Biophys Chem, 2000 Jul 31, 86(1), 79 - 83 Relating helix tilt in a bilayer to lipid disorder: a mean-field theory; Li L; We present a mean-field theory relating the helix tilt angle in a bilayer to lipid disorder . The theory provides a method to compare the rotational barriers for different helices in lipid bilayers . The results suggest that the helix tilt angle is strongly affected by both the hydrophobicity of the helix and the average lipid disorder . This leads us to point out future experiments that could shed light on lipid-protein interactions. J Biochem (Tokyo), 2000 Oct, 128(4), 679 - 86 Stereochemistry of the transamination reaction catalyzed by aminodeoxychorismate lyase from Escherichia coli: close relationship between fold type and stereochemistry; Jhee KH et al.; Aminodeoxychorismate lyase is a pyridoxal 5'-phosphate-dependent enzyme that converts 4-aminodeoxychorismate to pyruvate and p-aminobenzoate, a precursor of folic acid in bacteria . The enzyme exhibits significant sequence similarity to two aminotransferases, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase . In the present study, we have found that aminodeoxychorismate lyase catalyzes the transamination between D-alanine and pyridoxal phosphate to produce pyruvate and pyridoxamine phosphate . L-Alanine and other D- and L-amino acids tested were inert as substrates of transamination . The pro-R hydrogen of C4' of pyridoxamine phosphate was stereospecifically abstracted during the reverse half transamination from pyridoxamine phosphate to pyruvate . Aminodeoxychorismate lyase is identical to D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase in the stereospecificity of the hydrogen abstraction, and differs from all other pyridoxal enzymes that catalyze pro-S hydrogen transfer . Aminodeoxychorismate lyase is the first example of a lyase that catalyzes pro-R-specific hydrogen abstraction . The result is consistent with recent X-ray crystallographic findings showing that the topological relationships between the cofactor and the catalytic residue for hydrogen abstraction are conserved among aminodeoxychorismate lyase, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase {Nakai, T., Mizutani, H., Miyahara, I., Hirotsu, K., Takeda, S., Jhee, K.-H., Yoshimura, T., and Esaki, N . (2000) J . Biochem . 128, 29-38}. J Biochem (Tokyo), 2000 Oct, 128(4), 629 - 35 Monoclonal antibodies recognizing surface residues of the beta subunit of Escherichia coli F(1) ATPase: functional importance of the epitope residues; Kamauchi S et al.; Two monoclonal antibodies, beta 208 and beta 210, against the beta subunit of the F(1) ATPase from Escherichia coli reacted with an intact beta subunit and also a peptide corresponding to a portion of beta between residues 1 and 145 . Mutations at Ala-1, Val-15, Glu-16, Phe-17, Leu-29, Gly-65, or Leu-66, and His-110 or Arg-111 for beta 210 and beta 208, respectively, caused decreased antibody binding to beta, suggesting that these residues form the epitopes and are thought to lie close together on the surface of the beta subunit . The topological locations of the corresponding residues in the atomic structure of the bovine beta subunit agree well with these expectations, except for Ala-1 and Leu-29 . beta 210 binds to two beta strands including the epitope residues that are 50 residues apart, indicating that this antibody recognizes the tertiary structure of the N-terminal end region . Mutations in the epitope residues of beta 210 do not affect the F(1) ATPase activity, suggesting that surfaces of the two beta strands in the amino-terminal end region are not functionally essential . To analyze the functional importance around His-110 recognized by beta 208 we introduced site specific mutations at residues His-110 and Ile-109 . Ile-109 to Ala or Arg, and His-110 to Ala or Asp caused defective assembly of F(1) . However, the His-110 to Arg mutation had no effect on molecular assembly, suggesting that Ile-109 and His-110, especially the positive charge of His-110 are essential for the assembly of F(1) . The His-110 to Arg mutation caused a large decrease in F(1)-ATPase activity, suggesting that a subtle change in the topological arrangement of the positive charge of His-110 located on the surface of beta plays an important role in the catalytic mechanism of the F(1)-ATPase. J Biochem (Tokyo), 2000 Oct, 128(4), 601 - 7 Mouse T-cell antigen rt6.1 has thiol-dependent NAD glycohydrolase activity; Hara N et al.; Mouse Rt6.1 and Rt6.2, homologues of rat T-cell RT6 antigens, catalyze arginine-specific ADP-ribosylation . Without an added ADP-ribose acceptor, Rt6.2 shows NAD glycohydrolase (NADase) activity . However, Rt6.1 has been reported to be primarily an ADP-ribosyltransferase, but not an NADase . In the present study, we obtained evidence that recombinant Rt6.1 catalyzes NAD glycohydrolysis but only in the presence of DTT . The NADase activity of Rt6.1 observed in the presence of DTT was completely inhibited by N-ethylmaleimide (NEM) . Native Rt6.1 antigen, immunoprecipitated from BALB/c mouse splenocytes with polyclonal antibodies generated against recombinant RT6.1, also exhibited NADase activity in the presence of DTT . Compared with Rt6.2, Rt6.1 has two extra cysteine residues at positions 80 and 201 . When Cys-80 and Cys-201 in Rt6.1 were replaced with the corresponding residues of Rt6.2, serine and phenylalanine, respectively, Rt6.1 catalyzed the NADase reaction even in the absence of DTT . Conversely, replacing Ser-80 and Phe-201 in Rt6.2 with cysteines, as in Rt6.1, converted the thiol-independent Rt6.2 NADase to a thiol-dependent enzyme . Kinetic study of the NADase reaction revealed that the affinity of Rt6.1 for NAD and the rate of catalysis increased in the presence of DTT . Moreover, the NADase activity of Rt6.1 expressed on COS-7 cells was stimulated by culture supernatant from activated mouse macrophages, even in the absence of DTT . From these observations, we conclude that t!he Rt6.1 antigen has thiol-dependent NADase activity, and that Cys-80 and Cys-201 confer thiol sensitivity to Rt6.1 NADase . Our results also suggest that upon the interaction of T-cells expressing Rt6.1 with activated macrophages, the NADase activity of the antigen will be stimulated. J Reprod Immunol, 2000 Oct-Nov, 48(2), 99 - 105 Activation and deposition of human breast-milk complement C3 opsonins on serum sensitive Escherichia coli 0111; Ogundele MO; Little is known about the physiological roles and contribution of the human breast-milk complement system in the protection of both the maternal mammary gland and the nursing infant . The ability of a serum-sensitive Escherichia coli to activate the complement components of human breast-milk and colostrum was assessed in vitro . The consequent deposition of C3 fragments, C3b, iC3b and C3dg, on the bacteria was analysed, using a slight modification of a standard ELISA technique for the assessment of activated C3 fragments . The deposition C3 fragments from human milk were demonstrated on the killed bacteria, E . coli NCTC 8007, serotype 0111 K58(B4) H2, using buffers with and without detergent, supporting both the classical and alternative pathways of complement activation . The milk fat appears to competitively inhibit the deposition of these opsonins on the solid-phase bacteria . This study suggests that the complement system is able to contribute to the increased resistance of breast-fed infants against infections. Appl Environ Microbiol, 2000 Oct, 66(10), 4589 - 94 A bioluminescent whole-cell reporter for detection of 2, 4-dichlorophenoxyacetic acid and 2,4-dichlorophenol in soil; Hay AG et al.; A bioreporter was made containing a tfdRP(DII)-luxCDABE fusion in a modified mini-Tn5 construct . When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2, 4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 microM to 5.0 mM . This response was linear (R(2) = 0.9825) in the range of 2.0 microM to 1.1 x 10(2) microM . Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D . A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 x 10(2) microM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM . A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues. Appl Environ Microbiol, 2000 Oct, 66(10), 4555 - 8 Rapid and simple determination of the Escherichia coli phylogenetic group; Clermont O et al.; Phylogenetic analysis has shown that Escherichia coli is composed of four main phylogenetic groups (A, B1, B2, and D) and that virulent extra-intestinal strains mainly belong to groups B2 and D . Actually, phylogenetic groups can be determined by multilocus enzyme electrophoresis or ribotyping, both of which are complex, time-consuming techniques . We describe a simple and rapid phylogenetic grouping technique based on triplex PCR . The method, which uses a combination of two genes (chuA and yjaA) and an anonymous DNA fragment, was tested with 230 strains and showed excellent correlation with reference methods. Appl Environ Microbiol, 2000 Oct, 66(10), 4547 - 54 The presence of humic substances and DNA in RNA extracts affects hybridization results; Alm EW et al.; RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA . It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased . The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane . The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility . The presence of high amounts of DNA in RNA extracts resulted in membrane saturation . Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects. Appl Environ Microbiol, 2000 Oct, 66(10), 4366 - 71 Antisense downregulation of sigma(32) as a transient metabolic controller in Escherichia coli: effects on yield of active organophosphorus hydrolase; Srivastava R et al.; Plasmids containing an antisense fragment of the sigma(32) gene were constructed and introduced into Escherichia coli cells . Downregulation of the sigma(32)-mediated stress response was evaluated under heat shock and ethanol stress and during the production of organophosphorus hydrolase (OPH) . Northern blot analyses revealed that sigma(32) sense mRNA was virtually undetected in antisense-producing cultures from 5 to 20 min after antisense induction . However, lower-molecular-weight bands were found, presumably due to partial degradation of sigma(32) mRNA . While a >10-fold increase in sigma(32) protein level was found under ethanol stress in the control cultures, antisense producing cultures resulted in a <3-fold increase, indicating downregulation of sigma(32) . Correspondingly, antisense synthesis resulted in a decreased level of a sigma(32) regulated chaperone (GroEL) for the first 2 h after induction relative to control cultures without sigma(32) antisense mRNA . The total yield of OPH in the presence of sigma(32) antisense was, on average, 62% of the yield without antisense . However, during sigma(32) antisense production, a sixfold-higher specific OPH activity was observed compared to non-antisense-producing cultures. Biochemistry, 2000 Oct 3, 39(39), 12025 - 32 Folding of green fluorescent protein and the cycle3 mutant; Fukuda H et al.; Although the correct folding of green fluorescent protein (GFP) is required for formation of the chromophore, it is known that wild-type GFP cannot mature efficiently in vivo in Escherichia coli at 37 degrees C or higher temperatures that the jellyfish in the Pacific Northwest have never experienced . Recently, by random mutagenesis by the polymerase chain reaction (PCR) method, a mutant called Cycle3 was constructed . This mutant had three mutations, F99S, M153T, and V163A, on or near the surface of the GFP molecule and was able to mature correctly even at 37 degrees C {Crameri et al . (1996) Nat . Biotechnol . 143, 315-319} . In the present study, we investigated the differences in their folding behavior in vitro . We observed the folding and unfolding reactions of both wild-type GFP and the Cycle3 mutant by using green fluorescence as an indicator of the formation of the native structure and examining hydrogen-exchange reactions by Fourier transform infrared spectroscopy . Both proteins showed unusually slow refolding and unfolding rates, and their refolding rates were almost identical under the native state at 25 and at 35 degrees C . On the other hand, aggregation studies in vitro showed that wild-type GFP had a strong tendency to aggregate, while the Cycle3 mutant did not . These results indicated that the ability to mature efficiently in vivo at 37 degrees C was not due to the improved folding and that reduced hydrophobicity on the surface of the Cycle3 mutant was a more critical factor for efficient maturation in vivo. Biochemistry, 2000 Oct 3, 39(39), 11989 - 99 Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance; Neylon C et al.; The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication . To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K(D)) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB . The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined . The K(D) measured for the binding of wild type and His(6)-Tus to TerB was 0.5 nM in 250 mM KCl . Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences . Three of these substitutions (K89A, R198A, and Q250A) increased K(D) by 200-300-fold, whereas the A173T substitution increased K(D) by 4000-fold . Only the R198A substitution had a significant effect on binding to the nonspecific sequences . The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein. Biochemistry, 2000 Oct 3, 39(39), 11982 - 8 Cleavage of symmetric immobile DNA junctions by Escherichia coli RuvC; Sha R et al.; The Holliday junction is a key DNA intermediate in the process of genetic recombination . It consists of two double-helical domains composed of homologous strands that flank a branch point; two of the strands are roughly helical, and two form the crossover between the helices . RuvC is a Holliday junction resolvase that cleaves the helical strands at a symmetric sequence, leading to the production of two recombinant molecules . We have determined the position of the cleavage site relative to the crossover point by the use of symmetric immobile junctions; these are DNA molecules containing two crossover points, one held immobile by sequence asymmetry and the second a symmetric sequence, but held immobile by torsional coupling to the first junction . We have built five symmetric immobile junctions, in which the tetranucleotide recognition site is moved stepwise relative to the branch point . We have used kinetic analysis of catalysis, gel retardation, and hydroxyl radical hypersensitivity to analyze this system . We conclude that the internucleotide linkage one position 3' to the crossover point is the favored site of cleavage. Biochemistry, 2000 Oct 3, 39(39), 11939 - 47 Fluoride effects along the reaction pathway of pyrophosphatase: evidence for a second enzyme.pyrophosphate intermediate; Baykov AA et al.; The fluoride ion is a potent and specific inhibitor of cytoplasmic pyrophosphatase (PPase) . Fluoride action on yeast PPase during PP(i) hydrolysis involves rapid and slow phases, the latter being only slowly reversible {Smirnova, I . N., and Baykov, A . A . (1983) Biokhimiya 48, 1643-1653} . A similar behavior is observed during yeast PPase catalyzed PP(i) synthesis . The amount of enzyme.PP(i) complex formed from solution P(i) exhibits a rapid drop upon addition of fluoride, followed, at pH 7.2, by a slow increase to nearly 100% of the total enzyme . The slow reaction results in enzyme inactivation, which is not immediately reversed by dilution . These data show that fluoride binds to an enzyme.PP(i) intermediate during the slow phase and to an enzyme.P(i) intermediate during the rapid phase of the inhibition . In Escherichia coli PPase, the enzyme.PP(i) intermediate binds F(-) rapidly, explaining the lack of time dependence in the inhibition of this enzyme . The enzyme.PP(i) intermediate formed during PP(i) hydrolysis binds fluoride much faster (yeast PPase) or tighter (E . coli PPase) than the similar complex existing at equilibrium with P(i) . It is concluded that PPase catalysis involves two enzyme.PP(i) intermediates, of which only one (immediately following PP(i) addition and predominating at acidic pH) can bind fluoride . Simulation experiments have indicated that interconversion of the enzyme.PP(i) intermediates is a partially rate-limiting step in the direction of hydrolysis and an exclusively rate-limiting step in the direction of synthesis. Biochemistry, 2000 Oct 3, 39(39), 11928 - 38 (R)-3-hydroxybutyrate dehydrogenase: selective phosphatidylcholine binding by the C-terminal domain; Loeb-Hennard C et al.; (R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme that has a specific requirement of phosphatidylcholine (PC) for function . The C-terminal domain (CTBDH) of human heart BDH (residues 195-297) has now been expressed in Escherichia coli as a chimera with a soluble protein, glutathione S-transferase (GST), yielding GST-CTBDH, a novel fusion protein that has been purified and shown to selectively bind to PC vesicles . Both recombinant human heart BDH (HH-Histag-BDH) and GST-CTBDH (but not GST) form well-defined protein-lipid complexes with either PC or phosphatidylethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with digalactosyl diglyceride vesicles) as demonstrated by flotation in sucrose gradients . The protein-PC complexes are stable to 0.5 M NaCl, but complexes of either HH-Histag-BDH or GST-CTBDH with PE/DPG vesicles are dissociated by salt treatment . Thrombin cleavage of GST-CTBDH, either before or after reconstitution with PC vesicles, yields CTBDH (12 111 Da by MALDI mass spectrometry) which retains lipid binding without attached GST . The BDH activator, 1-palmitoyl-2-(1-pyrenyl)decanoyl-PC (pyrenyl-PC), at <2.5% of total phospholipid in vesicles, efficiently quenches a fraction (0.36 and 0.47, respectively) of the tryptophan fluorescence of both HH-Histag-BDH and GST-CTBDH with effective Stern-Volmer quenching constants, (K(Q))(eff), of 11 and 9.3 (%)(-)(1), respectively (half-maximal quenching at approximately 0.1% pyrenyl-PC) . Maximal quenching by pyrenyl-PC obtains at approximately stoichiometric pyrenyl-PC to protein ratios, reflecting high-affinity interaction of pyrenyl-PC with both HH-Histag-BDH and GST-CTBDH . The analogous pyrenyl-PE effects a similar maximal quenching of tryptophan fluorescence for both proteins but with approximately 15-fold lower (K(Q))(eff) (half-maximal quenching at approximately 1.5% pyrenyl-PE) referable to nonspecific interaction of pyrenyl-PE with HH-Histag-BDH or GST-CTBDH . Thus, the 103-residue CTBDH constitutes a PC-selective lipid binding domain of the PC-requiring BDH. Biochemistry, 2000 Oct 3, 39(39), 11913 - 20 Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification; Maytum R et al.; The yeast tropomyosin 1 gene (TPM1) encodes the major isoform of the two tropomyosins (Tm) found in yeast . The gene has been expressed in E . coli and the protein purified . The gene product (yTm1) is a 199-amino acid protein that has a low affinity for actin compared to the native yTm1 purified from yeast . Mass spectrometry shows that the native protein is acetylated while the recombinant protein is not . A series of yTm1 N-terminal constructs were made with either an Ala-Ser dipeptide extension previously shown to restore actin binding to skeletal muscle Tm or the natural extension found in fibroblast Tm 5a/b . All constructs bound actin tightly and showed similar CD spectra and thermal stability . All constructs induced cooperativity in the equilibrium binding of myosin subfragment 1, to actin but the binding curves differed significantly between the constructs . The apparent cooperative unit size (n) and closed/open equilibrium (K(T)) were determined using a fluorescence titration technique {Maytum et al . (1998) Biophys . J . 74, A347} . The data could be accounted for by changes in K(T) (0.1-1) with no change in n . Values of n were approximately twice the structural unit size (5 actin sites) . The presence of yTm on actin had little effect upon the overall affinity of S1 for actin despite showing an ability to regulate the acto-myosin interaction . These results show that the short yTm can aid our understanding of actomyosin regulation and that the N-terminus of Tm has a major influence upon its regulatory properties. Biochemistry, 2000 Oct 3, 39(39), 11865 - 75 Escherichia coli uracil DNA glycosylase: NMR characterization of the short hydrogen bond from His187 to uracil O2; Drohat AC et al.; Uracil DNA glycosylase (UDG) cleaves the glycosidic bond of deoxyuridine in DNA using a hydrolytic mechanism, with an overall catalytic rate enhancement of 10(12)-fold over the solution reaction . The nature of the enzyme-substrate interactions that lead to this large rate enhancement are key to understanding enzymatic DNA repair . Using (1)H and heteronuclear NMR spectroscopy, we have characterized one such interaction in the ternary product complex of Escherichia coli UDG, the short (2.7 A) H bond between His187 N(epsilon)(2) and uracil O2 . The H bond proton is highly deshielded at 15.6 ppm, indicating a short N-O distance and exhibits a solvent exchange rate that is 400- and 10(5)-fold slower than free imidazole at pH 7.5 and pH 10, respectively . Heteronuclear NMR experiments at neutral pH show that this H bond involves the neutral imidazole form of His187 and the N1-O2 imidate form of uracil . The excellent correspondence of the pK(a) for the disappearance of the H bond (pK(a) = 6.3 +/- 0.1) with the previously determined pK(a) = 6.4 for the N1 proton of enzyme-bound uracil indicates that the H bond requires negative charge on uracil O2 {Drohat, A . C., and Stivers, J . T . (2000) J . Am . Chem . Soc . 122, 1840-1841} . Although the above characteristics suggest a short strong H bond, the D/H fractionation factor of phi = 1.0 is more typical of a normal H bond . This unexpected observation may reflect a large donor-acceptor pK(a) mismatch or the net result of two opposing effects on vibrational frequencies: decreased N-H bond stretching frequencies (phi < 1) and increased bending frequencies (phi > 1) relative to the O-H bonds of water . The role of this H bond in catalysis by UDG and several approaches to quantify the H bond energy are discussed. Biochemistry, 2000 Oct 3, 39(39), 11845 - 54 Biochemical and biophysical characterization of OmpG: A monomeric porin; Conlan S et al.; A recombinant form of the porin OmpG, OmpGm, lacking the signal sequence, has been expressed in Escherichia coli . After purification under denaturing conditions, the protein was refolded in the detergent Genapol X-080, where it gained a structure rich in beta sheet as evidenced by a CD spectrum similar to that of the native form . Electrophoretic analysis and limited proteolysis experiments suggested that refolded OmpGm exists in at least three forms . Nevertheless, the recombinant protein formed uniform channels in planar bilayers with a conductance of 0.81 nS (1 M NaCl, pH 7.5) . Previous biochemical studies had suggested that OmpG is a monomeric porin, rather than the usual trimer . Bilayer recordings substantiated this proposal; voltage-induced closures occurred consistently in a single step, and channel block by Gd(3+) lacked the cooperativity seen with the trimeric porin OmpF . The availability of milligram amounts of a monomeric porin will be useful both for basic studies of porin function and for membrane protein engineering. Science, 2000 Sep 29, 289(5488), 2354 - 6 A specificity-enhancing factor for the ClpXP degradation machine; Levchenko I et al.; Events that stall bacterial protein synthesis activate the ssrA-tagging machinery, resulting in resumption of translation and addition of an 11-residue peptide to the carboxyl terminus of the nascent chain . This ssrA-encoded peptide tag marks the incomplete protein for degradation by the energy-dependent ClpXP protease . Here, a ribosome-associated protein, SspB, was found to bind specifically to ssrA-tagged proteins and to enhance recognition of these proteins by ClpXP . Cells with an sspB mutation are defective in degrading ssrA-tagged proteins, demonstrating that SspB is a specificity-enhancing factor for ClpXP that controls substrate choice. Crit Care Med, 2000 Sep, 28(9), 3166 - 70 Role of interleukin-10 on hyporesponsiveness of endotoxin during surgery; Ogata M et al.; OBJECTIVE: To examine whether surgical stress causes blood cells to lose their responsiveness to endotoxin during surgery . DESIGN: Prospective case series . SETTING: A university hospital . PATIENTS: Sixteen volunteers classified as American Society of Anesthesiologists physical status I-II who were scheduled for elective distal partial gastrectomy . INTERVENTIONS: We studied nine patients who underwent elective distal partial gastrectomy . Blood samples for tumor necrosis factor (TNF) and interleukin (IL)-10 assay were obtained before anesthesia, preincision, 2 hrs and 4 hrs postincision, postextubation, and 24 hrs postincision . The rest of each blood sample was diluted with 5 volumes of endotoxin-free saline, incubated for 4 hrs in the presence of lipopolysaccharide (LPS), centrifuged to remove cells, and assayed for TNF . In another seven patients, antihuman IL-10 antibody was added into the diluted whole blood sample before LPS stimulation . MEASUREMENTS AND MAIN RESULTS: TNF activity was not detected in the blood of any patient throughout the study . In contrast, plasma cortisol and IL-10 levels increased rapidly during surgery (p < .01, p < .05, respectively) . LPS-induced TNF activity in whole blood decreased significantly during surgery (p < .01) and recovered to control levels by 24 hrs postincision . The peak suppression of LPS-induced TNF and the peak value of plasma IL-10 levels occurred postextubation . Treatment with anti-IL-10 antibody partially restored the ability of LPS to induce TNF activity postextubation (p < .05) . CONCLUSIONS: Surgical trauma rapidly induces a transient hyporesponsiveness of blood cells to endotoxin . Plasma IL-10, which increases during surgery, participates in this hyporesponsiveness. Bioorg Khim, 2000 Jul, 26(7), 530 - 8 {Coupling of proteolysis with ATP hydrolysis by Escherichia coli Lon proteinase . I . Kinetic aspects of ATP hydrolysis}; Mel'nikov EE et al.; Some aspects of the ATPase function of the Escherichia coli Lon protease were studied around the optimum pH value . It was revealed that, in the absence of the protein substrate, the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg2+ ions in the area of their millimolar concentrations . Free components of the substrate complex (ATP-Mg)2- inhibit the enzyme ATPase activity . It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the "ADP-Mg-form" of the ATPase centers . It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)2- complex to the enzyme, by the elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the kcat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)- complex (without changing the Ki value) . Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment. FEBS Lett, 2000 Sep 22, 481(3), 281 - 4 Interaction of sigma 70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance; Ferguson AL et al.; The interaction between the core form of bacterial RNA polymerases and sigma factors is essential for specific promoter recognition, and for coordinating the expression of different sets of genes in response to varying cellular needs . The interaction between Escherichia coli core RNA polymerase and sigma 70 has been investigated by surface plasmon resonance . The His-tagged form of sigma 70 factor was immobilised on a Ni2+-NTA chip for monitoring its interaction with core polymerase . The binding constant for the interaction was found to be 1.9x10(-7) M, and the dissociation rate constant for release of sigma from core, in the absence of DNA or transcription, was 4x10(-3) s(-1), corresponding to a half-life of about 200 s. FEBS Lett, 2000 Sep 22, 481(3), 221 - 6 Functional involvement of a deoxy-D-xylulose 5-phosphate reductoisomerase gene harboring locus of Synechococcus leopoliensis in isoprenoid biosynthesis; Miller B et al.; The present work aimed to proof the functionality of the non-mevalonate pathway in cyanobacteria . It was intended to isolate the 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase gene (dxr), as this gene encodes the enzyme which catalyzes a pathway-specific, indicative step of this pathway . For this purpose, a segment of dxr was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions . Subsequent hybridization screening of a genomic cosmid library of S . leopoliensis with the PCR segment led to the identification of a 26 . 5 kbp locus on which a dxr homologous gene and two adjacent open reading frames organized in one operon were localized by DNA sequencing . The functionality of the gene was demonstrated expressing the gene in Escherichia coli and using the purified gene product in a photometrical NADPH dependent test based on the substrate DXP generating system . While the content of one of the central intermediates of the isoprenoid biosynthesis (dimethylallyl diphosphate=DMADP) was significantly (P</=0.001) increased in E . coli cells overexpressing the DXP synthase gene (dxs) of S . leopoliensis, overexpression of dxr does not lead to an elevated DMADP level . Since even in strains harboring an expression fusion of dxs the additional overexpression of dxr does not influence the DMADP content, it is concluded that Dxs but not Dxr catalyzes a rate limiting step of the non-mevalonate isoprenoid biosynthesis. Nephrol Dial Transplant, 2000 Oct, 15(10), 1638 - 46 Role of complement and platelet-activating factor in the stimulation of phagocytosis and reactive oxygen species production during haemodialysis; Gastaldello K et al.; BACKGROUND: Neutrophil phagocytic functions have been studied extensively in haemodialysis (HD) patients; however, results are contradictory and the mechanisms that modulate phagocytosis and oxidative burst during dialysis are not completely understood . METHODS: The present study investigated neutrophil functions in a selected population of patients before and during clinical dialysis with cuprophane, and polyacrylonitrile (AN69) membranes . We measured phagocytosis of Escherichia coli and intracellular hydrogen peroxide (H2O2) production by flow cytometry in whole blood . RESULTS: Before dialysis, neutrophils from HD patients showed normal phagocytic capability and H2O2 formation . Phagocytosis of FITC-E . coli was significantly stimulated in cuprophane but not AN69-treated patients . Spontaneous and stimulated H2O2 production was enhanced with both cuprophane and AN69 membranes . We then investigated in vitro the role of complement and platelet-activating factor (PAF) in the activation of neutrophils . Incubation of whole blood with C5a increased phagocytosis but not H2O2 production . On the contrary, the addition of synthetic PAF showed a markedly stimulated H2O2 production without increase in phagocytosis . Moreover, during dialysis with formaldehyde-reused cuprophane, complement activation was abolished and phagocytosis was no longer enhanced, while the stimulation of H2O2 production persisted . In addition, we also excluded a particular role of the membrane itself in the activation of neutrophils . CONCLUSION: We demonstrated that in a selected population of HD patients, neutrophils exhibit normal phagocytic capability and normal intracellular H2O2 production . During dialysis, the stimulation of phagocytosis observed with cuprophane is complement dependent, whereas the enhanced H2O2 production observed with both cuprophane and AN69 membranes might be related to PAF production. J Biol Chem, 2000 Dec 15, 275(50), 39332 - 8 Characterization of variants altered at the N-terminal proline, a novel heme-axial ligand in CooA, the CO-sensing transcriptional activator; Thorsteinsson MV et al.; CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding . The crystal structure shows that the N-terminal Pro(2) of one subunit (Met(1) is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer . To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the resulting CooA variants . While Pro(2) appears to be optimal for CooA function, it is not essential and a variety of studied variants at this position have substantial CO-sensing function . Surprisingly, even alterations that add a residue (where Pro(2) is replaced by Met(1)-Tyr(2), for example) accumulate heme-containing CooA with functional properties that are similar to those of wild-type CooA . Other nearby residues, such as Phe(5) and Asn(6) appear to be important for either the structural integrity or the function of CooA . These results are contrasted with those previously reported for alteration of the His(77) ligand on the opposite side of the heme. Int Immunol, 2000 Oct, 12(10), 1431 - 7 Differences in specificities of anti-centromere sera for the monomeric and dimeric C-terminal peptides of human centoromere protein C; Hayashi Y et al.; Centromere protein-C (CENP-C), one of the centromere autoantigens and components of the inner plate of the kinetochore, is suggested to make a dimer at the C-terminus . In order to investigate the presence of conformation-specific anti-centromere antibodies (ACA) to the dimer form, the C-terminal 124 amino acids (CF-124) were expressed in Escherichia coli, affinity purified and chemically cross-linked . Immunoblotting was utilized to compare the reactivities between the dimers and the monomers against 58 ACA(+) sera . The reactivities of the dimers were obviously higher in both IgG and IgM responses . The dimer was still more reactive than the glutathione S-transferase-fused monomer in some sera . Two kinds of CF-124 mutant (each contained one amino acid change at the N-terminal region of CF-124) and two cut segments of CF-124 (67 N-terminal amino acids and 58 C-terminal amino acids) were also examined . The former two mutants decreased the dimerization activity . The latter two mutants lost both activities except for the faint dimerization activity of the N-terminal half . Affinity-purified antibodies with CF-124 in a liquid phase containing the co-purified GroE protein of E . coli, GroEL, reacted to the centromere in culture cells . In conclusion, there are heterogeneous autoepitopes including some conformational epitopes at the C-terminal CENP-C. J Appl Physiol, 2000 Oct, 89(4), 1437 - 44 Diaspirin cross-linked hemoglobin improves oxygen extraction capabilities in endotoxic shock; Creteur J et al.; We studied the effects of diaspirin cross-linked hemoglobin (DCLHb), a cell-free hemoglobin derived from human erythrocytes, on blood flow distribution and tissue oxygen extraction capabilities in endotoxic shock . Eighteen pentobarbital sodium-anesthetized, mechanically ventilated dogs received 2 mg/kg of E . coli endotoxin, followed by saline resuscitation to restore cardiac filling pressures to baseline levels . The animals were randomly divided into three groups: six served as control, six received DCLHb at a dose of 500 mg/kg (group 1) and six DCLHb at a dose of 1,000 mg/kg (group 2) . Cardiac tamponade was then induced by saline injection in the pericardial sac to progressively reduce cardiac index and thereby allow study of tissue oxygen extraction capabilities . DCLHb had a dose-dependent vasopressor effect but did not significantly alter cardiac index or regional blood flow . During cardiac tamponade, critical oxygen delivery was 12.8 +/- 0.7 ml . kg(-1) . min(-1) in the control group, but 8.6 +/- 0.9 and 8.2 +/- 0.7 ml . kg(-1) . min(-1) in groups 1 and 2, respectively (both P < 0.05 vs . control group) . The critical oxygen extraction ratio was 39.1 +/- 3.1% in the control group but 58.7 +/- 12.8% and 60.2 +/- 9.0% in groups 1 and 2, respectively . We conclude that DCLHb can improve whole body oxygen extraction capabilities during endotoxic shock in dogs. Avian Dis, 2000 Jul-Sep, 44(3), 701 - 5 The effect of vitamin E on cellulitis in broiler chickens experiencing scratches in a challenge model; Macklin KS et al.; Two experiments are described; each experiment contained five treatments with each treatment consisting of a specific diet and vitamin E at 8.82 mg, 41.89 mg, 74.96 mg, 108.03 mg, or 141.10 mg vitamin E per kilogram of feed . Birds were raised with continuous feed containing the various levels of vitamin E available throughout the experiment . At 4 wk of age, the birds were scratched on the breast and placed onto avian cellulitis Escherichia coli-seeded litter . One week later, the birds were euthanatized and lesion presence was noted . There appeared to be a positive correlation between vitamin E and the inhibition of cellulitis formation when the birds were fed a diet containing 74.96 mg vitamin E/kg feed . Conflicting results were seen in the two experiments when the birds were fed 41.89 and 108.03 mg vitamin E/kg feed . Both experiments had a high incidence of cellulitis in birds whose diets consisted of 141.10 mg vitamin E/kg feed. Hawaii Med J, 2000 Aug, 59(8), 336 - 7 Emphysematous pyelonephritis; Hui L et al.; Emphysematous pyelonephritis is a rare, severe, necrotizing form of renal infection characterized by the presence of gas within the renal parenchyma or perinephric space . In patients suspected of emphysematous pyelonephritis, computed tomography scan should be done promptly . Based on the available data and this case, surgical intervention appears to be the preferred treatment. Mutat Res, 2000 Sep 20, 453(1), 97 - 103 High plating density improves Big Blue system efficiency without loss of sensitivity; Heinmoller PW et al.; To increase efficiency in the Big Blue system, the plating density was increased from 15000 to 30000 or 45000 plaque forming units (pfus) per plate by increasing the density of the E . coli lawn and decreasing individual plaque size . Small plaque size ensured minimal overlap of the plaques . Liver from one 3- and one 25-month-old mouse (low and high mutation frequencies, respectively) was analyzed and neither plating density nor plaque size affected mutant/mutation frequency and pattern . The color intensity of particular mutant plaques was not affected by plaque size or plating density . Optimal sensitivity is achieved by sequencing mutants to calculate the mutation frequency from the mutant frequency and to identify altered patterns of mutation . Detailed effort and cost accounting of the Big Blue system (including mouse handling, DNA extraction, plaque screening, plaque purification, and DNA sequencing) reveals that one-quarter of the total effort is devoted to plating and screening of plates . This effort is reduced two fold with high plating density . The total cost of the Big Blue system is reduced by 17% . The total cost of the High Plating Density Big Blue system is now only 12% more costly than a selectable assay and offers an extensively validated system with a large mutation database representing a decade of effort. J Biol Chem, 2000 Dec 15, 275(50), 39287 - 95 Modeling the late steps in HIV-1 retroviral integrase-catalyzed DNA integration; Brin E et al.; Model oligodeoxyribonucleotide substrates representing viral DNA integration intermediates with a gap and a two-nucleotide 5' overhang were used to examine late steps in human immunodeficiency virus, type 1 (HIV-1) retroviral integrase (IN)-catalyzed DNA integration in vitro . HIV-1 or avian myeloblastosis virus reverse transcriptase (RT) were capable of quantitatively filling in the gap to create a nicked substrate but did not remove the 5' overhang . HIV-1 IN also failed to remove the 5' overhang with the gapped substrate . However, with a nicked substrate formed by RT, HIV-1 IN removed the overhang and covalently closed the nick in a disintegration-like reaction . The efficiency of this closure reaction was very low . Such closure was not stimulated by the addition of HMG-(I/Y), suggesting that this protein only acts during the early processing and joining reactions . Addition of Flap endonuclease-1, a nuclease known to remove 5' overhangs, abolished the closure reaction catalyzed by IN . A series of base pair inversions, introduced into the HIV-1 U5 long terminal repeat sequence adjacent to and/or including the conserved CA dinucleotide, produced no or only a small decrease in the HIV-1 IN-dependent strand closure reaction . These same mutations caused a significant decrease in the efficiency of concerted DNA integration by a modified donor DNA in vitro, suggesting that recognition of the ends of the long terminal repeat sequence is required only in the early steps of DNA integration . Finally, a combination of HIV-1 RT, Flap endonuclease-1, and DNA ligase is capable of quantitatively forming covalently closed DNA with these model substrates . These results support the hypothesis that cellular enzyme(s) may catalyze the late steps of retroviral DNA integration. J Biol Chem, 2000 Dec 22, 275(51), 40324 - 8 A mutant Escherichia coli tyrosyl-tRNA synthetase utilizes the unnatural amino acid azatyrosine more efficiently than tyrosine; Hamano-Takaku F et al.; Alloproteins, proteins that contain unnatural amino acids, have immense potential in biotechnology and medicine . Although various approaches for alloprotein production exist, there is no satisfactory method to produce large quantities of alloproteins containing unnatural amino acids in specific positions . The tyrosine analogue azatyrosine, l-beta-(5-hydroxy-2-pyridyl)-alanine, can convert the ras-transformed phenotype to normal phenotype, presumably by its incorporation into cellular proteins . This provided the stimulus for isolation of a mutant tyrosyl-tRNA synthetase (TyrRS) capable of charging azatyrosine to tRNA . A plasmid library of randomly mutated Escherichia coli tyrS (encoding TyrRS) was made by polymerase chain reaction techniques . The desired TyrRS mutants were selected by screening for in vivo azatyrosine incorporation of E . coli cells transformed with the mutant tyrS plasmids . One of the clones thus isolated, R-6-A-7, showed a 17-fold higher in vivo activity for azatyrosine incorporation than wild-type TyrRS . The mutant tyrS gene contained a single point mutation resulting in replacement of phenylalanine by serine at position 130 in the protein . Structural modeling revealed that position 130 is located close to Asp(182), which directly interacts with tyrosyladenylate . Kinetic analysis of aminoacyl-tRNA formation by the wild-type and mutated F130S TyrRS enzymes showed that the specificity for azatyrosine, measured by the ratios of k(cat)/K(m) for tyrosine and the analogue, increased from 17 to 36 as a result of the F130S mutation . Thus, the high discrimination against azatyrosine is significantly reduced in the mutant enzyme . These results suggest that utilization of F130S TyrRS for in vivo protein biosynthesis may lead to efficient production of azatyrosine-containing alloproteins. Invest Ophthalmol Vis Sci, 2000 Oct, 41(11), 3590 - 9 Coordination between production and turnover of interphotoreceptor retinoid-binding protein in zebrafish; Cunningham LL et al.; PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP), which is secreted by the photoreceptors of most vertebrates, is the major soluble protein component of the interphotoreceptor matrix (IPM) . Recent studies suggest that IRBP is short lived in the IPM (half-life, approximately 11 hours) . The mechanisms coordinating the production and removal of IRBP are not known . Zebrafish provide a useful system to study the regulation of these two processes, because its IRBP mRNA levels are under circadian regulation . In the present study, the relationship between the quantity of IRBP, the rate of its turnover, and the expression of its mRNA in the zebrafish retina were examined . METHODS: Full-length zebrafish IRBP was expressed in Escherichia coli and an antiserum generated against purified recombinant IRBP . Western and protein dot blot analyses and indirect immunofluorescence were used to define the temporal and spatial patterns of IRBP expression in the adult zebrafish . In vivo and in vitro metabolic labeling experiments were used to examine the regulation of IRBP turnover by both environmental light and the light-dark cycle . RESULTS: Despite the known rhythmicity in IRBP mRNA expression, neither the amount of IRBP nor its localization changes significantly during the light-dark cycle . IRBP is rapidly removed from the zebrafish eye (half life, approximately 7 hours) . This rapid turnover is independent of environmental lighting conditions during subjective day and is more rapid during the day than at night . CONCLUSIONS: Because the amount of IRBP remains constant throughout the day, the enhanced daytime IRBP mRNA expression may function to compensate for an increased turnover of the protein during the day . These findings suggest that the processes of IRBP production and removal are coordinately regulated. Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 346 - 9 Enhancement of the activity of l-aspartase from Escherichia coli W by directed evolution; Wang LJ et al.; l-Aspartase was modified by directed evolution . After four rounds of error-prone PCR and three rounds of DNA shuffling, an evolved enzyme purified from the final round showed a 28-fold increased k(cat)/K(m) and 4.6-fold decreased K(m) . The thermostability and stable pH range were also enhanced . The DNA sequence of the evolved aspartase gene showed seven base changes, resulting in three amino acid changes from the native enzyme: N217K, T233R, V367G . The mechanism of the enhancement of activity was analyzed . Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 286 - 91 Molecular cloning of the crustacean DD4 cDNA encoding a Ca(2+)-binding protein; Endo H et al.; A cDNA, named DD4, was identified in the prawn Penaeus japonicus in a search for genes that were expressed during calcification of the crustacean exoskeleton . DD4 transcripts were detected in the epidermal cells underlying the exoskeleton specifically during the postmolt stage, when the calcification takes place . In the DD4 cDNA an open reading frame of 542 amino acids was found . The deduced protein was acidic and proline-rich, and exhibited similarity to the Drosophila Ca(2+)-binding protein calphotin in the amino acid sequence and composition . The DD4 cDNA was expressed in Escherichia coli to characterize Ca(2+)-binding of the encoded protein, and Ca(2+) was found to bind to a central segment of 186 amino acids . The DD4 protein is suggested to play a role in the calcification of the crustacean exoskeleton . Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 197 - 203 Characterization of l-asparaginase fused with a protective ScFv and the protection mechanism; Guo L et al.; A fusion protein of the protective scFv linked to the C-terminus of ASNase via (Gly(4)Ser)(6) peptide was constructed . The ASNase-scFv fusion protein expressed in Escherichia coli exists mainly in the form of inclusion bodies, and a small amount of it was soluble . The soluble form was purified by four-step purification and it has been demonstrated that ASNase-scFv fusion exists as a dimer . By assay of the stability against proteolysis, the ASNase-scFv fusion was found to be more stable than native ASNase but less stable than scFv-ASNase fusion . The results of immunological assay indicated that the immunogenicity of the fusion proteins increased while their binding capacity with the anti-ASNase serum decreased by comparison to the native ASNase . Moreover, here the comparison of the basic physical and chemical properties of the ASNase-scFv fusion, scFv-ASNase fusion, and native ASNase is presented . Based on the structural evidence and the biochemical analysis described in this paper, the protection mechanism proposed in our previous study was further supported . The scFv moiety of the fusion protein may confer the ASNase moiety resistance to proteolysis as a result of both steric hindrance such as blocking the cleavage sites of trypsin and a change in the electrostatic potential surface of the enzyme . Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 101 - 6 Three-hybrid strategy reveals a peptide segment that specifically binds to the 3'-untranslated region of NF-IL6 mRNA; He YZ et al.; The 3'UTR of eukaryotic mRNA is an important regulation region, on which many trans factors act . In recent years, a series of 3'UTRs were shown to have tumor suppressor function, including the 3'UTR of the human nuclear factor for interleukin-6 (NF-IL6 3'UTR) . To understand molecular basis for this function, we have tried to isolate genes encoding protein factors acting on the RNA of NF-IL6 3'UTR . Here we show that, by using a yeast three-hybrid system, a cDNA fragment was successfully isolated . This cDNA was allowed to express in E . coli, and its expression product, a polypeptide of ca . 70 amino acids long, was shown to specifically bind to the NF-IL6 3'UTR RNA . A search in GenBank did not reveal homologous sequences . Therefore, this cDNA fragment may be a part of the gene of a novel NF-IL6 3'UTR specific binding protein . Biotechnol Bioeng, 2000 Nov 20, 70(4), 467 - 72 The inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides-mathematical modeling; Cheng B et al.; Gene transcription is regulated by transcription factors that can bind to specific regions on DNA . Antigene oligonucleotides (oligos) can bind to specific regions on DNA and form a triplex with the double-stranded DNA . The triplex can competitively inhibit the binding of transcription factors and, as a result, transcription can be inhibited . A genetically structured model has been developed to quantitatively describe the inhibition of the Escherichia coli lac operon gene expression by triplex-forming oligos . The model predicts that the effect of triplex-forming oligos on the lac operon gene expression depends on their target sites . Oligonucleotides targeted to the operator are much more effective than those targeted to other regulatory sites on the lac operon . In some cases, the effect of oligo binding is similar to that of a mutation in the lac operon . The model provides insight as to the specific binding site to be targeted to achieve the most effective inhibition of gene expression . The model is also capable of predicting the oligo concentration needed to inhibit gene expression, which is in general agreement with results reported by other investigators . Biotechnol Bioeng, 2000 Nov 20, 70(4), 456 - 63 Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis; Guo L et al.; In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies . The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6) . Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E . coli and the expressed product was found to form inclusion bodies . We obtained a soluble fusion protein by refolding and purification . The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine . Importantly, the fusion protein was more stable than native ASNase . In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h) . The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package . On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme . Proc Natl Acad Sci U S A, 2000 Sep 26, 97(20), 10884 - 9 Roles of a conserved arginine residue of DsbB in linking protein disulfide-bond-formation pathway to the respiratory chain of Escherichia coli; Kadokura H et al.; The active-site cysteines of DsbA, the periplasmic disulfide-bond-forming enzyme of Escherichia coli, are kept oxidized by the cytoplasmic membrane protein DsbB . DsbB, in turn, is oxidized by two kinds of quinones (ubiquinone for aerobic and menaquinone for anaerobic growth) in the electron-transport chain . We describe the isolation of dsbB missense mutations that change a highly conserved arginine residue at position 48 to histidine or cysteine . In these mutants, DsbB functions reasonably well aerobically but poorly anaerobically . Consistent with this conditional phenotype, purified R48H exhibits very low activity with menaquinone and an apparent Michaelis constant (K(m)) for ubiquinone seven times greater than that of the wild-type DsbB, while keeping an apparent K(m) for DsbA similar to that of wild-type enzyme . From these results, we propose that this highly conserved arginine residue of DsbB plays an important role in the catalysis of disulfide bond formation through its role in the interaction of DsbB with quinones. J Biol Chem, 2000 Dec 22, 275(51), 40365 - 70 Molecular modeling and site-directed mutagenesis define the catalytic motif in human gamma -glutamyl hydrolase; Chave KJ et al.; Human gamma-glutamyl hydrolase (hGH) is a central enzyme in folyl and antifolylpoly-gamma-glutamate metabolism, which functions by catalyzing the cleavage of the gamma-glutamyl chain of substrates . We previously reported that Cys-110 is essential for activity . Using the sequence of hGH as a query, alignment searches of protein data bases were made using the SSearch and TPROBE programs . Significant similarity was found between hGH and the glutamine amidotransferase type I domain of Escherichia coli carbamoyl phosphate synthetase . The resulting hypothesis is that the catalytic fold of hGH is similar to the folding of this domain in carbamoyl phosphate synthetase . This model predicts that Cys-110 of hGH is the active site nucleophile and forms a catalytic triad with residues His-220 and Glu-222 . The hGH mutants C110A, H220A, and E222A were prepared . Consistent with the model, mutants C110A and H220A were inactive . However, the V(max) of the E222A hGH mutant was reduced only 6-fold relative to the wild-type enzyme . The model also predicted that His-171 in hGH may be involved in substrate binding . The H171N hGH mutant was found to have a 250-fold reduced V(max) . These studies to determine the catalytic mechanism begin to define the three dimensional interactions of hGH with poly-gamma-glutamate substrates. J Biol Chem, 2000 Dec 22, 275(51), 40003 - 13 Gene conversion (recombination) mediates expansions of CTG{middle dot}CAG repeats; Jakupciak JP et al.; Genetic recombination is a robust mechanism for expanding CTG.CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J . P., and Wells, R . D . (1999) J . Biol . Chem . 274, 23468-23479) . This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions . The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids . Expansions were much more frequent than deletions . Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171- or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths . Gene conversion, rather than crossing over, was the recombination mechanism . Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence . However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism . Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome. Am J Physiol Gastrointest Liver Physiol, 2000 Oct, 279(4), G781 - 90 Systemic lipopolysaccharide influences rectal sensitivity in rats: role of mast cells, cytokines, and vagus nerve; Coelho AM et al.; Intraperitoneal lipopolysaccharide (LPS) produces somatic hyperalgesia, releases interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha), and activates vagal afferents . The aim of this study was to evaluate the effect of peripheral LPS on rectal sensitivity and to specify the mechanisms involved . Abdominal muscle contractions were recorded in conscious rats equipped with intramuscular electrodes . Rectal distension (RD) was performed at various times after LPS or experimental treatments . In controls, RD significantly increased the number of abdominal contractions from a threshold volume of distension of 0.8 ml . At the lowest volume (0.4 ml), this number was increased after administration of LPS (3, 9, and 12 h later), recombinant human IL-1beta (from 3 to 9 h), recombinant bovine TNF-alpha (from 6 to 9 h), and BrX-537A (from 6 to 12 h), a mast cell degranulator . The effect of LPS was reduced by doxantrazole, Lys-D-Pro-Thr, and soluble recombinant TNF receptor . Vagotomy selectively amplified the response to LPS . We conclude that, in vivo, intraperitoneal LPS lowers visceral pain threshold (allodynia) through a mechanism involving mast cell degranulation and IL-1beta and TNF-alpha release and that the vagus nerve may exert a tonic protective role against LPS-induced rectal allodynia. Pflugers Arch, 2000, 440(5 Suppl), R83 - 5 Expression of human lymphotoxin alpha in Aspergillus niger; Krasevec N et al.; A gene-fusion expression strategy was applied for heterologous expression of human lymphotoxin alpha (LTalpha) in the Aspergillus niger AB1.13 protease-deficient strain . The LTalpha gene was fused with the A . niger glucoamylase GII-form as a carrier-gene, behind its transcription control and secretion signals . Special attention was paid to the influence of different codon usage on secretion of protein . In the case of human tumor necrosis factor alpha (TNFalpha) a dramatic change of secretion has been observed when human cDNA sequence was used instead of synthetic E . coli biased codons . In the case of LTalpha such a change of codon usage brought improvement at the RNA level, however, no increase in the quantity of secreted protein was observed, due to the proteolitic activity of the host organism . The estimated yield of secretion of LTalpha from A . niger into the soya medium was 50 pg l(-1) of culture. Int J Cancer, 2000 Oct 15, 88(2), 267 - 73 A novel recombinant fusion toxin targeting HER-2/NEU-over-expressing cells and containing human tumor necrosis factor; Rosenblum MG et al.; Over-expression of the proto-oncogene HER2/neu in breast cancer and certain other tumors appears to be a central mechanism that may be partly responsible for cellular progression of the neoplastic phenotype . Transfection of mammalian cells and over-expression of HER2/neu appears to result in reduced sensitivity to the cytotoxic effects of tumor necrosis factor (TNF) and reduced sensitivity to immune effector killing . The single-chain recombinant antibody sFv23 recognizes the cell-surface domain of HER2/neu . The cDNA for this antibody was fused to the cDNA encoding human TNF, and this sFv23/TNF fusion construct was cloned into a plasmid for expression in Escherichia coli . The fusion protein was expressed and purified by ion-exchange chromatography . SDS-PAGE demonstrated a single band at the expected m.w . (43 kDa) . Western analysis confirmed the presence of both the antibody component and the TNF component in the final fusion product . The fusion construct was tested for TNF activity against L-929 cells and found to have biological activity similar to that of authentic TNF (SA 420 nM) . The scFv23/TNF construct bound to SKBR-3 (HER2-positive) but not to A-375 human melanoma (HER2-negative) cells . Cytotoxicity studies against log-phase human breast carcinoma cells (SKBR-3-HP) over-expressing HER2/neu demonstrate that the sFv23/TNF fusion construct was 1, 000-fold more active than free TNF . Tumor cells expressing higher levels of HER2/neu (SKBR-3-LP) were relatively resistant to both the fusion construct and native TNF . These studies suggest that fusion constructs targeting the HER2/neu surface domain and containing TNF are more effective cytotoxic agents in vitro than native TNF and may be effective against tumor cells expressing intermediate, but not high, levels of HER2/neu . Biochim Biophys Acta, 2000 Aug 24, 1487(1), 106 - 11 Cloning and promoter analysis of the cotton lipid transfer protein gene Ltp3(1); Liu HC et al.; A cotton Ltp3 gene and its 5' and 3' flanking regions have been cloned with a PCR-based genomic DNA walking method . The amplified 2.6 kb DNA fragment contains sequences corresponding to GH3 cDNA which has been shown to encode a lipid transfer protein (LTP3) . The gene has an intron of 80 bp which is located in the region corresponding to the C-terminus of LTP3 . The Ltp3 promoter was systematically analyzed in transgenic tobacco plants by employing the Escherichia coli beta-glucuronidase gene (GUS) as a reporter . The results of histochemical and fluorogenic GUS assays indicate that the 5' flanking region of the Ltp3 gene contains cis-elements conferring the trichome specific activity of Ltp3 promoter. Biochim Biophys Acta, 2000 Aug 31, 1481(1), 131 - 8 Incorporation of nitrotyrosine into alpha-tubulin by recombinant mammalian tubulin-tyrosine ligase; Kalisz HM et al.; Tubulin-tyrosine ligase (TTL, EC 6.3.2.25) from porcine brain, which catalyses the readdition of tyrosine to the C-terminus of detyrosinated alpha-tubulin, was cloned and expressed in Escherichia coli as a glutathione S-transferase-fusion protein . Upon cleavage of the immobilised fusion protein, an electrophoretically homogeneous enzyme was obtained . Recombinant TTL, which exhibited similar catalytic properties as the mammalian enzyme purified from brain tissue, was capable of using nitrotyrosine as an alternative substrate in vitro . Incorporation of tyrosine into tubulin was competitively inhibited by nitrotyrosine with an apparent K(i) of 0.24 mM . The TTL-catalysed incorporation of nitrotyrosine as sole substrate into alpha-tubulin was clearly detectable at concentrations of 10 microM by immunological methods using nitrotyrosine specific antibodies . However, in competition with tyrosine 20-fold higher concentrations of nitrotyrosine were necessary before its incorporation became evident . Analysis of the C-terminal peptides of in vitro modified alpha-tubulin by MALDI-MS confirmed the covalent incorporation of nitrotyrosine into tubulin by TTL . In contrast to the C-terminal tyrosine, pancreatic carboxypeptidase A was incapable of cleaving nitrotyrosine from the modified alpha-tubulin. Biochim Biophys Acta, 2000 Aug 31, 1481(1), 45 - 54 Contributions of protein disulfide isomerase domains to its chaperone activity; Sun XX et al.; Protein disulfide isomerase (PDI), a member of the thioredoxin (Trx) superfamily, consists of five consecutive domains, a-b-b'-a'-c . Domain combinations, AB, A'C, B'A'C and AB-C, and hybrids of PDI domains with Trx, Trx-C and Trx-B'A'C, have been constructed and expressed in Escherichia coli to examine the contributions of PDI domains to its enzyme and chaperone activities . All the combination and hybrid products are considerably less active than intact PDI in their enzyme activities . Recombinant products containing C, at low concentrations, inhibit the reactivation of lysozyme in HEPES buffer, while those without C do not . Only the intact PDI molecule and the hybrid molecule, Trx-B'A'C, but to a much lower level, show general chaperone activity in assisting the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase . It is suggested that all domains of PDI contribute to the binding of target protein for its chaperone activity. Biochim Biophys Acta, 2000 Aug 31, 1481(1), 18 - 24 Escherichia coli is able to produce heterologous tetraheme cytochrome c(3) when the ccm genes are co-expressed; Herbaud ML et al.; The production of Desulfovibrio vulgaris Hildenborough cytochrome c(3) (M(r) 13000), which is a tetraheme cytochrome, in Escherichia coli was examined . This cytochrome was successfully produced in an E . coli strain co-expressing the ccmABCDEFGH genes involved in the cytochrome c maturation process . The apocytochrome c(3) was matured in either anaerobic or aerobic conditions, but aerobic growth in the presence of delta-aminolevulinic acid was found to be best for cytochrome c(3) production . Site-directed mutagenesis was performed to investigate the effect of the presence of four amino acids in between the two cysteines of the heme binding sites 2 and 4 on the maturation of holocytochrome c(3) in E . coli. Biochim Biophys Acta, 2000 Jul 14, 1480(1-2), 222 - 34 Active site studies of bovine alpha1-->3-galactosyltransferase and its secondary structure prediction; Shah PS et al.; The catalytic domain of bovine alpha1-->3-galactosyltransferase (alpha3GalT), residues 80-368, have been cloned and expressed, in Escherichia coli . Using a sequential purification protocol involving a Ni(2+) affinity column followed by a UDP-hexanolamine affinity column, we have obtained a pure and active protein from the soluble fraction which catalyzes the transfer of galactose (Gal) from UDP-Gal to N-acetyllactosamine (LacNAc) with a specific activity of 0.69 pmol/min/ng . The secondary structural content of alpha3GalT protein was analyzed by Fourier transform infrared (FTIR) spectroscopy, which shows that the enzyme has about 35% beta-sheet and 22% alpha-helix . This predicted secondary structure content by FTIR spectroscopy was used in the protein sequence analysis algorithm, developed by the Biomolecular Engineering Research Center at Boston University and Tasc Inc., for the assignment of secondary structural elements to the amino acid sequence of alpha3GalT . The enzyme appears to have three major and three minor helices and five sheet-like structures . The studies on the acceptor substrate specificity of the enzyme, alpha3GalT, show that in addition to LacNAc, which is the natural substrate, the enzyme accepts various other disaccharides as substrates such as lactose and Gal derivatives, beta-O-methylgalactose and beta-D-thiogalactopyranoside, albeit with lower specific activities . There is an absolute requirement for Gal to be at the non-reducing end of the acceptor molecule which has to be beta1-->4-linked to a second residue that can be more diverse in structure . The kinetic parameters for four acceptor molecules were determined . Lactose binds and functions in a similar way as LacNAc . However, beta-O-methylgalactose and Gal do not bind as tightly as LacNAc or lactose, as their K(ia) and K(A) values indicate, suggesting that the second monosaccharide is critical for holding the acceptor molecule in place . The 2' and 4' hydroxyl groups of the receiving Gal moiety are important in binding . Even though there is large structural variability associated with the second residue of the acceptor molecule, there are constraints which do not allow certain Gal-R sugars to be good acceptors for the enzyme . The beta1-->4-linked residue at the second position of the acceptor molecule is preferred, but the interactions between the enzyme and the second residue are likely to be non-specific. Biochim Biophys Acta, 2000 Jul 14, 1480(1-2), 191 - 200 Modifications of aclacinomycin T by aclacinomycin methyl esterase (RdmC) and aclacinomycin-10-hydroxylase (RdmB) from Streptomyces purpurascens; Wang Y et al.; The genes rdmB and rdmC of Streptomyces purpurascens encoding aclacinomycin modifying enzymes RdmB and RdmC were expressed in Streptomyces lividans TK24 . In contrast to the earlier suggestion that RdmC may be an esterase that causes the removal of the carbomethoxy group from the 10 position of aclacinomycins, RdmC functions as an aclacinomycin methyl esterase and catalyzes the removal of the methoxy group from the C-15 position of aclacinomycin T producing 15-demethoxyaclacinomycin T . RdmB acts upon C-10 of 15-demethoxyaclacinomycin T and is able to remove the carboxylic group from the C-10 position . It functions also as an aclacinomycin-10-hydroxylase being able to add a hydroxyl group at the same, C-10 position in vitro . Aclacinomycin methyl esterase was purified to apparent homogeneity from S . lividans carrying the rdmC and aclacinomycin-10-hydroxylase as a glutathione S-transferase fusion construct from Escherichia coli carrying the rdmB gene, respectively . Aclacinomycin methyl esterase functions as a monomer and aclacinomycin-10-hydroxylase as a tetramer . Aclacinomycin methyl esterase has an exceptionally high temperature stability and has an apparent K(m) for aclacinomycin T of 15.5 microM . The introduction of rdmC and rdmB in a Streptomyces galilaeus mutant HO38 produced the same modifications of aclacinomycin T in vivo as aclacinomycin methyl esterase and aclacinomycin-10-hydroxylase in vitro. Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 166 - 74 Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: pre-steady-state kinetic studies; Moore JA et al.; Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine . The mechanism of the enzyme-catalyzed reaction was studied by isotope trapping, pre-steady-state rapid quench, and single turnover experiments . Isotope trapping indicated that the enzyme.tRNA complex is catalytically competent, whereas the enzyme.DMAPP complex is not . The results are consistent with an ordered sequential mechanism for substrate binding where tRNA binds first . The association and dissociation rate constants for the enzyme.tRNA binary complex are 1 . 15+/-0.33x10(7) M(-1) s(-1) and 0.06+/-0.01 s(-1), respectively . Addition of DMAPP gives an enzyme.tRNA.DMAPP ternary complex in rapid equilibrium with the binary complex and DMAPP . Rapid quench studies yielded a linear profile (k(cat)=0.36+/-0.01 s(-1)) with no evidence for buildup of enzyme-bound product . Product release from DMAPP-tRNA transferase is therefore not rate-limiting . The Michaelis constant for tRNA and the equilibrium dissociation constant for DMAPP calculated from the individual rate constants determined here are consistent with values obtained from a steady-state kinetic analysis. Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 135 - 46 Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein; Gilbert SM et al.; The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens . They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences . The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli . This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined . In solution the peptide showed the presence of beta-turns and polyproline II-like structure . Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures . IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures . In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains . The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction. Biochim Biophys Acta, 2000 Jun 21, 1492(1), 247 - 51 Molecular characterization of Drosophila melanogaster dihydropteridine reductase; Park D et al.; Dihydropteridine reductase (DHPR) catalyzes the NAD(P)H-mediated reduction of quinonoid dihydropteridine as a part of pterin-dependent aromatic amino acid hydroxylation . We isolated a fragment of Drosophila DHPR gene by PCR using degenerate primers . By screening a cDNA library, we obtained full-length clones . The predicted amino acid sequence of the Drosophila DHPR protein was highly homologous to other species including human and mouse . In particular, the Tyr-(Xaa)(3)-Lys motif, known as the NAD(P)H binding domain, and most amino acids relevant to quinonoid dihydropteridine binding site are identical to human DHPR . The recombinant DHPR protein expressed in Escherichia coli showed DHPR enzyme activity . Northern blot analysis revealed two transcripts of 1.1 and 0.9 kb . Genomic DNA sequencing revealed that the gene consists of two exons interrupted by a single 96-bp intron . The two transcripts have alternative promoters, both having no putative TATA box or CAAT box, but sharing a common poly(A)(+) signal . The existence of two alternative promoters suggests that each transcript be regulated independently through different stimuli . Further study is needed to examine the expression and function of the two alternative transcripts. Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 521 - 7 A re-examination of the structural and functional consequences of mutation of alanine-128 of the b subunit of Escherichia coli ATP synthase to aspartic acid; Dunn SD et al.; The effects of mutation of residue Ala-128 of the b subunit of Escherichia coli ATP synthase to aspartate on the structure of the subunit and its interaction with the F(1) sector were analyzed . Determination of solution molecular weights by sedimentation equilibrium ultracentrifugation revealed that the A128D mutation had little effect on dimerization in the soluble b construct, b(34-156) . However, the mutation caused a structural perturbation detected through both a 12% reduction in the sedimentation coefficient and also a reduced tendency to form intersubunit disulfide bonds between cysteine residues inserted at position 132 . Unlike the wild-type sequence, the A128D mutant was unable to interact with F(1)-ATPase . These results indicate that the A128D mutation caused a structural change in the C-terminal region of the protein, preventing the binding to F(1) but having little or no effect on the dimeric nature of b. Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 331 - 8 What fuels polypeptide translocation? An energetical view on mitochondrial protein sorting; Herrmann JM et al.; Protein sorting into mitochondria is achieved by the concerted action of at least four translocation complexes . Vectorial transport of polypeptide chains by these complexes requires different driving forces . In particular, Deltapsi, matrix adenosine triphosphate and the free energy of the binding to other protein components are used in series to achieve sorting of proteins to the various mitochondrial subcompartments . The processes providing the translocation energy are presented in this review and their impact for protein sorting into and within mitochondria is discussed. Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 305 - 9 Characterization of two novel redox groups in the respiratory NADH:ubiquinone oxidoreductase (complex I); Friedrich T et al.; The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes . The bacterial complex consists of 14 different subunits . Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters . The remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane . Little is known about their function, but they are most likely involved in proton translocation . The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation . A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy . This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet . Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex . Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors . Electrochemical titration of this absorption reveals a midpoint potential of -80 mV . This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone. Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 284 - 90 The transmembrane domain and the proton channel in proton-pumping transhydrogenases; Bizouarn T et al.; Proton-pumping nicotinamide nucleotide transhydrogenases are composed of three main domains, the NAD(H)-binding and NADP(H)-binding hydrophilic domains I (dI) and III (dIII), respectively, and the hydrophobic domain II (dII) containing the assumed proton channel . dII in the Escherichia coli enzyme has recently been characterised with regard to topology and a packing model of the helix bundle in dII is proposed . Extensive mutagenesis of conserved charged residues of this domain showed that important residues are betaHis91 and betaAsn222 . The pH dependence of betaH91D, as well as betaH91C (unpublished), when compared to that of wild type shows that reduction of 3-acetylpyridine-NAD(+) by NADPH, i.e., the reverse reaction, is optimal at a pH essentially coinciding with the pK(a) of the residue in the beta91 position . It is therefore concluded that the wild-type transhydrogenase is regulated by the degree of protonation of betaHis91 . The mechanisms of the interactions between dI+dIII and dII are suggested to involve pronounced conformational changes in a 'hinge' region around betaR265. J Am Soc Nephrol, 2000 Oct, 11(10), 1848 - 56 Expression of endothelial nitric oxide synthase in human peritoneal tissue: regulation by Escherichia coli lipopolysaccharide; Arriero MM et al.; Changes in the expression of endothelial nitric oxide synthase (eNOS) in the peritoneum could be involved in the peritoneal dysfunction associated with peritoneal inflammation . Demonstrated recently in bovine endothelial cells was the existence of cytosolic proteins that bind to the 3'-untranslated region (3'-UTR) of eNOS mRNA and could be implicated in eNOS mRNA stabilization . The present work demonstrates that eNOS protein is expressed in human endothelial and mesothelial peritoneal cells . Escherichia coli lipopolysaccharide shortened the half-life of eNOS message, reducing eNOS protein expression in peritoneal mesothelial and endothelial cells . Moreover, under basal conditions, human peritoneal samples expressed cytosolic proteins that bind to the 3'-UTR of eNOS mRNA . The cytosolic proteins that directly bind to 3'-UTR were identified as a 60-kD protein . After incubation of human peritoneal samples with lipopolysaccharide, the binding activity of the cytosolic 60-kD protein increased in a time-dependent manner . Studies are now necessary to determine the involvement of this 60-kD protein in the regulation of eNOS expression in peritoneal cells and particularly its involvement in the peritoneal dysfunction associated with inflammatory reactions. J Bacteriol, 2000 Oct, 182(20), 5916 - 8 Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha; Handrick R et al.; Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival . Isolated native PHB granules of mobilized R . eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria . No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes . Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R . eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes. J Bacteriol, 2000 Oct, 182(20), 5898 - 901 Null mutation of the dam or seqA gene suppresses temperature-sensitive lethality but not hypersensitivity to novobiocin of muk null mutants; Onogi T et al.; Escherichia coli mukF, mukE, and mukB null mutants have common phenotypes such as temperature-dependent colony formation, anucleate cell production, chromosome cutting by septum closure, and abnormal localization of SeqA-DNA clusters . We show here that the associated muk null mutations cause hypersensitivity to novobiocin . Null mutation of either dam or seqA suppressed partially the temperature-sensitive lethality but failed to suppress the anucleate cell production and the hypersensitivity to novobiocin caused by muk null mutations. J Bacteriol, 2000 Oct, 182(20), 5872 - 9 The last RNA-binding repeat of the Escherichia coli ribosomal protein S1 is specifically involved in autogenous control; Boni IV et al.; The ssyF29 mutation, originally selected as an extragenic suppressor of a protein export defect, has been mapped within the rpsA gene encoding ribosomal protein S1 . Here, we examine the nature of this mutation and its effect on translation . Sequencing of the rpsA gene from the ssyF mutant has revealed that, due to an IS10R insertion, its product lacks the last 92 residues of the wild-type S1 protein corresponding to one of the four homologous repeats of the RNA-binding domain . To investigate how this truncation affects translation, we have created two series of Escherichia coli strains (rpsA(+) and ssyF) bearing various translation initiation regions (TIRs) fused to the chromosomal lacZ gene . Using a beta-galactosidase assay, we show that none of these TIRs differ in activity between ssyF and rpsA(+) cells, except for the rpsA TIR: the latter is stimulated threefold in ssyF cells, provided it retains at least ca . 90 nucleotides upstream of the start codon . Similarly, the activity of this TIR can be severely repressed in trans by excess S1, again provided it retains the same minimal upstream sequence . Thus, the ssyF stimulation requires the presence of the rpsA translational autogenous operator . As an interpretation, we propose that the ssyF mutation relieves the residual repression caused by normal supply of S1 (i.e., that it impairs autogenous control) . Thus, the C-terminal repeat of the S1 RNA-binding domain appears to be required for autoregulation, but not for overall mRNA recognition. J Bacteriol, 2000 Oct, 182(20), 5841 - 8 Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis; Cunningham FX Jr et al.; It is proposed that the lytB gene encodes an enzyme of the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) . A mutant of the cyanobacterium Synechocystis strain PCC 6803 with an insertion in the promoter region of lytB grew slowly and produced greenish-yellow, easily bleached colonies . Insertions in the coding region of lytB were lethal . Supplementation of the culture medium with the alcohol analogues of IPP and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely alleviated the growth impairment of the mutant . The Synechocystis lytB gene and a lytB cDNA from the flowering plant Adonis aestivalis were each found to significantly enhance accumulation of carotenoids in Escherichia coli engineered to produce these colored isoprenoid compounds . When combined with a cDNA encoding deoxyxylulose-5-phosphate synthase (dxs), the initial enzyme of the DOXP pathway, the individual salutary effects of lytB and dxs were multiplied . In contrast, the combination of lytB and a cDNA encoding IPP isomerase (ipi) was no more effective in enhancing carotenoid accumulation than ipi alone, indicating that the ratio of IPP and DMAPP produced via the DOXP pathway is influenced by LytB. J Bacteriol, 2000 Oct, 182(20), 5813 - 22 The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite; Wang H et al.; Escherichia coli possesses two distinct nitrite reductase enzymes encoded by the nrfA and nirB operons . The expression of each operon is induced during anaerobic cell growth conditions and is further modulated by the presence of either nitrite or nitrate in the cells' environment . To examine how each operon is expressed at low, intermediate, and high levels of either nitrate or nitrite, anaerobic chemostat culture techniques were employed using nrfA-lacZ and nirB-lacZ reporter fusions . Steady-state gene expression studies revealed a differential pattern of nitrite reductase gene expression where optimal nrfA-lacZ expression occurred only at low to intermediate levels of nitrate and where nirB-lacZ expression was induced only by high nitrate conditions . Under these conditions, the presence of high levels of nitrate suppressed nrfA gene expression . While either NarL or NarP was able to induce nrfA-lacZ expression in response to low levels of nitrate, only NarL could repress at high nitrate levels . The different expression profile for the alternative nitrite reductase operon encoded by nirBDC under high-nitrate conditions was due to transcriptional activation by either NarL or NarP . Neither response regulator could repress nirB expression . Nitrite was also an inducer of nirB and nrfA gene expression, but nitrate was always the more potent inducer by >100-fold . Lastly, since nrfA operon expression is only induced under low-nitrate concentrations, the NrfA enzyme is predicted to have a physiological role only where nitrate (or nitrite) is limiting in the cell environment . In contrast, the nirB nitrite reductase is optimally synthesized only when nitrate or nitrite is in excess of the cell's capacity to consume it . Revised regulatory schemes are presented for NarL and NarP in control of the two operons. J Bacteriol, 2000 Oct, 182(20), 5779 - 86 The torYZ (yecK bisZ) operon encodes a third respiratory trimethylamine N-oxide reductase in Escherichia coli; Gon S et al.; The bisZ gene of Escherichia coli was previously described as encoding a minor biotin sulfoxide (BSO) reductase in addition to the main cytoplasmic BSO reductase, BisC . In this study, bisZ has been renamed torZ based on the findings that (i) the torZ gene product, TorZ, is able to reduce trimethylamine N-oxide (TMAO) more efficiently than BSO; (ii) although TorZ is more homologous to BisC than to the TMAO reductase TorA (63 and 42% identity, respectively), it is located mainly in the periplasm as is TorA; (iii) torZ belongs to the torYZ operon, and the first gene, torY (formerly yecK), encodes a pentahemic c-type cytochrome homologous to the TorC cytochrome of the TorCAD respiratory system . Furthermore, the torYZ operon encodes a third TMAO respiratory system, with catalytic properties that are clearly different from those of the TorCAD and the DmsABC systems . The torYZ and the torCAD operons may have diverged from a common ancestor, but, surprisingly, no torD homologue is found in the sequences around torYZ . Moreover, the torYZ operon is expressed at very low levels under the conditions tested, and, in contrast to torCAD, it is not induced by TMAO or dimethyl sulfoxide. J Bacteriol, 2000 Oct, 182(20), 5757 - 64 Transport of C(4)-dicarboxylates in Wolinella succinogenes; Ullmann R et al.; C(4)-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane . W . succinogenes was found to transport C(4)-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms . The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration . The molar ratio of internal to external dicarboxylate concentration was up to 10(3) . The dicarboxylate antiport was either electrogenic or electroneutral . The electroneutral antiport required the presence of internal Na(+), whereas the electrogenic antiport also operated in the absence of Na(+) . In the absence of Na(+), no electrochemical proton potential (delta p) was measured across the membrane of cells catalyzing fumarate respiration . This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport . Three gene loci (dcuA, dcuB, and dctPQM) encoding putative C(4)-dicarboxylate transporters were identified on the genome of W . succinogenes . The predicted gene products of dcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration of Escherichia coli with external C(4)-dicarboxylates . The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates . A mutant (DcuA(-) DcuB(-)) of W . succinogenes lacking the intact dcuA and dcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates . The DcuA(-), DcuB(-), and DctQM(-) mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source . Cells of the DcuA(-) DcuB(-) mutant performed fumarate respiration without generating a proton potential even in the presence of Na(+) . This explains why the DcuA(-) DcuB(-) mutant does not grow by fumarate respiration . Growth by fumarate respiration appears to depend on the function of the Na(+)-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters . Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter. J Bacteriol, 2000 Oct, 182(20), 5706 - 14 Role of the Escherichia coli nucleotide excision repair proteins in DNA replication; Moolenaar GF et al.; DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication . Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium . Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC . In contrast, delta polA cells grow even better when the uvrC gene has been deleted . Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway . With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3' incision reaction . Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication . Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB . A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro . The same mutation, however, does give inviability in combination with the delta polA mutation . Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair . A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed. J Bacteriol, 2000 Oct, 182(20), 5671 - 5 Covariance of complementary rRNA loop nucleotides does not necessarily represent functional pseudoknot formation in vivo; Chernyaeva NS et al.; We examined mutationally a two-hairpin structure (nucleotides 57 to 70 and 76 to 110) in a region of domain I of Escherichia coli 23S rRNA that has been implicated in specific functions in protein synthesis by other studies . On the basis of the observed covariance of several nucleotides in each loop in Bacteria, Archaea, and chloroplasts, the two hairpins have been proposed to form a pseudoknot . Here, appropriate loop changes were introduced in vitro by site-directed mutagenesis to eliminate any possibility of base pairing between the loops . The bacterial cells containing each cloned mutant rRNA operon were then examined for cell growth, termination codon readthrough, and assembly of the mutant rRNAs into functional ribosomes . The results show that, under the conditions examined, the two hairpins do not form a pseudoknot structure that is required for the functioning of the ribosome in vivo and therefore that sequence covariance does not necessarily indicate the formation of a functional pseudoknot. Anesth Analg, 2000 Oct, 91(4), 1029 - 31, table of contents The anesthetic management of a patient with paroxysmal nocturnal hemoglobinuria; Kathirvel S et al.; Implications: This case report describes the anesthetic considerations for a patient with paroxysmal nocturnal hemoglobinuria . Specific strategies to be applied in the perioperative period to prevent hemolytic episodes and venous thrombosis are also discussed. Anesth Analg, 2000 Oct, 91(4), 896 - 903 Endotoxin augments cerebral hyperemic response to halothane by inducing nitric oxide synthase and cyclooxygenase; Okamoto H et al.; We examined the cerebral hyperemic response to halothane after treatment with bacterial lipopolysaccharide (LPS) . To determine the involvement of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), we tested whether the effect of LPS on halothane-induced hyperemia was altered by pretreatment with the selective iNOS inhibitor, aminoguanidine (100 mg/kg), COX-2 inhibitor, NS-398 (5 mg/kg), or enzyme expression inhibitor, dexamethasone (4 mg/kg) . Further, we examined whether the administration of a nitric oxide donor, diethylamine NONOate, would change the cerebral hyperemic response of halothane . Sprague-Dawley rats were anesthetized with 0.5 minimum alveolar anesthetic concentration of halothane and artificially ventilated . Regional cerebrocortical blood flow (rCBF) was assessed by laser-Doppler flowmetry . LPS (1 mg/kg) was administered intracerebroventricularly; artificial cerebrospinal fluid was used in controls . Four hours after LPS infusion, iNOS and COX-2 messenger ribonucleic acid (mRNA) levels (reverse transcription-polymerase chain reaction) and enzyme activities (arginine-citrulline conversion and prostaglandin E(2) enzyme immunoassay) were significantly increased . LPS enhanced halothane-induced 3.9 and 1.6-fold increases in rCBF at 1.0 and 1.5 minimum alveolar concentration, respectively . Co-treatment with NS-398 attenuated, but aminoguanidine or dexamethasone abolished the effect of LPS on halothane-induced rCBF increase . Diethylamine NONOate mimicked the enhanced rCBF response to halothane . These results suggest that LPS augmented halothane-induced cerebrocortical hyperemia by induction of iNOS and COX-2. Biochim Biophys Acta, 2000 Jul 26, 1475(3), 377 - 82 Role of alpha-helical conformation of histatin-5 in candidacidal activity examined by proline variants; Situ H et al.; Human salivary histatin-5 (Hsn-5) is a potent in vitro anticandidal agent . The aim of this study was to investigate the importance of alpha-helical structure of Hsn-5 for its candidacidal activity . The following three Hsn-5 variants, where one or more functionally nonessential residues were replaced with proline (potent alpha-helix breaker), were produced by Escherichia coli expression system: H21P (1P), H19P/H21P (2P), and E16P/H19P/H21P (3P) . The activities of purified proteins were determined by candidacidal assays, and the secondary structures by circular dichroism (CD) spectroscopy in trifluoroethanol (TFE) that is considered the helix-promoting solvent, and lysophosphatidyl-glycerol (LPG) micelles, the environment that more closely resembles the biological membranes . Our results indicated that 3P variant displayed a candidacidal activity which was similar to that of unaltered Hsn-5 (0P), while 1P and 2P variants showed lower cidal activity . The CD spectra in TFE indicated that 3P variant has less helical characteristics than the 0P, 1P and 2P . These results suggested that the alpha-helical content of Hsn-5 proline variants does not correlate with the candidacidal activity . Further, the CD spectral analysis of peptides in LPG micelles indicated the formation of beta-turn structures in 0P and 3P variants . In conclusion, 3P variant which exhibited comparable candidacidal activity to 0P contains lower percentage of alpha-helical structure than 1P and 2P variants, which exhibited lower candidacidal activity . This suggests alpha-helix may not be important for anticandidal activity of Hsn-5. Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 449 - 55 Fusion protein approach to improve the crystal quality of cytochrome bo(3) ubiquinol oxidase from Escherichia coli; Byrne B et al.; Crystals of cytochrome bo(3) ubiquinol oxidase from E . coli diffract X-rays to 3.5 A and the structure determination is in progress . The limiting factor to the elucidation of the structural detail is the quality of the crystals; the diffraction spots from the crystals are diffused which leads to difficulties in processing the data beyond 4.0 A . Weak protein-protein contacts within the crystal lattice is assumed to be the cause of this problem . To improve these contacts, we have introduced protein Z to the C-terminal end of the subunit IV of cytochrome bo(3) and expressed both proteins as a single fusion . We have successfully obtained crystals of this fusion protein . The spot shape problem has clearly been solved in the crystals of the fusion protein although further optimization is necessary to obtain higher resolution . We also discuss the potential applications of this approach to the crystallization of membrane proteins in general. Glycoconj J, 1999 Nov, 16(11), 697 - 705 Development of recombinant B subunit of Shiga-like toxin 1 as a probe to detect carbohydrate ligands in immunochemical and flowcytometric application; Miyashita S et al.; Measurement of carbohydrate binding activity of Escherichia coli Shiga-like toxin in a simple and quantitative way is an important step for evaluation of antibodies with therapeutic value and of effectiveness of vaccine treatment . We constructed a plasmid vector (pVT1-B5) to express carbohydrate binding (B) subunit of Shiga-like toxin 1 without expression of toxic (A) subunit, and established a simple method to purify the recombinant B subunit, which was then labeled with digoxigenin . The binding specificity of the digoxigenin-labeled B subunit for globotriaosylceramide was established by thin-layer chromatography immunostaining . We developed an enzyme-linked immunosorbent assay using immobilized glycolipids, demonstrating high sensitivity and clear-cut specificity of the assay . The digoxigenin-labeled B subunit was also readily applicable to the detection of cell surface carbohydrate ligands by flow cytometry. Arch Virol, 2000, 145(8), 1535 - 42 Immunodetection and subcellular localization of the proteins encoded by ORF 3 of grapevine viruses A and B; Saldarelli P et al.; Polyclonal antisera were raised to Escherichia coli-expressed ORF3 products (putative movement proteins) of Grapevine virus A (GVA) and Grapevine virus B (GVB) (genus Vitivirus), and used for their immunodetection in infected plants . Western blot analysis of subfractionated cellular compartments showed that the distribution of both proteins was comparable to that of plant virus movement proteins, as they were transiently present in a crude membrane fraction and accumulated in a cell wall-enriched fraction . The GVA ORF3-encoded protein, but not the comparable GVB protein, was also present in large amount in a cytoplasmic soluble fraction . Intracellular immunogold labelling localized these proteins in the cell wall and plasmodesmata of infected cells and, especially for GVA, in association with cytoplasmic virus aggregates. J Neurosci Res, 2000 Oct 1, 62(1), 28 - 39 Model for focal demyelination of the spinal dorsal columns of transgenic MBP-LacZ mice by phototargeted ablation of oligodendrocytes; Vanderluit JL et al.; Focal demyelination models provide powerful tools to study demyelination and remyelination in the central nervous system . In this report, we present a novel technique, which selectively targets oligodendrocytes within the spinal cord of transgenic mice to produce focal demyelination . Transgenic mice expressing the E . coli LacZ (beta-galactosidase) gene from the myelin basic protein promotor allowed for oligodendrocyte-specific cleavage of topically applied fluorescein-di-beta-galactopyranoside liberating photoactivatable fluorescein . Subsequent fluorescence illumination generated oxygen radicals that oxidized a second exogenous substrate, 3-amino-9-ethyl carbazole, to form a toxic precipitate within oligodendrocytes . Histochemical staining of the spinal cord dorsal columns 8 days following phototargeting revealed that the treated region no longer contained beta-galactosidase-positive cells . Focal demyelination of the dorsal columns was observed to a depth of 150 microm in transverse semithin plastic sections . Numerous bundles of naked axons interspersed with myelin, debris-laden macrophages, and reactive astrocytes were evident by electron microscopy . Remyelination of axons by both oligodendrocytes and invading Schwann cells was observed within the treated region 14 days after phototargeting . Newly generated oligodendrocytes were identified within the demyelinated region by their incorporation of bromodeoxyuridine . Thus, this novel focal demyelination protocol provides: (1) a method for selective targeted ablation of oligodendrocytes in vivo, (2) control over the extent of the demyelinated region, with (3) an environment that maintains its remyelination capacity . Phototargeted ablation of oligodendrocytes may therefore be a useful model for studying axon-glia interactions, axon regeneration within a demyelinated zone, and remyelination of axons . J Biol Chem, 2000 Sep 29, 275(39), 30157 - 62 The activity of the ATP synthase from Escherichia coli is regulated by the transmembrane proton motive force; Fischer S et al.; The ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid . The proteoliposomes were energized by an acid-base transition and a K(+)/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started . In the presence of ADP and P(i), the initial rate of ATP hydrolysis was up to 9-fold higher with pre-energized proteoliposomes than with proteoliposomes that had not seen an electrochemical proton gradient . After dissipating the electrochemical proton gradient, the high rate of ATP hydrolysis decayed to the rate without pre-energization within about 15 s . During this decay the enzyme carried out approximately 100 turnovers . In the absence of ADP and P(i), the rate of ATP hydrolysis was already high and could not be significantly increased by pre-energization . It is concluded that ATP hydrolysis is inhibited when ADP and P(i) are bound to the enzyme and that a high Delta mu(H(+)) is required to release ADP and P(i) and to convert the enzyme into a high activity state . This high activity state is metastable and decays slowly when Delta mu(H(+)) is abolished . Thus, the proton motive force does not only supply energy for ATP synthesis but also regulates the fraction of active enzymes. Biotechnol Appl Biochem, 2000 Oct, 32 ( Pt 2), 137 - 43 Expression and immunological evaluation of the Escherichia coli-derived hepatitis C virus envelope E1 protein; Lorenzo LJ et al.; Immunological response against envelope protein E1 is very important in natural hepatitis C virus (HCV) infection, although it is insufficient to clear the viraemia . The HCV genomic region encoding the first 149 amino acids of the envelope E1 protein (E1(340), amino acids 192-340) was expressed in Escherichia coli (to a level of 30% of the whole cellular proteins) and purified to 85% . We measured the immune response in rabbits and mice as well as the reactivity against 37 human sera raised against the whole recombinant protein and E1-encoding peptides . From this, 51.1% of human sera were found to react with E1(340) . High-level antibodies against E1(340) were obtained in rabbits and mice when immunized . These antibodies had a similar peptide-recognition pattern to that described previously for human sera . The most reactive region was located at the N-terminus of the E1 protein . Cellular immunity in mice was evaluated by delayed-type hypersensitivity assay . It revealed the induction of a CD4+ T-cell-mediated response by this protein . This E1(340) protein and the animal-derived anti-E1 sera are immunological tools that could aid in the monitoring and development of anti-HCV therapies. J Biol Chem, 2001 Feb 2, 276(5), 3341 - 7 Epub 2000 Sep 22. Identification and cDNA cloning of a novel RNA-binding protein that interacts with the cyclic nucleotide-responsive sequence in the Type-1 plasminogen activator inhibitor mRNA; Heaton JH et al.; Incubation of HTC rat hepatoma cells with 8-bromo-cAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator inhibitor (PAI-1) mRNA . We have reported previously that the 3'-most 134 nt of the PAI-1 mRNA is able to confer cyclic nucleotide regulation of message stability onto a heterologous transcript . R-EMSA and UV cross-linking experiments have shown that this 134 nt cyclic nucleotide-responsive sequence (CRS) binds HTC cell cytoplasmic proteins ranging in size from 38 to 76 kDa . Mutations in the A-rich region of the CRS both eliminate cyclic nucleotide regulation of mRNA decay and abolish RN-protein complex formation, suggesting that these RNA-binding proteins may be important regulators of mRNA stability . By sequential R-EMSA and SDS-PAGE we have purified a protein from HTC cell polysomes that binds to the PAI-1 CRS . N-terminal sequence analysis and a search of protein data bases revealed identity with two human sequences of unknown function . We have expressed one of these sequences in E . coli and confirmed that the recombinant protein interacts specifically with the PAI-1 CRS . Mutation of the A-rich portion of the PAI-1 CRS reduces binding by the recombinant PAI-1 RNA-binding protein . The amino acid sequence of this protein includes an RGG box and two arginine-rich regions, but does not include other recognizable RNA binding motifs . Detailed analyses of nucleic acid and protein data bases demonstrate that blocks of this sequence are highly conserved in a number of metazoans, including Arabidopsis, Drosophila, birds, and mammals . Thus, we have described a novel RNA-binding protein that identifies a family of proteins with a previously undefined sequence motif . Our results suggest that this protein, PAI-RBP1, may play a role in regulation of mRNA stability. J Inorg Biochem, 2000 Jul 15, 81(1-2), 111 - 7 DNA targeted platinum complexes: synthesis, cytotoxicity and DNA interactions of cis-dichloroplatinum(II) complexes tethered to phenazine-1-carboxamides; Perrin LC et al.; A series of intercalator-tethered platinum(II) complexes PtLCl2 have been prepared, where L are the diamine ligands N-{2-{(aminoethyl)amino}ethyl}-phenazine-1-carboxamide, N-{3-{(2-aminoethyl)amino}propyl}-phenazine-1-carboxamide, N-{4-{(2-aminoethyl)amino}butyl}-phenazine-1-carboxamide and N-{5-{(aminoethyl)amino}pentyl}-phenazine-1-carboxamide . Measurements of the time-course of unwinding of supercoiled pUC19 plasmid DNA by the phenazine complexes PtLCl2 reveal that the presence of the intercalator leads to enhanced rates of DNA platination when compared with the complex Pt(en)Cl2 . The platinum(II) complexes where the polymethylene linker chain contains three, four or five carbon atoms are considerably more cytotoxic against murine P388/W than either cisplatin, Pt(en)Cl2, or the metal-free ligands themselves. Hybridoma, 2000 Aug, 19(4), 317 - 21 Monoclonal antibodies generated against recombinant ATM support kinase activity; Alligood KJ et al.; We report on the rapid generation of two monoclonal antibodies, ATM A16.35 and ATM D16.11, that bind to the kinase domain of mutated ataxia telangiectasia (ATM) . These antibodies were generated against E . coli-expressed recombinant protein using the RIMMS strategy . We show that ATM A16.35 binds ATM by Western blot analysis, and ATM D16.11 forms immune complexes with