Escherichia coli

Protein Sci, 2000 Feb, 9(2), 353 - 60
Substrate- and pH-dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family; Szeltner Z et al.; Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family . Crystallographic studies (Fulop et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473 . To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli . The specificity rate constant, k(cat)/Km, for the modified enzyme decreased by a factor of 8-40 with highly specific substrates, Z-Gly-Pro-Nap, and a fluorogenic octapeptide . With these compounds, the decline in k(cat) was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin . With the less specific suc-Gly-Pro-Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (approximately 500-fold) in k(cat)/Km . The second-order rate constant for the reaction with Z-Pro-prolinal, a slow tight-binding transition-state analogue inhibitor, and the Ki values for a slow substrate and two product-like inhibitors were not significantly affected by the Tyr473 OH group . The mechanism of transition-state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms.

Protein Sci, 2000 Feb, 9(2), 325 - 31
Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin; Gasymov OK et al.; The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules . Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding . Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery . We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence . Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99 . For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra . The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin . Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL . Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins . These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets . Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues . Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand . Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion . The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography.

Protein Sci, 2000 Feb, 9(2), 242 - 51
Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme; Chou WY et al.; Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment . Site-specific mutagenesis was performed at these putative metal-binding sites . Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells . All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns . The mutants showed similar apparent Km,NADP values to that of the WT . The Km,Mal value was increased in the D141N and D194N mutants . The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy . Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N . Initial velocity experiments were performed to derive the various kinetic parameters . The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box . There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant {k(cat)/(Kd,Mn Km,Mal Km,NADP)} at least four orders of magnitude smaller than the WT value . This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency . Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194 . The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT . These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme . The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

Protein Sci, 2000 Feb, 9(2), 213 - 7
A novel method of affinity-purifying proteins using a bis-arsenical fluorescein; Thorn KS et al.; Genetically-encoded affinity tags constitute an important strategy for purifying proteins . Here, we have designed a novel affinity matrix based on the his-arsenical fluorescein dye FlAsH, which specifically recognizes short alpha-helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269-272) . We find that kinesin tagged with this cysteine-containing helix binds specifically to FlAsH resin and can be eluted in a fully active form . This affinity tag has several advantages over polyhistidine, the only small affinity tag in common use . The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis tagged kinesin . Moreover, unlike nickel affinity chromatography, which requires high concentrations of imidazole or pH changes for elution, protein bound to the FlAsH column can be completely eluted by dithiothreitol . Because of these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes.

Plant Cell, 2000 Mar, 12(3), 419 - 31
The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein; Lindahl M et al.; The photosystem II reaction center D1 protein is known to turn over frequently . This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy . However, the proteases responsible for D1 protein degradation remain unknown . In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process . The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH . Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH . In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover . Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate-functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

J Med Chem, 2000 Mar 9, 43(5), 971 - 83
Design, synthesis, and enzymatic evaluation of multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase; Esteban-Gamboa A et al.; A series of acyclic phosphonate derivatives of thymine has been synthesized and tested as multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase . The compounds synthesized include 1-(phosphonoalkyl)thymines with six to nine methylenes (1-4, respectively); 1-{(Z)-4-phosphonomethoxy-2-butenyl}thymine (5) and its butyl and 2,3-cis-dihydroxybutyl derivatives (6 and 7, respectively); 1-{(Z)-(4-(phosphonomethoxy)methoxy)-2-butenyl}thymine (8) and also its butyl and 2,3-cis-dihydroxybutyl analogues (9 and 10); and 1-{((Z)-4-(phosphonomethoxy)-2-butenoxy)methyl}thymine (11) . Evaluation of these compounds against E . coli revealed significant enzymatic inhibition by 2, 3, 4, 6, and 8 at a concentration of 1000 microM, 3 and 4 being the most potent . Replacement of the thymine base in 3 by 6-amino-5-bromouracil and 7-deazaxanthine afforded compounds 12 and 13, which showed a pronounced improvement of TPase inhibition, comparable to 7-deazaxanthine . When inorganic phosphate was used as a variable substrate, compounds 12 and 13 displayed competitive kinetics with respect to phosphate, indicating a direct interaction of these compounds with the phosphate binding site . Also compounds 12 and 13 were found to be competitive inhibitors of TPase against thymidine as a variable substrate . These results are consistent with the compounds being multisubstrate analogue inhibitors of E . coli TPase, and they represent the first example of such TPase inhibitors.

Biochemistry, 2000 Mar 21, 39(11), 3176 - 83
Modulation of MutS ATP hydrolysis by DNA cofactors; Bjornson KP et al.; Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics . At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM . Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM . The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs . The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair . Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis . Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate . In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis . Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly . These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.

Biochemistry, 2000 Mar 21, 39(11), 3169 - 75
Q-band ENDOR (electron nuclear double resonance) of the high-affinity ubisemiquinone center in cytochrome bo3 from Escherichia coli; Veselov AV et al.; Electron nuclear double resonance (ENDOR) was performed on the protein-bound, stabilized, high-affinity ubisemiquinone radical, QH*-, of bo3 quinol oxidase to determine its electronic spin distribution and to probe its interaction with its surroundings . Until this present work, such ENDOR studies of protein-stabilized ubisemiquinone centers have only been done on photosynthetic reaction centers whose function is to reduce a ubiquinol pool . In contrast, QH*- serves to oxidize a ubiquinol pool in the course of electron transfer from the ubiquinol pool to the oxygen-consuming center of terminal bo3 oxidase . As documented by large hyperfine couplings (>10 MHz) to nonexchangeable protons on the QH*- ubisemiquinone ring, we provide evidence for an electronic distribution on QH*- that is different from that of the semiquinones of reaction centers . Since the ubisemiquinone itself is physically nearly identical in both QH*- and the bacterial photosynthetic reaction centers, this electronic difference is evidently a function of the local protein environment . Interaction of QH*- with this local protein environment was explicitly shown by exchangeable deuteron ENDOR that implied hydrogen bonding to the quinone and by weak proton hyperfine couplings to the local protein matrix.

Biochemistry, 2000 Mar 21, 39(11), 3156 - 68
Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 A resolution: mechanistic implications; Howard BR et al.; The crystal structure of selenomethionine-substituted malate synthase G, an 81 kDa monomeric enzyme from Escherichia coli has been determined by MAD phasing, model building, and crystallographic refinement to a resolution of 2.0 A . The crystallographic R factor is 0.177 for 49 242 reflections observed at the incident wavelength of 1.008 A, and the model stereochemistry is satisfactory . The basic fold of the enzyme is that of a beta8/alpha8 (TIM) barrel . The barrel is centrally located, with an N-terminal alpha-helical domain flanking one side . An inserted beta-sheet domain folds against the opposite side of the barrel, and an alpha-helical C-terminal domain forms a plug which caps the active site . Malate synthase catalyzes the condensation of glyoxylate and acetyl-coenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A, requiring Mg(2+) . The structure reveals an enzyme-substrate complex with glyoxylate and Mg(2+) which coordinates the aldehyde and carboxylate functions of the substrate . Two strictly conserved residues, Asp631 and Arg338, are proposed to provide concerted acid-base chemistry for the generation of the enol(ate) intermediate of acetyl-coenzyme A, while main-chain hydrogen bonds and bound Mg(2+) polarize glyoxylate in preparation for nucleophilic attack . The catalytic strategy of malate synthase appears to be essentially the same as that of citrate synthase, with the electrophile activated for nucleophilic attack by nearby positive charges and hydrogen bonds, while concerted acid-base catalysis accomplishes the abstraction of a proton from the methyl group of acetyl-coenzyme A . An active site aspartate is, however, the only common feature of these two enzymes, and the active sites of these enzymes are produced by quite different protein folds . Interesting similarities in the overall folds and modes of substrate recognition are discussed in comparisons of malate synthase with pyruvate kinase and pyruvate phosphate dikinase.

Biochemistry, 2000 Mar 21, 39(11), 3134 - 40
Thiol cross-linking of cytoplasmic loops in the lactose permease of Escherichia coli; Kwaw I et al.; The N- and C-terminal halves of lactose permease, each with a single-Cys residue in a cytoplasmic loop, were coexpressed, and cross-linking was studied in the absence or presence of ligand . Out of the 68 paired-Cys mutants in cytoplasmic loops IV/V and VIII/IX or X/XI, three pairs in loop IV/V and X/XI, (i) Arg135 --> Cys/Thr338 --> Cys, (ii) Arg134 --> Cys/Val343 --> Cys, and (iii) Arg134 --> Cys/Phe345 --> Cys, form a spontaneous disulfide bond, indicating that loops IV/V and X/XI are in close proximity . In addition, specific paired-Cys residues in loop IV/V (132-138) and loop VIII/IX (282-290) or loop X/XI (335-345) cross-link with iodine and/or the homobifunctional cross-linking agents N, N'-o-phenylenedimaleimide, N,N'-p-phenylenedimaleimide, and 1, 6-bis(maleimido)hexane . The results demonstrate that loop IV/V is close to both loop VIII/IX and loop X/XI . On the other hand, similar though less extensive cross-linking studies indicate that neither the N terminus nor loop II/III appear to be close to loops VIII/IX or X/XI . The findings suggest that the longer cytoplasmic loops are highly flexible and interact in a largely random fashion . However, although a Cys residue at position 134 in loop IV/V, for example, is able to cross-link with a Cys residue at each position in loop VIII/IX or loop X/XI, Cys residues at other positions in loop IV/V exhibit markedly different cross-linking patterns . Therefore, although the domains appear to be very flexible, the interactions are not completely random, suggesting that there are probably at least some structural constraints that limit the degree of flexibility . In addition, evidence is presented suggesting that ligand binding induces conformational alterations between loop IV/V and loop VIII/IX or X/XI.

Biochemistry, 2000 Mar 21, 39(11), 2931 - 9
Topography of the surface of the Escherichia coli phosphotransferase system protein enzyme IIAglc that interacts with lactose permease; Sondej M et al.; The unphosphorylated form of enzyme IIAglc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system inhibits transport catalyzed by lactose permease . We (Seok et al . (1997) Proc . Natl . Acad . Sci . U.S.A . 94, 13515-13519) previously characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIAglc, using radioactive enzyme IIAglc . Subsequent studies (Sondej et al . (1999) Proc . Natl . Acad . Sci . U.S.A . 96, 3525-3530) suggested consensus binding sequences on proteins that interact with enzyme IIAglc . The present study characterizes a region on the surface of enzyme IIAglc that interfaces with lactose permease . Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIAglc, but not lactose permease, reduced the degree of interaction between the two proteins . To localize the lysine residue(s) on enzyme IIAglc that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized . Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr . Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIAglc in a test for binding to lactose permease . The Lys69 mutant was also defective in phosphoryl transfer to glucose permease . To derive further information concerning the contact surface, additional selected residues in the vicinity of Lys69 were mutagenized and tested for binding to lactose permease . On the basis of these studies, a model for the region of the surface of enzyme IIAglc that interacts with lactose permease is proposed.

J Mol Biol, 2000 Feb 25, 296(3), 911 - 9
The GxxxG motif: a framework for transmembrane helix-helix association; Russ WP et al.; In order to identify strong transmembrane helix packing motifs, we have selected transmembrane domains exhibiting high-affinity homo-oligomerization from a randomized sequence library based on the right-handed dimerization motif of glycophorin A . Sequences were isolated using the TOXCAT system, which measures transmembrane helix-helix association in the Escherichia coli inner membrane . Strong selection was applied to a large range of sequences ( approximately 10(7) possibilities) and resulted in the identification of sequence patterns that mediate high-affinity helix-helix association . The most frequent motif isolated, GxxxG, occurs in over 80% of the isolates . Additional correlations suggest that flanking residues act in concert with the GxxxG motif, and that size complementarity is maintained at the interface, consistent with the idea that the identified sequence patterns represent packing motifs . The convergent identification of similar sequence patterns from an analysis of the transmembrane domains in the SwissProt sequence database suggests that these packing motifs are frequently utilized in naturally occurring helical membrane proteins .

J Mol Biol, 2000 Feb 25, 296(3), 757 - 68
IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif; Haren L et al.; Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA . OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB . This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif . OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro . The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper . We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction . Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function . Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats . Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB{1-149}, that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex" . Preincubation of OrfAB{1-149} with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA . Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats .

J Mol Biol, 2000 Feb 25, 296(3), 739 - 42
A fragment of recombinant GABA(A) receptor alpha1 subunit forming rosette-like homo-oligomers; Xue H et al.; The type A gamma-aminobutyric acid (GABA(A)) receptor plays a major role in inhibitory synaptic transmission in the central nervous system . A fragment consisting of residues Cys166 to Leu296 of the alpha1 subunit of the GABA(A) receptor was overexpressed in Escherichia coli and was found to have stable beta-rich structures . Here, results from laser scattering, gel electrophoresis and electron microscopy demonstrated that this recombinant protein formed rosette-like homo-oligomers, mainly pentamers in solution . Therefore, the fragment apparently provides a valuable model system for studying the pentameric holoreceptor assembly . Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fragment showed that disulfide bonds formed between monomers contributed to the oligomerization of the fragment . The fact that this fragment alone could form pentamers in vitro strongly suggests that amino acid residues located within the Cys166-Leu296 region of the alpha1 subunit may contribute to the oligomerization of GABA(A) receptor in vivo .

J Nucl Med, 2000 Mar, 41(3), 463 - 9
Radiolabeled interleukin-8: specific scintigraphic detection of infection within a few hours; van der Laken CJ et al.; Several small receptor-binding agents have been tested for imaging of infection and inflammation . The potential of chemotactic peptides and of interleukins is promising and superior to that of conventional agents . In this study, we investigated the potential of interleukin-8 (IL-8) to image infection in rabbits . METHODS: IL-8 was labeled with 123I using the Bolton-Hunter method . Twenty-fours hours after induction of Escherichia coli abscesses in the left thigh muscle, rabbits were injected intravenously with 18.5 MBq 123I-IL-8 . Gamma camera images were obtained at 5 min and at 1, 4, and 8 h after injection . Biodistribution was determined 8 h after injection . RESULTS: 123I-IL-8 rapidly cleared from the blood . Accumulation of 123I-IL-8 in the abscess was visible as early as 1 h after injection . The highest abscess uptake was obtained 4 h after injection (2.6+/-0.2 percentage injected dose {%ID}), whereas 123I-IL-8 rapidly cleared from all other tissues . This resulted in increases in abscess-to-background ratios to 13.0+/-0.7 (8 h after injection), as determined by quantification of the images . In tissue biodistribution (8 h after injection), the abscess uptake was 0.057+/-0.011 %ID/g with abscess-to-contralateral muscle ratios of 114.7+/-23.0 . The radioiodination method clearly affected the in vivo biodistribution of IL-8 because IL-8 iodinated using the lodo-Gen method cleared significantly slower from the blood and most other organs, resulting in poor visualization of the abscess . CONCLUSION: The superior characteristics of IL-8 radioiodinated using the Bolton-Hunter method--i.e., high abscess uptake and rapid background clearance within a few hours--make IL-8 a promising agent to image infection and inflammation.

Circulation, 2000 Mar 14, 101(10), 1158 - 64
Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa; Peter K et al.; BACKGROUND: Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot . METHODS AND RESULTS: The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA . To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly(4)Ser)(3) was introduced by overlap PCR . After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the Bbeta15-22 peptide of fibrin (beta-peptide) . Hirudin was genetically fused to the C-terminus of the variable region of the light chain . To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv(59D8) and hirudin . The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to beta-peptide, and by thrombin inhibition after Xa cleavage . Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin . CONCLUSIONS: A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa . This recombinant anticoagulant effectively inhibits appositional clot growth in vitro . Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.

J Bacteriol, 2000 Apr, 182(7), 2033 - 6
Dephosphorylation of the Escherichia coli transcriptional antiterminator BglG by the sugar sensor BglF is the reversal of its phosphorylation; Chen Q et al.; The Escherichia coli BglF protein catalyzes transport and phosphorylation of beta-glucosides . In addition, BglF is a membrane sensor which reversibly phosphorylates the transcriptional regulator BglG, depending on beta-glucoside availability . Therefore, BglF has three enzymatic activities: beta-glucoside phosphotransferase, BglG phosphorylase, and phospho-BglG (BglG-P) dephosphorylase . Cys-24 of BglF is the active site which delivers the phosphoryl group either to the sugar or to BglG . To characterize the dephosphorylase activity, we asked whether BglG-P can give the phosphoryl group back to Cys-24 of BglF . Here we provide evidence which is consistent with the interpretation that Cys-24-P is an intermediate in the BglG-P dephosphorylation reaction . Hence, the dephosphorylation reaction catalyzed by BglF proceeds via reversal of the phosphorylation reaction.

J Bacteriol, 2000 Apr, 182(7), 1995 - 2000
Cooperative action of the catabolite activator protein and AraC in vitro at the araFGH promoter; Johnson CM et al.; Full activation of transcription of the araFGH promoter, p(FGH), requires both the catabolite activator protein (CAP) and AraC protein . At p(FGH), the binding site for CAP is centered at position -41.5, an essential binding site for AraC is centered at position -79.5, and a second, nonessential binding site is centered at position -154.5 . In this work, we used the minimal promoter region required for in vivo activation of p(FGH) to examine the roles of CAP and AraC in stimulating formation of open complexes at p(FGH) . Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-p(FGH) complex and that the order of addition of proteins to the initiating complex is important . Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit . Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true p(FGH) promoter.

J Bacteriol, 2000 Apr, 182(7), 1969 - 77
Regulation of rRNA transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters; Bartlett MS et al.; We recently identified Escherichia coli RNA polymerase (RNAP) mutants (RNAP beta' Delta215-220 and beta RH454) that form extremely unstable complexes with rRNA P1 (rrn P1) core promoters . The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of rrn P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo . Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed . We report here that the rrn transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs . FIS activates the mutant RNAPs, at least in part, by reducing the apparent K(ATP) for the initiating NTP . This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding . FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of rrn P1 transcription in the mutant strains . These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.

J Bacteriol, 2000 Apr, 182(7), 1964 - 8
Repair of DNA lesions induced by hydrogen peroxide in the presence of iron chelators in Escherichia coli: participation of endonuclease IV and Fpg; Galhardo RS et al.; In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product . Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions . Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions . Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions . We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.

J Bacteriol, 2000 Apr, 182(7), 1812 - 8
Reinvestigation of the proteolytic activity of neocarzinostatin; Heyd B et al.; Neocarzinostatin (NCS) is the most studied member of a family of chromoproteins secreted by a range of actinomycetes species . It has been proposed that in addition to their antitumoral activity related to the bound chromophores, this group of related proteins could be a secreted proteases superfamily . With the aim of dissecting the molecular basis of the proteolytic activity of NCS, an expression system allowing efficient expression of apo-NCS in Escherichia coli was constructed . The recombinant protein was properly folded and functional . Its histone-specific proteolytic activity was similar to the activity described for the natural protein . Further analyses unambiguously demonstrated that the proteolytic activity could be physically separated from NCS . This activity is therefore due not to NCS itself but to minor contaminating proteases, the nature of which differed in the recombinant and natural NCS preparations . The histone degradation test commonly used to monitor proteolytic activity is extremely sensitive and may easily generate false-positive results . These results strongly suggest that the possible proteolytic activity of the proteins of this family should be critically reconsidered.

J Bacteriol, 2000 Apr, 182(7), 1788 - 93
Flavodoxin mutants of Escherichia coli K-12; Gaudu P et al.; The flavodoxins are flavin mononucleotide-containing electron transferases . Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon . An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it . A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB . Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen . A high-copy-number fldB(+) plasmid did not complement the fldA mutation . Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I . An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon . However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites . The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion . Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds . However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.

J Inorg Biochem, 2000 Jan 15, 78(1), 83 - 7
The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays; Karlsson A et al.; Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center . Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form . Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum . Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.

Bone Marrow Transplant, 2000 Mar, 25(5), 571 - 3
Constrictive pericarditis post allogeneic bone marrow transplant for Philadelphia-positive acute lymphoblastic leukaemia; Cavet J et al.; We describe two cases of severe constrictive pericarditis arising after allogeneic BMT conditioning involving total body irradiation and melphalan to treat Philadelphia-chromosome positive ALL . Both patients required pericardectomy, resulting in marked improvement in ventricular filling . However, a degree of right-sided cardiac failure persisted in both patients secondary to restrictive cardiomyopathy . Constrictive pericarditis has not been previously described after BMT, but has been observed following thoracic radiotherapy for malignancy, usually involving a substantially higher radiation dose . Pericardial constriction and restrictive cardiomyopathy should be considered as causes of breathlessness and/or oedema occurring late after BMT . Bone Marrow Transplantation (2000) 25, 571-573.

Biochemistry (Mosc), 2000 Feb, 65(2), 237 - 43
Influence of nucleic acids and polysaccharides on phosphotransferase activity of preparations of secretory immunoglobulin A from human milk; Kit YY et al.; The influence of nucleic acids (DNA, tRNA), synthetic oligonucleotides, and polysaccharides (lipopolysaccharides from Escherichia coli, heparin) on protein kinase and lipid kinase activities of preparations of human secretory immunoglobulin A (sIgA) has been studied . The preparations of sIgA were isolated from human milk by chromatography on the column with Protein A-Sepharose and DEAE-sorbent (sIgA1), by affinity chromatography of sIgA1 on DNA-cellulose (sIgA2), and by gel-filtration of sIgA1 in buffer containing 5% dioxane (sIgA3) . Two 32P-labeled products with high and low electrophoretic mobility in polyacrylamide gel containing SDS were found after incubation of sIgA1 and sIgA2 with {gamma-32P}ATP . The product with low electrophoretic mobility was degraded in 10% trichloroacetic acid giving a radioactive background in lanes of the polyacrylamide gel . 32P-Labeled phospholipids were found among the phosphorylation products . Soluble and immobilized DNA increase lipid kinase activity of preparations of sIgA . In this case the secretory component and H-chains of sIgA were degraded . Fractions possessing lipid kinase activity were precipitated in the presence of heparin (1 mg/ml), and lipid kinase activity was separated from sIgA by gel-filtration in buffer containing 5% dioxane . 32P-Labeled products were formed in the presence of {gamma-32P}ATP as well as {32P}ortho-phosphoric acid . The influence of heparin and synthetic deoxy- and ribooligonucleotides on casein kinase activity of sIgA3 was studied . It was observed that deoxyribooligonucleotides in micromolar concentrations increased the rate of casein phosphorylation in the presence of sIgA3 and {gamma-32P}ATP . It has been proposed that catalytically active sIgA have an affinity to DNA (anti-DNA sIgA) and can be present in human milk as a part of lipoprotein complexes.

Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 372 - 5
Arabidopsis thaliana peroxidase N: structure of a novel neutral peroxidase; Mirza O et al.; The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5% . ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d . of 0.82 A when compared with horseradish peroxidase C (HRP C) . HRP C is 54% identical to ATP N in sequence . When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule . The architecture of the haem pocket of ATP N is very similar to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases . The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel . Since there are fewer hydrogen bonds to haem C17 propionate O atoms in ATP N than in HRP C, it is suggested that ATP N will lose haem more easily than HRP C . Unlike almost all other class III plant peroxidases, ATP N has a free cysteine residue at a similar position to the suggested secondary substrate-binding site in lignin peroxidase.

Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 359 - 62
Crystallization and preliminary X-ray analysis of the catalytic domain of the adenylate cyclase GRESAG4.1 from Trypanosoma brucei; Bieger B et al.; Adenylate cyclases (ACs) are involved in signal transduction by generating the second messenger, cAMP . In Trypanosoma brucei, 3', 5'-cyclic adenosine monophosphate (cAMP) controls the life cycle of this unicellular parasite . cAMP is generated by a class of adenylate cyclases which are either constitutively (GRESAG4.1-4.3) or transiently expressed (ESAG4) during the life cycle . Unlike mammalian ACs, the trypanosomal ACs have a simple topology comprising of a large extracellular region, a transmembrane helix and a cytosolic catalytic region . Two orthorhombic crystal forms of the catalytic AC domain of GRESAG4.1 (residues Ala884-Thr1132) were generated by the hanging-drop vapour-diffusion method . X-ray diffraction data from GRESAG4.1 crystals were collected at 1.9 A resolution using synchrotron radiation . Furthermore, two heavy-metal derivative data sets were collected from crystal form A; heavy-atom sites were subsequently located in difference Patterson maps.

Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 357 - 8
Crystallization and preliminary x-ray crystallographic analysis of NAD+-dependent DNA ligase from Thermus filiformis; Lee JY et al.; A highly thermostable DNA ligase from Thermus filiformis has been crystallized at room temperature using methoxypolyethylene glycol 5000 as a precipitant . The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 90.63, b = 117.80, c = 98 . 65 A, beta = 115.56 degrees . Two molecules of DNA ligase are present in the asymmetric unit, giving a crystal volume per protein mass (V(m)) of 3.1 A(3) Da(-1) and a solvent content of 61% . A native data set extending to 3.0 A resolution has been collected at 100 K using synchrotron X-rays.

Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 354 - 6
Crystallization and preliminary X-ray analysis of insect antifreeze protein from the beetle Tenebrio molitor; Liou YC et al.; Hyperactive antifreeze protein from the beetle Tenebrio molitor (TmAFP) was produced in Escherichia coli and purified by gel-permeation chromatography and HPLC . An iodinated derivative was prepared by incubating the 8.5 kDa TmAFP with N-iodosuccinimide . Native and iodinated TmAFP produced two different crystal forms when crystallized using the hanging-drop vapor-diffusion technique . Native crystals were rectangular plates that diffracted to approximately 2.5 A resolution . They were monoclinic and belonged to the space group P2(1), with unit-cell dimensions a = 38.4, b = 73.4, c = 59.3 A, beta = 97.0 degrees . Crystals of iodinated TmAFP formed elongated hexagons that allowed data to be collected to approximately 1.4 A . These crystals belonged to the space group P6(1) (or P6(5)), with unit-cell dimensions a = 73.85, b = 73.85, c = 53.15 A . There were two molecules per asymmetric unit, which corresponds to V(m) = 2.46 A(3) Da(-1) and 51% solvent content . A twofold non-crystallographic symmetry was evident from self-rotation calculations.

Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 348 - 50
Structural studies of MIP synthase; Stein AJ et al.; The conversion of glucose 6-phosphate to 1-L-myo-inositol 1--phosphate (MIP) by 1-L-myo-inositol 1-phosphate synthase (MIP synthase) is the first committed and rate-limiting step in the de novo biosynthesis of inositol in all eukaryotes . The importance of inositol-containing molecules both as membrane components and as critical second messenger signal-transduction species make the function and regulation of this enzyme important for a host of biologically important cellular functions including proliferation, neurostimulation, secretion and contraction . MIP synthase has been overexpressed in Esherichia coli and purified to homogeneity by chromatographic methods . Two crystal forms of MIP synthase were obtained by the hanging-drop vapor-diffusion method . Native data sets for both crystal forms were collected in-house on a Rigaku R-AXIS IIC imaging-plate detector . Crystal form I belongs to space group C2, with unit-cell parameters a = 153.0, b = 96.6, c = 122.6 A, beta = 126.4 degrees, and diffracts to 2.5 A resolution . Crystal form II belongs to space group P2(1), with unit-cell parameters a = 94.5, b = 186.2, c = 86.5 A, beta = 110.5 degrees, and diffracts to 2.9 A resolution.

Gene, 2000 Mar 7, 245(1), 75 - 9
Cloning and characterization of cDNA encoding zebrafish Danio rerio NM23-B gene; Lee JS et al.; A full-length zebrafish NM23-B cDNA was cloned and sequenced . The zebrafish NM23-B cDNA consists of 624bp with an open reading frame of 153 amino acids . NM23-B mRNA of approximately 0.7kb is present in adult zebrafish tissues . Zebrafish NM23-B his-tagged protein (17kDa) was produced in E . coli and characterized by binding and UV-cross-linking to a single-stranded telomeric repeat (TTAGGG)(6) . This is the first report to show that fish have a NM23-H2 homologue that is similar to that in humans.

Gene, 2000 Mar 7, 245(1), 59 - 63
A T-extended vector using a green fluorescent protein as an indicator; Ito Y et al.; T-extended vector (T-vector) is a useful tool for cloning PCR products directly . We exploited a novel T-vector using a green fluorescent protein (GFP) as an indicator based on insertional inactivation . The brightest GFP mutant was used for easy detection even under daylight . The 100bp and 0.9kb of PCR products were cloned, and the transformant colonies with inserts were adjudged by the fluorescent green-white screening . The GFP system was more sensitive to insertional inactivation than the beta-galactosidase system at the conventional insertion sites.

FEMS Microbiol Lett, 2000 Mar 15, 184(2), 231 - 5
Evidence for the transport of zinc(II) ions via the pit inorganic phosphate transport system in Escherichia coli; Beard SJ et al.; A locus involved in zinc(II) uptake in Escherichia coli K-12 was identified through the generation of a zinc(II)-resistant mutant by transposon (Tn10dCam) mutagenesis . The mutation was located within the pitA gene, which encodes the low-affinity inorganic phosphate transport system (Pit) . The pitA mutant accumulated reduced amounts of zinc(II) when exposed to 0.5-2.0 mM ZnSO(4) during growth in Luria-Bertani medium.

FEMS Microbiol Lett, 2000 Mar 15, 184(2), 225 - 9
Identification of iron-regulated genes of Helicobacter pylori by a modified fur titration assay (FURTA-Hp); Fassbinder F et al.; The Escherichia coli-based Fur titration assay (FURTA), although a powerful tool for identification of genes regulated by the ferric uptake regulator (Fur), was unsuccessful for the gastric pathogen Helicobacter pylori . The FURTA was modified by construction of an E . coli indicator strain producing H . pylori Fur only . The promoter regions of the ferric citrate receptor homolog fecA2 and the riboflavin synthesis gene ribBA were both positive in the modified FURTA, but negative in the original FURTA . Transcription of fecA2 and ribBA was demonstrated to be iron-repressed in H . pylori . This type of modification should allow FURTA analysis for bacteria with Fur binding sequences poorly recognized by E . coli Fur.

FEMS Microbiol Lett, 2000 Mar 15, 184(2), 165 - 71
Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli; Kitagawa M et al.; To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain . The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress . However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain . The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB . These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress.

J Biol Chem, 2000 Mar 17, 275(11), 8213 - 9
Stoichiometry of P1 plasmid partition complexes; Bouet JY et al.; The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site . This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS . We have investigated the assembly of ParB at parS and its stoichiometry of binding . Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1 . We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex . Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex . Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB . Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS . Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.

J Biol Chem, 2000 Mar 17, 275(11), 8196 - 205
Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function; Xu L et al.; Escherichia coli strains lacking PriA are severely compromised in their ability to repair UV-damaged DNA and to perform homologous recombination . These phenotypes arise because of a lack of PriA-directed replication fork assembly at recombination intermediates such as D-loops . Naturally arising suppressor mutations in dnaC restore strains carrying the priA2::kan null allele to wild-type function . We have cloned one such gene, dnaC810, and overexpressed, purified, and characterized the DnaC810 protein . DnaC810 can support a PriA-independent synthesis of phiX174 complementary strand DNA . This can be attributed to its ability, unlike wild-type DnaC, to catalyze a SSB-insensitive general priming reaction with DnaB and DnaG on any SSB-coated single-stranded DNA . Gel mobility shift analysis revealed that DnaC810 could load DnaB directly to SSB-coated single-stranded DNA as well as to D loop DNA . This explains the ability of DnaC810 to bypass the requirement for PriA, PriB, PriC, and DnaT during replication fork assembly at recombination intermediates.

J Biol Chem, 2000 Mar 17, 275(11), 8103 - 13
Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli; Zechiedrich EL et al.; DNA supercoiling is essential for bacterial cell survival . We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli . Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (sigma) of -0.015 at an initial rate of 0.8 links min(-1) . Topoisomerase I relaxed DNA at a faster rate, 5 links min(-1), but only to a sigma of -0.05 . Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to sigma = -0.08) . The role of topoisomerase IV was revealed by two functional assays . Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (sigma = -0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by lambda integrase site-specific recombination.

J Biol Chem, 2000 Mar 17, 275(11), 8097 - 102
In the human malaria parasite Plasmodium falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase; Muller S et al.; The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation . Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections . Usually both enzymes are individually transcribed and highly regulated as monofunctional proteins . We have isolated a cDNA from the malaria parasite Plasmodium falciparum that encodes both proteins on a single open reading frame, with the AdoMetDC domain in the N-terminal region connected to a C-terminal ODC domain by a hinge region . The predicted molecular mass of the entire transcript is 166 kDa . The ODC/AdoMetDC coding region was subcloned into the expression vector pASK IBA3 and transformed into the AdoMetDC- and ODC-deficient Escherichia coli cell line EWH331 . The resulting recombinant protein exhibited both AdoMetDC and ODC activity and co-eluted after gel filtration on Superdex S-200 at approximately 333 kDa, which is in good agreement with the molecular mass of approximately 326 kDa determined for the native protein from isolated P . falciparum . SDS-polyacrylamide gel electrophoresis analysis of the recombinant ODC/AdoMetDC revealed a heterotetrameric structure of the active enzyme indicating processing of the AdoMetDC domain . The data presented describe the occurrence of a unique bifunctional ODC/AdoMetDC in P . falciparum, an organization which is possibly exploitable for the design of new antimalarial drugs.

J Biol Chem, 2000 Mar 17, 275(11), 8044 - 50
The role of ATP binding and hydrolysis by UvrB during nucleotide excision repair; Moolenaar GF et al.; We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads . With this method the UvrB-DNA preincision complex remains stable even in the absence of ATP . For the binding of UvrC to the UvrB-DNA complex no cofactor is needed . The subsequent induction of 3' incision does require ATP binding by UvrB but not hydrolysis . This ATP binding induces a conformational change in the DNA, resulting in the appearance of the DNase I-hypersensitive site at the 5' side of the damage . In contrast, the 5' incision is not dependent on ATP binding because it occurs with the same efficiency with ADP . We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule . A DNA substrate containing damage close to the 5' end of the damaged strand is specifically bound by UvrB in the absence of UvrA and ATP (Moolenaar, G . F., Monaco, V., van der Marel, G . A., van Boom, J . H., Visse, R., and Goosen, N . (2000) J . Biol . Chem . 275, 8038-8043) . To initiate the formation of an active UvrBC-DNA incision complex, however, UvrB first needs to hydrolyze ATP, and subsequently a new ATP molecule must be bound . Implications of these findings for the mechanism of the UvrA-mediated formation of the UvrB-DNA preincision complex will be discussed.

J Biol Chem, 2000 Mar 17, 275(11), 8038 - 43
The effect of the DNA flanking the lesion on formation of the UvrB-DNA preincision complex . Mechanism for the UvrA-mediated loading of UvrB onto a DNA damaged site; Moolenaar GF et al.; The UvrB-DNA preincision complex plays a key role in nucleotide excision repair in Escherichia coli . To study the formation of this complex, derivatives of a DNA substrate containing a cholesterol adduct were constructed . Introduction of a single strand nick into either the top or the bottom strand at the 3' side of the adduct stabilized the UvrB-DNA complex, most likely by the release of local stress in the DNA . Removal of both DNA strands up to the 3' incision site still allowed formation of the preincision complex . Similar modifications at the 5' side of the damage, however, gave different results . The introduction of a single strand nick at the 5' incision site completely abolished the UvrA-mediated formation of the UvrB-DNA complex . Deletion of both DNA strands up to the 5' incision site also prevented the UvrA-mediated loading of UvrB onto the damaged site, but UvrB by itself could bind very efficiently . This demonstrates that the UvrB protein is capable of recognizing damage without the matchmaker function of the UvrA protein . Our results also indicate that the UvrA-mediated loading of the UvrB protein is an asymmetric process, which starts at the 5' side of the damage.

J Biol Chem, 2000 Mar 17, 275(11), 7958 - 63
Mitochondrial import and processing of wild type and type III mutant isovaleryl-CoA dehydrogenase; Volchenboum SL et al.; Isovaleric acidemia is a rare inborn error of metabolism caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD), a nucleus-encoded, homotetrameric, mitochondrial flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA . We have previously identified a nucleotide deletion in the gene for IVD in fibroblasts from a patient with isovaleric acidemia leading to a shift in reading frame and premature termination of translation . The mutant IVD precursor is imported and processed to mature size, but no active enzyme is detected in mutant fibroblasts or expressed in Escherichia coli . Examination of the crystal structure of human IVD reveals that the C terminus is involved in tetramer stability . In vitro mitochondrial import experiments show that wild type IVD protein rapidly and stably forms mature homotetramer following import, whereas Type III mutant protein never forms stable oligomers . An additional series of mutant proteins with truncations and/or alterations in the C-terminal sequence implicates the C terminus of IVD in both enzyme activity and tetramer stability . Importantly, a dimeric intermediate in the folding pathway for wild type IVD has been identified in the in vitro mitochondrial import experiments, the first report of such an intermediate in the biogenesis of an acyl-CoA dehydrogenase.

J Biol Chem, 2000 Mar 17, 275(11), 7779 - 86
Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli; Silberg JJ et al.; Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis . To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides . Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones . The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C . ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0 . 1 s(-1)) . Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)) . ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)) . These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange . Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.

J Biol Chem, 2000 Mar 17, 275(11), 7708 - 12
Structural studies of lacUV5-RNA polymerase interactions in vitro . Ethylation interference and missing nucleoside analysis; Noel RJ Jr et al.; Substantial effort has been made to investigate the interactions that the Escherichia coli RNA polymerase makes with promoter DNA during transcription initiation . The lacUV5 promoter has been the object of many of these studies, and to date, an incredible wealth of information exists on how RNA polymerase interacts with this promoter . We have sought to expand current knowledge by the use of two chemical interference protocols, phosphate ethylation and missing nucleoside . We have added to existing information with the identification of additional phosphates, for example, at the start site of the template strand that, when ethylated, perturb the binding of RNA polymerase . We have also discovered a number of positions, most remarkably -37 to -34 of the nontemplate strand, where nucleoside loss decreases binding . Finally, we have discovered positions of ethylation and/or nucleoside loss that can stimulate binding . In particular, missing nucleosides and phosphate ethylation near the transcription start site enhance RNA polymerase binding.

J Biol Chem, 2000 Mar 17, 275(11), 7547 - 52
Identification and functional characterization of thioredoxin from Trypanosoma brucei brucei; Reckenfelderbaumer N et al.; Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead . The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system . As presented here, this is not the case . A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness . The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite . The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major . The relationship to other thioredoxins is low . T . brucei thioredoxin is unusual in having a calculated pI value of 8.5 . The gene has been overexpressed in Escherichia coli . The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase . T . brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T . brucei ribonucleotide reductase . The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.

Chem Biol, 2000 Mar, 7(3), 185 - 96
Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates; Jarret C et al.; BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen . Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product . According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first . Two different mechanisms for the first bond-forming step between the two substrates have been proposed . The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion . RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site . These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors . Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior . CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction . Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.

Mol Phylogenet Evol, 2000 Mar, 14(3), 342 - 52
The distribution of group I introns in lichen algae suggests that lichenization facilitates intron lateral transfer; Friedl T et al.; The nuclear-encoded small subunit ribosomal DNA gene of many lichen-forming green algae in the genus Trebouxia contains a group I intron at Escherichia coli genic position 1512 . We studied the evolutionary history of the 1512 intron in Trebouxia spp . (Trebouxiophyceae) by analyzing intron and "host" cell phylogenies . The host trees were constructed by comparing internal transcribed spacer regions of rDNA . Maximum-likelihood, maximum-parsimony, and distance analyses suggest that the 1512 intron was present in the common ancestor of the green algal classes Trebouxiophyceae, Chlorophyceae, and Ulvophyceae . The 1512 intron, however, was laterally transferred at least three times among later-diverging Trebouxia spp . that form lichen partnerships . Intron secondary structure analyses are consistent with this result . Our results support the hypothesis that lichenization may facilitate 1512 group I intron lateral transfer through the close cell-to-cell contact that occurs between the lichen algal and fungal symbionts in the developing lichen thallus .

Mol Cell Biol, 2000 Apr, 20(7), 2343 - 9
Modulation of DNA binding protein affinity directly affects target site demethylation; Lin IG et al.; It has recently been shown that in Xenopus, DNA demethylation at promoter regions may involve protein-DNA interactions, based on the specificity of the demethylated sites . Utilizing a stable episomal system in human cells, we recently mapped the sites and dissected the steps of demethylation at oriP sites bound by EBNA1 protein . Although it is clear that protein binding is required for demethylation of the oriP sites, it is uncertain whether this is a unique feature of the replication origin or whether it is a general phenomenon for all DNA sequences to which sequence-specific proteins are bound . In the present study, we utilize the well-defined Escherichia coli lac repressor/operator system in human cells to determine whether protein binding to methylated DNA, in a region that is neither a replication origin nor a promoter, can also lead to demethylation of the binding sites . We found that demethylation specified by protein binding is not unique to the replication origin or to the promoter . We also found that transcriptional activity does not influence demethylation of the lac operator . Isopropyl-beta-D-thiogalactopyranoside (IPTG), an inhibitor of the lac repressor, can prevent demethylation of the lac operator DNA sites and can modulate demethylation of the lac operator by affecting the binding affinity of the lac repressor . Using this system, a titration of protein binding can be done . This titration permits one to infer that protein binding site occupancy is the determinant of demethylation at DNA sites and permits a determination of how this process progresses over time.

Mutat Res, 2000 Feb 16, 459(1), 81 - 7
Dominant sensitization variants of human O(6)-methylguanine-DNA-methyltransferase obtained by a mutational screen of surface residues; Brown LR et al.; A scanning mutagenesis experiment was performed on human O(6)-methylguanine methyltransferase (hMGMT), directed largely at non-conserved surface residues that have not previously been studied . Variants typically contained two or more substitutions . Two of the 16 variants characterized in detail are inactive for methyltransfer, but increase the cytotoxicity and mutagenic effects of methylating agents . This phenotype is reminiscent of a variant (C145A) that has a mutation in the methyl-accepting cysteine . C145A is inactive, but reportedly binds methylated DNA and confers sensitivity to methylating agents . The sensitization phenotype of the two new variants is more striking in strains that are wild-type for DNA repair than in strains that are deficient for repair, suggesting that these proteins inhibit functional DNA repair proteins by competitively binding to methylated DNA . Both variants have multiple substitutions in the last helix of the protein . These results suggest that the C-terminal helix is necessary for methyltransfer activity, but not for methylguanine-specific binding.

Zhonghua Gan Zang Bing Za Zhi, 2000 Feb, 8(1), 33 - 4
{Transfection and expression of HCV-NS(5)B gene in Huh-7 cells}; Fang J et al.; OBJECTIVE: Hepatitis C virus (HCV) is the major cause of blood-borne non-A, non-B hepatitis . An RNA-dependent RNA polymerase is encoded by HCV non-structural protein 5B (NS(5)B) gene . A full-length HCV RNA has been transfected into human hepatoma cells, and HCV NS(5)B has been expressed in E.Coli purified . But human hepatocyte line with high expression of NS(5)B has not been established yet . METHODS: The NS(5)B gene has been transfected into Huh-7 cells by Lipofectamine . The results of transfection were confirmed by PCR and Southern blot analysis, and the level of the NS(5)B protein in Huh-7 cells was detected by Western blot analysis . RESULTS: There were the NS(5)B sequence and the expression of HCV-RNA polymerase in Huh-7 cells transfected NS(5)B plasmids . CONCLUSIONS: We have established a HCV RNA polymerase expression system in Huh-7 cells that can be used to study the mechanism of HCV replication and to test gene therapy of hepatitis C.

Zhonghua Gan Zang Bing Za Zhi, 2000 Feb, 8(1), 18 - 20
{Expression and immunological reactivity of recombinant HCV-core protein}; Dai W et al.; OBJECTIVE: To express HCV-core proteins in E.coli and to develop effective HCV-core DNA-based vaccine . METHODS: The vector that expresses the highly conserved HCV core genes were constructed . The pGEX-3X HCVCore constructs contained the 1-201 ncls (1-67aa, C201), 1-402 ncls (1-134aa, C402) and 1-591ncls ( 1-197aa, C591), then expressed in E.coli cells . RESULTS: The products of HCV C201 and C402 genes were expressed as a fusion protein with glutathione-S-transferase (GST, 26kDa) whose molecular weight were 3.1 x 10(4) and 3.9 x 10(4) separately . C591 gene was not effectively expressed in E.coli . The expressed proteins were sequestered within inclusion bodies (IB) and a variety of procedures designed to minimize IB formation proved unsuccessful . The method finally adopted involved the purification of inclusion bodies followed by the solubilization, purification, and refolding of the expressed protein . The purified C402 protein was antigenically reactive with serum from chronically infected HCV patients . BALB/C mice were immunized by a subcutaneous injection of C402 protein together with Freund's complete adjuvant which produced strong anti-HCV core humoral immune responses . CONCLUSION: It is important for the study of gene vaccine to construct a certain length of HCV core gene.

Biotechnol Bioeng, 2000 Apr 20, 68(2), 218 - 30
Synthesis of bioprocesses using physical properties data; Steffens MA et al.; The aim of this article is to illustrate and evaluate a synthesis procedure which has been extended to tackle bioprocesses . Physical property information is used to screen candidate units thereby reducing the size of the synthesis problem . In this way, only units which exploit large property differences between components in a stream are selected . This is important for bioprocesses because of the large number of components and wide range of unit operations which are available . The screening technique and bioprocess-unit-design methodologies have been incorporated within an implicit enumeration algorithm which was developed for chemical process synthesis and is implemented in Java programming language . An important advantage is the ability of the bioprocess synthesis software to generate a ranked list of flowsheets which may subsequently be analyzed in more detail . Two case studies are used to evaluate the bioprocess-synthesis technique . The first system involves a product which is secreted from the host organism . The second has significantly different characteristics in that the product is intracellular and forms inclusion bodies . The latter case study, in particular, is a large synthesis problem with 12 unit operations and 20 contaminant compounds . The results show that the synthesis methodology identifies a set of economically optimal flowsheets in a reasonable computational time which demonstrates its ability to deal with large synthesis problems . Using the synthesis methodology we can generate bioprocesses which are optimal in a system-wide, rather than unit-by-unit, sense .

Biotechnol Bioeng, 2000 Apr 20, 68(2), 136 - 41
Production of Sm37-GAPDH, a major therapeutical target in human schistosomiasis; Argiro L et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic metabolism and the production of energy . This probably explains why GAPDH was evidenced as a major therapeutical target in several parasitic diseases; either as a vaccine candidate or as a target for chemotherapeutic treatments . Schistosoma mansoni GAPDH (Sm37-GAPDH) is one of the main schistosome vaccine candidates . The production of recombinant Sm37-GAPDH is essential to evaluate the ability of this molecule to induce protective immunity in animals and possibly in humans . The cDNA encoding Sm37-GAPDH has been cloned and sequenced . In addition, five B cell (including the major B-cell epitope Sm35-5) and two T cell epitopes have been localized on the molecule . Different expression systems have been evaluated in respect with the production yield and the GAPDH enzymatic activity . Some of them have led to either a high production of insoluble material (E . coli) or to an inactive enzyme (Pischia pastoris) . The present article describes the production setting of rSm37-GAPDH using the baculovirus-insect cell system . Large amounts of soluble rSm37-GAPDH with enzymatic activity were obtained . Most sera from individuals living in an area endemic for S . mansoni recognised the rSm37 molecule and inhibited its catalytic activity .

Mol Microbiol, 2000 Mar, 35(5), 1235 - 43
Rapid inactivation of the Escherichia coli Kdp K+ uptake system by high potassium concentrations; Roe AJ et al.; The Kdp K+ uptake system of Escherichia coli is induced by limitation for K+ and/or high osmolarity . In the present study, the regulation of the activity of the Kdp system has been investigated in E . coli mutants possessing only the Kdp system as the mechanism of K+ accumulation . Cells grown in the presence of low K+ (0.1-1 mM) exhibit normal growth . However, growth inhibition results from exposure of cells to moderate levels of external K+ (> 5 mM) . Measurement of the cytoplasmic pH, of K+ pools and of transport via the Kdp system demonstrates that the Kdp system is rapidly and irreversibly inhibited by moderate external K+ . Concentrations of K+ greater than 2 mM are sufficient to cause inhibition of Kdp . At pH 6, this results in rapid lowering of the capacity for pH homeostasis, but at pH 7 the intracellular pH is unaffected . Parallel analysis of the expression of the Kdp system in a Kdp+/kdpFABC-lacZ strain shows that levels of K+ that are sufficient to inhibit Kdp activity also repress expression . As a result, growth inhibition of strains solely possessing Kdp arises jointly from inhibition of Kdp activity and repression of Kdp gene expression . These data identify an important aspect of the regulation of potassium transport via the Kdp system and also provide support for a model of regulation of Kdp expression via at least two mechanisms: sensing of both turgor and external K+ concentration.

Mol Microbiol, 2000 Mar, 35(5), 1202 - 10
Two types of cold sensitivity associated with the A184-->V change in the DnaA protein; Nyborg M et al.; Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation . The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation . The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid . Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid . In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain . The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings . Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently . The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth . The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift . However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication . Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature . The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement . In this case, the cold-sensitive phenotype may be due to DNA overproduction.

Mol Microbiol, 2000 Mar, 35(5), 1065 - 78
Identification of the Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter; Makui H et al.; The Escherichia coli mntH (formerly yfeP) gene encodes a putative membrane protein (MntH) highly similar to members of the eukaryotic Nramp family of divalent metal ion transporters . To determine the function of E . coli MntH, a null mutant was created and MntH was overexpressed both in wild-type E . coli and in the metal-dependent mutant hflB1(Ts) . At the restrictive temperature 42 degrees C, the mntH null mutation reduces the suppression of hflB1(Ts) thermosensitivity by exogenous divalent metals . Conversely, overexpression of MntH restores growth at 42 degrees C, increases suppression of the ts phenotype by Fe(II) and Ni(II) and renders hflB1(Ts) cells hypersensitive to Mn(II) . Transport studies in intact cells show that MntH selectively facilitates uptake of 54Mn(II) and 55Fe(II) in a temperature-, time- and proton-dependent manner . Competition studies in uptake assays and growth inhibition experiments in hflB1(Ts) mutants together indicate that MntH is a divalent metal cation transporter of broad substrate specificity . The functional characteristics of MntH suggest that it corresponds to the previously described manganese transporter of E . coli . This study indicates that proton-dependent divalent metal ion uptake has been preserved in the Nramp family from bacteria to humans.

Mol Microbiol, 2000 Mar, 35(5), 961 - 73
Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent; Brock M et al.; Aspergillus nidulans was used as a model organism to investigate the fungal propionate metabolism and the mechanism of growth inhibition by propionate . The fungus is able to grow slowly on propionate as sole carbon and energy source . Propionate is oxidized to pyruvate via the methylcitrate cycle . The key enzyme methylcitrate synthase was purified and the corresponding gene mcsA, which contains two introns, was cloned, sequenced and overexpressed in A . nidulans . The derived amino acid sequence of the enzyme shows more than 50% identity to those of most eukaryotic citrate synthases, but only 14% identity to the sequence of the recently detected bacterial methylcitrate synthase from Escherichia coli . A mcsA deletion strain was unable to grow on propionate . The inhibitory growth effect of propionate on glucose medium was enhanced in this strain, which led to the assumption that trapping of the available CoA as propionyl-CoA and/or the accumulating propionyl-CoA itself interferes with other biosynthetic pathways such as fatty acid and polyketide syntheses . In the wild-type strain, however, the predominant inhibitor may be methylcitrate . Propionate (100 mM) not only impaired hyphal growth of A . nidulans but also synthesis of the green polyketide-derived pigment of the conidia, whereas in the mutant pigmentation was abolished with 20 mM propionate.

Eur J Biochem, 2000 Mar, 267(6), 1858 - 68
Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases; Zgiby SM et al.; Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate . A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase {Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A . & Hunter, W.N . (1999) J . Mol . Biol . 287, 383-394} in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism . The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate . From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis . We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate . In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role . Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates . Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation . Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme . In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E . coli was cloned, expressed and kinetically characterized . Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold . This produces an overall discrimination factor of almost 5 x 105 between the two enzymes . Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site . These residues might potentially be important in substrate recognition . As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated . Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed . This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.

Eur J Biochem, 2000 Mar, 267(6), 1777 - 83
Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis; Nayak SK et al.; Restrictocin, produced by the fungus Aspergillus restrictus, is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA . It is a nonglycosylated, single-chain, basic protein of 149 amino acids . The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136 . To map the catalytic activity in the restrictocin molecule, and to study the role of N- and C-terminus in its activity, we have systematically deleted amino-acid residues from both the termini . Three N-terminal deletions removing 8, 15 and 30 amino acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids were constructed . The deletion mutants were expressed in Escherichia coli, purified to homogeneity and functionally characterized . Removal of eight N-terminal or four C-terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin . The study demonstrates that intact N- and C-termini are required for the optimum functional activity of restrictocin.

Eur J Biochem, 2000 Mar, 267(6), 1732 - 42
The role of amino-acid residues in the hydrophobic patch surrounding the haem group of cytochrome f in the interaction with plastocyanin; Gong XS et al.; Soluble turnip cytochrome f has been purified from the periplasmic fraction of Escherichia coli expressing a truncated petA gene encoding the precursor protein lacking the C-terminal 33 amino-acid residues . The protein is identical {as judged by 1H-NMR spectroscopy, midpoint redox potential (+ 365 mV) and electron transfer reactions with plastocyanin} to cytochrome f purified from turnip leaves . Several residues in the hydrophobic patch surrounding the haem group have been changed by site-directed mutagenesis, and the proteins purified from E . coli . The Y1F and Q7N mutants showed only minor changes in the plastocyanin-binding constant Ka and the second-order rate constant for electron transfer to plastocyanin, whereas the Y160S mutant showed a 30% decrease in the overall rate of electron transfer caused in part by a 60% decrease in binding constant and partially compensated by an increased driving force due to a 27-mV decrease in redox potential . In contrast, the F4Y mutant showed increased rates of electron transfer which may be ascribed to an increased binding constant and a 14-mV decrease in midpoint redox potential . This indicates that subtle changes in the hydrophobic patch can influence rates of electron transfer to plastocyanin by changing the binding constants and altering the midpoint redox potential of the cytochrome haem group.

Eur J Biochem, 2000 Mar, 267(6), 1723 - 31
Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli; Avliyakulov NK et al.; Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity . In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro . L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe . The cloned gene was overexpressed and purified as a his-tag fusion protein in E . coli . Radioligand binding experiments, using {gamma-35S}ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50-100 nM), while it has no ATPase activity . Furthermore, we showed that L3 has more than 500-fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates . Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N-terminal 296 residues . Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of {gamma-35S}ATP at subclinical levels . Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and binds strongly to L3 in nearly stoichiometric amounts.

Eur J Biochem, 2000 Mar, 267(6), 1565 - 70
Quantitative analysis of gene expression with an improved green fluorescent protein . p6; Scholz O et al.; The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities . We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+ . Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli . The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo . However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity . This effect is probably due to the higher absorbance of cells containing GFP+ . Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene . Possibilities of using GFP variants beyond this range are discussed.

Eur J Biochem, 2000 Mar, 267(6), 1533 - 49
New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system; van de Werve G et al.; The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46) . Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm . P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin . P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36 . Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter . Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively . Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion . The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined . The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.

Plant Physiol, 2000 Mar, 122(3), 887 - 94
Analysis of reductant supply systems for ferredoxin-dependent sulfite reductase in photosynthetic and nonphotosynthetic organs of maize; Yonekura-Sakakibara K et al.; Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide in chloroplasts and root plastids using ferredoxin (Fd) as an electron donor . Using purified maize (Zea mays L.) SiR and isoproteins of Fd and Fd-NADP(+) reductase (FNR), we reconstituted illuminated thylakoid membrane- and NADPH-dependent sulfite reduction systems . Fd I and L-FNR were distributed in leaves and Fd III and R-FNR in roots . The stromal concentrations of SiR and Fd I were estimated at 1.2 and 37 microM, respectively . The molar ratio of Fd III to SiR in root plastids was approximately 3:1 . Photoreduced Fd I and Fd III showed a comparable ability to donate electrons to SiR . In contrast, when being reduced with NADPH via FNRs, Fd III showed a several-fold higher activity than Fd I . Fd III and R-FNR showed the highest rate of sulfite reduction among all combinations tested . NADP(+) decreased the rate of sulfite reduction in a dose-dependent manner . These results demonstrate that the participation of Fd III and high NADPH/NADP(+) ratio are crucial for non-photosynthetic sulfite reduction . In accordance with this view, a cysteine-auxotrophic Escherichia coli mutant defective for NADPH-dependent SiR was rescued by co-expression of maize SiR with Fd III but not with Fd I.

Plant Physiol, 2000 Mar, 122(3), 635 - 44
Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed; Metz JG et al.; The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve . This is in contrast to the triglycerides found in seeds of other plants . We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme . Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols . The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development . When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils . Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production . In addition to free alcohols, novel wax esters were detected in the transgenic seed oils . In vitro assays revealed that B . napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis . Thus, introduction of a single cDNA into B . napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.

Am J Respir Crit Care Med, 2000 Mar, 161(3 Pt 1), 982 - 9
Endothelin receptor blockade attenuates lipopolysaccharide-induced pulmonary nitric oxide production; Fujii Y et al.; Increased nitric oxide (NO) synthesis by the inducible nitric oxide synthase (iNOS) has been shown to contribute to the development of acute lung injury and delayed hypotension in animals injected with bacterial lipopolysaccharides (LPS) . Recent evidence indicates that endothelin-1 (ET-1) is also elevated in septic humans and in animals . To assess the contribution of ETs to LPS-induced pulmonary NO production and iNOS expression, we used P1/fl, a 22 amino acid peptide, to selectively antagonize endothelin-A receptors . Anesthetized, mechanically ventilated rats were injected with either saline or LPS (E . coli endotoxin, 20 mg/kg) and studied for 5 h . Two other groups of rats were pretreated 15 min earlier with P1/fl peptide (20 microg/kg) . Unlike saline-treated rats, rats injected with LPS showed a progressive decline in arterial pressure and a significant rise in plasma ET concentration and serum nitrite-nitrate level . In the lungs, LPS injection elicited a several-fold rise in lung iNOS activity and exhaled NO concentration and increased lung wet/dry ratio significantly . Pretreatment with P1/fl peptide eliminated the decline in arterial pressure, the rise in lung wet/dry ratio, lung NOS activity, and iNOS protein expression and significantly attenuated the increase in pulmonary exhaled NO production but had no effect on plasma ET concentration . We conclude that activation of ET-A receptors by rising ET-1 concentration enhances NO production and iNOS expression in the respiratory and vascular systems and contributes to both LPS-induced hypotension and acute lung injury.

Microbiol Immunol, 2000, 44(1), 1 - 7
Genetic and biochemical properties of a hemolysin (pyolysin) produced by a swine isolate of Arcanobacterium (Actinomyces) pyogenes; Ikegami M et al.; Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor . This hemolysin was purified from the culture supernatant of A . pyogenes swine isolate . The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis, and its isoelectric point was 9.2 . The activity of this hemolysin was not enhanced by the addition of L-cysteine or sodium thioglycolate, but it was inhibited by cholesterol . The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coli by means of ZAP Express vector . Analysis by SDS-polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E . coli is the same as that of the hemolysin purified from A . pyogenes . Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da . The nucleotide sequence of the hemolysin gene from A . pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A . pyogenes strain BBR1 reported by Billington et al (J . Bacteriol . 179: 6100-6106, 1997) . The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol-activated cytolysins (cholesterol-binding cytolysins), and is replaced with alanine . Therefore, the hemolysin of A . pyogenes seems to be a novel member of the thiol-activated cytolysin family.

Obstet Gynecol, 2000 Mar, 95(3), 453 - 6
Preterm delivery in mice with renal abscess; Mussalli GM et al.; OBJECTIVE: Our purpose was to develop a mouse model of renal abscess to study the effect of extrauterine infection on preterm delivery . METHODS: Escherichia coli or sterile medium was injected into the left kidney of 70 pregnant mice that had completed approximately 75% of gestation . Preterm delivery rates were recorded for various inocula . Kidney specimens were obtained and examined grossly and histologically for abscess formation . RESULTS: Thirty-one of 51 animals (60.8%) infected with 1 x 10(5)-9 x 10(6) bacteria and none of 19 uninfected animals delivered prematurely (P < .001) . Renal abscess was induced in 100% of mice receiving bacterial inoculation but in none receiving sterile medium . CONCLUSION: Kidney injection provides a reliable method for inducing renal abscess in pregnant mice . Renal abscess induces preterm delivery at a stable rate across a wide range of bacterial inocula . This model of extrauterine infection may be particularly useful in investigations of infection-induced preterm delivery.

Dig Dis Sci, 2000 Feb, 45(2), 230 - 6
Effect of bile and pancreatic juice on adenoviral-mediated gene delivery: implications on the feasibility of gene delivery through ERCP; Xie X et al.; Current research in gene delivery to the liver is focused on the intravenous, intraarterial, intraportal, or intratumoral route . Another possible route for gene delivery is via the common bile duct through endoscopic retrograde cholangiopancreatography (ERCP) . Whether bile and pancreatic juice have any effect on gene delivery is not established . To evaluate the effect of bile and pancreatic juice on adenoviral-mediated gene delivery, liver and pancreatic cell lines were infected with a recombinant adenovirus expressing an E . coli beta-galactosidase gene under the control of a cytomegalovirus promoter (rAdCMVpLacZ) in the absence or presence of various concentrations of bile and pancreatic juice . The proportion of cells infected was evaluated through X-gal staining . The toxicity of bile and pancreatic juice was also evaluated through cell morphology and detachment . Bile appeared to induce significant cytotoxicity in HepG2 and Huh7 cells (50% viability with 15 min of incubation) . Neither bile nor pancreatic juice affected transgene expression . In the absence of bile/pancreatic juice, HepG2 (15-25%) and PANC-1 cells (10-18%) were less susceptible to rAdCMVpLacZ compared to Huh7 cells (75-84%, vs HepG2, P < 0.001) and BxPc-3 (82-95%, vs PANC-1, P < 0.001) at a multiplicity of infection (MOI) of 5 . Bile reduced the transduction efficiency, but 5-10% HepG2 and 5-42% of Huh7 cells were still transduced in the presence of 80% bile for up to 10 min . Adenoviral-mediated gene delivery was reduced in the presence of pancreatic juice with a low multiplicity of infection (MOI of 5), but this effect was negated with an MOI of 50 . These data provide encouragement to develop adenoviral-mediated gene delivery through ERCP.

Genetica, 1999, 106(1-2), 141 - 7
Exploring structure space . A protein structure initiative; Terwilliger TC et al.; The genome projects are changing biology by providing the genetic blueprints of entire organisms . The blueprints are tantalizing but we cannot deduce everything we need to know from them, including the structures and detailed functions of proteins . In this paper we describe an approach for obtaining structural information about proteins on a genomic scale . We describe how structural and functional information might eventually be put together to form a basis for describing life at many levels . We then describe how structural information fits into this picture and classes of proteins for which structural information would be useful in a genomic context . We conclude with a proposal for an initiative to determine protein structures on a very large scale.

Genetica, 1999, 106(1-2), 85 - 92
Single-chain 434 repressors with altered DNA-binding specificities . Isolation of mutant single-chain repressors by phenotypic screening of combinatorial mutant libraries; Simoncsits A et al.; Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities . Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker . The mutant domain contains randomized amino acids in place of the DNA-contacting residues . The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA--6 base-pairs--NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain . An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator . Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence . Some of the isolated mutants bound with very high affinities (10-50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target.

Genetica, 1999, 106(1-2), 49 - 55
Superhelical DNA studied by solution scattering and computer models; Langowski J et al.; We present here recent results on the structure of superhelical DNA and its changes with salt concentration between 0.01 and 1.5 M NaCl . Scattering curves of two different superhelical DNAs were determined by static light scattering . The measured radii of gyration do not change significantly with salt concentration . Small-angle neutron scattering, together with calculations from a Monte Carlo model, allows to determine the superhelix diameter . Measured and simulated scattering curves agreed almost quantitatively . Experimentally we find that the diameter decreases from 16.0 +/- 0.9 nm at 10 mM to 9.0 +/- 0.7 nm at 100 mM NaCl . The superhelix diameter from the simulated conformations decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM NaCl . At higher salt concentrations up to 1.5 M NaCl, the diameter stays constant at 9 nm.

Mol Cell, 2000 Jan, 5(1), 1 - 11
The activation loop of phosphatidylinositol phosphate kinases determines signaling specificity; Kunz J et al.; Phosphatidylinositol-4,5-bisphosphate plays a pivotal role in the regulation of cell proliferation and survival, cytoskeletal reorganization, and membrane trafficking . However, little is known about the temporal and spatial regulation of its synthesis . Higher eukaryotic cells have the potential to use two distinct pathways for the generation of phosphatidylinositol-4,5-bisphosphate . These pathways require two classes of phosphatidylinositol phosphate kinases, termed type I and type II PIP kinases . While highly related by sequence, these kinases localize to different subcellular compartments, phosphorylate distinct substrates, and are functionally nonredundant . Here, we show that a 20- to 25-amino acid loop spanning the catalytic site, termed the activation loop, determines both enzymatic specificity and subcellular targeting of PIP kinases . Therefore, the activation loop controls signaling specificity and PIP kinase function at multiple levels.

J Biomol NMR, 1999 Nov, 15(3), 251 - 64
HYPER: a hierarchical algorithm for automatic determination of protein dihedral-angle constraints and stereospecific C beta H2 resonance assignments from NMR data; Tejero R et al.; A new computer program, HYPER, has been developed for automated analysis of protein dihedral angle values and C beta H2 stereospecific assignments from NMR data . HYPER uses a hierarchical grid-search algorithm to determine allowed values of phi, psi, and chi 1 dihedral angles and C beta H2 stereospecific assignments based on a set of NMR-derived distance and/or scalar-coupling constraints . Dihedral-angle constraints are valuable for restricting conformational space and improving convergence in three-dimensional structure calculations . HYPER computes the set of phi, psi, and chi 1 dihedral angles and C beta H2 stereospecific assignments that are consistent with up to nine intraresidue and sequential distance bounds, two pairs of relative distance bounds, thirteen homo- and heteronuclear scalar coupling bounds, and two pairs of relative scalar coupling constant bounds . The program is designed to be very flexible, and provides for simple user modification of Karplus equations and standard polypeptide geometries, allowing it to accommodate recent and future improved calibrations of Karplus curves . The C code has been optimized to execute rapidly (0.3-1.5 CPU-sec residue-1 using a 5 degrees grid) on Silicon Graphics R8000, R10000 and Intel Pentium CPUs, making it useful for interactive evaluation of inconsistent experimental constraints . The HYPER program has been tested for internal consistency and reliability using both simulated and real protein NMR data sets.

Appl Microbiol Biotechnol, 2000 Feb, 53(2), 209 - 18
Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli; Reiser SE et al.; An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity from Rhodospirillum rubrum . Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide . The gene encoding the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the gene in E . coli led to the purification of the enzyme to homogeneity . The purified enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3-hexenoyl-CoA with approximately equal specificity as substrates in the hydration reaction . However, no activity was observed using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration . Based on the nucleotide sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined by gel filtration chromatography and native PAGE . The hydratase was expressed together with the PHA synthase from Thiocapsa pfennigii in E . coli strain DH5alpha . Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) .

Appl Microbiol Biotechnol, 2000 Feb, 53(2), 201 - 8
Production characteristics of interferon-alpha using an L-arabinose promoter system in a high-cell-density culture; Lim HK et al.; Using high-cell-density culture of Escherichia coli under the control of an L-arabinose promoter (ParaB), several factors affecting the production of recombinant protein and the formation of inclusion bodies were studied . The inducer, L-arabinose, showed a maximal induction level above 10.7 mM in the final concentration . The concentration of inducer also affected the partition of interferon-alpha (IFN-alpha) into the soluble form and inclusion bodies . Induction kinetics of the rate of accumulation of IFN-alpha on the ParaB promoter showed a slower rate than those of other promoter systems, for example T7, lac or tac . These innate characteristics of ParaB enabled cells to grow continuously in spite of the metabolic burden induced by the expression of foreign protein . The duration time of induction could control the expression of both soluble and insoluble protein . The ratio of yeast extract to glycerol (N/C ratio) in feeding media significantly affected both the production level of recombinant protein and inclusion body formation . The reason for decreasing specific bioactivity during induction can be explained by the increased proportion of inclusion bodies in the total expressed IFN-alpha.

Mutat Res, 2000 Feb 16, 465(1-2), 39 - 44
Mutagenicity of benzo{a}pyrene-deoxyadenosine adducts in a sequence context derived from the p53 gene; Khalili H et al.; Mutations in the human p53 tumor suppressor gene are prominently linked to sporadic cancers in breast, lung and other tissues . Recent research has shown that tobacco-associated cancer in the human lung is related to mutation of the p53 gene mediated by the carcinogen benzo{a}pyrene (BaP), and the mutations are targeted to DNA "hot spots" at specific codons . In order to gain insight into the relation between the structures of the adducts formed by BaP at these sites and their mutagenic activities, we have synthesized site-specifically modified oligo-nucleotide adducts of the active BaP diol epoxide metabolite (anti-BaPDE) . This manuscript reports on the mutagenic consequences of replication past anti-BaPDE-deoxyadenosine adducts located within a sequence context related to codon 157 in exon 5 of the p53 gene . In this sequence context, the adduct derived from the carcinogenic 7R,8S-dihydrodiol 9S,10R-epoxide was much more active as a mutagen than the adduct derived from the noncarcinogenic 7S,8R-dihydrodiol 9R,10S-epoxide and the mutation found most frequently was an A-->G transition . Since previous studies in other sequence contexts have yielded somewhat different findings, these studies further emphasize the key role played by sequence context in determining the mutational properties of carcinogen-DNA adducts.

Biochim Biophys Acta, 2000 Mar 7, 1477(1-2), 157 - 67
Structure and function of the methionine aminopeptidases; Lowther WT et al.; The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP) . Substantial progress has recently been made in determining the structures of several members of this family . The identification of human MetAP as the target of putative anti-cancer drugs reiterates the importance of this family of enzymes . Determination of the modes of binding to E . coli MetAP of a substrate-like bestatin-based inhibitor, as well as phosphorus-containing transition-state analogs and reaction products has led to a rationalization of the substrate specificity and suggested the presumed catalytic mechanism . The conservation of key active site residues and ligand interactions between the MetAPs and other enzyme of the same fold suggest that avoidance of cross-reactivity may be an important consideration in the design of inhibitors directed toward a single member of the family.

FEBS Lett, 2000 Mar 3, 469(1), 105 - 10
Trimeric ring-like structure of ArsA ATPase; Wang HW et al.; ArsA protein is the soluble subunit of the Ars anion pump in the Escherichia coli membrane which extrudes arsenite or antimonite from the cytoplasm . The molecular weight of the subunit is 63 kDa . In the cell it hydrolyzes ATP, and the energy released is used by the membrane-bound subunit ArsB to transport the substrates across the membrane . We have obtained two-dimensional crystals of ArsA in the presence of arsenite on negatively-charged lipid monolayer composed of DMPS and DOPC . These crystals have been studied using electron microscopy of negatively-stained specimens followed by image processing . The projection map obtained at 2.4 nm resolution reveals a ring-like structure with threefold symmetry . Many molecular assemblies with the same ring-shape and dimensions were also seen dispersed on electron microscopy grids, prepared directly from purified ArsA protein solution . Size-exclusion chromatography of the protein sample with arsenite present revealed that the majority of the protein particles in solution have a molecular weight of about 180 kDa . Based on these experiments, we conclude that in solution the ArsA ATPase with substrate bound is mainly in a trimeric form.

FEBS Lett, 2000 Mar 3, 469(1), 9 - 13
Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon; Zhao KH et al.; The structure of phycoviolobilin, the photoactive chromophore of alpha-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a Delta3, 3(1)-double bond of the phycobilin . The two putative phycoerythrocyanin lyase genes of Mastigocladus laminosus, pecE and pecF, were overexpressed in Escherichia coli . Their action has been studied on the addition reaction of phycocyanobilin to apo-alpha-phycoerythrocyanin (PecA) . In the absence of the components of alpha-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (lambda(max)=641 nm) and low photoactivity (<10%) . In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (lambda(max)=565 nm) and high photoreversible photochromism (100% type I) has been identified as integral alpha-phycoerythrocyanin . We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore.

FEBS Lett, 2000 Mar 3, 469(1), 5 - 8
Lactoferrin inhibits the binding of lipopolysaccharides to L-selectin and subsequent production of reactive oxygen species by neutrophils; Baveye S et al.; The activation of leukocytes by lipopolysaccharides (LPS), resulting in the oxidative burst, contributes to the pathogenesis of septic shock . The binding of LPS to L-selectin, which was reported as a serum-independent LPS receptor on neutrophils, induces the production of oxygen free radicals . Human lactoferrin (hLf), an anti-inflammatory glycoprotein released from neutrophil granules during infection, binds to LPS . In this study, we investigated the capacity of hLf to inhibit the L-selectin-mediated activation of neutrophils . Our experiments revealed that hLf prevents the binding of LPS to L-selectin in a concentration-dependent manner . Inhibition was maximum (87.7+/-0.5%) at a concentration of 50 microg/ml of hLf . Furthermore, hLf inhibited up to 55.4+/-0.5% of the intracellular hydrogen peroxide production induced by LPS in neutrophils . These findings suggest that the anti-inflammatory properties of hLf are due, at least in part, to their ability to prevent the binding of LPS to neutrophil L-selectin.

Acta Trop, 2000 Mar 25, 75(2), 203 - 10
Plasticity of the histone H2A genes in a Brazilian and six Colombian strains of Trypanosoma cruzi; Thomas MC et al.; The analysis of three recombinant clones containing the histone H2A locus isolated from a genomic library of Trypanosoma cruzi DNA shows that the H2A gene loci are formed by 1.2 and 0.76 kb long intercalated units organized in a head-to-tail tandem array . The difference in length between the two gene units is due to the presence of a short interspersed nucleotide element (SINE)-like DNA sequence inserted at the 3' end of some of these units . Southern, northern and chromosomal blot analysis of a Brazilian Y strain and six Colombian strains demonstrated the existence of polymorphisms regarding the relative copy number of the H2A gene units, the relative abundance of the H2A transcripts and their chromosomal location . These results show the existence of a dynamic organization in the H2A loci among T . cruzi strains in which a SINE-like sequence may be involved and support the fact that T . cruzi has a high degree of plasticity in its genome.

Protein Eng, 2000 Feb, 13(2), 105 - 12
The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase; Jeffery CJ et al.; In previous kinetic studies of Escherichia coli aspartate aminotransferase, it was determined that some substitutions of conserved cysteine 191, which is located outside of the active site, altered the kinetic parameters of the enzyme (Gloss,L.M., Spencer,D . E . and Kirsch,J.F., 1996, Protein Struct . Funct . Genet., 24, 195-208) . The mutations resulted in an alkaline shift of 0.6-0.8 pH units for the pK(a) of the internal aldimine between the PLP cofactor and Lys258 . The change in the pK(a) affected the pH dependence of the k(cat)/K(m) (aspartate) values for the mutant enzymes . To help to understand these observations, crystal structures of five mutant forms of E.coli aspartate aminotransferase (the maleate complexes of C191S, C191F, C191Y and C191W, and C191S without maleate) were determined at about 2 A resolution in the presence of the pyridoxal phosphate cofactor . The overall three-dimensional fold of each mutant enzyme is the same as that of the wild-type protein, but there is a rotation of the mutated side chain around its C(alpha)-C(beta) bond . This side chain rotation results in a change in the pattern of hydrogen bonding connecting the mutant residue and the protonated Schiff base of the cofactor, which could account for the altered pK(a) of the Schiff base imine nitrogen that was reported previously . These results demonstrate how residues outside the active site can be important in helping determine the subtleties of the active site amino acid geometries and interactions and how mutations outside the active site can have effects on catalysis . In addition, these results help explain the surprising result previously reported that, for some mutant proteins, replacement of a buried cysteine with an aromatic side chain did not destabilize the protein fold . Instead, rotation around the C(alpha)-C(beta) bond allowed each large aromatic side chain to become buried in a nearby pocket without large changes in the enzyme's backbone geometry.

Biochem Biophys Res Commun, 2000 Mar 16, 269(2), 451 - 6
Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a {3Fe-4S}(+) cluster; Broderick JB et al.; Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E . coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-l-methionine (AdoMet) . Although PFL-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center . We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions . Iron and sulfide analysis as well as UV-visible, EPR, and resonance Raman data support the presence of a {3Fe-4S}(+) cluster in the purified enzyme . The isolated native enzyme, but not apo-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity .

Genomics, 2000 Feb 15, 64(1), 111 - 3
BAC trimming: minimizing clone overlaps; Hill F et al.; Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing . Long overlaps between some clones lead to considerable redundant effort . A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones . This procedure, called trimming, was carried out in the recA(-) BAC host . We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced . This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process . BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases .

Nucleic Acids Res . 2000 Apr 1;28(7):E24.
Phage display of ScFv peptides recognizing the thymidine(6-4)thymidine photoproduct; Zavala AG et al.; Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers . To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed . One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods . To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, {alpha}UVssDNA-1 (mAb C3B6), recognizing the thymidine(6-4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid . This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage . In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure-function studies and application to human investigations.

Nucleic Acids Res, 2000 Apr 1, 28(7), 1640 - 6
2-Hydroxy-dATP is incorporated opposite G by Escherichia coli DNA polymerase III resulting in high mutagenicity; Kamiya H et al.; Four kinds of oxidatively damaged DNA precursors, 8-hydroxydeoxyguanosine 5'-triphosphate (8-OH-dGTP), 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), 5-hydroxydeoxycytidine 5'-triphosphate (5-OH-dCTP) and 5-formyldeoxyuridine 5'-triphosphate (5-CHO-dUTP), were employed in in vitro gap-filling reactions of the supF gene conducted by the Escherichia coli DNA polymerase III holoenzyme, and these treated DNAs were transfected into various E.coli strains . When the manipulated DNAs were transfected into the repair-proficient strain, supF mutants were obtained much more frequently by the purine nucleotides than by the pyrimidine nucleotides (2-OH-dATP > 8-OH-dGTP >> 5-OH-dCTP approximately 5-CHO-dUTP) . This result is in contrast to our previous observation that these four oxidatively damaged nucleotides induce chromosomal gene mutations with similar frequencies when incorporated directly into E.coli . 2-OH-dATP elicited G-->T transversions, indicating the formation of G*2-OH-dATP pairs . These results demonstrate that 2-OH-dATP was highly mutagenic in this assay system containing the in vitro DNA synthesis by the E.coli replicative DNA polymerase, in addition to in the in vivo assay system reported previously . Slight increases in the mutant frequencies were observed when alkA (for 8-OH-dGTP and 2-OH-dATP) and mutY (for 2-OH-dATP) strains were used as hosts . This is the first report that clearly shows the formation of G*2-OH-dATP pairs.

Nucleic Acids Res, 2000 Apr 1, 28(7), 1555 - 63
In vitro DNA synthesis opposite oxazolone and repair of this DNA damage using modified oligonucleotides; Duarte V et al.; Emphasis was placed in this work on the assessment of biological features of 2,2,4-triaminooxazolone, a major one-electron and( . )OH-mediated oxidation product of guanine . For this purpose, two oligonucleotides that contain a unique oxazolone residue were synthesized . Herein we report the mutagenic potential of oxazolone during in vitro DNA synthesis and its behavior towards DNA repair enzymes . Nucleotide insertion opposite oxazolone, catalyzed by Klenow fragment exo(-)and Taq polymerase indicates that the oxazolone lesion induces mainly dAMP insertion . This suggests that the formation of oxazolone in DNA may lead to G-->T transversions . On the other hand, oxazolone represents a blocking lesion when DNA synthesis is performed with DNA polymerase beta . Interestingly, DNA repair experiments carried out with formamidopyrimidine DNA N -glycosylase (Fpg) and endonuclease III (endo III) show that oxazolone is a substrate for both enzymes . Values of k (cat)/ K (m)for the Fpg-mediated removal of oxidative guanine lesions revealed that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than oxazolone . In the case of endo III-mediated cleavage of modified bases, the present results suggest that oxazolone is a better substrate than 5-OHC, an oxidized pyrimidine base . Finally, MALDI-TOF-MS analysis of the DNA fragments released upon digestion of an oxazolone-containing oligonucleotide by Fpg gave insights into the enzymatic mechanism of oligonucleotide cleavage.

Nucleic Acids Res, 2000 Apr 1, 28(7), 1542 - 7
A missense mutation in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase suppresses a mitochondrial tRNA(Asp) mutation; Chiang CS et al.; The nuclear suppressor allele NSM3 in strain FF1210-6C/170-E22 (E22), which suppresses a mutation of the yeast mitochondrial tRNA(Asp)gene in Saccharomyces cerevisiae, was cloned and identified . To isolate the NSM3 allele, a genomic DNA library using the vector YEp13 was constructed from strain E22 . Nine YEp13 recombinant plasmids were isolated and shown to suppress the mutation in the mitochondrial tRNA(Asp)gene . These nine plasmids carry a common 4 . 5-kb chromosomal DNA fragment which contains an open reading frame coding for yeast mitochondrial aspartyl-tRNA synthetase (AspRS) on the basis of its sequence identity to the MSD1 gene . The comparison of NSM3 DNA sequences between the suppressor and the wild-type version, cloned from the parental strain FF1210-6C/170, revealed a G to A transition that causes the replacement of amino acid serine (AGU) by an asparagine (AAU) at position 388 . In experiments switching restriction fragments between the wild type and suppressor versions of the NSM3 gene, the rescue of respiratory deficiency was demonstrated only when the substitution was present in the construct . We conclude that the base substitution causes the respiratory rescue and discuss the possible mechanism as one which enhances interaction between the mutated tRNA(Asp)and the suppressor version of AspRS.

Microbiology, 2000 Feb, 146 ( Pt 2), 527 - 36
Oxidase and periplasmic cytochrome assembly in Escherichia coli K-12: CydDC and CcmAB are not required for haem-membrane association; Cook GM et al.; The mechanism(s) that bacteria use to transport haem into and across the cytoplasmic membrane to complete the assembly of periplasmic cytochromes is unknown . The authors have tested directly the role(s) of two ATP-binding cassette (ABC) transporters - the cydDC and ccmAB gene products - in Escherichia coli by measuring haem uptake in everted (inside-out) membrane vesicles . If haem is exported to the periplasm in vivo, the same process should result in active accumulation in such everted vesicles . {14C}Haemin (chloride) with bovine serum albumin (BSA) as a carrier protein was accumulated in intact everted membrane vesicles by an energy-independent mechanism . The kinetics of this process were biphasic: rapid uptake/binding was followed by a slower uptake of haem, which was inhibited by a large excess of unlabelled haemin-BSA, but not by BSA . However, accumulated haemin was not chased out of the vesicles by unlabelled haemin-BSA, suggesting specific binding of haemin with the membrane or transport into the lumen of the vesicle . Neither ATP nor a protonmotive force (delta(p)) generated by lactate oxidation was required for haemin binding or subsequent transport, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium vanadate and monensin had no effect on haemin transport . The rate of haemin uptake following the initial rapid binding was proportional to the external haemin concentration, suggesting that the uptake process was driven by the haemin concentration gradient across the cell membrane . The kinetics of {14C}haemin uptake were similar in wild-type and cydD1 or delta(ccmA) mutants, suggesting that the activity of neither the CydDC nor CcmAB transporters is essential for haem export to the periplasm . Cytochrome d levels were unaffected by mutations in trxB (encoding thioredoxin reductase), trxA (thioredoxin), or grx (glutaredoxin), suggesting that the CydDC transporter does not export these components of reducing pathways for cytochrome assembly.

Microbiology, 2000 Feb, 146 ( Pt 2), 497 - 507
A flagellar gene cluster from the oral spirochaete Treponema maltophilum; Heuner K et al.; A flagellar gene cluster from the oral spirochaete Treponema maltophilum ATCC 51939T was cloned . Sequence analysis revealed six putative ORFs, two of which encode the flagellar subunit proteins FlaB2 (286 aa) and FlaB3 (285 aa) . Northern blot analysis revealed two flagellin transcripts with the expected size of monocistronic mRNAs . Sequence analysis and primer extension experiments indicated that the transcription of the flaB2 gene is directed by a sigma28-like FliA factor . Using fliA and fliA+ Escherichia coli K-12 strains, it was shown that flaB2 expression in E . coli required the sigma28 factor using an initiation site identical to that in Treponema maltophilum . Primer extension analysis revealed two transcriptional start sites 5' of the flaB3 gene, a strong promoter with a sigma28-like -10 promoter element and a weak promoter with a putative sigma54 promoter consensus sequence . Downstream of flaB3, a putative fliD homologue was found, probably encoding the flagellar cap protein of Treponema maltophilum . Flagellin-gene-specific DNA probes hybridized to all 13 Treponema strains investigated, whereas a fliD-specific DNA probe only hybridized to Treponema maltophilum, other treponemal group IV isolates and Treponema brennaborense.

Microbiology, 2000 Feb, 146 ( Pt 2), 305 - 13
Analysis of the internal replication region of a mycobacterial linear plasmid; Picardeau M et al.; Linear plasmids have previously been identified by the authors in mycobacteria, the telomeres of which have terminal inverted repeats and covalently attached proteins . In this study, the replication of these unusual molecules was investigated by studying a 25 kb linear plasmid from the slow-growing species Mycobacterium celatum called pCLP . An internal region of pCLP responsible for replication in Mycobacterium smegmatis was identified . The nucleotide sequence of the minimum replication region of pCLP, which was 2.8 kb long, contained a putative replication gene, rep, and a putative origin of replication consisting of an 18 bp direct repeat and an AT-rich region . A short section of the pCLP replication region was also found to have sequence identity with the replication regions of mycobacterial circular plasmids, suggesting that these linear and circular plasmids are related . It was found that pCLP replicated in Mycobacterium bovis BCG and was compatible in M . smegmatis with pAL5000- and pJAZ38-derived plasmids from Mycobacterium fortuitum, which belong to two different compatibility groups . Thus, this new Escherichia coli-mycobacteria shuttle vector may be used in both slow- and fast-growing mycobacteria and in co-transformation experiments with other mycobacterial vectors.

Crit Care Med, 2000 Feb, 28(2), 496 - 503
Spontaneous high systemic oxygen delivery increases survival rate in awake sheep during sustained endotoxemia; Pittet JF et al.; OBJECTIVE: To study the natural evolution of systemic oxygen delivery (Do2) and oxygen consumption (Vo2) in sheep infused with low or high doses of endotoxin . DESIGN: Prospective, controlled experimental study . SETTING: Animal research laboratory at a medical university . SUBJECTS: Twenty-nine chronically instrumented awake sheep (25-35 kg) . INTERVENTIONS: Awake animals were continuously infused with saline (n = 8) or two doses of Escherichia coli endotoxin (20 or 40 ng/kg/min; n = 21) for 72 hrs . No attempt was made to increase Do2, but respiratory failure was treated by mechanical ventilation and metabolic acidosis was corrected . MEASUREMENTS AND MAIN RESULTS: The mortality rate was 25% in the group infused with the low dose and 89% in the group infused with the high dose of endotoxin . During the first 12 hrs of endotoxemia, both surviving (S group; n = 10) and nonsurviving (NS group; n = 11) sheep developed similar pulmonary hypertension, left ventricular failure, and hypotension with low systemic vascular resistance . However, S sheep had less interstitial lung edema (pulmonary lymph protein clearance at 8 hrs was 13+/-3 mL/hr vs . 27+/-6 mL/hr in the NS group and 4+/-1 mL/hr in the control group) . During this early phase of endotoxemia, Do2, Vo2, and oxygen extraction ratio did not change significantly in any group . After this phase, animals that ultimately survived had a persistent hyperdynamic syndrome with high cardiac output and hypotension . In this group, the Do2 increase was greater than the Do2 measured in controls and remained steady up to 48 hrs after the start of the endotoxin infusion . Because systemic Vo2 did not change significantly, oxygen extraction ratio decreased progressively to values less than those measured in controls . In contrast, animals that ultimately died had a hypotensive and normokinetic syndrome associated with pulmonary hypertension, persistent depressed left ventricular function, hypothermia, and a progressive deterioration of gas exchange . Systemic Do2 was not significantly different from that in the control group . In contrast, Vo2 decreased progressively to values significantly lower than those measured in controls and remained low until death . CONCLUSIONS: Our results indicate that in the absence of treatment such as fluid challenge or inotropic drugs in sheep infused with endotoxin, the occurrence of spontaneous hyperdynamic syndrome and high Do2 improves the survival rate.

Crit Care Med, 2000 Feb, 28(2), 462 - 6
Accuracy of mucosal pH and mucosal-arterial carbon dioxide tension for detecting mesenteric hypoperfusion in acute canine endotoxemia; Kellum JA et al.; OBJECTIVE: To determine the level of mucosal-arterial Pco2 (Pco2 gap) that is both sensitive and specific for the detection of mesenteric hypoperfusion as defined by either a >50% reduction in portal blood flow or release of lactate by the gut . DESIGN: Animal experiment . SUBJECTS: Seven anesthetized, intubated, mechanically ventilated, and surgically instrumented mongrel dogs . INTERVENTION: Escherichia coli endotoxin (1 mg/kg) given intravenously for 5 mins . MEASUREMENTS AND MAIN RESULTS: Tonometric Pco2, arterial blood gases, arterial and portal venous lactates, and portal and systemic hemodynamic variables were measured . Mucosal pH (pHi) was calculated according to the manufacturers' instructions . From these data, receiver operating characteristics were calculated . Although animals were resuscitated to maintain a constant cardiac output, portal flow decreased from 350+/-101 to 152+/-75 mL/min (p<.01) and the gut released lactate into the portal circulation in all animals . Pco2 gap increased from 13.1+/-3.9 to 40.2+/-39.2 torr (p<.01) and was inversely correlated with portal blood flow (r2 = .20; p<.05) . For detection of a >50% reduction in portal blood flow, a Pco2 gap of 20 torr yielded a maximum accuracy of 67% (sensitivity, 55%; specificity, 73%) and was less accurate than a pHi of 7.20, which yielded a maximum accuracy of 76% (sensitivity, 90%; specificity, 70%), although this difference was not significant (p = .24) . There was also a correlation between pHi and portal blood flow (r2 = .31; p<.01) . For detection of lactate release by the gut, a Pco2 gap of 20 torr was also 67% accurate (sensitivity, 53%; specificity, 78%), whereas a pHi of 7.10 achieved an accuracy of 64% (sensitivity, 40%; specificity, 83%), which was not significantly different . CONCLUSION: Pco2 gap measurements are neither sensitive nor specific for mesenteric hypoperfusion with regard to total gut blood flow reductions of >50% or the release of lactate into the portal circulation.

Amino Acids, 1999, 17(4), 347 - 55
Evidence that taurine modulates osmoregulation by modification of osmolarity sensor protein ENVZ--expression; Moenkemann H et al.; Although the involvement of taurine in osmoregulation is well-documented and widely accepted, no detailed mechanism for this function has been reported so far . We used subtractive hybridization to study mRNA steady state levels of genes up- or downregulated by taurine . Rats were fed taurine 100 mg/kg body weight per day for a period of three days and hearts (total ventricular tissue) of experimental animals and controls were pooled and used for mRNA extraction . mRNAs from two groups were used for subtractive hybridization . Clones of the subtractive library were sequenced and the obtained sequences were identified by gen bank assignment . Two clones were found to contain sequences which could be assigned to the osmolarity sensor protein envZ, showing homologies of 61 and 65% . EnvZ is an inner membrane protein in bacteria, important for osmosensing and required for porine gene regulation . It undergoes autophosphorylation and subsequently phosphorylates OmpR, which in turn binds to the porine (outer membrane protein) promoters to regulate the expression of OmpF and OmpC, major outer membrane porines . This is the first report of an osmosensing mechanism in the mammalian system, which was described in bacteria only . Furthermore, we are assigning a tentative role for taurine in the osmoregulatory process by modifying the expression of the osmoregulatory sensor protein ENVZ.

Vet Clin North Am Food Anim Pract, 2000 Mar, 16(1), 175 - 85
Edema disease; Moxley RA; Edema disease is a common cause of illness and death loss in pigs during the first 2 weeks after weaning . The disease is an enterotoxemia caused by strains of E . coli that colonize the small intestine and produce Stx2e . Bacterial colonization is mediated by F18ab fimbriae . Susceptibility to disease is determined by presence of receptors for these fimbriae on small intestinal epithelial cells and is inherited as a dominant trait . Clinical signs and lesions are largely the result of Stx2e, which causes necrosis of endothelial and smooth muscle cells in small arteries and arterioles . Vascular damage in the brain stem with resultant infarction and malacia is the main cause of death in affected pigs . Studies conducted by veterinary researchers in the 1950s and 1960s identified the cause of the disease and provided future scientists with hypotheses to test regarding the pathogenesis . In the last two decades, studies using molecular-based techniques have allowed for the definitive identification of bacterial virulence factors that mediate intestinal colonization and vascular damage, that is, F18ab fimbriae and Stx2e . Identification of these virulence factors has provided a basis for current and future development of effective preventative measures, for example, vaccines.

Vaccine, 2000 Apr 3, 18(19), 2049 - 54
Humoral immunoresponse to varicella-zoster virus pernasally coadministered with Escherichia coli enterotoxin in mice; Tsuji T et al.; It is evaluated whether Escherichia coli enterotoxin is useful for induction of immunity to varicella-zoster virus (VZV) as a mucosal adjuvant in mice . When a commercially available live varicella vaccine (Oka strain) and toxin were administered simultaneously via a nasal route three times at 2 or 6 month intervals, an antibody neutralizing VZV was detected in half or all of the mice vaccinated, respectively . The antibody specific to the vaccine strain of VZV reacted to five proteins, molecular weights of which were 110 K, 100 K, 62 K, 54 K and 46 K . These proteins were composed of glycosylated products of all kinds of glycoproteins . These results suggest that although a nasal administration of the vaccine without the adjuvant has little immunogenicity in mice, the simultaneous administration of the live vaccine and the toxin over a long period induces a specific humoral immunity to VZV.

Anal Biochem, 2000 Mar 15, 279(2), 218 - 25
Ultrasensitive fluorescence-based detection of nascent proteins in gels; Gite S et al.; The most common method of analysis of proteins synthesized in a cell-free translation system (e.g., nascent proteins) involves the use of radioactive amino acids such as {(35)S}methionine or {(14)C}leucine . We report a sensitive, nonisotopic, fluorescence-based method for the detection of nascent proteins directly in polyacrylamide gels . A fluorescent reporter group is incorporated at the N-terminus of nascent proteins using an Escherichia coli initiator tRNA(fmet) misaminoacylated with methionine modified at the alpha-amino group . In addition to the normal formyl group, we find that the protein translational machinery accepts BODIPY-FL, a relatively small fluorophore with a high fluorescent quantum yield, as an N-terminal modification . Under the optimal conditions, fluorescent bands from nanogram levels of in vitro-produced proteins could be detected directly in gels using a conventional UV-transilluminator . Higher sensitivity ( approximately 100-fold) could be obtained using a laser-based fluorescent gel scanner . The major advantages of this approach include elimination of radioactivity and the rapid detection of the protein bands immediately after electrophoresis without any downstream processing . The ability to rapidly synthesize nascent proteins containing an N-terminal tag facilitates many biotechnological applications including functional analysis of gene products, drug discovery, and mutation screening .

Anal Biochem, 2000 Mar 15, 279(2), 209 - 17
Temperature dependence of DNA affinity chromatography of transcription factors; Jarrett HW; Oligonucleotides bound by the CAAT enhancer binding protein (C/EBP), the lactose repressor, and Gal4 were chemically coupled to cyanogen bromide-activated Sepharose and the temperature dependence of transcription factor chromatography was characterized . Each transcription factor was applied to the appropriate column and eluted using a salt gradient at several temperatures . Each transcription factor showed a unique behavior . As temperature was increases, less salt was required to elute C/EBP, more salt was required to elute lac repressor, while Gal4 showed a biphasic dependency with the amount of salt first decreasing between 4 and 19 degrees C and then increasing above 19 degrees C . This temperature dependence is not due to protein or DNA unfolding but rather is a property of complex formation . By loading a column, washing it at a permissive temperature, and then rapidly changing the column temperature, highly selective elution can be obtained . The thermodynamics of this temperature effect are different for the binding of specific and nonspecific DNA sequences, making chromatography at different temperatures a potentially important way of purifying transcription factors .

J Virol, 2000 Apr, 74(7), 3273 - 83
The T-cell receptor zeta chain contains two homologous domains with which simian immunodeficiency virus Nef interacts and mediates down-modulation; Schaefer TM et al.; We have recently demonstrated that simian immunodeficiency virus (SIV) Nef binds to the zeta chain of the T-cell receptor (TCR), leading to its down-modulation from T-cell surfaces (I . Bell, C . Ashman, J . Maughan, E . Hooker, F . Cook, and T . A . Reinhart, J . Gen . Virol . 79:2717-2727, 1998) . Using a panel of human as well as rhesus macaque TCR zeta cytoplasmic domain mutants, we have identified in this report two linear peptides in the cytoplasmic domain of TCR zeta which independently interact with SIV Nef . Each SIV Nef interaction domain was sufficient in the absence of the other for interaction with SIV Nef in a yeast two-hybrid assay . In parallel, we demonstrated that Nef down-modulation of CD8-TCR zeta fusion proteins containing full-length or truncated portions of the TCR zeta cytoplasmic domain occurs in transiently transfected 293T cells . Furthermore, using proteins expressed in Escherichia coli, a glutathione S-transferase-Nef fusion protein coprecipitated histidine-tagged portions of the TCR zeta cytoplasmic domain which contained SNID-1 or SNID-2 . The peptides targeted by SIV Nef, YNELNL and YSEIGMKGERRR, are portions of the first and second of three immunoreceptor tyrosine-based activation motifs which are important in signal transduction, thymocyte development, and TCR biogenesis . These results demonstrate that SIV Nef binds to two distinct domains on TCR zeta in the absence of other T-cell-specific factors, and that interaction with either domain is sufficient to cause down-modulation of TCR zeta.

J Virol, 2000 Apr, 74(7), 3093 - 104
A protein kinase activity associated with Epstein-Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro; Chen MR et al.; The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases . In order to investigate this potential kinase activity, BGLF4 was expressed in Escherichia coli and the purified protein was used to generate a specific antiserum . Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter . Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay . In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells . Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity . BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay . BGLF4 can phosphorylate histone and casein in vitro . Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro . Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.

Hepatology, 2000 Mar, 31(3), 606 - 14
Gene therapy of hepatocellular carcinoma in vitro and in vivo in nude mice by adenoviral transfer of the Escherichia coli purine nucleoside phosphorylase gene; Mohr L et al.; Expression of viral or bacterial enzymes in tumor cells to convert nontoxic prodrugs into highly toxic metabolites is an attractive gene-therapeutic approach for the treatment of hepatocellular carcinoma (HCC) . The Escherichia coli purine nucleoside phosphorylase (PNP) converts purine analogs into freely diffusible metabolites, which are highly toxic to dividing and nondividing cells . We investigated the antitumor effects of PNP in the human HCC cell lines, HepG2, Hep3B, and HuH-7, and performed a comparison with herpes simplex thymidine kinase (TK) . The genes for PNP, TK, and enhanced green fluorescent protein (EGFP) were delivered to HCC cells by identical adenoviral vectors . Fludarabine and ganciclovir (GCV) served as prodrugs for PNP and TK, respectively . Expression of PNP highly sensitized HCC cells to fludarabine treatment . Fludarabine concentrations between 0.5 and 1 microg/mL killed 100% of the cells expressing PNP with no detectable toxicity in control cells expressing EGFP . Expression of PNP in as few as 10% of HCC cells induced efficient killing of most bystander cells . Expression of TK followed by GCV treatment produced a potent growth inhibition but failed to kill all TK-expressing HCC cells . More importantly, the TK system exhibited a lower degree of bystander effect . Adenoviral delivery of PNP followed by fludarabine administration prevented subcutaneous and intrahepatic tumor formation in nude mice and was also effective for the treatment of established tumors . These results demonstrate the potential of the PNP/fludarabine system for the treatment of HCC.

Nature, 2000 Feb 24, 403(6772), 859 - 66
Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA; Bruner SD et al.; Spontaneous oxidation of guanine residues in DNA generates 8-oxoguanine (oxoG) . By mispairing with adenine during replication, oxoG gives rise to a G x C --> T x A transversion, a frequent somatic mutation in human cancers . The dedicated repair pathway for oxoG centres on 8-oxoguanine DNA glycosylase (hOGG1), an enzyme that recognizes oxoG x C base pairs, catalysing expulsion of the oxoG and cleavage of the DNA backbone . Here we report the X-ray structure of the catalytic core of hOGG1 bound to oxoG x C-containing DNA at 2.1 A resolution . The structure reveals the mechanistic basis for the recognition and catalytic excision of DNA damage by hOGG1 and by other members of the enzyme superfamily to which it belongs . The structure also provides a rationale for the biochemical effects of inactivating mutations and polymorphisms in hOGG1 . One known mutation, R154H, converts hOGG1 to a promutator by relaxing the specificity of the enzyme for the base opposite oxoG.

Chem Pharm Bull (Tokyo), 2000 Feb, 48(2), 223 - 30
Mechanism of superoxide dismutase-like activity of Fe(II) and Fe(III) complexes of tetrakis-N,N,N',N'(2-pyridylmethyl)ethylenediamine; Hirano T et al.; The superoxide dismutase (SOD) activity of iron(II) tetrakis-N,N,N',N'(2-pyridylmethyl)ethylenediamine complex (Fe-TPEN) was reexamined using a pulse radiolysis method . In our previous study (J . Biol . Chem., 264, 9243-9249 (1989)), we reported that this complex has a potent SOD activity in a cyt . c (cytochrome c)-based system (IC50 = 0.8 microM) and protects E . coli cells against paraquat toxicity . The present pulse radiolysis experiment revealed that Fe(II)TPEN reacts stoichiometrically with superoxide to form Fe(III)TPEN with a second-order rate constant of 3.9 x 10(6) M-1 S-1 at pH 7.1, but superoxide did not reduce Fe(III)TPEN to Fe(II)TPEN . The reaction of Fe(III)TPEN and superoxide was biphasic . In the fast reaction, an adduct (Fe(III)TPEN-superoxide complex) was formed at the second-order rate constant of 8.5 x 10(5) M-1 S-1 at pH 7.4 . In the slow one, the adduct reacted with another molecule of the adduct, regenerating Fe(III)TPEN . In the cyt . c method with catalase, this Fe(III)TPEN-superoxide complex showed cyt . c oxidation activity, which had led to overestimation of its SOD activity . Based on the titration data, the main species of complex in aqueous media at neutral pH was indicated to be Fe(III)TPEN(OH-) . A spectral change after the reduction with hydrated electron indicates that the OH- ion coordinates directly to Fe(III) by displacing one of the pyridine rings . The X-ray analysis of {Fe(II)TPEN}SO4 supported this structure . From the above results we propose a novel reaction mechanism of FeTPEN and superoxide which resembles a proton catalyzed dismuting process, involving Fe(III)TPEN-superoxide complex.

Biosci Biotechnol Biochem, 2000 Jan, 64(1), 80 - 8
Sequence of egV and properties of EgV, a Ruminococcus albus endoglucanase containing a dockerin domain; Ohara H et al.; The Ruminococcus albus F-40 egV gene, encoding endoglucanase V (EGV), consists of an open reading frame of 1,833 nucleotides and encodes 611 amino acids with a deduced molecular weight of 67,103 . The deduced EGV is a modular enzyme composed of a catalytic domain of family 5 of glycosyl hydrolases, a domain of unknown function, and a dockerin domain responsible for cellulosome assembly, suggesting that R . albus F-40 produces a cellulosome, and EGV is a component of the cellulosome . A truncated form of EGV with an apparent molecular weight of 42,000 was purified from a recombinant Escherichia coli and characterized since EGV suffered from partial proteolysis by E . coli protease(s) . The truncated EGV was active toward carboxylmethyl cellulose, xylan, lichenan, and acid-swollen cellulose . The pH and temperature optima of the enzyme were 7.0 and 40 degrees C, respectively . By Western blot analysis using the antiserum raised against the truncated enzyme, EGV was detected in the whole cells but not in the culture supernatant of R . alubus F-40, suggesting that EGV was located on the cell surface.

Bioinformatics, 1999 Oct, 15(10), 785 - 98
A new method to predict the consensus secondary structure of a set of unaligned RNA sequences; Bouthinon D et al.; MOTIVATION: To predict the consensus secondary structure, possibly including pseudoknots, of a set of RNA unaligned sequences . RESULTS: We have designed a method based on a new representation of any RNA secondary structure as a set of structural relationships between the helices of the structure . We refer to this representation as a structural pattern . In a first step, we use thermodynamic parameters to select, for each sequence, the best secondary structures according to energy minimization and we represent each of them using its corresponding structural pattern . In a second step, we search for the repeated structural patterns, i.e . the largest structural patterns that occur in at least one sequence, i.e . included in at least one of the structural patterns associated to each sequence . Thanks to an efficient encoding of structural patterns, this search comes down to identifying the largest repeated word suffixes in a dictionary . In a third step, we compute the plausibility of each repeated structural pattern by checking if it occurs more frequently in the studied sequences than in random RNA sequences . We then suppose that the consensus secondary structure corresponds to the repeated structural pattern that displays the highest plausibility . We present several experiments concerning tRNA, fragments of 16S rRNA and 10Sa RNA (including pseudoknots); in each of them, we found the putative consensus secondary structure.

Fetal Diagn Ther, 2000 Jan-Feb, 15(1), 50 - 3
Does amniotomy influence the prognosis of babies in cases with severe chorioamnionitis? Report of a twin pregnancy with varying outcome; Suzuki Y et al.; We report our experience in a woman with a twin pregnancy . The patient suffered severe Escherichia coli chorioamnionitis and the outcomes were different between the two babies after birth . The first baby had only a mild infection, but the second suffered sepsis and subsequent perinatal death . These differences in outcome appeared to be due to amniotomy performed for the first baby after late labor stage I to augment uterus contractions . Removal of infectious amniotic fluid from the amniotic cavity might thus have prevented the spread of the chorioamnionitis . E . coli sometimes causes severe infection during pregnancy and the perinatal period . In this case, a large number of enteropathogenic E . coli (serotype O-6) was cultured from blood, stool, pharyngeal swab, gastric juice and puncture fluid from the thoracic cavity of the second baby . O-6 is classified an enterotoxigenic strain mainly causing diarrhea because of endotoxin released from bacteria . O-6 has not hitherto been reported as a cause of severe infection in chorioamnionitis and perinatal sepsis .

Int J Parasitol, 2000 Feb, 30(2), 125 - 8
Cloning and characterisation of a peroxiredoxin from the swine roundworm Ascaris suum; Tsuji N et al.; Antioxidant enzymes in parasites play an important role in protection against the oxygen radicals by generating during aerobic metabolism, as well as in defence against host immune cell assault . Here we report the cloning and characterisation of a cDNA encoding peroxiredoxin from Ascaris suum (AsPrx) . AsPrx is 776bp long and contains the nematode 22bp splice leader sequence at the 5' end and polyadenylation signal followed by poly(A) tail at the 3' end . AsPrx codes a full-length protein with a predicted molecular mass of 22 . 6kDa, and possesses two cysteine residues at amino acid 49 and 168 that are conserved among Prx proteins . GenBank() analysis showed that the deduced amino acid sequence had significant similarity to parasite and mammalian Prx at the amino acid level . DNA nicking revealed that Escherichia coli-expressed recombinant AsPrx (rAsPrx) is enzymatically inhibited to form oxidative-nicking of supercoiled plasmid DNA . Two-dimensional immunoblot analysis with mouse anti-rAsPrx serum reacted two major constituent protein spots in extracts of adult female worms, suggesting that the native AsPrx might be function as a major antioxidant enzyme in Ascaris suum.

Int J Parasitol, 2000 Feb, 30(2), 113 - 8
Overcoming codon bias: a method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli; Baca AM et al.; Parasite genes often use codons which are rarely used in the highly expressed genes of Escherichia coli, possibly resulting in translational stalling and lower yields of recombinant protein . We have constructed the "RIG" plasmid to overcome the potential codon-bias problem seen in Plasmodium genes . RIG contains the genes that encode three tRNAs (Arg, Ile, Gly), which recognise rare codons found in parasite genes . When co-transformed into E . coli along with expression plasmids containing parasite genes, RIG can greatly increase levels of overexpressed protein . Codon frequency analysis suggests that RIG may be applied to a variety of protozoan and helminth genes.

Virology, 2000 Mar 15, 268(2), 430 - 9
The human papillomavirus type 11 E1E4 protein is phosphorylated in genital epithelium; Bryan JT et al.; The most abundant viral transcript in human papillomavirus (HPV) 11-infected xenograft tissue has been shown to encode the E1(wedge)E4 protein . The function of E1(wedge)E4 protein has not been determined . Several potential phosphorylation sequence motifs were identified in the HPV 11 E1(wedge)E4 protein, including potential sites of phosphorylation by mitogen-activated protein kinase (MAPK), cAMP-dependent protein kinase (PKA), casein kinase II, and protein kinase C . To test phosphorylation of the HPV 11 E1(wedge)E4 protein, a soluble maltose binding protein (MBP) fusion was produced in Escherichia coli . Only MAPK and PKA phosphorylated the E1(wedge)E4 protein . Phosphoamino acid analysis showed that one or more threonine residues were phosphorylated by MAPK, and both serine and threonine residues were phosphorylated by PKA . MBP-E1(wedge)E4 mutant proteins were designed to delineate the E1(wedge)E4 phosphoacceptor residues . MAPK was shown to phosphorylate E1(wedge)E4 on threonine 53 within a MAPK consensus phorphorylation sequence motif . PKA was shown to phosphorylate E1(wedge)E4 at two residues: threonine 36 within a consensus motif and serine 44 within a variant of the PKA consensus phosphorylation sequence motif . HPV 11-infected human genital tissue grown as a xenograft in an athymic mouse was labeled with {(32)P}orthophosphate . Phosphoamino acid analysis of E1(wedge)E4 protein immunoprecipitated from (32)P-labeled tissue revealed that both serine and threonine residues were phosphorylated . Analysis by liquid chromatography-mass spectrophotometry was consistent with phosphorylation of residues within the PKA and MAPK phosphorylation sequence motifs . Expression of E1(wedge)E4 protein containing phosphorylation substitution mutations showed that the PKA mutant did not differ from wild-type E1(wedge)E4 protein in intracellular distribution . In contrast, the MAPK mutant did not localize exclusively to the cytoplasm nor did it colocalize with wild-type E1(wedge)E4 protein . We conclude that HPV 11 E1(wedge)E4 protein is phosphorylated in vitro and in vivo . Our data are consistent with phosphorylation of HPV 11 E1(wedge)E4 protein by MAPK and PKA in infected tissue .

J Mol Biol, 2000 Mar 17, 297(1), 233 - 49
Assessing annotation transfer for genomics: quantifying the relations between protein sequence, structure and function through traditional and probabilistic scores; Wilson CA et al.; Measuring in a quantitative, statistical sense the degree to which structural and functional information can be "transferred" between pairs of related protein sequences at various levels of similarity is an essential prerequisite for robust genome annotation . To this end, we performed pairwise sequence, structure and function comparisons on approximately 30,000 pairs of protein domains with known structure and function . Our domain pairs, which are constructed according to the SCOP fold classification, range in similarity from just sharing a fold, to being nearly identical . Our results show that traditional scores for sequence and structure similarity have the same basic exponential relationship as observed previously, with structural divergence, measured in RMS, being exponentially related to sequence divergence, measured in percent identity . However, as the scale of our survey is much larger than any previous investigations, our results have greater statistical weight and precision . We have been able to express the relationship of sequence and structure similarity using more "modern scores," such as Smith-Waterman alignment scores and probabilistic P-values for both sequence and structure comparison . These modern scores address some of the problems with traditional scores, such as determining a conserved core and correcting for length dependency; they enable us to phrase the sequence-structure relationship in more precise and accurate terms . We found that the basic exponential sequence-structure relationship is very general: the same essential relationship is found in the different secondary-structure classes and is evident in all the scoring schemes . To relate function to sequence and structure we assigned various levels of functional similarity to the domain pairs, based on a simple functional classification scheme . This scheme was constructed by combining and augmenting annotations in the enzyme and fly functional classifications and comparing subsets of these to the Escherichia coli and yeast classifications . We found sigmoidal relationships between similarity in function and sequence, with clear thresholds for different levels of functional conservation . For pairs of domains that share the same fold, precise function appears to be conserved down to approximately 40 % sequence identity, whereas broad functional class is conserved to approximately 25 % . Interestingly, percent identity is more effective at quantifying functional conservation than the more modern scores (e.g . P-values) . Results of all the pairwise comparisons and our combined functional classification scheme for protein structures can be accessed from a web database at 2000 Academic Press.

J Mol Biol, 2000 Mar 17, 297(1), 179 - 92
Molecular clocks reduce plasmid loss rates: the R1 case; Paulsson J et al.; Plasmids control their replication so that the replication frequency per plasmid copy responds to the number of plasmid copies per cell . High sensitivity amplification in replication response to copy number deviations generally reduces variation in copy numbers between different single cells, thereby reducing the plasmid loss rate in a cell population . However, experiments show that plasmid R1 has a gradual, insensitive replication control predicting considerable copy number variation between single cells . The critical step in R1 copy number control is regulation of synthesis of a rate-limiting cis-acting replication protein, RepA . De novo synthesis of a large number of RepA molecules is required for replication, suggesting that copy number control is exercised at multiple steps . In this theoretical kinetic study we analyse R1 multistep copy number control and show that it results in the insensitive replication response found experimentally but that it at the same time effectively prohibits the existence of only one plasmid copy in a dividing cell . In combination with the partition system of R1, this can lead to very high segregational stability . The R1 control mechanism is compared to the different multistep copy number control of plasmid ColE1 that is based on conventional sensitivity amplification . This implies that while copy number control for ColE1 efficiently corrects for fluctuations that have already occurred, R1 copy number control prevents their emergence in cells that by chance start their cycle with only one plasmid copy . We also discuss how regular, clock-like, behaviour of single plasmid copies becomes hidden in experiments probing collective properties of a population of plasmid copies because the individual copies are out of phase . The model is formulated using master equations, taking a stochastic approach to regulation, but the mathematical formalism is kept to a minimum and the model is simplified to its bare essence . This simplicity makes it possible to extend the analysis to other replicons with similar design principles .

J Mol Biol, 2000 Mar 17, 297(1), 39 - 47
Mutational analysis of archaeal histone-DNA interactions; Soares DJ et al.; Site-specific mutagenesis of the hmfB gene cloned from the archaeon Methanothermus fervidus, followed by expression in Escherichia coli, has been used to generate approximately 90 recombinant (r) variants of the archaeal histone HMfB . The abilities of these variants to form stable archaeal nucleosome-containing complexes with linear pBR322 DNA, and with an 89 bp restriction fragment of this DNA have been determined . Variants that failed to form such complexes, based on negative gel-shift assays, had substitutions at the N terminus or within the alpha1, L1 and L2 regions of the rHMfB histone fold, at sites predicted to be homologous to eucaryal histone fold residues that contact the DNA in the eucaryal nucleosome . Variants that failed to give gel shifts were further assayed for their abilities to facilitate ligase-catalyzed circularization of a linear 88 bp DNA molecule, and to reduce the ellipticity of a DNA solution at 275 nm (theta(275)) . Consistent with cooperative but independent sites of DNA binding, a combination of three residue substitutions, one each in alpha1, L1 and L2, was required to generate a rHMfB variant with no detectable DNA binding based on gel shift, circularization and theta(275) reduction assays .

J Mol Biol, 2000 Mar 17, 297(1), 25 - 37
A minimal system for Tn7 transposition: the transposon-encoded proteins TnsA and TnsB can execute DNA breakage and joining reactions that generate circularized Tn7 species; Biery MC et al.; In the presence of ATP and Mg(2+), the bacterial transposon Tn7 translocates via a cut and paste mechanism executed by the transposon-encoded proteins TnsA+TnsB+TnsC+TnsD . We report here that in the presence of Mn(2+), TnsA+TnsB alone can execute the DNA breakage and joining reactions of Tn7 recombination . ATP is not essential in this minimal system, revealing that this cofactor is not directly involved in the chemical steps of recombination . In both the TnsAB and TnsABC+D systems, recombination initiates with double-strand breaks at each transposon end that cut Tn7 away from flanking donor DNA . In the minimal system, breakage occurs predominantly at a single transposon end and the subsequent end-joining reactions are intramolecular, with the exposed 3' termini of a broken transposon end joining near the other end of the Tn7 element in the same donor molecule to form circular transposon species . In contrast, in TnsABC+D recombination, breaks occur at both ends of Tn7 and the two ends join to a target site on a different DNA molecule to form an intermolecular simple insertion . This demonstration of the capacity of TnsAB to execute breakage and joining reactions supports the view that these proteins form the Tn7 transposase .

J Theor Biol, 2000 Mar 21, 203(2), 117 - 33
How to make a biological switch; Cherry JL et al.; Some biological regulatory systems must "remember" a state for long periods of time . A simple type of system that can accomplish this task is one in which two regulatory elements negatively regulate one another . For example, two repressor proteins might control one another's synthesis . Qualitative reasoning suggests that such a system will have two stable states, one in which the first element is "on" and the second "off", and another in which these states are reversed . Quantitative analysis shows that the existence of two stable steady states depends on the details of the system . Among other things, the shapes of functions describing the effect of one regulatory element on the other must meet certain criteria in order for two steady states to exist . Many biologically reasonable functions do not meet these criteria . In particular, repression that is well described by a Michaelis-Menten-type equation cannot lead to a working switch . However, functions describing positive cooperativity of binding, non-additive effects of multiple operator sites, or depletion of free repressor can lead to working switches .

Biochemistry, 2000 Mar 14, 39(10), 2792 - 804
Self-assembly of recombinant prion protein of 106 residues; Baskakov IV et al.; The central event in the pathogenesis of prion diseases is a profound conformational change of the prion protein (PrP) from an alpha-helical (PrP(C)) to a beta-sheet-rich isoform (PrP(Sc)) . The elucidation of the mechanism of conformational transition has been complicated by the challenge of collecting high-resolution biophysical data on the relatively insoluble aggregation-prone PrP(Sc) isoform . In an attempt to facilitate the structural analysis of PrP(Sc), a redacted chimeric mouse-hamster PrP of 106 amino acids (MHM2 PrP106) with two deletions (Delta23-88 and Delta141-176) was expressed and purified from Escherichia coli . PrP106 retains the ability to support PrP(Sc) formation in transgenic mice, implying that it contains all regions of PrP that are necessary for the conformational transition into the pathogenic isoform {Supattapone, S., et al . (1999) Cell 96, 869-878} . Unstructured at low concentrations, recombinant unglycosylated PrP106 (rPrP106) undergoes a concentration-dependent conformational transition to a beta-sheet-rich form . Following the conformational transition, rPrP106 possesses properties similar to those of PrP(Sc)106, such as high beta-sheet content, defined tertiary structure, resistance to limited digestion by proteinase K, and high thermodynamic stability . In GdnHCl-induced denaturation studies, a single cooperative conformational transition between the unstructured monomer and the assembled beta-oligomer was observed . After proteinase K digestion, the oligomers retain an intact core with unusually high beta-sheet content (>80%) . Using mass spectrometry, we discovered that the region of residues 134-215 of rPrP106 is protected from proteinase K digestion and possesses a solvent-independent propensity to adopt a beta-sheet-rich conformation . In contrast to the PrP(Sc)106 purified from the brains of neurologically impaired animals, multimeric beta-rPrP106 remains soluble, providing opportunities for detailed structural studies.

Biochemistry, 2000 Mar 14, 39(10), 2778 - 83
Escherichia coli ATP synthase alpha subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state; Le NP et al.; The three catalytic sites of the F(O)F(1) ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis . Replacement of the conserved alpha subunit Arg-376 in the Escherichia coli F(1) catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 10(3)-fold lower than that of the wild type . Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site . Direct monitoring of nucleotide binding in the alphaR376A and alphaR376K mutant F(1) by a tryptophan in place of betaTyr-331 (Weber et al . (1993) J . Biol . Chem . 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric . These results indicate that alphaArg-376, which is close to the beta- or gamma-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.

Biochemistry, 2000 Mar 14, 39(10), 2733 - 9
Factors determining vesicular lipid mixing induced by shortened constructs of influenza hemagglutinin; LeDuc DL et al.; The HA2 subunit of influenza hemagglutinin is responsible for fusion of the viral and host-cell membranes during infection . An N-terminal 127 amino acid construct of HA2, FHA2-127, is shown to induce lipid mixing of large unilamellar vesicles under endosomal low pH conditions . Thus, FHA2 could serve as a good model system for biophysical studies of membrane fusion . With FHA2, we began to develop a mechanistic model which could explain how this short construct facilitates membrane fusion . In this endeavor, we studied the possible role of the kinked loop region (amino acids 105-113) . A construct missing this loop, FHA2-90, although able to induce lipid mixing, has lost the sharp pH-dependent transition seen with FHA2-127 and native HA . In addition, FHA2-127 promotes extensive vesicle aggregation more effectively than FHA2-90 upon acidification . These data suggest that the kinked loop may play a pH-dependent regulatory role . To test this, we compared bis-ANS binding to the two constructs and observed that binding to FHA2-127 increases at a faster rate than FHA2-90 as the pH is decreased, indicating that the kinked loop not only is an ANS-binding site, but that it binds better at low pH . The pH dependence of this transition directly correlates with that observed in lipid mixing . Further, cysteine mutations of acidic residues in the kinked region are both fusion inactive and bind much less ANS, whereas a similar mutation of a threonine residue had little effect on fusion activity or ANS binding . This evidence lends further support to our idea that the kinked loop serves a regulatory role . To test the physiological relevance of the FHA2-127 fusion mechanism, we studied the effects of a G1E mutation, known to abolish fusion in native HA . We found that G1E-127 is fusion inactive as expected . This evidence indirectly suggests that the mechanism of FHA2-127 is perhaps physiologically relevant and from its study, we can learn much about the mechanism of native HA.

Biochemistry, 2000 Mar 14, 39(10), 2652 - 8
Kinetic parameters for tmRNA binding to alanyl-tRNA synthetase and elongation factor Tu from Escherichia coli; Barends S et al.; Aminoacylation and transportation of tmRNA to stalled ribosomes constitute prerequisite steps for trans-translation, a process facilitating the release of stalled ribosomes from 3' ends of truncated mRNAs and the degradation of incompletely synthesized proteins . Kinetic analysis of the aminoacylation of tmRNA indicates that tmRNA has both a lower affinity and a lower turnover number than cognate tRNA(Ala) for alanyl-tRNA synthetase, resulting in a 75-fold lower k(cat)/K(M) value . The association rate constant of Ala-tmRNA for elongation factor Tu in complex with GTP is about 150-fold lower than that of Ala-tRNA(Ala), whereas its dissocation rate constant is about 5-fold lower . These observations can be interpreted to suggest that additional factors facilitate tmRNA binding to ribosomes.

Biochemistry, 2000 Mar 14, 39(10), 2633 - 8
Reevaluation of transcriptional regulation by TATA-binding protein oligomerization: predominance of monomers; Campbell KM et al.; The TATA-binding protein (TBP) plays an important role in transcriptional initiation by all three nuclear RNA polymerases . TBP contains a conserved C-terminal domain (cTBP) that binds DNA . Crystallographic studies of cTBP (i.e., TBP without the N-terminal domain) from various species and molecular biology studies of cTBP and mixed cTBP/TBP species have led to the view that DNA binding by TBP is regulated by TBP dimerization . Using sedimentation equilibrium, we show that yeast cTBP forms dimers in solution at 5 degrees C with a dissociation constant of 7 +/- 1 microM . This observation of cTBP dimers in solution is in accord with the dimeric state observed in crystal structures of cTBP . In contrast, physiologically relevant, full-length yeast TBP is monomeric at 5 degrees C and forms dimers at 30 degrees C with a dissociation constant of 51 +/- 16 microM . This dissociation constant precludes formation of stable full-length TBP dimers at physiological concentrations . In addition, we tested for yeast TBP oligomerization in the presence of TBP-associated factors in the context of TFIID . No evidence for TBP oligomers was found using immunoprecipitation techniques from yeast whole-cell extracts . We conclude that yeast TBP is predominantly monomeric under physiological conditions, arguing against a role for TBP dimerization in the regulation of transcriptional initiation.

Biochemistry, 2000 Mar 14, 39(10), 2626 - 32
Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I; Morales JC et al.; We describe studies aimed at evaluating the physical factors governing the rate of 3'-end proofreading by the Klenow fragment of E . coli DNA polymerase I . Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene (F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mimics of thymine and adenine, respectively, are used to investigate the effects of base pair geometry and stability on the rate of this exonuclease activity . Steady-state kinetics measurements show that complementary T.A base pairs at the end of a primer-template duplex are edited 14-40-fold more slowly than mismatches . By contrast, a 3'-end T residue in a T . Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact that it resembles a T.A pair in structure . Similarly, the A in an A.F pair is edited as rapidly as a mismatched pair despite its close structural mimicry of an A.T pair . Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicating specific interactions between the exonuclease domain and the base of the nucleotide being edited . Finally, thermal denaturation studies are carried out to investigate the relationship between editing and the ease of unwinding of the duplex . The rapid editing of bases opposite F or Z residues at the duplex terminus seems to correlate well with the stability of these base pairs when placed in a context resembling a primer-template duplex . In general, the rate of 3'-end editing appears to be governed by the rate of fraying of the DNA terminal pair, and base pair geometry appears to have little effect.

Biochemistry, 2000 Mar 14, 39(10), 2612 - 8
C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding; Li VS et al.; We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding . Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC) . We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences . In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected . These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding . We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.

Cell Motil Cytoskeleton, 2000 Mar, 45(3), 211 - 22
Terminal regions of mouse nebulin: sequence analysis and complementary localization with N-RAP; Herrera AH et al.; The regions of mouse nebulin extending from the ends of the super repeats to the C-terminus and N-terminus were cloned and sequenced . Comparison of the mouse sequence with the previously published human sequence shows that the terminal regions of nebulin are highly conserved . The four phosphorylation motifs and SH3 domain found at the C-terminus of mouse nebulin are identical to those found in human nebulin, with the exception of four conservative substitutions . The modules linking this C-terminal region to the super repeats have deletions relative to both fetal and adult human nebulins that correspond to integral numbers of modules, making the mouse C-terminal simple repeat region among the shortest observed to date . The N-terminal region and the C-terminal modules were expressed in Escherichia coli and used for antibody production . Immunofluorescent labeling of these regions of nebulin in isolated myofibrils demonstrates that they are located near the center of the sarcomere and near the Z-line, respectively . Immunogold labeling with antibodies raised against the N-terminal nebulin sequence localizes this region in the A-band near the tips of the thin filaments . Nebulin localization is complementary to that of N-RAP, another muscle-specific protein containing nebulin-like super repeats; nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendinous junction . Cell Motil . Cytoskeleton 3:211-222, 2000 Published 2000 Wiley-Liss, Inc.

Biofactors, 2000, 11(1-2), 39 - 41
Cloning and expression of rat glucocorticoid-receptor DNA-binding domain; Okamoto K et al.; The inability to obtain sufficient quantities of purified glucocorticoid receptor (GR) causes difficulties in studying in vitro the interactions of GR with other proteins which contribute to the transcriptional activation of chromatin . Thus, we overexpressed the DNA-binding domains (DBD) of GR in E . coli using glutathione S-transferase (GST)-fusion protein expression vector . The DBD-GST protein (about 57 kDa), recovered in the soluble fraction, was purified by glutathione-agarose column . However, during the chromatography, more than 90% of the DBD-GST protein was cleaved into three species with molecular sizes of about 40, 35 and 30 kDa . The 40 kDa and 35 kDa proteins were immunostained with the anti-GR antibody, and the 30 kDa protein was stained with anti-GST antibody . Among these proteins, only the 57 kDa DBD-GST protein bound to the GRE-agarose . These data indicate that the properly folded 57 kDa protein has a DNA binding function and is resistant to intrinsic cleavage of the protein.

Eur Cytokine Netw, 2000 Mar, 11(1), 47 - 52
Interleukin-18 stimulates HIV-1 replication in a T-cell line; Klein SA et al.; Interleukin-18 (IL-18) is a recently identified proinflammatory cytokine . Its ability to induce interferon-g suggests a potential virustatic effect . On the other hand, it stimulates NFkB - an activator of HIV replication . Recently, stimulation of HIV-1 in monocytic cells has been demonstrated . In the present study, the influence of IL-18 on HIV-1 replication in lymphatic cells was investigated . Hut78 cells were infected with HIV-1 in the presence of recombinant human IL-18 expressed either in E . coli or eucaryotically by baculovirus in Sf9 cells . HIV-1 replication was monitored by p24 ELISA and endpoint titration of culture supernatants on C8166 cells . The addition of IL-18 led to a 3- to 15-fold enhancement of HIV replication in Hut78 cells . By addition of neutralising monoclonal anti-IL-18 antibodies, this effect of IL-18 was reduced by 75% . Exposure of Hut78 to IL-18 prior to HIV infection could exclude the possibility that IL-18 promotes infection of cells . Taken together, these data provide direct evidence for an IL-18-mediated enhancement of HIV-1 replication in lymphatic cells.

Eur Cytokine Netw, 2000 Mar, 11(1), 39 - 46
Regulation of lipopolysaccharide-induced NO synthase expression in the major organs in a mouse model . The roles of endogenous interferon-gamma, tumor necrosis factor-alpha and interleukin-10; ter Steege JC et al.; Elevated NO production mediated by activation of the enzyme iNOS is thought to play a central role in the development of tissue damage observed during septic shock . IFN-gamma, TNF-alpha and IL-10 have been shown to be involved in the regulation of LPS-induced serum levels of the NO-oxidation products nitrate and nitrite . Therefore, in the present study, we investigated the role of endogenous IFN-gamma, TNF-alpha and IL-10 in the regulation of LPS-induced tissue iNOS expression in the major organs . To this end, mice were pre-treated with anti-IFN-gamma, anti-TNF-alpha, anti-IL-10 monoclonal antibodies, or combinations of these, two hours before intraperitoneal LPS-challenge . Immunohistochemical staining for iNOS and determination of iNOS activity indicated that iNOS expression was mainly upregulated in the small intestine, lung and heart, and that IFN-gamma, TNF-alpha as well as IL-10 are involved in the regulation of iNOS expression and enzyme activity . Whereas blocking either IFN-gamma or TNF-alpha did not affect iNOS expression, iNOS enzymatic activity seems to be inhibited . In contrast, blocking both mediators nearly completely prevents iNOS expression after LPS challenge, suggesting that the presence of either IFN-gamma or TNF-alpha is essential for LPS-induced iNOS expression in these organs . Combined treatment of these monoclonal antibodies revealed that whereas on the one hand IL-10 inhibits LPS-induced iNOS expression, on the other hand IL-10 or an IL-10 inducible factor is also involved in the upregulation of iNOS expression after LPS challenge.

Mediators Inflamm, 1999, 8(2), 107 - 13
Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment; Alencar NM et al.; The natural physiological ligands for selectins are oligosaccharides found in glycoprotein or glycolipid molecules in cell membranes . In order to study the role of sugar residues in the in vivo lectin anti-inflammatory effect, we tested three leguminous lectins with different carbohydrate binding affinities in the peritonitis and paw oedema models induced by carrageenin in rats . L . sericeus lectin was more anti-inflammatory than D . virgata lectin, the effects being reversed by their specific binding sugars (N-acetylglucosamine and alpha-methylmannoside, respectively) . However, V . macrocarpa, a galactose-specific lectin, was not anti-inflammatory . The proposed anti-inflammatory activity of lectins could be due to a blockage of neutrophil-selectin carbohydrate ligands . Thus, according to the present data, we suggest an important role for N-acetylglucosamine residue as the major ligand for selectins on rat neutrophil membranes.

Mediators Inflamm, 1999, 8(2), 77 - 84
Upregulation of ICAM-1 expression on J774.2 macrophages by endotoxin involves activation of NF-kappaB but not protein tyrosine kinase: comparison to induction of iNOS; Ruetten H et al.; This study compares the signal transduction pathway which leads to the upregulation of intercellular adhesion molecule-1 (ICAM-1) expression with that of the increase in the expression of inducible nitric oxide synthase (iNOS) protein and activity caused by endotoxin in cultured J774.2 macrophages . Treatment of J774.2 cells with lipopolysaccharide E . coli (LPS) induced a concentration-dependent increase in the expression of ICAM-1 on the cell surface within 4 h and an increase in iNOS protein and activity at 24 h . The upregulation of ICAM-1 expression on J774.2 macrophages caused by LPS was significantly inhibited by pretreatment of the cells with inhibitors of the activation of the nuclear transcription factor NF-kappaB, such as L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK), pyrrolidine dithiocarbamate (PDTC), rotenone or calpain inhibitor I, but not by the tyrosine kinase inhibitors, tyrphostin AG126 or genistein . In contrast, genistein or tyrphostin AG126 also prevented the induction of iNOS protein and activity in J774.2 macrophages elicited by LPS . Thus, the increase in the expression of ICAM-1 on J774.2 macrophages by endotoxin involves the activation of NFkappaB, but not of protein tyrosine kinase.

Gen Physiol Biophys, 1999 Oct, 18 Spec No, 70 - 4
Use of single cell gel electrophoresis (comet assay) modifications for analysis of DNA damage; Horvathova E et al.; Single cell gel electrophoresis (SCGE) or comet assay is a rapid and sensitive fluorescent microscopic method which allows measurement of DNA strand breaks in individual cells . Modifications of SCGE conditions permitted to detect different types of DNA damage . In order to characterize DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2) in Chinese hamster V79 cells, two approaches were used: (1) two pH values of unwinding and electrophoresis solutions (pH > or = 13.0 and pH = 12.1) to specify the type of DNA lesions {the alkali-labile sites and true DNA single-strand breaks (ssb)} and (2) DNA glycosylases {endonuclease III (EndoIII) and formamidopyrimidine-DNA glycosylase (FaPy)} or DNA inhibitors {hydroxyurea (HU) + 1-(beta-D-arabinofuranosyl)cytosine (AraC)} to characterize the types of DNA damage . Our results showed that the lesions induced by H2O2 represented mainly the true DNA ssb, while MNNG formed predominantly alkali-labile sites, which were converted to DNA ssb under strong alkaline conditions (pH > or = 13.0) . The effects of DNA repair enzymes and DNA inhibitors were more significant under lower pH (pH = 12.1) of unwinding and electrophoresis solution . Both, DNA glycosylases and DNA inhibitors increased the level of DNA ssb.

Mol Gen Mikrobiol Virusol, 2000, (1), 20 - 3
{Transposition and inheritance of Bordetella Tn-element in Escherichia coli K12}; Nechaeva EV et al.; B . pertussis genetically mobile element TnBp3 integrates the plasmid in E . coli chromosome . During culturing under nonselective conditions the majority of cells of some E . coli strains lose the kanamycin resistance marker, which indicates the instability of TnBp3 inheriting . The stability of inheriting the integrated structure is higher in E . coli cells with recB-21 recC-12 sbcB-2 mutations . The role of RecBC recombination system in extrusion of TnBp3 is discussed.

J Biotechnol, 2000 Feb 28, 78(1), 61 - 7
Mass-spectral analysis of human interferon-gamma and chloramphenicol acetyltransferase I produced in two Escherichia coli strains; Vassileva-Atanassova A et al.; Recombinant human interferon-gamma and chloramphenicol acetyltransferase I were isolated from two Escherichia coli strains, E . coli LE329 and E . coli XL1-blue and characterized by electrospray ionization mass spectrometry (ESI-MS) . The ESI-MS analysis showed higher masses in comparison with the theoretically calculated for both proteins as well as unexpected molecular heterogeneity . The ESI-MS spectral patterns of the proteins depended on the host strain used and were more heterogenous for the proteins isolated from E . coli LE392 . One of the proteins (human interferon-gamma obtained from E . coli XL1-blue) was further subjected to BrCN cleavage . The ESI-MS analysis of the polypeptide mixture revealed shift in the molecular mass for two peptides including the last 26 amino acids of the human interferon-gamma molecule.

J Biotechnol, 2000 Feb 28, 78(1), 49 - 59
Synthesis and purification of a deleted human growth hormone, hGH delta 135-146: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities; Alam KS et al.; Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity . In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system . The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro . The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay . In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH . In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations . By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH . These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.

Clin Diagn Lab Immunol, 2000 Mar, 7(2), 265 - 73
Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli; Johnson JR et al.; Repetitive-element PCR (rep-PCR) fingerprinting is a promising molecular typing tool for Escherichia coli, including for discriminating between pathogenic and nonpathogenic clones, but is plagued by irreproducibility . Using the ERIC2 and BOXA1R primers and 15 E . coli strains from the ECOR reference collection (three from each phylogenetic group, as defined by multilocus enzyme electrophoresis {MLEE}, including virulence-associated group B2), we rigorously assessed the effect of extremely elevated annealing temperatures on rep-PCR's reproducibility, discriminating power, and ability to reveal MLEE-defined phylogenetic relationships . Modified cycling conditions significantly improved assay reproducibility and discriminating power, allowing fingerprints from different cyclers to be analyzed together with minimal loss of resolution . The correspondence of rep-PCR with MLEE with respect to tree structure and regression analysis of distances was substantially better with modified than with standard cycling conditions . Nonetheless, rep-PCR was only a fair surrogate for MLEE, and when fingerprints from different days were compared, it failed to distinguish between different clones within all-important phylogenetic group B2 . These findings indicate that although the performance and phylogenetic fidelity of rep-PCR fingerprinting can be improved substantially with modified assay conditions, even when so improved rep-PCR cannot fully substitute for MLEE as a phylogenetic typing method for pathogenic E . coli.

Vet Microbiol, 2000 Feb, 71(3-4), 201 - 10
Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein; Rosati S et al.; The gene encoding the P48 major surface lipoprotein of M . agalactiae has been recently characterised . Since its product plays an important role in the immune response of infected animals, in this study we analysed a recombinant P48 expressed in E . coli . Multiple point mutations were introduced by site directed mutagenesis in order to convert four tryptophan TGA codons, which are a typical feature of the mycoplasma genetic code, into the standard TGG . The mutated p48 gene was subcloned into pGex-2T and expressed in fusion with glutathione-S transferase . Following purification steps, P48 was eluted from carrier protein by thrombin digestion and used in Western blot and indirect ELISA using well-characterised sheep sera . Results demonstrate that specific antibodies against P48 are detected 3 weeks after onset of clinical disease and the recombinant P48 is a diagnostically relevant marker of M . agalactiae infection.

Domest Anim Endocrinol, 2000 Jan, 18(1), 133 - 43
Effect of endotoxin challenge on hepatic 5'-deiodinase activity in cattle; Kahl S et al.; Thyroid status is compromised in a variety of acute and chronic infections and toxin-mediated disease states . Conversion of thyroxine (T4) into the metabolically active hormone, triiodothyronine (T3), is catalyzed by 5'-deiodinase (5'D) . Our objective was to determine the effect of endotoxin (LPS) challenge with and without L-arginine (Arg) infusion on hepatic activity of 5'D and plasma concentrations of T4 and T3 . In a 2 x 2 factorial, beef heifers (275-310 kg b.wt.) were fed low (8% CP; 6.5 kg/d) or high (14% CP; 7.2 kg/d) isocaloric protein diets (1.96 Mcal/kg DM) for 10 d before LPS challenge . L-Arginine in saline (0.5 g/kg b.wt.) or saline alone was infused i.v . throughout an 8 hr period starting 2 hr before bolus LPS injection (Escherichia coli, 055: B5; 0.2 microg/kg; i.v.) . Blood samples were collected at -2, 0, 3, 6, 12, and 24 hr relative to LPS injection . Liver samples were obtained 20 hr before, and then 6 and 24 hr after LPS challenge using a biopsy needle . Plasma T4 and T3 concentrations were not affected by dietary CP or Arg . Compared with levels at 0 hr, LPS challenge decreased plasma T4 (P < 0.01) and T3 (P < 0.001), respectively, 8.4% and 28.9% at 6 hr and 19.7% and 31.3% at 24 hr . Consistent with these changes, the T3:T4 ratio was lower than that at 0 hr (P < 0.001) 22.0% at 6 hr and 13.5% at 24 hr . Hepatic 5'D activities 20 hr before LPS injection were 2.80 +/- 0.11 nmol I- x hr(-1) x mg protein(-1) and decreased 24 hr after LPS, respectively, 45.4% (P < 0.01) and 17.6% (P < 0.05) in saline- and Arg-infused heifers . The results indicate that mild LPS challenge in cattle inhibits hepatic generation of T3 and decreases plasma concentrations of thyroid hormones . The data also suggest that the impact of LPS on 5'D activity in liver can be altered by Arg supplementation.

Domest Anim Endocrinol, 2000 Jan, 18(1), 71 - 82
Physiological response to acute endotoxemia in swine: effect of genotype on energy metabolites and leptin; Leininger MT et al.; Certain high lean gain swine genotypes have greater sensitivity to pathogen and nonpathogen stressors evident by reduced productivity and increased mortality during disease stress or in suboptimal production environments . Saline (control) and an immunologic challenge (LPS; 25 microg lipopolysaccharide/kg BW) were administered to three genetic populations (each pig used as its own control): high lean (H), moderate lean terminal cross (MT), and moderate lean maternal cross (MM) . LPS induced anorexia, and significantly increased body temperature and circulating TNF-alpha, cortisol, and NEFA in all genotypes (P < 0.0004) . LPS reduced circulating glucose, insulin, and IGF-1 in all genotypes (P < 0.05) . The LPS-induced hypoglycemia was significantly greater in MM versus MT and H pigs (P < 0.03) . The hypoinsulinemia was significantly greater in MM versus H pigs (P < 0.02) . MM pigs recovered from hypoinsulinemia slower than MT pigs (P < 0.03) . Control insulin was higher in H versus MT pigs (P < 0.08), but relative to basal, the insulin response to LPS was similar . Plasma haptoglobin response to LPS was lower for MM versus MT and H pigs (P < 0.02), and tended to be lower in MT versus H pigs (P < 0.09) . LPS treatment caused similar decreases in plasma IGF-1 concentrations among genotypes . Ten hours after LPS treatment, leptin mRNA abundance in adipose tissue was significantly reduced (relative to control) in MM and H pigs (P < 0.02) but not in MT pigs (P > 0.05) . Physiological differences in leptin, a potent regulator of food intake and energy metabolism, may be important factors in the genetic variation in sensitivity to environmental stress.

Anal Chem, 2000 Feb 15, 72(4), 777 - 90
Tuning of an electrospray ionization source for maximum peptide-ion transmission into a mass spectrometer; Geromanos S et al.; We describe assembly and optimization of a continuous flow nanoelectrospray source for high-performance analysis on a routine basis . It is derived from an inJection adaptable Fine Ionization Source ("JaFIS"), previously shown to be durable and easy to use (Geromanos, S.; et al . Rapid Commun . Mass Spectrom . 1998, 12, 551-556) and now modified for maximum sensitivity . Proper design, manufacturing, and quality control of spray needles with specific orifice diameters, in combination with precisely controlled helium backpressure and applied voltage, enable stable flows at 1-2 nL/min . Needle positioning and ion spray potential are hereby exceedingly important, as shifts by 0.5 mm or 25 V, respectively, cause significant reduction in signal strength . In addition to prolonged analysis times, ultralow flows also yield higher sensitivity, the result of an improved "overall ion transfer efficiency" measured to be approximately 5% at 1.6 nL/min . Used in combination with a "microtip" (Erdjument-Bromage, H.; et al . J . Chromatogr . A 1998, 826, 167-181), the optimized JaFIS implements infusion-style ESI-MS at sensitivities approaching capillary LC-MS . Spraying times in excess of 20 h allow for any number of tandem mass spectrometric analysis routines to be performed, and to average thousands of scans in every experiment, thereby further improving sensitivity . This was fully illustrated by extensive analysis of a 2-fmol peptide mixture, in a 2-microL volume, using a multimode MS approach.

Arch Biochem Biophys, 2000 Mar 15, 375(2), 385 - 8
Functional expression of an Arabidopsis cDNA clone encoding a flavonol 3'-O-methyltransferase and characterization of the gene product; Muzac I et al.; We report that the cDNA clone (Accession No . U70424), previously isolated from Arabidopsis thaliana as encoding a caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT) (1), has now been overexpressed in Escherichia coli BL21 and its recombinant protein identified as a novel flavonol 3'-OMT . It is, therefore, renamed AtOMT1 . This cDNA clone has previously been identified on the basis of its 88% amino acid sequence similarity and 80% identity to the aspen bispecific lignin OMT (2), the type member of the group involved in lignin biosynthesis . Our data indicate that this novel OMT uses the flavonol quercetin as the preferred substrate, but neither of the hydroxycinnamic acids, caffeic or 5-hydroxyferulic, to any significant extent . This indicates that the high sequence similarity/identity of AtOMT1 to that of the aspen lignin OMT (2) is not sufficient to assign the function of this gene product .

Arch Biochem Biophys, 2000 Mar 15, 375(2), 364 - 70
Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation; Lukacin R et al.; Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the course of flavonol/anthocyanin or catechin biosynthesis . The enzyme from Petunia hybrida consists of a 41,655-Da polypeptide that is prone to rapid proteolysis in crude plant extracts as well as on expression in Escherichia coli, and commercial protease inhibitors were inefficient in stopping the degradation . To pinpoint the primary site of proteolysis and to improve the activity yields, two revised schemes of purification were developed for the recombinant polypeptides . Applying a four-step protocol based on extraction and ion-exchange chromatography at pH 7.5, the primary, catalytically inactive proteolytic enzyme fragment (1.1 mg) was isolated and shown to cross-react on Western blotting as one homogeneous band of about 38 kDa . Mass spectrometric analysis assigned a mass of 37,820 +/- 100 Da to this fragment, and partial sequencing revealed an unblocked amino terminus identical to that of the native 3beta-hydroxylase . Thus, the native enzyme had been degraded by proteolysis of a small carboxy-terminal portion, and the primary site of cleavage must be assigned most likely to the Glu 337-Leu 338 bond, accounting for a loss of about 3800 Da . Alternatively, the enzyme degradation was greatly reduced when the extraction of recombinant bacteria was carried out with phosphate buffer at pH 5.5 followed by size exlusion and anion-exchange chromatography . This rapid, two-step purification resulted in a homogeneous 3beta-hydroxylase of high specific acitivity (about 32 mkat/kg) at roughly 5% yield, and the procedure is a major breakthrough in mechanistic investigations of this class of labile dioxygenases .

Arch Biochem Biophys, 2000 Mar 15, 375(2), 261 - 9
Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-beta-ocimene synthase; Bohlmann J et al.; The Arabidopsis genome project has recently reported sequences with similarity to members of the terpene synthase (TPS) gene family of higher plants . Surprisingly, several Arabidopsis terpene synthase-like sequences (AtTPS) share the most identity with TPS genes that participate in secondary metabolism in terpenoid-accumulating plant species . Expression of a putative Arabidopsis terpene synthase gene, designated AtTPS03, was demonstrated by amplification of a 392-bp cDNA fragment using primers designed to conserved regions of plant terpene synthases . Using the AtTPS03 fragment as a hybridization probe, a second AtTPS cDNA, designated AtTPS10, was isolated from a jasmonate-induced cDNA library . The partial AtTPS10 cDNA clone contained an open reading frame of 1665 bp encoding a protein of 555 amino acids . Functional expression of AtTPS10 in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate (C(10)) into the acyclic monoterpenes beta-myrcene and (E)-beta-ocimene and small amounts of cyclic monoterpenes . Based on sequence relatedness, AtTPS10 was classified as a member of the TPSb subfamily of angiosperm monoterpene synthases . Sequence comparison of AtTPS10 with previously cloned monoterpene synthases suggests independent events of functional specialization of terpene synthases during the evolution of terpenoid secondary metabolism in gymnosperms and angiosperms . Functional characterization of the AtTPS10 gene was prompted by the availability of Arabidopsis genome sequences . Although Arabidoposis has not been reported to form terpenoid secondary metabolites, the unexpected expression of TPS genes belonging to the TPSb subfamily in this species strongly suggests that terpenoid secondary metabolism is active in the model system Arabidopsis .

Nat Struct Biol, 2000 Mar, 7(3), 209 - 14
A closer view of the conformation of the Lac repressor bound to operator; Bell CE et al.; Crystal structures of the Lac repressor, with and without isopropyithiogalactoside (IPTG), and the repressor bound to operator have provided a model for how the binding of the inducer reduces the affinity of the repressor for the operator . However, because of the low resolution of the operator-bound structure (4.8 A), the model for the allosteric transition was presented in terms of structural elements rather than in terms of side chain interactions . Here we have constructed a dimeric Lac repressor and determined its structure at 2.6 A resolution in complex with a symmetric operator and the anti-inducer orthonitrophenylfucoside (ONPF) . The structure enables the induced (IPTG-bound) and repressed (operator-bound) conformations of the repressor to be compared in atomic detail . An extensive network of interactions between the DNA-binding and core domains of the repressor suggests a possible mechanism for the allosteric transition.

Nat Struct Biol, 2000 Mar, 7(3), 205 - 9
Substrate-induced exposure of an energy-coupling motif of a membrane transporter; Merianos HJ et al.; BtuB is an outer membrane protein responsible for the uptake of vitamin B12 by Escherichia coli . It belongs to a family of bacterial transport proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling protein TonB . Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB . In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter . Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm . This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion . In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate . These data suggest that the TonB box extends into the periplasm and interacts with TonB only in

Nat Struct Biol, 2000 Mar, 7(3), 196 - 9
Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli; McCarthy AA et al.; DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm . DsbC is a 2 x 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity . We report the 1.9 A resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds . The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain . The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.

Nat Biotechnol, 2000 Mar, 18(3), 317 - 20
Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine; May O et al.; Using directed evolution, we have improved the hydantoinase process for production of L-methionine (L-met) in Escherichia coli . This was accomplished by inverting the enantioselectivity and increasing the total activity of a key enzyme in a whole-cell catalyst . The selectivity of all known hydantoinases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer leads to the accumulation of intermediates and reduced productivity for the L-amino acid . We used random mutagenesis, saturation mutagenesis, and screening to convert the D-selective hydantoinase from Arthrobacter sp . DSM 9771 into an L-selective enzyme and increased its total activity fivefold . Whole E . coli cells expressing the evolved L-hydantoinase, an L-N-carbamoylase, and a hydantoin racemase produced 91 mM L-met from 100 mM D,L-MTEH in less than 2 h . The improved hydantoinase increased productivity fivefold for >90% conversion of the substrate . The accumulation of the unwanted intermediate D-carbamoyl-methionine was reduced fourfold compared to cells with the wild-type pathway . Highly D-selective hydantoinase mutants were also discovered . Enantioselective enzymes rapidly optimized by directed evolution and introduced into multienzyme pathways may lead to improved whole-cell catalysts for efficient production of chiral compounds.

Int J Cancer, 2000 Feb 15, 85(4), 571 - 7
Catalytic activity of an in vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein; Bhatia J et al.; Antibody-directed enzyme prodrug therapy (ADEPT) targets an enzyme selectively to a tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug . Previous clinical work using antibody-enzyme chemical conjugates has been limited by the moderate efficiency of tumor targeting of these molecules . To address this a recombinant fusion protein composed of MFE-23, an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody, fused to the amino-terminus of the enzyme carboxypeptidase G2 (CPG2) has been constructed to achieve ADEPT in CEA-producing tumors . MFE-23::CPG2 fusion protein was overexpressed in Escherichia coli and purified using CEA affinity chromatography . Efficacy of MFE-23::CPG2 delivery to tumors in vivo was assessed by measuring catalytic activity after intravenous injection of purified MFE-23::CPG2 into nude mice bearing CEA-positive LS174T human colon adenocarcinoma xenografts . Recombinant MFE-23::CPG2 cleared rapidly from circulation and catalytic activity in extracted tissues showed tumor to plasma ratios of 1.5:1 (6 hr), 10:1 (24 hr), 19:1 (48 hr) and 12:1 (72 hr) . (125)I-MFE-23::CPG2 was retained in kidney, liver and spleen but MFE-23::CPG2 catalytic activity was not, resulting in excellent tumor to normal tissue enzyme ratios 48 hr after injection . These were 371:1 (tumor to liver), 450:1 (tumor to lung), 562:1 (tumor to kidney), 1,477:1 (tumor to colon) and 1,618:1 (tumor to spleen) . Favorable tumor : normal tissue ratios occurred at early time points when there was still 21% (24 hr) and 9.5% (48 hr) of the injected activity present per gram of tumor tissue . The high tumor concentrations and selective tumor retention of active enzyme delivered by MFE-23::CPG2 establish that this recombinant fusion protein has potential to give improved clinical efficiency for ADEPT .

Biotechnol Bioeng, 2000 Apr 5, 68(1), 115 - 20
Development and characterization of an oxygen-dependent inducible promoter system, the modified nar promoter in a mutant Escherichia coli; Han SJ et al.; A nar promoter system (a modified nar promoter in a mutant host Escherichia coli (pMW618/W3110narL(-))), which is maximally induced under microaerobic conditions, was developed and characterized through batch and fed-batch culture to see whether the modified nar promoter can be used as an oxygen-dependent inducible promoter in the absence of nitrate ion . The modified nar promoter (pMW618) derived by mutations at -10 and -35 regions of the wild-type nar promoter does not require nitrate ion for the full induction, while a mutant host E . coli, W3110narL(-), does not express nitrate-dependent regulatory protein, NARL, from the host chromosome . In this study, it was found from fed-batch culture that the specific beta-galactosidase activity expressed from the lacZ gene fused to the modified nar promoter in the absence of nitrate ion was maximal when E . coli was grown under aerobic conditions (dissolved oxygen (DO) at 80%) to absorbance at 600 nm (OD(600)) of 35, and then the modified nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic conditions . The maximal specific beta-galactosidase activity became 58,000 Miller at OD(600) of 160 with an induction ratio of 20 . On the basis of these results, we conclude that the modified nar promoter system (pMW618/W3110narL(-)), requiring only reduction of DO for the full induction, provides a convenient and effective high-level expression system under conditions of fed-batch culture .

Biotechnol Bioeng, 2000 Mar 20, 67(6), 827 - 40
Computer model for glucose-limited growth of a single cell of Escherichia coli B/r-A . Reprinted from Biotechnology and Bioengineering, Vol . 26, Issue 3, Pp 203-216 (1984)
Domach MM, Leung SK, Cahn RE, Cocks GG, Shuler ML.
A computer model is described which is capable of predicting changes in cell composition, cell size, cell shape, and the timing of chromosome synthesis in response to changes in external glucose limitation . The model is constructed primarily from information on unrestricted growth in glucose minimal medium . The ability of the model to make reasonable quantitative predictions under glucose-limitation is a test of the plausibility of the basic biochemical mechanisms included in the model . Such a model should be of use in differentiating among competing hypotheses for biological mechanisms and in suggesting as yet unobserved phenomena . The last two points are illustrated with the testing of a mechanism for the control of the initiation of DNA synthesis and predictions on cell-width variations during the division cycle.

Biotechnol Bioeng, 2000 Mar 20, 67(6), 805 - 12
Analysis of growth rate effects on productivity of recombinant Escherichia coli populations using molecular mechanism models . Reprinted from Biotechnology and Bioengineering, Vol . 26, Issue 1, Pages 66-73 (1984)
Lee SB, Bailey JE.
The influence of growth rate on Escherichia coli plasmid content and expression of a cloned-gene product has been described by a mathematical model based upon the molecular mechanism of lambdadv plasmid replication and known relationships between growth rate and transcription and translation activities of the host cell . The model simulates correctly decreases in plasmid content with increasing growth rate as observed experimentally for pBR322, NR1, R1, and Col E1 plasmids . A maximum with respect to growth rate in intracellular product accumulation is indicated by the model, as is a transient overshoot in product concentration following a shift from smaller to larger growth rate . Available data, although very limited, show the same trends . These results, obtained without parameter or kinetic form adjustments or manipulation, clearly illustrate the advantages of kinetic descriptions of recombinant systems based upon the pertinent molecular mechanisms.

J Microbiol Methods, 2000 Apr, 40(2), 153 - 62
Critical factors influencing the recovery and integrity of rRNA extracted from environmental samples: use of an optimized protocol to measure depth-related biomass distribution in freshwater sediments; Alm EW et al.; A protocol was developed for the efficient recovery of intact, high molecular weight rRNA from different environmental matrices . Critical variables were identified in sample processing that influenced yield and integrity of recovered nucleic acid . Most notably, the order of addition and the buffer to sample volume ratio profoundly influenced the efficiency of nucleic acid recovery from sediment material when utilizing a guanidine thiocyanate-beta-mercaptoethaol extraction buffer . Addition of one sample volume to five buffer volumes contributed to an order of magnitude increase in recovery relative to reverse order of addition (buffer addition to sample) . An optimized extraction protocol was used to evaluate rRNA yield by seeding samples with whole cells and radiolabeled nucleic acid . Recovery of intact rRNA was confirmed by polyacrylamide gel electrophoresis, which was also used to provide another estimate of quantity . This optimized protocol was used to measure depth-related changes in biomass distribution in Lake Michigan deep-water sediments . This revealed a biomodal biomass distribution; a maximum near the water/sediment interface and a secondary peak associated with the oxic/suboxic boundary . A significant portion of the community at the oxic/suboxic boundary was composed of non-methanogenic Archaea.

J Immunol Methods, 2000 Mar 6, 236(1-2), 147 - 65
Characterizing the functionality of recombinant T-cell receptors in vitro: a pMHC tetramer based approach; Tissot AC et al.; The very low affinity of the T-cell receptor (TCR) for the peptide-major histocompatibility complex (pMHC) has made it very challenging to design assays for testing the functionality of these molecules on small scales, which in turn has severely hampered the progress in developing expression and refolding methodologies for the TCR . We have now developed an ELISA assay for detecting pMHC binding to functional recombinant TCRs . It uses tetramers of biotinylated pMHCs bound to a neutravidin-horseradish peroxidase conjugate and detects the presence of functional TCR, bound in a productive orientation to an immobilized anti-Cbeta antibody . Specificity can be stringently demonstrated by inhibition with monomeric pMHCs . The assay is very sensitive and specific, and requires only very small amounts of protein . It has allowed us to study the unstable recombinant TCR P14, which we expressed and refolded from Escherichia coli . The TCR P14 is directed against the most abundant epitope of LCMV . We have confirmed the specificity of the interaction by BIAcore, and were able to determine the dissociation constant of the interaction of the P14 TCR and of the gp33-pMHC as 6 microM . This affinity ranks it among the tighter ones of TCR-pMHC interactions, and unusually low affinity thus does not seem to be the cause of the modest protective power of these T-cells, compared to others elicited in the anti-LCMV response . This strategy of multimerizing one partner and immobilizing the other in both a native form and productive orientation should be generally useful for characterizing the weak interactions of cell-surface molecules.

J Immunol Methods, 2000 Mar 6, 236(1-2), 133 - 46
Sheep monoclonal antibody fragments generated using a phage display system; Li Y et al.; Monoclonal sheep antibodies have great potential for biomedical, veterinary and agricultural purpose . Although conventional sheep monoclonal antibodies can be generated by a modified hybridoma technology, the procedures are not routine . Here, we describe a method to generate recombinant sheep antibody fragments from immunised animals using a modified phage display system . Total RNA from pooled spleens of sheep immunised with the model antigens human serum albumin and conalbumin were used to amplify immunoglobulin V gene repertoires and an efficient two-step cloning method was employed to rapidly construct a phage display single-chain Fv (scFv) library . A total of 14 different scFvs were isolated and characterised . Sequence analysis indicated typical ovine immunoglobulin characteristics . Thirteen Vlambda and 11 VH genes were identified that could be grouped into the sheep Vlambda families I, II, VI and a single VH family . Soluble monomeric scFvs, produced in the periplasm of Escherichia coli, were subjected to affinity measurement via surface plasmon resonance analysis and affinities typical of the secondary immune response were observed . The method described here should be of value for the study of sheep immunology as well as for biorecognition in general.

Vaccine, 2000 Mar 17, 18(18), 1944 - 51
Effectiveness of intranasal immunization with HIV-gp160 and an HIV-1 env CTL epitope peptide (E7) in combination with the mucosal adjuvant LT(R192G); Morris CB et al.; LT(R192G) is a novel mucosal adjuvant that induces protective immunity when co-administered with certain whole inactivated bacteria or viruses or with subunits of relevant virulence determinants from these pathogens . LT(R192G) stimulates antigen-specific humoral and cellular immune responses, both systemically and in mucosal compartments, and is safe and nontoxic at adjuvant effective doses . Intranasal (IN) immunization of mice with LT(R192G) in conjunction with oligomeric HIV-1 gp160 elevates antigen-specific systemic and mucosal IgG and IgA production and Th1- and Th2-type cytokine responses . Isotype characterization of induced IgG reveals that gp160 alone fails to stimulate IgG2a responses in the absence of adjuvant . Both IgG1 and IgG2a are induced by immunization in the presence of LT(R192G) . Additionally, intranasal immunization with a 15-amino acid peptide corresponding to an HIV-1 Env CTL determinant and LT(R192G) induces systemic, peptide-specific CTL activity and Th1 and Th2 cytokine responses that are absent when the adjuvant is excluded from the immunizations . These studies show that LT(R192G) quantitatively and qualitatively enhances cellular and humoral HIV-specific immune responses and that this adjuvant may offer significant advantages toward vaccine development against HIV.

Vaccine, 2000 Mar 6, 18(17), 1802 - 10
Reversible protection of disulfide bonds followed by oxidative folding render recombinant hCGbeta highly immunogenic; Mukhopadhyay A; Active immunization of women against human chorionic gonadotropin (hCG) has been considered as a promising option for contraception . However, prototype hCG vaccines based on natural sources of antigen are expected to be costlier for use by common people . In the present report, a functionally active, cost-effective antigen of bacterial origin has been described . Sulfonation of thiol groups of the protein, anion-exchange purification, refolding with concomitant formation of disulfide bonds in the presence of cysteamine-cystamine redox buffer, and slow removal of denaturant resulted in 95% homogeneous, monomeric form of the antigen . The recombinant processed antigen {CGbeta(p)} obtained this way was highly immunopotent . Cellular DNA and endotoxin contaminants were appreciably low in the final product . The immunogenic response was drastically reduced with the unprocessed antigen . This finding envisages better prospect of a cost-effective hCG vaccine for birth control.

Mol Biochem Parasitol, 2000 Mar 5, 106(2), 213 - 24
Expression and characterisation of Plasmodium falciparum acidic basic repeat antigen expressed in Escherichia coli; Kushwaha A et al.; The acidic basic repeat antigen (ABRA) of Plasmodium falciparum has been localised on the merozoite surface and in the parasitophorous vacuole . It is one of the antigens enriched in the clusters of merozoites formed with growth inhibitory immune serum and possesses chymotrypsin-like activity . Chymostatin, an inhibitor of chymotrypsin, inhibits malaria invasion as well as autoproteolysis of ABRA . Based on these characteristics of ABRA, it seems important for invasion and should be investigated as a target for vaccine and drug design . For the functional characterisation of this protein, the full-length mature ABRA protein and its fragments with/without the putative protease active site were cloned, expressed and purified from Escherichia coli . The polyclonal serum raised against recombinant ABRA fragment recognised a parasite protein with a mobility of 101 kDa in an immunoblot assay and showed immunofluorescence activity with a schizont-rich preparation of P . falciparum . Using a partially purified fragment containing the putative active site and fluorogenic and chromogenic substrates, we established that the protease activity of ABRA resides in the N-terminal portion of the protein and the highly charged C-terminal part of the protein is not required for this activity . The protease activity of ABRA was inhibited with serine protease inhibitors like chymostatin and phenyl methyl sulfonyl fluoride (PMSF) whereas leupeptin was not able to inhibit this enzyme activity . These results clearly indicated that ABRA is a protease with chymotrypsin-like specificity . This is the first report describing the expression and characterisation of recombinant ABRA protein.

FASEB J, 2000 Mar, 14(3), 603 - 11
Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display; Goldman E et al.; An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage . The sequence encoded a peptide that bound to the growth hormone binding protein . Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame . Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences . In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene . These sequences have been cloned in all three reading frames relative to the reporter . The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames . A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame . By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half . We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.

EMBO J, 2000 Mar 1, 19(5), 1148 - 56
Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing; Mazin AV et al.; The repair of potentially lethal DNA double-stranded breaks (DSBs) by homologous recombination requires processing of the broken DNA into a resected DNA duplex with a protruding 3'-single-stranded DNA (ssDNA) tail . Accordingly, the canonical models for DSB repair require invasion of an intact homologous DNA template by the 3'-end of the ssDNA, a characteristic that the bacterial pairing protein RecA possesses . Unexpectedly, we find that for the eukaryotic homolog, Rad51 protein, the 5'-end of ssDNA is more invasive than the 3'-end . This pairing bias is unaffected by Rad52, Rad54 or Rad55-57 proteins . However, further investigation reveals that, in contrast to RecA protein, the preferred DNA substrate for Rad51 protein is not ssDNA but rather dsDNA with ssDNA tails . This important distinction permits the Rad51 proteins to promote DNA strand invasion using either 3'- or 5'-ends with similar efficiency.

EMBO J, 2000 Mar 1, 19(5), 1130 - 7
Escherichia coli promoter opening and -10 recognition: mutational analysis of sigma70; Fenton MS et al.; The opening of specific segments of DNA is required for most types of genetic readout, including sigma70-dependent transcription . To learn how this occurs, a series of single point mutations were introduced into sigma70 region 2 . These were assayed for duplex DNA binding, DNA opening and DNA double strand-single strand fork junction binding . Band shift assays for closed complex formation implicated a series of arginine and aromatic residues within a minimal 26 amino acid region . Permanganate assays implicated two additional aromatic residues in DNA opening, known to form a parallel stack of the type that can accept a flipped-out base . Substitution for either of these aromatics had no effect on duplex probe recognition . However, when a single unpaired -11 nucleotide is added to the probe, the mutants fail to bind appropriately to give heparin resistance . A model for DNA opening is presented in which duplex recognition by regions 2.3, 2.4 and 2.5 of sigma positions the pair of aromatic amino acids, which then create the fork junction required for stable opening.

EMBO J, 2000 Mar 1, 19(5), 1045 - 54
Structure of the RXR-RAR DNA-binding complex on the retinoic acid response element DR1; Rastinejad F et al.; The 9-cis retinoic acid receptor (retinoid X receptor, RXR) forms heterodimers with the all-trans retinoic acid receptor (RAR) and other nuclear receptors on DNA regulatory sites composed of tandem binding elements . We describe the 1.70 A resolution structure of the ternary complex of RXR and RAR DNA-binding regions in complex with the retinoic acid response element DR1 . The receptors recognize identical half-sites through extensive base-specific contacts; however, RXR binds exclusively to the 3' site to form an asymmetric complex with the reverse polarity of other RXR heterodimers . The subunits associate in a strictly DNA-dependent manner using the T-box of RXR and the Zn-II region of RAR, both of which are reshaped in forming the complex . The protein-DNA contacts, the dimerization interface and the DNA curvature in the RXR-RAR complex are distinct from those of the RXR homodimer, which also binds DR1 . Together, these structures illustrate how the nuclear receptor superfamily exploits conformational flexibility and locally induced structures to generate combinatorial transcription factors.

EMBO J, 2000 Mar 1, 19(5), 852 - 61
SecYEG assembles into a tetramer to form the active protein translocation channel; Manting EH et al.; Translocase mediates preprotein translocation across the Escherichia coli inner membrane . It consists of the SecYEG integral membrane protein complex and the peripheral ATPase SecA . Here we show by functional assays, negative-stain electron microscopy and mass measurements with the scanning transmission microscope that SecA recruits SecYEG complexes to form the active translocation channel . The active assembly of SecYEG has a side length of 10.5 nm and exhibits an approximately 5 nm central cavity . The mass and structure of this SecYEG as well as the subunit stoichiometry of SecA and SecY in a soluble translocase-precursor complex reveal that translocase consists of the SecA homodimer and four SecYEG complexes.

EMBO J, 2000 Mar 1, 19(5), 831 - 42
X-ray structure of MalY from Escherichia coli: a pyridoxal 5'-phosphate-dependent enzyme acting as a modulator in mal gene expression; Clausen T et al.; MalY represents a bifunctional pyridoxal 5'-phosphate-dependent enzyme acting as a beta-cystathionase and as a repressor of the maltose regulon . Here we present the crystal structures of wild-type and A221V mutant protein . Each subunit of the MalY dimer is composed of a large pyridoxal 5'-phosphate-binding domain and a small domain similar to aminotransferases . The structural alignment with related enzymes identifies residues that are generally responsible for beta-lyase activity and depicts a unique binding mode of the pyridoxal 5'-phosphate correlated with a larger, more flexible substrate-binding pocket . In a screen for MalY mutants with reduced mal repressor properties, mutations occurred in three clusters: I, 83-84; II, 181-189 and III, 215-221, which constitute a clearly distinguished region in the MalY crystal structure far away from the cofactor . The tertiary structure of one of these mutants (A221V) demonstrates that positional rearrangements are indeed restricted to regions I, II and III . Therefore, we propose that a direct protein-protein interaction with MalT, the central transcriptional activator of the maltose system, underlies MalY-dependent repression of the maltose system.

EMBO J, 2000 Mar 1, 19(5), 807 - 18
Crystal structure of ribosomal protein L4 shows RNA-binding sites for ribosome incorporation and feedback control of the S10 operon; Worbs M et al.; Ribosomal protein L4 resides near the peptidyl transferase center of the bacterial ribosome and may, together with rRNA and proteins L2 and L3, actively participate in the catalysis of peptide bond formation . Escherichia coli L4 is also an autogenous feedback regulator of transcription and translation of the 11 gene S10 operon . The crystal structure of L4 from Thermotoga maritima at 1.7 A resolution shows the protein with an alternating alpha/beta fold and a large disordered loop region . Two separate binding sites for RNA are discernible . The N-terminal site, responsible for binding to rRNA, consists of the disordered loop with flanking alpha-helices . The C-terminal site, a prime candidate for the interaction with the leader sequence of the S10 mRNA, involves two non-consecutive alpha-helices . The structure also suggests a C-terminal protein-binding interface, through which L4 could be interacting with protein components of the transcriptional and/or translational machineries.

Appl Environ Microbiol, 2000 Mar, 66(3), 1205 - 8
A new Shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons; Schmidt H et al.; We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx(2f) . This gene is most similar to sltIIva of patient E . coli O128:B12 isolate H.I.8 . Stx2f reacted only weakly with commercial immunoassays . The prevalence of STEC organisms carrying the stx(2f) gene in pigeon droppings was 12.5% . The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E . coli pathogenic to humans may occur.

Appl Environ Microbiol, 2000 Mar, 66(3), 966 - 75
Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs; Gilbert B et al.; The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp . strain M, have been cloned and sequenced . Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp . strain M . Immediately upstream of the putative start site, consensus sequences for sigma(70) promoters were identified, suggesting that these pmo genes are recognized by sigma(70) and negatively regulated under low-copper conditions . The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host . Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs . The complete DNA sequence of one copy of pmo genes from each organism is reported here . The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma-Proteobacteria, Methylococcus capsulatus Bath . The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices . Hybridization experiments showed that there are two copies of pmoC in both M . trichosporium OB3b and Methylocystis sp . strain M, and not three copies as found in M . capsulatus Bath.

Appl Environ Microbiol, 2000 Mar, 66(3), 943 - 7
Expression of outer membrane proteins in Escherichia coli growing at acid pH; Sato M et al.; It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression . In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased . In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH . Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values . However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity . Thus, it appears that E . coli has a different mechanism for porin expression at acid pH . A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5) . The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.

Appl Environ Microbiol, 2000 Mar, 66(3), 884 - 9
Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli; Nishihara K et al.; To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones . The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme . The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding . Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo . Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme . These results attest to the usefulness of the present expression plasmids for improving protein production in E . coli.

Science, 2000 Mar 3, 287(5458), 1658 - 60
Resonant formation of DNA strand breaks by low-energy (3 to 20 eV) electrons; Boudaiffa B et al.; Most of the energy deposited in cells by ionizing radiation is channeled into the production of abundant free secondary electrons with ballistic energies between 1 and 20 electron volts . Here it is shown that reactions of such electrons, even at energies well below ionization thresholds, induce substantial yields of single- and double-strand breaks in DNA, which are caused by rapid decays of transient molecular resonances localized on the DNA's basic components . This finding presents a fundamental challenge to the traditional notion that genotoxic damage by secondary electrons can only occur at energies above the onset of ionization, or upon solvation when they become a slowly reacting chemical species.

Biochem J, 2000 Mar 15, 346 Pt 3, 857 - 64
Molecular cloning and expression of novel sulphotransferase-like cDNAs from human and rat brain; Falany CN et al.; The sulphotransferase (SULT) gene family is involved with the conjugation of many small drugs, xenobiotics and endogenous compounds . In this report, we describe the cloning and expression of novel cDNAs from human and rat brain, which are structurally related to the SULTs . These cDNAs have been termed 'brain sulphotransferase-like' (BR-STL), because of their similarity to the SULTs and their selective expression in brain tissue . The proteins encoded by the human and rat BR-STL cDNAs (hBR-STL-1 and rBR-STL cDNA respectively), denoted as hBR-STL and rBR-STL, are 98% identical and 99% similar in sequence . The hBR-STL-1 cDNA contains an 852-nt open reading frame encoding a 284-amino-acid protein with a calculated molecular mass of 33083 Da . Northern-blot analyses of RNA isolated from eight human tissues indicate that the hBR-STL message is selectively expressed in brain . Characterization of different brain regions showed that message levels were highest in cortical brain regions . rBR-STL message levels were relatively low in brains of 1-day-old male and female rats, but increased to adult levels in RNA from 7-day-old rats, and remained at that level in adult animals . The hBR-STL and rBR-STL cDNAs were expressed in both Escherichia coli and Sf9 insect cells in the presence or absence of an N-terminal histidine-affinity tag (His-tag) . Polyclonal antibodies were raised in chickens against purified His-tagged hBR-STL, and were used to detect the presence of rBR-STL in adult male and female rat brain cytosol . The high degree of sequence conservation, and the selective localization of the BR-STL message in brain, suggest an important function in the central nervous system.

Biochem J, 2000 Mar 15, 346 Pt 3, 737 - 42
Deuterium-labelled isotopomers of 2-C-methyl-D-erythritol as tools for the elucidation of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis; Charon L et al.; Escherichia coli synthesizes its isoprenoids via the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway . The MC4100dxs::CAT strain, defective in deoxyxylulose-5-phosphate synthase, which is the first enzyme in this metabolic route, exclusively synthesizes its isoprenoids from exogenous 2-C-methyl-D-erythritol (ME) added to the culture medium . The fate of the hydrogen atoms in the MEP pathway was followed by the incorporation of {1,1-(2)H(2)}ME and {3,5,5,5-(2)H(4)}ME . The two C-1 hydrogen atoms of ME were found without any loss in the prenyl chain of menaquinone and/or ubiquinone on the carbon atoms derived from C-4 of isopentenyl diphosphate (IPP) and on the E-methyl group of dimethylallyl diphosphate (DMAPP), the C-5 hydrogen atoms on the methyl groups derived from IPP C-5 methyl group and the Z-methyl group of DMAPP . This showed that no changes in the oxidation state of these carbon atoms occurred in the reaction sequence between MEP and IPP . Furthermore, no deuterium scrambling was observed between the carbon atoms derived from C-4 and C-5 of IPP or DMAPP, suggesting a completely stereoselective IPP isomerase or no significant activity of this enzyme . The C-3 deuterium atom of {3,5,5,5-(2)H(4)}ME was preserved only in the DMAPP starter unit and was completely missing from all those derived from IPP . This finding, aided by the non-essential role of the IPP isomerase gene, suggests the presence in E . coli of two different routes towards IPP and DMAPP, starting from a common intermediate derived from MEP.

J Pharm Biomed Anal, 1999 May, 19(6), 847 - 54
Enzyme inhibition XI: glutamate decarboxylase activity relationship with the reaction products as determined by the colorimetric and radioisotopic methods; Sethi ML; The relationship of glutamate acid decarboxylase (GAD) activity with the reaction products was developed . It was based on incubating sodium glutamate substrate (S) with GAD enzyme (E) when the enzyme-substrate-complex (ES) product was obtained along with gamma aminobutyric acid (GABA) and unreacted sodium glutamate . The reaction products were separated by paper chromatography . The ES, GABA and S products were sprayed with ninhydrin reagent when ninhydrin-colored-complex (NCC) was formed on the paper chromatogram . The products were extracted with 75% ethanol containing 0.5% cupric sulfate . The NCC absorption readings of ES and S products were measured by a spectrophotometer . A standard curve was prepared by plotting absorption readings against different concentrations of sodium glutamate . This curve was the basis of determining GAD activity of E . coli and C . welchii . It was observed that NCC absorption of ES and S products was directly related with the enzyme activity . The qualities of ES and S products in the reaction mixture increased as the enzyme activity increased with the incubation time . On the other hand, some products in the reaction mixture decreased in the presence of an inhibitor of GAD activity . The relationship of reaction products with GAD activity was also established by the radioisotopic method . The results obtained by the chromatographic separation of products followed by the spectrophotometric method of determining GAD activity is a simple, safe and less expensive compared to the other methods.

Bioorg Med Chem Lett, 2000 Feb 7, 10(3), 293 - 5
Synthesis and study of a new adenine-acridine tandem, inhibitor of exonuclease III; Belmont P et al.; A new heterodimer adenine-chain-acridine containing a mixed amido-guanidinium linker chain was synthesized . To achieve the synthesis a new method of introduction of aminoalkyl chain at position 9 of adenine was designed . The heterodimer interacts specifically with the abasic sites in DNA and inhibits the major base excision repair enzyme in Escherichia coli, Exonuclease III.

Acta Biochim Pol, 1999, 46(3), 785 - 99
Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA; Tudek B et al.; The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua) . We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions . We have analyzed which lesions lead to these mutations . We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E . coli . Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions . Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of {3H}MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions . Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of {3H}MNU alkylated poly(A) and poly(dA) . After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua . Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde . Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.

Endocrinology, 2000 Mar, 141(3), 1050 - 8
Prostaglandins mediate the endotoxin-induced suppression of pulsatile gonadotropin-releasing hormone and luteinizing hormone secretion in the ewe; Harris TG et al.; Five experiments were conducted to test the hypothesis that PGs mediate the endotoxin-induced inhibition of pulsatile GnRH and LH secretion in the ewe . Our approach was to test whether the PG synthesis inhibitor, flurbiprofen, could reverse the inhibitory effects of endotoxin on pulsatile LH and GnRH secretion in ovariectomized ewes . Exp 1-4 were cross-over experiments in which ewes received either flurbiprofen or vehicle 2 weeks apart . Jugular blood samples were taken for LH analysis throughout a 9-h experimental period . Depending on the specific purpose of the experiment, flurbiprofen or vehicle was administered after 3.5 h, followed by endotoxin, vehicle, or ovarian steroids (estradiol plus progesterone) at 4 h . In Exp 1, flurbiprofen reversed the endotoxin-induced suppression of mean serum LH concentrations and the elevation of body temperature . In Exp 2, flurbiprofen prevented the endotoxin-induced inhibition of pulsatile LH secretion and stimulation of fever, reduced the stimulation of plasma cortisol and progesterone, but did not affect the rise in circulating tumor necrosis factor-alpha . In Exp 3, flurbiprofen in the absence of endotoxin had no effect on pulsatile LH secretion . In Exp 4, flurbiprofen failed to prevent suppression of pulsatile LH secretion induced by luteal phase levels of the ovarian steroids progesterone and estradiol, which produce a nonimmune suppression of gonadotropin secretion . In Exp 5, flurbiprofen prevented the endotoxin-induced inhibition of pulsatile GnRH release into pituitary portal blood . Our finding that this PG synthesis inhibitor reverses the inhibitory effect of endotoxin leads to the conclusion that PGs mediate the suppressive effects of this immune/inflammatory challenge on pulsatile GnRH and LH secretion.

J Am Soc Mass Spectrom, 2000 Mar, 11(3), 237 - 43
Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of the recombinant human macrophage colony stimulating factor beta and derivatives; Maier CS et al.; The potential of electrospray ionization (ESI) Fourier transform ion cyclotron mass spectrometry (FTICR-MS) to assist in the structural characterization of monomeric and dimeric derivatives of the macrophage colony stimulating factor beta (rhM-CSF beta) was assessed . Mass spectrometric analysis of the 49 kDa protein required the use of sustained off-resonance irradiation (SORI) in-trap cleanup to reduce adduction . High resolution mass spectra were acquired for a fully reduced and a fully S-cyanylated monomeric derivative (approximately 25 kDa) . Mass accuracy for monomeric derivatives was better than 5 ppm, after applying a new calibration method (i.e., DeCAL) which eliminates space charge effects upon high accuracy mass measurements . This high mass accuracy allowed the direct determination of the exact number of incorporated cyanyl groups . Collisionally induced dissociation using SORI yielded b- and y-fragment ions within the N- and C-terminal regions for the monomeric derivatives, but obtaining information on other regions required proteolytic digestion, or potentially the use of alternative dissociation methods.

Drug Dev Ind Pharm, 2000 Feb, 26(2), 199 - 204
Comparison of the effect of lipid A analog E5531 and the lipid A from Escherichia coli on phospholipid membrane properties; Asai Y et al.; The effect of the lipid A analog E5531 on the phospholipid membrane was compared with that of the lipid A from Escherichia coli (EC) . E5531 decreased the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) membrane and increased the fluidity and micropolarity . On the other hand, the effect of EC on the membrane was contradictory . These results suggested that the reason for the difference of biological effects of these two lipid A would be caused by the differences from the effect on the cell membranes.

Anticancer Res, 1999 Nov-Dec, 19(6B), 5319 - 21
Enhancement of mitomycin C efficiency by vitamin C, E-acetate and beta-carotene under irradiation . A study in vitro; Kammerer C et al.; Using E.coli bacteria (AB 1157) and leukemia cells (HL 60) as a model for in vitro studies it was established that the efficiency of mitomycin C (MMC) can be influenced in the presence of antioxidant vitamins . This synergistic effect of the vitamins C, E-acetate and beta-carotene on MMC activity is rather strong for E.coli bacteria under irradiation (15 and 50 Gy) in the presence of air . Vitamin C contributes more efficiently to the MMC-activity in leukemia cells than the other two vitamins . The effect is explained by a cascade electron transfer process from the vitamins to MMC, where vitamin C is acting as a major electron source . These results might be of importance in cancer therapy.

J Trauma, 2000 Feb, 48(2), 215 - 22; discussion 222-3
Chlorpromazine modulates cytokine expression in the liver and lung after burn injury and endotoxemia; Clancy KD et al.; BACKGROUND: Previous data from our laboratory have demonstrated that alterations in cytokine production occur in the lung and liver as the result of a two-hit model of injury, i.e., burn with subsequent endotoxin administration . The purpose of this study was to determine whether the phenothiazine derivative chlorpromazine would alter cytokine production in a sequential model of injury . METHODS: By using a sublethal burn/endotoxemia model, B2D6F1 mice (n = 40) were assigned to two groups and subjected to a 15% full-thickness burn . Three days after burn injury, one group (BURN/ETX) received 2.5 mg/kg Escherichia coli endotoxin intraperitoneally, and the other group (CPZ) received 4 mg/kg chlorpromazine 1 hour before the administration of 2.5 mg/kg E . coli endotoxin intraperitoneally . At selected time points, the animals were killed and lung and liver were removed and processed for protein and total RNA . Northern blots and enzyme-linked immunosorbent assays were used to assess the production of tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, and interleukin-10 . RESULTS: Chlorpromazine significantly reduced tumor necrosis factor-alpha mRNA and protein expression in the liver . Macrophage inflammatory protein-1alpha mRNA was reduced by chlorpromazine in both liver and lung . Interleukin-10 production was not altered by chlorpromazine . CONCLUSION: The reduction of tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha by chlorpromazine in the liver and lungs may have potential as a pharmaceutical agent that may dampen the inflammatory response in a model of sequential injury.

Insect Biochem Mol Biol, 2000 Feb, 30(2), 173 - 82
Cloning, sequence analysis and expression in Escherichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata lugens; Hongoh Y et al.; A urate oxidase (uricase; EC 1.7.3.3) gene of the yeast-like fungal endosymbiont of the brown planthopper, Nilaparvata lugens, was cloned, and sequenced together with its flanking regions . The gene comprised a open reading frame of 987 bp, that was split into two parts by a single 96 bp intron . The encoded uricase was 296 amino acids with 62% sequence identity with that of Aspergillus flavus . The molecular weight deduced was 32,882, and the predicted isoelectric point was 6.06 . The symbiont's uricase conserved all the known consensus motifs, except the C-terminal PTS-1, Ser-basic-Leu . The leucine at the third position of PTS-1 was replaced by serine in the C-terminus of the symbiont's uricase . The symbiont's uricase gene was successfully expressed in Escherichia coli, and the product, tagged with histidine residues, was purified . The symbiont's uricase, thus produced, was as active as those from plants and animals, but less active than those from other fungi.

Insect Biochem Mol Biol, 2000 Feb, 30(2), 153 - 61
Isolation and sequencing of a cDNA encoding for a ribonuclease from the insect Ceratitis capitata; Sideris DC et al.; Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5' RACE . The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids . The calculated M(r) of the mature protein was found to be 13.7 kDa . Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity . Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved . Furthermore, expression of the clone in E . coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies . These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily.

Can J Microbiol, 1999 Dec, 45(12), 1017 - 26
Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1; Rioux S et al.; Lipopolysaccharide (LPS) has previously been identified as the major adhesin of Actinobacillus pleuropneumoniae involved in adherence to porcine respiratory tract cells . The purpose of the present study was to isolate and characterize mutants in LPS biosynthesis by using a mini-Tn10 transposon mutagenesis system . Seven mutants appeared to possess a rough LPS (among which two had similar Southern blot profiles) while one mutant (#5.1) expressed the high-molecular-mass LPS, but as visualized by Tricine SDS-PAGE, showed an additional band in the core-lipid A region . The LPS mutants showed sensitivity to pig serum to various degrees, while the parent strain was serum-resistant . Use of piglet frozen tracheal sections indicated that, surprisingly, the rough LPS mutants adhered similarly or in greater numbers than the parent strain . However, the LPS mutant #5.1 adhered significantly less than the parent strain and was also less virulent in pigs . The gene affected by mini-Tn10 in LPS mutant #5.1 is galU, the structural gene for UTP-alpha-D-glucose-1-phosphate uridylyltransferase, involved in LPS core biosynthesis . Complementation analysis confirmed that the phenotypic characteristics of LPS mutant #5.1 are the result of the inactivation of the galU gene . Our data suggest that although the presence of O-antigen does not seem to be essential, an intact core-lipid A region might be required for adherence of A . pleuropneumoniae to porcine respiratory tract cells . To the best of our knowledge, these mutants represent the first isogenic mutants of A . pleuropneumoniae defective in LPS biosynthetic genes.

Acta Virol, 1999 Apr-Jun, 43(2-3), 174 - 80
Identification and structural analysis of a MDV gene encoding a protein kinase; Reddy SM et al.; DNA sequence analysis of the BamHI-C fragment of Marek's Disease Virus (MDV) reveals the presence of a 513 amino acid open reading frame (ORF) . This ORF codes for a protein with an estimated M(r) of 58,901 . Comparison of the amino acid sequence with those available in the Swiss-Prot database indicates extensive homology with a protein kinase (PK) of herpes simplex virus (HSV) and varicella-zoster virus (VZV) . In Northern blot hybridization, a transcript of 2.0 kb was detected in MDV (GA strain) infected duck embryo fibroblasts (DEFs) . A portion of the ORF was expressed in Escherichia coli as a trpE-fusion protein and used to generate antiserum in New Zealand rabbits . This antiserum specifically detected a protein of 60 kDa in MDV serotype 1, 2 and 3 infected DEFs or chicken embryo fibroblasts (CEFs) by Western blot analysis . This ORF codes for a functional PK.

Acta Virol, 1999 Apr-Jun, 43(2-3), 152 - 8
Identification and characterization of glycoprotein H of MDV-1 GA strain; Wu P et al.; A 2439 bp open reading frame (ORF) was identified from the DNA sequence of BamHI-F and -K2 fragments of Marek's disease virus of serotype 1 (MDV-1) GA strain, which predicts an 813 amino acid polypeptide . This peptide is homologous to HSV-1 gH, and has typical glycoprotein features . There are nine potential N-linked glycosylation sites within the extracellular domain . A fragment of the gH ORF was cloned into pGEX vector in frame with glutathione S-transferase (GST) to produce a GST-gH fusion protein in Escherichia coli . The GST-gH fusion protein was used to develop gH monoclonal and polyclonal antibodies . Expression of gH was detected in duck embryo fibroblasts (DEFs) infected with MDV-1 GA strain by immunofluorescence assay (IFA) with these antibodies . Virus neutralization and plaque-forming inhibition analyses were conducted with the gH antiserum . There were no neutralization and plaque-forming inhibition activities of gH antiserum . Comparison of the DNA sequence of gH gene between GA and RB1B strains of MDV-1 revealed major difference in the upstream control elements of gH ORF.

Somat Cell Mol Genet, 1998 Sep, 24(5), 307 - 11
Chromosomal localization and genomic structure of the human arsenite-stimulated ATPase (hASNA-I); Kurdi-Haidar B et al.; The hASNA-I is a novel human arsenite-stimulated ATPase identified as the human paralogue of the ATPase component of the arsenite efflux system in E . coli . The hASNA-I has distinct biochemical properties and a dual nuclear and cytoplasmic distribution . Immunohistochemical staining showed a distinct pattern of hASNA-I expression in cells within normal tissues, and its overexpression in breast cancer . Recently, the yeast two-hybrid system has identified hASNA-I as a cellular partner of metallothionein II suggesting an additional role in Zn homeostasis and cellular detoxification . This report describes the assignment of hASNA-I to human chromosome 19 by somatic-cell hybrid PCR mapping, the isolation of a chromosome 19-specific cosmid clone, and the genomic structure and exon-intron boundaries of hASNA-I . Our results indicate that the coding region of hASNA-I consists of 4 exons spanning 6 kb on band 19q13.3 . These data will facilitate molecular analysis of the role of hASNA-I in human disease.

Somat Cell Mol Genet, 1998 Sep, 24(5), 263 - 72
Nucleolin promotes homologous DNA pairing in vitro; Thyagarajan B et al.; We purified to near homogeneity a previously identified 100 kDa mammalian homologous DNA pairing protein . The purified 100 kDa protein also catalyzed high levels of cell-free homologous DNA recombination activity . This ATP-dependent activity was capable of forming conservative recombinant products between two circular, double-stranded DNA molecules . We were unable to detect any DNA polymerase, DNA ligase, or 5' or 3' exonuclease activity associated with this purified material . The purified 100 kDa protein bound silver nitrate as well as a monoclonal antibody specific for nucleolin . A recombinant protein comprised of the Escherichia coli maltos-ebinding protein fused to the carboxyl-terminal two-thirds of human nucleolin possessed homologous DNA pairing activity . These data indicate that the 100 kDa homologous DNA pairing protein is nucleolin . The observation that nucleolin can carry out homologous DNA strand pairing in vitro raises the prospect that it may function similarly in vivo.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1808 - 12
Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli; Kornberg HL et al.; From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (K(m) for growth approximately 8 mM but with V(max) for generation time approximately 1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak) . The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme II(Glc) of the PTS, which we designate ptsG-F . Exconjugants that had acquired ptsG(+) from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (V(max) approximately 7 h 20 min), even though they were rich in Mak activity . A mutant of E . coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme II(Man) (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose . Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl alpha-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1477 - 82
Folding and activity of circularly permuted forms of a polytopic membrane protein; Beutler R et al.; The transmembrane subunit of the Glc transporter (IICB(Glc)), which mediates uptake and concomitant phosphorylation of glucose, spans the membrane eight times . Variants of IICB(Glc) with the native N and C termini joined and new N and C termini in the periplasmic and cytoplasmic surface loops were expressed in Escherichia coli . In vivo transport/in vitro phosphotransferase activities of the circularly permuted variants with the termini in the periplasmic loops 1 to 4 were 35/58, 32/37, 0/3, and 0/0% of wild type, respectively . The activities of the variants with the termini in the cytoplasmic loops 1 to 3 were 0/25, 0/4 and 24/70, respectively . Fusion of alkaline phosphatase to the periplasmic C termini stabilized membrane integration and increased uptake and/or phosphorylation activities . These results suggest that internal signal anchor and stop transfer sequences can function as N-terminal signal sequences in a circularly permuted alpha-helical bundle protein and that the orientation of transmembrane segments is determined by the amino acid sequence and not by the sequential appearance during translation . Of the four IICB(Glc) variants with new termini in periplasmic loops, only the one with the discontinuity in loop 4 is inactive . The sequences of loop 4 and of the adjacent TM7 and TM8 are conserved in all phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system transporters of the glucose family.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1456 - 60
Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein; Lin X et al.; The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced . The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain . Memapsin 2 mRNA is abundant in human brain . The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified . Recombinant memapsin 2 specifically hydrolyzed peptides derived from the beta-secretase site of both the wild-type and Swedish mutant beta-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter . Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the beta-secretase site of APP intracellularly . These and other results suggest that memapsin 2 fits all of the criteria of beta-secretase, which catalyzes the rate-limiting step of the in vivo production of the beta-amyloid (Abeta) peptide leading to the progression of Alzheimer's disease . Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency . Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S(1)' subsite, which prefers small side chains such as Ala, Ser, and Asp.

J Biol Chem, 2000 Mar 10, 275(10), 7390 - 4
Molecular and biochemical characterization of rat gamma-trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine biosynthesis; Vaz FM et al.; The penultimate step in carnitine biosynthesis is mediated by gamma-trimethylaminobutyraldehyde dehydrogenase (EC 1.2.1.47), a cytosolic NAD(+)-dependent aldehyde dehydrogenase that converts gamma-trimethylaminobutyraldehyde into gamma-butyrobetaine . This enzyme was purified from rat liver, and two internal peptide fragments were sequenced by Edman degradation . The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA containing an open reading frame of 1485 base pairs encoding a polypeptide of 494 amino acids with a calculated molecular mass of 55 kDa . Expression of the coding sequence in Escherichia coli confirmed that the cDNA encodes gamma-trimethylaminobutyraldehyde dehydrogenase . The previously identified human aldehyde dehydrogenase 9 (EC 1.2.1.19) has 92% identity with rat trimethylaminobutyraldehyde dehydrogenase and has been reported to convert substrates that resemble gamma-trimethylaminobutyraldehyde . When aldehyde dehydrogenase 9 was expressed in E . coli, it exhibited high trimethylaminobutyraldehyde dehydrogenase activity . Furthermore, comparison of the enzymatic characteristics of the heterologously expressed human and rat dehydrogenases with those of purified rat liver trimethylaminobutyraldehyde dehydrogenase revealed that the three enzymes have highly similar substrate specificities . In addition, the highest V(max)/K(m) values were obtained with gamma-trimethylaminobutyraldehyde as substrate . This indicates that human aldehyde dehydrogenase 9 is the gamma-trimethylaminobutyraldehyde dehydrogenase, which functions in carnitine biosynthesis.

J Biol Chem, 2000 Mar 10, 275(10), 7351 - 8
Epitope randomization redefines the functional role of glutamic acid 110 in interleukin-5 receptor activation; Wu SJ et al.; Sequence randomization through functional phage display of single chain human interleukin (IL)-5 was used to investigate the limits of replaceability of the Glu(110) residues that form a part of the receptor-binding epitope . Mutational analysis revealed unexpected affinity for IL-5 receptor alpha chain with variants containing E110W or E110Y . Escherichia coli-expressed Glu(110) variants containing E110W in the otherwise sequence-intact N-terminal half, including a variant with an E110A replacement in the sequence-disabled C-terminal half, were shown by their CD spectra to be folded into secondary structures similar to that of single chain human IL-5 (scIL-5) . Biosensor kinetics analysis revealed that (E110W/A5)scIL-5 and (E110W/A6)scIL-5 had receptor alpha chain binding affinities similar to that of (wt/A5)scIL-5 . However, (E110W/A6)scIL-5 had a significantly reduced bioactivity in TF-1 cell proliferation compared with both (wt/A5)scIL-5 and (E110W/A5)scIL-5, and this activity reduction was disproportionately greater than the much smaller effect of Glu(110) mutation on receptor binding affinity . The marked and disproportionate decrease in TF-1 proliferation observed with (E110W/A6)scIL-5 suggests a role for Glu(110) in the biological activity mediated by the signal transducing receptor betac subunit of the IL-5 receptor . This is also consistent with the lack of stimulation of JAK2 phosphorylation by the (E110W/A6)scIL-5 mutant in recombinant 293T cells, as compared with the concentration-dependent stimulation seen for scIL-5 . The results reveal the dispensability of charge in the Glu(110) locus of IL-5 for receptor alpha chain binding and, in contrast, its heretofore underappreciated importance for receptor activation.

J Biol Chem, 2000 Mar 10, 275(10), 7327 - 36
A unique organization of the protein subunits of the DNA polymerase clamp loader in the archaeon Methanobacterium thermoautotrophicum deltaH; Kelman Z et al.; Replication factor C (RFC, also called activator 1), in conjunction with proliferating cell nuclear antigen (PCNA), is responsible for processive DNA synthesis catalyzed by the eukaryotic replicative DNA polymerases delta and epsilon . Here we report the isolation and characterization of homologues of RFC and PCNA from the archaeon, Methanobacterium thermoautotrophicum DeltaH . In contrast to the five subunit RFC complex isolated from eukaryotic cells, the mthRFC contains only two subunits . The two genes encoding the RFC subunits called, mthRFC1 and mthRFC3, were cloned, and the proteins (54.4 and 36.8 kDa, respectively) were overexpressed in Escherichia coli and purified individually and as a complex . The gene encoding PCNA was also cloned, and the protein was purified after overexpression in E . coli . Based on sizing column elution and subunit composition, the mthRFC complex appears to be a hexamer consisting of two mthRFC1 protomers and four mthRFC3 protomers . Although mthRFC differs in organization from its eukaryotic counterpart, it was shown to be functionally similar to eukaryotic RFC in: (i) catalyzing DNA-dependent ATP hydrolysis; (ii) binding preferentially to DNA primer ends; (iii) loading mthPCNA onto singly nicked circular DNA; and (iv) supporting mthPolB-catalyzed PCNA-dependent DNA chain elongation . The importance and roles of RFC and PCNA in M . thermoautotrophicum DeltaH replication are discussed.

J Biol Chem, 2000 Mar 10, 275(10), 7321 - 6
Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase; Elias MD et al.; Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses . Of these, critical mutants were further characterized after purification or by different amino acid substitutions . H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2) . W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ . D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ . Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).

J Biol Chem, 2000 Mar 10, 275(10), 7117 - 24
The yeast mitochondrial citrate transport protein . Probing the roles of cysteines, Arg(181), and Arg(189) in transporter function; Xu Y et al.; Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae . Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys(73) with Val . Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles . During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined . The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents . Experiments conducted with triple Cys replacement mutants (i.e . Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold . Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein . Finally, utilizing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined . Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function . The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not . Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.

J Biol Chem, 2000 Mar 10, 275(10), 6975 - 9
Building a thermostable membrane protein; Zhou Y et al.; The poor stability of membrane proteins in detergent solution is one of the main technical barriers to their structural and functional characterization . Here we describe a solution to this problem for diacylglycerol kinase (DGK), an integral membrane protein from Escherichia coli . Twelve enhanced stability mutants of DGK were obtained using a simple screen . Four of the mutations were combined to create a quadruple mutant that had improved stability in a wide range of detergents . In n-octylglucoside, the wild-type DGK had a thermal inactivation half-life of 6 min at 55 degrees C, while the quadruple mutant displayed a half-life of 35 min at 80 degrees C . In addition, the quadruple mutant had improved thermodynamic stability . Our approach should be applicable to other membrane proteins that can be conveniently assayed.

J Biol Chem, 2000 Mar 10, 275(10), 6885 - 93
The roles of specific template nucleosides in the formation of stable transcription complexes by Escherichia coli RNA polymerase; Levin JR et al.; We have examined the effects of removing individual template nucleosides on promoter escape by Escherichia coli RNA polymerase in vitro . The ability of DNA templates containing random single nucleoside gaps generated by hydroxyl radical treatment to support the production of stable ternary transcription complexes was analyzed . On two templates containing different promoter and initial transcribed regions, we found that removal of nucleosides on the template strand in the region from -13 to at least +8 relative to the transcription start site interfered with ternary complex formation . The downstream border of this region varied for the two templates, suggesting an effect of the specific nucleotide sequence on the stability of intermediates in the promoter escape process . On the nontemplate strand, removal of nucleosides in the vicinity of the -10 consensus promoter element interfered with escape, whereas removal of nucleosides in the vicinity of the transcription start site actually enhanced the yield of ternary complexes . On one template, removal of nucleosides in an A-tract containing region upstream of the promoter caused a significant decrease in promoter escape, consistent with previous suggestions that contacts between this region and the RNA polymerase play a role in promoter binding and/or initiation.

J Biol Chem, 2000 Mar 10, 275(10), 6806 - 12
Thymidine diphosphate-6-deoxy-L-lyxo-4-hexulose reductase synthesizing dTDP-6-deoxy-L-talose from Actinobacillus actinomycetemcomitans; Nakano Y et al.; The serotype c-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans NCTC 9710 contains an unusual sugar, 6-deoxy-L-talose, which has been identified as a constituent of cell wall components in some bacteria . Two genes coding for thymidine diphosphate (dTDP)-6-deoxy-L-lyxo-4-hexulose reductases were identified in the gene cluster required for biosynthesis of serotype c-specific polysaccharide . Both dTDP-6-deoxy-L-lyxo-4-hexulose reductases were overproduced and purified from Escherichia coli transformed with the plasmids containing these genes . The sugar nucleotides converted by both reductases were purified by reversed-phase high performance liquid chromatography and identified by (1)H nuclear magnetic resonance and gas-liquid chromatography . The results indicated that one of two reductases produced dTDP-6-deoxy-L-talose and the other produced dTDP-L-rhamnose (dTDP-6-deoxy-L-mannose) . The amino acid sequence of the dTDP-6-deoxy-L-lyxo-4-hexulose reductase forming dTDP-6-deoxy-L-talose shared only weak homology with that forming dTDP-L-rhamnose, despite the fact that these two enzymes catalyze the reduction of the same substrate and the products are determined by the stereospecificity of the reductase activity . Neither the gene for dTDP-6-deoxy-L-talose biosynthesis nor its corresponding protein product has been found in other bacteria; this biosynthetic pathway is identified here for the first time.

J Biol Chem, 2000 Mar 10, 275(10), 6798 - 805
Resonance Raman characterization of biotin sulfoxide reductase . Comparing oxomolybdenum enzymes in the ME(2)SO reductase family; Garton SD et al.; Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sphaeroides biotin sulfoxide reductase expressed in Escherichia coli . On the basis of (18)O/(16)O labeling studies involving water and the alternative substrate dimethyl sulfoxide and the close correspondence to the resonance Raman spectra previously reported for dimethyl sulfoxide reductase (Garton, S . D., Hilton, J., Oku, H., Crouse, B . R., Rajagopalan, K . V., and Johnson, M . K . (1997) J . Am . Chem . Soc . 119, 12906-12916), vibrational modes associated with a terminal oxo ligand and the two molybdopterin dithiolene ligands have been assigned . The results indicate that the enzyme cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) forms with both molybdopterin dithiolene ligands remaining coordinated in both redox states . Direct evidence for an oxygen atom transfer mechanism is provided by (18)O/(16)O labeling studies, which show that the terminal oxo group at the molybdenum center is exchangeable with water during redox cycling and originates from the substrate in substrate-oxidized samples . Biotin sulfoxide reductase is not reduced by biotin or the nonphysiological products, dimethyl sulfide and trimethylamine . However, product-induced changes in the Mo=O stretching frequency provide direct evidence for a product-associated mono-oxo-Mo(VI) catalytic intermediate . The results indicate that biotin sulfoxide reductase is thermodynamically tuned to catalyze the reductase reaction, and a detailed catalytic mechanism is proposed.

J Mol Biol, 2000 Mar 10, 296(5), 1169 - 73
A cruciform structural transition provides a molecular switch for chromosome structure and dynamics; Shlyakhtenko LS et al.; The interaction between specific sites along a DNA molecule is often crucial for the regulation of genetic processes . However, mechanisms regulating the interaction of specific sites are unknown . We have used atomic force microscopy to demonstrate that the structural transition between cruciform conformations can act as a molecular switch to facilitate or prevent communication between distant regions in DNA . Cruciform structures exist in vivo and they are critically involved in the initiation of replication and the regulation of gene expression in different organisms . Therefore, structural transitions of the cruciform may play a key role in these processes .

Eur Respir J, 2000 Jan, 15(1), 85 - 91
Respiratory effects of lipopolysaccharide-induced inflammatory lung injury in mice; Faffe DS et al.; The pathogenic mechanisms of lipopolysaccharide (LPS)-induced lung injury have not been classified . This study examined the physiological changes after endotoxin inhalation and related those to features of pulmonary inflammation in mice . Pulmonary mechanics, histopathology, and bronchoalveolar lavage fluid (BALF) from BALB/c mice were analysed at different occasions (3, 24, 48 and 72 h) after inhalation of saline or LPS from Escherichia coli (0.3 (L0.3) or 10 mg x mL(-1) (L10)) . Mice were sedated, anaesthetized, and ventilated . After chest wall resection static (Est) and dynamic (Edyn) elastances, deltaE (Edyn-Est), resistive (deltaP1) and viscoelastic/inhomogeneous pressures (deltaP2), and deltaP1+deltaP2 (deltaPtot) were obtained by end-inflation occlusion method . Lungs were prepared for histopathology . In parallel groups, tumour necrosis factor (TNF)-alpha, neutrophils, and protein were evaluated in the BALF . L0.3 and L10 showed a time-dependent production of TNF-alpha preceding a massive neutrophil infiltration . In L10 BALF there was an increase in protein level at 24 and 48 h . Est and Edyn increased early in L0.3 (65%, 63%) and L10 (41%, 51%) . In L10 deltaE, deltaP2, and deltaPtot showed a gradual rise . At 72 h all groups were similar . L0.3 showed an early increase in cellularity, which returned to normal at 72 h . L10 presented the same pattern with the cell count remaining elevated until 72 h . In conclusion, lipopolysaccharide inhalation led to elastic and viscoelastic pulmonary changes together with tumour necrosis factor-alpha production and neutrophil infiltration in mouse lung.

Vet Immunol Immunopathol, 2000 Jan 31, 73(1), 31 - 44
Molecular cloning, phylogenetic analysis and expression of beluga whale (Delphinapterus leucas) interleukin 6; St-Laurent G et al.; Interleukin 6 (IL-6) is a cytokine produced primarily by the monocytes/macrophages with regulatory effects in hematopoiesis, acute phase response, and multiple aspects of the immune response . IL-6 exerts its activity through its binding to specific high affinity receptors at the surface of target cells . As yet, no molecular data have been reported for the beluga whale IL-6 . In this study, we cloned and determined the entire beluga whale IL-6-encoding cDNA sequence by reverse transcription-polymerase chain reaction (RT-PCR) sequencing, and analysed its genetic relationship with those from several mammalian species including human, rodent, ruminant, carnivore and other marine species . The identity levels of beluga whale IL-6 nucleic and deduced amino acid sequences with those from these mammalian species ranged from 62.3 to 97.3%, and 42.9 to 95.6%, respectively . Phylogenetic analysis based on amino acid sequences showed that the beluga whale IL-6 was most closely related to that of the killer whale . Thereafter, beluga whale IL-6-encoding sequence was successfully expressed in Escherichia coli by using the pTHIOHisA expression vector for the production of a recombinant fusion protein . The immunogenicity of the recombinant fusion protein was then confirmed as determined by the production of a beluga whale IL-6-specific rabbit antiserum.

Med Pediatr Oncol, 2000 Mar, 34(3), 200 - 5
Pegaspargase-induced pancreatitis; Alvarez OA et al.; BACKGROUND: The purpose of this study is to report the incidence of pancreatitis in patients treated with pegaspargase in our hospital during a 2-year period . PROCEDURE: We identified episodes of pancreatitis related to the intramuscular administration of pegaspargase 2,500 IU/m(2) for the treatment of childhood hematological malignancies during a 2-year period (May 1996-April 1998) . Patients were evaluated clinically and by sequential serum amylase and lipase determinations and radiographic examinations . For comparison, episodes of pancreatitis in patients who only received native Escherichia coli L-asparaginase were examined during the same time period . RESULTS: Nine children with acute lymphoblastic leukemia (ALL) of 50 (18%) patients who received pegaspargase were diagnosed to have pancreatitis . All had prior therapy with native L-asparaginase . These children developed symptoms consisting of abdominal pain, nausea, vomiting, and decreased appetite within a median of 15 days from the onset of pegaspargase administration . Six patients became symptomatic after their initial dose . Seven patients developed severe or unacceptable toxicity (grades 3 and 4), measured by increased amylase (>2 times normal) and lipase levels or radiographic evidence of pancreatic inflammation or pseudocyst . One patient also developed hyperammonemia and encephalopathy . In contrast, only one out of 52 (1.9%) ALL patients who received native E . coli L-asparaginase during the same time period developed pancreatitis (P= 0.007) . CONCLUSION: Clinicians should be aware of a possible higher incidence of pancreatitis associated with pegaspargase .

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2023 - 8
Structure of Escherichia coli ribosomal protein L25 complexed with a 5S rRNA fragment at 1.8-A resolution; Lu M et al.; The crystal structure of Escherichia coli ribosomal protein L25 bound to an 18-base pair portion of 5S ribosomal RNA, which contains "loop E," has been determined at 1.8-A resolution . The protein primarily recognizes a unique RNA shape, although five side chains make direct or water-mediated interactions with bases . Three beta-strands lie in the widened minor groove of loop E formed by noncanonical base pairs and cross-strand purine stacks, and an alpha-helix interacts in an adjacent widened major groove . The structure of loop E is largely the same as that of uncomplexed RNA (rms deviation of 0.4 A for 11 base pairs), and 3 Mg(2+) ions that stabilize the noncanonical base pairs lie in the same or similar locations in both structures . Perhaps surprisingly, those residues interacting with the RNA backbone are the most conserved among known L25 sequences, whereas those interacting with the bases are not.

Br J Pharmacol, 2000 Mar, 129(5), 975 - 83
Role for endogenous endothelin in the regulation of plasma volume and albumin escape during endotoxin shock in conscious rats; Filep JG; To explore the role of endogenous endothelin (ET) in the regulation of vascular functions, we studied the effects endothelin receptor blockade on blood pressure, plasma volume and albumin escape during endotoxin shock in conscious, chronically catheterized rats . Red blood cell volume and plasma volume were determined by using chromium-51-tagged erythrocytes and iodine-125-labelled albumin, respectively . Intravenous injection of lipopolysaccharide (LPS, 10 mg kg(-1)) resulted in hypotension, haemoconcentration, and increased total-body albumin escape, which is reflected by a 30% reduction in plasma volume . Plasma ET-1 concentrations increased 2.1 fold and 5.4 fold at 30 and 120 min post-LPS, respectively . LPS-induced losses in plasma volume and albumin escape were significantly attenuated by pretreatment of animals with the dual ET(A)/ET(B) receptor antagonist bosentan (17.4 micromol kg(-1), i.v . 15 min prior to LPS) or the ET(A) receptor antagonist FR 139317 (3.8 micromol kg(-1), i.v.) during both the immediate and delayed phases of endotoxin shock . The inhibitory actions of bosentan and FR 139317 were similar . Both antagonists augmented the hypotensive action of LPS . Administration of bosentan or FR 139317 70 min after injection of LPS also attenuated further losses in plasma volume and increases in total body and organ albumin escape rates with the exception of the lung and kidney . These results indicate a role for endogenous endothelin in mediating losses in plasma volume and albumin escape elicited by LPS predominantly through activation of ET(A) receptors, and suggest that by attenuating these events, ET(A) or dual ET(A)/ET(B) receptor blockers may be useful agents in the therapy of septic shock.

Biochemistry, 2000 Mar 7, 39(9), 2362 - 9
Unfolding thermodynamics of the tetrameric chaperone, SecB; Panse VG et al.; SecB is a cytosolic tetrameric chaperone in Escherichia coli, which maintains polypeptides, destined for export in a translocation competent state . The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods . The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers . Increasing the pH decreases the stability of the tetramer significantly, the T(m) changing from 341.3 K at pH 6.5 to 332.6 K at pH 9.5 . The value of DeltaC(p) obtained from measurements of DeltaH(m) as a function of T(m) was 10.7 +/- 0.7 kcal mol(-1) K(-1) . The value of DeltaC(p) is among the highest measured for a multimeric protein . At 298 K, pH 7.4, the DeltaG degrees (u) for the SecB tetramer is 27.9 +/- 2 kcal mol(-1) . Denaturant-mediated unfolding of SecB was found to be irreversible . The reactivity of the four solvent-exposed free thiols in tetrameric SecB is salt dependent . The kinetics of reactivity suggests that these four cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments . The thermodynamic data suggest that SecB is a stable, well-folded, and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity.

Biochemistry, 2000 Mar 7, 39(9), 2307 - 15
A molecular coupling mechanism for the oxaloacetate decarboxylase Na+ pump as inferred from mutational analysis; Jockel P et al.; The oxaloacetate decarboxylase Na+ pump consists of subunits alpha, beta, and gamma, and contains biotin as the prosthetic group . Membrane-bound subunit beta catalyzes the decarboxylation of carboxybiotin coupled to Na+ translocation, and consumes a periplasmically derived proton . Site-directed mutagenesis of conserved amino acids of transmembrane helix VIII indicated that residues N373, G377, S382, and R389 are functionally important . The polar side groups of these amino acids may constitute together with D203 a network of ionizable groups which promotes the translocation of Na+ and the oppositely oriented H+ across the membrane . Evidence is presented that two Na+ ions are bound simultaneously to subunit beta during transport with D203 and S382 acting as binding sites . Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin, and a conformational switch exposes the bound Na+ ions toward the periplasm . After dissociation of Na+ and binding of H+, the cytoplasmically exposed conformation is regained.

Biochemistry, 2000 Mar 7, 39(9), 2193 - 200
Evaluation by site-directed mutagenesis of aspartic acid residues in the metal site of pig heart NADP-dependent isocitrate dehydrogenase; Grodsky NB et al.; Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis . On the basis of affinity cleavage studies {Soundar and Colman (1993) J . Biol . Chem . 268, 5267} and analysis of the crystal structure of E . coli NADP-isocitrate dehydrogenase {Hurley et al . (1991) Biochemistry 30, 8671}, the residues Asp(253), Asp(273), Asp(275), and Asp(279) were selected as potential ligands of the divalent metal cation in the pig heart enzyme . Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn . The wild-type and mutant enzymes were expressed in Escherichia coli and purified . D253N has a specific activity, K(m) values for Mn(2+), isocitrate, and NADP, and also a pH-V(max) profile similar to those of the wild-type enzyme . Thus, Asp(253) is not involved in enzyme function . D273N has an increased K(m) for Mn(2+) and isocitrate with a specific activity 5% that of wild type . The D273N mutation also prevents the oxidative metal cleavage seen with Fe(2+) alone in the wild-type enzyme . As compared to wild type, D275N has greatly increased K(m) values for Mn(2+) and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pK(a) for the enzyme-substrate complex . D279N has only small increases in K(m) for Mn(2+) and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH-V(max) profile . These results suggest that Asp(273) and Asp(275) contribute to metal binding, whereas Asp(279), as well as Asp(275), is critical for catalysis . Asp(279) may function as the catalytic base . Using the Modeler program of Insight II, a structure for porcine NADP-isocitrate dehydrogenase was built based on the X-ray coordinates of the E . coli enzyme, allowing visualization of the metal-isocitrate site.

Biochemistry, 2000 Mar 7, 39(9), 2131 - 9
Simultaneous binding of two DNA duplexes to the NtrC-enhancer complex studied by two-color fluorescence cross-correlation spectroscopy; Rippe K; The transcription activator protein NtrC (nitrogen regulatory protein C, also termed NR(I)) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma(54) factor (RNAP x sigma(54)) from the closed complex (RNAP x sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA) . This process involves phosphorylation of NtrC (NtrC-P), assembly of an octameric NtrC-P complex at the enhancer DNA sequence, interaction of this complex with promoter-bound RNAP x sigma(54) via DNA looping, and hydrolysis of ATP . Here it is demonstrated by two-color fluorescence cross-correlation spectroscopy measurements of 6-carboxyfluorescein and 6-carboxy-X-rhodamine-labeled DNA oligonucleotide duplexes that the NtrC-P complex can bind two DNA duplexes simultaneously . This suggests a model for the conformation of the looped intermediate that is formed between NtrC-P and RNAP . sigma(54) at the glnAp2 promoter during the activation process.

Biochem Biophys Res Commun, 2000 Mar 5, 269(1), 14 - 20
Characterization of Pen n 13, a major allergen from the mold Penicillium notatum; Chow LP et al.; Penicillium notatum is a well-known indoor aeroallergen and is frequently included in skin test panels for allergic diagnosis . On two-dimensional immunoblotting using patients' sera containing IgE and monoclonal antibody D7B8 specific for Pen c 1 of P . citrinum, two allergens with a molecular mass of 33 kDa but different isoelectric points were identified . A novel cDNA coding for Pen n 13 was cloned and sequenced . The nucleotide sequence codes for a protein 397 amino acids including a putative signal peptide of 25 amino acids and a propeptide of 90 amino acids . The allergen is an alkaline serine protease that shares more than 39% identical residues with other kinds of mold allergens . The coding cDNA of Pen n 13 was cloned into vector pQE-30 and expressed in E . coli M15 as a His-tag fusion protein and purified to homogeneity . The fusion protein reacted with monoclonal antibodies of Pen c 1 and with IgE from Penicillium-allergic patients . Furthermore, it also cross-reacted strongly with IgE specific for the natural Pen c 1, indicating that similar IgE binding epitopes may exist in the allergens of P . notatum and P . citrinum . Antigenicity index plots indicated that there are several similar epitope regions of high antigenic indices in Pen c 1 and Pen n 13, corroborating that mold allergens belonging to the alkaline serine protease family possess similar protein structure and strong antigenic cross-reactivity .

Am J Obstet Gynecol, 2000 Feb, 182(2), 401 - 8
Effects of antenatal endotoxin and glucocorticoids on the lungs of preterm lambs; Jobe AH et al.; OBJECTIVE: We hypothesized that the proinflammatory response to intra-amniotic endotoxin would induce lung maturation in preterm lambs . STUDY DESIGN: Ewes were randomly assigned to receive 20 mg Escherichia coli endotoxin by intra-amniotic injection, maternal betamethasone (0.5 mg/kg), or sodium chloride solution . Preterm lambs were delivered at 125 days' gestation and underwent ventilation to assess lung function . Lung gas volume, surfactant concentrations, and inflammation were subsequently evaluated, with data analyzed by analysis of variance . RESULTS: Fetal endotoxin exposure 6 days before delivery increased compliance by 59%, increased lung gas volume 2.3-fold, increased concentrations of surfactant lipids, increased surfactant A and B protein levels, and increased messenger ribonucleic acid expressions for surfactant proteins (all P <.01, vs control group) . Betamethasone exposure resulted in less consistent effects . White blood cell counts were increased in fetal membranes and lungs after endotoxin exposure, but there was no severe inflammation . CONCLUSION: A single fetal exposure to endotoxin resulted in large improvements in postnatal lung function and increases in surfactant concentrations after preterm delivery . These effects were qualitatively larger than those achieved with betamethasone.

Epidemiol Infect, 1999 Dec, 123(3), 413 - 21
Characterization of enteroaggregative Escherichia coli isolated from outbreaks of diarrhoeal disease in England; Spencer J et al.; Twenty-two strains of enteroaggregative Escherichia coli (EAggEC), isolated from four outbreaks of diarrhoeal disease in England, were examined for a range of phenotypic attributes including the ability to produce fimbriae, haemolysins and siderophores, and cell-surface properties such as surface charge and hydrophobicity . Strains of EAggEC isolated from two of these outbreaks belonged to a diverse range of serotypes and were heterogeneous in phenotype . Strains of EAggEC isolated from the other two outbreaks belonged predominantly to serotypes 086:H34 and 098:H-, respectively . Only two strains expressed fimbriae and two strains produced an 18 kDa membrane associated protein (MAP), suggesting that EAggEC express a range of adhesion mechanisms to produce the cell arrangement recognized as the 'stacked brick' formation . The possible explanation for the diversity of EAggEC serotypes is discussed.

Free Radic Res, 1999 Dec, 31 Suppl, S213 - 8
Characterisation of a cDNA encoding gamma-glutamylcysteine synthetase in Medicago truncatula; Frendo P et al.; A gamma-ECS cDNA from Medicago truncatula was isolated using an Arabidopsis thaliana cDNA as probe . The analysis of the amino acid sequence deduced from this cDNA revealed 80% identity with the gamma-ECS from A . thaliana and Brassica juncea and suggested a plastidial localisation for the enzyme . Gamma-ECS activity and high level of GSH were detected in the gamma-ECS-deficient E . coli strain expressing a fusion protein containing the M . truncatula gamma-ECS protein . Southern blot analysis suggests that gamma-ECS is encoded by a small multigenic family in M . truncatula and shows that homologous genes are present in two other leguminous plants, Medicago sativa and Pisum sativum . Gamma-ECS gene expression was analysed by Northern blot in seedlings, plantlets and mature plants.

J Biol Chem, 2000 Mar 3, 275(9), 6207 - 13
Distant downstream sequence determinants can control N-tail translocation during protein insertion into the endoplasmic reticulum membrane; Nilsson I et al.; We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes . Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an "inverted" topology where normally nontranslocated parts are translocated and vice versa . N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment . We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail . We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction.

J Bacteriol, 2000 Mar, 182(6), 1768 - 73
Membrane association of the Escherichia coli enterobactin synthase proteins EntB/G, EntE, and EntF; Hantash FM et al.; The cytosolic proteins EntE, EntF, and EntB/G, which are Escherichia coli enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock . Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations . Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.

J Bacteriol, 2000 Mar, 182(6), 1761 - 3
Activation of SoxR by overproduction of desulfoferrodoxin: multiple ways to induce the soxRS regulon; Gaudu P et al.; The soxRS response, which protects cells against superoxide toxicity, is triggered by the oxidation of SoxR, a transcription factor . Superoxide excess and NADPH depletion induce the regulon . Unexpectedly, we found that the overproduction of desulfoferrodoxin, a superoxide reductase from sulfate-reducing bacteria, also induced this response . We suggest that desulfoferrodoxin interferes with the reducing pathway that keeps SoxR in its inactive form.

J Bacteriol, 2000 Mar, 182(6), 1659 - 70
Analysis of Escherichia coli RecA interactions with LexA, lambda CI, and UmuD by site-directed mutagenesis of recA; Mustard JA et al.; An early event in the induction of the SOS system of Escherichia coli is RecA-mediated cleavage of the LexA repressor . RecA acts indirectly as a coprotease to stimulate repressor self-cleavage, presumably by forming a complex with LexA . How complex formation leads to cleavage is not known . As an approach to this question, it would be desirable to identify the protein-protein interaction sites on each protein . It was previously proposed that LexA and other cleavable substrates, such as phage lambda CI repressor and E . coli UmuD, bind to a cleft located between two RecA monomers in the crystal structure . To test this model, and to map the interface between RecA and its substrates, we carried out alanine-scanning mutagenesis of RecA . Twenty double mutations were made, and cells carrying them were characterized for RecA-dependent repair functions and for coprotease activity towards LexA, lambda CI, and UmuD . One mutation in the cleft region had partial defects in cleavage of CI and (as expected from previous data) of UmuD . Two mutations in the cleft region conferred constitutive cleavage towards CI but not towards LexA or UmuD . By contrast, no mutations in the cleft region or elsewhere in RecA were found to specifically impair the cleavage of LexA . Our data are consistent with binding of CI and UmuD to the cleft between two RecA monomers but do not provide support for the model in which LexA binds in this cleft.

J Bacteriol, 2000 Mar, 182(6), 1564 - 74
Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems; Grahn AM et al.; During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell . A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport . For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins . We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera . Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein . The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E . coli membranes . The Tra1 region is known to encode the components of the RP4 relaxosome . Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction . This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E . coli cell envelope . The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.

Biophys J, 2000 Mar, 78(3), 1207 - 15
Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity; Berry RM et al.; Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum . The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E . coli sensory proteins . We tethered photosynthetically grown cells of R . sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity . The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E . coli but similar to the probability of not tumbling in swimming E . coli . Otherwise, the step and impulse responses were the same as those of tethered E . coli to chemical attractants . This indicates that the single motor and multiple sensory signaling pathways in R . sphaeroides generate the same swimming response as several motors and a single pathway in E . coli, and that the response of the single motor is directly observable in the swimming pattern . Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.

FEBS Lett, 2000 Feb 25, 468(2-3), 220 - 4
Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT; Kramer RA et al.; Escherichia coli outer membrane protease OmpT has been characterised as a serine protease based on its inhibitor profile, but serine protease consensus sequences are absent . By site-directed mutagenesis we substituted all conserved serines and histidines . Substitution of His(101) and His(212) by Ala, Asn or Gln resulted in variant enzymes with 0.01 and 9-20% residual enzymatic activity towards a fluorogenic pentapeptide substrate, respectively . The mutations S140A and S201A did not decrease activity, while variants S40A and S99A yielded 0.5 and 0.2% residual activities, respectively . When measured with a dipeptide substrate the variant S40A demonstrated full activity, whereas variant S99A displayed at least 500-fold reduced activity . We conclude that Ser(99) and His(212) are essential active site residues . We propose that OmpT is a novel serine protease with Ser(99) as the active site nucleophile and His(212) as general base.

FEBS Lett, 2000 Feb 25, 468(2-3), 215 - 9
Isolation of recombinant BMP receptor IA ectodomain and its 2:1 complex with BMP-2; Kirsch T et al.; Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily which induces bone formation and regeneration, and important steps during early embryonic development . BMP-2 signals via oligomerization of type I and type II serine/threonine kinase receptors . We report here expression of the extracellular domain of the human type IA receptor for BMP-2 (BMPR-IA) in Escherichia coli . This soluble form of BMPR-IA (sBMPR-IA) was purified employing a BMP-2 affinity column . Gel filtration experiments and analysis of gel filtration fractions by polyacrylamide electrophoresis and densitometry reveal that BMP-2 forms a defined 1:2 complex with sBMPR-IA that can be purified and hopefully used for crystallization studies.

FEBS Lett, 2000 Feb 25, 468(2-3), 155 - 8
Semi-synthetic Rab proteins as tools for studying intermolecular interactions; Iakovenko A et al.; Rab GTPases play a key role in the regulation of membrane traffic . Posttranslational geranylgeranylation is critical for their biological activity and is conferred by a Rab geranylgeranyl transferase (RabGGTase) . To study the interactions between Rab proteins and RabGGTase, we used in vitro ligation methodology to generate a fluorescent semi-synthetic Rab7 protein . The obtained protein was functionally active and was used to demonstrate a micromolar affinity interaction of Rab7 with the RabGGTase in the absence of Rab escort protein (REP) . This finding is consistent with an earlier proposed model according to which RabGGTase possesses two independent weak binding sites for REP and Rab proteins.

J Biol Chem, 2000 Mar 3, 275(9), 6673 - 9
Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants; Munch-Petersen B et al.; The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster (Dm-dNK) with remarkably broad substrate specificity has recently been indicated (Munch-Petersen, B., Piskur, J., and Sondergaard, L . (1998) J . Biol . Chem . 273, 3926-3931) . To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partially sequenced cDNA clones, originating from the Berkeley Drosophila genome sequencing project, were searched for homology with human deoxyribonucleoside kinases . The total sequence of one cDNA clone and the corresponding genomic DNA was determined and expressed in Escherichia coli as a glutathione S-transferase fusion protein . The purified and thrombin cleaved recombinant protein phosphorylated the four deoxyribonucleosides with high turnover and K(m) values similar to those of the native Dm-dNK, as well as the four ribonucleosides and many therapeutical nucleoside analogs . Dm-dNK has apparently the same origin as the mammalian kinases, thymidine kinase 2, deoxycytidine kinase, deoxyguanosine kinase, and the herpes viral thymidine kinases, but it has a unique C terminus that seems to be important for catalytic activity and specificity . The C-terminal 20 amino acids were dispensable for phosphorylation of deoxyribonucleosides but necessary for full activity with purine ribonucleosides . Removal of the C-terminal 20 amino acids increased the specific activity 2-fold, but 99% of the activity was lost after removal of the C-terminal 30 amino acids.

J Biol Chem, 2000 Mar 3, 275(9), 6546 - 52
Identification and characterization of Elongin A2, a new member of the Elongin family of transcription elongation factors, specifically expressed in the testis; Aso T et al.; The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template . Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, the latter of which bind stably to each other to form a binary complex that interacts with Elongin A and strongly induces its transcriptional activity . To further understand the roles of Elongin in transcriptional regulation, we attempted to identify Elongin-related proteins . Here, we report on the cloning, expression, and characterization of human Elongin A2, a novel transcription elongation factor that exhibited 47% identity and 61% similarity to Elongin A . Biochemical studies have shown that Elongin A2 stimulates the rate of transcription elongation by RNA polymerase II and is capable of forming a stable complex with Elongin BC . However, in contrast to Elongin A, its transcriptional activity is not activated by Elongin BC . Northern blot analysis revealed that Elongin A2 mRNA was specifically expressed in the testis, suggesting that Elongin A2 may regulate the transcription of testis-specific genes.

J Biol Chem, 2000 Mar 3, 275(9), 6537 - 45
5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms; Li L et al.; S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2.1.1.6) has traditionally been thought to catalyze the methylation of caffeate and 5- hydroxyferulate for the biosynthesis of syringyl monolignol, a lignin constituent of angiosperm wood that enables efficient lignin degradation for cellulose production . However, recent recognition that coniferyl aldehyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of COMT during monolignol biosynthesis . Based on recombinant aspen (Populus tremuloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl aldehyde is both the preferred AldOMT substrate and an inhibitor of caffeate and 5-hydroxyferulate methylation, as measured by K(m) and K(i) values . 5-Hydroxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate methylation activities of xylem proteins from various angiosperm tree species . The evidence that syringyl monolignol biosynthesis is independent of caffeate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate 5-hydroxylation and at the same time ensures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde . Together, our results provide conclusive evidence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hydroxylation/methylation pathway that directs syringyl monolignol biosynthesis in angiosperms.

J Biol Chem, 2000 Mar 3, 275(9), 6490 - 8
The role of the conserved box E residues in the active site of the Escherichia coli type I signal peptidase; Klenotic PA et al.; Type I signal peptidases are integral membrane proteins that function to remove signal peptides from secreted and membrane proteins . These enzymes carry out catalysis using a serine/lysine dyad instead of the prototypical serine/histidine/aspartic acid triad found in most serine proteases . Site-directed scanning mutagenesis was used to obtain a qualitative assessment of which residues in the fifth conserved region, Box E, of the Escherichia coli signal peptidase I are critical for maintaining a functional enzyme . First, we find that there is no requirement for activity for a salt bridge between the invariant Asp-273 and the Arg-146 residues . In addition, we show that the conserved Ser-278 is required for optimal activity as well as conserved salt bridge partners Asp-280 and Arg-282 . Finally, Gly-272 is essential for signal peptidase I activity, consistent with it being located within van der Waals proximity to Ser-278 and general base Lys-145 side-chain atoms . We propose that replacement of the hydrogen side chain of Gly-272 with a methyl group results in steric crowding, perturbation of the active site conformation, and specifically, disruption of the Ser-90/Lys-145 hydrogen bond . A refined model is proposed for the catalytic dyad mechanism of signal peptidase I in which the general base Lys-145 is positioned by Ser-278, which in turn is held in place by Asp-280.

J Biol Chem, 2000 Mar 3, 275(9), 6428 - 38
A novel phosphatidylinositol-phospholipase C of Trypanosoma cruzi that is lipid modified and activated during trypomastigote to amastigote differentiation; Furuya T et al.; The phosphoinositide (PI)-specific phospholipase C gene (TcPI-PLC) of the protozoan parasite Trypanosoma cruzi was cloned, sequenced, expressed in Escherichia coli, and the protein product (TcPI-PLC) was shown to have enzymatic characteristics similar to those of mammalian delta-type PI-PLCs . The TcPI-PLC gene is expressed at high levels in the epimastigote and amastigote stages of the parasite, and its expression is induced during the differentiation of trypomastigotes into amastigotes, where TcPI-PLC associates with the plasma membrane and increases its catalytic activity . In contrast to other PI-PLCs described so far, the deduced amino acid sequence of TcPI-PLC revealed some unique features such as an N-myristoylation consensus sequence at its amino-terminal end, lack of an apparent pleckstrin homology domain and a highly charged linker region between the catalytic X and Y domains . TcPI-PLC is lipid modified in vivo, as demonstrated by metabolic labeling with {(3)H}myristate and {(3)H}palmitate and fatty acid analysis of the immunoprecipitated protein, and may constitute the first example of a new group of PI-PLCs.

J Biol Chem, 2000 Mar 3, 275(9), 6241 - 5
Identification and characterization of a novel cAMP receptor protein in the cyanobacterium Synechocystis sp . PCC 6803; Yoshimura H et al.; Three open reading frames of Synechocystis sp . PCC 6803 encoding a domain homologous with the cAMP binding domain of bacterial cAMP receptor protein were analyzed . These three open reading frames, sll1371, sll1924, and slr0593, which were named sycrp1, sycrp2, and sypk, respectively, were expressed in Escherichia coli as His-tagged or glutathione S-transferase fusion proteins and purified, and their biochemical properties were investigated . The results obtained for equilibrium dialysis measurements using these recombinant proteins suggest that SYCRP1 and SYPK show a binding affinity for cAMP while SYCRP2 does not . The dissociation constant of His-tagged SYCRP1 for cAMP is approximately 3 microM . A cross-linking experiment using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide revealed that His-tagged SYCRP1 forms a homodimer, and the presence or absence of cAMP does not affect the formation of the homodimer . The amino acid sequence reveals that SYCRP1 has a domain similar to the DNA binding domain of bacterial cAMP receptor protein in the COOH-terminal region . Consistent with this, His-tagged SYCRP1 forms a complex with DNA that contains the consensus sequence for E . coli cAMP receptor protein in the presence of cAMP . These results strongly suggest that SYCRP1 is a novel cAMP receptor protein.

J Biol Chem, 2000 Mar 3, 275(9), 6234 - 40
Substitution of leucine 28 with histidine in the Escherichia coli transcription factor FNR results in increased stability of the {4Fe-4S}(2+) cluster to oxygen; Bates DM et al.; To understand the role of the {4Fe-4S}(2+) cluster in controlling the activity of the Escherichia coli transcription factor FNR (fumarate nitrate reduction) during changes in O(2) availability, we have characterized a mutant FNR protein containing a substitution of Leu-28 with His (FNR-L28H) which, unlike its wild type (WT) counterpart, is functional under aerobic growth conditions . The His-28 substitution appears to stabilize the {4Fe-4S}(2+) cluster of FNR-L28H in the presence of O(2) because air-exposed FNR-L28H did not undergo the rapid {4Fe-4S}(2+) to {2Fe-2S}(2+) cluster conversion or concomitant loss in site-specific DNA binding and dimerization, which are characteristic of WT-FNR under these conditions . This increased cluster stability was not a result of His-28 replacing the WT-FNR cluster ligands because substitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in {4Fe-4S}(2+) cluster-deficient preparations of FNR-L28H . The Mossbauer spectra of FNR-L28H indicated that the coordination environment of the {4Fe-4S}(2+) cluster did not differ from that of WT-FNR . Whole cell Mossbauer spectroscopy showed that aerobically grown cells overexpressing FNR-L28H had levels of the FNR species containing the {4Fe-4S}(2+) cluster similar to those of cells grown under anaerobic conditions . Thus, the increase in cluster stability is sufficient to allow accumulation of the {4Fe-4S}(2+) cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under aerobic growth conditions . From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions.

J Biol Chem, 2000 Mar 3, 275(9), 6195 - 200
cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase . Candidate for selenium delivery protein in selenoprotein synthesis; Mihara H et al.; Selenocysteine lyase (SCL) (EC 4.4.1.16) is a pyridoxal 5'-phosphate-dependent enzyme that specifically catalyzes the decomposition of L-selenocysteine to L-alanine and elemental selenium . The enzyme was proposed to function as a selenium delivery protein to selenophosphate synthetase in selenoprotein biosynthesis (Lacourciere, G . M., and Stadtman, T . C . (1998) J . Biol . Chem . 273, 30921-30926) . We purified SCL from pig liver and determined its partial amino acid sequences . Mouse cDNA clones encoding peptides resembling pig SCL were found in the expressed sequence tag data base, and their sequences were used as probes to isolate full-length mouse liver cDNA . The cDNA for mouse SCL (mSCL) was determined to be 2,172 base pairs in length, containing an open reading frame encoding a polypeptide chain of 432 amino acid residues (M(r) 47, 201) . We also determined the sequence of the N-terminal region of putative human SCL . These enzymes were shown to be distantly related in primary structure to NifS, which catalyzes the desulfurization of L-cysteine to provide sulfur for iron-sulfur clusters . The recombinant mSCL overproduced in Escherichia coli was a homodimer with the subunit M(r) of 47,000 . The enzyme was pyridoxal phosphate-dependent and highly specific to L-selenocysteine (the k(cat)/K(m) value for L-selenocysteine was about 4,200 times higher than that for L-cysteine) . Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that mSCL is cytosolic and predominantly exists in the liver, kidney, and testis, where mouse selenophosphate synthetase is also abundant, supporting the view that mSCL functions in cooperation with selenophosphate synthetase in selenoprotein synthesis . This is the first report of the primary structure of mammalian SCL.

J Biol Chem, 2000 Mar 3, 275(9), 6101 - 6
Evidence for interactions between helices 5 and 8 and a role for the interdomain loop in tetracycline resistance mediated by hybrid Tet proteins; Saraceni-Richards CA et al.; An interdomain hybrid Tet protein consisting of a class C alpha domain and a class B beta domain (Tet(C/B)) lacks detectable efflux ability and provides only minimal levels of resistance to tetracycline (Tc) (3 microg/ml) compared with intact class B (256 microg/ml) and class C (64 microg/ml) . Twenty-one independently isolated mutants of the Tet(C/B) protein with increased Tc resistance were generated by random chemical mutagenesis . Nine mutants with a Glu substitution for Gly-152 in helix 5 of the class C alpha domain produced a resistance of 48 microg/ml, whereas another 9 with an Asp replacement of Gly-247 in helix 8 of the class B beta domain mediated resistance at 32 microg/ml . The third type of mutation, found in 3 mutants expressing 24 microg/ml resistance, was a S202F replacement in the putative interdomain cytoplasmic loop of Tet(C/B) . The latter underscores a previously unappreciated function of the interdomain cytoplasmic loop . All three types of Tet(C/B) mutant proteins were expressed in amounts comparable with that of the original protein and demonstrated restored energy-dependent efflux of tetracycline . Site-directed mutational analysis demonstrated that a Gly-247 to Asn mutation could also facilitate Tc resistance by the Tet(C/B) hybrid, and a negatively charged side chain at position 152 was required for Tet(C/B) activity . These mutations appear to promote the necessary functional interactions between the interclass domains that do not occur in the Tet(C/B) hybrid protein and suggest a direct association between helix 5 and helix 8 in the function of Tet efflux proteins.

Environ Mol Mutagen, 2000, 35(1), 48 - 56
Mutagenic potential of adenine N(6) adducts of monoepoxide and diolepoxide derivatives of butadiene; Carmical JR et al.; To determine the biological effects of specific DNA adducts resulting from the interaction of 1,3-butadiene metabolites with DNA, deoxyoligonucleotides have been synthesized with four different adducts at the N(6) position of adenine, centrally located within the human N-ras codon 61 . The adducts are those arising from adduction by either the R or S stereoisomer of the monoepoxide (BDO) or the (R,R) or (S,S) isomer of the diolepoxide (BDE) . The diolepoxide can arise from partial hydrolysis of the diepoxide (BDO(2)) or from epoxidation of hydrolyzed monoepoxide . These adducted oligonucleotides were used in in vivo and in vitro assays designed both to determine their mutagenic potency and to examine specific interactions with Escherichia coli polymerases . Each adducted oligonucleotide was ligated into a single-stranded vector M13mp7L2 that was subsequently used to transfect E . coli . The resulting mutagenic spectrum for these modified DNAs was stereoisomer specific . Both monoepoxide lesions were nonmutagenic, but the mutagenic spectra for the modified DNAs containing BDE adducts were stereoisomer specific . The mutations generated by adducts of the R,R enantiomer of the diolepoxide were exclusively A --> G, whereas adducts of the S,S enantiomer of the diolepoxide yielded exclusively A --> C mutations . None of the four modifications resulted in significant blocks to in vivo phage replication, as evidenced by no decrease in plaque-forming ability . Consistent with these data, when each of three purified E . coli polymerases was used to replicate DNAs containing these adducted deoxyoligonucleotides, the individual polymerases appeared to be virtually unaffected, such that all lesions were readily bypassed . Whereas previous animal model studies identified the mutagenic spectrum related to butadiene exposure, these studies begin to establish the specific lesions responsible for mutagenesis . This is the first report of stereoselectivity related to butadiene-induced mutagenesis .

Environ Mol Mutagen, 2000, 35(1), 22 - 30
Oxidative mutagenesis in Escherichia coli strains lacking ROS-scavenging enzymes and/or 8-oxoguanine defenses; Ruiz-Laguna J et al.; Escherichia coli strains with different combinations of null mutations in the katG, katE, sodA, sodB, fpg, and mutY genes were constructed to compare their spontaneous mutation frequencies and sensitivities to various oxidants with those of bacteria solely deficient in catalase (katG katE) or cytosolic superoxide dismutase (sodA sodB) and the parental strain possessing a full complement of these enzymes . The MutY DNA glycosylase represented the major protection against the mutagenic consequences of processes associated with normal aerobic metabolism . Spontaneous mutagenesis in MutY-lacking bacteria was not influenced by the absence of (A)BC excinuclease or the presence of MucAB proteins, a result consistent with 8-oxoguanine being a principal premutational lesion . In contrast, catalase and SOD represented the major protection against the genotoxic consequences of bursts of oxidative stress caused by reactive-oxygen-generating compounds . Therefore, only bacteria simultaneously defective in both katG and katE or sodA and sodB genes were hypersensitive with respect to mutability by peroxide and superoxide, respectively . These data suggest that oxidative lesions other than 8-oxoguanine contribute to mutagenesis by hydrogen peroxide and redox-cycling chemicals .

Pathol Int, 2000 Jan, 50(1), 34 - 40
Endotoxin priming and liver damage by experimental duodenal obstruction in the rat; Pirisi M et al.; To verify whether endotoxin (LPS) might act as a priming cofactor of liver injury caused by obstructing the duodenum, four groups of male Wistar rats were studied . The first two groups comprised rats in which a closed duodenal loop (CDL) was created: CDL, n = 6 and CDL + LPS, n = 7; the next two groups comprised sham-operated animals: Sham n = 6 and Sham + LPS, n = 6 . LPS, 400 microg/kg bodyweight, was administered i.p . to the rats belonging to groups CDL + LPS and Sham + LPS, 24 h before laparotomy . Twenty-four hours after laparotomy the animals were killed . Damage to bile ducts, extent and grading of coagulative and lytic spotty necrosis in liver tissue were evaluated morphologically . Coagulative necrosis was severe in four of seven rats of the group CDL + LPS, mild in six of six rats of group CDL, and absent in four of six and five of six rats of groups Sham and Sham + LPS (chi2 32.8, P = 0.0001) . The animals of group CDL + LPS had more frequently diffuse lytic spotty necrosis than the animals in the three other groups (chi2 9.57 P<0.01) . The results of our study indicate that, in rodents subjected to a closed duodenal loop, priming with LPS exacerbates liver injury due to cholate stasis.

Mol Microbiol, 2000 Feb, 35(4), 888 - 95
Identification of a unique domain essential for Escherichia coli DNA topoisomerase III-catalysed decatenation of replication intermediates; Li Z et al.; A 17-amino-acid residue domain has been identified in Escherichia coli DNA topoisomerase III (Topo III) that is essential for Topo III-mediated resolution of DNA replication intermediates in vitro . Deletion of this domain reduced Topo III-catalysed resolution of DNA replication intermediates and decatenation of multiply linked plasmid DNA dimers by four orders of magnitude, whereas reducing Topo III-catalysed relaxation of negatively supercoiled DNA substrates only 20-fold . The presence of this domain has been detected in multiple plasmid-encoded topoisomerases, raising the possibility that these enzymes may also be decatenases.

Mol Microbiol, 2000 Feb, 35(4), 877 - 87
Phase variation of Ag43 in Escherichia coli: Dam-dependent methylation abrogates OxyR binding and OxyR-mediated repression of transcription; Haagmans W et al.; It has been shown previously that phase variation of the outer membrane protein Antigen43 (Ag43) of Escherichia coli requires the DNA-methylating enzyme deoxyadenosine methyltransferase (Dam) and the global regulator OxyR . In this study, we analysed the regulation of the Ag43 encoding gene (agn) using isolates containing a fusion of the agn regulatory region to the reporter gene lacZ . Our results indicate that phase variation of Ag43 is regulated at the level of transcription . Repression of agn'-lacZ transcription required OxyR, whereas activation required Dam . The regulatory region of agn contains three GATC sequences, which are target sites for Dam-dependent methylation . In vivo, the methylation state of these GATC sequences correlated with the transcription state of agn'-lacZ . These GATC sequences were not protected from Dam-dependent methylation in an oxyR background, suggesting that OxyR binding results in Dam-dependent methylation protection in OFF cells . In vitro, both oxidized OxyR and OxyR(C199S), which is locked in the reduced conformation, bound to the agn regulatory region, but methylation of the three GATC sequences abrogated this binding . In vivo, OxyR(C199S) was sufficient to repress Ag43 transcription . Our data support a model in which OxyR-mediated repression of transcription is alleviated by methylation of three GATC sequences in its binding site . In addition, we show that, in an oxyR background, Dam was still required for full activation, suggesting that the model concerning the role of Dam in agn regulation is incomplete . These results show that Dam-dependent phase variation in E . coli is not limited to the previously identified regulatory system of the family of pap-like fimbrial operons.

Mol Microbiol, 2000 Feb, 35(4), 845 - 53
Involvement of differential efficiency of transcription by esigmas and esigma70 RNA polymerase holoenzymes in growth phase regulation of the Escherichia coli osmE promoter; Bordes P et al.; Transcription of the gene osmE of Escherichia coli is inducible by elevated osmotic pressure and during the decelerating phase of growth . osmE expression is directed by a single promoter, osmEp . Decelerating phase induction of osmEp is dependent on the sigmas (RpoS) factor, whereas its osmotic induction is independent of sigmas . Purified Esigmas and Esigma70 were both able to transcribe osmEp in vitro on supercoiled templates . In the presence of rpoD800, a mutation resulting in a thermosensitive sigma70 factor, a shift to non-permissive temperature abolished induction of osmEp after an osmotic shock during exponential phase, but did not affect the decelerating phase induction . Point mutations affecting osmEp activity were isolated . Down-promoter mutations decreased transcription in both the presence and the absence of sigmas, indicating that the two forms of RNA polymerase holoenzyme recognize very similar sequence determinants on the osmE promoter . Three up-promoter mutations brought osmEp closer to the consensus of Esigma70-dependent promoters . The two variant promoters exhibiting the highest efficiency became essentially independent of sigmas in vivo . Our data suggest that Esigmas transcribes wild-type osmEp with a higher efficiency than Esigma70 . A model in which an intrinsic differential recognition contributes to growth phase-dependent regulation is proposed . Generalization of this model to other sigmas-dependent promoters is discussed.

Mol Microbiol, 2000 Feb, 35(4), 835 - 44
IHF redistributes bound initiator protein, DnaA, on supercoiled oriC of Escherichia coli; Grimwade JE et al.; In Escherichia coli, initiation of chromosome replication requires that DnaA binds to R boxes (9-mer repeats) in oriC, the unique chromosomal replication origin . At the time of initiation, integration host factor (IHF) also binds to a specific site in oriC . IHF stimulates open complex formation by DnaA on supercoiled oriC in cell-free replication systems, but it is unclear whether this stimulation involves specific changes in the oriC nucleoprotein complex . Using dimethylsulphate (DMS) footprinting on supercoiled oriC plasmids, we observed that IHF redistributed prebound DnaA, stimulating binding to sites R2, R3 and R5(M), as well as to three previously unidentified non-R sites with consensus sequence (A/T)G(G/C) (A/T)N(G/C)G(A/T)(A/T)(T/C)A . Redistribution was dependent on IHF binding to its cognate site and also required a functional R4 box . By reducing the DnaA level required to separate DNA strands and trigger initiation of DNA replication at each origin, IHF eliminates competition between strong and weak sites for free DnaA and enhances the precision of initiation synchrony during the cell cycle.

Clin Exp Allergy, 2000 Mar, 30(3), 426 - 32
Airborne endotoxin exposure and the development of airway antigen-specific allergic responses; Wan GH et al.; BACKGROUND: AND OBJECTIVE: Repeated exposure of aerosolized antigen via respiratory tract can induce immunoglobulin (Ig) E isotype-specific tolerance to this antigen . However, the atopic individuals often produce a higher titre of IgE in response to airborne environmental allergens . The mechanisms of this differential regulation of airway allergen-specific immune responses are not fully understood . This study investigated the role of airborne endotoxin on the initiation of antigen-specific airway allergic responses . METHODS: ELISA methods for detection of isotypes of antigen-specific antibodies and competitive reverse transcription polymerase chain reaction for detection mRNA of cytokines were used . In addition, Liu stain method was used to analyse the amounts of eosinophils in bronchoalveolar lavage fluid . RESULTS: Mice pre-exposed with airborne endotoxin mounted significantly higher amounts of OVA-specific IgE antibody responses to inhaled OVA than those OVA-only sensitized mice . Inhaled endotoxin could downregulate repeated airway antigen exposure-induced IgE isotype-specific tolerance and increase antigen-induced lung eosinophils infiltration . CONCLUSIONS: These data show that airborne endotoxin exposure could potentiate allergen-specific airway inflammation . The results should have potential implications for understanding the development of allergen-induced airway allergic responses.

Clin Exp Allergy, 2000 Mar, 30(3), 324 - 32
Complementary DNA cloning and expression of a newly recognized high molecular mass allergen phl p 13 from timothy grass pollen (Phleum pratense); Suck R et al.; BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass . Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa . These proteins have not yet been fully characterized on the protein and DNA level . OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning . METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing . Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR . The cloned cDNA and deduced amino acid sequence were compared with sequence data bases . Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects . The IgE-binding capacity of natural and recombinant allergen was determined using EAST . RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases . Four potential sites for glycosylation and 16 cysteine residues were found . The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive . CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens . A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.

J Agric Food Chem, 2000 Feb, 48(2), 571 - 5
Immunological characterization of recombinant soy protein allergen produced by Escherichia coli expression system; Babiker EE et al.; To elucidate the molecular mechanism of the allergenicity of soybean P34 protein recognized as the most allergenic protein in soybean, the protein was expressed in Escherichia coli transformed with a plasmid carrying P34 cDNA . SDS-PAGE pattern showed that the molecular weight of the recombinant P34 was approximately 2 kDa less than that of the native soybean P34 . The difference in the molecular mass between these two proteins could be due to the native P34 in soybean being glycosylated at position Asn(170), whereas the recombinant protein generated in E . coli lacks this post-translational modification . Immunoblot analysis showed that both soybean and recombinant P34 proteins cross-reacted not only with polyclonal and monoclonal antibodies produced against P34 and crude soybean protein but also with patients' sera . The results suggest that the recombinant P34 is immunologically reactive, indicating that both proteins have similar epitope structures . Thus, the recombinant P34 produced by the E . coli expression system can be used as a standard allergen for molecular design to reduce the allergenic structure.

Med Microbiol Immunol (Berl), 1999 Aug, 188(1), 31 - 40
The ferric uptake regulator (Fur) homologue of Helicobacter pylori: functional analysis of the coding gene and controlled production of the recombinant protein in Escherichia coli; Bereswill S et al.; A homologue of the ferric uptake regulator protein Fur has recently been identified within the Helicobacter pylori genome . The promoterless gene on a plasmid did partially complement a fur-negative mutant of Escherichia coli, and was strongly positive in the Fur titration assay (FURTA) . The genetic and functional characterization of the complete fur homologue performed in this study revealed that the gene is conserved among H . pylori strains ( > 95% identity), and does not carry nucleotide transitions in iron-resistant mutants of H . pylori . The fur homologue on a plasmid mediated full iron-dependent ferric uptake regulator activity in the fur-deficient mutant strains H1681 and H1780 of E . coli . Immunoblot analysis revealed that Fur from H . pylori cross-reacts with antibodies raised against Fur from E . coli . The fact that inactivation of the fur gene abolished the FURTA-positive phenotype in the E . coli indicator strain H1717, indicated that this phenotype is rather caused by the encoded protein than by real Fur titration . Subcloning of the fur gene into an expression vector allowed controlled production in E . coli, and purification of a recombinant version of the H . pylori Fur protein . In summary, the results confirm the function of the H . pylori Fur homologue as iron-dependent transcriptional repressor by its ability to interact with the Fur-regulated promoters of the genes fiu and fhuF in E . coli.

IUBMB Life, 1999 Sep, 48(3), 327 - 32
Substitution of surface-exposed framework residues alters secretion of recombinant fusion protein Fv/tumor necrosis factor in Escherichia coli; Yang J et al.; Substitution of hydrophobic residues with hydrophilic ones at surface-exposed positions may influence the yield of antibody fragment expression in Escherichia coli by reducing its aggregation potential . We introduced such substitutions at 8 surface-exposed framework region residues of a fusion protein Fv/Tumor Necrosis Factor, which resulted in the expression of 10 mutant fusion proteins (Mut 1-10) in E . coli . Our results showed that expression levels of Mut 1-3, with mutations of A9S, T18K, and G41D, respectively, decreased by 4- to 8-fold, whereas expression levels of Mut 4, 9, and 10, with mutations of S60D/S70D, T28D, and G65D, respectively, were not affected . In contrast, mutation of F83A at light-chain residue 83, which is usually buried at the variable/constant domain interface of antibody molecules but becomes surface-exposed in recombinant Fv molecules, increased the expression level of Mut 5 by 4-fold . Our results suggest that this important substitution of a hydrophobic residue with a hydrophilic one may also be applied to increase the secretion of other recombinant Fv molecules in E . coli periplasm.

IUBMB Life, 1999 Sep, 48(3), 247 - 9
Is the HemK family of putative S-adenosylmethionine-dependent methyltransferases a "missing" zeta subfamily of adenine methyltransferases? A hypothesis; Bujnicki JM et al.; Previous comparative studies revealed close similarity among various groups of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases), indicating their common evolutionary origin . We present evidence for a remarkable similarity between the sequence and predicted structure of HemK (a widespread family of putative proteins encoded in genomes from bacteria to humans) and the catalytic domain of the gamma-subfamily of adenine-specific DNA MTases (N6mA MTases) . We predict the structure and function of the putative catalytic domain of HemK proteins and speculate that the target-recognizing function may be conferred by the N-terminal variable region.

Hepatogastroenterology, 2000 Jan-Feb, 47(31), 83 - 9
Treatment of ulcerative colitis; Ludwig D et al.; In recent years new standards for the treatment of ulcerative colitis have evolved . This review updates evidence based therapy for the various clinical situations as well as some novel approaches . The literature search was based on Medline, Cochrane database (CD-ROM) and handsearch of relevant papers including quoted literature . There is clear-cut evidence-based support for the use of local 5-aminosalicylates in mild/moderate distal and oral 5-aminosalicylates in extensive ulcerative colitis . The administration of corticosteroids is definitely indicated in severe disease . Fulminant attacks are treated by intravenous cyclosporine or colectomy . In chronic active disease azathioprine is probably helpful . Relapse prevention again is a domain of 5-aminosalicylates or, as a novel development, E . coli Nissle . The various meta-analyses as well as the controlled trials performed in the various clinical situations typical for the manifestations of ulcerative colitis form a solid base of evidence to guide individual treatment decisions.

Annu Rev Genet, 1999, 33, 57 - 88
Mechanisms of stationary phase mutation: a decade of adaptive mutation; Foster PL; A decade of research on adaptive mutation has revealed a plethora of mutagenic mechanisms that may be important in evolution . The DNA synthesis associated with recombination could be an important source of spontaneous mutation in cells that are not proliferating . The movement of insertion elements can be responsive to environmental conditions . Insertion elements not only activate and inactivate genes, they also provide sequence homology that allows large-scale genomic rearrangements . Some conjugative plasmids can recombine with their host's chromosome, and may acquire chromosomal genes that could then spread through the population and even to other species . Finally, a subpopulation of transient hypermutators could be a source of multiple variant alleles, providing a mechanism for rapid evolution under adverse conditions.

J Travel Med, 2000 Jan, 7(1), 27 - 9
Double-blind, randomized, placebo controlled pilot study evaluating efficacy and reactogenicity of an oral ETEC B-subunit-inactivated whole cell vaccine against travelers' diarrhea (preliminary report); Wiedermann G et al.; Diarrhea caused by enterotoxigenic E.coli (ETEC) is an important health problem in developing countries and in travelers to these areas . In previous trials formulations of ETEC vaccines containing the B-subunit of cholera toxin, which is antigenically similar to the heat labile enterotoxin of ETEC, and the most prevalent colonization factor antigens of ETEC, were shown to stimulate relevant mucosal immune responses in volunteers from Sweden and Egypt.

FEMS Microbiol Lett, 2000 Mar 1, 184(1), 119 - 25
Transcriptional regulation of the pas gene of enterohemorrhagic Escherichia coli; Beltrametti F et al.; The Pas protein plays a key role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC), being required for the secretion of the Esp proteins . Here, the transcriptional regulation of the pas gene was analyzed through the construction of a pas::lacZ translational fusion . When bacteria were grown in Luria Bertani medium or tissue culture medium supplemented with HEPES, a bimodal activation curve was observed . The early phase of induction was not significantly modified by the incubation temperature (either 25 or 37 degrees C), whereas the second phase, which overlaps with the late exponential growth phase, was enhanced at 37 degrees C . The early phase was also stimulated by growth on tissue culture medium and by the addition of Ca(2+), Mn(2+)or Mg(2+) to the M9-glucose minimal medium . Primer extension analysis showed the presence of two major starts of transcription, which were located 58 and 60 bp upstream of the ATG-start codon of the Pas protein, respectively . Although these sites are very close to each other, the transcripts produced during the early induction phase mainly start on the -60 position, whereas the -58 start was activated during the second induction phase.

FEMS Microbiol Lett, 2000 Mar 1, 184(1), 47 - 52
Isolation and characterization of mutated Fh1A proteins which activate transcription of the hyc operon (formate hydrogenlyase) of Escherichia coli in the absence of molybdate(1); Self WT et al.; Escherichia coli growing under anaerobic conditions produces H(2) and CO(2) by the enzymatic cleavage of formate catalyzed by formate hydrogenlyase (FHL) consisting of a molybdoenzyme formate dehydrogenase H (fdhF), hydrogenase 3 (hyc), and intermediate electron carriers (hyc) . Transcription of both the fdhF and hyc operons requires the activator, FhlA protein, as well as formate and molybdate . Several fhlA mutants with an altered response to the required effector molybdate were isolated and these FhlA mutated proteins activated hyc transcription in the absence of molybdate, but only in the presence of formate . Mutated protein FhlA126 carries a single mutation (R495C) in the conserved central domain of the modular, sigma(54)-dependent, enhancer-binding protein . FhlA57 contains two mutations; one in the unique N-terminal domain (E205K) and a second in the central domain (P442S) . Both mutations in FhlA132 are located in the N-terminal domain (A42T and E363K) . Both FhlA126 and FhlA132 proteins activated the hyc operon even in the absence of ModE and MoeA, two components of Mo-metabolism which are required for hyc-lac expression in wild-type E . coli . Based on these results, a model is proposed in which the native FhlA protein interacts with a unique form of Mo (MoeA product?) as a second effector for optimum expression of the hyc operon in E . coli.

Vaccine, 2000 Feb 25, 18(16), 1675 - 80
Mucosal vaccination and immune responses in the elderly; Fujihashi K et al.; To develop a mucosal vaccine strategy for the elderly, we have compared mucosal and systemic immune responses between aged (12-14 months) and young adult (8-12 weeks) mice . Both aged and young mice were immunized weekly with three oral doses of 1 mg of ovalbumin (OVA) and 10 microg of cholera toxin as adjuvant . Although elevated levels of OVA-specific systemic IgG and mucosal IgA Ab responses were seen in young mice, aged mice showed impaired antigen-specific mucosal and systemic immune responses . These results suggest that mucosal immunity is down regulated in aged mice.

Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2497 - 502
Physical association of ubiquitin ligases and the 26S proteasome; Xie Y et al.; The ubiquitin (Ub) system recognizes degradation signals of the target proteins through the E3 components of E3-E2 Ub ligases . A targeted substrate bears a covalently linked multi-Ub chain and is degraded by the ATP-dependent 26S proteasome, which consists of the 20S core protease and two 19S particles . The latter mediate the binding and unfolding of a substrate protein before its transfer to the interior of the 20S core . It is unclear how a targeted substrate is delivered to the 26S proteasome, inasmuch as Rpn10p, the only known proteasomal subunit that binds multi-Ub chains, has been found to be not essential for degradation of many proteins in the yeast Saccharomyces cerevisiae . Here we show that Ubr1p and Ufd4p, the E3 components of two distinct Ub ligases, directly interact with the 26S proteasome . Specifically, Ubr1p is shown to bind to the Rpn2p, Rpt1p, and Rpt6p proteins of the 19S particle, and Ufd4p is shown to bind to Rpt6p . These and related results suggest that a substrate-bound Ub ligase participates in the delivery of substrates to the proteasome, because of affinity between the ligase's E3 component and specific proteins of the 19S particle.

Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2521 - 6
Mammalian thioredoxin reductase: oxidation of the C-terminal cysteine/selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity; Lee SR et al.; Mammalian cytosolic thioredoxin reductase (TrxR) has a redox center, consisting of Cys(59)/Cys(64) adjacent to the flavin ring of FAD and another center consisting of Cys(497)/selenocysteine (SeCys)(498) near the C terminus . We now show that the C-terminal Cys(497)-SH/SeCys(498)-Se(-) of NADPH-reduced enzyme, after anaerobic dialysis, was converted to a thioselenide on incubation with excess oxidized Trx (TrxS(2)) or H(2)O(2) . The Cys(59)-SH/Cys(64)-SH pair also was oxidized to a disulfide . At lower concentrations of TrxS(2), the Cys(59)-SH/Cys(64)-SH center was still converted to a disulfide, presumably by reduction of the thioselenide to Cys(497)-SH/SeCys(498)-Se(-) . Specific alkylation of SeCys(498) completely blocked the TrxS(2)-induced oxidation of Cys(59)-SH/Cys(64)-SH, and the alkylated enzyme had negligible NADPH-disulfide oxidoreductase activity . The effect of replacing SeCys(498) with Cys was determined by using a mutant form of human placental TrxR1 expressed in Escherichia coli . The NADPH-disulfide oxidoreductase activity of the purified Cys(497)/Cys(498) mutant enzyme was 6% or 11% of that of wild-type rat liver TrxR1 with 5, 5'-dithiobis(2-nitrobenzoic acid) or TrxS(2), respectively, as substrate . Disulfide formation induced by excess TrxS(2) in the mutant form was 12% of that of the wild type . Thus, SeCys has a critical redox function during the catalytic cycle, which is performed poorly by Cys.

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 1993 - 8
Molecular basis of a progressive juvenile-onset hereditary cataract; Pande A et al.; In a recent paper, patients with a progressive juvenile-onset hereditary cataract have been reported to have a point mutation in the human gammaD crystallin gene (Stephan, D . A., Gillanders, E., Vanderveen, D., Freas-Lutz, D., Wistow, G., Baxevanis, A . D., Robbins, C . M., VanAuken, A., Quesenberry, M . I., Bailey-Wilson, J., et al . (1999) Proc . Natl . Acad . Sci . USA 96, 1008-1012) . This mutation results in the substitution of Arg-14 in the native protein by a Cys residue . It is not understood how this mutation leads to cataract . We have expressed recombinant wild-type human gammaD crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution . Eventually, R14C precipitates . In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers . These data strongly suggest that the observed cataract is triggered by the thiol-mediated aggregation of R14C . The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one . Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities . Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2373 - 8
The movement protein NSm of tomato spotted wilt tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins; Soellick T et al.; The nonstructural NSm protein of tomato spotted wilt tospovirus (TSWV) represents a putative viral movement protein involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures . To study the molecular basis of NSm function, we expressed the protein in Escherichia coli and investigated protein-protein and protein-RNA interactions of NSm protein in vitro . NSm specifically interacts with TSWV N protein and binds single-stranded RNA in a sequence-nonspecific manner . Using NSm as a bait in a yeast two-hybrid screen, we identified two homologous NSm-binding proteins of the DnaJ family from Nicotiana tabacum and Arabidopsis thaliana.

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