3) gene from Candida utilis AS2.117 was cloned by PCR amplification with primers derived from conserved regions of published uicase DNA sequence . The DNA sequence of cloned uricase gene was determined and a high homology compared to the reported gene was found . The cloned gene was inserted into Bam H I and Nde I sites of pET21a to create the recombinant plasmid pURO . In Escherichia coli BL21(DE3) host, the expression lever of uricase reached to about 40% of total soluble proteins of the cell . The western blot analysis confirmed the result of expression . Properties of the enzyme protein produced by E . coli BL21(DE3)/pURO were determined and similar with those of original protein from Candida utilis AS2.117 . Furthermore, the thermostability of the expressed protein was enhanced . The purified recombinant uricase was used in serum uric acid analysis.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 59 - 63
{High cell density culture of phosphotransacetylase mutants of Escherichia coli BL21(DE3)}; Zhang WC et al.; Cell culture, organic acid production and foreign protein (TNF) expression of E . coli BL21(DE3) and its pta mutant were investigated . Under shaking conditions, TNF expression in pta mutant increased by 23% . During the fed-batch culture without limitation of specific growth rates, the mutant reached a cell density as high as 32.5 g(DCW)/L and total TNF expression at 2.8 g/L, while the parental strain only obtained 19.5 g(DCW)/L and 0.84 g/L . The results indicate that utility of pta mutant as a host is advantageous in foreign protein expression and high cell density culture . Meanwhile, the analysis data of organic acids accumulated during fed-batch culture showed that as the decrease of acetate production(42% of the parental strain), the accumulation of other organic acids(mainly pyruvate, lactate and succinate) obviously increased . As a result, the amount of total organic acids increased by 123% over its parent . The lactate production may be the main obstacle in further growth of the cells.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 50 - 4
{A three-domain antibody fragment VH/L specific to tumor blood vessels}; Wu XP et al.; AA98 is a specificaally anti-angiogenic antibody generated in our lab . The heavy chain variable region (VH) attached with mutagenized 36 nucleotides sequence derived from the heavy chain constant region1 (CH1) was amplified VH and light chain (L) were inserted into high-level expression vector pET21a successively, thus pET21a-VH/L was constructed . VH/L was expressed in E . coli BL21 (DE3) after induction with IPTG . The expression of VH/L was 20% of the total bacterial proteins . The refolding of VH/L was conducted by dilution and gel filtration chromatography . The refolded VH/L could bind to HUVEC specifically . Its affinity to the antigen is similar to that of recombinant AA98Fab, but lower than that of the parent antibody AA98.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 46 - 9
{Gene chimeric fusion and expression of nucleocapsid NS3 regions and NS4 regions of hepatitis C virus genome}; Shen XC et al.; Genes encoding HCV core and NS4 antigen epitopes and C33c antigen were cloned from HCV genome by PCR, respectively . Two fused genes were constructed . One contained these three genes, another contained genes encoding C33c antigen and NS4 antigen epitopes . These fused genes were cloned into expression plasmid pET-24(a)+ and pET-22(b)+ under T7 promoter and transformed into E . coli BL21 (DE3) respectively . SDS-PAGE analysis revealed that these fused antigens CCN, CN were highly expressed after the induction by 1 mmol/L IPTG . These Expression products were detected by western blotting with anti-HCV serum.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 16 - 9
{Study on the expression of pig zona pellucida-3 beta excluding N-terminus signal peptide and C-terminus transmembrance-like domain in Escherichia coli}; Xu WX et al.; Pig oocytes are surrounded by an acellular translucent envelope-zona pellucida(pZP), which is comprised of three biochemically and immunologically distinct glycoproteins(pZP1, pZP3 alpha and pZP3 beta) . Due to the cross-reactivity of anti-pZP3 beta antibodies with human ZP in vitro, pZP3 beta has been considered as potential one of immunogens for developing human contraceptive vaccine . In order to express pZP3 beta directly in E . coli; we amplified the core fragment of pZP3 beta cDNA by PCR that was deleted 5'- and 3'-terminal sequences of coding for signal peptide and transmembrane-like domain . The DNA sequenced EcoRI and SalI restriced core fragment was cloned in a frame downstream of PRPL promoter in the pBV221 vector . SDS-PAGE analysis showed that pZP3 beta protein was expressed especially in E . coli after thermal induction, which the expression band displayed the molecular weight of about 38 kD matching with its deduced molecular weight(36.5 kD) . In addition, the specially expressed protein band on SDS-PAGE gel showed specific immunological reaction with anti-pig ZP IgGs of rabbit in Western blot.

Nippon Ganka Gakkai Zasshi, 2001 Apr, 105(4), 230 - 6
{Effects of topical prostaglandin analogues on the aqueous flare intensity in rabbit eyes at an early phase of endotoxin-induced uveitis}; Kiuchi Y et al.; PURPOSE: We examined the effects of prostaglandin analogues on the blood-aqueous barrier(BAB) permeability in rabbit eyes at an early phase of endotoxin-induced uveitis(EIU) . SUBJECTS AND METHODS: One drop of 0.005% latanoprost or 0.12% unoprostone were applied to rabbit eyes . Escherichia coli lipopolysaccharides were injected to induce uveitis . The changes in flare intensity in normal eyes and EIU eyes after application of eye drops were evaluated . The effect of cyclooxygenase inhibitor on the flare intensity changes caused by the application of unoprostone was also examined . RESULTS: Flare intensity increased significantly after a single instillation of unoprostone, and the increase was not prevented by pretreatment with cyclooxygenase inhibitor . In eyes with EIU, unoprostone caused an additional increase of flare intensity to uveitis induced flare change . Latanoprost had no effects on BAB in eyes with normal and with uveitic conditions . CONCLUSION: Latanoprost and unoprostone did not cause an excessive inflammatory reaction in rabbit eyes at an early phase of EIU.

J Radiat Res (Tokyo), 2000 Dec, 41(4), 355 - 66
Redox reactions of sanazole (AK-2123) in aqueous solutions: a pulse radiolysis study; Kapoor S et al.; The redox chemistry of sanazole, an efficient hypoxic cell radiosensitizer, generally referred to as AK-2123, was studied by pulse radiolysis with eaq-, CO2-., 2-propanol radicals and CH2OH radicals . AK-2123 reacts with eaq-, CO2- . and 2-propanol radicals at almost diffusion-controlled rates, producing a nitro radical anion (lambda max = 290 nm) within a few microseconds . The decay kinetics of the radical anion was independent of the pH . The radical anion reacts with oxygen with a rate constant of 3.4 x 10(6) dm3 mol-1 s-1 . An electron-transfer reaction was observed from the thymine radical anion to AK-2123 . From redox equilibria with methyl viologen, the one-electron reduction potential of AK-2123 in aqueous solution, determined by pulse radiolysis, was estimated to be -0.33 +/- 0.02 V vs . NHE . Depletion of intracellular nonprotein thiols did not mitigate the radiosensitizing affect of the hypoxic radiosensitizer, AK-2123.

J Radiat Res (Tokyo), 2000 Dec, 41(4), 349 - 54
2-hydroxyadenine in DNA is a very poor substrate of the Escherichia coli MutY protein; Kamiya H et al.; To test the possibility that the Escherichia coli MutY or MutM protein acts as a 2-hydroxyadenine (2-OH-Ade) glycosylase, we treated double-stranded oligodeoxyribonucleotides containing 2-OH-Ade with the E . coli MutY or MutM protein in vitro . We found that a strand with 2-OH-Ade was a very poor substrate of MutY, irrespective of the base in the complementary strand . Moreover, a strand containing adenine or guanine opposite 2-OH-Ade was also rarely cleaved by MutY . The cleavage of oligonucleotides with 2-OH-Ade by MutM was not observed . These results indicate that neither MutY nor MutM plays an important role in the removal of 2-OH-Ade from DNA.

Clin Diagn Lab Immunol, 2001 May, 8(3), 652 - 7
Oral administration of influenza vaccine in combination with the adjuvants LT-K63 and LT-R72 induces potent immune responses comparable to or stronger than traditional intramuscular immunization; Barackman JD et al.; Mucosal immunization strategies are actively being pursued in the hopes of improving the efficacy of vaccines against the influenza virus . Our group investigated the oral immunization of mice via intragastric gavage with influenza hemagglutinin (HA) combined with mutant Escherichia coli heat-labile enterotoxins K63 (LT-K63) and R72 (LT-R72) . These oral immunizations resulted in potent serum antibody and HA inhibition titers, in some cases stronger than those obtained with traditional intramuscular administration, in addition to HA-specific immunoglobulin A in the saliva and nasal secretions . This study demonstrates that it may be possible to develop effective oral influenza vaccines.

Clin Diagn Lab Immunol, 2001 May, 8(3), 637 - 40
Prevalence of known P-fimbrial G alleles in Escherichia coli and identification of a new adhesin class; Manning SD et al.; Screening a large Escherichia coli collection for P-fimbrial adhesin classes identified 20 unclassifiable strains . Cloning and sequencing of papG from an unclassifiable strain identified another G allele . The novel adhesin gene has 65% identity to the class I adhesin gene, 44% identity to the class II adhesin gene, and 43% identity to the class III adhesin gene.

Biochemistry, 2001 Feb 20, 40(7), 2282 - 90
Effects of benzo{a}pyrene DNA adducts on Escherichia coli DNA polymerase I (Klenow fragment) primer-template interactions: evidence for inhibition of the catalytically active ternary complex formation; Alekseyev YO et al.; Benzo{a}pyrene diol epoxide (B{a}PDE) adducts are strong blocks of DNA replication in vitro, allowing the rare incorporation of a nucleotide across from the lesion and negligibly small extent of further bypass . To study the mechanistic details of this process, a gel-retardation assay was used to measure the dissociation constants for the binding of DNA polymerase I (Klenow fragment) (KF) to the primer-templates containing a (+)-trans- or (+)-cis-B{a}P-N(2)-dG adduct . When the primer was terminated one nucleotide before the adduct, the presence of a (+)-trans-B{a}P-N(2)-dG adduct did not affect the binding while a (+)-cis-B{a}P-N(2)-dG adduct caused a slight decrease in affinity . The presence of any dNTP decreased the affinity of KF to the modified primer-templates . (In contrast, a strong increase of the affinity to unmodified primer-templates was observed in the presence of the next correct dNTP.) Limited protease digestion experiments indicated that a closed ternary complex of KF with the modified primer-templates was not detectable in the presence of any dNTP, whereas it was clearly observed with unmodified template in the presence of the next correct nucleotide . These findings suggest that these adducts may interfere with the conformational change to the catalytically active closed ternary complex and/or cause significant destabilization of this complex . When the primers extended to the position across from the adduct, the affinity of KF was significantly decreased irrespective of the identity of the base across from the adduct, possibly explaining the low bypass of the lesion . Interestingly, the stability of these DNA-polymerase complexes correlated with nucleotide insertion kinetics for the unmodified and (+)-trans-B{a}PDE-modified templates.

Biochemistry, 2001 Feb 20, 40(7), 2276 - 81
Escherichia coli transcription termination factor Rho binds and hydrolyzes ATP using a single class of three sites; Stitt BL; Escherichia coli transcription termination factor Rho uses the energy of ATP hydrolysis to travel 5' --> 3' along RNA . We previously showed that the hexameric Rho protein binds three molecules of ATP in active sites and that hydrolysis of the three bound ATP molecules upon RNA binding is sequential . Other models of Rho ATP hydrolysis activity have arisen from reports of additional ATP binding sites on Rho . Here we present further evidence from binding, isotope partitioning, and rapid mix/chemical quench experiments, in support of the presence of only three equivalent ATP binding sites on Rho that are catalytic sites and that fire sequentially . These results are incorporated into a proposed mechanism for directional Rho tracking along RNA.

Biochemistry, 2001 Feb 20, 40(7), 2148 - 54
Amyloid-induced aggregation and precipitation of soluble proteins: an electrostatic contribution of the Alzheimer's beta(25-35) amyloid fibril; Konno T; Amyloid-induced aggregation and precipitation of soluble proteins were investigated in vitro using the amyloid fibrils of the beta(25--35) peptide, a cytotoxic fragment of the Alzheimer's beta-peptide at positions 25--35 . The aggregation rate of firefly luciferase was found to be modulated by both a chaperone molecule DnaK and the beta(25--35) amyloid, but their effects were opposite in direction . The amyloid fibril drastically facilitated the luciferase aggregation, which may define a kind of anti-chaperone activity . The effect of the beta(25--35) amyloid to promote protein aggregation and precipitation was further demonstrated for a wide variety of target proteins . The amount of coprecipitation was well correlated with the predicted isoelectric point of the target proteins, indicating that the interaction between the beta(25--35) amyloid and the target was driven by an electrostatic force between them . This view was confirmed by the experiments using an electrically neutral mutant peptide, beta(25--35)KA . It was also found that clustering of the beta(25--35) peptide to form amyloid and the conformation of the target protein are additional factors that determine the strength of the amyloid-protein interaction . Spectroscopic and electron microscopic methods have revealed that the proteins coprecipitated with the beta(25--35) amyloid formed amorphous aggregates deposited together with the amyloid fibrils . The conformation of protein molecules left in the residual soluble fraction was also damaged in the amyloid-containing solution . As a summary, this study has proposed a scheme for events related to the nonspecific amyloid-protein interaction, which may play substantial roles in in vivo conditions.

Biochemistry, 2001 Feb 20, 40(7), 2104 - 12
Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function; Hitchcock-DeGregori SE et al.; Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform . Periodic repeats in the sequence have been proposed to correspond to actin binding sites . To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5) . Recombinant Tms (unacetylated) were expressed in Escherichia coli . Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn) . dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition . Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding . dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1 . In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding . The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding . The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA . In the presence of Ca(2+), relief of inhibition by these Tms was incomplete . We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different.

Biochemistry, 2001 Feb 20, 40(7), 1996 - 2003
Functional estimation of loop-helix boundaries in the lactose permease of Escherichia coli by single amino acid deletion analysis; Wolin CD et al.; Mutants with single amino acid deletions in the loops of lactose permease retain activity, while mutants with single deletions in transmembrane helices are inactive, and the loop--helix boundaries of helices IV, V, VII, VIII, and IX have been approximated functionally by the systematic deletion of single residues {Wolin, C . D., and Kaback, H . R . (1999) Biochemistry 38, 8590-8597} . The experimental approach is applied here to the remainder of the permease . Periplasmic and cytoplasmic loop-helix boundaries for helices I, II, X, XI, and XII and the cytoplasmic boundary of helix III are in reasonable agreement with structural predictions . In contrast, the periplasmic end of helix III appears to be five to eight residues further into the transmembrane domain than predicted . Taken together with the previous findings, the analysis estimates that 11 of the 12 transmembrane helices have an average length of 21 residues . Surprisingly, deletion analysis of loop V/VI, helix VI, and loop VI/VII does not yield an activity profile typical of the rest of the protein, as individual deletion of only three residues in this region abolishes activity . Thus, transmembrane domain VI which is probably on the periphery of the 12-helix bundle may make few functionally important contacts.

Biochemistry, 2001 Feb 20, 40(7), 1984 - 95
The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase: kinetic and structural effects of mutations of Phe-50; Czerwinski RM et al.; The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers because of its unusually low pK(a) of 6.4 +/- 0.2, which is 3 units lower than that of the model compound, proline amide . Recent studies show that this abnormally low pK(a) is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) {Czerwinski, R . M., Harris, T . K., Johnson, Jr., W . H., Legler, P . M., Stivers, J . T., Mildvan, A . S., and Whitman, C . P . (1999) Biochemistry 38, 12358-12366} . Hence, it may result solely from a low local dielectric constant of 14.7 +/- 0.8 at the otherwise hydrophobic active site . Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 A from Pro-1 and is one of 12 apolar residues within 9 A of Pro-1 . Replacing Phe-50 with Tyr does not significantly alter k(cat) or K(m) and results in a pK(a) of 6.0 +/- 0.1 for Pro-1 as determined by (15)N NMR spectroscopy, comparable to that observed for wild type . (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme . In the F50Y mutant, the pK(a) of Tyr-50 is increased by two units from that of a model compound N-acetyl-tyrosine amide to 12.2 +/- 0.3, as determined by UV and (1)H NMR titrations, yielding a local dielectric constant of 13.4 +/- 1.7, in agreement with the value of 13.7 +/- 0.3 determined from the decreased pK(a) of Pro-1 in this mutant . In the F50A mutant, the pK(a) of Pro-1 is 7.3 +/- 0.1 by (15)N NMR titration, comparable to the pK(a) of 7.6 +/- 0.2 found in the pH vs k(cat)/K(m) rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 +/- 2.6 . A loss of structure of the beta-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT . (1)H-(15)N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone (15)N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 degrees C and their disappearance at 43 degrees C due to rapid exchange with solvent . These observations confirm that the active site of the F50A mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pK(a) of Pro-1 . In addition, the F50A mutation decreased k(cat) 167-fold and increased K(m) 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pK(a) of Pro-1 . The F50I and F50V mutations destabilize the protein and decrease k(cat) by factors of 58 and 1.6, and increase K(m) by 3.3- and 3.8-fold, respectively.

Biochemistry, 2001 Feb 20, 40(7), 1956 - 63
Determination of the redox properties of human NADPH-cytochrome P450 reductase; Munro AW et al.; Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains . Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) {oxidized/semiquinone} = -286 +/- 6 mV, E(2) {semiquinone/reduced} = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) {FMN} = -66 +/- 8 mV, E(2) {FMN} = -269 +/- 10 mV; E(1) {FAD} = -283 +/- 5 mV, E(2) {FAD} = -382 +/- 8 mV) . The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains . Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples . Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain . In both cases, large amounts of the higher potential FMN are in the semiquinone form . The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase . However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes.

Biochemistry, 2001 Feb 20, 40(7), 1913 - 21
Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites; Olsen LR et al.; N-Acetylglucosamine-1-PO(4) uridyltransferase (GlmU) is a trimeric bifunctional enzyme that catalyzes the last two sequential reactions in the de novo biosynthetic pathway for UDP-GlcNAc . The X-ray crystal structure of Escherichia coli GlmU in complex with UDP-GlcNAc and CoA has been determined to 2.1 A resolution and reveals a two-domain architecture that is responsible for these two reactions . The C-terminal domain is responsible for the CoA-dependent acetylation of Glc-1-PO(4) to GlcNAc-1-PO(4) and displays the longest left-handed parallel beta-helix observed to date . The acetyltransferase active site defined by the binding site for CoA makes use of residues from all three subunits and is positioned beneath an open cavity large enough to accommodate the Glc-1-PO(4) acetyl acceptor . The N-terminal domain catalyzes uridyl transfer from UTP to GlcNAc-1-PO(4) to form the final products UDP-GlcNAc and pyrophosphate . This domain is composed of a central seven-stranded beta-sheet surrounded by six alpha-helices in a Rossmann fold-like topology . A Co(2+) ion binds to just one of the two independent pyrophosphorylase active sites present in the crystals studied here, each of which nonetheless binds UDP-GlcNAc . The conformational changes of the enzyme and sugar nucleotide that accompany metal binding may provide a window into the structural dynamics that accompany catalysis.

Biochemistry, 2001 Feb 20, 40(7), 1897 - 902
Human thymidylate synthase is in the closed conformation when complexed with dUMP and raltitrexed, an antifolate drug; Phan J et al.; Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms while raltitrexed (Tomudex, ZD1694) is an antifolate inhibitor of TS approved for clinical use in several European countries . The crystal structure of the complex between recombinant human TS, dUMP, and raltitrexed has been determined at 1.9 A resolution . In contrast to the situation observed in the analogous complex of the rat TS, the enzyme is in the closed conformation and a covalent bond between the catalytic Cys 195 and dUMP is present in both subunits . This mode of ligand binding is similar to that of the analogous complex of the Escherichia coli enzyme . The only major differences observed are a direct hydrogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by about 2 A . The thiophene ring of the drug is disordered between two parallel positions.

J Biol Chem, 2001 Jul 20, 276(29), 26852 - 9 Epub 2001 Apr 27.
Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli; Assarsson M et al.; The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component . The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein . The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state . We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present . Paramagnetic intermediates, i.e . a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions . A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined . Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204 . A general scheme for the reconstitution reaction is presented based on EPR and structure results . This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.

DNA Seq, 2000, 11(5), 419 - 31
MglA and mglB of Treponema denticola; similarity to ABC transport and spa genes; Lepine G et al.; The mglA and mglB genes (td-mglA and td-mglB) of the oral spirochete Treponema denticola were sequenced . These two T . denticola genes are highly homologous to the E . coli and Treponema pallidum mglA and mglB genes which are part of the three gene beta-methylgalactoside transport operon, mglBAC . Both Td-mglA and td-mglB are also homologous to the high affinity ABC-type transporters for ribose and arabinose, and surface presentation antigens (spa) locus, part of the type III secretion systems in enteropathogens . Td-mglB and td-mglA are co-transcribed as a single mRNA in T . denticola as well as in E . coli cells as determined by reverse transcription PCR (RT-PCR) . Homology to td-mglB and its expressed protein was found in other oral spirochetes as determined by Southern and western blot analysis.

DNA Seq, 2000, 11(5), 395 - 404
Identification and characterization of the gene encoding the mitochondrial elongation factor G in rice; Kato A et al.; A plant nuclear gene coding for a mitochondrial elongation factor G (mEF-G) was cloned from a cDNA library and genomic library of rice (Oryza sativa L.) . This DNA sequence predicts a 757-amino-acid protein exhibiting 79%, 55% and 49% homology to Arabidopsis thaliana, Saccharomyces cerevisiae and rat mEF-G respectively, 53% homology to the elongation factor G in Escherichia coli and 43% homology to soybean chloroplast elongation factor G . The deduced amino acid sequence contains the characteristic motifs shared by all GTP binding proteins . Comparison of the sequence of the genomic clone to that of the cDNA clone revealed that this gene is split nineteen times by introns, although the gene of Arabidopsis is split seventeen times by introns . Some of the introns found in the rice genome are relatively long and they result in a long gene with a size of approximately 15 kb.

J Biochem (Tokyo), 2001 May, 129(5), 761 - 8
The amino acid residues affecting the activity and azole susceptibility of rat CYP51 (sterol 14-demethylase P450); Nitahara Y et al.; The amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation . Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli . Substitution of highly conserved Y131, E369, R372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues . Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the "conserved threonine" of CYP51 . H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species . A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole . Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51 . Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51.

J Biochem (Tokyo), 2001 May, 129(5), 709 - 16
Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression; Yubisui T et al.; A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5) . The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs . The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5) . A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues . The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration . The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase . The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5) . Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5 . Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low . This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.

Br J Haematol, 2001 Apr, 113(1), 115 - 9
Drug monitoring of low-dose PEG-asparaginase (Oncaspar) in children with relapsed acute lymphoblastic leukaemia; Vieira Pinheiro JP et al.; Use of asparaginase (ASNase) in the treatment of relapsed childhood acute lymphoblastic leukaemia (ALL) is associated with a high rate of hypersensitive reactions . 'Silent' inactivation may additionally reduce treatment intensity . Therefore, PEG-ASNase (Oncaspar), a polyethylene glycol conjugate of the native Escherichia coli-ASNase, was introduced into the Berlin-Frankfurt-Munster (BFM) 96 treatment protocol for relapsed ALL under drug monitoring conditions . A single i.v . dose of 500 IU/m2 PEG-ASNase, substituted for the native ASNases, was administered to supply a plasma activity of 100 IU/l for 1 week . From November 1997 to March 2000, 35 patients from 23 BFM-associated hospitals, with or without a previous allergic reaction to one or both native preparations, underwent monitoring . After 82 applications, a total of 270 samples were submitted to be tested for ASNase activity . The ASNase activity on the day of the administration and the following day ranged between < 20 and 693 IU/l, with a median of 413 IU/l (53 samples) . The median on d 7 +/- 1 was 199 IU/l (range <20--421 IU/l; 41 samples) and on d 14 +/- 1, 105 IU/l (range <20--188 IU/l; 19 samples) . An ASNase activity of > 100 IU/l was seen on d 7 in 36 activity time courses of 52 interpretable applications (69%) . Intraindividual variability of activity time courses was low . However, a rapid decrease in ASNase activity after repeated applications was observed in 4 out of 20 children . Previously experienced allergic reactions to native ASNases did not influence PEG-ASNase pharmacokinetics . PEG-ASNase is a useful alternative to the native ASNases in children with relapsed ALL . Whenever possible, drug monitoring should be performed to identify patients with 'silent' inactivation.

Biochemistry, 2001 Feb 27, 40(8), 2588 - 98
HU binding to DNA: evidence for multiple complex formation and DNA bending; Wojtuszewski K et al.; HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA . The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes . The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation . These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex . The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length . Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity . The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe . These results are suggestive of a local bending or unwinding of the DNA . On the basis of these results we propose a model in which bending of DNA accompanies HU binding . Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes . We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.

Biochemistry, 2001 Feb 27, 40(8), 2502 - 10
Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer; Mizuno H et al.; The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated . The recombinant DsRed expressed in E . coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm . Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm . Incubation of E . coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak . In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R) . Light-scattering analysis revealed that DsRed proteins expressed in E . coli and HeLa cells form a stable tetramer complex . DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm . The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm) . When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin . Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved . Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy . Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant.

Biochemistry, 2001 Feb 13, 40(6), 1844 - 9
N-terminal intramolecularly conserved histidines of three domains in Gonyaulax luciferase are responsible for loss of activity in the alkaline region; Li L et al.; Gonyaulax luciferase is a single-chain ( approximately 137 kDa) polypeptide comprising 111 N-terminal amino acids followed by three contiguous homologous domains (377 amino acids each) . Each domain has luciferase activity, accounting for the earlier observation that proteolytic fragments ( approximately 35 kDa) of luciferase are active . The activity of the full-length native enzyme is maximal at pH 6.3, dropping to near zero at pH 8; the activity of fragments also peaks at pH 6.3 but remains high at 8 . While the activity loss at higher pH might be thought to be associated with the conformation of the full-length protein, we show here that this is a property of individual domains . The three intramolecularly homologous domains, separately cloned and expressed in Escherichia coli as fusion proteins, exhibit pH-activity curves similar to that of the full-length enzyme . For each domain the removal of approximately 50 N-terminal amino acids resulted in an increase in the ratio of luciferase activity at pH 8 relative to that at pH 6.3, such that their pH-activity profiles mimicked that of the proteolytic fragments reported earlier . Replacement of N-terminal histidines by alanine by site-directed mutagenesis identified four that are involved in the loss of activity at high pH . This system illustrates an unusual, possibly unique mechanism for pH regulation of enzyme activity, which has been postulated to be responsible for the control of the characteristic flashes of bioluminescence.

Biochemistry, 2001 Feb 13, 40(6), 1835 - 43
Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein; Ding H et al.; SecA ATPase is an essential component of the Sec-dependent protein translocation machinery . Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane . To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions . Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure . Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins . Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands . Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide . These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation.

Biochemistry, 2001 Feb 13, 40(6), 1804 - 11
Resonance energy transfer between tryptophan 57 in the epsilon subunit and pyrene maleimide labeled gamma subunit of the chloroplast ATP synthase; Johnson EA et al.; The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM) . The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1 . CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subunits in which phenylalanine replaced tryptophan at position 15 (epsilonW15F) and position 57 (epsilonW15/57F) . CF1(epsilonW15F) containing a single tryptophan, epsilonW57, was labeled with PM at gammaC322 . Resonance energy transfer (RET) from epsilonW57 to PM on gammaC322 occurred with an efficiency of energy transfer of 20% . RET was also observed from epsilonW57 to PM attached to the disulfide thiols of the gamma subunit (gammaC199,205) with an efficiency of approximately 45% . The R(o) (the distance at which the efficiency of energy transfer is 50%) for the epsilonW57 and PM donor/acceptor pair is 30 A, indicating that both gammaC322 and gammaC199,205 must be within 40 A of epsilonW57 . These RET measurements show that both gammaC322 and gammaC199,205 are located near the base of the alpha/beta hexamer . This places the C-terminus of CF1 gamma much closer to epsilon than hypothesized based on homology to crystal structures of mitochondrial F1 . These new RET measurements also allow the alignment of the predicted epsilon subunit structure . The orientation is similar to that predicted from cross-linking and mutational studies for the epsilon subunit of Escherichia coli F1.

Biochemistry, 2001 Feb 13, 40(6), 1796 - 803
Evidence in Escherichia coli that N3-methyladenine lesions induced by a minor groove binding methyl sulfonate ester can be processed by both base and nucleotide excision repair; Shah D et al.; It has been previously reported that a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (MeOSO(2)-lex) selectively affords N3-methyladenine lesions . To study the interaction of the neutral lex dipeptide with calf thymus DNA, we have prepared stable, nonmethylating sulfone analogues of MeOSO(2)-lex that are neutral and cationic . Thermodynamic studies show that both the neutral and monocationic sulfone compounds bind to DNA with K(b)'s of 10(5) in primarily entropy-driven reactions . To determine how the cytotoxic N3-methyladenine adduct generated from MeOSO(2)-lex is repaired in E . coli, MeOSO(2)-lex was tested for toxicity in wild-type E . coli and in mutant strains defective in base excision repair (tag and/or alkA glycosylases or apn endonuclease), nucleotide excision repair (uvrA), and both base and nucleotide excision repair (tag/alkA/uvrA) . The results clearly demonstrate the cellular toxicity of the N3-methyladenine lesion, and the protective role of base excision glycosylase proteins . A novel finding is that in the absence of functional base excision glycosylases, nucleotide excision repair can also protect cells from this cytotoxic minor groove lesion . Interaction between base and nucleotide excision repair systems is also seen in the protection of cells treated with cis-diamminedichloroplatinum(II) but not with anti-(+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene.

Biochemistry, 2001 Feb 13, 40(6), 1764 - 73
Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p; Thual C et al.; The {URE3} factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2 . The molecular basis of the propagation of this phenotype is unknown . We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded . Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils {Thual et al . (1999) J . Biol . Chem . 274, 13666-13674} . To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions . We show here that Ure2p dimerizes through its C-terminal region . We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils . A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain . Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein . This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils . Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies.

Biochemistry, 2001 Feb 13, 40(6), 1734 - 40
Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: site-directed mutagenesis of highly conserved residues; Soderberg T et al.; Dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes alkylation of the exocyclic amine of adenosine at position 37 in some tRNAs by the hydrocarbon moiety of dimethylallyl diphosphate (DMAPP) . A multiple-sequence alignment of 28 gene sequences encoding DMAPP-tRNA transferases from various organisms revealed considerable homology, including 11 charged, 12 polar, and four aromatic amino acids that are highly conserved or conservatively substituted . Site-directed mutants were constructed for all of these amino acids, and a tripeptide Glu-Glu-Phe alpha-tubulin epitope was appended to the C-terminus of the protein to facilitate separation by immunoaffinity chromatography of overproduced mutant enzymes from coexpressed chromosomally encoded wild-type DMAPP-tRNA transferase . Steady-state kinetic constants were measured for wild-type DMAPP-tRNA transferase and the site-directed mutants using DMAPP and a 17-base RNA oligoribonucleotide corresponding to the stem-loop region of tRNA(Phe) as substrates . Substantial changes in k(cat), K(m)(DMAPP), and/or K(m)(RNA) were seen for several of the mutants, suggesting possible roles for these residues in substrate binding and catalysis.

Biochemistry, 2001 Feb 13, 40(6), 1679 - 87
Activation of prostate-specific antigen precursor (pro-PSA) by prostin, a novel human prostatic serine protease identified by degenerate PCR; Takayama TK et al.; A novel serine protease was found in human prostate by degenerate oligonucleotide PCR amplification and cloned . The zymogen form of this enzyme, named prostinogen, is composed of 240 amino acid residues with an amino-terminal propiece of 5 residues and a 235-residue mature enzyme . The transcript has a signal peptide of 15 amino acid residues . The mature enzyme has 41% sequence identity with prostate specific antigen (PSA) . Prostinogen was expressed in Escherichia coli and refolded from inclusion bodies . The zymogen, with a molecular mass of 28 kDa, was readily activated by agarose-immobilized trypsin to generate prostin, a serine protease, which cleaves the chromogenic substrate (N-benzoyl-L-Ile-L-Glu-L-Gly-L-Arg-p-nitroaniline hydrochloride) (S-2222) . Recombinant prostin readily activates the precursor of PSA (pro-PSA) by cleavage of the amino terminal Arg(7)-Ile(8) peptide bond . These results indicate that prostin may be a physiological activator of pro-PSA following its own proteolytic activation, as part of a cascade system involving a series of serine protease precursor proteins in the prostate.

Methods, 2001 May, 24(1), 71 - 80
Site- and time-specific gene targeting in the mouse; Metzger D et al.; The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases . We have established a conditional site-specific recombination system in mice using a new version of the Cre/lox system . The Cre recombinase has been fused to a mutated ligand binding domain of the human estrogen receptor (ER), resulting in a tamoxifen-dependent Cre recombinase, Cre-ER(T), that is activated by tamoxifen, but not by estradiol . Transgenic mice were generated expressing Cre-ER(T) under the control of a cytomegalovirus promoter . Administration of tamoxifen to these transgenic mice induced excision of a chromosomally integrated gene flanked by loxP sites in a number of tissues, whereas no excision could be detected in untreated animals . However, the efficiency of excision varied between tissues, and the highest level (approximately 40%) was obtained in the skin . To determine the efficiency of excision mediated by Cre-ER(T) in a given cell type, Cre-ER(T)-expressing mice were crossed with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination . The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice . Site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER(T) . These results indicate that cell-specific expression of Cre-ER(T) in transgenic mice can be used for efficient tamoxifen-dependent Cre-mediated recombination at loci containing loxP sites, to generate site-specific somatic mutations in a spatiotemporally controlled manner . This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting .

J Mol Biol, 2001 May 4, 308(3), 541 - 53
Conformational spread in a ring of proteins: a stochastic approach to allostery; Duke TA et al.; We recently suggested that the sensitivity and range of a cluster of membrane receptors in bacteria would be enhanced by cooperative interactions between neighbouring proteins . Here, we examine the consequences of this "conformational spread" mechanism for an idealised one-dimensional system comprising a closed ring of identical allosteric protomers (protein molecules, or a group of protein domains operating as a unit) . We show analytically and by means of Monte Carlo simulations that a ring of allosteric protomers can exhibit a switch-like response to changes in ligand concentration . We derive expressions for the sensitivity and cooperativity of switching and show that the maximum sensitivity is proportional to the number of protomers in the ring . A ring of this kind can reproduce the sensitivity and kinetics of the switch complex of a bacterial flagellar motor, for example, which is based on a ring of 34 FliM proteins . We also compare smaller rings of conformationally coupled protomers to classical allosteric proteins such as haemoglobin and show that the canonical MWC and KNF models arise naturally as limiting cases . Conformational spread appears to be a natural extension of the familiar mechanism of allostery: a physically realistic mechanism that should apply widely to many structures built from protein molecules .

J Mol Biol, 2001 May 4, 308(3), 527 - 39
Expression of the Fabs of human auto-antibodies in Escherichia coli: optimization and determination of their fine binding characteristics and cross-reactivity; Kumar S et al.; The Fabs of three human auto-antibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned and expressed in Escherichia coli, and their relative binding to single-stranded or double-stranded DNA or to cardiolipin has been assessed in the presence of modulators (salts and serum) . We describe optimized conditions that have led to significant improvement in the quality and quantity of the purified auto-antibodies . Protein expression of the assembled and functionally active Fabs was achievable with a yield of up to 5 to 9 mg/l of culture . The comparative DNA/cardiolipin-binding analyses of the nine Fabs in the presence of modulators demonstrated that B3 and 33H11 L chains possess both anti-DNA and anti-cardiolipin activities . This is the first report of the demonstration that both anti-DNA and anti-cardiolipin activities may lie on the same light chain of a human auto-antibody . We provide evidence that the auto-antibodies that appeared to be similar, in that they bound DNA or cardiolipin in conventional ELISA immunoassays, exhibited significant difference in their cross-reactivity and binding to the antigen in the presence of modulators . Such auto- antigen specificity and/or cross-reactivity may dictate the potential of an auto-antibody to cause pathogenicity and may provide an explanation as to why apparently similar auto-antibodies behave differently in vivo .

J Mol Biol, 2001 May 4, 308(3), 457 - 63
Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction; Vila-Sanjurjo A et al.; Interactions within the decoding center of the 30 S ribosomal subunit have been investigated by constructing all 15 possible mutations at nucleotides C1402 and A1500 in helix 44 of 16 S rRNA . As expected, most of the mutations resulted in highly deleterious phenotypes, consistent with the high degree of conservation of this region and its functional importance . A total of seven mutants were viable under conditions where the mutant ribosomes comprised 100 % of the ribosomal pool . A suppressor mutation specific for the C1402U-A1500G mutant was isolated at position 1520 in helix 45 of 16 S rRNA . In addition, lack of dimethylation of A1518/A1519 caused by mutation of the ksgA methylase enhanced the deleterious effect of many of the 1402/1500 mutations . These data suggest that a higher-order interaction between helices 44 and 45 in 16 S rRNA is important for the proper functioning of the ribosome . This is consistent with the recent high-resolution crystal structures of the 30 S subunit, which show a tertiary interaction between the 1402/1500 region of helix 44 and the dimethyl A stem loop .

J Mol Biol, 2001 Apr 27, 308(2), 409 - 22
Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin; Kim JM et al.; Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium . The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore . In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations . These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles . Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements . In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band . The position of the equilibrium depended on temperature, SDS and relative DMPC concentration . The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity . All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced . These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein . An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants . This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles . The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable .

J Mol Biol, 2001 Apr 27, 308(2), 131 - 45
Efficient transcriptional antitermination from the Escherichia coli cytoplasmic membrane; Gorke B et al.; The BglG protein is a transcriptional antiterminator acting within the beta-glucoside operon of Escherichia coli by binding to a specific sequence motif in the growing mRNA . Binding of BglG prevents formation of the terminator stem-loop structure, thereby causing the RNA polymerase to continue transcription . Activity of BglG is modulated in a complex way by antagonistically acting phosphorylations in response to the availability of beta-glucosidic substrates and to the catabolic state of the cell . The enzymes responsible for these phosphorylations are members of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) that represents a central carbohydrate uptake and signal transduction system . As these enzymes are believed to all form higher-order complexes associated with the cytoplasmic membrane, we tested whether or not BglG would remain active when artificially anchored to its presumptive site of regulation, the inner membrane . We show that the membrane-anchored protein indeed efficiently catalyzes transcriptional antitermination . Moreover, the membrane-attached BglG remains regulated by the PTS . Thus, a membrane-bound regulatory RNA binding protein can potentially interact fast enough with its target within the nascent transcript and cause the transcriptional machinery to proceed, before transcriptional termination would occur . Consequently, there is no principal necessity for an RNA-binding transcriptional regulator like BglG to leave the inner membrane, a potential regulatory site, and migrate to the site of transcription, the nucleoid .

Bioorg Med Chem Lett, 2001 Apr 23, 11(8), 965 - 8
Vanilloid and isovanilloid analogues as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases; Lee J et al.; As aminoacyl adenylate surrogates, a series of methionyl and isoleucyl phenolic analogues containing bioisosteric linkers mimicking ribose have been investigated . Inhibition of synthesized compounds to the aminoacylation reaction by the corresponding Escherichia coli methionyl-tRNA and isoleucyl-tRNA synthetases indicated that 18 was found to be a potent inhibitor of isoleucyl-tRNA synthetase . A molecular modeling study demonstrated that in 18, isovanillate and hydroxamate served as proper surrogates for adenine and ribose in isoleucyl adenylate, respectively.

Bioorg Med Chem Lett, 2001 Apr 23, 11(8), 961 - 4
Ester and hydroxamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases; Lee J et al.; The structure activity relationship on a series of ester and hydroxamate analogues of methionyl and isoleucyl adenylate has been investigated through introducing linkers between the 1'-position of ribose and adenine surrogates as methionyl-tRNA, and isoleucyl-tRNA synthetase inhibitors, respectively . The results indicate that ester analogue 23 was found to be a potent inhibitor of Escherichia coli methionyl-tRNA synthetase, and its interaction with the active site was proposed by a molecular modeling study.

IUBMB Life, 2000 Dec, 50(6), 355 - 9
Genetic instabilities of triplet repeat sequences by recombination; Jakupciak JP et al.; The expansion of triplet repeat sequences is an initial step in the disease etiology of a number of hereditary neurological disorders in humans . Diseases such as myotonic dystrophy, Huntington's, several spinocerebellar ataxias, fragile X syndrome, and Friedreich's ataxia are caused by the expansions of CTG.CAG, CGG.CCG, or GAA.TTC repeats . The mechanisms of the expansion process have been investigated intensely in E . coli, yeast, transgenic mice, mammalian cell culture, and in human clinical cases . Whereas studies from 1994-1999 have implicated DNA replication and repair at the paused synthesis sites due to the unusual conformations of the triplet repeat sequences, recent work has shown that homologous recombination (gene conversion) is a powerful mechanism for generating massive expansions, in addition to, or in concert with, replication and repair.

Am J Hum Genet, 2001 Jun, 68(6), 1353 - 60 Epub 2001 Apr 20.
Missense mutations in the N-terminal domain of human phenylalanine hydroxylase interfere with binding of regulatory phenylalanine; Gjetting T et al.; Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene . Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation . In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective . Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway . Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH . To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2-120) of human PAH were expressed as fusion proteins in Escherichia coli . Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity . Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency.

Neoplasia, 2001 Jan-Feb, 3(1), 53 - 61
Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer . Implications for tumor angiogenesis and metastasis; Gallo O et al.; We evaluated the role of COX-2 pathway in 35 head and neck cancers (HNCs) by analyzing COX-2 expression and prostaglandin E2 (PGE2) production in relation to tumor angiogenesis and lymph node metastasis . COX-2 activity was also correlated to vascular endothelial growth factor (VEGF) mRNA and protein expression . COX-2 mRNA and protein expression was higher in tumor samples than in normal mucosa . PGE2 levels were higher in the tumor front zone in comparison with tumor core and normal mucosa (P<.0001) . Specimens from patients with lymph node metastasis exhibited higher COX-2 protein expression (P=.0074), PGE2 levels (P=.0011) and microvessel density (P<.0001) than specimens from patients without metastasis . A significant correlation between COX-2 and tumor vascularization (r(s)=0.450, P=.007) as well as between COX-2 and microvessel density with VEGF expression in tumor tissues was found (r(s)=0.450, P=.007; r(s)=0.620, P=.0001, respectively) . The induction of COX-2 mRNA and PGE2 synthesis by EGF and Escherichia coli lipopolysaccharide (LPS) in A-431 and SCC-9 cell lines, resulted in an increase in VEGF mRNA and protein production . Indomethacin and celecoxib reversed the EGF- and LPS-dependent COX-2, VEGF, and PGE2 increases . This study suggests a central role of COX-2 pathway in HNC angiogenesis by modulating VEGF production and indicates that COX-2 inhibitors may be useful in HNC treatment.

Science, 2001 Apr 27, 292(5517), 730 - 3
Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins; Toulokhonov I et al.; DNA, RNA, and regulatory molecules control gene expression through interactions with RNA polymerase (RNAP) . We show that a short alpha helix at the tip of the flaplike domain that covers the RNA exit channel of RNAP contacts a nascent RNA stem-loop structure (hairpin) that inhibits transcription, and that this flap-tip helix is required for activity of the regulatory protein NusA . Protein-RNA cross-linking, molecular modeling, and effects of alterations in RNAP and RNA all suggest that a tripartite interaction of RNAP, NusA, and the hairpin inhibits nucleotide addition in the active site, which is located 65 angstroms away . These findings favor an allosteric model for regulation of transcript elongation.

J Clin Microbiol, 2001 May, 39(5), 1932 - 7
The common ovine Shiga toxin 2-containing Escherichia coli serotypes and human isolates of the same serotypes possess a Stx2d toxin type; Ramachandran V et al.; Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease . In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models . We have characterized the stx(2) subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans . The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep . The major stx(2) subtype in the ovine isolates was shown to be stx(2d-Ount) (119 of 146 {81.5%}) and was predominantly associated with serotypes O75:H(-)/H8/H40, O91:H(-), O123:H(-), O128:H2, and OR:H2 . However, 17 of 18 (94.4%) ovine isolates of serotype O5:H(-) possessed a stx(2d-O111/OX3a) subtype . Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a stx(2d) (stx(2d-Ount/O111/OX3a)) subtype . These studies suggest that a specific stx(2) subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E . coli during outbreaks of STEC-associated diseases in humans.

J Biol Chem, 2001 Jul 6, 276(27), 25294 - 301 Epub 2001 Apr 26.
X-ray crystal structure of the trimeric N-terminal domain of gephyrin; Sola M et al.; Gephyrin is a ubiquitously expressed protein that, in the central nervous system, forms a submembraneous scaffold for anchoring inhibitory neurotransmitter receptors in the postsynaptic membrane . The N- and C-terminal domains of gephyrin are homologous to the Escherichia coli enzymes MogA and MoeA, respectively, both of which are involved in molybdenum cofactor biosynthesis . This enzymatic pathway is highly conserved from bacteria to mammals, as underlined by the ability of gephyrin to rescue molybdenum cofactor deficiencies in different organisms . Here we report the x-ray crystal structure of the N-terminal domain (amino acids 2-188) of rat gephyrin at 1.9-A resolution . Gephyrin-(2-188) forms trimers in solution, and a sequence motif thought to be involved in molybdopterin binding is highly conserved between gephyrin and the E . coli protein . The atomic structure of gephyrin-(2-188) resembles MogA, albeit with two major differences . The path of the C-terminal ends of gephyrin-(2-188) indicates that the central and C-terminal domains, absent in this structure, should follow a similar 3-fold arrangement as the N-terminal region . In addition, a central beta-hairpin loop found in MogA is lacking in gephyrin-(2-188) . Despite these differences, both structures show a high degree of surface charge conservation, which is consistent with their common catalytic function.

J Biol Chem, 2001 Jul 20, 276(29), 27178 - 87 Epub 2001 Apr 26.
GGA*TCC-interrupted triplets in long GAA*TTC repeats inhibit the formation of triplex and sticky DNA structures, alleviate transcription inhibition, and reduce genetic instabilities; Sakamoto N et al.; Large expansions of GAA.TTC repeats in the first intron of the frataxin (X25) gene are the principal mutation responsible for Friedreich's ataxia (FRDA) . Sticky DNA, based on R.R.Y triplexes, was found at the expanded GAA.TTC repeats from FRDA patients . The (GAAGGA.TCCTTC)(65) repeat occurs in the same frataxin locus but is nonpathogenic and does not form sticky DNA . To elucidate the behavior of sticky DNA, we introduced various extents of GGA.TCC interruptions into the long GAA.TTC repeat . More than 20% of GGA.TCC interruptions abolished the formation of sticky DNA . However, the GAA.TTC repeats with less than 11% of GGA.TCC interruptions formed triplexes and/or sticky DNA similar to the uninterrupted repeat sequence . These triplexes showed different P1 nuclease sensitivities, and the GGA.TCC interruptions were slightly more sensitive than the surrounding GAA.TTC repeats . Furthermore, genetic instability investigations in Escherichia coli revealed that a small number (4%) of interruptions substantially stabilized the long GAA.TTC tracts . Furthermore, the greater the extent of interruptions of the GAA.TTC repeats, the less inhibition of in vitro transcription was observed, as expected, based on the capacity of interruptions to inhibit the formation of sticky DNA . We propose that the interruptions introduce base mismatches into the R.R.Y triplex, which explains the observed chemical and biological properties.

J Biol Chem, 2001 Jun 29, 276(26), 23349 - 56 Epub 2001 Apr 26.
Calmodulin activates intersubunit electron transfer in the neuronal nitric-oxide synthase dimer; Panda K et al.; Neuronal nitric oxide synthase (nNOS) is composed of an oxygenase domain that binds heme, (6R)-tetrahydrobiopterin, and Arg, coupled to a reductase domain that binds FAD, FMN, and NADPH . Activity requires dimeric interaction between two oxygenase domains and calmodulin binding between the reductase and oxygenase domains, which triggers electron transfer between flavin and heme groups . We constructed four different nNOS heterodimers to determine the path of calmodulin-induced electron transfer in a nNOS dimer . A predominantly monomeric mutant of rat nNOS (G671A) and its Arg binding mutant (G671A/E592A) were used as full-length subunits, along with oxygenase domain partners that either did or did not contain the E592A mutation . The E592A mutation prevented Arg binding to the oxygenase domain in which it was present . It also prevented NO synthesis when it was located in the oxygenase domain adjacent to the full-length subunit . However, it had no effect when present in the full-length subunit (i.e . the subunit containing the reductase domain) . The active heterodimer (G671A/E592A full-length subunit plus wild type oxygenase domain subunit) showed remarkable similarity with wild type homodimeric nNOS in its catalytic responses to five different forms and chimeras of calmodulin . This reveals an active involvement of calmodulin in supporting transelectron transfer between flavin and heme groups on adjacent subunits in nNOS . In summary, we propose that calmodulin functions to properly align adjacent reductase and the oxygenase domains in a nNOS dimer for electron transfer between them, leading to NO synthesis by the heme.

J Bacteriol, 2001 May, 183(10), 3149 - 59
The phosphoryl transfer domain of UhpB interacts with the response regulator UhpA; Wright JS 3rd et al.; Bacterial two-component regulatory systems control the expression of target genes through regulated changes in protein phosphorylation . Signal reception alters the ability of a membrane-bound histidine kinase (HK) protein to transfer phosphate from ATP to a highly conserved histidine residue . The transfer of phosphate from the histidine to an aspartate residue on the cognate response regulator (RR) changes the ability of the latter protein to bind to target DNA sequences and to alter gene transcription . UhpB is the HK protein which controls production of the sugar phosphate transporter UhpT . Elevated expression of full-length UhpB or of a soluble hybrid protein, GST-Bc, which is glutathione S-transferase (GST) fused to the cytoplasmic C-terminal portion of UhpB, results in complete blockage of uhpT expression in a uhp(+) strain . This dominant-negative interference could result from the ability of GST-Bc to bind and sequester the RR UhpA and to accelerate its dephosphorylation . The portion of GST-Bc responsible for the interference phenotype was localized using truncation, linker insertion, and point mutations to the region between residues 293 and 366 flanking His-313, the putative site of autophosphorylation . Point mutations which allow GST-Bc to activate uhpT expression or which relieve the interference phenotype were obtained at numerous sites throughout this region . This region of UhpB is related to the phosphoryl transfer domain of EnvZ, which forms half of an interdimer four-helix bundle and is responsible for dimerization of its cytoplasmic domain . The expression of GST fusion proteins carrying the corresponding portions of EnvZ strongly interfered with the activation of porin gene expression by OmpR . The GST-Bc protein accelerated dephosphorylation of P-UhpA . Reverse transfer of phosphate from P-UhpA to GST-Bc was observed in the presence of the metal chelator EDTA and depended on the presence of His-313 . Phosphate transfer from P-UhpA to the liberated phosphoryl transfer domain also occurred . Taken together, these results indicate that the phosphoryl transfer-dimerization domain of UhpB participates in the specific binding of UhpA, in the control of autokinase activity, and in the dephosphorylation of P-UhpA.

Circ Res, 2001 Apr 27, 88(8), 832 - 8
Attenuation of hypoxic pulmonary vasoconstriction by endotoxemia requires 5-lipoxygenase in mice; Ichinose F et al.; Sepsis and endotoxemia impair hypoxic pulmonary vasoconstriction (HPV), thereby reducing systemic oxygenation . To assess the role of leukotrienes (LTs) in the attenuation of HPV during endotoxemia, the increase in left lung pulmonary vascular resistance (LPVR) before and during left mainstem bronchus occlusion (LMBO) was measured in mice with and without a deletion of the gene encoding 5-lipoxygenase (5-LO) . LMBO increased the LPVR equally in saline-challenged wild-type and 5-LO-deficient mice (96+/-20% and 94+/-19%, respectively) . Twenty-two hours after challenge with Escherichia coli endotoxin, the ability of LMBO to increase LPVR was markedly impaired in wild-type mice (27+/-7%; P<0.05) but not in 5-LO-deficient mice (72+/-9%) or in wild-type mice pretreated with MK886, an inhibitor of 5-LO activity (76+/-10%) . Compared with wild-type mice, endotoxin-induced disruption of lung structures and inflammatory cell influx in the lung were markedly attenuated in 5-LO-deficient mice . Administration of MK571, a selective cysteinyl LT(1) receptor antagonist, 1 hour before endotoxin challenge preserved HPV and attenuated pulmonary injury in wild-type mice but did not prevent the endotoxin-induced increase in pulmonary myeloperoxidase activity . Taken together, these findings demonstrate that a 5-LO product, most likely a cysteinyl LT, contributes to the attenuation of HPV and to pulmonary injury after challenge with endotoxin.

Biophys J, 2001 May, 80(5), 2396 - 408
Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy; Harms GS et al.; The spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells . We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis . It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of approximately 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm2, and photobleaching yields from 10(-4) to 10(-5) . Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo . This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research . From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.

Biophys J, 2001 May, 80(5), 2198 - 206
Chemically charging the pore constriction opens the mechanosensitive channel MscL; Yoshimura K et al.; MscL is a bacterial mechanosensitive channel that protects the cell from osmotic downshock . We have previously shown that substitution of a residue that resides within the channel pore constriction, MscL's Gly-22, with all other 19 amino acids affects channel gating according to the hydrophobicity of the substitution () . Here, we first make a mild substitution, G22C, and then attach methanethiosulfonate (MTS) reagents to the cysteine under patch clamp . Binding MTS reagents that are positively charged ({2-(trimethylammonium)ethyl} methanethiosulfonate and 2-aminoethyl methanethiosulfonate) or negatively charged (sodium (2-sulfonatoethyl)methanethiosulfonate) causes MscL to gate spontaneously, even when no tension is applied . In contrast, the polar 2-hydroxyethyl methanethiosulfonate halves the threshold, and the hydrophobic methyl methanethiolsulfonate increases the threshold . These observations indicate that residue 22 is in a hydrophobic environment before gating and in a hydrophilic environment during opening to a substate, a finding consistent with our previous study . In addition, we have found that cysteine 22 is accessible to reagents from the cytoplasmic side only when the channel is opened whereas it is accessible from the periplasmic side even in the closed state . These results support the view that exposure of hydrophobic surfaces to a hydrophilic environment during channel opening serves as the barrier to gating.

Chem Biol, 2001 Apr, 8(4), 301 - 12
Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI; Chen H et al.; BACKGROUND: Coumarin group antibiotics, such as novobiocin, coumermycin A1 and clorobiocin, are potent inhibitors of DNA gyrase . These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxy-coumarin moiety as their structural core . Prior labeling experiments on novobiocin established that the coumarin moiety was derived from L-tyrosine, probably via a beta-hydroxy-tyrosine (beta-OH-Tyr) intermediate . Recently the novobiocin gene cluster from Streptomyces spheroides was cloned and sequenced and allows analysis of the biosynthesis of the coumarin at the biochemical level using overexpressed and purified proteins . RESULTS: Two open reading frames (ORFs), NovH and NovI, from the novobiocin producer S . spheroides have been overexpressed in Escherichia coli, purified and characterized for tyrosine activation and oxygenation which are the initial steps in coumarin formation . The 65 kDa NovH has two predicted domains, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent of non-ribosomal peptide synthetases . Purified NovH catalyzes L-tyrosyl-AMP formation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme intermediate on the holo PCP domain . The second enzyme in the pathway, NovI, is a 45 kDa heme protein that functions as a cytochrome P450-type monooxygenase with specificity for the tyrosyl-S-NovH acyl enzyme . The product beta-OH-tyrosyl-S-NovH was detected by alkaline release and high performance liquid chromatography analysis of radioactive {3H}beta-OH-Tyr and by mass spectrometry . Also detected was 4-OH-benzaldehyde, a retro aldol breakdown product of beta-OH-Tyr . The amino acid released was (3R,2S)-3-OH-Tyr by comparison with authentic standards . CONCLUSIONS: This work establishes that NovH and NovI are responsible for the formation of a beta-OH-Tyr intermediate that is covalently tethered to NovH in novobiocin biosynthesis . Comparable A-PCP/P450 pairs for amino acid beta-hydroxylation are found in various biosynthetic gene clusters, such as ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in the coumermycin A1 cluster for tyrosine, and NikP1/NikQ in the nikkomycin cluster for histidine . This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification by downstream tailoring enzymes, could represent a common strategy for controlling the partitioning of the amino acid for incorporation into secondary metabolites.

Biochim Biophys Acta, 2001 May 3, 1526(2), 168 - 74
Characterization of monoclonal antibodies against ascorbate peroxidase isoenzymes: purification and epitope-mapping using immunoaffinity column chromatography; Yoshimura K et al.; We have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX) isoenzymes to analyze the cross-reactivity and the structure of the epitopes for each monoclonal antibody . All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others . Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the absorption on the corresponding immunoaffinity column . The cyt-mAb1 reacted with a peptide fragment containing the distal His region obtained by the lysyl endopeptidase digestion . The chl-mAb6 was capable of binding to the fragment, D-I-K-E-K-R, which is consistent with an inherent region of chloroplastic isoenzymes . No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage . We concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.

J Anim Sci, 2001 Apr, 79(4), 907 - 11
Factors influencing consumer demand for U.S . pork exported to the Republic of Korea (South Korea); Vonada ML et al.; The potential market for single-ribbed bellies and Boston butts in South Korea was characterized and quantitative selection criteria were identified for use by U.S . packers when selecting pork for export . South Korean retail meat market managers and traders/wholesalers in Seoul and Pusan were interviewed and asked to identify the quality attributes that are considered when making pork-purchasing decisions . In addition, pork labeling characteristics and meat display case measurements and space allocations were recorded in each retail store . Data from box labels were recorded in retail storage coolers to characterize pork products currently being merchandized in South Korea . Sample retail packages of belly and butt slices were collected and sent to a commercial laboratory for analysis of iodine values, ether-extractable fat content, total aerobic plate count (APC), total coliform count (TCC), and generic Escherichia coli count (ECC) . No quality attributes of U.S . products exceeded the expectations of retailers . Quality attributes of U.S . pork products that exceeded the expectations of traders included presence of foreign material, marbling, tenderness, juiciness, flavor, and overall eating satisfaction . Traders/wholesalers assigned negative ratings for overall workmanship and adherence to purchase criteria for U.S . pork products . Retail APC for South Korean belly samples were higher (P < 0.05) than APC for U.S . belly samples . Retail TCC and ECC for butts and belly samples and APC for butt samples did not differ by country of origin . Retail prices for South Korean bellies were higher (P < 0.05) than prices for retail U.S . and Danish bellies . Pork butt prices did not differ (P > 0.05) by country of origin . Beef, pork, and poultry products comprised 66.8, 27.8, and 5.4%, respectively, of the total meat display case frontage . U.S . beef products occupied, on average, 18% of the total beef display area, whereas U.S . pork products comprised 2.6% of the total pork display case area.

Cell Biochem Biophys, 2000, 33(3), 241 - 52
Role of modified nucleosides of yeast tRNA(Phe) in ribosomal binding; Ashraf SS et al.; Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition . The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined . Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)) . The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding . In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability . With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop . The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop . With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL . Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site . In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe) . Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop . Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.

Cell Biochem Biophys, 2000, 33(2), 149 - 69
Thermodynamic molecular switch in macromolecular interactions; Chun PW; It is known that most living systems can live and operate optimally only at a sharply defined temperature, or over a limited temperature range, at best, which implies that many basic biochemical interactions exhibit a well-defined Gibbs free energy minimum as a function of temperature . The Gibbs free energy change, deltaG(o) (T), for biological systems shows a complicated behavior, in which deltaG(o)(T) changes from positive to negative, then reaches a negative value of maximum magnitude (favorable), and finally becomes positive as temperature increases . The critical factor in this complicated thermodynamic behavior is a temperature-dependent heat capacity change (deltaCp(o)(T) of reaction, which is positive at low temperature, but switches to a negative value at a temperature well below the ambient range . Thus, the thermodynamic molecular switch determines the behavior patterns of the Gibbs free energy change, and hence a change in the equilibrium constant, Keq, and/or spontaneity . The subsequent, mathematically predictable changes in deltaH(o)(T), deltaS(o)(T), deltaW(o)(T), and deltaG(o)(T) give rise to the classically observed behavior patterns in biological reactivity, as demonstrated in three interacting protein systems: the acid dimerization reaction of alpha-chymotrypsin at low pH, interaction of chromogranin A with the intraluminal loop peptide of the inositol 1,4,5-triphosphate receptor at pH 5.5, and the binding of L-arabinose and D-galactose to the L-arabinose binding protein of Escherichia coli . In cases of protein unfolding of four mutants of phage T4 lysozyme, no thermodynamic molecular switch is observed.

Nat Struct Biol, 2001 May, 8(5), 467 - 72
The structure of ADP-ribose pyrophosphatase reveals the structural basis for the versatility of the Nudix family; Gabelli SB et al.; Regulation of cellular levels of ADP-ribose is important in preventing nonenzymatic ADP-ribosylation of proteins . The Escherichia coli ADP-ribose pyrophosphatase, a Nudix enzyme, catalyzes the hydrolysis of ADP-ribose to ribose-5-P and AMP, compounds that can be recycled as part of nucleotide metabolism . The structures of the apo enzyme, the active enzyme and the complex with ADP-ribose were determined to 1.9 A, 2.7 A and 2.3 A, respectively . The structures reveal a symmetric homodimer with two equivalent catalytic sites, each formed by residues of both monomers, requiring dimerization through domain swapping for substrate recognition and catalytic activity . The structures also suggest a role for the residues conserved in each Nudix subfamily . The Nudix motif residues, folded as a loop-helix-loop tailored for pyrophosphate hydrolysis, compose the catalytic center; residues conferring substrate specificity occur in regions of the sequence removed from the Nudix motif . This segregation of catalytic and recognition roles provides versatility to the Nudix family.

Nat Struct Biol, 2001 May, 8(5), 459 - 66
Crystal structure of proteolytic fragments of the redox-sensitive Hsp33 with constitutive chaperone activity; Kim SJ et al.; Heat shock protein 33 (Hsp33) inhibits aggregation of partially denatured proteins during oxidative stress . The chaperone activity of Hsp33 is unique among heat shock proteins because the activity is reversibly regulated by cellular redox status . We report here the crystal structure of the N-terminal region of Hsp33 fragments with constitutive chaperone activity . The structure reveals that the N-terminal portion of Hsp33 forms a tightly associated dimer formed by a domain crossover . A concave groove on the dimeric surface contains an elongated hydrophobic patch that could potentially bind denatured protein substrates . The termini of the subunits are located near the hydrophobic patch, indicating that the cleaved C-terminal domain may shield the hydrophobic patch in an inactive state . Two of the four conserved zinc-coordinating cysteines are in the end of the N-terminal domain, and the other two are in the cleaved C-terminal domain . The structural information and subsequent biochemical characterizations suggest that the redox switch of Hsp33 occurs by a reversible dissociation of the C-terminal regulatory domain through oxidation of zinc-coordinating cysteines and zinc release.

Nat Struct Biol, 2001 May, 8(5), 432 - 6
Gal repressosome contains an antiparallel DNA loop; Geanacopoulos M et al.; Gal repressosome assembly and repression of the gal operon in Escherichia coli occurs when two dimeric GalR proteins and the histone-like HU protein bind to cognate sites causing DNA looping . Structure-based genetic analysis defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure . Stereochemical models of the four possible DNA loops compatible with the GalR tetramer configuration were constructed using the sequence-dependent structural parameters of the interoperator DNA and conformation changes caused by GalR and asymmetric HU binding . Evaluation of their DNA elastic energies gave unambiguous preference to a loop structure in which the two gal operators adopt an antiparallel orientation causing undertwisting of DNA.

Nat Struct Biol, 2001 May, 8(5), 427 - 32
Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange; Brehmer D et al.; The Hsp70 chaperone activity in protein folding is regulated by ATP-controlled cycles of substrate binding and release . Nucleotide exchange plays a key role in these cycles by triggering substrate release . Structural searches of Hsp70 homologs revealed three structural elements within the ATPase domain: two salt bridges and an exposed loop . Mutational analysis showed that these elements control the dissociation of nucleotides, the interaction with exchange factors and chaperone activity . Sequence variations in the three elements classify the Hsp70 family members into three subfamilies, DnaK proteins, HscA proteins and Hsc70 proteins . These subfamilies show strong differences in nucleotide dissociation and interaction with the exchange factors GrpE and Bag-1.

Nat Struct Biol, 2001 May, 8(5), 423 - 6
Direct structural evidence for a concerted allosteric transition in Escherichia coli aspartate transcarbamoylase; Macol CP et al.; Regulation of protein function, often achieved by allosteric mechanisms, is central to normal physiology and cellular processes . Although numerous models have been proposed to account for the cooperative binding of ligands to allosteric proteins and enzymes, direct structural support has been lacking . Here, we used a combination of X-ray crystallography and small angle X-ray scattering in solution to provide direct structural evidence that the binding of ligand to just one of the six active sites of Escherichia coli aspartate transcarbamoylase induces a concerted structural transition from the T to the R state.

J Biol Chem, 2001 Jun 29, 276(26), 23589 - 98 Epub 2001 Apr 25.
Site-specific charge interactions of alpha-conotoxin MI with the nicotinic acetylcholine receptor; Papineni RV et al.; We have tested the importance of charge interactions for alpha-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR) . Ionic residues on alpha-conotoxin MI were altered by site-directed mutagenesis or by chemical modification . In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2-4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR . It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium . Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the alphadelta site but not at the alphagamma site . It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the alphadelta site, and the two sites have distinct arrangements of charged residues . Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium . Thus, alpha-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.

Toxicol Lett, 2001 Mar 31, 120(1-3), 187 - 98
New models for assessing carcinogenesis: an ongoing process; Sills RC et al.; Traditionally, the use of rodent models in assessing the carcinogenic potential of chemicals has been expensive and lengthy, and the relevance of the carcinogenic effect to humans is often not fully understood . Today, however, with the rapid advances in molecular biology, genetically altered mice containing genes relevant to humans (e.g . oncogenes, tumor suppressor genes) and reporter genes (e.g . lacI) provide powerful tools for examining specific chemical-gene interactions thereby allowing a better understanding of the mechanisms of carcinogenesis in a shorter period of time . This paper will cover an overview of ongoing validation efforts, followed by examples of studies using several genetically engineered models including the p53def mouse model and the Big Blue transgenic mouse model . Specifically, examples where transgenic models were integrated into the testing program based on specific hypotheses dealing with genetic alterations in cancer genes and reporter genes will be discussed . The examples will highlight possible ways genetically altered mice may be integrated into a comprehensive research and testing strategy and thereby provide an improved estimation of human health risks.

Plasmid, 2001 Mar, 45(2), 63 - 74
Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1; Tian QB et al.; The hig (host inhibition of growth) gene system of plasmid Rts1 belongs to the plasmid-encoded proteic killer gene family . Among the proteic killer genes described so far, hig is unique in that the toxin gene (higB) exists upstream of the antidote gene (higA) . There are two promoters in the hig locus, Phig and PhigA, and only the former, which expresses both higB and higA genes, is negatively controlled by HigA and HigB proteins . In this study, we purified HigA protein by means of GST fusion . The electrophoretic mobility shift assay using the purified protein revealed that HigA specifically bound to the Phig region, but not to PhigA . The HigA-binding sequence was determined by DNase I footprinting assay to be a 56-bp sequence that completely covered the -35 and -10 boxes of Phig . The presence of two inverted repeats in the binding sequence and the identification of a dimer form of HigA by cross-linking experiment suggested that the protein bound to the Phig region as a dimer . HigB was purified as a GST fusion protein as well, though it was achieved only in the presence of HigA . HigA and GST-HigB formed a highly stable complex where the two proteins were present in an equimolar ratio .

Biochem Biophys Res Commun, 2001 Apr 27, 283(1), 219 - 23
Tertiary structures of the Escherichia coli and human chromosome 21 molecules of DNA; Hanzalek P et al.; Using the NDB database, we calculated geometrical parameters that were needed to reproduce crystal structures of short DNA fragments in a phosphorus atom representation . The geometrical parameters were included in a software generating tertiary structures of, for example, the Escherichia coli and human chromosome 21 molecules of DNA whose complete nucleotide sequences are deposited in the EMBL and related databases . Both molecules were found to be heavily folded and composed of domains . A more elaborate version of the present approach will make analysis and comparison possible of tertiary structures of genomic DNA molecules of various chromosomes to identify the chromosome evolutionary and functional relationships .

Biochem Biophys Res Commun, 2001 Apr 27, 283(1), 93 - 6
Noncompetitive inhibition of plant protein Ser/Thr phosphatase PP7 by phosphate; Kutuzov MA et al.; Changes in the cytoplasmic inorganic phosphate (P(i)) concentrations are an important cue for the plant cells to regulate their metabolism and phosphate homeostasis . However, phosphate sensors/receptors involved in this regulation are largely unknown . P(i) is a common nonspecific competitive inhibitor of phosphatases, usually in millimolar range . Here we report a procedure to refold recombinant Arabidopsis thaliana protein Ser/Thr phosphatase PP7 and demonstrate that PP7 is inhibited by submillimolar P(i) concentrations (IC(50) = 0.66 +/- 0.14 mM) via a mainly noncompetitive mechanism . The results indicate that PP7 may possess a specific P(i)-binding site responsible for its allosteric regulation, and suggest a possible phosphate sensor function for this protein phosphatase .

Int J Food Microbiol, 2001 Apr 11, 65(1-2), 93 - 103
Uptake of choline from salmon flesh and its conversion to glycine betaine in response to salt stress in Shewanella putrefaciens; Leblanc L et al.; When cultured in M63 minimal medium plus 0.6 M NaCl, the growth of Shewanella putrefaciens was strongly inhibited . The addition of an extract from smoked salmon to this medium restored the growth almost to the unstressed level . A comparison of the 13C NMR spectra of intracellular solutes extracted from S . putrefaciens cells cultured in both conditions revealed the accumulation of glycine betaine (GB) from the smoked salmon extract (SSE) . Analysis of the osmoprotective properties of this extract for several strains of Escherichia coli (which differ from each other in their ability to accumulate GB (i) from the surrounding environment, and (ii) from its hydroxylated precursor choline), demonstrated the absence of GB in the SSE . From the overall results, we inferred that salt-stressed S . putrefaciens cells accumulated GB from choline present in the SSE . Furthermore, the use of {14C}-labeled betaines gave evidence that S . putrefaciens (i) oxidised choline to GB, (ii) accumulated GB as a non-metabolisable osmolyte (up to 1300 nmol (mg dw)(-1) when cultured in a medium containing 0.5 M NaCl and either 1 mM choline or 1 mM GB), and (iii) both choline and GB uptake activities were osmotically upregulated (both activities were increased more than 50-fold in media containing 0.4 to 0.6 M NaCl) . In all, our results suggest that in salted smoked salmon, S . putrefaciens imports and oxidises choline, leading to the intracellular accumulation of GB.

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 273 - 83
The diheme cytochrome b subunit (Narl) of Escherichia coli nitrate reductase A (NarGHI): structure, function, and interaction with quinols; Rothery RA et al.; Significant recent advances have been made in studies of the major dissimilatory nitrate reductase (NarGHI) of Escherichia coli . This enzyme is a complex iron-sulfur ({Fe-S}) molybdoenzyme that oxidizes menaquinol or ubiquinol at a periplasmically oriented Q-site (Qp site), and reduces nitrate at a cytoplasmically-oriented molybdo-(bismolybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor . The Qp site, as well as two hemes, termed bL and bH, are localized in a hydrophobic diheme cytochrome b(Narl) that: (i) provides a conduit for electron-transfer from the periplasmically-oriented Qp-site; (ii) provides a membrane anchoring functionality for the membrane-extrinsic subunits (NarGH) that coordinate the Mo-bisMGD (NarG) and four {Fe-S} clusters (NarH); and (iii) helps ensure the separation of sites of H+-yielding and H+-consuming reactions such that enzyme turnover leads to the generation of a proton-electrochemical potential across the cytoplasmic membrane . This minireview focuses on recent advances and future prospects for the diheme cytochrome b subunit (Narl) of NarGHI.

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 215 - 8
AcrAB and related multidrug efflux pumps of Escherichia coli; Nikaido H et al.; The AcrAB system of Escherichia coli is a multidrug efflux system composed of an RND-type transporter AcrB and a periplasmic accessory protein AcrA, and pumps out a wide variety of lipophilic and amphiphilic inhibitors directly into the medium, presumably through the TolC outer membrane channel . AcrA, a highly elongated protein, is thought to bring the outer and inner membranes closer . It forms a trimer that interacts with a monomeric AcrB, which was shown by in vitro reconstitution to be a proton antiporter . Details of interaction between the

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 155 - 62
Precious things come in little packages; Schuldiner S et al.; The 110-amino acid multidrug transporter from E . coli, EmrE, is a member of the family of MiniTexan or Smr drug transporters . EmrE can transport acriflavine, ethidium bromide, tetraphenylphosphonium (TPP+), benzalkonium and several other drugs with relatively high affinities . EmrE is an H+/drug antiporter, utilizing the proton electrochemical gradient generated across the bacterial cytoplasmic membrane by exchanging two protons with one substrate molecule . The EmrE multidrug transporter is unique in its small size and hydrophobic nature . Hydropathic analysis of the EmrE sequence predicts four alpha-helical transmembrane segments . This model is experimentally supported by FTIR studies that confirm the high alpha-helicity of the protein and by high-resolution heteronuclear NMR analysis of the protein structure . The TMS of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by Cysteine scanning experiments . These results suggest the existence of a hydrophobic pathway through which the substrates are translocated . EmrE is functional as a homo-oligomer as suggested by several lines of evidence, including co-reconstitution experiments of wild-type protein with inactive mutants in which negative dominance has been observed . EmrE has only one membrane embedded charged residue, Glu-14, that is conserved in more than fifty homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters . We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site . Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions . We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons . The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes . Because of some of its properties and its size, EmrE provides a unique system to understand mechanisms of substrate recognition and translocation.

J Physiol Pharmacol, 2001 Mar, 52(1), 107 - 26
Involvement of cyclooxygenase-derived prostaglandin E2 and nitric oxide in the protection of rat pancreas afforded by low dose of lipopolysaccharide; Jaworek J et al.; Prostaglandins (PG), the products of arachidonate metabolism through cyclooxygenase (COX) pathway, protect the pancreas from the acute damage . The existence of two isoforms of COX was documented including: COX-1, present in normal tissues and COX-2, expressed at the site of inflammation, such as induced by bacterial lipopolysaccharide (LPS) . Pretreatment with low dose of LPS and activation of nitric oxide (NO) synthase (NOS) has been shown to prevent the injury caused by caerulein-induced pancreatitis (CIP) in the rat . The aim of this study was to investigate the role of COX-1 and COX-2 in the LPS-induced protection of the pancreas against CIP and the involvement of NOS in the activation of COX-PG system in the rats with CIP . CIP was produced by subcutaneous (s.c.) infusion of caerulein (5 microg/kg-h for 5 h) to the conscious rats . Protective dose of LPS, from Escherichia coli, (1 mg/kg) was given intraperitoneally (i.p.) 15 min prior to the start of CIP . Nonselective inhibitor of COX; indomethacin (5 or 10 mg/kg), selective inhibitor of COX-1: resveratrol, or a highly selective inhibitors of COX-2: rofecoxib or NS-398 (2 or 10 mg/kg) were injected i.p . 15 min prior to the administration of LPS . COX-1 or COX-2 mRNA was determined by reverse transcription-polimerase chain reaction (RT-PCR) in the pancreatic tissue . Pancreatic blood flow (PBF) was measured by a laser Doppler flowmetry . PGE2 content in the pancreas was measured by radioimmunoassay . CIP was manifested by an increase of pancreatic weight and plasma amylase activity (by 500% and 700%, respectively) and it was confirmed by histological examination . CIP slightly increased pancreatic PGE2 generation (by 12%) and diminished PBF (by about 40%) . LPS (1 mg/kg i.p.), given prior to the start of CIP, increased PGE2 generation in the pancreas (by 45%), reversed the histological manifestations of pancreatitis, reduced the rise in amylase blood level and improved PBF . Administration of nonselective inhibitor of COX; indomethacin (5 or 10 mg/kg i.p.) prior to the injection of LPS abolished its protective effects on CIP and reduced pancreatic PGE2 generation . Selective inhibitor of COX-1; resveratrol (10 mg/kg i.p.) given prior to the injection of LPS reversed its protective effects against CIP . Pretreatment with a selective inhibitors of COX-2: rofecoxib or NS-398 (10 mg/kg) attenuated LPS-induced pancreatic protection in the CIP rats . COX-1 expression was detected in the intact pancreas and was not significantly changed by CIP, LPS, indomethacin, NS-389 and their combination, while COX-2 mRNA expression appeared in the pancreas of ratssubjected to CIP and was significantly increased after LPS injection to these rats . Addition of selective COX-2 inhibitor; NS-389, or nonselective inhibitor of COX; indomethacin, enhanced COX-2 mRNA expression in the rats with CIP pretreated with LPS . Pretreatment of the rats with inhibitor of NOS; L-NNA (20 mg/kg i.p.), given together with LPS, 15 min prior to the start of caerulein overstimulation, resulted in complete reversion of LPS-induced pancreatic protection and decreased PGE2 generation stimulated by LPS . Addition to L-NNA of the substrate for NOS; L-arginine (100 mg/kg i.p.), restored pancreatic protection afforded by low dose of LPS and increased pancreatic PGE2 level in the rats with CIP . We conclude that: 1 . increased pancreatic PGE2 generation, induced by low dose LPS pretreatment, contributes to the pancreatic resistance to acute damage produced by caerulein overstimulation and 2 . the NO-system is involved in above stimulation of PGE2 generation and pancreatic protection against acute damage.

Clin Hemorheol Microcirc, 2000, 23(2-4), 177 - 83
Immunostimulating effect of pule (Alstonia scholaris L . R.Br., Apocynaceae) bark extracts; Iwo MI et al.; The immunostimulating effect of "Pule" (Alstonia scholaris (L.) R.Br., Apocynaceae) bark extracts was studied in BALB/c mouse . The extracts were administered orally, once a day for 7 consecutive days . The results showed that at the same doses (50, 100 and 200 mg/kg b.w.) the aqueous extract had higher phagocytic index (1.39-1.79) than the ethanolic extracts (0.81-0.93) in normal mice . The aqueous extract at 50 mg/kg b.w . also enhanced phagocytic activity of immunosuppressed mice significantly (P < 0.01) . At 50 and 100 mg/kg b.w . the extract prevents the decrease of immune system induced by prednisone . The aqueous extract at 100 mg/kg b.w . increased lytic activity of peritoneal exudate cells against Escherichia coli significantly (P < 0.05) . At the doses of 50 and 100 mg/kg b.w . the aqueous extract had no effect on primary antibody level . The aqueous extract at 50 mg/kg b.w . induced the cellular immune response while at 100 mg/kg b.w . inhibited the delayed type of hypersensitivity reaction.

J Pediatr Gastroenterol Nutr, 2001 Feb, 32(2), 122 - 6
Enteropathogenic Escherichia coli strain RDEC-1 produces a novel electrogenic factor active on rabbit ileum in vitro; Raimondi F et al.; BACKGROUND: Attaching and effacing Escherichia coli demonstrate marked species specificity in inducing diarrhea, although its mechanism remains largely unclear . The purpose of this study was to investigate the existence of a soluble, species-specific factor that induces diarrhea in an in vitro model . METHODS: Stripped rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after the addition of bacterial culture supernatant . RESULTS: The culture supernatant from rabbit-specific strain RDEC-1, but not from human-specific enteropathogenic Escherichia coli strain E2348/69, induced an increase in potential difference and short-circuit current in rabbit ileum mounted in Ussing chambers . This electrical signal was related to chloride ion secretion, was absent in colonic tissue, and was retained in the 30 to 100-KDa fraction of the supernatant . Preliminary experiments failed to show an involvement of calcium or cyclic nucleotides as intracellular messengers . RDEC-1 cured of a 42-MDa plasmid lost the enterotoxicity whereas conjugation of the plasmid into the negative E . coli recipient HB101 resulted in the expression of toxicity . CONCLUSIONS: The authors describe a novel, species-specific factor that helps to explain RDEC-1 diarrhea, which may be relevant to the pathogenesis of enteropathogenic Escherichia coli infection.

Inflammation, 2001 Apr, 25(2), 75 - 81
Downregulation of lipopolysaccharide-induced intercellular adhesion molecule-1 expression via EP2/EP4 receptors by prostaglandin E2 in human fibroblasts; Noguchi K et al.; In the present study, the effect of prostaglandin E2 (PGE2) on intercellular adhesion molecule-1 (ICAM-1) expression in human gingival fibroblasts (HGF) stimulated with lipopolysaccharides (LPS) was investigated . LPS were isolated from periodontopathic bacteria, Actinobacillus actinomycetemcomitans (A . actinomycetemcomitans) and Porphyromonas gingivalis (P . gingivalis), by the phenol-water method and Escherichia coli (E . coli) LPS was used as a control . PGE2 significantly inhibited A . actinomycetemcomitans-, P . gingivalis- and E . coli-LPS-induced ICAM-1 expression . Next, of four PGE2 receptor subtypes (EP1, EP2, EP3 and EP4), we examined which subtype(s) was involved in inhibition of LPS-elicited ICAM-1 expression by PGE2 . Eleven-deoxy-PGE1, a selective EP2/EP4 agonist, and butaprost, a selective EP2 agonist, attenuated A . actinomycetemcomitans-, P . gingivalis- and E . coli-LPS-elicited ICAM-1 expression, although butaprost was less potent than PGE2 and 11-deoxy-PGE1 . Sulprostone, an EP1/EP3 agonist, and ONO-AP-324, an EP3 agonist, was inert to the LPS-elicited ICAM-1 expression . Furthermore, dibutyryl cAMP, a cAMP analogue, and forskolin, an adenylate cyclase activator, downregulated A . actinomycetemcomitans-, P . gingivalis- and E . coli-LPS-elicited ICAM-1 expression in HGF . Our data suggest that PGE2 downregulates A . actinomycetemcomitans- and P . gingivalis-LPS-induced ICAM-1 expression in HGF, via EP2/EP4 receptors by cAMP-dependent signaling pathways . The cAMP-elevating agents such as EP2/EP4 receptor activators may serve to control inflammatory and immune responses in periodontal disease.

Prep Biochem Biotechnol, 2001 Feb, 31(1), 59 - 70
Polyethylene glycol enhanced refolding of the recombinant human tissue transglutaminase; Ambrus A et al.; Tissue transglutaminase forms cross-links between lysine and glutamine side-chains of polypeptide chains in a Ca2+-dependent reaction; its structural basis is still not clarified . In this study, we demonstrate that the refolding of the human recombinant enzyme molecule to its catalytically active form from inclusion bodies needs the presence of a helper material with higher molecular mass, but only in the initiation phase . Ca2+ and nucleotides are ascribed as affector molecules also in the early phase of structural reconstitution . Two optimal concentrations of polyethylene glycol and a relatively long time scale for the evolution of the final structure were identified . The optimized refolding procedure is reported.

Prep Biochem Biotechnol, 2001 Feb, 31(1), 49 - 57
Expression of anti-neuroexcitation peptide (ANEP) of scorpion Buthus martensii Karsch in Escherichia coli; Zhang JH et al.; According to the cDNA sequence of anti-neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti-neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by a PCR method, then was cloned into expression plasmid pET28a, fused with His tag at its 3' end . When expressed in E . coli BL21 (DE3), the expression of recombinant ANEP was 15% of total cellular proteins, while most recombinant ANEP products existed in the form of insoluble inclusion bodies . Coexpression of molecular chaperones or protein disulfide isomerase could not improve its solubility . The recombinant ANEP in the cell lysate was purified to homogeneity by metal chelating affinity chromatography and Superdex 30 chromatography . In bioassay with convulsive mice model induced by thiosemicarbazide, recombinant ANEP could apparently delay the convulsion seizure of model animals by 18% and showed anti-neuroexcitatory activity.

J Ind Microbiol Biotechnol, 2000 Dec, 25(6), 310 - 314
Development of an Escherichia coli expression system and thermostability screening assay for libraries of mutant xylanase; Ebanks R et al.; A thermostability screening assay was developed using an Escherichia coli expression system to express Streptomyces lividans xylanase A (XlnA) . The screening system was tested using mutants randomized at position 49 of the S . lividans XlnA gene, a position previously shown to confer thermostability with a I49P point mutation . The library was cloned into an E . coli expression vector and transformed into XL1-blue bacteria . The resulting clones were screened for increased thermostability with respect to wild-type XlnA . Using this assay, we isolated the I49P mutant previously shown to be thermostable, as well as novel I49A and I49C mutants . The I49A and I49C mutants were shown to have 2.8- to 8-fold increase in thermostability over that of wild-type XlnA . The results show that the screening assay can selectively enrich for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000-20,000 clones.

Acta Crystallogr D Biol Crystallogr, 2001 May, 57(Pt 5), 759 - 62 Epub 2001 Apr 24.
Post-translational modification of the N-terminal His tag interferes with the crystallization of the wild-type and mutant SH3 domains from chicken src tyrosine kinase; Kim KM et al.; Structural studies of the wild type and mutants of the src SH3 domain were initiated to elucidate the correlation of the native-state topology with protein thermostability and folding kinetics . An extra mass of 178 Da arising from the post-translational modification at the N-terminal His tag was observed . The spontaneous alpha-N-6 gluconoylation at the amino group of the His-tagged SH3 domain contributed to the observed extra mass . The partial modification of the N-terminal His-tag produced heterogeneity, both in size and in charge, in the Escherichia coli expressed SH3 domain . The removal of the His tag from the SH3 domain was essential for the crystallization of both wild-type and mutant src SH3 . Both the wild type and the W43I mutant were crystallized by hanging-drop vapor diffusion and are in the hexagonal space group P6(5)22 with one molecule in the asymmetric unit . Data sets were collected to 1.8 and 1.95 A resolution for the the wild type and the W43I mutant, respectively.

Acta Crystallogr D Biol Crystallogr, 2001 May, 57(Pt 5), 728 - 30 Epub 2001 Apr 24.
Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli RbsD, a component of the ribose-transport system with unknown biochemical function; Kim MS et al.; The Escherichia coli high-affinity ribose-transport system consists of six proteins encoded by the rbs operon (rbsD, rbsA, rbsC, rbsB, rbsK and rbsR) . Of the six components, RbsD is the only one whose function is unknown . In order to gain insights into the function of RbsD by structural analysis, we overexpressed and crystallized the protein as a first step toward this goal . RbsD was overexpressed in E . coli and crystallized using the hanging-drop vapour-diffusion method at 296 K . The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 285.9, b = 92.3, c = 93.3 A, beta = 105.0 degrees . The unit cell is likely to contain 64 molecules of RbsD, with a crystal volume per protein mass (V(M)) of 2.43 A(3) Da(-1) and a solvent content of about 49.3% by volume . An equilibrium centrifugation analysis demonstrated that RbsD (MW = 15 292 Da) exists as an octamer in solution, suggesting that the asymmetric unit contains two octameric assemblies of RbsD . A native data set to 2.7 A resolution was obtained from a flash-cooled crystal.

Acta Crystallogr D Biol Crystallogr, 2001 May, 57(Pt 5), 722 - 4 Epub 2001 Apr 24.
Crystallization and preliminary X-ray analysis of the complex of the epsilon-subunit and the central domain of the gamma-subunit from the Escherichia coli ATP synthase; Rodgers A et al.; A complex of the epsilon-subunit and the central domain of the gamma-subunit from the ATP synthase of Escherichia coli has been purified and crystallized and preliminary X-ray analysis has been carried out . The crystals belong to space group C222(1), with unit-cell parameters a = 76.7, b = 176.1, c = 67.1 A (at 100 K) . Determination of the structure of this protein complex promises to greatly improve the understanding of energy coupling between the F(0) and F(1) sectors within the enzyme complex.

Acta Crystallogr D Biol Crystallogr, 2001 May, 57(Pt 5), 714 - 6 Epub 2001 Apr 24.
Crystallographic characterization of the PDZ1 domain of the human Na+/H+ exchanger regulatory factor; Webster G et al.; The Na(+)/H(+) exchanger regulatory factor (NHERF) contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes . The human NHERF PDZ1 domain, which spans residues 11-99, interacts specifically with carboxy-terminal residues of the beta2 adrenergic receptor and the cystic fibrosis transmembrane conductance regulator . The NHERF PDZ1 was expressed in Escherichia coli as a soluble protein, purified and crystallized in the unbound form using the vapor-diffusion method with 2 M ammonium sulfate as the precipitant . Diffraction data were collected to 1.5 A resolution using synchrotron radiation . The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 51.6, c = 58.9 A, and one molecule in the asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2001 May, 57(Pt 5), 664 - 9 Epub 2001 Apr 24.
Structure of Escherichia coli fragment TR2C from calmodulin to 1.7 A resolution; Olsson LL et al.; Fragment TR2C is the C-terminal part of the calcium-binding protein calmodulin, including residues 78-148 . The crystal structure of TR2C was solved by molecular replacement and refined to a conventional R value of 21.8% (R(free) = 22.0%), using all data in the resolution range 20.0-1.7 A . This study shows that the secondary structure of TR2C, a pair of EF-hand motifs with two calcium-binding sites, is similar to the corresponding motifs in intact calmodulin . However, it also indicates that the N-terminus of helix E is closer to the C-terminus of helix H in TR2C than in the intact protein and that the loop connecting the EF-hands shows different conformations in the two structures . The crystal structure of TR2C was further found to be similar to the set of NMR structures of this fragment, although some pronounced differences exist.

Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 4966 - 71
The preferred stoichiometry of c subunits in the rotary motor sector of Escherichia coli ATP synthase is 10; Jiang W et al.; The stoichiometry of c subunits in the H(+)-transporting F(o) rotary motor of ATP synthase is uncertain, the most recent suggestions varying from 10 to 14 . The stoichiometry will determine the number of H(+) transported per ATP synthesized and will directly relate to the P/O ratio of oxidative phosphorylation . The experiments described here show that the number of c subunits in functional complexes of F(o)F(1) ATP synthase from Escherichia coli can be manipulated, but that the preferred number is 10 . Mixtures of genetically fused cysteine-substituted trimers (c(3)) and tetramers (c(4)) of subunit c were coexpressed and the c subunits crosslinked in the plasma membrane . Prominent products corresponding to oligomers of c(7) and c(10) were observed in the membrane and purified F(o)F(1) complex, indicating that the c(10) oligomer formed naturally . Oligomers larger than c(10) were also observed in the membrane fraction of cells expressing c(3) or c(4) individually, or in cells coexpressing c(3) and c(4) together, but these larger oligomers did not copurify with the functional F(o)F(1) complex and were concluded to be aberrant products of assembly in the membrane.

Microbiology, 2001 May, 147(Pt 5), 1315 - 22
The bio operon on the acquired symbiosis island of Mesorhizobium sp . strain R7A includes a novel gene involved in pimeloyl-CoA synthesis; Sullivan JT et al.; The symbiosis island of Mesorhizobium sp . strain R7A is a 500 kb chromosomal genetic element that upon transfer converts nonsymbiotic mesorhizobia to symbionts able to nodulate and fix nitrogen with Lotus corniculatus . Four genomic species of nonsymbiotic mesorhizobia have been isolated . All were auxotrophic for thiamin and biotin and three were auxotrophic for nicotinate, whereas derivatives of the strains containing the symbiosis island were prototrophic for all three vitamins . In this work, a 13.2 kb region of the island that converts the nonsymbionts to nicotinate and biotin prototrophy was characterized . The region contained orthologues of the Escherichia coli bioBFD and A genes arranged in an operon with a novel gene, bioZ, a nadABC operon, the nitrogen-fixation regulatory gene nifA, and a homologue of the pantothenate biosynthesis gene panD . The bioZ gene product was similar to beta-ketoacyl-acyl carrier protein synthase III (FabH) . bioZ::Tn5 mutants grew poorly in the absence of biotin and the bioZ gene complemented an E . coli bioH mutant, suggesting that its product is involved in the synthesis of pimeloyl-COA: The bio operon was not required for symbiosis, as only mutants in the nifA gene were impaired in symbiosis, and a bioA::Tn5 mutant was not impaired in rhizosphere colonization . The rationale for the vitamin biosynthetic loci being located on an acquired genetic element that is absent from nonsymbiotic mesorhizobia remains to be determined.

Microbiology, 2001 May, 147(Pt 5), 1291 - 301
Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase of Streptomyces aureofaciens requires GapR, a member of the AraC/XylS family of transcriptional activators; Sprusansky O et al.; Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is developmentally regulated, and induced by glucose in Streptomyces aureofaciens . A gene, gapR, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators was identified upstream of gap . The gapR gene was constitutively expressed from a single promoter during the course of differentiation . By integrative transformation, via double crossover, a stable null mutant of the gapR gene was obtained . The mutation only slightly affected growth, and had no effect on differentiation of S . aureofaciens . However, transcription of the GAPDH-encoding gap gene was substantially reduced in the S . aureofaciens DeltagapR null mutant, irrespective of carbon source used . Though GAPDH activity was about 1.5-fold lower in the mutant, the substantial enzyme activity remained, suggesting the presence of a second GAPDH which is sufficient to ensure growth . The GapR protein, overproduced in Escherichia coli, was shown to bind upstream of the gap-P promoter region . The results indicate a direct role of GapR in regulation of gap expression in S . aureofaciens.

Microbiology, 2001 May, 147(Pt 5), 1267 - 76
Lethality of glnD null mutations in Azotobacter vinelandii is suppressible by prevention of glutamine synthetase adenylylation; Colnaghi R et al.; GlnD is a pivotal protein in sensing intracellular levels of fixed nitrogen and has been best studied in enteric bacteria, where it reversibly uridylylates two related proteins, PII and GlnK . The uridylylation state of these proteins determines the activities of glutamine synthetase (GS) and NtrC . Results presented here demonstrate that glnD is an essential gene in Azotobacter vinelandii . Null glnD mutations were introduced into the A . vinelandii genome, but none could be stably maintained unless a second mutation was present that resulted in unregulated activity of GS . One mutation, gln-71, occurred spontaneously to give strain MV71, which failed to uridylylate the GlnK protein . The second, created by design, was glnAY407F (MV75), altering the adenylylation site of GS . The gln-71 mutation is probably located in glnE, encoding adenylyltransferase, because introducing the Escherichia coli glnE gene into MV72, a glnD(+) derivative of MV71, restored the regulation of GS activity . GlnK-UMP is therefore apparently required for GS to be sufficiently deadenylylated in A . vinelandii for growth to occur . The DeltaglnD GS(c) isolates were Nif(-), which could be corrected by introducing a nifL mutation, confirming a role for GlnD in mediating nif gene regulation via some aspect of the NifL/NifA interaction . MV71 was unexpectedly NtrC(+), suggesting that A . vinelandii NtrC activity might be regulated differently than in enteric organisms.

J Biol Chem, 2001 Jul 6, 276(27), 24704 - 12 Epub 2001 Apr 24.
Insertion of mitochondrial DNA-encoded F1F0-ATPase subunit 8 across the mitochondrial inner membrane in vitro; Ii M et al.; Cytochrome oxidase subunits I, II, and III, the mitochondrial DNA-encoded proteins, are inserted across the inner membrane by the Oxa1p-containing translocator in a membrane potential-dependent manner . Oxa1p is also involved in the insertion of the cytoplasmically synthesized precursor of Oxa1p itself into the inner membrane from the matrix via the conservative sorting pathway . The mechanism of insertion of the other mitochondrially synthesized proteins, however, is unexplored . The insertion of the mitochondrial DNA-encoded subunit 8 of F(1)F(0)-ATPase (Su8) across the inner membrane was analyzed in vitro using the inverted inner membrane vesicles and the Escherichia coli lysate-synthesized substrate . This assay revealed that the N-terminal segment of Su8 inserted across the membrane to the intermembrane space and assumed the correct trans-cis topology depending on the mitochondrial matrix fraction . This translocation reaction was similar to those of Sec-independent, direct insertion pathways of E . coli and chloroplast thylakoid membranes . (i) It required neither nucleotide triphosphates nor membrane potential, and hydrophobic forces drove the process . (ii) It did not require protease-sensitive membrane components facing the matrix space . (iii) It could be inserted across liposomes in the correct topology in a matrix fraction-dependent manner . Thus, a novel mechanism conserved in bacteria and chloroplasts also functions in the insertion of Su8 across the mitochondrial inner membrane.

Anal Biochem, 2001 May 1, 292(1), 139 - 47
An integrated system to study multiply substituted human immunodeficiency virus type 1 reverse transcriptase; Boretto J et al.; We describe a gene system allowing the facile production of multiply substituted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic background of the macrophagetropic, non-laboratory-adapted human immunodeficiency virus type 1 (HIV-1) AD8 strain . Thirteen unique silent restriction sites were introduced in the pol gene encoding HIV-1 RT, allowing easy introduction of mutations . To simplify genetic manipulation and generate p66/p51 heterodimers in Escherichia coli, a gene construct of the viral protease alone was optimized for expression from a separate vector carrying a p15A origin of replication . Active-site titration experiments using pre-steady-state kinetics showed that our system yields a higher proportion of active enzyme than that obtained by alternate methods . To facilitate phenotype/genotype correlations, the modified RT gene was designed to be easily reintroduced into a recombinant proviral AD8 HIV-1 DNA . Infectious viruses made from this vector were undistinguishable from wild-type AD8 HIV-1, an isolate able to infect peripheral blood mononuclear cells and macrophages . Thus, the pol gene can tolerate many silent mutations in the polymerase domain without affecting the functionality of the HIV-1 genome . The system was validated biochemically and virologically using the V75T substitution associated with stavudine resistance.

Anal Biochem, 2001 May 1, 292(1), 1 - 7
Microplate enzyme-linked immunosorbent assay for the detection of primary DNA alterations based on the interaction with UvrA/UvrB; Bose G et al.; An enzyme-linked microplate immunoassay for the analysis of primary DNA lesions is described . The assay principle is based on the interaction of the bacterial DNA repair proteins UvrA and UvrB with DNA and on the immunodetection of UvrB forming a stable complex with covalently modified nucleotides . Using this technique we were able to detect damages in genomic DNA induced by uv light and by several different genotoxic agents . The detection sensitivity of the method reaches down to the nanomolar range of the mutagenic compound depending on the type of the DNA alteration . The method might be used in automated high-throughput studies.

J Infect Dis, 2001 May 15, 183(10), 1526 - 9 Epub 2001 Apr 06.
Ampicillin-resistant Escherichia coli in gestational pyelonephritis: increased occurrence and association with the colonization factor Dr adhesin; Hart A et al.; The pattern of ampicillin resistance and possible association with virulence factors of 78 Escherichia coli isolates taken from 78 pregnant women with pyelonephritis were evaluated . The current incidence of ampicillin resistance among pyelonephritis isolates (46%) was significantly higher than that reported in 1985 (22%) . Resistance was found more frequently during the first (60%) and third (53%) trimesters than during the second trimester (33%) . Of all dra(+) E . coli isolates, 75% were ampicillin resistant, whereas dra(+) isolates of O75 serotype E . coli accounted for 87% of ampicillin-resistant strains . The significant increase of ampicillin resistance among gestational pyelonephritis E . coli and the association with the dra gene cluster encoding colonization and invasive capacity may warrant further study involving obstetric and neonate wards, with the latter being at the higher risk for potential problems.

J Infect Dis, 2001 May 15, 183(10), 1494 - 500 Epub 2001 Apr 16.
Characterization of the antibody response to pneumococcal glycoconjugates and the effect of heat-labile enterotoxin on IGg subclasses after intranasal immunization; Jakobsen H et al.; The antibody response to pneumococcal glycoconjugate (Pnc) was characterized by analyzing pneumococcal polysaccharide (PPS)-- and protein carrier-specific IgG subclass profiles and their relationship . Mice were immunized intranasally (inl) or subcutaneously (sc) with Pnc with mutants of Escherichia coli heat-labile enterotoxin, LT-R72 and LT-K63, as mucosal adjuvants . Subcutaneous immunization with Pnc alone induced predominantly IgG1, whereas native PPS administered sc induced very low IgG titers that were exclusively of the IgG3 subclass . Compared with sc immunization with Pnc alone, inl immunization with Pnc and LT mutants induced significantly higher systemic IgG2a, IgG3, and IgA antibodies to both PPS and the carrier, whereas the IgG1 titers were comparable . There also was a significant correlation between PPS- and protein carrier--specific antibody responses for all IgG subclasses . This demonstrates that LT mutants can be used to both enhance and modulate the antibody response to the PS moiety of glycoconjugate vaccines.

Int J Obes Relat Metab Disord, 2001 Apr, 25(4), 586 - 9
Does obesity affect febrile responsiveness?
Ivanov AI, Kulchitsky VA, Romanovsky AA.
BACKGROUND AND OBJECTIVE: A decreased resistance to infection and impairments of immunity are common in obese humans and in rodents with hereditary obesity . Since brown fat thermogenesis is also suppressed in obese rodents, we hypothesized that obesity leads to a decreased febrile responsiveness . METHODS: We compared the fever responses to intravenous E . coli lipopolysaccharide (10 microg/kg) between Zucker fa/fa (obese due to a defective leptin receptor) and Fa/? (lean) rats and between Otsuka Long-Evans Tokushima Fatty (OLETF; obese due to the lacking cholecystokinin-A receptor) and Long-Evans Tokushima Otsuka (lean) rats . Obesity of Zucker fa/fa and OLETF rats was verified by increased body mass and fat content, hypertriglyceridemia and hypercholesterolemia . RESULTS: Neither fa/fa nor OLETF animals exhibited a decreased febrile responsiveness; if anything, their fevers tended to be higher than those in their lean counterparts . CONCLUSION: Obesity per se does not lead to antipyresis.

In Vitr Mol Toxicol, 2000 Winter, 13(4), 237 - 48
N-acetyl-L-cysteine simultaneously increases mitogenesis and suppresses apoptosis in mitogen-stimulated B-lymphocytes from p53 haploinsufficient Tg.AC (v-Ha-ras) mice; Martin KR et al.; Recent epidemiological evidence suggests that antioxidants may enhance carcinogenesis by promoting cellular proliferation and/or impeding programmed cell death . We examined the effect of N-acetyl-l-cysteine (NAC) on mitogenesis and apoptosis in splenocytes from p53 haploinsufficient Tg.AC (v-Ha-ras) mice . This model contains genetic lesions found frequently in human cancer and is predisposed to develop carcinogen-induced cancer . Splenocytes were incubated with NAC alone or with the B- and T-cell-specific mitogens Concanavalin A (Con A) and E . coli lipopolysaccharide (LPS), respectively . Mitogenesis increased 17-fold in mitogen-stimulated cultures and 10-fold in cultures incubated with NAC alone . Co-incubation with both NAC (1000 microg/mL) and mitogen increased mitogenesis by 33-fold without changing apoptosis rates . Strikingly, incubation with NAC and LPS attenuated LPS-induced apoptosis . Mitogen alone did not affect GSH levels but NAC-induced increases were significantly depleted by co-incubation with mitogen . Furthermore, NAC increased the number of CD45R+ B cells, but decreased CD3+ T cells showing enhanced survival of B cells under these conditions . These results demonstrate concurrent reduced apoptosis and increased mitogenesis in B lymphocytes that may favor clonal selection of preneoplastic cells.

Mol Biol Evol, 2001 May, 18(5), 750 - 6
Structural constraints and emergence of sequence patterns in protein evolution; Parisi G et al.; The aim of this work was to study the relationship between structure conservation and sequence divergence in protein evolution . To this end, we developed a model of structurally constrained protein evolution (SCPE) in which trial sequences, generated by random mutations at gene level, are selected against departure from a reference three-dimensional structure . Since at the mutational level SCPE is completely unbiased, any emergent sequence pattern will be due exclusively to structural constraints . In this first report, it is shown that SCPE correctly predicts the characteristic hexapeptide motif of the left-handed parallel beta helix (LbetaH) domain of UDP-N-acetylglucosamine acyltransferases (LpxA).

J Biol Chem, 2001 Jul 13, 276(28), 25687 - 91 Epub 2001 Apr 23.
Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta; Asami T et al.; Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species . The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells . The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells . In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position . DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed . Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity . The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives . In brassinazole-treated A . thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs . These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.

J Biol Chem, 2001 Jun 22, 276(25), 22604 - 7 Epub 2001 Apr 23.
Iron-sulfur cluster assembly: characterization of IscA and evidence for a specific and functional complex with ferredoxin; Ollagnier-de-Choudens S et al.; The synthesis of iron-sulfur clusters in Escherichia coli is believed to require a complex protein machinery encoded by the isc (iron-sulfur cluster) operon . The product of one member of this operon, IscA, has been overexpressed, purified, and characterized . It can assemble an air-sensitive {2Fe-2S} cluster as shown by UV-visible and resonance Raman spectroscopy . The metal form but not the apoform of IscA binds ferredoxin, another member of the isc operon, selectively, allowing transfer of iron and sulfide from IscA to ferredoxin and formation of the {2Fe-2S} holoferredoxin . These results thus suggest that IscA is involved in ferredoxin cluster assembly and activation . This is an important function because a functional ferredoxin is required for maturation of other cellular Fe-S proteins.

J Biol Chem, 2001 Jun 29, 276(26), 24170 - 6 Epub 2001 Apr 23.
Design, engineering, and production of human recombinant t cell receptor ligands derived from human leukocyte antigen DR2; Chang JW et al.; Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells . Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2 . Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501) . Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients . We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (alpha1 and beta1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G . G., Chang, J . W., Bachinger, H.-P., Bourdette, D . N., Wegmann, K . W., Offner, H., and Vandenbark A . A . (1999) Protein Eng . 12, 771-778; Burrows, G . G., Adlard, K . L., Bebo, B . F., Jr., Chang, J . W., Tenditnyy, K., Vandenbark, A . A., and Offner, H . (2000) J . Immunol . 164, 6366-6371) . Single-chain human recombinant T cell receptor ligands (RTLs) of approximately 200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides . Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native HLA-DR2 heterodimer . The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85-99)) showed increased stability to thermal unfolding relative to the empty DR2-derived RTLs . These novel molecules represent a new class of small soluble ligands for modulating the behavior of T cells and provide a platform technology for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.

J Biol Chem, 2001 Jul 6, 276(27), 24667 - 73 Epub 2001 Apr 23.
Expression and characterization of recombinant rat Acyl-CoA synthetases 1, 4, and 5 . Selective inhibition by triacsin C and thiazolidinediones; Kim JH et al.; Inhibition by triacsins and troglitazone of long chain fatty acid incorporation into cellular lipids suggests the existence of inhibitor-sensitive and -resistant acyl-CoA synthetases (ACS, EC ) that are linked to specific metabolic pathways . In order to test this hypothesis, we cloned and purified rat ACS1, ACS4, and ACS5, the isoforms present in liver and fat cells, expressed the isoforms as ACS-Flag fusion proteins in Escherichia coli, and purified them by Flag affinity chromatography . The Flag epitope at the C terminus did not alter the kinetic properties of the enzyme . Purified ACS1-, 4-, and 5-Flag isoforms differed in their apparent K(m) values for ATP, thermolability, pH optima, requirement for Triton X-100, and sensitivity to N-ethylmaleimide and phenylglyoxal . The ACS inhibitor triacsin C strongly inhibited ACS1 and ACS4, but not ACS5 . The thiazolidinedione (TZD) insulin-sensitizing drugs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, troglitazone, rosiglitazone, and pioglitazone, strongly and specifically inhibited only ACS4, with an IC(50) of less than 1.5 microm . Troglitazone exhibited a mixed type inhibition of ACS4 . alpha-Tocopherol, whose ring structure forms the non-TZD portion of troglitazone, did not inhibit ACS4, indicating that the thiazolidine-2,4-dione moiety is the critical component for inhibition . A non-TZD PPARgamma ligand, GW1929, which is 7-fold more potent than rosiglitazone, inhibited ACS1 and ACS4 poorly with an IC(50) of greater than 50 microm, more than 100-fold higher than was required for rosiglitazone, thereby demonstrating the specificity of TZD inhibition . Further, the PPARalpha ligands, clofibrate and GW4647, and various xenobiotic carboxylic acids known to be incorporated into complex lipids had no effect on ACS1, -4, or -5 . These results, together with previous data showing that triacsin C and troglitazone strongly inhibit triacylglycerol synthesis compared with other metabolic pathways, suggest that ACS1 and ACS4 catalyze the synthesis of acyl-CoAs used for triacylglycerol synthesis and that lack of inhibition of a metabolic pathway by triacsin C does not prove lack of acyl-CoA involvement . The results further suggest the possibility that the insulin-sensitizing effects of the thiazolidinedione drugs might be achieved, in part, through direct interaction with ACS4 in a PPARgamma-independent manner.

J Biol Chem, 2001 Jun 29, 276(26), 23547 - 53 Epub 2001 Apr 23.
Investigating the structure of human RNase H1 by site-directed mutagenesis; Wu H et al.; In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis . The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E . coli RNase enzyme . The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E . coli RNase H1 region by a 62-amino acid sequence . These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate . Deletion of the region between the dsRNA-binding domain and the conserved E . coli RNase H1 domain resulted in a significant loss in the RNase H activity . Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate . The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme . Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity . Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.

J Biol Chem, 2001 Jun 15, 276(24), 20821 - 3 Epub 2001 Apr 23.
PAS domain receptor photoactive yellow protein is converted to a molten globule state upon activation; Lee BC et al.; Biological signaling generally involves the activation of a receptor protein by an external stimulus followed by protein-protein interactions between the activated receptor and its downstream signal transducer . The current paradigm for the relay of signals along a signal transduction chain is that it occurs by highly specific interactions between fully folded proteins . However, recent results indicate that many regulatory proteins are intrinsically unstructured, providing a serious challenge to this paradigm and to the nature of structure-function relationships in signaling . Here we study the structural changes that occur upon activation of the blue light receptor photoactive yellow protein (PYP) . Activation greatly reduces the tertiary structure of PYP but leaves the level secondary structure largely unperturbed . In addition, activated PYP exposes previously buried hydrophobic patches and allows significant solvent penetration into the core of the protein . These traits are the distinguishing hallmarks of molten globule states, which have been intensively studied for their role in protein folding . Our results show that receptor activation by light converts PYP to a molten globule and indicate stimulus-induced unfolding to a partially unstructured molten globule as a novel theme in signaling.

Appl Environ Microbiol, 2001 May, 67(5), 2270 - 5
Degradation of substituted phenylurea herbicides by Arthrobacter globiformis strain D47 and characterization of a plasmid-associated hydrolase gene, puhA; Turnbull GA et al.; Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil . This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621) . Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid . The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid . Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A . globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source . The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture . A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron . Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity . A smaller subclone of this gene (2.5 kb) expressed in E . coli coded for the protein that degraded diuron . This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca . 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).

Appl Environ Microbiol, 2001 May, 67(5), 2176 - 82
Purification of Synechocystis sp . strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity; Aboulmagd E et al.; Synechocystis sp . strain PCC6308 cyanophycin synthetase was purified 72-fold in three steps by anion exchange chromatography on Q Sepharose, affinity chromatography on the triazine dye matrix Procion Blue HE-RD Sepharose, and gel filtration on Superdex 200 HR from recombinant cells of Escherichia coli . The native enzyme, which catalyzed the incorporation of arginine and aspartic acid into cyanophycin, has an apparent molecular mass of 240 +/- 30 kDa and consists of identical subunits of 85 +/- 5 kDa . The K(m) values for arginine (49 microM), aspartic acid (0.45 mM), and ATP (0.20 mM) indicated that the enzyme had a high affinity towards these substrates . During in vitro cyanophycin synthesis, 1.3 +/- 0.1 mol of ATP per mol of incorporated amino acid was converted to ADP . The optima for the enzyme-catalyzed reactions were pH 8.2 and 50 degrees C, respectively . Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), beta-hydroxy aspartic acid (35 and 37%), aspartic acid beta-methyl ester (38 and 40%), norvaline (0 and 3%), citrulline (9 and 7%), and asparagine (2 and 0%) exhibited an almost equal inhibitory effect on the incorporation of both arginine and aspartic acid, respectively, when these compounds were added to the complete reaction mixture . In contrast, the incorporation of arginine was diminished to a greater extent than that of aspartic acid, respectively, with canavanine (82 and 53%), lysine (36 and 19%), agmatine (33 and 25%), D-aspartic acid (37 and 30%), L-glutamic acid (13 and 5%), and ornithine (23 and 11%) . On the other hand, canavanine (45% of maximum activity) and lysine (13%) stimulated the incorporation of aspartic acid, whereas aspartic acid beta-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine . {(3)H}lysine (15% of maximum activity) and {(3)H}canavanine (13%) were incorporated into the polymer, when they were either used instead of arginine or added to the complete reaction mixture, whereas L-glutamic acid was not incorporated . No effect on arginine incorporation was obtained by the addition of other amino acids (i.e., alanine, histidine, leucine, proline, tryptophan, and glycine) . Various samples of chemically synthesized poly-alpha,beta-D,L-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-alpha-L-aspartic acid was almost inactive.

Appl Environ Microbiol, 2001 May, 67(5), 2044 - 50
Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase; Nyyssola A et al.; Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure . Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor . These methyltransferases were expressed in Escherichia coli and purified, and some of their enzymatic properties were characterized . Both enzymes had high substrate specificities and pH optima near the physiological pH . No evidence of cofactors was found . The enzymes showed Michaelis-Menten kinetics for their substrates . The apparent K(m) and V(max) values were determined for all substrates when the other substrate was present in saturating concentrations . Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine . Betaine inhibited the methylation reactions only at high concentrations.

Plant J, 2001 Mar, 25(6), 687 - 98
Maize endosperm secretes a novel antifungal protein into adjacent maternal tissue; Serna A et al.; A series of endosperm transfer layer-specific transcripts has been identified in maize by differential screening of a cDNA library of transcripts at 10 days after pollination . Sequence comparisons revealed among this class of cDNAs a novel, small gene family of highly diverged sequences encoding basal layer antifungal proteins (BAPs) . The bap genes mapped to two loci on chromosomes 4 and 10 . So far, bap-homologous sequences have been detected only in maize, teosinte and sorghum, and are not present in grasses outside the Andropogoneae tribe . BAP2 is synthesized as a pre-proprotein, and is processed by successive removal of a signal peptide and a 29-residue prodomain . The proprotein can be detected exclusively in microsomal membrane-containing fractions of kernel extracts . Immunolocalization reveals BAP2 to be predominantly located in the placentochalazal cells of the pedicel, adjacent to the basal endosperm transfer layer (BETL) cells, although the BAP2 transcript is found only in the BETL cells . The biological roles of BAP2 propeptide and mature peptide have been investigated by heterologous expression of the proprotein in Escherichia coli, and by tests of its fungistatic activity and that of the fully processed form in vitro . The mature BAP2 peptide exhibits potent broad-range activity against a range of filamentous fungi, including several plant pathogens.

Plant J, 2001 Mar, 25(6), 651 - 61
Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties; Wang X et al.; The orchid Gastrodia elata depends on the fungus Armillaria mellea to complete its life cycle . In the interaction, fungal hyphae penetrate older, nutritive corms but not newly formed corms . From these corms, a protein fraction with in vitro activity against plant-pathogenic fungi has previously been purified . Here, the sequence of gastrodianin, the main constituent of the antifungal fraction, is reported . Four isoforms that encoded two different mature proteins were identified at the cDNA level . Another isoform was detected in sequenced peptides . Because the antifungal activity of gastrodianins produced in and purified from Escherichia coli and Nicotiana tabacum was comparable to that of gastrodianin purified from the orchid, gastrodianins are the active component of the antifungal fractions . Gastrodianin accumulation is probably an important part of the mechanism by which the orchid controls Armillaria penetration . Gastrodianin was found to be homologous to monomeric mannose-binding proteins of other orchids, of which at least one (Epipactis helleborine mannose-binding protein) also displayed in vitro antifungal activity . This establishes the gastrodianin-like proteins (GLIPs) as a novel class of antifungal proteins.

Physiol Plant, 2001 May, 112(1), 87 - 94
Cloning, characterization and functional expression of a phospholipase Dalpha cDNA from tomato fruit; Whitaker BD et al.; Phospholipase D (PLD; EC 3.1.4.4) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL . Phosphatidic acid (PA), the product of PLD hydrolysis of PL, increases in pericarp tissue during ripening of tomato (Lycopersicon esculentum Mill.) fruit, suggesting that increased PLD activity may be involved in loss of membrane function associated with ripening . However, a recent report showed a decline in soluble PLD activity in both normal and nonripening mutant fruit over the span that encompasses full ripening . To directly assess the role of PLD in tomato ripening, we have initiated a molecular genetic approach . Using a PLDalpha cDNA from castor bean as a probe, a PLDalpha cDNA (LEPLD2) was isolated from a tomato fruit library . It has an open reading frame of 2 421 nucleotides, predicted to encode a polypeptide of 807 amino acids, with a molecular mass of 91.9 kDa . These values are close to those of PLDalphas from 11 plant species and LEPLD2 has >/=73% nucleotide sequence identity with PLDalpha cDNAs from castor bean and tobacco, as well as another tomato cDNA . LEPLD2 transcript was detected in all tissues of the tomato plant by RNA gel-blot analysis . Levels were very low in roots, low in stems, moderate in leaves, high in flowers and increased in fruit during development and ripening . Expression of LEPLD2 in Escherichia coli yielded phosphatidylcholine-hydrolyzing enzyme, and cells transformed with a pFLAG-MAC vector construct produced a FLAG-PLDalpha fusion protein that migrated close to the calculated 94 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Biochemistry, 2001 May 1, 40(17), 5275 - 82
Parameters affecting the restoration of activity to inactive mutants of thymidylate synthase via subunit exchange: further evidence that thymidylate synthase is a half-of-the-sites activity enzyme; Saxl RL et al.; In a previous study we demonstrated that Escherichia coli thymidylate synthase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R(126)E, another inactive mutant {Maley, F., Pedersen-Lane, J., and Changchien, L.-M . (1995) Biochemistry 34, 1469-1474} . Since only one of the enzyme's two subunits possessed a functional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was proposed that thymidylate synthase was a half-of-the-sites activity enzyme . We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity . For this purpose, we employed two mutants with marginal thymidylate synthase activity, Y(94)A and R(126)E . The parameters that were examined included pH, concentration of protein, temperature, and urea concentration, all of which influenced the rate of activity restoration . It was found, surprisingly, that by maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly enhanced . Increasing the pH from 6.0 to 9.0 markedly increased the rate at which the optimal activity was restored, as did increasing the temperature from 4 to 40 degrees C . A similar effect was obtained when the incubation of the mutants was conducted at 4 degrees C in the presence of 1.5 M urea, a temperature at which activity is restored extremely slowly . Raising the pH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degrees C . The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene glycol, and glycerol suggests that changes in subunit interaction and enzyme conformation are in part responsible for the observed differences . Most significantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitory . At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory . Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombination to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis . Sequence analysis of the isolated homo- and heterodimers clearly demonstrated that the active enzyme is a product of subunit exchange, one that is very efficient relative to the wild-type enzyme, which did not exchange subunits unless denatured.

Biochemistry, 2001 May 1, 40(17), 5260 - 8
Electron transfer from heme bL to the {3Fe-4S} cluster of Escherichia coli nitrate reductase A (NarGHI); Rothery RA et al.; We have investigated the functional relationship between three of the prosthetic groups of Escherichia coli nitrate reductase A (NarGHI): the two hemes of the membrane anchor subunit (NarI) and the {3Fe-4S} cluster of the electron-transfer subunit (NarH) . In two site-directed mutants (NarGHI(H56R) and NarGHI(H205Y)) that lack the highest potential heme of NarI (heme b(H)), a large negative DeltaE(m,7) is elicited on the NarH {3Fe-4S} cluster, suggesting a close juxtaposition of these two centers in the holoenzyme . In a mutant retaining heme b(H), but lacking heme b(L) (NarGHI(H66Y)), there is no effect on the NarH {3Fe-4S} cluster redox properties . These results suggest a role for heme b(H) in electron transfer to the {3Fe-4S} cluster . Studies of the pH dependence of the {3Fe-4S} cluster, heme b(H), and heme b(L) E(m) values suggest that significant deprotonation is only observed during oxidation of the latter heme (a pH dependence of -36 mV pH(-1)) . In NarI expressed in the absence of NarGH {NarI(DeltaGH)}, apparent exposure of heme b(H) to the aqueous milieu results in both it and heme b(L) having E(m) values with pH dependencies of approximately -30 mV pH(-1) . These results are consistent with heme b(H) being isolated from the aqueous milieu and pH effects in the holoenzyme . Optical spectroscopy indicates that inhibitors such as HOQNO and stigmatellin bind and inhibit oxidation of heme b(L) but do not inhibit oxidation of heme b(H) . Fluorescence quench titrations indicate that HOQNO binds with higher affinity to the reduced form of NarGHI than to the oxidized form . Overall, the data support the following model for electron transfer through the NarI region of NarGHI: Q(P) site --> heme b(L) --> heme b(H) --> {3Fe-4S} cluster.

Biochemistry, 2001 May 1, 40(17), 5151 - 60
Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism; Schmidt A et al.; 2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine . It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA . The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes . We present the three-dimensional structure of E . coli KBL determined at 2.0 A resolution . KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase . Its closest structural homologue is E . coli 8-amino-7-oxononanoate synthase . Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal . There are two active sites per dimer, located at the dimer interface . Both monomers contribute side chains to each active/substrate binding site . Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure . The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL . A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.

Biochemistry, 2001 May 1, 40(17), 5111 - 8
Conformationally specific misfolding of an integral membrane protein; Oxenoid K et al.; Membrane protein misfolding is related to the etiology of many diseases, but is poorly understood, particularly from a structural standpoint . This study focuses upon misfolding of a mutant form of diacylglycerol kinase (s-DAGK), a 40 kDa homotrimeric protein having nine transmembrane segments . Preparations of s-DAGK sometimes contain a kinetically trapped misfolded population, as evidenced by lower-than-expected enzyme activity (with no accompanying change in substrate K(m)) and by the appearance of a second band in electrophoresis gels . Misfolding of s-DAGK may take place during cellular overexpression, but can also be reproduced using the purified enzyme . TROSY NMR spectra of s-DAGK as a 100 kDa complex with detergent micelles exhibit a single additional set of resonances from the misfolded form, indicating a single misfolded conformational state . The relative intensities of these extra resonances correlate with the percent reduction in enzyme activity below the maximum observed for fully folded s-DAGK . Misfolded s-DAGK exhibits a modest difference in its far-UV CD spectrum compared to the folded enzyme, consistent with a small degree of variance in secondary structural content between the two forms . However, differences in NMR chemical shift dispersion and temperature-dependent line widths exhibited by folded and misfolded s-DAGK support the notion that they represent very different structural states . Cross-linking experiments indicate that both the correctly folded enzyme and the kinetically trapped misfolded form are homotrimers . This work appears to represent the first documentation of conformationally specific misfolding of an integral membrane protein.

Clin Immunol, 2001 May, 99(2), 291 - 7
Autoantibody to DNA excision repair enzyme hMYH in a patient with rheumatic disease; Lai FP et al.; We screened a human HepG2 cell cDNA expression library using serum from a patient with rheumatic disease . This serum had immunofluorescence reactivity to nuclei with a homogeneous staining pattern and to punctuate nuclear aggregates, chromosomal metaphase plate, midbody, and cytoplasmic bridge . YT1, the longest cDNA clone isolated, has sequence identity to hMYH, the human homologue of the Escherichia coli excision repair enzyme, DNA adenine glycosylase MutY . YT1 is a truncated cDNA of 1619 bp, encoding amino acids 22-535, and contains a full-length 3'-UTR sequence . We were unable to express a bacterial malE fusion protein incorporating amino acids 22 to 535 of hMYH . Consequently, we generated two additional malE fusion proteins of hMYH encoding amino acids 1-120 (pMAL-c2:hMYH(1-120)) and amino acids 121-535 (pMAL-c2:hMYH(121-535)) . The patient serum immunoblotted only pMAL-c2:hMYH(1-120), suggesting that the autoepitope(s) is restricted to amino acids 22-120 of hMYH, and detected a protein of approximately 59-kDa in total HeLa and nuclear extracts consistent with reactivity to hMYH . Affinity-purified autoantibodies to pMAL-c2:hMYH(1-120) reacted by immunoblot to pMAL-c2:hMYH(1-120), with no reactivity to pMAL-c2:hMYH(121-535) . By immunofluorescence, these antibodies displayed staining of nuclei . This is the first report of autoantibodies to hMYH in a patient with rheumatic disease . We were able to identify hMYH reactivity in relatively small cohorts of sera collected from rheumatoid factor-positive patients (6 of 18) and dsDNA-positive patients (1 of 18), with no reactivity detected in serum collected from 9 healthy subjects .

J Parasitol, 2001 Apr, 87(2), 237 - 41
Gut-associated immunolocalization of the Schistosoma mansoni cysteine proteases, SmCL1 and SmCL2; Bogitsh BJ et al.; Transcripts encoding discrete, cathepsin L-like cysteine proteases, known as SmCL1 and SmCL2, have been reported from the adult stages of the human blood fluke Schistosoma mansoni . However, the physiological roles of these 2 enzymes and their natural substrates remain uncertain and controversial . To determine their localization in adult S . mansoni by immunocytochemical procedures, and thereby to gain insight into their likely functions, polymerase chain reaction-based cDNAs encoding mature SmCL1 and SmCL2 were ligated into Escherichia coli . The bacterially expressed recombinant proteins (bSmSL1, bSmCL2) were used to generate monospecific rabbit antisera . For light microscopy, paraffin-embedded sections were visualized with the fluorophore Cy3 . For transmission electron microscopy (TEM), LR White-embedded tissue was visualized with 15 nm gold . Under light microscopy, fluorescence was visible on the luminal surface of the gastrodermis in both sexes for both proteins . For bSmCL1 and bSmCL2, TEM revealed gold particles primarily associated with amorphous deposits within superficial digestive vacuoles on the gastrodermal surface of males and females . Some bSmCL1 reaction product was observed in vesicles within the gastrodermis, and very sparse gastrodermal activity was observed with bSmCL2 . By contrast, neither enzyme was immunolocalized in the reproductive organs, vitelline glands, nor gynecorphoric canal . The gut-associated immunolocalization of SmCL1 and SmCL2 indicates that both these endopeptidases participate in hemoglobin proteolysis.

In Vivo, 2001 Mar-Apr, 15(2), 145 - 9
Diverse biological activity of polycaphenol; Jiang Y et al.; Millimolar concentrations of alkaline extract of Cacao husk (polycaphenol) were more cytotoxic to human oral tumor cells (human oral squamous cell carcinoma HSC-2, human salivary gland tumor HSG), than to human gingival fibroblast (HGF), suggesting its tumor-specific action . Polycaphenol enhanced the radical intensity and cytotoxic activity of vitamin K3 more effectively than that of sodium ascorbate (vitamin C) . Polycaphenol effectively scavenged the superoxide anion, produced by the hypoxanthine-xanthine oxidase reaction, indicating bimodal (prooxidant and antioxidant) action of polycaphenol . Polycaphenol inhibited the cytopathic effect of HIV (human immunodeficiency virus) infection in MT-4 cells, to a comparable extent as that achieved by lignin . Pretreatment of mice with polycaphenol protected them from lethal infection of Eschericia coli . These data suggest the medicinal efficacy of polycaphenol.

Environ Mol Mutagen, 2001, 37(3), 203 - 14
Gene- and tissue-specificity of mutation in Big Blue rats treated with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene; Chen T et al.; In a previous study, we found that treating transgenic Big Blue rats with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) produced the same major DNA adduct in the target liver and the nontarget spleen lymphocytes and bone marrow cells, induced lacI mutants in the liver, and induced much lower frequencies of lacI and hprt mutants in spleen lymphocytes . In the present study, sequence analysis was conducted on lacI DNA and hprt cDNA from the mutants, to determine the mutational specificity of N-OH-AAF in the rat . All the mutation spectra from N-OH-AAF-treated rats differed significantly from corresponding mutation profiles from untreated animals (P = 0.02 to P < 0.0001) . Although there were similarities among the mutational patterns derived from N-OH-AAF-treated rats (e.g., G:C --> T:A transversion was the most common mutation in all mutation sets), there were significant differences in the patterns of basepair substitution and frameshift mutation between the liver and spleen lymphocyte lacI mutants (P = 0.02) and between the spleen lymphocyte lacI and hprt mutants (P = 0.04) . Also, multiplex PCR analysis of genomic DNA from the hprt mutants indicated that 12% of mutants from treated rats had major deletions in the hprt gene; no corresponding incidence of large deletions was evident among lacI mutations . All the mutation profiles reflect the general mutational specificity of the major DNA adduct formed by N-OH-AAF . The differences between N-OH-AAF mutation in the endogenous gene and transgene can be partially explained by the structures of the two genes . The tissue-specificity of the mutation spectra may contribute to targeting tumor formation to the liver . Environ . Mol . Mutagen . 37:203-214, 2001 . Published 2001 Wiley-Liss, Inc.

Environ Mol Mutagen, 2001, 37(3), 195 - 202
Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats; Chen T et al.; N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene . In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues . Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p . injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing . Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes . At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals . Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6) . Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling . Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments . N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined . Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses . In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively . The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat . Environ . Mol . Mutagen . 37:195-202, 2001 . Published 2001 Wiley-Liss, Inc.

Transfusion, 2001 Apr, 41(4), 470 - 6
Immune response to autologous transfusion in healthy volunteers: WB versus packed RBCs and FFP; Frietsch T et al.; BACKGROUND: Storage of blood as packed RBCs and FFP is standard practice in allogeneic transfusion . Separation into components has been proposed for autologous transfusion, as well, but beneficial effects have not yet been shown . STUDY DESIGN AND METHODS: Twenty-four healthy male volunteers were randomly assigned to receive 1 unit of either autologous RBCs and FFP (RCP group) or WB (WB group) after 49 or 35 days of storage, respectively . The immune response was analyzed by ELISA for IL-6, C3a, terminal complement complex SC5b-9, TNF-alpha, and neopterin . Differential WBC counts and the phagocytosis of neutrophils and monocytes were measured by flow cytometry . RESULTS: Cell counts of monocytes (0.85 x 10(3) ng/microL) {corrected} and neutrophils (6.9 x 10(3) ng/microL) {corrected} increased 30 minutes after WB transfusion and then returned to close to the baseline values seen in the RCP group (0.47 and 2.9 x 10(3) ng/microL {corrected}, respectively) throughout the monitored period (p<0.05) . C3a (169 vs . 116 ng/microL) {corrected} and IL-6 (29 vs . 6 pg/mL) reached higher plasma concentrations in the WB group (n = 11) than in the RCP group (n = 10) . Phagocytosis of opsonized Escherichia coli was increased in neutrophils and monocytes and lasted up to 7 days after the transfusion of whole blood . CONCLUSION: Autologous WB induces a modest immunomodulation, but this effect is not observed upon transfusion of autologous blood components.

Protein Sci, 2001 May, 10(5), 1023 - 31
Frequencies of amino acid strings in globular protein sequences indicate suppression of blocks of consecutive hydrophobic residues; Schwartz R et al.; Patterns of hydrophobic and hydrophilic residues play a major role in protein folding and function . Long, predominantly hydrophobic strings of 20-22 amino acids each are associated with transmembrane helices and have been used to identify such sequences . Much less attention has been paid to hydrophobic sequences within globular proteins . In prior work on computer simulations of the competition between on-pathway folding and off-pathway aggregate formation, we found that long sequences of consecutive hydrophobic residues promoted aggregation within the model, even controlling for overall hydrophobic content . We report here on an analysis of the frequencies of different lengths of contiguous blocks of hydrophobic residues in a database of amino acid sequences of proteins of known structure . Sequences of three or more consecutive hydrophobic residues are found to be significantly less common in actual globular proteins than would be predicted if residues were selected independently . The result may reflect selection against long blocks of hydrophobic residues within globular proteins relative to what would be expected if residue hydrophobicities were independent of those of nearby residues in the sequence.

Protein Sci, 2001 May, 10(5), 988 - 96
Crystal structure of a deletion mutant of human thymidylate synthase Delta (7-29) and its ternary complex with Tomudex and dUMP; Almog R et al.; The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 A and 2.5 A resolution, respectively . The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type enzyme . The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands . This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation . While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 A away from the catalytic cysteine in the open conformation . As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E . coli enzyme, is not formed . This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule . Despite the absence of the closed conformation, Tomudex inhibits human TS ten-fold more strongly than E . coli TS . These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.

J Biol Chem, 2001 Jul 6, 276(27), 25208 - 11 Epub 2001 Apr 20.
Characterization of a novel carotenoid cleavage dioxygenase from plants; Schwartz SH et al.; The plant hormone abscisic acid is derived from the oxidative cleavage of a carotenoid precursor . Enzymes that catalyze this carotenoid cleavage reaction, nine-cis epoxy-carotenoid dioxygenases, have been identified in several plant species . Similar proteins, whose functions are not yet known, are present in diverse organisms . A putative cleavage enzyme from Arabidopsis thaliana contains several highly conserved motifs found in other carotenoid cleavage enzymes . However, the overall homology with known abscisic acid biosynthetic enzymes is low . To determine the biochemical function of this protein, it was expressed in Escherichia coli and used for in vitro assays . The recombinant protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' positions . In most instances, the enzyme cleaves the substrate symmetrically to produce a C(14) dialdehyde and two C(13) products, which vary depending on the carotenoid substrate . Based upon sequence similarity, orthologs of this gene are present throughout the plant kingdom . A similar protein in beans catalyzes the same reaction in vitro . The characterization of these activities offers the potential to synthesize a variety of interesting, natural products and is the first step in determining the function of this gene family in plants.

J Biol Chem, 2001 Jul 13, 276(28), 25797 - 803 Epub 2001 Apr 20.
Reaction mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis; Nakajima J et al.; In the conversion from colorless leucoanthocyanidin to colored anthocyanidin 3-glucoside, at least two enzymes, anthocyanidin synthase (ANS) and UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT), are postulated to be involved . Despite the importance of this reaction sequence for coloring in anthocyanin biosynthesis, the biochemical reaction mechanism has not been clarified, and the possible involvement of a dehydratase has not been excluded . Here we show that recombinant ANSs from several model plant species, snapdragon, petunia, torenia, and maize, catalyze the formation of anthocyanidin in vitro through a 2-oxoglutarate-dependent oxidation of leucoanthocyanidin . Crude extracts of Escherichia coli, expressing recombinant ANSs from these plant species, and purified recombinant enzymes of petunia and maize catalyzed the formation of anthocyanidin in the presence of ferrous ion, 2-oxoglutarate, and ascorbate . The in vitro formation of colored cyanidin 3-glucoside from leucocyanidin, via a cyanidin intermediate, was demonstrated using petunia ANS and 3-GT . The entire reaction sequence did not require any additional dehydratase but was dependent on moderate acidic pH conditions following the enzymatic steps . The present study indicated that the in vivo cytosolic reaction sequence involves an ANS-catalyzed 2-oxoglutarate-dependent conversion of leucoanthocyanidin (flavan-3,4-cis-diol) to 3-flaven-2,3-diol (pseudobase), most probably through 2,3-desaturation and isomerization, followed by glucosylation at the C-3 position by 3-GT.

J Biol Chem, 2001 Jul 6, 276(27), 25582 - 8 Epub 2001 Apr 20.
c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro; Lively TN et al.; c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation . We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors . Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID) . Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250 . The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids . This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID . We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription . In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites . Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250 . In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

J Pineal Res, 2001 Apr, 30(3), 147 - 56
Melatonin prevents endotoxin-induced circulatory failure in rats; Wu CC et al.; The pineal secretory product melatonin was found to exert protective effects in septic shock . In a host infected by bacterial lipopolysaccharide (LPS), the expression and release of proinflammatory tumor necrosis factor-alpha (TNF-alpha) is rapidly increased, suggesting that TNF-alpha is associated with the etiology of endotoxic shock . Recent reports show that the expression of NO synthase (NOS) II and the production of superoxide anion (O2*-) also contribute to the pathophysiology of septic shock . In the present study we demonstrate that melatonin prevents circulatory failure in rats with endotoxemia and improves survival in mice treated with a lethal dose of LPS . The beneficial hemodynamic effects of melatonin in the endotoxemic animal appear to be associated with the inhibition of (i) the release of TNF-alpha in plasma, (ii) the expression of NOS II in liver, and (iii) the production of O2*- in aortae . In addition, the infiltration of polymorphonuclear neutrophils into the liver from the surviving LPS mice treated with melatonin was reduced . Thus, our results support the clinical use of melatonin in endotoxemia.

Arthritis Rheum, 2001 Apr, 44(4), 838 - 45
Implication of cartilage intermediate layer protein in cartilage destruction in subsets of patients with osteoarthritis and rheumatoid arthritis; Tsuruha J et al.; OBJECTIVE: To investigate whether cartilage intermediate layer protein (CILP), a protein recently cloned from human articular cartilage, is recognized as an autoantigen in patients with osteoarthritis (OA) and rheumatoid arthritis (RA), and whether the immune response against CILP is involved in disease pathogenesis . METHODS: Recombinant fusion proteins, which contain the first half (C1), second half (C2), or 3 fragments within the C2 region (designated C2F1, C2F2, and C2F3) of the non-porcine nucleotide pyrophosphohydrolase-homologous region of CILP, were prepared using Escherichia coli . Autoantibodies to these proteins in serum samples from patients with OA or RA and from age-matched healthy individuals were detected by enzyme-linked immunosorbent assay and Western blotting . In addition, mice were immunized with a mixture of the C1 and C2 fusion proteins to assess the arthrogenicity of CILP . RESULTS: Production of antibodies to the C2 region was detected in 10.5% (11 of 105) of the tested OA patients and in 8.0% (7 of 88) of the tested RA patients, although antibodies to the C1 region were rarely detected in either patient group . All C2F1, C2F2, and C2F3 fragments were found to carry autoepitopes . The C2F2 fusion protein was recognized most frequently in the tested OA patients, whereas the C2F3 fusion protein was dominantly recognized in the tested RA patients . All 4 mice strains, DBA/1J, ICR, C57BL/6, and BALB/c, immunized with the CILP fusion proteins developed chronic arthritis; in particular, the ICR mice developed polyarthritis that was characterized by infiltration of mononuclear cells in the synovium and exfoliation of the surface of cartilage . CONCLUSION: The immune response to CILP may play a role in the pathogenesis of inflammatory joint destruction . Our results support the role of an immune-mediated process in the joint destruction present in chronic arthropathies such as OA and RA . The results suggest that suppression of immune responses to various components of the cartilage, such as CILP, might be therapeutically beneficial in these chronic arthropathies.

J Biol Inorg Chem, 2001 Mar, 6(3), 315 - 23
Crystal structures of oxidized dinuclear manganese centres in Mn-substituted class I ribonucleotide reductase from Escherichia coli: carboxylate shifts with implications for O2 activation and radical generation; Hogbom M et al.; The di-iron carboxylate proteins constitute a diverse class of non-heme iron enzymes performing a multitude of redox reactions . These reactions usually involve high-valent Fe-oxo species and are thought to be controlled by carboxylate shifts . Owing to their short lifetime, the intermediate structures have so far escaped structural characterization by X-ray crystallography . In an attempt to map the carboxylate conformations available to the protein during different redox states and different ligand environments, we have studied metal-substituted forms of the R2 protein of ribonucleotide reductase from Escherichia coli . In the present work we have solved the crystal structures of Mn-substituted R2 oxidized in two different ways . Oxidation was performed using either nitric oxide or a combination of hydrogen peroxide and hydroxylamine . The two structures are virtually identical, indicating that the oxidation states are the same, most likely a mixed-valent MnII-MnIII centre . One of the carboxylate ligands (D84) adopts a new, so far unseen, conformation, which could participate in the mechanism for radical generation in R2 . E238 adopts a bridging-chelating conformation proposed to be important for proper O2 activation but not previously observed in the wild-type enzyme . Probable catalase activity was also observed during the oxidation with H2O2, indicating mechanistic similarities to the di-Mn catalases.

Can J Microbiol, 2001 Mar, 47(3), 179 - 87
Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis; Vetrivel KS et al.; Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography . The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S . lividans 66, S . coelicolor A3(2), S . plicatus, and S . thermoviolaceus OPC-520 . Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S . peucetius chitinase . A genomic library of SPVI constructed in E . coli using lambda DASH II was probed with chiC of S . lividans 66 to screen for the chitinase gene . A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced . The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S . lividans 66 chiC . The N-terminal amino acid sequence of the purified S . peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only . A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.

J Bone Joint Surg Am, 2001, 83-A Suppl 1(Pt 2), S99 - 104
In vitro and in vivo studies of a bone morphogenetic protein-2 expressing adenoviral vector; Okubo Y et al.; BACKGROUND: Bone morphogenetic proteins (BMPs) play important roles in the migration of osteoblast progenitor cells, the proliferation of mesenchymal cells, and their differentiation into chondrogenic and osteogenic cells . However, the optimum procedure to deliver BMPs remains unknown . To examine the effectiveness of a gene transfer procedure for the delivery of BMP-2, we constructed a human BMP-2-expressing replication-deficient adenoviral vector, AxCAOBMP-2, and evaluated its osteoinductive activity in vitro and in vivo . METHODS: C2C12 myoblasts were infected in vitro with this viral vector or an Escherichia coli LacZ gene-expressing control adenovirus vector (AxCALacZ) . Twenty-four hours after the infection, indirect immunofluorescence was performed . On day 5 after the infection, alkaline phosphatase (ALP) in the cells and osteocalcin in the culture medium were measured . Furthermore, to examine the effectiveness of gene transfer of BMP-2 in vivo, we evaluated osteoinduction by AxCAOBMP-2, under transient immunosuppression with cyclophosphamide, given at a dose of 125 mg/kg intraperitoneally the day before injection of the adenoviral vector . Twenty-five microliters of AxCAOBMP-2 (8.75 x 10(8) plaque-forming units {pfu}, Group I) and AxCALacZ (1.75 x 10(8) pfu, control group) and 5 microl of AxCAOBMP-2 (1.75 x 10(8) pfu, Group II) were injected into a right calf muscle of Wistar rats . On day 21, bone formation in each group was investigated radiologically and histologically . RESULTS: Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence . C2C12 cells transferred with the BMP-2 gene by this vector produced ALP in the cells and also produced and secreted osteocalcin in the culture medium . Osteoinduction was found only in the AxCAOBMP-2 treated groups with immunosuppression . Osteoinduction activity was higher in Group I than in Group II . CONCLUSION: This study demonstrated the osteoinductive activity in vitro and in vivo by an adenoviral vector carrying the BMP-2 gene . CLINICAL RELEVANCE: Gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for bone reconstruction.

J Craniofac Surg, 2000 Jul, 11(4), 371 - 5; discussion
Spontaneously infected cephalohematoma: case report and review of the literature; Goodwin MD et al.; Spontaneously infected cephalohematomas are rare occurrences; only five cases have been reported previously . Uninfected cephalohematomas are common and usually resolve without treatment . However, physicians should be aware that cephalohematomas are potential sites for infection and may require aspiration for diagnosis and treatment . Untreated infected cephalohematomas may lead to osteomyelitis, epidural abscess, or subdural empyema . We present a case of a spontaneously infected cephalohematoma with an associated osteomyelitis which was successfully managed with drainage and long-term antibiotics . A review of the literature is also presented.

Biotechniques, 2001 Apr, 30(4), 852 - 6, 858, 860 passim
Isothermal amplification and multimerization of DNA by Bst DNA polymerase; Hafner GJ et al.; We have demonstrated the isothermal in vitro amplification and multimerization of several different linear DNA targets using only two primers and the strongly strand-displacing exonuclease-negative Bst DNA polymerase . This reaction has been termed linear target isothermal multimerization and amplification (LIMA) . LIMA has been compared with cascade rolling-circle amplification and has been found to be less sensitive but to yield similar variable-length multimeric dsDNA molecules . Products from several different LIMA reactions were characterized by restriction analysis and partial sequence determination . They were found to be multimers of subsets of the target sequence and were not purely primer derived . The sensitivities with respect to target concentration of several different LIMA reactions were determined, and they varied from 0.01 amol to 1 fmol . The sensitivity and specificity of LIMA were further tested using E . coli genomic DNA, and the selective amplification of a transposon fragment was demonstrated . A successful strategy for reducing LIMA-dependent background DNA synthesis in rolling-circle amplification embodiments was devised . This entailed the affinity purification of circular DNA templates before amplification.

Biotechniques, 2001 Apr, 30(4), 846 - 50
Continuous spectrophotometric assay for beta-glucuronidase; Aich S et al.; A continuous spectrophotometric assay has been developed for detecting beta-glucuronidase activity . In the assay, Para-nitrophenyl beta-D-glucuronide is cleaved to yield a chromophoric product . With the commercial E . coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm . The method is highly sensitive and able to detect less than 1.4 x 10(-4) U/mL of the enzyme activity in solution . Such a new assay offers significant advantages over the existing discontinuous methods and should be useful for both routine enzyme assay and accurate kinetic studies.

Biotechniques, 2001 Apr, 30(4), 776 - 7, 780-1
Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates; Nazarenko DA et al.; beta-galactosidase (beta-gal), the product of the E . coli LacZ gene, has been used extensively as a reporter in numerous systems . Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form a blue precipitate . However, when increased sensitivity is needed, many investigators now turn to alternate substrates that produce fluorescent or luminescent products upon cleavage by beta-gal . These products are much more easily quantified than X-gal . The luminescent and fluorometric assays work very well in cultured cells but are often less sensitive in whole tissue lysates . In this study, we have evaluated the sensitivity of a fluorescent and a luminescent substrate in whole tissue lysates cleared of red blood cells or washed with PBS only . We have found that both assays show increased low-end sensitivity in tissues with reduced levels of hemoglobin (Hb) . Hb is apparently able to quench luminescent and, to a lesser degree, fluorescent reporter light emission . Therefore, steps should be taken to reduce Hb levels either by lysis, perfusion, or both to enhance the sensitivity of these assays.

Gene Ther, 2001 Feb, 8(4), 332 - 9
Combination suicide/cytokine gene therapy as adjuvants to a defective herpes simplex virus-based cancer vaccine; Toda M et al.; We have used syngeneic, established bilateral subcutaneous tumor models to examine the antitumor activity of herpes simplex virus (HSV) vectors, including the induction of an immune response against non-inoculated distant tumors . In such a model with CT26 murine colon adenocarcinoma, unilateral intratumoral inoculation of replication-deficient HSV-1 tsK inhibited the growth of both the inoculated and noninoculated established tumors . To enhance this limited antitumor immune response, we generated a defective HSV vector, dvIL12-tk encoding both interleukin-12 (IL-12) and HSV thymidine kinase (TK), with tsK as the helper virus . In a 'suicide gene' strategy, ganciclovir (GCV) treatment after intratumoral inoculation of dvlacZ-tk/tsK, encoding E . coli lacZ instead of IL-12, resulted in enhanced antitumor activity . Antitumor activity was also enhanced by local expression of IL-12 from dvIL12-tk/tsK . The combination of IL-12 cytokine therapy with GCV treatment was the most efficacious approach, with significantly greater inhibition of tumor growth than IL-12 or TK + GCV alone . These results illustrate the power of combining different cancer therapy approaches; 'suicide gene' therapy, cytokine therapy, and HSV vector infection . HSV vectors are particularly well suited to this because they can accommodate the insertion of large and multiple gene sequences.

Gene Ther, 2001 Feb, 8(4), 274 - 81
Tumour-specific therapeutic adenovirus vectors: repression of transgene expression in healthy cells by endogenous p53; Lipinski KS et al.; Approximately 50% of human tumours lack functional p53 suppressor protein . A promoter that is repressed by p53 in healthy cells could thus provide tumour-specific gene expression for a huge subset of tumours . In this report we describe a double recombinant adenovirus vector, 'Ad.p53R', encoding a therapeutic gene that is indirectly repressed by endogenous wild-type p53 . Ad.p53R contains two independent expression cassettes; (1) the E . coli nitroreductase gene (NTR) driven by the human hsp70 promoter containing LacI binding sites (hsp70lacO-NTR) and (2) a p53-inducible lac repressor gene (tkGC3-lacI) . In p53 null cells (Hep3B), Ad.p53R directed the same level of NTR expression as Ad.p53NR which lacks the tkGC3-lacI cassette . Moreover, injection of SW480 xenografts (mutated p53) with Ad.p53R resulted in a clear inhibition of growth in response to the prodrug CB1954 . In cells retaining wt p53 (HepG2 and primary human endothelial cells), Ad.p53R expressed significantly less NTR (approximately 70%) than Ad.p53NR . Ad.p53R administered by i.v . injection also produced significantly less NTR than Ad.p53NR in normal tissues in vivo . Finally, adenovirus infection per se of cultured HepG2 cells at low MOI induced p53 stabilisation suggesting that adenovirus-mediated gene delivery may contribute to p53-based selectivity.

Science, 2001 Apr 20, 292(5516), 501 - 4
Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway; Doring V et al.; Aminoacyl transfer RNA (tRNA) synthetases establish the rules of the genetic code by catalyzing the aminoacylation of tRNAs . For some synthetases, accuracy depends critically on an editing function at a site distinct from the aminoacylation site . Mutants of Escherichia coli that incorrectly charge tRNA(Val) with cysteine were selected after random mutagenesis of the whole chromosome . All mutations obtained were located in the editing site of valyl-tRNA synthetase . More than 20% of the valine in cellular proteins from such an editing mutant organism could be replaced with the noncanonical aminobutyrate, sterically similar to cysteine . Thus, the editing function may have played a central role in restricting the genetic code to 20 amino acids . Disabling this editing function offers a powerful approach for diversifying the chemical composition of proteins and for emulating evolutionary stages of ambiguous translation.

Science, 2001 Apr 20, 292(5516), 498 - 500
Expanding the genetic code of Escherichia coli; Wang L et al.; A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli . When introduced into E . coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-l-tyrosine into protein in response to an amber nonsense codon . The fidelity of translation is greater than 99%, as determined by analysis of dihydrofolate reductase containing the unnatural amino acid . This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.

Mol Cell Biol, 2001 May, 21(10), 3416 - 24
Protein binding protects sites on stable episomes and in the chromosome from de novo methylation; Han L et al.; We have utilized the Escherichia coli lac repressor-operator system to test whether protein binding can interfere with de novo DNA methylation in mammalian cells . We find that a DNA binding protein can protect sites on the episome as well as in the genome from the de novo methylation activity of Dnmt3a . Transcriptional machinery moving through the binding sites does not affect the de novo methylation of these sites, and it does not affect the binding protein protection of these sites from de novo methylation . This study and previous studies provide a possible mechanism for the observation that an Sp1 site can serve as a cis-acting signal for demethylation and for preventing de novo methylation of the CpG island upstream of the mouse adenine phosphoribosyltransferase (Aprt) gene . These findings also support the hypothesis that protein binding may play a crucial role in changes of CpG methylation pattern in mammalian cells.

J Biol Chem, 2001 Jun 29, 276(26), 23881 - 7 Epub 2001 Apr 19.
A Gal4-sigma 54 hybrid protein that functions as a potent activator of RNA polymerase II transcription in yeast; Chen BS et al.; The bacterial final sigma(54) protein associates with core RNA polymerase to form a holoenzyme complex that renders cognate promoters enhancer-dependent . Although unusual in bacteria, enhancer-dependent transcription is the paradigm in eukaryotes . Here we report that a fragment of Escherichia coli final sigma(54) encompassing amino acid residues 29-177 functions as a potent transcriptional activator in yeast when fused to a Gal4 DNA binding domain . Activation by Gal4-final sigma(54) is TATA-dependent and requires the SAGA coactivator complex, suggesting that Gal4-final sigma(54) functions by a normal mechanism of transcriptional activation . Surprisingly, deletion of the AHC1 gene, which encodes a polypeptide unique to the ADA coactivator complex, stimulates Gal4-final sigma(54)-mediated activation and enhances the toxicity of Gal4-final sigma(54) . Accordingly, the SAGA and ADA complexes, both of which include Gcn5 as their histone acetyltransferase subunit, exert opposite effects on transcriptional activation by Gal4-final sigma(54) . Gal4-final sigma(54) activation and toxicity are also dependent upon specific final sigma(54) residues that are required for activator-responsive promoter melting by final sigma(54) in bacteria, implying that activation is a consequence of final sigma(54)-specific features rather than a structurally fortuitous polypeptide fragment . As such, Gal4-final sigma(54) represents a novel tool with the potential to provide insight into the mechanism by which natural activators function in eukaryotic cells.

J Biol Chem, 2001 Jun 29, 276(26), 24348 - 51 Epub 2001 Apr 19.
TIP47 is not a component of lipid droplets; Barbero P et al.; TIP47 functions in the delivery of mannose 6-phosphate receptors from endosomes to the trans-Golgi network both in vitro and in vivo . It binds directly and very specifically to the cytoplasmic domains of both the cation-independent and cation-dependent mannose 6-phosphate receptors . TIP47 is 43% identical to a lipid droplet-associated protein named adipophilin; much of the identity resides near the N termini of these proteins . It was recently reported in this journal, in a study using antiserum from this laboratory, that TIP47 is a constituent of lipid droplets (Wolins, N . E., Rubin, B., and Brasaemle, D . L . (2001) J . Biol . Chem . 276, 5101-5108) . We show here that the findings of Wolins et al . were likely due to either a cross-reactive, unidentified protein in HeLa cells that is recognized by our antiserum and/or the fact that our serum also cross-reacts with the adipophilin protein itself, shown directly by expression of adipophilin in Escherichia coli . Using antibodies specific for residues 152-434 of TIP47, we show that TIP47 is not a constituent of lipid droplets.

J Biol Chem, 2001 Jul 13, 276(28), 26568 - 76 Epub 2001 Apr 19.
Functional significance of the beta hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a; Tedford HW et al.; omega-Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels . In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions . First, we constructed a Hairpinless mutant in which the C-terminal beta-hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin . The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin . We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial "hits" from the alanine scan . This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity . We propose that this surface of the beta-hairpin is a key site for interaction of the toxin with insect calcium channels.

J Biol Chem, 2001 Jul 13, 276(28), 26405 - 10 Epub 2001 Apr 19.
Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana; Takei K et al.; The initial step in the de novo biosynthesis of cytokinin in higher plants is the formation of isopentenyladenosine 5'-monophosphate (iPMP) from AMP and dimethylallylpyrophosphate (DMAPP), which is catalyzed by adenylate isopentenyltransferase (IPT) . Although cytokinin is an essential hormone for growth and development, the nature of the enzyme for its biosynthesis in higher plants has not been identified . Herein, we describe the molecular cloning and biochemical identification of IPTs from Arabidopsis thaliana . Eight cDNAs encoding putative IPT, designated as AtIPT1 to AtIPT8, were picked up from A . thaliana . The Escherichia coli transformants expressing the recombinant proteins excreted cytokinin species into the culture medium except for that expressing AtIPT2 that is a putative tRNA IPT . A purified recombinant AtIPT1 catalyzed the formation of iPMP from DMAPP and AMP . These results indicate that the small multigene family contains both types of isopentenyltransferase, which could synthesize cytokinin and mature tRNA.

J Biol Chem, 2001 Jun 29, 276(26), 24075 - 81 Epub 2001 Apr 19.
Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA; Rajamohan F et al.; Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step . Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP . Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA . Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA . These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket . The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.

J Biol Chem, 2001 Jun 29, 276(26), 24097 - 107 Epub 2001 Apr 19.
cAmp regulation of arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87): a new cell line (1E7) provides evidence of intracellular AANAT activation; Coon SL et al.; Arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase, AANAT, EC ) is the penultimate enzyme in melatonin synthesis . As described here, a cell line (1E7) expressing human AANAT (hAANAT) has been developed to study the human enzyme . 1E7 hAANAT is detectable in immunoblots as a 23-kDa band and is immunocytochemically visualized in the cytoplasm . The specific concentration of hAANAT in homogenates is comparable to that of the night rat pineal gland . Kinetics of AANAT extracted from 1E7 cells are the same as those of bacterially expressed hAANAT; both preparations of hAANAT are equally sensitive to the inhibitor CoA-S-N-acetyltryptamine . Studies of cAMP regulation indicate that treatment with forskolin, dibutyryl cAMP, isobutylmethylxanthine, or isoproterenol activate cellular hAANAT within intact 1E7 cells approximately 8-fold without markedly increasing the abundance of AANAT protein or the activity of AANAT in broken cell preparations; and, that forskolin, isobutylmethylxanthine and isoproterenol elevate cyclic AMP production . These observations extend our understanding of cAMP regulation of AANAT activity, because it is currently thought that this only involves changes in the steady-state levels of AANAT protein . This previously unrecognized switching mechanism could function physiologically to control melatonin production without changing AANAT protein levels.

Gene, 2001 Apr 18, 267(2), 213 - 20
Expression plasmid with a very tight two-step control: Int/att-mediated gene inversion with respect to the stationary promoter; Sektas M et al.; A very tightly controlled expression vector was constructed, which was originally designed as to be able to use any promoter, constitutive or regulated . Moreover, in vector pNH46T1, the repressible P(tac)/P(lac) promoters were used to transcribe genes cloned in the proximal multiple cloning site (MCS), which was flanked by convergent attB and attP sites . The gene of interest was cloned into MCS in the OFF orientation, i.e . facing the promoter(s) . In such OFF orientation, the cloned gene could not be expressed, and only its anti-sense mRNA could be produced . Four strong rrnBT1 terminators, in a tandem arrangement and proximal to the N-terminal end of the cloned non-inverted gene, were protecting it from any inadvertent transcription originating in the vector . Moreover, the P(tac)/P(lac) promoters/operators are controlled by the LacI(q)ts and LacI(+) repressor(s) that further reduce the basal gene expression in the uninduced state . When induced, the total vector population is converted to the ON orientation by expression of the Int function that inverts the attB and attP-flanked MCS including the cloned gene . This places the gene under direct control of the P(tac)/P(lac) promoters, and thus results in very high expression . An additional feature is the anti-termination system that consists of the promoter-proximal nutL site and the inducible gene N, whose role in the ON state is to overcome the rrnBT1 terminators and any other adventitiously cloned terminators.

Gene, 2001 Apr 18, 267(2), 183 - 91
Identification of a PD-(D/E)XK-like domain with a novel configuration of the endonuclease active site in the methyl-directed restriction enzyme Mrr and its homologs; Bujnicki JM et al.; The Escherichia coli K-12 restriction enzyme Mrr recognizes and cleaves N6-methyladenine- and 5-methylcytosine-containing DNA . Its amino acid sequence has been subjected to structure prediction and comparison with other sequences from publicly available sources . The results obtained suggest that Mrr and related putative endonucleases possess a cleavage domain typical for all the so far structurally characterized type II restriction enzymes, however with an unusual glutamine residue at the central position of the (D/E)-(D/E)XK hallmark of the active site . The "missing" acidic side chain was instead found anchored in a different, unusual position, suggesting that Mrr represents a third topological variant of the endonuclease active site in addition to the two alternatives determined previously (Skirgaila et al., 1998 . J . Mol . Biol . 279, 473-481) . One of the newly identified putative endonucleases from the Mrr family is composed of two diverged cleavage domains, which possess both the "typical" D-EXK and the "Mrr-like" D-QXK variants of the active site . Among the Mrr homologs there are also proteins from yeast, in which restriction phenotype has not been observed, suggesting that the free-standing Eukaryotic PD-(D/E)XK superfamily members might be implicated in other cellular processes involving enzymatic DNA cleavage.

FEMS Microbiol Lett, 2001 Apr 13, 197(2), 187 - 93
Outer sheath associated proteins of the oral spirochete Treponema maltophilum; Heuner K et al.; We recently cloned the major outer membrane protein of Treponema maltophilum {Heuner, K., Choi, B.K., Schade, R., Moter, A., Otto, A., Gobel, U.B., J . Bacteriol . 181, 1025-1029} . Here we report the localization of the major sheath protein (Msp)A protein in T . maltophilum by immunogold electron microscopy and its expression . Northern blot analysis revealed that mspA is expressed constitutively as a monocistronic unit . The transcription initiation site of the mspA gene was identified by primer extension analysis . A further screening of a genomic library of T . maltophilum with an anti-outer membrane fraction antibody was done . We were able to clone DNA regions of T . maltophilum encoding putative sugar transport operons and putative outer membrane proteins of this oral treponeme which has a high prevalence in periodontal lesions.

Biotechnol Prog, 2001 Mar-Apr, 17(2), 252 - 7
Effects of FIS overexpression on cell growth, rRNA synthesis, and ribosome content in Escherichia coli; Richins R et al.; The Escherichia coli DNA binding protein FIS is a transcriptional modulator involved in the regulation of many cellular processes, including the activation of rRNA synthesis . High-level overproduction of FIS in early, mid, or late log cultures resulted in growth-phase- and media-specific variations in cell growth, rRNA synthesis, and ribosome content . FIS overproduction caused a pronounced increase in rRNA synthesis for late-exponential cultures but a substantial reduction in cell growth and ribosome content . The addition of simple sugars such as glucose or fructose reversed these phenomena, consistent with the functional role of FIS in carbon metabolism.

Microb Pathog, 2001 Apr, 30(4), 221 - 8
Molecular cloning, sequence and characterization of cjsT, a putative protease from Rickettsia rickettsii; Temenak JJ et al.; The cloning and sequencing of a gene from Rickettsia rickettsii which confers haemolytic activity on Escherichia coli strain TB1 is described . The open reading frame of the haemolysis-promoting gene, cjsT, is 1041 bp and encodes a putative protein with a molecular mass of 33 825 Da . CjsT has high sequence similarity to several bacterial proteases, particularly type IV signal peptidases . Cell lysates from an E . coli clone containing cjsT in pUC19 (pJON1) exhibited greater protease activity in functional assays than found in E . coli containing pUC19 alone . Disruption of the cjsT gene by insertional inactivation with a kanamycin cassette reduced both the protease and haemolytic activities conferred by cjsT . The protease inhibitors antipain and diisopropylfluorophosphate (DFP) both reduced the proteolytic activity of pJON1 . The mechanism by which the R . rickettsii cjsT promotes haemolysis in E . coli remains unclear .

J Virol, 2001 May, 75(10), 4752 - 60
Human papillomavirus virus-like particles are efficient oral immunogens when coadministered with Escherichia coli heat-labile enterotoxin mutant R192G or CpG DNA; Gerber S et al.; Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide . HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects . Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines . Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R . C . Rose, C . Lane, S . Wilson, J . A . Suzich, E . Rybicki, and A . L . Williamson, Vaccine 17:2129-2135, 1999) . Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity . We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with Escherichia coli heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions . Our results also suggest that LT R192G may be superior to CpG DNA in this ability . These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted.

J Biol Chem, 2001 Jul 6, 276(27), 24574 - 80 Epub 2001 Apr 18.
Comparative in vitro studies on native and recombinant human cationic trypsins . Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis; Szilagyi L et al.; Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene . The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG) . In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical . We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin . Both forms were equally stable . Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin . Mesotrypsin did not activate either form . The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+ . At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG . The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well . Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5-3 times as fast as Hu1Asn21 TG . The presence of hPSTI did not prevent the activation of zymogens by cathepsin B . Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.

Int Immunol, 2001 May, 13(5), 615 - 23
H-2 allele specificity of the NK cell C-type lectin-like MHC class I receptor Ly49A visualized by soluble Ly49A tetramer; Matsumoto N et al.; Ly49A is a C-type lectin-like receptor on NK cells that recognizes MHC class I ligands, H-2D(d) and D(k) . The engagement of Ly49A with the ligands inhibits activation of NK cells and protects target cells from lysis by NK cells . Here we express the extracellular region of Ly49A with an N-terminal biotinylation tag in Escherichia coli to obtain soluble Ly49A (sLy49A) after refolding . sLy49A is indistinguishable from native Ly49A expressed on NK cells serologically and in the ability to specifically bind H-2D(d) after tetramerization with R-phycoerythrin-coupled streptavidin . The fluorescently labeled tetramer of sLy49A is applied to explore MHC class I haplotype specificity of Ly49A . We demonstrate the hierarchical reactivity of Ly49A with H-2 of various alleles in the order of d > k, r > p > v > q > s > z . Reactivity of sLy49A tetramer to spleen lymphocytes from B10.QBR mice (H-2K(b), I(b), D(q), Qa-1/Tla(b)) but not from C57BL/10 mice (H-2(b)) identifies H-2D(q) and L(q) as candidates for a Ly49A ligand . Binding of sLy49A tetramer to H-2D(q)- or L(q)-transfected cell lines demonstrates that the two highly related MHC class I molecules, H-2D(q) and L(q), are ligands for Ly49A . sLy49A tetramer staining also demonstrates preferential expression of Ly49A ligand on a subset of B cells in P/J mice . These results provide the basis to examine the molecular mechanism by which Ly49A discriminates polymorphic MHC class I molecules.

Vaccine, 2001 Apr 30, 19(23-24), 3131 - 6
Long-term persistence of cellular immunity to Oka vaccine virus induced by pernasal co-administration with Escherichia coli enterotoxin in mice; Kamiya N et al.; A mutant of Escherichia coli enterotoxin induced cellular immunity to a live varicella vaccine (the Oka strain) as a mucosal adjuvant in mice . The persistence of this cellular immunity was investigated . A commercially available live Oka vaccine virus and toxin were administered once simultaneously via the nasal route, in mice . Ten or 12 months later, a delayed-type hypersensitivity to the vaccine virus was detected by footpad test, but an antibody neutralizing the varicella-zoster virus was not . When spleen cells from mice immunized with the vaccine and toxin were re-stimulated by live vaccine in vitro, their thymidine uptake and IL-2 production were higher than those from mice immunized with the vaccine alone, but lower than those of spleen cells prepared from mice 2 months after nasal administration . Production of IL-4 in these cells, however, was not induced by re-stimulation in vitro . These results suggest that although humoral immunity for Oka vaccine virus is only weakly induced by one co-administration of the vaccine and toxin, cellular immunity is induced and maintained over 1 year, though it declines with age . The nasal administration of the vaccine and toxin might be effective for maintaining cellular immunity to the varicella-zoster virus long term.

Vaccine, 2001 Apr 30, 19(23-24), 3116 - 23
Recombinant Semliki Forest virus vector exhibits potential for avian virus vaccine development; Phenix KV et al.; The Semliki Forest virus (SFV) expression system was evaluated as a basis for avian vaccine development . Initial studies indicated that 1-day-old specific pathogen-free (SPF) chicks were susceptible to infection with an infectious strain of SFV, producing SFV-specific antibodies but no clinical disease . One-day-old SPF chicks immunised intramuscularly with recombinant replication-defective SFV (rSFV) particles expressing the Escherichia coli (E . coli) lacZ reporter gene developed high titres of beta-gal- specific antibodies at 4 weeks p.i . after two inoculations . In contrast, significantly lower antibody levels were elicited in chicks immunised with a recombinant SFV-based DNA construct or a conventional CMV promoter-based DNA plasmid . rSFV particles encoding the protective VP2 protein or the VP2/VP4/VP3 polyprotein of infectious bursal disease virus (IBDV) were produced and the expressed antigens were characterised in cell culture . Proteins of the correct size were generated and found to react against a range of IBDV-specific monoclonal antibodies . Immunisation of 1-day-old SPF chicks with rSFV particles encoding the IBDV proteins resulted in specific antibodies being elicited in all birds, neutralising antibodies being induced in some but not all birds.

Vaccine, 2001 Apr 30, 19(23-24), 3067 - 75
DNA vaccination by immersion and ultrasound to trout viral haemorrhagic septicaemia virus; Fernandez-Alonso M et al.; This work reports preliminary data on the application of a novel method, ultrasound, for the DNA vaccination of rainbow trout . First, the best formulations were selected that increased the transfer by immersion of a plasmid coding for the green fluorescent protein (GFP) gene into trout fry . Quantification of GFP expression by fluorescence in the fin cells was used to study time course, DNA concentration dependence and comparison of different formulations . The best GFP expression results were obtained with short pulses of ultrasound, DOTAP liposomes and recombinant bacteria or bactofection . Other liposomes or microencapsulation formulations resulted in a GFP fluorescence similar to background values . Second, DNA immersion-vaccination of immunocompetent fingerling trout with the selected formulations was performed by using a plasmid coding for the glycoprotein G gene of the viral haemorrhagic septicaemia virus (VHSV) . The immunization of fingerling trout was estimated by measuring humoral antibody, lymphoproliferation and VHSV challenge responses . Short pulses of low intensity ultrasound were the only method by which both humoral antibody responses and survival after VHSV challenge were obtained . Immersion DNA-vaccination using short pulses of ultrasound could eventually lead to a practical way to vaccinate small fish.

FEMS Microbiol Ecol, 2001 May, 35(3), 313 - 321
Strain-specific differentiation of environmental Escherichia coli isolates via denaturing gradient gel electrophoresis (DGGE) analysis of the 16S-23S intergenic spacer region; Buchan A et al.; Denaturing gradient gel electrophoresis (DGGE) was applied to the 16S-23S rRNA intergenic spacer region (ISR) as a means to evaluate strain level differences in Escherichia coli . The ISRs of 81 environmental E . coli isolates obtained from bovine, poultry, and human sources yielded a total of 41 unique DGGE banding patterns, with identical patterns and common bands within each source and no overlapping patterns among sources . An additional 51 isolates from two nearby streams yielded 45 unique banding patterns with no overlap between sites . However, two of the isolates from the streams had identical banding patterns to those from two of the source isolates, resulting in a total of 84 unique DGGE banding patterns out of 132 isolates identified in this study . These results revealed high diversity among environmental E . coli isolates, which made it difficult to unambiguously ascribe strains found in water samples to specific host organisms.

J Microbiol Methods, 2001 Jun, 45(2), 103 - 11
A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response sigma(E) in Escherichia coli; Rezuchova B et al.; We have previously established a two-plasmid system in Escherichia coli for identification of promoters recognized by RNA polymerase containing a heterologous sigma factor . Attempts to optimize this system for identification of promoters recognized by RNA polymerase containing E . coli extracytoplasmic stress response sigma(E) failed owing to high toxicity of the expressed rpoE . A new system for identification of sigma(E)-cognate promoters was established, and verified using the two known sigma(E)-dependent promoters, rpoEp2 and degPp . Expression of the sigma(E)-encoding rpoE gene was under the control of the AraC-dependent P(BAD) promoter . A low level of arabinose induced a non-toxic, however, sufficient level of sigma(E) to interact with the core enzyme of RNA polymerase . Such an RNA polymerase holoenzyme recognized both known sigma(E)-dependent promoters, rpoEp2 and degPp, which were cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZ alpha reporter gene . This new system has proved to be useful for identification of E . coli sigma(E)-cognate promoters . Moreover, the system could be used for identification of ECF sigma-cognate promoters from other bacteria.

J Virol Methods, 2001 Apr, 93(1-2), 49 - 56
Construction of recombinant swinepox viruses and expression of the classical swine fever virus E2 protein; Hahn J et al.; To explore the swinepox virus (SPV) as a potential live vector for immunization, a vector was developed for the construction of a recombinant SPV carrying foreign genes . In this system, a foreign gene placed under the strong vaccinia virus promoter P(11) can be inserted into the viral thymidine kinase (TK) gene, and the recombinant virus can be isolated in a non-selective medium by the co-expression of E . coli lacZ gene . Compared with the wild type virus, the TK(-)recombinant SPV showed a modest level of attenuation in porcine cells while more attenuation was observed in monkey or human cells . Using this system, a recombinant virus expressing the E2 glycoprotein of classical swine fever virus (CSFV) was produced . Engineered with the gX signal sequence of the pseudorabies virus, and transmembrane domain of E2, the E2 protein was expressed as a dimeric form in the cytoplasm of the infected cells.

FEBS Lett, 2001 Apr 13, 494(3), 213 - 9
APAP, a sequence-pattern recognition approach identifies substance P as a potential apoptotic peptide; del Rio G et al.; We have previously described a novel cancer chemotherapeutic approach based on the induction of apoptosis in targeted cells by homing pro-apoptotic peptides . In order to improve this approach we developed a computational method (approach for detecting potential apoptotic peptides, APAP) to detect short PAPs, based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point . PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly . Among other peptides, substance P was identified as a PAP and subsequently demonstrated to be a pro-apoptotic peptide experimentally . APAP thus provides a method to detect and ultimately improve pro-apoptotic peptides for chemotherapy.

FEBS Lett, 2001 Apr 13, 494(3), 165 - 9
Monomeric state and ligand binding of recombinant GABA transporter from Escherichia coli; Li XD et al.; The gamma-aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture . The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His- and myc-tags, and size-exclusion chromatography . The purified transporter exists as a monomer in FOS-Choline 12 detergent, with a Stokes radius of 45 A for the protein-detergent complex . In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching . Its dissociation constants (K(d)) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 microM, respectively . This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter.

Development, 2001 May, 128(10), 1899 - 909
even skipped is required to produce a trans-acting signal for larval neuroblast proliferation that can be mimicked by ecdysone; Park Y et al.; Development of a multicellular organism requires precise coordination of cell division and cell type determination . The selector homeoprotein Even skipped (Eve) plays a very specific role in determining cell identity in the Drosophila embryo, both during segmentation and in neuronal development . However, studies of gene expression in eve mutant embryos suggest that eve regulates the embryonic expression of the vast majority of genes . We present here genetic interaction and phenotypic analysis showing that eve functions in the trol pathway to regulate the onset of neuroblast division in the larval CNS . Surprisingly, Eve is not detected in the regulated neuroblasts, and culture experiments reveal that Eve is required in the body, not the CNS . Furthermore, the effect of an eve mutation can be rescued both in vivo and in culture by the hormone ecdysone . These results suggest that eve is required to produce a trans-acting factor that stimulates cell division in the larval brain.

Development, 2001 May, 128(10), 1805 - 15
Groucho augments the repression of multiple Even skipped target genes in establishing parasegment boundaries; Kobayashi M et al.; Groucho acts as a co-repressor for several Drosophila DNA binding transcriptional repressors . Several of these proteins have been found to contain both Groucho-dependent and -independent repression domains, but the extent to which this distinction has functional consequences for the regulation of different target genes is not known . The product of the pair-rule gene even skipped has previously been shown to contain a Groucho-independent repression activity . In the Even skipped protein, outside the Groucho-independent repression domain, we have identified a conserved C-terminal motif (LFKPY), similar to motifs that mediate Groucho interaction in Hairy, Runt and Huckebein . Even skipped interacts with Groucho in yeast and in vitro, and groucho and even skipped genetically interact in vivo . Even skipped with a mutated Groucho interaction motif, which abolished binding to Groucho, showed a significantly reduced ability to rescue the even skipped null phenotype when driven by the complete even skipped regulatory region . Replacing this motif with a heterologous Groucho interaction motif restored the rescuing function of Even skipped in segmentation . Further functional assays demonstrated that the Even skipped C terminus acts as a Groucho-dependent repression domain in early Drosophila embryos . This novel repression domain was active on two target genes that are normally repressed by Even skipped at different concentrations, paired and sloppy paired . When the Groucho interaction motif is mutated, repression of each target gene is reduced to a similar extent, with some activity remaining . Thus, the ability of Even skipped to repress different target genes at different concentrations does not appear to involve differential recruitment or function of Groucho . The accumulation of multiple domains of similar function within a single protein may be a common evolutionary mechanism that fine-tunes the level of activity for different regulatory functions.

Biochem J, 2001 May 1, 355(Pt 3), 609 - 15
The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bepsilon at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase; Woods YL et al.; The substrate specificity of glycogen synthase kinase 3 (GSK3) is unusual in that efficient phosphorylation only occurs if another phosphoserine or phosphothreonine residue is already present four residues C-terminal to the site of GSK3 phosphorylation . One such substrate is the epsilon-subunit of rat eukaryotic protein-synthesis initiation factor 2B (eIF2Bepsilon), which is inhibited by the GSK3-catalysed phosphorylation of Ser(535) . There is evidence that GSK3 is only able to phosphorylate eIF2Bepsilon at Ser(535) if Ser(539) is already phosphorylated by another protein kinase . However, no protein kinases capable of phosphorylating Ser(539) have so far been identified . Here we show that Ser(539) of eIF2Bepsilon, which is followed by proline, is phosphorylated specifically by two isoforms of dual-specificity tyrosine phosphorylated and regulated kinase (DYRK2 and DYRK1A), but only weakly or not at all by other 'proline-directed' protein kinases tested . We also establish that phosphorylation of Ser(539) permits GSK3 to phosphorylate Ser(535) in vitro and that eIF2Bepsilon is highly phosphorylated at Ser(539) in vivo . The DYRK isoforms also phosphorylate human microtubule-associated protein tau at Thr(212) in vitro, a residue that is phosphorylated in foetal tau and hyperphosphorylated in filamentous tau from Alzheimer's-disease brain . Phosphorylation of Thr(212) primes tau for phosphorylation by GSK3 at Ser(208) in vitro, suggesting a more general role for DYRK isoforms in priming phosphorylation of GSK3 substrates.

Acta Biochim Pol, 2000, 47(3), 841 - 6
An optimized recipe for cloning of the polymerase chain reaction-amplified DNA inserts into plasmid vectors; Topcu Z; This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells . The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E . coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration . The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 microg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4 degrees C.

Acta Biochim Pol, 2000, 47(3), 807 - 14
Preliminary crystallographic studies of Y25F mutant of periplasmic Escherichia coli L-asparaginase; Kozak M et al.; Periplasmic Escherichia coli L-asparaginase II with Y25F mutation in the active-site cavity has been obtained by recombinant techniques . The protein was crystallized in a new hexagonal form (P6(5)22) . Single crystals of this polymorph, suitable for X-ray diffraction, were obtained by vapor diffusion using 2-methyl-2,4-pentanediol as precipitant (pH 4.8) . The crystals are characterized by a = 81.0, c = 341.1 A and diffract to 2.45 A resolution . The asymmetric unit contains two protein molecules arranged into an AB dimer . The physiologically relevant ABA'B' homotetramer is generated by the action of the crystallographic 2-fold axis along {1, -1, 0} . Kinetic studies show that the loss of the phenolic hydroxyl group at position 25 brought about by the replacement of Y with F strongly impairs kcat without significantly affecting Km.

Bioorg Med Chem, 2001 Mar, 9(3), 629 - 36
New polycyclic pyrimidine derivatives with antiplatelet in vitro activity: synthesis and pharmacological screening; Bruno O et al.; The preparation and the pharmacological screening of novel anti-aggregatory/antiphlogistic polycyclic pyrimidine derivatives are described . The compounds were developed starting from bioactive 2-aminobenzopyranopyrimidine derivatives in order to assess the importance of the benzopyrano{4,3-d}pyrimidine structure and the role of an amino basic moiety in position 2 . Antiplatelet activity was assessed in vitro against ADP and arachidonic acid-induced aggregation in guinea-pig plasma . Anti-inflammatory/analgesic/antipyretic activities were studied in rat paw oedema, mouse writhing test and E . coli-induced rat fever . Ulcerogenic and gastroprotective effects were also investigated in vivo on rat gastric mucosa . Among the tested compounds, the 5-substituted benzopyranopyrimidine derivatives 3d and 4d proved to be the most active antiplatelet agents as potent as acetylsalicylic acid against arachidonic acid-stimulated aggregation . Furthermore the 2-methylthio derivative 4d was endowed with greater efficacy against ADP aggregation suggesting that additional non-TXA2 dependent mechanisms are involved in its biological activity . Orally administered at 100 mg kg(-1) in rats this latter compound displayed antiphlogistic acitivity comparable to indomethacin (10 mg kg(-1)) coupled with an unusual gastroprotective effect on ethanol-induced ulcers . In conclusion, these findings indicate that the 5-pyrrolidino-2-methylthiobenzopyrano{4,3-d}pyrimidine 4d fulfils the chemical requirements to exhibit antiplatelet activity associated with gastroprotective effect.

Biochem Cell Biol, 2001, 79(2), 183 - 93
His-391 of beta-galactosidase (Escherichia coli) promotes catalyses by strong interactions with the transition state; Huber RE et al.; His-391 of beta-galactosidase (Escherichia coli) was substituted by Phe, Glu, and Lys . Homogeneous preparations of the substituted enzymes were essentially inactive unless very rapid purifications were performed, and the assays were done immediately . The inactive enzymes were tetrameric, just like wild-type beta-galactosidase and their fluorescence spectra were identical to the fluorescence spectrum of wild-type enzyme . Analyses of two of the substituted enzymes that were very rapidly purified to homogeneity and rapidly assayed while they were still active (at only a few substrate concentrations so that the data could be rapidly obtained), showed that the kinetic values were very similar to the values obtained with the same enzymes that were only partially purified . This showed that the kinetics were not affected by the degree of purity and allowed kinetic analyses with partially purified enzymes so that large numbers of points could be used for accuracy . The data showed that His-391 is a very important residue . It interacts strongly with the transition state and promotes catalysis by stabilizing the transition state . Activation energy differences (deltadelta G(S) double dagger), as determined by differences in the kcat/Km values, indicated that substitutions for His-391 caused very large destabilizations (22.8-35.9 kJ/mol) of the transition state . The importance of His-391 for transition state stabilization was confirmed by studies that showed that transition state analogs are very poor inhibitors of the substituted enzymes, while inhibition by substrate analogs was only affected in a small way by substituting for His-391 . The poor stabilities of the transition states caused significant decreases of the rates of the glycolytic cleavage steps (galactosylation, k2) . Degalactosylation (k3) was not decreased to the same extent.

Int J Med Microbiol, 2001 Mar, 290(8), 683 - 91
Intimin from Shiga toxin-producing Escherichia coli and its isolated C-terminal domain exhibit different binding properties for Tir and a eukaryotic surface receptor; Deibel C et al.; The outer membrane protein intimin plays a crucial role in the attaching and effacing process employed by different enteropathogens to colonize the epithelial surface of their hosts . In this study we have characterized the C-terminal binding domain of intimin from the Shiga toxin-producing Escherichia coli strain 413/89-1, that belongs to the beta-subtype of intimins . We found that a fusion of this domain to the maltose-binding protein binds efficiently to both the translocated intimin receptor (Tir) and the surface of uninfected eukaryotic host cells . In contrast, no such binding was observed with the full-length protein localized on the bacterial surface . As the C-terminal domain of intimin and the full-length protein differ in their binding activity, we suggest that the intimin-binding domain might be controlled by the N-terminal portion of the molecule to prevent unproductive interactions with molecules in the lumen of the gut.

Pacing Clin Electrophysiol, 2001 Mar, 24(3), 282 - 7
Ancillary tools in pacemaker and defibrillator lead extraction using a novel lead removal system; Manolis AS et al.; A previous report described our preliminary experience with a highly successful pacing lead removal system (VasoExtor) . Extending this experience, we found it necessary to use additional tools to enhance the success of percutaneous lead extraction with this system . In the present series, we used the standard locking stylets (S and K), and recently, one newer type of stylet (Magic) over the last 3 years in 34 patients to extract 48 pacemaker leads in 31 patients and 3 defibrillator (ICD) leads in 3 patients . Lead extraction was carried out in 23 men and 11 women (aged 64 +/- 17 years) because of pacemaker infection (n = 21), pacemaker (n = 8) or ICD (n = 3) lead malfunction, or prior to ICD implant (n = 2) . Leads were in place for 3.5 +/- 3.7 years . Infections, involving pocket and lead(s), were due to S . epidermidis (n = 13), S . aureus (n = 6), S . aureus plus E . coli (n = 1), for fungi (n = 1) . Of the 48 pacing leads, 31 were ventricular, 15 atrial, and 2 were VDD leads . The ICD leads were two double-coil leads (CPI) and one single-coil lead (Telectronics) . Using the S (n = 12), K (n = 8), or Magic (n = 3) stylets, all pacing leads in 23 patients and the ICD leads in 2 patients were successfully removed from a subclavian approach using the locking stylets . However, in nine (26.5%) patients ancillary tools were required . In four patients, lead fragments were captured with use of a noose catheter, a pigtail catheter, and a bioptome from a right femoral approach . In two patients, locking could not be effected and a noose catheter from the right femoral vein was used, aided by a pigtail and an Amplatz catheter and a bioptome to remove three leads . In a patient with an ICD lead, a combined subclavian (stylet S) and right femoral approach (noose catheter) was required . In a patient with a dysfunctional ventricular lead 12 years old, a motor drive unit was used to facilitate the exchange of locking stylets, but extraction failed . In another patient, a fragment of a dysfunctional ventricular lead remained intravascularly despite resorting to a femoral approach . Finally, lead removal was completely (32/34, 94%) or partially (1/34, 3%) successful in 33 (97%) of 34 patients for 50 (98%) of 51 leads without complications . In conclusion, to enhance the success of pacing or ICD lead extraction with use of the VascoExtor locking stylets, an array of ancillary tools were required in more than one fourth of patients.

Am J Hum Genet, 2001 May, 68(5), 1130 - 8 Epub 2001 Apr 16.
Characterization of mutations in the CPO gene in British patients demonstrates absence of genotype-phenotype correlation and identifies relationship between hereditary coproporphyria and harderoporphyria; Lamoril J et al.; Hereditary coproporphyria (HCP) is the least common of the autosomal dominant acute hepatic porphyrias . It results from mutations in the CPO gene that encodes the mitochondrial enzyme, coproporphyrinogen oxidase . A few patients have also been reported who are homoallellic or heteroallelic for CPO mutations and are clinically distinct from those with HCP . In such patients the presence of a specific mutation (K404E) on one or both alleles produces a neonatal hemolytic anemia that is known as "harderoporphyria"; mutations on both alleles elsewhere in the gene give rise to the "homozygous" variant of HCP . The molecular relationship between these disorders and HCP has not been defined . We describe the molecular investigation and clinical features of 17 unrelated British patients with HCP . Ten novel and four previously reported CPO mutations, together with three previously unrecognized single-nucleotide polymorphisms, were identified in 15 of the 17 patients . HCP is more heterogeneous than other acute porphyrias, with all but one mutation being restricted to a single family, with a predominance of missense mutations (10 missense, 2 nonsense, 1 frameshift, and 1 splice site) . Of the four known mutations, one (R331W) has previously been reported to cause disease only in homozygotes . Heterologous expression of another mutation (R401W) demonstrated functional properties similar to those of the K404E harderoporphyria mutation . In all patients, clinical presentation was uniform, in spite of the wide range (1%-64%) of residual coproporphyrinogen oxidase activity, as determined by heterologous expression . Our findings add substantially to knowledge of the molecular epidemiology of HCP, show that single copies of CPO mutations that are known or predicted to cause "homozygous" HCP or harderoporphyria can produce typical HCP in adults, and demonstrate that the severity of the phenotype does not correlate with the degree of inactivation by mutation of coproporphyrinogen oxidase.

Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 5019 - 24 Epub 2001 Apr 17.
Balanced branching in transcription termination; Harrington KJ et al.; The theory of stochastic transcription termination based on free-energy competition {von Hippel, P . H . & Yager, T . D . (1992) Science 255, 809-812 and von Hippel, P . H . & Yager, T . D . (1991) Proc . Natl . Acad . Sci . USA 88, 2307-2311} requires two or more reaction rates to be delicately balanced over a wide range of physical conditions . A large body of work on glasses and large molecules suggests that this balancing should be impossible in such a large system in the absence of a new organizing principle of matter . We review the experimental literature of termination and find no evidence for such a principle, but do find many troubling inconsistencies, most notably, anomalous memory effects . These effects suggest that termination has a deterministic component and may conceivably not be stochastic at all . We find that a key experiment by Wilson and von Hippel {Wilson, K . S . & von Hippel, P . H . (1994) J . Mol . Biol . 244, 36-51} thought to demonstrate stochastic termination was an incorrectly analyzed regulatory effect of Mg(2+) binding.

Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 5128 - 33 Epub 2001 Apr 17.
An essential amino acid residue in the protein translocation channel revealed by targeted random mutagenesis of SecY; Mori H et al.; The SecY/Sec61alpha family of membrane proteins are the central subunits of the putative protein translocation channel . We introduced random mutations into a segment of Escherichia coli SecY within its cytoplasmic domain 5, which was shown previously to be important for the SecA-dependent translocation activity . Mutations were classified into those retaining function and those gaining a dominant-interfering ability caused by a loss of function . These analyses showed that Arg-357, Pro-358, Gly-359, and Thr-362 are functionally important; Arg-357, conserved in almost all organisms, was identified as an indispensable residue.

J Biol Chem, 2001 Jul 6, 276(27), 25351 - 8 Epub 2001 Apr 17.
Isolation of hyperactive mutants of the MAPK p38/Hog1 that are independent of MAPK kinase activation; Bell M et al.; Mitogen-activated protein kinases (MAPKs) play pivotal roles in growth, development, differentiation, and apoptosis . The exact role of a given MAPK in these processes is not fully understood . This question could be addressed using active forms of these enzymes that are independent of external stimulation and upstream regulation . Yet, such molecules are not available . MAPK activation requires dual phosphorylation, on neighboring Tyr and Thr residues, catalyzed by MAPK kinases (MAPKKs) . It is not known how to force MAPK activation independent of MAPKK phosphorylation . Here we describe a series of nine hyperactive (catalytically and biologically), MAPKK-independent variants of the MAPK Hog1 . Each of the active molecules contains just a single point mutation . Six mutations are in the conserved L16 domain of the protein . The active Hog1 mutants were obtained through a novel genetic screen that could be applied for isolation of active MAPKs of other families . Equivalent mutations, introduced to the human p38alpha, rendered the enzyme active even when produced in Escherichia coli, showing that the mutations increased the intrinsic catalytic activity of p38 . It implies that the activating mutations could be directly used for production of active forms of MAPKs from yeasts to humans and could open the way to revealing their biological functions.

Plant J, 2001 Mar, 25(5), 487 - 98
Peroxisomal alanine : glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana; Liepman AH et al.; At least two glyoxylate aminotransferases are hypothesized to participate in the steps of photorespiration located in peroxisomes . Until recently, however, genes encoding these enzymes had not been identified . We describe the isolation and characterization of an alanine : glyoxylate aminotransferase (AGT1, formerly AGT) cDNA from Arabidopsis thaliana . Southern blot analysis confirmed that Arabidopsis AGT1 is encoded by a single gene . Homologs of this class IV aminotransferase are also known in other plants, animals, and methylotrophic bacteria, suggesting an ancient evolutionary origin of this enzyme . AGT1 transcripts were present in all tissues of Arabidopsis, but were most abundant in green, leafy tissues . Purified, recombinant Arabidopsis AGT1 expressed in Escherichia coli catalyzed three transamination reactions using the following amino donor : acceptor combinations: alanine : glyoxylate, serine : glyoxylate, and serine : pyruvate . AGT1 had the highest specific activity with the serine : glyoxylate transamination, and apparent Km measurements indicate that this is the preferred in vivo reaction . In vitro import experiments and subcellular fractionations localized AGT1 to peroxisomes . Sequence analysis of the photorespiratory sat mutants revealed a single nucleotide substitution in the AGT1 gene from these plants . This transition mutation is predicted to result in a proline-to-leucine substitution at residue 251 of AGT1 . When this mutation was engineered into the recombinant AGT1 protein, enzymatic activity using all three donor : acceptor pairs was abolished . We conclude that Arabidopsis AGT1 is a peroxisomal photorespiratory enzyme that catalyzes transamination reactions with multiple substrates.

Mol Microbiol, 2001 Apr, 40(2), 498 - 507
The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome; Weigel C et al.; We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome . Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination . The mutations were subsequently transferred to a wild-type host by transduction . oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function . The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells . Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry . In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous . Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication . We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.

Mol Microbiol, 2001 Apr, 40(2), 440 - 50
A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC --> YojN --> RcsB signalling pathway implicated in capsular synthesis and swarming behaviour; Takeda S et al.; In this study, we re-investigated the previously characterized RcsC (sensor His-kinase) --> RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli . The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus . As judged from the current general view as to the His --> Asp phosphorelay, this RcsC --> RcsB framework is somewhat puzzling, because RcsC appears to contain both a His-kinase domain and a receiver domain, but not a histidine (His)-containing phosphotransmitter domain (e.g . HPt domain) . We thus suspected that an as yet unknown mechanism might be underlying in this particular His --> Asp phosphorelay system . Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His-containing phosphotransmitter (named YojN) is essential for this signalling system . A revised model is proposed in which the multistep RcsC --> YojN --> RcsB phosphorelay is implicated . It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E . coli cells during colony formation on the surface of agar plates, namely swarming.

Mol Microbiol, 2001 Apr, 40(2), 397 - 413
Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol; Tomoyasu T et al.; We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol . In DeltarpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5-10% and 20-30% of total protein aggregated at 30 degrees C and 42 degrees C respectively . The aggregates contained 350-400 protein species, of which 93 were identified by mass spectrometry . The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30 degrees C, and showed strong overlap with previously identified DnaK substrates . Overproduction of the DnaK system, or low-level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to DeltarpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival . In rpoH+ cells, DnaK depletion did not lead to protein aggregation at 30 degrees C, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins . Lon was the most efficient protease in degrading misfolded proteins in DnaK-depleted cells . At 42 degrees C, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42 degrees C.

Mol Microbiol, 2001 Apr, 40(2), 376 - 86
DNA replication-coupled inactivation of DnaA protein in vitro: a role for DnaA arginine-334 of the AAA+ Box VIII motif in ATP hydrolysis; Su'etsugu M et al.; The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein . We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication-stimulated hydrolysis of DnaA-bound ATP yields the ADP-bound inactive form . The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7-fold higher than the number formed during incubation without replication . These results support the notion that coupling of DnaA-ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation . We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro . Whereas ATP bound to wild-type DnaA is hydrolysed in a DNA-dependent intrinsic manner or in a sliding clamp-dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions . This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis . This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.

Mol Microbiol, 2001 Apr, 40(2), 332 - 46
Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway; Fernandez LA et al.; In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges . To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion) . The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e . to preserve its antigen-binding activity) . We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed . Neither periplasmic and inner membrane-bound Dsb enzymes (e.g . DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation . However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins . These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process . Taken together, these results indicate not only that the type I system tolerates secretion of disulphide-containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.

Mol Microbiol, 2001 Apr, 40(2), 323 - 31
Identification of a twin-arginine leader-binding protein; Oresnik IJ et al.; The transport and targeting of a number of periplasmic proteins is carried out by the Sec-independent Mtt (also referred to as Tat) protein translocase . Proteins using this translocase have a distinct twin-arginine-containing leader . We hypothesized that specific leader-binding proteins exist to escort proteins to the translocase complex . A fusion was constructed with the twin-arginine leader from dimethyl sulphoxide (DMSO) reductase, subunit DmsA, to the N-terminus of glutathione-S-transferase . This leader fusion was bound to a glutathione affinity column through which an Escherichia coli anaerobic cell-free extract was passed . Proteins that bound to the leader were then separated and identified by N-terminal sequencing, which identified DnaK and a protein originating from the uncharacterized reading frame ynfI . This gene has been designated dmsD based on the findings presented in this paper . DmsD was purified as a His6 fusion and was shown to interact with preprotein forms of DmsA and TorA (trimethyl amine N-oxide reductase) . A strain carrying a dmsD knock-out mutation showed a loss of anaerobic growth on glycerol-DMSO medium and reduced growth on glycerol-fumarate medium . This work suggests that DmsD is a twin-arginine leader-binding protein.

Mol Microbiol, 2001 Apr, 40(2), 314 - 22
Biogenesis of inner membrane proteins in Escherichia coli; de Gier JW et al.; For a long time, it was generally assumed that the biogenesis of inner membrane proteins in Escherichia coli occurs spontaneously, and that only the translocation of large periplasmic domains requires the aid of a protein machinery, the Sec translocon . However, evidence obtained in recent years indicates that most, if not all, inner membrane proteins require the assistance of protein factors to reach their native conformation in the membrane . Here, we review and discuss recent advances in our understanding of the biogenesis of inner membrane proteins in E . coli.

J Microencapsul, 2001 May-Jun, 18(3), 385 - 95
Biological cells as templates for hollow microcapsules; Neu B et al.; Microcapsules in the micrometer size range with walls of nanometer thickness are of both scientific and technological interest, since they can be employed as micro- and nano-containers . Liposomes represent one example, yet their general use is hampered due to limited stability and a low permeability for polar molecules . Microcapsules formed from polyelectrolytes offer some improvement, since they are permeable to small polar molecules and resistant to chemical and physical influences . Both types of closed films are, however, limited by their spherical shape which precludes producing capsules with anisotropic properties . Biological cells possess a wide variety of shapes and sizes, and, thus, using them as templates would allow the production of capsules with a wide range of morphologies . In the present study, human red blood cells (RBC) as well as Escherichia coli bacteria were used; these cells were fixed by glutardialdehyde prior to layer-by-layer (LbL) adsorption of polyelectrolytes . The growth of the layers was verified by electrophoresis and flow cytometry, with morphology investigated by atomic force and electron microscopy; the dissolution process of the biological template was followed by confocal laser scanning microscopy . The resulting microcapsules are exact copies of the biological template, exhibit elastic properties, and have permeabilities which can be controlled by experimental parameters; this method for microcapsule fabrication, thus, offers an important new approach for this area of biotechnology.

Biol Chem, 2001 Feb, 382(2), 291 - 7
Biosynthesis of N-acetylneuraminic acid in cells lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase; Hinderlich S et al.; The first two steps in mammalian biosynthesis of N-acetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase . A subclone of the human B lymphoma cell line BJA-B K20, lacking UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates . Here we show that this cell line surprisingly still retains N-acetylmannosamine kinase activity . A gel filtration analysis of BJA-B K88 control cells, which express UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, revealed two N-acetylmannosamine kinase activity peaks, one co-eluting with UDP-N-acetylglucosamine 2-epimerase activity and one co-eluting with N-acetylglucosamine kinase . For this enzyme previous studies already showed a ManNAc kinase activity in vitro . In contrast, the hyposialylated BJA-B K20 subclone displayed only the N-acetylmannosamine kinase peak, co-migrating with N-acetylglucosamine kinase . The CMP-N-acetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supplementing the medium with N-acetylmannosamine . This N-acetylmannosamine-induced increase was drastically reduced by co-supplementation with N-acetylglucosamine only in K20 cells . We therefore propose the phosphorylation of N-acetylmannosamine as a hitherto unrecognized role of N-acetylglucosamine kinase in living cells.

Biol Chem, 2001 Feb, 382(2), 195 - 207
The liver flukes Fasciola gigantica and Fasciola hepatica express the leucocyte cluster of differentiation marker CD77 (globotriaosylceramide) in their tegument; Wuhrer M et al.; Glycosphingolipids from the parasitic liver flukes Fasciola gigantica and Fasciola hepatica were isolated and their carbohydrate moieties were structurally analysed by methylation analysis, exoglycosidase treatment, on-target exoglycosidase cleavage and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry . For both liver fluke species, the ceramide monohexosides Gal1-ceramide and Glc1-ceramide were found in relative amounts of 1.0 to 0.1, respectively . From F . gigantica, the ceramide dihexoside was isolated in sufficient amounts to be structurally determined as lactosylceramide, Gal beta4-Glc1-ceramide, while for both liver fluke species the ceramide trihexoside was shown to be Gal alpha4Gal beta4-Glc1-ceramide, which is designated as either globotriaosylceramide, Pk-blood group antigen or CD77 leucocyte cluster of differentiation antigen . To our knowledge, this is the first report on the expression of globo-series glycosphingolipids in non-mammalian species . Ceramide analysis of ceramide monohexosides yielded as major components octadecanoic and 2-hydroxyoctadecanoic fatty acids together with C18- and C20-phytosphingosines . By the use of an anti-CD77 monoclonal antibody and the Escherichia coli Shiga toxin B1 subunit, globotriaosylceramide could be immunolocalised to the tegument of F . hepatica cryosections . The sharing of CD77 between liver flukes and their mammalian hosts fits in with the concept of molecular mimicry, which is closely parallel to the established imitation of host CD15 (Lewis X) displayed by the blood fluke Schistosoma mansoni.

J Protein Chem, 2000 Nov, 19(8), 663 - 9
A functional raw starch-binding domain of barley alpha-amylase expressed in Escherichia coli; Tibbot BK et al.; The mature form of barley seed low-pI alpha-amylase (BAA1) possesses a raw starch-binding site in addition to the catalytic site . A truncated cDNA encoding the C-terminal region (aa 281-414) and containing the proposed raw starch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Escherichia coli as an N-terminal His-Tag fusion protein . SBD was produced in the form of insoluble inclusion bodies that were extracted with urea and successfully refolded into a soluble form via dialysis . To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligand cross-linked to Sepharose . This work demonstrates that a SBD is located in the C-terminal region and retains sufficient function in the absence of the N-terminal, catalytic, and Trp278/279 regions.

Int Arch Allergy Immunol, 2001 Jan-Mar, 124(1-3), 406 - 10
The influence of CpG motifs on a protein or DNA-based Th2-type immune response against major pollen allergens Bet v 1a, Phl p 2 and Escherichia coli-derived beta-galactosidase; Hochreiter R et al.; BACKGROUND: DNA immunization and protein immunization with CpG motifs as adjuvants represent promising approaches in allergen-specific immunotherapy . Objective: We investigated the effect of coinjection or prepriming with CpG-ODN on Th2-type responses induced by gene gun and protein immunization . METHODS: BALB/c mice were immunized with the gene gun using plasmid DNA containing the cDNAs coding for the genes of Bet v 1a, Phl p 2 and beta-galactosidase or with the purified Al(OH)(3)-adsorbed proteins . In addition, CpG-ODN were applied by coinjection or by prepriming treatment . Antibody and cytokine responses were measured by ELISA, proliferative and cytotoxic responses were determined by standard labeling procedures . Furthermore, the allergenic activity of sera was measured by passive cutaneous anaphylaxis . RESULTS: Gene gun immunization and protein immunization induced a clear Th2-type response for all antigens . The Th1-promoting effect of CpG-ODN coinjection together with gene gun immunization was restricted to beta-galactosidase as indicated by the increase of IgG2a and a marked expression of IFN-gamma . CpG motifs also increased the specific cytotoxic response against beta-galactosidase . Prepriming with CpG-ODN and gene gun or protein immunization with Bet v 1a exhibited no significant difference to the non-CpG control group . However, sera from mice preprimed with CpG-ODN induced no anaphylaxis with gene gun immunization, but with protein immunization . CONCLUSIONS: The effect of CpG motifs in vivo depends on a variety of parameters like the nature of the antigen and the immunization modality . Furthermore, our studies indicate that a combination of CpG + DNA immunization may be more effective in antagonizing Th2 responses than the combination of CpG + protein immunization .

Int Arch Allergy Immunol, 2001 Jan-Mar, 124(1-3), 61 - 3
Sequence polymorphisms and antibody binding to the group 2 dust mite allergens; Smith AM et al.; BACKGROUND: The group 2 allergens Der p 2, Der f 2 and Eur m 2 are 14-kD proteins with > 80% sequence identity . Isoforms within each genus have been identified which differ by 3-4 amino acids . The aim of this study was to investigate the importance of these substitutions to antibody binding . METHODS: Recombinant allergens were expressed and purified from Escherichia coli . ELISA and skin testing were used to evaluate antibody binding . Molecular modeling of the tertiary structure was preformed to examine the location of substitutions . RESULTS: The three Der f 2 isoforms and two of three of the Der p 2 isoforms reacted with all monoclonal antibodies (mAb) . Der p 2.0101, the isoform with aspartate at position 114, bound all mAb except 1D8 . Substitution of asparagine for aspartate restored binding of rDer p 2.0101 to mAb 1D8 and increased the correlation coefficient for IgE binding from 0.72 to 0.77 . The three Der p 2 isoforms showed comparable skin test reactivity to nDer p 2 and commercial extract . rEur m 2.0101 bound to all mAb except 7A1 and when compared with rDer p 2 for IgE binding, r(2) = of 0.58 (n = 72) . Lep d 2 did not react with mAb or with Dermatophagoides spp . allergic sera . Modeling revealed that Eur m 2, Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2 and the substitutions are on the surface . CONCLUSIONS: mAb could distinguish isoform substitutions . IgE binding showed a good correlation among all isoforms, thus the recombinant allergens are useful for diagnosis .

Int Arch Allergy Immunol, 2001 Jan-Mar, 124(1-3), 55 - 6
Identification of a novel cat allergen--cystatin; Ichikawa K et al.; BACKGROUND: Cat allergen is an important cause of sensitization among children with asthma in Japan . Although there is good evidence that cats produce other allergens, only one major allergen, Fel d 1, has been studied in detail . AIMS: To identify and define the molecular structure of the other potential cat allergens . METHODS: A cat skin cDNA library was screened using IgE antibodies to cat dander and selected clones were sequenced and expressed . RESULTS: One cDNA clone contained an open reading frame encoding a 98-amino acid residue protein . Sequence homology searches revealed a high degree of identity with bovine and human cystatin A, 79 and 75%, respectively . This cat cystatin clone contained the conserved cysteine protease motif and two of three lipocalin motifs . By plaque immunoassay, 60-90% of cat allergic sera had IgE Ab to cat cystatin . This cysteine protease inhibitor motif was partially conserved in dog allergens, Can f 1 and Can f 2, which are lipocalins . Recombinant cystatin was produced in Escherichia coli cells and purified as an 11-kD protein, corresponding to the predicted MW of cystatin . The structure of cat cystatin was modeled on human cystatin B using the SWISS-MODEL . CONCLUSION: A newly identified allergen, cystatin, has been cloned from cat skin and is a member of the cysteine protease inhibitor family .

Mol Pharmacol, 2001 May, 59(5), 1181 - 6
Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases; Van Rompay AR et al.; Uridine-cytidine kinases (UCK) have important roles for the phosphorylation of nucleoside analogs that are being investigated for possible use in chemotherapy of cancer . We have cloned the cDNA of two human UCKs . The approximately 30-kDa proteins, named UCK1 and UCK2, were expressed in Escherichia coli and shown to catalyze the phosphorylation of Urd and Cyd . The enzymes did not phosphorylate deoxyribonucleosides or purine ribonucleosides . UCK1 mRNA was detected as two isoforms of approximately 1.8 and approximately 2.7 kb . The 2.7-kb band was ubiquitously expressed in the investigated tissues . The band of approximately 1.8 kb was present in skeletal muscle, heart, liver, and kidney . The two isoforms of UCK2 mRNA of 1.2 and 2.0 kb were only detected in placenta among the investigated tissues . The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.2-9q34.3 and 1q22-1q23.2, respectively . We tested 28 cytidine and uridine nucleoside analogs as possible substrates of the enzymes . The enzymes phosphorylated several of the analogs, such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N(4)-acetylcytidine, N(4)-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N(4)-anisoylcytidine . The cloning and recombinant expression of the two human UCKs will be important for development of novel pyrimidine ribonucleoside analogs and the characterization of their pharmacological activation.

Cancer Res, 2001 Apr 1, 61(7), 3022 - 6
Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing; Black ME et al.; Cancer suicide gene therapy affords the prospect of using the most optimal genes available because the source of the therapeutic gene is often irrelevant . Currently, there are numerous preclinical and clinical trials to develop tumor ablative therapies that use viral, yeast, or bacterial genes . One such gene, the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) is widely used as a suicide gene in combination with ganciclovir . In the study reported here, a restricted set of random sequences (semi-random) was introduced into the active site of HSV-1 TK, and the resulting variants were selected on the basis of their ability to confer increased ganciclovir or acyclovir sensitivity to Escherichia coli . Sequence analysis demonstrated that functional mutants contained three to five amino acid substitutions that are unique and novel combinations . On the basis of enzyme assay results, three mutants were identified for further analysis in vitro . These three mutants conferred substantial increased sensitivity to both ganciclovir and acyclovir when compared with IC50s of wild-type TK expressing rat C6 glioma cells . One mutant, SR39, was further evaluated in a xenograft tumor model in nude mice . Expression of SR39 in tumors was shown to prevent tumor growth at prodrug dosages that did not affect wild-type HSV-1 TK-expressing tumors . The use of any of these mutants as a suicide gene should provide a more effective and safer alternative to wild-type TK, because lower, less immunosuppressive doses of ganciclovir will be necessary for tumor ablation, and the use of acyclovir may now be possible.

Cancer Res, 2001 Apr 1, 61(7), 2983 - 95
Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo; Jacobs A et al.; To evaluate the efficiency of gene delivery in gene therapy strategies for malignant brain tumors, it is important to determine the distribution and magnitude of transgene expression in target tumor cells over time . Here, we assess the time- and vector dose-dependent kinetics of recombinant herpes simplex virus (HSV)-1 vector-mediated gene expression and vector replication in culture and in vivo by a recently developed radiotracer method for noninvasive imaging of gene expression (J . G . Tjuvajev et al., Cancer Res., 55: 6126-6132, 1995) . The kinetics of viral infection of rat 9L gliosarcoma cells by the replication-conditional HSV-1 vector, hrR3, was studied by measuring the accumulation rate of 2-{14C}-fluoro-5-iodo-1-beta-D-arabinofuranosyl-uracil (FIAU), a selective substrate for viral thymidine kinase (TK) . The level of viral TK activity in 9L cells was monitored by the radiotracer assay to assess various vector doses and infection times, allowing vector replication and spread . In parallel, viral yields and levels of Escherichia coli beta-galactosidase activity were assessed quantitatively . To study vector replication, spread and HSV-1-tk and lacZ gene coexpression in vivo, first- or second-generation recombinant HSV-1 vectors (hrR3 or MGH-1) were injected into s.c . growing rat 9L or human U87 deltaEGFR gliomas in nude rats at various times (8 h to 8 days) and at various vector doses {1 x 10(6) to 2 x 10(9) plaque-forming units (PFUs)} prior to imaging . For noninvasive assessment of HSV-1-tk gene expression (124I-labeled FIAU % dose/g), 0.15 mCi of 124I-labeled FIAU was injected i.v . 8 h after the last vector administration, and FIAU positron emission tomography (PET) was performed 48 h later . For the assessment of HSV-1-tk and lacZ gene coexpression, 0.2 mCi of 131I-labeled FIAU was injected i.v . 24 h after the last vector administration . Forty-eight h later, animals were killed, and tumors were dissected for quantitative autoradiographical and histochemical assessment of regional distribution of radioactivity (TK expression measured as 131I-labeled FIAU % dose/g) and coexpressed lacZ gene activity . The rates of FIAU accumulation (Ki) in hrR3-infected 9L cells in culture, which reflect the levels of HSV-1-tk gene expression, ranged between 0.12 and 3.4 ml/g/min . They increased in a vector dose- and infection time-dependent manner and correlated with the virus yield (PFUs/ml), where the PFUs:Ki ratios remained relatively constant over time . Moreover, a linear relationship was observed between lacZ gene expression and FIAU accumulation 5-40 h after infection of 9L cells with a multiplicity of infection of 1.5 . At later times (> 52 h postinjection), high vector doses (multiplicity of infection, 1.5) led to a decrease of FIAU accumulation rates, viral yield, and cell pellet weights, indicating vector-mediated cell toxicity . Various levels of HSV-1-tk gene expression could be assessed by FIAU-PET after in vivo infection of s.c . tumors . The levels of FIAU accumulation were comparatively low (approximately ranging from 0.00013 to 0.003% injected dose/g) and were spatially localized; this may reflect viral-induced cytolysis of infected tumor cells and limited lateral spread of the virus . Image coregistration of tumor histology, HSV-1-tk related radioactivity (assessed by autoradiography), and lacZ gene expression (assessed by beta-galactosidase staining) demonstrated a characteristic pattern of gene expression around the injection sites . A rim of lacZ gene expression immediately adjacent to necrotic tumor areas was observed, and this zone was surrounded by a narrow band of HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue . These results demonstrate that recombinant HSV-1 vector-mediated HSV-1-tk gene expression can be monitored noninvasively by PET, where the areas of FIAU-derived radioactivity identify the viable portion of infected tumor tissue that retains FIAU accumulation ability, and that the accumulation rate of FIAU in culture, Ki, reflects the number of HSV-1 viral particles in the infected tumor cell population {4.1 +/- 0.6 x 10(6) PFUs/Ki unit (PFUs divided by ml/min/g)} . Moreover, time-dependent and spatial relationships of HSV-1-tk and lacZ gene coexpression in culture and in vivo indicate the potential for indirect in vivo imaging of therapeutic gene expression in tumor tissue infected with any recombinant HSV-1 vector where a therapeutic gene is substituted for the lacZ gene.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 871 - 8
Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38; Sciotti M et al.; Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme . Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes . We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity . Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency (k(cat)/K(m,NADPH)) by more than three orders of magnitude . Similarly, substitution of Asp for Ala-37 decreased k(cat)/K(m,NADPH) 1000-fold but had little effect on k(cat)/K(m,NADH) . The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 863 - 70
Cloning and expression of cDNA encoding hamster liver 3-hydroxyhexobarbital/17beta(3alpha)-hydroxysteroid dehydrogenase 1; Takenoshita R et al.; Using RACE techniques we have cloned and sequenced one of the hamster liver 3-hydroxy-hexobarbital dehydrogenases which catalyze not only cyclic alcohols but also 17beta-hydroxy-steroids and 3alpha-hydroxysteroids . The gene specific primers to 3-hydroxyhexobarbital dehydrogenase 1 (G2) were synthesized on the basis of its partial peptide sequences . The sequence of full length cDNA generated by 3'- and 5'-RACE PCR consisted of 1225 nucleotides including an open reading frame of 972 nucleotides encoding a protein of 323 amino acids . The deduced amino acid sequence matched exactly with the partial peptide sequences of hamster liver 3-hydroxyhexobarbital dehydrogenase 1 (G2) . The sequence showed 84.5% identity to mouse liver 17beta-dehydrogenase(A-specific), and 74-76% identity to human liver bile acid binding protein/3alpha-hydroxysteroid dehydrogenase (DD2), human liver 3alpha-hydroxysteroid dehydrogenase type I (DD4) and type II (DD3), and rabbit ovary 20alpha-hydroxysteroid dehydrogenase . The protein contains catalytic residues of aldo-keto reductases, Asp50, Tyr55, Lys84, His117 . These results suggest that the hamster liver 3-hydroxyhexobarbital/17beta(3alpha)-hydroxysteroid dehydrogenase belongs to aldo-keto reductase superfamily . The insert containing the full-length cDNA of 3-hydroxyhexobarbital dehydrogenase and vector specific overhang produced by PCR was annealed with pET-32 Xa/LIC vector . The plasmid was transformed into BL21 (DE3) cells containing pLysS . The recombinant enzyme was induced 1 mM IPTG . The expressed enzyme was produced as fusion protein and purified by nickel chelating affinity chromatography followed by POROS CM column chromatography and superdex 75 gel filtration . Molecular weight of the recombinant enzyme fused thioredoxin and his*tag was about 55000 and that was 35000 after Factor Xa protease treatment . The recombinant enzyme dehydrogenated 3-hydroxy-hexobarbital, 1-acenaphthenol, 2-cyclohexen-1-ol, testosterone, glycolithocholic acid as well as the native enzyme purified from hamster liver.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 847 - 61
Characterization of multiple Chinese hamster carbonyl reductases; Terada T et al.; Carbonyl reductase (CR) is an enzyme which can catalyze the oxidoreduction of various carbonyl compounds in the presence of NAD(P)H . With the PCR method, using primers carrying the conserved nucleotide sequence among mammalian CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which encode a unique carbonyl reductase from the Chinese hamster . The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu1102I, BspEI and XmaI restriction enzymes . The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2 . The predicted double-wound betaalphabetaalpha-structures of the CHCRs suggests the presence of a typical NADP(+)-binding motif and is similar to the corresponding region of 3alpha,20beta-hydroxysteroid dehydrogenase and mouse lung tetrameric carbonyl reductase . The deduced amino acid sequence of CHCR1 showed a high homology to CHCR2 (>96%) and the other mammalian CRs (>81%) . However, CHCR3 showed a high homology to human CBR3 (>86%) and a relatively lower homology to the other CHCRs (<76%) . Bacterial recombinant CHCRs showed typical carbonyl reductase activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4-carboxyaldehyde . These three CRs showed not only 3-keto reductase of steroids, but also 20-keto reductase . However, these CRs did not show any activity of 17-keto reductase activity . Both CHCR1 and CHCR2 have prostaglandin 9-keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE(2) and PGF(2alpha) from the analyses of enzymatic reaction products . The results of Western blotting and RT-PCR suggest these CHCRs have a tissue-dependent-distribution in the Chinese hamster.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 833 - 45
Site-directed mutagenesis studies of bovine liver cytosolic dihydrodiol dehydrogenase: the role of Asp-50, Tyr-55, Lys-84, His-117, Cys-145 and Cys-193 in enzymatic activity; Terada T et al.; A previous report on the cloning, bacterial expression and purification of bovine liver cytosolic dihydrodiol dehydrogenase (DD3) cDNA (1,330 bp in full length) using pKK223-3 expression vector characterize the properties of the recombinant DD3 in the aspects of substrate specificity and inhibitor sensitivity (Terada et al., Adv . Exp . Biol . Res . 414 (1997) 543-53) . The nucleotide sequence of this DD3 cDNA completely matches that of bovine liver-type prostaglandin F synthase (PGFS) (Suzuki et al., J . Biol . Chem . 274 (1999) 241-8) . In the present study, a large amount of recombinant DD3 (rDD3) was expressed in Escherichia coli BL21 (DE3) with a pET28a expression vector . The recombinant DD3 (rDD3) was easily and quickly purified to an apparent homogeneity with one step column chromatography of Ni(2+)-affinity resin . The rDD3 showed essentially the same substrate specificity and inhibitor sensitivity as purified liver DD3 (DD3) . To analyze the role of amino acid residues of DD3 in its enzymatic activity, site-directed mutagenesis of DD3 with PCR method was performed . The results of the analyses of these mutants in the aspects of substrate specificity and cofactor-binding suggested a variety of functions in the enzymatic activity: as an active site Tyr-55 may act as a general acid and Asp-50, Lys-84 and His-117 may play an important role in the control of protonation of Tyr-55 as a general acid in the dehydrogenase activity under higher pH conditions, though these residues may not be involved in reductase activity under lower pH conditions . Though the mutated DD3s (Cys to Ser) did not show significant differences in their substrate specificities, these mutants showed different sensitivities to SH-reagents . Present results indicate that Cys-193 may play an important role in the modulation of enzymatic activity under redox conditions generated with GSH+GSSG among five cysteines in DD3.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 785 - 91
New developments in our understanding of the beta-hydroxyacid dehydrogenases; Njau RK et al.; The beta-hydroxyacid dehydrogenases are a structurally conserved family of enzymes that catalyze the NAD(+) or NADP(+)-dependent oxidation of specific beta-hydroxyacid substrates like beta-hydroxyisobutyrate . These enzymes share distinct domains of amino acid sequence homology, most of which now have assigned putative functions . 6-phosphogluconate dehydrogenase and beta-hydroxyisobutyrate dehydrogenase, the most well-characterized members, both appear to be readily inactivated by chemical modifiers of lysine residues, such as 2,4,6-trinitrobenzene sulfonate (TNBS) . Peptide mapping by ESI-LCMS showed that inactivation of beta-hydroxyisobutyrate dehydrogenase with TNBS occurs with the labeling of a single lysine residue, K248 . This lysine residue is completely conserved in all family members and may have structural importance relating to cofactor binding . The structural framework of the beta-hydroxyacid dehydrogenase family is shared by many bacterial homologues . One such homologue from E . coli has been cloned and expressed as recombinant protein . This protein was found to have enzymatic activity characteristic of tartronate semialdehyde reductase, an enzyme required for bacterial biosynthesis of D-glycerate . A homologue from H . influenzae was also cloned and expressed as recombinant protein . This protein was active in the oxidation of D-glycerate, but showed approximately ten-fold higher activity with four carbon substrates like beta-D-hydroxybutyrate and D-threonine . This enzyme might function in H . influenzae, and other species, in the utilization of polyhydroxybutyrates, an energy storage form specific to bacteria . Cloning and characterization of these bacterial beta-hydroxyacid dehydrogenases extends our knowledge of this enzyme family.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 723 - 36
A model on the regulation of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni; Xiong G et al.; 3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds . The enzyme has recently been cloned and characterized by our group . A strong induction of enzyme activity is observed in the presence of steroids like testosterone . In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3alpha-HSD/CR gene (hsdA) expression . Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter . The deletion of this cis-regulating sequence significantly increases hsdA expression . About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted . To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichia coli . Rep1 does not bind to DNA but may bind to 3alpha-HSD/CR mRNA as predicted by its secondary structure . Concluding from our data, induction of 3alpha-HSD/CR in C . testosteroni by steroids in fact appears to be a de-repression, where the steroidal 'inducer' prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 47 - 56
Subunit communication in tetrameric class 2 human liver aldehyde dehydrogenase as the basis for half-of-the-site reactivity and the dominance of the oriental subunit in a heterotetramer; Weiner H et al.; Data has been published showing that in heterotetrameric liver mitochondrial aldehyde dehydrogenase composed of the active (E487) and the inactive Oriental-variant (K487) subunit, the Oriental variant was dominant and caused the inactivation of the E487 subunit . The published structures of the enzyme showed that the glutamate at position 487 is salt bonded to an arginine (475) in a different subunit . Arg475 was mutated to a glutamine to test for its importance in causing the Oriental variant to be an enzyme with a high Km for NAD and a low specific activity . Unexpectedly, the R475Q mutant exhibited positive cooperativity in NAD binding with a Hill coefficient of 2 . Individual heterotetramers composed of subunits of E487 and K487 were produced by making changes to two residues on the surface of the enzyme and then co-expressing both cDNAs in E . coli . The E(3)K form had essentially 50% the activity of the E(4) homotetrameric form while EK(3) had essentially the same properties as did the homotetrameric K(4) Oriental variant . This showed that in a dimer pair composed of one K- and one E- subunit the K-subunit became dominant and caused the inactivation of its E-partner . Further, pre-steady state burst data and steady state kinetic data make it appear that there was one functioning active subunit in each of the dimer pairs that made up the tetrameric enzyme . Thus, the half-of-the-site reactivity is a result of having one functioning and one non-functioning subunit in each dimer pair . The actual structural basis for this is still not understood, but could be related to the E487-R475 inter-dimer salt bond.

Biochemistry, 2001 Apr 24, 40(16), 5102 - 10
Mechanism of action of Escherichia coli ribonuclease III . Stringent chemical requirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant; Sun W et al.; Escherichia coli ribonuclease III (EC 3.1.24) is a double-strand- (ds-) specific endoribonuclease involved in the maturation and decay of cellular, phage, and plasmid RNAs . RNase III is a homodimer and requires Mg(2+) to hydrolyze phosphodiesters . The RNase III polypeptide contains an N-terminal catalytic (nuclease) domain which exhibits eight highly conserved acidic residues, at least one of which (Glu117) is important for phosphodiester hydrolysis but not for substrate binding {Li and Nicholson (1996) EMBO J . 15, 1421-1433} . To determine the side chain requirements for activity, Glu117 was changed to glutamine or aspartic acid . The mutant proteins were purified as (His)(6)-tagged species, and both exhibited normal homodimeric behavior as shown by chemical cross-linking . The Glu117Gln mutant is unable to cleave substrate in vitro under all tested conditions but can bind substrate . The Glu117Asp mutant also is defective in cleavage while able to bind substrate . However, low level activity is observed at extended reaction times and high enzyme concentrations, with an estimated catalytic efficiency approximately 15 000-fold lower than that of RNase III . The activity of the Glu117Asp mutant but not that of the Glu117Gln mutant can be greatly enhanced by substituting Mn(2+) for Mg(2+), with the catalytic efficiency of the Glu117Asp-Mn(2+) holoenzyme approximately 400-fold lower than that of the RNase III-Mn(2+) holoenzyme . For RNase III, a Mn(2+) concentration of 1 mM provides optimal activity, while concentrations >5 mM are inhibitory . In contrast, the Glu117Asp mutant is not inhibited by high concentrations of Mn(2+) . Finally, high concentrations of Mg(2+) do not inhibit RNase III nor relieve the Mn(2+)-dependent inhibition . In summary, these experiments establish the stringent functional requirement for a precisely positioned carboxylic acid group at position 117 and reveal two classes of divalent metal ion binding sites on RNase III . One site binds either Mg(2+) or Mn(2+) and supports catalysis, while the other site is specific for Mn(2+) and confers inhibition . Glu117 is important for the function of both sites . The implications of these findings on the RNase III catalytic mechanism are discussed.

Biochemistry, 2001 Apr 24, 40(16), 5056 - 64
Quantitation of rate enhancements attained by the binding of cobalamin to methionine synthase; Bandarian V et al.; Cobalamin-dependent methionine synthase (MetH) catalyzes the methylation of homocysteine using methyltetrahydrofolate as the methyl donor . The cobalamin cofactor serves as an intermediate carrier of the methyl group from methyltetrahydrofolate to homocysteine . In the two half-reactions that comprise turnover for MetH, the cobalamin is alternatively methylated by methyltetrahydrofolate and demethylated by homocysteine to form methionine . Upon binding to the protein, the usual dimethylbenzimidazole ligand is replaced by the imidazole side chain of His759 {Drennan, C . L., Huang, S., Drummond, J . T., Matthews, R . G., and Ludwig, M . L . (1994) Science 266, 1669-1674} . Despite the ligand replacement that accompanies binding of cobalamin to the holo-MetH protein, a MetH(2-649) fragment of methionine synthase that contains the regions that bind homocysteine and methyltetrahydrofolate utilizes exogenously supplied cobalamin in methyl transfer reactions akin to those of the catalytic cycle . However, the interactions of MetH(2-649) with endogenous cobalamin are first order in cobalamin, while the half-reactions catalyzed by the holoenzyme are zero order in cobalamin, so rate constants for reactions of bound and exogenous cobalamins cannot be compared . In this paper, we investigate the catalytic rate enhancements generated by binding cobalamin to MetH after dividing the protein in half and reacting MetH(2-649) with a second fragment, MetH(649-1227), that harbors the cobalamin cofactor . The second-order rate constant for demethylation of methylcobalamin by Hcy is elevated 60-fold and that for methylation of cob(I)alamin is elevated 120-fold . Thus, binding of cobalamin to MetH is essential for efficient catalysis.

Biochemistry, 2001 Apr 24, 40(16), 5032 - 40
Superoxide reductase from Desulfoarculus baarsii: reaction mechanism and role of glutamate 47 and lysine 48 in catalysis; Lombard M et al.; Superoxide reductase (SOR) is a small metalloenzyme that catalyzes reduction of O(2)(*)(-) to H(2)O(2) and thus provides an antioxidant mechanism against superoxide radicals . Its active site contains an unusual mononuclear ferrous center, which is very efficient during electron transfer to O(2)(*)(-) {Lombard, M., Fontecave, M., Touati, D., and Niviere, V . (2000) J . Biol . Chem . 275, 115-121} . The reaction of the enzyme from Desulfoarculus baarsii with superoxide was studied by pulse radiolysis methods . The first step is an extremely fast bimolecular reaction of superoxide reductase with superoxide, with a rate constant of (1.1 +/- 0.3) x 10(9) M(-1) s(-1) . A first intermediate is formed which is converted to a second one at a much slower rate constant of 500 +/- 50 s(-1) . Decay of the second intermediate occurs with a rate constant of 25 +/- 5 s(-1) . These intermediates are suggested to be iron-superoxide and iron-peroxide species . Furthermore, the role of glutamate 47 and lysine 48, which are the closest charged residues to the vacant sixth iron coordination site, has been investigated by site-directed mutagenesis . Mutation of glutamate 47 into alanine has no effect on the rates of the reaction . On the contrary, mutation of lysine 48 into an isoleucine led to a 20-30-fold decrease of the rate constant of the bimolecular reaction, suggesting that lysine 48 plays an important role during guiding and binding of superoxide to the iron center II . In addition, we report that expression of the lysine 48 sor mutant gene hardly restored to a superoxide dismutase-deficient Escherichia coli mutant the ability to grow under aerobic conditions.

Biochemistry, 2001 Apr 24, 40(16), 4957 - 65
A structural characterization of the interactions between titin Z-repeats and the alpha-actinin C-terminal domain; Joseph C et al.; Titin and alpha-actinin, two modular muscle proteins, are with actin the major components of the Z-band in vertebrate striated muscles where they serve to organize the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation . Interactions between titin and alpha-actinin have been mainly localized in a 45-amino acid multiple motif (Z-repeat) in the N-terminal region of titin and the C-terminal region of alpha-actinin . In this study, we provide the first quantitative characterization of alpha-actinin-Z-repeat recognition and dissect the interaction to its minimal units . Different complementary techniques, such as circular dichroism, calorimetry, and nuclear magnetic spectroscopy, were used . Two overlapping alpha-actinin constructs (Act-EF34 and Act-EF1234) containing two and four EF-hand motifs, respectively, were produced, and their folding properties were examined . Complex formation of Act-EF34 and Act-EF1234 with single- and double-Z-repeat constructs was studied . Act-EF34 was shown quantitatively to be necessary and sufficient for binding to Z-repeats, excluding the presence of additional high-affinity binding sites in the remaining part of the domain . The binding affinities of the different Z-repeats for Act-EF34 range from micromolar to millimolar values . The strongest of these interactions are comparable to those observed in troponin C-troponin I complexes . The binding affinities for Act-EF34 are maximal for Zr1 and Zr7, the two highly homologous sequences present in all muscle isoforms . No cooperative or additional contributions to the interaction were observed for Z-repeat double constructs . These findings have direct relevance for evaluating current models of Z-disk assembly.

Biochemistry, 2001 Apr 24, 40(16), 4932 - 9
Lack of catalytic activity of a murine mRNA cytoplasmic serine hydroxymethyltransferase splice variant: evidence against alternative splicing as a regulatory mechanism; Liu X et al.; Mammalian serine hydroxymethyltransferase (SHMT) is a tetrameric, pyridoxal phosphate-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate . This reaction generates single-carbon units for purine, thymidine, and methionine biosynthesis . Cytoplasmic SHMT (cSHMT) has been postulated to channel one-carbon substituted folates to various folate-dependent enzymes, and alternative splicing of the cSHMT transcript may be a mechanism that enables specific protein-protein interactions . The cytoplasmic isozyme is expressed from species-specific and tissue-specific alternatively spliced transcripts that encode proteins with modified carboxy-terminal domains, while the mitochondrial isozyme is expressed from a single transcript . While the full-length mouse and human cSHMT proteins are 91% identical, their alternatively spliced transcripts differ . The murine cSHMT gene is expressed as two transcripts . One transcript encodes a full-length 55 kDa active enzyme (cSHMT), while the other transcript encodes a 35 kDa protein (McSHMTtr) . The McSHMTtr protein present in mouse liver and kidney does not bind 5-formyltetrahydrofolate, nor does it oligomerize with the full-length cSHMT enzyme . While recombinant cSHMT-glutathione S-transferase fusion proteins form tetramers and are catalytically active, McSHMTtr-glutathione S-transferase fusion proteins are catalytically inactive, do not form heterotetramers, and do not bind pyridoxal phosphate . Analysis of the murine cSHMT crystal structure indicates that the active site lysine that normally binds pyridoxal phosphate in the cSHMT protein is exposed to solvent in the McSHMTtr protein, preventing stable formation of a Schiff base with pyridoxal phosphate . Modeling studies suggest that the human cSHMT proteins expressed from alternatively spliced transcripts are inactive as well . Therefore, channeling mechanisms enabling specific protein-protein interactions of active enzymes are not based on cSHMT alternative splicing.

Anal Chem, 2001 Mar 15, 73(6), 1134 - 9
A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays; Grigorenko V et al.; Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays . Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry . We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme . The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure . As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction . The recombinant conjugate was fully active {650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate} and obtained in a yield of 12 mg/L of E . coli culture, which is better than that for recombinant peroxidase alone . The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format . It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making.

Ned Tijdschr Geneeskd, 2001 Mar 31, 145(13), 620 - 5
{Hemolytic uremic syndrome in children}; Heuvelink AE et al.; In Europe, haemolytic uraemic syndrome (HUS) is the most frequent cause of acute renal insufficiency during childhood . In the Netherlands about 20 children per year are seriously affected . Following a prodromal phase of mostly bloody diarrhoea, the main symptoms become evident, namely, haemolytic anaemia, thrombocytopenia and renal failure . The syndrome is caused by a verocytotoxin producing Escherichia coli, which damages the endothelium of the arterioles and glomeruli in the kidney . In humans, the most important cause of infection is the consumption of infected food-stuffs . After transport through the intestinal epithelium, the toxin becomes attached to the neutrophils, which transport it through the circulatory system, mainly to the kidneys where it has a toxic effect . Why the endothelial cells in these organs are the main targets for this toxin is as yet unknown . A symptomatic treatment is given to the 10% of infected patients who develop HUS . Increasing knowledge about the pathogenesis has resulted in new forms of treatment that will be studied during the next few years.

Biopolymers, 2000, 55(6), 416 - 24
DNA recognition of a 24-mer peptide derived from RecA protein; Sugimoto N; A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA . The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA . However, truncated 15-mer peptide showed no ssDNA binding activity . In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure . The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively . This result is useful to design synthetic peptides as functional materials for DNA recognition .

J Biol Chem, 2001 Jun 22, 276(25), 22183 - 90 Epub 2001 Apr 13.
The importance of structural transitions of the switch II region for the functions of elongation factor Tu on the ribosome; Knudsen C et al.; Elongation factor Tu (EF-Tu) undergoes a large conformational transition when switching from the GTP to GDP forms . Structural changes in the switch I and II regions in the G domain are particularly important for this rearrangement . In the switch II region, helix alpha2 is flanked by two glycine residues: Gly(83) in the consensus element DXXG at the N terminus and Gly(94) at the C terminus . The role of helix alpha2 was studied by pre-steady-state kinetic experiments using Escherichia coli EF-Tu mutants where either Gly(83), Gly(94), or both were replaced with alanine . The G83A mutation slows down the association of the ternary complex EF-Tu.GTP.aminoacyl-tRNA with the ribosome and abolishes the ribosome-induced GTPase activity of EF-Tu . The G94A mutation strongly impairs the conformational change of EF-Tu from the GTP- to the GDP-bound form and decelerates the dissociation of EF-Tu.GDP from the ribosome . The behavior of the double mutant is dominated by the G83A mutation . The results directly relate structural transitions in the switch II region to specific functions of EF-Tu on the ribosome.

J Biol Chem, 2001 Jun 29, 276(26), 24309 - 14 Epub 2001 Apr 13.
ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei; Aro N et al.; A novel yeast-based method to isolate transcriptional activators was applied to clone regulators binding to the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei (Hypocrea jecorina) . This led to the isolation of the cellulase activator ace2 encoding for a protein belonging to the class of zinc binuclear cluster proteins found exclusively in fungi . The DNA-binding domain of ACEII was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and ACEII was shown to bind in vitro to the 5'-GGCTAATAA site present in the cbh1 promoter . This site also contains the proposed binding sequence of the xylanase activator XlnR of Aspergillus niger . Mutation of the GGC triplet abolished ACEII binding . The function of ACEII was studied by analyzing the effects of ace2 deletion in the hypercellulolytic T . reesei strain ALKO2221 . Deletion of the ace2 gene led to lowered induction kinetics of mRNAs encoding the major cellulases cellobiohydrolases I and II and endoglucanases I and II and to 30-70% reduced cellulase activity when the fungus was grown on medium containing Solka floc cellulose . The expression level of the gene encoding xylanase was also affected . ace2 deletion led to lowered xyn2 expression in cellulose-induced cultivation . Cellulase induction by sophorose was not affected by ace2 deletion.

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