Escherichia coli

Genes Cells, 2000 Dec, 5(12), 991 - 9
Genetic dissection of SecA: suppressor mutations against the secY205 translocase defect; Matsumoto G et al.; BACKGROUND: The driving force for protein translocation across the bacterial plasma membrane is provided by SecA ATPase, which undergoes striking conformational changes characterized by the membrane insertion and deinsertion cycle . This action of SecA requires the membrane-embedded SecYEG complex . Previously, we have identified a cold-sensitive secY mutation (secY205), affecting the most carboxy-terminal cytosolic domain, that did not allow an ATP-dependent insertion of a SecA-preprotein complex . Thus, this mutant provides an excellent system for genetic analysis of the SecY-SecA interaction . RESULTS: We carried out a systematic isolation of secA mutations that suppressed secY205 cold-sensitivity . A total of 40 independent suppressor mutations were classified into: (i) allele-specific suppressors, acting only against secY205, and (ii) 'super active' suppressors, acting against almost any sec defects . The former class of mutations, presumably with specific effects on the SecY-SecA interaction, clustered in two regions close to the Walker motif A sequences of the two ATP-binding domains . The latter mutations, enhancing general SecA activities, were mostly in or around the minor ATP-binding domain . CONCLUSIONS: The Walker motif A regions of SecA are important for the SecA-SecY interaction that leads to the SecA conformational changes required for insertion into the SecYEG channel . The minor ATP-binding domain is important for the down-regulation of SecA activities.

Genes Cells, 2000 Dec, 5(12), 965 - 74
Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli; Maki Y et al.; BACKGROUND: Ribosomes in Escherichia coli change their composition and conformation in the stationary phase . Ribosome modulation factor (RMF) and ribosomal protein S22 are known to be associated with stationary phase ribosomes . RMF association causes the loss of translational activity and the dimerization of 70S ribosomes into 100S ribosomes, which may increase cell survival in the stationary phase . RESULTS: Two weakly acidic proteins having related amino acid sequences were found to be associated with E . coli ribosomes in the stationary phase . These proteins are the products of ORFs named yfiA and yhbH . The sum of the copy numbers of their product proteins, YfiA and YhbH, in the ribosomal particles was low in the log phase, but increased to nearly one in the stationary phase . YfiA was found in the 70S ribosomal fraction rather than the 100S . On the other hand, YhbH was detected exclusively in the 100S ribosomal fraction . When the stationary phase cells were transferred to fresh medium, YfiA and YhbH were found in the 70S ribosomal fraction, but not in the polysome fraction . CONCLUSIONS: Two proteins, YfiA and YhbH, associated with E . coli ribosomes were found to accumulate in the stationary phase, leading to the formation of several types of ribosomes . They are not likely to have roles in the elongation step of the translation in log phase cells, but are likely to be involved in the stabilization and preservation of ribosomes in the stationary phase, which might be necessary for cell survival.

Eur J Biochem, 2001 Feb, 268(3), 826 - 31
Critical role of glutamic acid 202 in the enzymatic activity of stromelysin-1 (MMP-3); Arza B et al.; To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn(2+) in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site-specific mutagenesis . Wild-type proMMP-3, proMMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity . Whereas 33-kDa wild-type proMMP-3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercuric acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistant to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa species . In contrast, treatment with plasmin quantitatively converted the wild-type and the three mutant proMMP-3 moieties into the corresponding 24-kDa MMP-3 moieties . Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild-type and mutant proMMP-3 (K(a) of 2.6-6.3 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 10(6) M(-1)) moieties . The affinity for binding to single-chain urokinase-type plasminogen activator (scu-PA) was also similar for wild-type and mutant proMMP-3 (K(a) of 5.0-6.9 x 10(6) M(-1)) or MMP-3 (K(a) of 37-72 x 10(6) M(-1)) moieties . However, MMP-3(E202A) and MMP-3(E202K) did not hydrolyze plasminogen whereas MMP-3(E202D) showed an activity of 20--30% of wild-type MMP-3 . All three mutants were inactive towards scu-PA under conditions where this was quantitatively cleaved by wild-type MMP-3 . Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive toward a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the activity of wild-type MMP-3 . Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP-3.

Eur J Biochem, 2001 Feb, 268(3), 800 - 8
Interaction of Escherichia coli hemolysin with biological membranes . A study using cysteine scanning mutagenesis; Schindel C et al.; Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins . Lytic activity depends on binding of Ca2(+) to the C-terminus of the molecule . The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain . In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan) . The fluorescence emission of the label was examined in soluble and membrane-bound toxin . Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer . The emission shifts occurred both in the presence and absence of Ca2(+), suggesting that Ca2(+) is not required for the toxin to enter membranes . However, binding of Ca2(+) to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation . Our data indicate that binding of Ca2(+) to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.

Eur J Biochem, 2001 Feb, 268(3), 743 - 51
Mapping DNA-binding sites of HIV-1 integrase by protein footprinting; Dirac AM et al.; The HIV-1 integrase protein catalyzes integration of the viral genome into host cell DNA . Whereas the structures of the three domains of integrase have been solved separately, both the structural organization of the full-length protein and its interaction with DNA remain unresolved . A protein footprinting approach was employed to investigate the accessibility of residues in the full-length soluble integrase mutant, INF(185K,C280S), to proteolytic attack in the absence and presence of DNA . The N-terminal and C-terminal domains were relatively more accessible to proteolytic attack than the core domain . The susceptibility to proteolytic attack was specifically affected by DNA at residues Lys34, in the N-terminal domain, Lys111, Lys136, Glu138, Lys156-Lys160, Lys185-Lys188, in the core domain, and Asp207, Lys 215, Glu246, Lys258 and Lys273 in the linker and C-terminal domain, suggesting that these regions are involved in, or shielded by, DNA binding . Lys34 is positioned in a putative dimerization domain, consistent with the notion that DNA stabilizes the dimeric state of integrase.

Eur J Biochem, 2001 Feb, 268(3), 737 - 42
Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process; Ciriolo MR et al.; Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion . By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134) . The effects of these mutations on the rate of superoxide dismutation were not dramatic . In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme . The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions . On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione {Cu(I)-GSH} complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme . The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.

Eur J Biochem, 2001 Feb, 268(3), 722 - 6
Aldehyde dehydrogenase . Maintaining critical active site geometry at motif 8 in the class 3 enzyme; Hempel J et al.; Alignment of all known, diverse members of the aldehyde dehydrogenase (ALDH) extended family revealed only two strictly conserved, nonglycine residues, a glutamate and a phenylalanine residue . Both occur in one of the highly conserved 'motif' segments and both occupy strategic locations in the tertiary structure at the bottom of the catalytic funnel . In class 3 ALDH, these are Glu333 and Phe335 . In addition, Asp247, which is not highly conserved but is characteristic of class 3 ALDHs, hydrogen bonds the main chain between Glu333 and Phe335 . These three residues were mutated conservatively . Michaelis constants determined for both NAD/propanal and NADP/benzaldehyde substrate pairs show all three residues to be crucial to effective catalysis, and suggest that the hydrogen bond to Asp247 is a key element in maintaining precise geometry of key elements at the active site.

Eur J Biochem, 2001 Feb, 268(3), 694 - 702
Construction and functional evaluation of a single-chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector; Devaux C et al.; 9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom . It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals . Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv) . This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L-agarose beads yielding 1-2 mg.L(-1) of bacterial culture . scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI . The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range . Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes . Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1 . Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.

Eur J Biochem, 2001 Feb, 268(3), 686 - 93
The cysteine synthase complex from plants . Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction; Wirtz M et al.; Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex . Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases . Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases . The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure . This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization . The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.

Eur J Biochem, 2001 Feb, 268(3), 565 - 72
Determination of binding constant of transcription factor AP-1 and DNA . Application of inhibitors; Kwon H et al.; The equilibrium binding and association kinetics of the fos-jun dimer (basic and leucine zipper domain) to the AP-1 DNA were studied using a quantitative assay . The basic-region and leucine zipper (bZip) domain of c-fos was expressed as a fusion protein with glutathione S-transferase, and it was bound to glutathione-agarose . The GST-fused fos bZip region was allowed to form a heterodimer with the bZip domain of c-jun, to which radiolabeled AP-1 nucleotides were added . After thorough washing, the gel-bound radioactivity was counted . The binding and dissociation rate constants (k(1) and k-(1)) of the fos-jun dimer and DNA could be obtained from a time-course experiment . The association binding constant (K(1)) was determined using both a thermodynamic equation and kinetic parameters . Nordihydroguaiaretic acid (NDGA), momordin I, natural product inhibitors of the fos-jun/DNA complex formation, was applied to this jun-GST-fused fos system and it was found to decrease the apparent equilibrium binding of dimer and DNA . The thermodynamic constant of dimer and inhibitor binding was also determined.

Eur J Biochem, 2001 Feb, 268(3), 544 - 53
Unmasking a hyaluronan-binding site of the BX(7)B type in the H3 heavy chain of the inter-alpha-inhibitor family; Jean L et al.; The inter-alpha-inhibitor (I alpha I) family gathers together several plasma protease inhibitors such as I alpha I and pre-alpha-inhibitor (P alpha I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P . In addition to their protease inhibitory activity, a major physiological function of I alpha I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types . Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis . However, how HA and I alpha I family members first recognize each other has so far remained elusive . The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue . This domain has now been identified in the N-terminal end of H3P that is a precursor of P alpha I . A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion . Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end . The latter observation was confirmed with a natural, mature H3 chain purified from human plasma . Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not . Therefore, in vivo, the HA-binding activity of the mature H3 chain within P alpha I may vary with the folding and/or fragmentation of this protein.

Eur J Biochem, 2001 Jan, 268(2), 429 - 36
The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation . Role of disulfide bonds; Roher N et al.; The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT) . This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment with dithiothreitol . Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation . This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar ratios of 4 : 1 . The presence of dithiothreitol markedly reduced the anti-aggregation effects of grp94-CT on CK2alpha without altering the solubility of the chaperone . It is concluded that the chaperone activity of the C-terminal domain of human grp94 requires the maintenance of its quaternary structure (dimers and oligomers), which seems to be stabilised by disulphide bonds.

Eur J Biochem, 2001 Jan, 268(2), 396 - 404
Zinc-dependent conformational changes in domain D5 of high molecular mass kininogen modulate contact activation; Herwald H et al.; Human high molecular mass kininogen (HK) participates as nonenzymatic cofactor in the contact system . Here, we show that recombinant domain D5 of HK (rD5) prolongs the clotting time of the intrinsic pathway of coagulation and attenuates the generation of bradykinin . Further studies indicate that a correct fold of domain D5 within HK is required for the activation of the contact system . The folding of rD5 seems to be modulated by the metal ions Zn2+, Ni2+, and Cu2+ as a specific antibody directed against the zinc-binding site in HK binds to HK and rD5 in a metal ion concentration dependent manner . The finding that these three metal ions specifically affect contact activation suggests that they regulate the accessibility of rD5 for negatively charged surfaces . Support for the assumption that the observed phenomena are due to conformational changes was obtained by fluorescence spectroscopy of rD5, demonstrating that its fluorescence spectrum was changed in the presence of ZnCl2 . Moreover, negative staining electron microscopy experiments suggest that the zinc-induced changes in D5 also affect the conformation of the entire HK protein . The present data emphasize the role of zinc and other metal ions in the regulation of contact activation.

Eur J Biochem, 2001 Jan, 268(2), 310 - 6
Phosphorylation of 1-deoxy-D-xylulose by D-xylulokinase of Escherichia coli; Wungsintaweekul J et al.; 1-deoxy-D-xylulose 5-phosphate serves as a precursor for the biosynthesis of the vitamins thiamine and pyridoxal and for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate via the nonmevalonate pathway of terpenoid biosynthesis . Earlier studies had shown that Escherichia coli incorporates unphosphorylated 1-deoxy-D-xylulose into the terpenoid side chain of ubiquinones with high efficacy . We show that D-xylulokinase of E . coli (EC 2.7.1.17) catalyzes the phosphorylation of 1-deoxy-D-xylulose at the hydroxy group of C-5 at a rate of 1.6 micromol.mg min-1 . This reaction constitutes a potential salvage pathway for the generation of 1-deoxy-D-xylulose 5-phosphate from exogenous or endogenous 1-deoxy-D-xylulose as starting material for the biosynthesis of terpenoids, thiamine and pyridoxal.

Eur J Biochem, 2001 Jan, 268(2), 287 - 94
Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology; Eriksson TL et al.; The dust mite Lepidoglyphus destructor is a common species in Europe and a major cause of dust mite allergy in rural surroundings, but it also contributes to dust mite allergy in urban areas . One major allergen, Lep d 2, has been expressed as a recombinant protein and evaluated both in vivo and in vitro and shown to detect 60% or more of L . destructor-sensitized subjects . Additional recombinant allergens are needed to obtain a reliable diagnostic tool for L . destructor allergy . The aim of this study was to clone and express new allergens from L . destructor and determine their recognition frequency among sensitized individuals . A phage display cDNA expression library was constructed and screened with sera from L . destructor-sensitized individuals . The cDNAs encoding the allergens were cloned into the pET17b vector and subsequently expressed in Escherichia coli as C-terminal His6-tagged proteins . Immunoblotting of the recombinant proteins was performed using sera from 45 subjects allergic to L . destructor . Three new allergens from L . destructor, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were identified and recognized by 4/45 (9%), 28/45 (62%) and 6/45 (13%) sera from L . destructor-sensitized subjects, respectively.

Eur J Biochem, 2001 Jan, 268(2), 278 - 86
Functional interactions of an Escherichia coli ribosomal ATPase; Kiel MC et al.; The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified . The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein . RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits . Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis . RbbA is reported to have affinity for EF-Tu but not for EF-G . Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1 . Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes . A possible role for RbbA in protein-chain elongation is proposed.

Eur J Biochem, 2001 Jan, 268(2), 260 - 7
Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29 . Effects of zeaxanthin, pH and phosphorylation; Crimi M et al.; Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants . The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo . We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein . These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics . We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins) . Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin . The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins) . When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics . The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.

Clin Microbiol Infect, 2000 Feb, 6(2), 82 - 7
Epidemiologic typing of Escherichia coli using RAPD analysis, ribotyping and serotyping; Vogel L et al.; OBJECTIVE: To compare random amplified polymorphic DNA (RAPD) analysis and ribotyping with serotyping for epidemiologic typing of Escherichia coli . METHODS: Thirty-two epidemiologically unrelated strains, nine cerebrospinal fluid isolates with the O7K1 serotype from nine patients, and nine sets of epidemiologically related E . coli isolates from nine patients were typed by RAPD analysis, ribotyping and serotyping . RESULTS: Among the 32 epidemiologically unrelated E . coli isolates, 29 types were distinguished by RAPD analysis, 25 by ribotyping and 27 by serotyping . Indistinguishable patterns were obtained by RAPD analysis and ribotyping within the collection of nine cerebrospinal fluid isolates . For the epidemiologically related isolates, intrapatient variation was only found by RAPD analysis among the isolates of one set and by ribotyping among the isolates of two sets . No interpatient variation was observed between three sets of isolates . With serotyping, the epidemiologically related isolates yielded similar typing relationships to those obtained by RAPD analysis and ribotyping . CONCLUSIONS: RAPD analysis had the highest discriminatory capacity for typing E . coli isolates . RAPD analysis, ribotyping and serotyping can all be used for assessment of strain relationships.

Acta Anaesthesiol Scand, 2001 Feb, 45(2), 213 - 20
Effects of different preparations of propofol, diazepam, and etomidate on human neutrophils in vitro; Heine J et al.; BACKGROUND: Intravenous anaesthetics and sedatives can influence polymorphonuclear cell (PMN) functions . Some of the drugs for sedation and anaesthesia have been alternatively dissolved in lipid solutions containing medium (MCT) and/or long chain (LCT) triglycerides . The in vitro effects of two different diazepam (benzyl-alcohol, LCT/MCT), etomidate (propylene-glycol, LCT/MCT), and propofol (LCT, LCT/MCT) preparations on respiratory burst (RB) and phagocytosis of human PMNs were studied . METHODS: Diazepam (2, 20 microg ml(-1)), etomidate (0.5, 5 microg ml(-1)), and propofol (6, 60 microg ml(-1)) were investigated in clinical and 10-fold concentrations with flow cytometric assays . The RB was measured with the fluorescent dye rhodamine after induction with Escherichia coli or formyl-methionyl-leucylphenylalanine (FMLP) following priming with tumour necrosis factor alpha (TNF-alpha) . Phagocytosis of PMNs was carried out in whole blood after incubation with fluorescein-labelled E . coli . RESULTS: LCT-propofol at 60 microg ml(-1) reduced the percentage of PMNs with RB activity after induction with E . coli (52.8+/-20.4) and TNF-alpha/FMLP (10.8+/-5.1)) as well as the percentage of phagocytosing PMNs (48.9+/-19.5) in contrast to LCT/MCT-propofol, which augmented all parameters (85.4+/-10.1, 50.3+/-12.7, 66.5+/-12.5) . Also the higher concentrations of LCT/MCT-diluted etomidate and diazepam increased the percentage of RB positive PMNs compared to the alternative compositions . The percentage of phagocytosing PMNs was less reduced with 20 microg ml(-1) LCT/MCT-diazepam (85.2+/-6.9) than with the same concentration of benzyl-alcohol diluted diazepam (68.8+/-12.2) compared to the control . CONCLUSION: The in vitro effects of diazepam, etomidate, and propofol are dependent on the solvent applied . The tested LCT/MCT preparations reduce the inhibitory effects on the bacterial killing capacity of PMNs found after incubation with propyleneglycol, benzyl-alcohol, or LCT preparations, respectively.

Int J Biochem Cell Biol, 2001 Jan, 33(1), 87 - 94
Purification and characterization of a cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli; Ye XY et al.; A cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli was purified 88-fold by chromatography on High Q and hydroxyapatite . N-terminal amino acid sequence analyses confirmed that the cellulase represented the product of the cellulase gene Cel B2 . The purified enzyme possessed high activity toward barley beta-glucan, lichenan, carboxymethyl cellulose (CMC), xylan, but not toward laminarin and pachyman . In addition, the cloned enzyme was able to hydrolyze p-nitrophenyl (PNP)-cellobioside, PNP-cellotrioside, PNP-cellotetraoside, PNP-cellopentaoside, but not PNP-glucopyranoside . The specific activity of the cloned enzyme on barley beta-glucan was 297 units/mg protein . The purified enzyme appeared as a single band in SDS-polyacrylamide gel electrophoresis and the molecular mass of this enzyme (58000) was consistent with the value (56463) calculated from the DNA sequence . The optimal pH of the enzyme was 5.5, and the enzyme was stable between pH 5.0 and pH 7.5 . The enzyme had a temperature optimum at 40 degrees C . The K(m) values estimated for barley beta-glucan and CMC were 0.32 and 0.50 mg/ml, respectively.

Gene, 2000 Dec 31, 261(2), 383 - 90
In vivo expansion of trinucleotide repeats yields plasmid and YAC constructs for targeting and transgenesis; Sopher BL et al.; Production of mouse models of inherited neurodegenerative diseases is an important step towards understanding the mechanism of neurotoxicity and for testing potential therapies . We are interested in creating a mouse model for X-linked spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by expansion of a CAG repeat within the androgen receptor (AR) gene . To permit generation of mice that will show a SBMA phenotype within their life span, we decided to obtain a yeast artificial chromosome (YAC) carrying the AR gene and introduce CAG repeat mutations numbering 100 or more triplets . SBMA patients with more than 70 CAGs have never been observed; therefore, we chose to expand a 59 CAG repeat tract in vivo in Escherichia coli . Although we set out to expand this repeat tract using a recombination paradigm involving two plasmid co-propagation, we did not observe large expansions . We were instead able to incrementally generate repeat tracts from 100 to 200 CAGs in a yeast integrating plasmid vector by taking advantage of replication instability . In the course of our experiments that yielded these CAG repeat tracts, we evaluated the role of repeat orientation, vector co-propagation, and recA function on the expansion process . We then used one of the yeast integrating vectors to successfully produce an AR YAC construct carrying 100 CAG repeats . AR YAC CAG100 will serve as a valuable reagent for the production of a SBMA mouse.

Gene, 2000 Dec 31, 261(2), 289 - 98
Control of copper homeostasis in Escherichia coli by a P-type ATPase, CopA, and a MerR-like transcriptional activator, CopR; Petersen C et al.; We have isolated and characterized a copper sensitive Escherichia coli mutant that is deficient in the copper transporting P-type ATPase encoded by the copA gene (previously ybaR) . Measurements of uptake and efflux of 64Cu by wild-type and mutant cells implicated the CopA protein in copper efflux from the cytoplasm, and further demonstrated that cell-associated copper in intact E . coli cells is distributed between two kinetically distinguishable pools, the ratio of which was dramatically disturbed by the copA mutation . Using a copA-lacZ gene fusion the copA promoter was found to be specifically induced by copper, and this induction was shown to be dependent on a MerR-like transcriptional activator encoded by a previously uncharacterized gene, copR (previously ybbI) . In the copA deficient background the copA-lacZ fusion was super induced to very high levels even in the absence of copper addition to the medium, and this induction was dependent on CopR . These results indicated that the cytoplasmic copper concentration was dramatically increased in the copA mutant, in agreement with the 64Cu uptake experiments . Moreover, they implied, that the copper concentration in wild type cells is determined primarily by the CopA efflux pump, while copper is taken up by an essentially constitutive mechanism.

Vaccine, 2001 Feb 8, 19(13-14), 1747 - 52
Detoxification of endotoxin by aluminum hydroxide adjuvant; Shi Y et al.; Langmuir adsorption isotherms of endotoxin and aluminum-containing adjuvants at pH 7.4 and 25 degrees C revealed that aluminum hydroxide adjuvant has a greater adsorption capacity (283 microg/mg Al) and adsorption coefficient (1.3x10(4) ml/miccrog) than aluminum phosphate adjuvant (3.0 microg/mg Al, 0.20 ml/microg) . The difference in endotoxin adsorption was related to two adsorption mechanisms: electrostatic attraction and covalent bonding . The isoelectric point (iep) of endotoxin is approximately 2 . An electrostatic attractive force will be present with aluminum hydroxide adjuvant (iep=11.4), and an electrostatic repulsive force will operate with aluminum phosphate adjuvant (iep=4.6) . Endotoxin contains two phosphate groups in the lipid A portion . Covalent bonding occurs with surface aluminum in aluminum hydroxide adjuvant but is inhibited by surface phosphate in aluminum phosphate adjuvant . In-vitro desorption experiments using components of interstitial fluid showed that endotoxin adsorbed by aluminum hydroxide adjuvant was not desorbed by interstitial anions (5 mM phosphate or 2.7 mM citrate) or interstitial proteins (25 mg albumin/ml) . The effect of aluminum-containing adjuvants on the systemic response of Sprague-Dawley rats to a 15 microg/kg subcutaneous dose of endotoxin was determined by measuring the serum concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) . TNF-alpha and IL-6 were observed in the group which received an endotoxin solution or endotoxin and aluminum phosphate adjuvant . No TNF-alpha or IL-6 was detected in the group that received endotoxin and aluminum hydroxide adjuvant . Aluminum hydroxide adjuvant detoxifies endotoxin by adsorbing it in the vaccine and then not releasing it in interstitial fluid upon administration.

Antiviral Res, 2001 Jan, 49(1), 35 - 47
High efficient production of Pr55(gag) virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A; Buonaguro L et al.; The main goal of this study was to investigate a novel approach for an efficient and reproducible production of Virus-Like Particles (VLPs) expressing multiple HIV-1 epitopes . The HIV-1 Pr55(gag)-based VLPs have been produced in a Baculovirus expression system, using a transfer vector able to support the independent expression of different open reading frames (ORFs) . In this regard, the gp120 derived from 94UG018 HIV-1(A) isolate, previously studied in our laboratory, has been packaged into the VLPs together with nef and pol ORFs . In particular, the gp120(UG) sequence shows a 90% homology in the V3 region compared to African HIV-1 strains of the A-clade . This novel approach is extremely effective for the production of VLPs expressing all the epitopes, as confirmed by Western Blot characterization . Furthermore, the resulting HIV-VLP(A)s show the expected density (1.14--1.18 g/ml) on a 10--60% sucrose gradient and the morphology of an immature virion at standard transmission electron microscopy . Our results demonstrate that this strategy is highly efficient for expressing a balanced amount of multiple epitopes and their packaging in VLP structures, without affecting the Pr55(gag) autoassembling capacities . Furthermore, the genetic transposition performed in a modified E . coli represents a methodological improvement, allowing a faster and more reproducible identification of recombinant Baculovirus DNA molecules.

Curr Opin Chem Biol, 2001 Feb, 5(1), 74 - 7
Peptide inhibitors expressed in vivo; Kamb A et al.; Peptide inhibitors isolated from libraries either through genetic screens or binding assays have gained visibility in the past year - especially with the publication of four studies in model systems (two in yeast, two in Escherichia coli) . These and other studies demonstrate that forward and reverse genetic experiments with peptides can be extremely efficient in validating candidate drug targets and in defining elements of biochemical pathways.

Trends Biochem Sci, 2001 Feb, 26(2), 131 - 6
The Rad51 and Dmc1 recombinases: a non-identical twin relationship; Masson JY et al.; A double-strand break in genomic DNA that remains unrepaired can be lethal for a cell . Indeed, the integrity of the genome is paramount for survival . It is therefore surprising that some cells deliberately introduce double-strand breaks at certain times during their life cycle . Why might they do this? What are the benefits? How are these breaks repaired? The answers to these questions lie in understanding the basis of meiotic recombination, the process that leads to genetic variation . This review summarizes the key roles played by the two recombinases, Dmc1 and Rad51, in the faithful repair of DNA breaks.

Trends Biochem Sci, 2001 Feb, 26(2), 93 - 9
The evolution of ribonucleotide reduction revisited; Stubbe J et al.; Ribonucleotide reductases (RNRs) catalyze the conversion of both purine and pyrimidine nucleotides to deoxynucleotides in all organisms and provide all the monomeric precursors essential for both DNA replication and repair . RNRs have been divided into three classes on the basis of their unique metallo-cofactors . The exquisitely controlled free radical chemistry used by all RNRs, and the commonality of the structures of the subunits where the nucleotide reduction process occurs, together provide compelling evidence for the importance of chemistry in the divergent evolution of RNRs from a common progenitor.

Plant Sci, 2001 Feb 5, 160(3), 463 - 472
Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily; Ostergaard L et al.; Two tandem genes were identified on Arabidopsis chromosome II (AtCRL1 and AtCRL2) encoding proteins with homology to members of the dihydroflavonol-4-reductase (DFR) superfamily . The encoded CRL1 and CRL2 proteins share 87% mutual amino acid sequence identity whereas their promoter regions are highly divergent, suggesting differential regulation of the CRL genes . Phylogenetic analysis placed CRL1 and CRL2 in a separate branch of the DFR superfamily . Northern blotting showed strong AtCRL1 induction by abscisic acid (ABA), drought, and heat shock, and high expression level in seeds, thus resembling the expression pattern of late embryogenic abundant ABA-responsive genes . Differential expression of the two genes during plant development was confirmed in plants expressing transcriptional fusions between the two promoters and the Escherichia coli beta-glucuronidase reporter gene . This showed that, whereas high expression of AtCRL1 in mature seeds declines during subsequent vegetative growth, transcriptional activity from the AtCRL2 promoter increases during vegetative growth . Expression of both genes is restricted to vascular tissue . Based upon their homology to proteins involved in lignin synthesis, we propose that AtCRL2 is involved in generating conducting tissue late in development, while AtCRL1 is involved in vascular tissue differentiation and/or synthesis in the germinating embryos.

Mutat Res, 2001 Feb 20, 473(2), 229 - 47
An aerobic recA-, umuC-dependent pathway of spontaneous base-pair substitution mutagenesis in Escherichia coli; Bhamre S et al.; Antimutator alleles indentify genes whose normal products are involved in spontaneous mutagenesis pathways . Mutant alleles of the recA and umuC genes of Escherichia coli, whose wild-type alleles are components of the inducible SOS response, were shown to cause a decrease in the level of spontaneous mutagenesis . Using a series of chromosomal mutant trp alleles, which detect point mutations, as a reversion assay, it was shown that the reduction in mutagenesis is limited to base-pair substitutions . Within the limited number of sites than could be examined, transversions at AT sites were the favored substitutions . Frameshift mutagenesis was slightly enhanced by a mutant recA allele and unchanged by a mutant umuC allele . The wild-type recA and umuC genes are involved in the same mutagenic base-pair substitution pathway, designated "SOS-dependent spontaneous mutagenesis" (SDSM), since a recAumuC strain showed the same degree and specificity of antimutator activity as either single mutant strain . The SDSM pathway is active only in the presence of oxygen, since wild-type, recA, and umuC strains all show the same levels of reduced spontaneous mutagenesis anaerobically . The SDSM pathway can function in starving/stationary cells and may, or may not, be operative in actively dividing cultures . We suggest that, in wild-type cells, SDSM results from basal levels of SOS activity during DNA synthesis . Mutations may result from synthesis past cryptic DNA lesions (targeted mutagenesis) and/or from mispairings during synthesis with a normal DNA template (untargeted mutagenesis) . Since it occurs in chromosomal genes of wild-type cells, SDSM may be biologically significant for isolates of natural enteric bacterial populations where extended starvation is often a common mode of existence.

Mutat Res, 2001 Feb 20, 473(2), 219 - 28
Different characteristics distinguish early versus late arising adaptive mutations in Escherichia coli FC40; Powell SC et al.; The Escherichia coli strain FC40 has frequently been employed to investigate the mechanism of adaptive mutations . The strain cannot utilize lactose due to a +1 frameshift mutation that reduces beta-galactosidase to about 1% of normal levels . Cells undergo a high rate of mutation from Lac- to Lac+ when cells are grown with lactose as the sole energy source . Almost all Lac+ colonies arising 3-6 days after plating result from a base pair deletion in runs of iterated base pairs within a 130-bp target region . In this study we characterized Lac+ colonies arising 3-10 days after plating . Temperature gradient gel electrophoresis (TGGE) was used to detect mutations in the target region as a function of the day a colony appears . TGGE results confirmed the occurrence of mutations within the target region in 36 of 37 FC40 Lac+ colonies arising on days 3-7 . However, mutations in this region were not detected in 23 of 37 Lac+ colonies arising from days 8-10 . Sequencing data verified the TGGE results . Half of the Lac+ mutants arising on days 8-10 with no base pair change in the target region were unstable and exhibited a Lac- phenotype after successive growth cycles in rich medium . The results suggest that amplification of the lac operon region is a common factor in late arising colonies, and that different characteristics distinguish early and late arising Lac+ colonies.

Mutat Res, 2001 Feb 20, 473(2), 201 - 10
Analyses of spontaneous mutations of cloned gene 49 of phage T4; Hartung M et al.; Holliday structure resolving enzyme endonuclease VII (endo VII) of phage T4 is highly toxic for E . coli when expressed outside of the phage infection environment . As a consequence, plasmids with a mutated gene 49, the gene which encodes for endo VII, can be easily isolated and characterised . We have isolated and characterised 400 survivors from independent transformations with a plasmid carrying gene 49 under the control of the T7 promoter . The majority had mutated gene 49 by IS10 insertions which almost exclusively mapped to a distinct site . When this site was mutated other insertion sites were observed as well as an increase in other mutational events including large deletions . Neither of the observed insertion sites mapped matched the consensus IS10 sequence completely . Additionally when the level of expression of gene 49 was altered the distribution of mutations was changed suggesting that other elements apart from the target sequence are necessary for determining IS10 insertion . The expression of gene 49 in E . coli provides a particularly useful tool for the analysis of mutational events.

Mutat Res, 2001 Jan 25, 473(1), 109 - 19
Effect of endogenous carotenoids on "adaptive" mutation in Escherichia coli FC40; Bridges BA et al.; The appearance over many days of Lac(+) frameshift mutations in Escherichia coli strain FC40 incubated on lactose selection plates is a classic example of apparent "adaptive" mutation in an episomal gene . We show that endogenously overproduced carotenoids reduce adaptive mutation under selective conditions by a factor of around two . Carotenoids are known to scavenge singlet oxygen suggesting that the accumulation of oxidative base damage may be an integral part of the adaptive mutation phenomenon . If so, the lesion cannot be 7,8-dihydro-8-oxoguanine since adaptive mutation in FC40 is unaffected by mutM and mutY mutations . If active oxygen species such as singlet oxygen are involved in adaptive mutation then they should also induce frameshift mutations in FC40 under non-selective conditions . We show that such mutations can be induced under non-selective conditions by protoporphyrin photosensitisation and that this photodynamic induction is reduced by a factor of just over two when endogenous carotenoids are present . We argue that the involvement of oxidative damage would in no way be inconsistent with current understanding of the mechanism of adaptive mutation and the role of DNA polymerases.

Mutat Res, 2001 Jan 25, 473(1), 37 - 49
Lung-specific mutagenicity and mutational spectrum in B6C3F1 lacI transgenic mice following inhalation exposure to 1,2-epoxybutene; Saranko CJ et al.; 1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice . BD inhalation induces an increased frequency of specific base substitution mutations in the bone marrow and spleen of B6C3F1 lacI transgenic mice . BD is bioactivated to at least three mutagenic metabolites: 1,2-epoxybutene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB), however, the contribution of these individual metabolites to the in vivo mutational spectrum of BD is uncertain . In the present study, lacI transgenic mice were exposed by inhalation (6h per day, 5 days per week for 2 weeks) to 0 or 29.9ppm of the BD metabolite, EB to assess its contribution to the in vivo mutational spectrum of BD . No increase in lacI mutant frequency was observed in the bone marrow or spleen of EB-exposed mice . The lack of mutagenicity in the bone marrow or spleen likely relate to insufficient levels of EB reaching these tissues . The lacI mutant frequency was increased 2.7-fold in the lungs of EB-exposed mice (mean+/-S.D., 9.9+/-3.0x10(-5)) compared to air control mice (3.6+/-0.7x10(-5)) . DNA sequence analysis of 65 and 66 mutants from the lungs of air control and EB-exposed mice, respectively, revealed an increase in the frequency of two categories of base substitution mutation and deletions . Like mice exposed to BD, EB-exposed mice had an increased frequency of A:T-->T:A transversions . However, in contrast to the BD mutational spectra, G:C-->A:T transitions at 5'-CpG-3' sequences, occurred with increased frequency in the EB-exposed mice . The increased frequency of deletions as well as the induction of two tandem mutations and a tandem deletion in the lungs of EB-exposed mice are also inconsistent with previous mutational spectra from BD-exposed mice or EB-exposed cells in culture . We hypothesize that the direct in vivo mutagenicity and further in situ metabolism of EB in the lungs of EB-exposed mice played a prominent role in the generation of the current mutational spectrum.

Cancer Lett, 2001 Feb 26, 163(2), 239 - 44
Molecular cloning, expression and purification of truncated midkine and its growth stimulatory activity on Wilms' tumor (G401) cells; Paul S et al.; Midkine (MK) is a heparin binding growth factor identified as a product of a retinoic acid-responsive gene; it is frequently expressed at high levels in many human carcinomas . Although the expression of the mRNA encoding truncated MK (tMK) in unique human cancer cells has been reported, the tMK polypeptide itself has not yet been identified . In order to clarify the biological role of tMK, recombinant tMK was expressed in Escherichia coli and purified . Recombinant tMK was purified as a single band in SDS-PAGE under reducing conditions showing an apparent molecular mass of 10 kDa . Purified recombinant tMK showed the same extent of proliferative activity towards Wilms' tumor (G401) cells as full length human MK . These results suggest that the structure of this recombinant tMK is same as the native polypeptide.

Trends Biochem Sci, 2001 Jan, 26(1), 36 - 40
The alpha and the beta: protein translocation across mitochondrial and plastid outer membranes; Gabriel K et al.; In the evolution of mitochondria and plastids from endosymbiotic bacteria, most of the proteins that make up these organelles have become encoded by nuclear genes and must therefore be transported across the organellar membranes, following synthesis in the cytosol . The core component of the protein translocation machines in both the mitochondrial and plastid outer membranes appears to be a beta-barrel protein, perhaps a relic from their bacterial ancestry, distinguishing these translocases from the alpha-helical-based protein translocation pores found in all other eukaryotic membranes.

Mech Dev, 2001 Feb, 100(2), 291 - 301
The Drosophila homolog of human AF10/AF17 leukemia fusion genes (Dalf) encodes a zinc finger/leucine zipper nuclear protein required in the nervous system for maintaining EVE expression and normal growth; Bahri SM et al.; Sibling neurons in the embryonic central nervous system (CNS) of Drosophila can adopt distinct states as judged by gene expression and axon projection . In the NB4-2 lineage, two even-skipped (eve)-expressing sibling neuronal cells, RP2 and RP2sib, are formed in each hemineuromere . Throughout embryogenesis, only RP2, but not RP2sib, maintains eve expression . In this report, we describe a P-element induced mutation that alters the expression pattern of EVE in RP2 motoneurons in the Drosophila embryonic CNS . The mutation was mapped to a Drosophila homolog of human AF10/AF17 leukemia fusion genes (alf), and therefore named Dalf . Like its human counterparts, Dalf encodes a zinc finger/leucine zipper nuclear protein that is widely expressed in embryonic and larval tissues including neurons and glia . In Dalf mutant embryos, the RP2 motoneuron no longer maintains EVE expression . The effect of the Dalf mutation on EVE expression is RP2-specific and does not affect other characteristics of the RP2 motoneuron . In addition to the embryonic phenotype, Dalf mutant larvae are retarded in their growth and this defect can be rescued by the ectopic expression of a Dalf transgene under the control of a neuronal GAL4 driver . This indicates a requirement for Dalf function in the nervous system for maintaining gene expression and the facilitation of normal growth.

Int J Parasitol, 2001 Jan, 31(1), 67 - 71
Isolation of Neospora caninum genes detected during a chronic murine infection; Atkinson RA et al.; In order to isolate genes coding for antigens of Neospora caninum which are recognised by the host immune system during a chronic murine infection, a cDNA library was immunoscreened with pooled sera from mice which survived three independent infections by N . caninum . Two new genes from N . caninum were isolated and expressed in Escherichia coli . The genes identified include one homologous to GRA1 of Toxoplasma gondii, plus another (NCP20) previously unknown in any taxon . Both genes encode small polypeptides which induced an IgG response in the mouse and were also recognised by IgG from a cow chronically infected with N . caninum . These results are consistent with the hypothesis that the polypeptides encoded by these genes are a target for the host immune system during chronic infections of N . caninum.

Int J Parasitol, 2001 Jan, 31(1), 63 - 5
Successful reinfection of chronically infected mice by a different Toxoplasma gondii genotype; Dao A et al.; It is generally assumed that primary infection by Toxoplasma gondii protects from reinfection . A recent study using a murine model has questioned this dogma using indirect procedures to detect the reinfecting strain . We have reinvestigated this issue using a transfected strain of T . gondii (Prugniaud beta galactosidase: Pru beta gal) which expresses Escherichia coli beta-galactosidase . Detection of enzyme activity on fixed parasites allows a direct distinction between transfected and untransfected strains . We have found that in OF1 mice primary infection with the 76 K strain of T . gondii fully protects mice against tissue cyst production upon reinfection with the Pru beta gal T . gondii strain whereas primary infection with the Pru beta gal T . gondii strain does not impair tissue cyst formation upon reinfection with the Ned strain of T . gondii, which belongs to another T . gondii genotype . These results suggest that the immune protection conferred by one strain of T . gondii can be breached by reinfection with a strain belonging to another genotype; which can have significant consequences in human or veterinary medicine.

FEBS Lett, 2001 Feb 2, 489(2-3), 229 - 32
Spectroscopic studies on the active site of hydroperoxide lyase; the influence of detergents on its conformation; Noordermeer MA et al.; Expression of high quantities of alfalfa hydroperoxide lyase in Escherichia coli made it possible to study its active site and structure in more detail . Circular dichroism (CD) spectra showed that hydroperoxide lyase consists for about 75% of alpha-helices . Electron paramagnetic resonance (EPR) spectra confirmed its classification as a cytochrome P450 enzyme . The positive influence of detergents on the enzyme activity is paralleled by a spin state transition of the heme Fe(III) from low to high spin . EPR and CD spectra showed that detergents induce a subtle conformational change, which might result in improved substrate binding . Because hydroperoxide lyase is thought to be a membrane bound protein and detergents mimic a membrane environment, the more active, high spin form likely represents the in vivo conformation . Furthermore, the spin state appeared to be temperature-dependent, with the low spin state favored at low temperature . Point mutants of the highly conserved cysteine in domain D indicated that this residue might be involved in heme binding.

FEBS Lett, 2001 Feb 2, 489(2-3), 182 - 6
Phosphorylation of vimentin head domain inhibits interaction with the carboxyl-terminal end of alpha-helical rod domain studied by surface plasmon resonance measurements; Gohara R et al.; The amino-terminal head domain of vimentin is the target site for several protein kinases and phosphorylation induces disassembly of the vimentin intermediate filaments in vivo and in vitro . To better understand molecular mechanisms involved in phosphorylation-dependent disassembly, we examined domain interactions involving the head domain and the effect of phosphorylation on the interaction, using surface plasmon resonance . We observed that the head domain binds to the carboxyl-terminal helix 2B in the rod domain, under physiological ionic strength . This interaction was interfered with by A-kinase phosphorylation of the head domain . Deletion of the carboxyl-terminal 20 amino acids of helix 2B resulted in loss of the interaction . Furthermore, peptide representing the carboxyl-terminal 20 residues of helix 2B had a substantial affinity with the head domain but not with the phosphorylated one . These findings support the idea that the interaction between the head domain and the last 20 residues of helix 2B is essential for association of vimentin tetramers into the intermediate filaments and that the phosphorylation-dependent disassembly is the result of loss of the interaction.

Mol Cell Endocrinol, 2001 Jan 22, 171(1-2), 137 - 49
Structure-function aspects and inhibitor design of type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3); Penning TM et al.; 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) type 5 has been cloned from human prostate and is identical to type 2 3alpha-HSD and is a member of the aldo-keto reductase (AKR) superfamily; it is formally AKR1C3 . In vitro the homogeneous recombinant enzyme expressed in Escherichia coli functions as a 3-keto-, 17-keto- and 20-ketosteroid reductase and as a 3alpha-, 17beta- and 20alpha-hydroxysteroid oxidase . The enzyme will reduce 5alpha-DHT, Delta(4)-androstene-3,17-dione, estrone and progesterone to produce 3alpha-androstanediol, testosterone, 17beta-estradiol and 20alpha-hydroxprogesterone, respectively . It will also oxidize 3alpha-androstanediol, testosterone, 17beta-estradiol and 20alpha-hydroxyprogesterone to produce 5alpha-androstane-3,17-dione, Delta(4)-androstene-3,17-dione, and progesterone, respectively . Many of these properties are shared by the related AKR1C1, AKR1C2 and AKR1C4 isoforms . RT-PCR shows that AKR1C3 is dominantly expressed in the human prostate and mammary gland . Examination of k(cat)/K(m) for these reactions indicates that as a reductase it prefers 5alpha-dihydrotestosterone and 5alpha-androstane-3,17-dione as substrates to Delta(4)-androstene-3,17-dione, suggesting that in the prostate it favors the formation of inactive androgens . Its concerted reductase activity may, however, lead to a pro-estrogenic state in the breast since it will convert estrone to 17beta-estradiol; convert Delta(4)-androstene-3,17-dione to testosterone (which can be aromatized to 17beta-estradiol); and it will reduce progesterone to its inactive metabolite 20alpha-hydroxyprogesterone . Drawing on detailed structure-function analysis of the related rat 3alpha-HSD (AKR1C9), which shares 69% sequence identity with AKR1C3, it is predicted that AKR1C3 catalyzes an ordered bi bi mechanism, that the rate determining step is k(chem), and that an oxyanion prevails in the transition state . Based on these relationships steroidal-based inhibitors that compete with the steroid product would be desirable since they would act as uncompetitive inhibitors . With regards to transition state analogs steroid carboxylates and pyrazoles may be preferred while 3alpha, 17beta or 20alpha-spiro-oxiranes may act as mechanism-based inactivators.

Plant Sci, 2000 Dec 7, 160(1), 121 - 128
Detection and characterization of a 36-kDa peptide in C-terminal region of a 24-kDa vacuolar protein (VP24) precursor in anthocyanin-producing sweet potato cells in suspension culture; Xu W et al.; A 24-kDa vacuolar protein (VP24) was found to accumulate in anthocyanin-producing sweet potato cells (Ipomoea batatas Lam.) in suspension culture {Nozue et al., Plant Physiol . 115 (1997) 1065} . VP24 cDNA (accession No . AB025531) encodes a 96.3-kDa large precursor protein with a C-terminal propeptide which contains the eight putative transmembrane domains . The mature VP24 is probably involved in the formation of intravacuolar pigmented globules (cyanoplasts) in highly anthocyanin-containing vacuoles, but the biological function of the C-terminal region including the putative transmembrane domains is unknown . To confirm the expression and characterize the C-terminal region in the VP24 protein precursor in the anthocyanin-producing cells, polyclonal antibodies were developed against the fusion protein, including the C-terminal region, expressed in Escherichia coli . Western blot analysis showed that a 36-kDa peptide (VP36) localized in anthocyanin-containing vacuoles was expressed under continuous illumination, but not in darkness . The expression pattern of VP36 showed high similarity to VP24 . These results suggested that VP36 may be derived from the large VP24 protein precursor; it includes several of the hydrophobic domains in the C-terminal region.

J Virol Methods, 2001 Jan, 91(1), 93 - 8
Rapid coliphage detection assay; Stanek JE et al.; A rapid coliphage detection assay was developed, based on the phage-induced release of beta-galactosidase from cells of Escherichia coli . The assay could detect as few as five coliphage per sample without an overnight incubation period . The range of acceptable assay parameters was identified.

FEMS Microbiol Lett, 2001 Jan 15, 194(2), 187 - 92
Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter; Bes MT et al.; A gene coding for a Fur (ferric uptake regulation) protein from the cyanobacterium Anabaena PCC 7119 has been cloned and overexpressed in Escherichia coli . DNA sequence analysis confirmed the presence of a 151-amino-acid open reading frame that showed homology with the Fur proteins reported for the unicellular cyanobacteria Synechococcus 7942 and Synechocystis PCC 6803 . Two putative Fur-binding sites were detected in the promoter regions of the fur gene from Anabaena . Partially purified recombinant Fur binds to the flavodoxin promoter as well as its own promoter . This suggests that the Fur gene is autoregulated in Anabaena.

J Biotechnol, 2000 Dec 28, 84(3), 285 - 9
Production and purification of SV40 major capsid protein (VP1) in Escherichia coli strains deficient for the GroELS chaperone machine; Wrobel B et al.; Production of the major capsid protein of SV40, VP1, is of great interest for the study on capsid assembly in vitro . Production of soluble His6-VP1 in Escherichia coli strains deficient in the GroELS chaperone machine was substantially higher than in the wild-type strain . The His6-VP1 produced in a groEL mutant strain was readily purified . The protein was able to form higher-order structures as evidenced by analysis of the soluble fraction by gel filtration, by sedimentation in sucrose gradient, and by electron microscopy . We propose the use of groE mutants for the production of the major capsid protein of SV40 and perhaps also other papovaviruses.

J Biotechnol, 2000 Dec 28, 84(3), 273 - 84
Modification of the carboxy-terminal amino acid sequence alters the Escherichia coli expression of a gene encoding multiple repeats of a bovine growth hormone releasing factor analog; Trepod CM et al.; Since investigations into the determinants of intracellular protein degradation have shown that the carboxy terminal sequence can be a critical factor for protein expression in Escherichia coli, we attempted to increase the expression of a protein containing multiple repeats of a bovine growth hormone releasing factor analog (bGRF30) by modifying the carboxy terminus with the addition of short amino acid extensions derived from stable E . coli proteins . Extensions capable of increasing bGRF30 per liter titers up to four-fold, as well as extensions that completely abolished bGRF30 expression were identified . Select C-terminal extensions were investigated further to determine the mechanism by which they affected bGRF30 expression . Analysis of mRNA levels and protein production titers suggests that extensions which increase bGRF30 titers primarily affect protein stability and ribosomal release . Negative extensions exert their influence through a more complex mechanism, appearing to interfere with the ability of ribosomes to be efficiently released from their cognate mRNA.

Int J Biol Macromol, 2001 Jan 10, 28(2), 143 - 50
Head-to-tail and side-by-side oligomerization of human carbonic anhydrase II: a small angle X-ray scattering study; Ceolin M et al.; Solvent-induced directional aggregation of human carbonic anhydrase II (hCA) was studied by small angle X-ray scattering and fluorescence and fourth-derivative ultraviolet absorption spectroscopy . We propose that hCA at 5 mg ml(-1) in pure water forms head-to-tail oligomers built up, on average, by four to five monomers . At higher protein concentrations, the oligomers associate pair-wise and side-by-side . Spectroscopic evidence suggests that the subunits forming the aggregates are tightly folded, but with a structure that differs, at least locally, from the native state . A more complex aggregation pattern was observed under solvent conditions that favor the removal of zinc from the enzyme-active site, conditions under which the subunits are significantly less compact than in water . hCA may provide a useful model to investigate the effects of additives and genetic manipulation on protein aggregation.

Gene, 2000 Dec 23, 259(1-2), 217 - 22
Computer analysis of potential stem structures of rRNA operons in various procaryote genomes; Saito R et al.; The processing of 16S rRNA and 23S rRNA by RNase III in E.coli is known to involve stem structures formed by both ends of the rRNA . Indeed, complementary nucleotide sequences are usually found at both ends of 16S rRNA and 23S rRNA . However, whether or not this phenomenon exists in various other bacteria has not yet been adequately studied . We have conducted computer analyses of potential stem structures of rRNA operons in 12 bacterial and 3 archaeal genomes, and compared characteristics of the stem structures among these species . We systematically computed free energy values by exhaustively 'annealing' sequences around the 5' end and sequences around the 3' end of both 16S rRNA and 23S rRNA genes, in order to predict potential stem structures.The results suggest that rRNAs in most species form stem structures at both ends . Some species, such as A.aeolicus, seem to form unusually stable stem structures . On the other hand, some rRNAs, such as rRNAs of D.radiodurans, seem not to form solid stem structures . This suggests that rRNA processing in those species must employ a reliable targeting mechanism other than recognizing stem structures by RNase III.

Gene, 2000 Dec 23, 259(1-2), 123 - 7
Rapid and simple detection of PCR product DNA: a comparison between Southern hybridization and fluorescence polarization analysis; Kido C et al.; The essential aim of this study was to compare two different methods, Southern hybridization and fluorescence polarization (FP) assay . They both detect specific hybridization and were examined using common asymmetric PCR products and probes . FP assay clearly showed the hybridization of probe DNAs with the asymmetric PCR products of their target genes . Southern blot patterns presented excellent consistency with the results of FP assay . In both methods, two types of Shiga toxin (vero toxin) genes held in enterohaemorrhagic Escherichia coli (EHEC) were used as target genes . For detection of the two genes, stx1 and stx2, two respective DNA probes were synthesized . Both in FP assay and in Southern hybridization, the probe for stx1 hybridized only with the product of stx1 and vice versa . The results of the DNA detection using different methods were completely in agreement . Moreover, FP assay makes it possible to detect the hybridization rapidly . In our high NaCl concentration condition, hybridization between the probes and the asymmetric PCR products could be monitored within about 15min.

Immunol Lett, 2000 Dec 1, 75(1), 33 - 40
Bacterially expressed human Fc gamma RIIb is soluble and functionally active after in vitro refolding; Kurucz I et al.; A recombinant soluble form of the human Fc gamma receptor was produced by engineering a cDNA construct containing the extracellular part of the mature protein . After expression in bacteria as inclusion body, the polypeptide was highly purified and was refolded in vitro with a method that was developed for the renaturation of immunoglobulin fragments . With this method oxidation of the disulfide bridges within the domains of the protein is done in the presence of an artificial 'chaperone' which protects the polypeptide molecules from unwanted protein protein interactions thereby inhibiting the incorrect oxidation of the SH-groups . and misfolding of the protein . The refolded recombinant soluble Fc gamma RIIb showed several characteristics of the native receptor: (i) it was recognized by a series of monoclonal antibodies specific for, and in most cases produced against the native cell-surface receptor: (ii) it is bound to its ligand (the Fc-region of different immunoglobulins) under very diverse conditions: and (iii) it is competed strongly and specifically with the native cell surface receptor for both ligand and antibody binding in experiments with distinct read-outs; (iv) monoclonal antibodies produced against the recombinant protein specifically recognized Fc gamma RIIb on different cells . From these data it was concluded that the recombinant soluble Fc-receptor was in a native, functionally active form, and its function was not affected by the lack of glycosylation.

FEBS Lett, 2001 Jan 12, 488(1-2), 55 - 8
The role of pyridoxal phosphate in the function of EspB, a protein secreted by enteropathogenic Escherichia coli; Taylor KA et al.; The sequence of EspB, a secreted protein required for virulence of enteropathogenic Escherichia coli (EPEC), reveals a motif common to enzymes that bind pyridoxal phosphate . Pyridoxal phosphate was not found by fluorometry in concentrated supernatants of EPEC cultures that contain EspB . Plasmids containing cloned espB, in which the lysine residue conserved in the motif was substituted with either an arginine or methionine residue, remained capable of complementing an espB deletion mutant to restore EspB function . The results of these studies do not support a role for pyridoxal phosphate in EspB function.

FEBS Lett, 2001 Jan 12, 488(1-2), 9 - 12
Carbamoylphosphate requirement for synthesis of the active center of {NiFe}-hydrogenases; Paschos A et al.; The iron of the binuclear active center of {NiFe}-hydrogenases carries two CN and one CO ligands which are thought to confer to the metal a low oxidation and/or spin state essential for activity . Based on the observation that one of the seven auxiliary proteins required for the synthesis and insertion of the {NiFe} cluster contains a sequence motif characteristic of O-carbamoyl-transferases it was discovered that carbamoyl phosphate is essential for formation of active {NiFe}-hydrogenases in vivo and is specifically required for metal center synthesis suggesting that it is the source of the CO and CN ligands . A chemical path for conversion of a carbamoyl group into cyano and carbonyl moieties is postulated

Schizophr Res, 2001 Jan 15, 47(1), 27 - 36
Prenatal exposure to maternal infection alters cytokine expression in the placenta, amniotic fluid, and fetal brain; Urakubo A et al.; Prenatal exposure to infection appears to increase the risk of schizophrenia and other neurodevelopmental disorders . We have hypothesized that cytokines, generated in response to maternal infection, play a key mechanistic role in this association . E16 timed pregnancy rats were injected i.p . with Escherichia coli lipopolysaccharide (LPS) to model prenatal exposure to infection . Placenta, amniotic fluid and fetal brains were collected 2 and 8h after LPS exposure . There was a significant treatment effect of low-dose (0.5mg/kg) LPS on placenta cytokine levels, with significant increases of interleukin (IL)-1beta (P<0.0001), IL-6 (P<0.0001), and tumor necrosis factor-alpha (TNF-alpha) (P=0.0001) over the 2 and 8h time course . In amniotic fluid, there was a significant effect of treatment on IL-6 levels (P=0.0006) . Two hours after maternal administration of high-dose (2.5mg/kg) LPS, there were significant elevations of placenta IL-6 (P<0.0001), TNF-alpha (P<0.0001), a significant increase of TNF-alpha in amniotic fluid (P=0.008), and a small but significant decrease in TNF-alpha (P=0.035) in fetal brain . Maternal exposure to infection alters pro-inflammatory cytokine levels in the fetal environment, which may have a significant impact on the developing brain.

Mol Immunol, 2000 Aug, 37(10), 579 - 90
Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features; Harmsen MM et al.; In addition to conventional antibodies (Abs), camelids possess Abs consisting of only heavy chains . The variable domain of such a heavy-chain Ab (VHH) is fully capable of antigen (Ag) binding . Earlier analysis of 47 VHHs showed sequence features unique to VHH domains . These include the presence of characteristic amino acid substitutions in positions which, in conventional VH domains are involved in interdomain interactions, and the presence of a long third complementarity-determining region (CDR3) which is frequently constrained by an interloop disulphide bond . Here, we describe a large (152) set of Lama glama VHH cDNAs . Based on amino acid sequence similarity, these and other published camelid VHHs were classified into four subfamilies . Three subfamilies are absent in dromedaries, which have been the primary source of VHHs thus far . Comparison of these subfamilies to conventional VH regions reveals new features characteristic of VHHs and shows that many features earlier regarded as characteristic of VHHs in general are actually subfamily specific . A long CDR3 with a concomitant putative additional disulphide bond is only observed in two VHH subfamilies . Furthermore, we identified new VHH-characteristic residues at positions forming interdomain sites in conventional VH domains . The VHH subfamilies also differ from each other and conventional VH domains in the canonical structure of CDR1 and CDR2, mean CDR3 length, and amino acid residue variability . Since different VHH-characteristic residues are observed in all four subfamilies, these subfamilies must have evolved independently from classical VH domains.

FEBS Lett, 2001 Jan 5, 487(3), 327 - 32
Interdomain interactions in oligomeric enzymes: creation of asymmetry in homo-oligomers and role in metabolite channeling between active centers of hetero-oligomers; Nagradova NK; Interdomain interactions play an important role in the structural organization of many enzymes and the conformational flexibility of their molecules . In this review, the role of intrasubunit and intersubunit domain-domain interactions in the origins of pre-existent asymmetry of homo-oligomeric D-glyceraldehyde-3-phosphate dehydrogenase and tryptophanyl-tRNA synthetase is discussed on the basis of recent X-ray data and other available information about the properties of these and related enzymes . In addition, a novel key function of interdomain interactions is considered: their potential contribution to intramolecular channeling of intermediates between active centers located on different subunits of a hetero-oligomeric enzyme (alpha,beta-heterodimeric carbamoyl phosphate synthetase).

Neuron, 2000 Dec, 28(3), 911 - 25
Molecular organization of a zinc binding n-terminal modulatory domain in a NMDA receptor subunit; Paoletti P et al.; Ionotropic glutamate receptors (iGluRs) bind agonists in a domain that has been crystallized and shown to have a bilobed structure . Eukaryotic iGluRs also possess a second extracellular N-terminal domain related to the bacterial periplasmic binding protein LIVBP . In NMDA receptors, the high-affinity Zn inhibition is eliminated by mutations in the LIVBP-like domain of the NR2A subunit . Using LIVBP structure, we have modeled this domain as two lobes connected by a hinge and show that six residues controlling Zn inhibition form two clusters facing each other across a central cleft . Upon Zn binding the two lobes close tightly around the divalent cation . Thus, the extracellular region of NR2A consists of a tandem of Venus flytrap domains, one binding the agonist and the other a modulatory ligand . Such a functional organization may apply to other eukaryotic iGluRs.

Mol Cell, 2000 Dec, 6(6), 1515 - 21
Visualization of substrate binding and translocation by the ATP-dependent protease, ClpXP; Ortega J et al.; Binding and internalization of a protein substrate by E . coli ClpXP was investigated by electron microscopy . In sideviews of ATP gamma S-stabilized ClpXP complexes, a narrow axial channel was visible in ClpX, surrounded by protrusions on its distal surface . When substrate lambda O protein was added, extra density attached to this surface . Upon addition of ATP, this density disappeared as lambda O was degraded . When ATP was added to proteolytically inactive ClpXP-lambda O complexes, the extra density transferred to the center of ClpP and remained inside ClpP after separation from ClpX . We propose that substrates of ATP-dependent proteases bind to specific sites on the distal surface of the ATPase, and are subsequently unfolded and translocated into the internal chamber of the protease.

Curr Opin Genet Dev, 2001 Feb, 11(1), 64 - 70
Is the MCM2-7 complex the eukaryotic DNA replication fork helicase?
Labib K, Diffley JF.
The MCM2-7 complex is essential for both the initiation and elongation phases of eukaryotic chromosome replication . There is some evidence that MCM2-7 proteins may act as a DNA helicase; at the same time, a variety of other DNA helicases have also been implicated in the replication of eukaryotic chromosomes.

Curr Opin Cell Biol, 2001 Feb, 13(1), 55 - 60
The FtsZ protofilament and attachment of ZipA--structural constraints on the FtsZ power stroke; Erickson HP; Bacterial cell division protein FtsZ forms protofilaments in vitro that can shift from a straight to a curved conformation . The inside of the curved protofilaments, which corresponds to the carboxyl terminus, should face the center of the cell as curvature increases during constriction of the Z-ring . ZipA, a membrane-tethered division protein, binds to a highly conserved short peptide on the carboxyl terminus of FtsZ . A model is proposed here for how membrane-bound ZipA can reach around the FtsZ protofilament to bind the carboxy-terminal peptide, which faces away from the membrane.

Virology, 2001 Feb 1, 280(1), 41 - 51
Interaction of poliovirus-encoded 2C/2BC polypeptides with the 3' terminus negative-strand cloverleaf requires an intact stem-loop b; Banerjee R et al.; The poliovirus-encoded, membrane-associated polypeptide 2C and its precursor, 2BC, is believed to be required for initiation and elongation of viral RNA synthesis . Previous studies have shown that the 2C polypeptide specifically interacts with the 3'-terminal sequence of poliovirus negative-strand RNA . This interaction is facilitated by the presence of the conserved sequence UGUUUU in the "stem a" within the 3'-terminal cloverleaf structure . We show that the 2C precursor, 2BC, also interacts with the 3'-terminal cloverleaf of the negative-strand RNA . We also demonstrate here that interaction of 2C/2BC with the negative strand 3'-terminal sequence not only depends on an intact "stem a" containing the UGUUUU sequence but is equally influenced by the presence of an intact "stem b" within the negative-strand cloverleaf . The results presented here suggest that the spatial configuration of stem a UGUUUU sequence with respect to an intact stem-b is crucial for 2C/2BC interaction with the 3'-terminal negative-strand cloverleaf structure .

J Struct Biol, 2000 Nov, 132(2), 133 - 41
The 6.9-A structure of GlpF: a basis for homology modeling of the glycerol channel from Escherichia coli; Stahlberg H et al.; The three-dimensional structure of GlpF, the glycerol facilitator of Escherichia coli, was determined by cryo-electron microscopy . The 6.9-A density map calculated from images of two-dimensional crystals shows the GlpF helices to be similar to those of AQP1, the erythrocyte water channel . While the helix arrangement of GlpF does not reflect the larger pore diameter as seen in the projection map, additional peripheral densities observed in GlpF are compatible with the 31 additional residues in loops C and E, which accordingly do not interfere with the inner channel construction . Therefore, the atomic structure of AQP1 was used as a basis for homology modeling of the GlpF channel, which is predicted to be free of bends, wider, and more vertically oriented than the AQP1 channel . Furthermore, the residues facing the GlpF channel exhibit an amphiphilic nature, being hydrophobic on one side and hydrophilic on the other side . This property may partially explain the contradiction of glycerol diffusion but limited water permeation capacity .

J Struct Biol, 2000 Nov, 132(2), 106 - 11
Crystallization of the F41 fragment of flagellin and data collection from extremely thin crystals; Samatey FA et al.; Flagellin, which constructs supercoiled filaments of the bacterial flagellum, is very difficult to crystallize because of its strong tendency to polymerize . We therefore crystallized the F41 fragment of flagellin, which does not polymerize because terminal regions that play important roles in polymerization are cleaved off . F41 was crystallized by the hanging drop vapor diffusion method in a mixture of polyethylene glycol, glycerol, and isopropanol, with a reservoir solution covered with silicon oil . The two key factors for success in growing sufficiently large crystals were isopropanol and silicon oil, which worked well to reduce the otherwise very high nucleation rate that resulted in hundreds of tiny crystals . The crystals were grown to very thin plates with thickness less than 10 microm, which made the collection of diffraction data very difficult . Freezing and annealing of the crystals and irradiation at synchrotron beamlines had to be carried out by specific methods and under specific conditions for its structure analysis at 2.0-A resolution .

Biochem Biophys Res Commun, 2001 Feb 9, 280(5), 1385 - 8
Magnetic field exposure induces DNA degradation; Li SH et al.; In our earlier experiments, we discovered that magnetic field exposure could bring both stabilizing and destabilizing effects to the DNA of Escherichia coli, depending on our parameters of assessment, and both of these effects were associated with the induced synthesis of the heat shock proteins Hsp70/Hsp40 (DnaK/DnaJ) . These contradicting results prompted us to explore in this study the effect of magnetic field exposure on the DNA stability in vivo when the heat shock response of the cell was suppressed . By using plasmid pUC18 in E . coli as the indicator, we found that without the protection of the heat shock response, magnetic field exposure indeed induced DNA degradation and this deleterious effect could be diminished by the presence of an antioxidant, Trolox C . In our in vitro test, we also showed that the magnetic field could potentiate the activity of oxidant radicals.

Biochem Biophys Res Commun, 2001 Jan 26, 280(3), 914 - 7
Continuous-exchange cell-free protein-synthesizing system: synthesis of HIV-1 antigen Nef; Chekulayeva MN et al.; HIV-1 antigen Nef, Green Fluorescent Protein (GFP), and the fusion protein GFP-Nef ("Green Fluorescent Nef") were synthesized in bacterial cell-free system with continuous supply of substrates and continuous removal of low-molecular-weight products through a dialysis membrane during incubation, the so-called continuous-exchange cell-free (CECF) system . The identity of synthesized proteins was confirmed by electrophoretic mobility, Western blotting, and GFP fluorescence . The system produced several nanomoles (hundreds of microg) of each protein per ml of the reaction mixture . Construction of GFP-fused proteins is considered as a general strategy for visualization and monitoring of cell-free production of the proteins that have no easily testable functions .

Biochem Biophys Res Commun, 2001 Jan 26, 280(3), 842 - 4
Random PCR-based screening for soluble domains using green fluorescent protein; Kawasaki M et al.; A novel approach to screen soluble protein domains is presented, by combining tagged random primer polymerase chain reaction (PCR) method and protein-folding assay using green fluorescent protein . Tagged random primer PCR method was used to amplify random DNA fragments from a template cDNA . The PCR products were fused to the green fluorescent protein (GFP) gene . Then solubilities of their translation products were rapidly monitored by the fluorescence of transformed Escherichia coli colonies on plates . We succeeded in cloning four soluble domains from Vav protein using this method . The present method is applicable to all proteins when cDNAs are available .

Biochem Biophys Res Commun, 2001 Jan 26, 280(3), 595 - 604
Anti-8-oxo-2'-deoxyguanosine phage antibodies: isolation, characterization, and relationship to disease states; Seedhouse CH et al.; We have used human single chain Fv (scFv) phage display antibody libraries to isolate recombinant antibodies against the DNA adduct 8-oxo-2'-deoxyguanosine (8-oxodG) . One of these scFvs (175G) bound to several 8-oxodG-containing oligonucleotides whilst demonstrating no cross-reactivity with G-containing control oligonucleotides, and bound to 8-oxodG lesions introduced into DNA by treatment with methylene blue and white light . In addition, 175G inhibited the cleavage of an 8-oxodG-containing oligonucleotide by the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg) . The nucleotide sequence of the 175G V(H) gene segment was 98% homologous to the published V(H) sequence of a human hybridoma derived from a patient with systemic lupus erythematosus (SLE) . Sera from two SLE patients bound to damaged DNA, and this binding could be inhibited by 175G . The use of human scFv phage display libraries has thus produced a unique reagent with specificity for 8-oxodG, which may have a role in damage detection and quantitation and in modifying DNA repair activity . 175G also offers support to the hypothesis that SLE might be associated with oxidative damage to DNA .

Biochem Biophys Res Commun, 2001 Jan 19, 280(2), 567 - 73
No isochores in the human chromosomes 21 and 22?
Haring D, Kypr J.
The human genome is described in the literature as being composed of the isochores, i.e., long (hundreds of kilobases) segments with a homogeneous (G + C) content . We calculated the (G + C) content variations along the DNA molecules of the human chromosomes 21 and 22 and found the variations to be higher everywhere compared to the randomized sequences . Hence the (G + C) content is certainly not homogeneous on the isochore scale in the two human chromosomes . In addition, we found no significant difference between the two human molecules and the genome of E . coli regarding the (G + C) content variations . Hence no isochores are either present in the DNA molecules of the human chromosomes 21 and 22, or the isochores are also present in the genome of Escherichia coli . In any case, the present communication demonstrates that the isochores should be defined in unambiguous molecular terms if they are to be used for an up-to-date genome structure characterization .

Biochem Biophys Res Commun, 2001 Jan 19, 280(2), 466 - 70
Intersubunit crosslinking of the heterotetrameric proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli defines intersubunit contacts between transmembrane helices of the beta subunits; Hou C et al.; The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer . The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation . Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and nine transmembrane helices at the amino-terminal region of the beta subunit . We have introduced pairs of cysteine residues into all of the loops connecting the transmembrane helices of domain II of the beta subunit . Crosslinking between the two beta subunits of the tetramer was induced spontaneously, or by treatment with cupric 1,10-phenanthrolinate or o-phenylenedimaleimide . Crosslinks between pairs of betaA114C, betaS183C, and betaA262C residues were observed, suggesting that pairs of domain II transmembrane helices 11, 12, and 14 were in proximity . These results, together with previous data (Bragg and Hou (2000) Biochem . Biophys . Res . Commun . 273, 955-959) suggest that the transhydrogenase tetramer is formed by apposition of alpha(2) and beta(2) dimers . Crosslinking between pairs of cysteine residues in the same beta subunit was not observed, possibly because the interhelical loops of the domain II region of the beta subunit were too short to allow correct orientation of the sulfhydryl groups for crosslinking .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 396 - 400
Directed evolution of operon of trehalose-6-phosphate synthase/phosphatase from Escherichia coli; Kong X et al.; Trehalose is a nonspecific protective agent for biomacromolecules . Trehalose-6-phosphate synthase (OtsA)/phosphatase (OtsB), which is encoded by the gene operon otsBA located at -42 of the Escherichia coli genome, is the main enzyme system that catalyzes the synthesis of trehalose in E . coli . We cloned the operon and modified it by directed evolution . Unlike in the previously reported work, we modified the whole operon and screened the positive mutant simultaneously . Thus we believe that the gene complex solves the negative effects between two enzymes if one of them diversifies its structure or functions and finds the form most suitable for trehalose synthesis . It thus mimics the natural process, in which the functional improvement of organisms is related to alterations in coordinated enzymes . The evolution procedure was carried out in a sequence of error-prone PCR, shuffling PCR, and then strict screening of the mutants . After screening of a library of more than 4000 colonies, about 15 positive colonies were analyzed, resulting in a higher concentration of trehalose than control . One of them, E . coli TS7, shows 12.3-fold higher trehalose synthesis ability than E . coli DH5alpha . In contrast, we introduced the cDNA sequence of the tps1 gene from Saccharomyces cerevisiae, which has 54% identity with the gene otsA, as one of the templates in shuffling PCR . By hybrid evolution and screening, we obtained 10 positive colonies with higher concentrations of trehalose than control . E . coli TS22 appears to have 5.3-fold higher trehalose synthesis ability than E . coli DH5alpha and 1.6-fold more than E . coli DEF3(pOTS11) . This result demonstrated that coevolution and hybrid evolution, as powerful protocols in protein engineering, are effective in modifying enzyme . It indicates that repeating the process of genomic evolution in nature is feasible .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 358 - 62
Sensitivity of metallothionein-null mice to LPS/D-galactosamine-induced lethality; Kimura T et al.; Mice treated with lipopolysaccharide (LPS)/D-galactosamine (GalN) selectively develop hepatic failure . The acute-phase protein alpha(1)-acid glycoprotein (AGP) has been demonstrated to protect mice from LPS/GalN-induced lethality . Metallothionein (MT), which is a low-molecular weight, cysteine-rich, metal-binding protein, is also induced in the acute-phase reaction . However, the specific function of MT in acute-phase response remain to be elucidated . We showed that MT-null mice were more sensitive to LPS/GalN-induced lethality than wild-type mice . The increase in vital mediator levels, TNF-alpha and NO were of similar levels in wild-type and MT-null mice . A remarkable increase in plasma platelet-activating factor levels was not observed in our experimental conditions . On the other hands, the mRNA level of AGP in the response to LPS/GalN was decreased in MT-null mice compared to wild-type mice . These results indicated that MT may have the potential to prevent LPS/GalN-induced lethality, at least through the attenuation of AGP induction .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 151 - 7
Imaging and mapping protein-binding sites on DNA regulatory regions with atomic force microscopy; Moreno-Herrero F et al.; Regulation of gene expression is fundamental in biological systems . A systematic search for protein binding sites in gene promoters has been done in recent years . Biochemical techniques are easy and reliable when analysing protein interactions with short pieces of DNA, but are difficult and tedious when long pieces of DNA have to be analysed . Here we propose AFM as a reliable and easy technique for identifying protein interaction sites in long DNA molecules like gene promoters . We support this idea using a well-known model: the interaction of the Pho4 protein with the PHO5 gene promoter . We have also applied the technique to demonstrate that Mig1 protein binds to two motifs in the promoter of HXK2 gene . Our results allow us to define Mig1p as a new factor probably contributing to the carbon source-dependent transcription regulation of HXK2 gene .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 9 - 13
c-Fos is surface active and interacts differentially with phospholipid monolayers; Borioli GA et al.; The transcription factor c-Fos forms stable Gibbs and Langmuir monolayers at the air-buffer interface . Its marked surface activity is enhanced by penetration into phospholipid films above the protein's own maximum adsorption surface pressure to a lipid-free interface . The protein-phospholipid stabilizing interactions at the interface depend on the lipid polar head group and the increases of lateral surface pressure generated are comparable to those of membrane-active proteins . The surface activity of c-Fos is strong enough to thermodynamically drive and retain c-Fos at the membrane interface where it may exert direct or indirect effects .

Biochem Biophys Res Commun, 2000 Dec 29, 279(3), 949 - 54
Reduction of phenoxyl radicals mediated by monodehydroascorbate reductase; Sakihama Y et al.; Monodehydroascorbate (MDA) reductase catalyzes the reduction of MDA, the only organic radical substrate for the enzyme reported so far . Here, we show that cucumber MDA reductase is also capable of reducing phenoxyl radicals which are generated by horseradish peroxidase (HRP) with H2O2 . The addition of MDA reductase plus NADH suppressed the HRP/H2O2 dependent oxidation of quercetin, accompanied by the oxidation of NADH . The quenching of the quercetin radical by MDA reductase plus NADH was confirmed by ESR . MDA reductase with NADH also suppressed the HRP/H2O2 dependent oxidation of hydroxycinnamates, including ferulic acid, coniferyl alcohol, and chlorogenic acid . Thus, the phenoxyl radicals of plant phenols can be reduced to their respective parent phenols by MDA reductase via a mechanism similar to the reduction of MDA.

Biochem Biophys Res Commun, 2000 Dec 29, 279(3), 942 - 8
Cloning and characterization of the pknA gene from Streptomyces coelicolor A3(2), coding for the Mn2+ dependent protein Ser/Thr kinase; Petrickova K et al.; A gene pknA, coding for an eukaryotic-type protein Ser/Thr kinase, was cloned from the Streptomyces coelicolor A3(2) chromosome . The PknA protein kinase, containing the C-terminal eukaryotic-type kinase domain with an N-terminal extension, was expressed in Escherichia coli and Streptomyces lividans . The affinity purified MBP-PknA fusion protein was assayed for kinase activity that showed its ability to autophosphorylate in vitro in the presence of {gamma-32P}ATP . The activity was Mn2+ dependent . The preautophosphorylated kinase phosphorylated at least two proteins (sizes 30 and 32 kDa) in the S . coelicolor J1501 cell-free extracts of all developmental stages . The larger of them was also phosphorylated in vitro by an endogenous protein kinase in late stages extracts, but not earlier . Although Mn2+ dependent protein phosphorylation has previously been described in Streptomyces, this is the first report of a gene encoding such an enzyme in this genus.

Biochem Biophys Res Commun, 2000 Dec 29, 279(3), 853 - 7
Serine 27, a human retinoid X receptor alpha residue, phosphorylated by protein kinase A is essential for cyclicAMP-mediated downregulation of RXRalpha function; Harish S et al.; Retinoid X Receptor alpha (RXRalpha), a member of the steroid-thyroid hormone receptor super family, is phosphorylated in vitro by protein kinase A (PKA) and this phosphorylation is inhibited in presence of PKA inhibitory peptide . Analysis of various deletion mutants of RXRalpha indicate that the amino-terminal A/B domain is the target for PKA phosphorylation . An RXRalpha mutant in which serine residue 27 is mutated to alanine is no longer phosphorylated by PKA . In vivo transfection experiments in COS cells indicate that cyclic AMP represses retinoic acid-mediated transcriptional activation of RXRalpha and this repression is mediated by serine 27 . These results indicate that serine 27 of RXRalpha is an unique target for phosphorylation by PKA in vitro and it has an important role in the crosstalk between RXRalpha and cyclic AMP signalling pathways.

Protein Expr Purif, 2001 Feb, 21(1), 243 - 50
High yield expression and single step purification of human thionein/metallothionein; Hong S et al.; Human metallothionein (MT), isoform 2, was expressed in Escherichia coli as an intein (protein splicing element) fusion protein in the absence of added metals and purified by intein-mediated purification with an affinity chitin-binding tag (IMPACT system) . This procedure constitutes a novel and simple strategy to prepare thionein (T), the metal-free form, or MT when reconstituting T with metals in vitro . The yield was 8 mg of T or 6 mg of pure Cd(7)- or Zn(7)-MT from a 1-L culture, significantly higher than yields from any other expression system . Purified recombinant protein is indistinguishable from the native protein on the basis of its metal-binding ability, titration of its sulfhydryls, and UV and CD spectra . The MALDI-TOF mass spectrum is consistent with that of T with a free N-terminus .

Protein Expr Purif, 2001 Feb, 21(1), 224 - 34
A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli; Tan S; To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid . The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector . Restriction sites present in the polycistronic expression vector allow both affinity tagged and untagged complexes to be overexpressed . I demonstrate successful use of the expression system for binary and ternary complexes, including the reconstitution of the VHL-elonginC-elonginB complex in E . coli and purification of the complex by affinity and ion-exchange chromatography . This polycistronic expression system should provide an important alternative to in vitro reconstitution of multicomponent complexes .

Protein Expr Purif, 2001 Feb, 21(1), 220 - 3
Rapid evaluation and optimization of recombinant protein production using GFP tagging; Rucker E et al.; The isolation of recombinant proteins from bacterial or eukaryotic systems often requires a laborious optimization of expression and purification conditions . To greatly facilitate this procedure we included the green fluorescent protein (GFP) in bacterial expression vectors . This approach allowed us to sensitively detect the GFP hybrid proteins already in intact bacterial cells using a fluorescence microscope . To rapidly analyze a variety of conditions essential for protein expression, the GFP signal, indicative of expression levels, was directly quantitated in live bacterial suspensions using a fluorescence plate reader . Thus, GFP tagging not only allows one to directly monitor protein expression in general but also appears to increase protein stability or solubility .

Protein Expr Purif, 2001 Feb, 21(1), 210 - 9
High-yield expression, purification, and characterization of the recombinant diisopropylfluorophosphatase from Loligo vulgaris; Hartleib J et al.; Organophosphate degrading enzymes are of great interest in light of their ability to detoxify chemical warfare agents . The diisopropylfluorophosphatase (DFPase) from Loligo vulgaris is characterized by its high stability and broad substrate specifity . Here we report the production of large amounts of active, recombinant DFPase using an Escherichia coli expression system . The enzyme was purified to homogeneity using a combination of immobilized metal affinity and ion exchange chromatography . CD-spectroscopy indicates a well folded protein with a high amount of beta-sheet structure . Limited proteolysis was used to gain a deeper insight into the structural organization of the protein . DFPase possesses an internal protease-sensitive region located between amino acids 146 and 149 . The two proteolytic fragments remain as a tight complex retaining a DFPase activity comparable to the intact enzyme . Overexpression clones for each fragment were constructed with the expression resulting in the formation of inclusion bodies . Upon isolation and refolding active protein is only formed when both fragments are present . Thus, the two proteolytic fragments are probably part of a stable single-domain protein with multiple contacts between them and only one protease accessible surface loop .

Protein Expr Purif, 2001 Feb, 21(1), 134 - 40
Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli; Ward RJ et al.; Hydrolysis of phospholipids by Group II phospholipase A2 enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region . Bothropstoxin-I (BthTX-I) is a PLA(2) variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism . In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli . The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E . coli BL21(DE3) . The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix . After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained . After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein . Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes .

Protein Expr Purif, 2001 Feb, 21(1), 129 - 33
Purification and in vitro folding of recombinant human thrombopoietin receptor expressed in Escherichia coli; Ng CK et al.; Thrombopoietin receptor (TPOR) is a member of the cytokine receptor superfamily expressed primarily on hematopoietic cells . TPOR plays an important role in regulating platelet production . Due to its low expression level in human tissue, studies on the biochemical and biophysical properties of TPOR have been limited . In the present study, an extracellular domain of recombinant human TPOR (rh TPOR-EN) was expressed in Escherichia coli as inclusion bodies . Purification was achieved by metal chelated chromatography under denaturing condition and was refolded by gel filtration chromatography . Far UV circular dichroism spectroscopy and surface plasmon resonance experiments were performed to demonstrate that the protein was in a refolded state and could bind with its ligand . Thus, a production and purification scheme was developed by which sufficient quantities of rh TPOR-EN can be made available for biochemical and biophysical characterizations .

Protein Expr Purif, 2001 Feb, 21(1), 99 - 104
Expression and purification of the DNA-binding domain of the human transcription factor E2F1; Zeng W et al.; The DNA-binding domain of the human transcription factor E2F1 was expressed in Escherichia coli . Through a single purification step using a Ni2+ column, 40-50 mg of the highly purified recombinant protein was obtained from 1 liter of bacterial culture . In addition, it was shown that the recombinant protein had higher stability and solubility under acidic conditions than at a neutral or alkaline pH . The gel shift assay showed that the recombinant E2F1 DNA-binding domain was active in binding a fragment containing E2F sites . Circular dichroism measurements revealed that the recombinant protein approximately contains 33% alpha-helix, 11% beta-sheet, 5% turn, and 51% random coil at pH 7.0, and there was no obvious change for the secondary structure of the recombinant protein between pH 4.0 and pH 9.0 . A 3D model was obtained by comparative protein modeling with a homologous protein whose structure was known by program Modeller 4 . With the program DSSP, the predicted secondary structure content of the model was consistent with the result of circular dichroism spectrum .

Protein Expr Purif, 2001 Feb, 21(1), 87 - 91
Expression and purification of cytokine receptor homology domain of human granulocyte-colony-stimulating factor receptor fusion protein in Escherichia coli; Tatsuda D et al.; Direct expression of the cytokine receptor homology (CRH) domain of granulocyte-colony-stimulating factor (G-CSF) receptor is lethal to Escherichia coli . For the efficient and stable production of an active CRH domain in E . coli, we fused the CRH domain with different proteins, such as maltose-binding protein (MalE), glutathione S-transferase, and thioredoxin (Trx) . Among these, Trx appeared to be the best in terms of the protein expression level, purification efficiency by affinity chromatography, and binding activity to its ligand, G-CSF . The yield of active Trx-CRH fusion protein increased about 200-fold compared to that of previously reported MalE-CRH fusion .

Protein Expr Purif, 2001 Feb, 21(1), 49 - 59
Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization; Lohmann K et al.; All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli . The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E . coli . The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC) . The complex was isolated and purified by three chromatographic steps . Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained . Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart . By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex .

Protein Expr Purif, 2001 Feb, 21(1), 40 - 8
Expression and purification of recombinant Giardia fibrillarin and its interaction with small nuclear RNAs; Ghosh S et al.; Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing . The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing . This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein . The condition for production of soluble protein was standardized . The expressed protein was purified by using Ni-chelation chromatography and used for functional studies . The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR . Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract . The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization . Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D . Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs .

Protein Expr Purif, 2001 Feb, 21(1), 30 - 9
Individual expression of recombinant alpha- and beta-tubulin from Haemonchus contortus: polymerization and drug effects; Oxberry ME et al.; Three tubulin isotypes from the parasitic nematode Haemonchus contortus were individually expressed in Escherichia coli, purified, and induced to polymerize into microtubules in the absence of microtubule-associated proteins . The effect of different conditions on the rate of polymerization of pure tubulin was assessed . This is the first time that recombinant alpha-tubulin has been shown to be capable of polymerization into microtubule-like structures when incubated with recombinant beta-tubulin . In addition, the present study has shown that: (1) microtubule-associated proteins are not required for tubulin polymerization; and (2) pure beta-tubulin isotype, beta12-16, alone was capable of forming microtubule-like structures in the absence of alpha-tubulin . Polymerization of the recombinant invertebrate tubulin, as measured by a spectrophotometric assay, was found to be enhanced by a concentration of tubulin >0.25 mg/mL; temperature > or =20 degrees C; 2 mM GTP; glycerol; EGTA; and Mg(2+) . Polymerization was inhibited by GTP (>2 mM) and albendazole . Calcium ions and a pH range of 6 to 8.5 had no measurable effect on polymerization . Individual isotypes of tubulin polymerized to approximately the same extent as an alpha-/beta-tubulin mixture . Samples of tubulin assembled under the above conditions for 60 min were also examined under a transmission electron microscope . Although the spectrophotometric assay indicated polymerization, it did not predict the structure of the polymer . In many cases tubulin sheets, folded sheets, and rings were observed in addition to, or instead of, microtubule-like structures .

Protein Expr Purif, 2001 Feb, 21(1), 8 - 12
Affinity purification of Csk protein tyrosine kinase based on its catalytic requirement for divalent metal cations; Sun G et al.; Protein tyrosine kinase Csk requires two Mg2+ ions for activity: one magnesium is part of the ATP-Mg complex, and the second free Mg2+ ion is required as an essential activator . Zn2+ can bind to this site to replace Mg2+, which inhibits Csk kinase activity . The binding is reversible and removal of Zn2+ results in an active Csk apoenzyme . In this communication, we report that this tight binding can be used as a mechanism for affinity purification of Csk . When bacterial cell lysate containing overexpressed GST-Csk was applied to a column of Zn2+-iminodiacetic acid immobilized to agarose, Csk was specifically retained by the column . Since the binding of Csk to Zn2+ is not affected by up to 200 mM NaCl, high ionic strength conditions were used in the purification procedure, minimizing nonspecific binding due to ionic interactions . Washing the column with 200 mM NaCl and 50 mM imidazole removed virtually all other proteins from the column while Csk remained bound . The retained Csk enzyme was eluted with 1 M imidazole . The 1 M imidazole-eluted fraction contained pure Csk that had a specific activity similar to the enzyme purified by a glutathione-agarose affinity column .

J Mol Biol, 2001 Jan 26, 305(4), 905 - 15
The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A; Diez J et al.; We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose . TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis . In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification . The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein . The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli . It consists of two similar lobes, each formed by a parallel beta-sheet flanked by alpha-helices on both sides . Both are connected by a hinge region consisting of two antiparallel beta-strands and an alpha-helix . As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions . However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced . To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations . Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities .

J Mol Biol, 2001 Jan 26, 305(4), 891 - 904
Structural basis for oligosaccharide recognition by Pyrococcus furiosus maltodextrin-binding protein; Evdokimov AG et al.; A maltodextrin-binding protein from Pyrococcus furiosus (PfuMBP) has been overproduced in Escherichia coli, purified, and crystallized . The crystal structure of the protein bound to an oligosaccharide ligand was determined to 1.85 A resolution . The fold of PfuMBP is very similar to that of the orthologous MBP from E . coli (EcoMBP), despite the moderate level of sequence identity between the two proteins (27 % identity, 46 % similarity) . PfuMBP is extremely resistant to heat and chemical denaturation, which may be attributed to a number of factors, such as a tightly packed hydrophobic core, clusters of isoleucine residues, salt-bridges, and the presence of proline residues in key positions . Surprisingly, an attempt to crystallize the complex of PfuMBP with maltose resulted in a structure that contained maltotriose in the ligand-binding site . The structure of the complex suggests that there is a considerable energy gain upon binding of maltotriose in comparison to maltose . Moreover, isothermal titration calorimetry experiments demonstrated that the binding of maltotriose to the protein is exothermic and tight, whereas no thermal effect was observed upon addition of maltose at three temperatures . Therefore, PfuMBP evidently is designed to bind oligosaccharides composed of three or more glucopyranose units .

J Mol Biol, 2001 Jan 26, 305(4), 851 - 61
Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in Escherichia coli and of the W171F variant that eliminates the redox active tryptophan 171 . Implications for the reaction mechanism; Blodig W et al.; The heme enzyme lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium contains a solvent exposed redox active tryptophan residue (Trp171) that carries a unique hydroxy group stereo-specifically attached to its C(beta) atom . A Trp171Phe mutant has no activity at all towards the substrate veratryl alcohol . The mechanism of veratryl alcohol oxidation involving beta-hydroxy-Trp171 is largely unknown . Here, we present the first crystal structures of LiP isozyme H8 at high resolution in its pristine non-hydroxylated form, of the C(beta)-hydroxylated form, and of the Trp171Phe mutant using recombinantly expressed and refolded protein produced from Escherichia coli . As a consequence, all structures are unglycosylated . Structural changes in response to the mutation are marginal and allow us to attribute the complete lack of activity exclusively to the absence of the redox active indole side-chain . The origin of the stereospecificity of the Trp171 hydroxylation can be explained on structural grounds . A reaction mechanism involving Trp171 is proposed and the possible function of the modification is discussed . Another important result regarding the ongoing debate on the co-ordination state of the heme iron in the resting state is that the iron is six co-ordinate in all cases the data being collected at room temperature . The mean distance from the iron to the distal water ligand is 2.18(+/-0.08) A . The radical scavenger orcinol was found to decrease radiation damage to the crystals, during data collection at room temperature .

J Mol Biol, 2001 Jan 26, 305(4), 805 - 16
Disruption of the helix-u-turn-helix motif of MutS protein: loss of subunit dimerization, mismatch binding and ATP hydrolysis; Biswas I et al.; The DNA mismatch repair protein, MutS, is a dimeric protein that recognizes mismatched bases and has an intrinsic ATPase activity . Here, a series of Taq MutS proteins having C-terminal truncations in the vicinity of a highly conserved helix-u-turn-helix (HuH) motif are assessed for subunit oligomerization, ATPase activity and DNA mismatch binding . Those proteins containing an intact HuH region are dimers; those without the HuH region are predominantly monomers in solution . Steady-state kinetics of truncated but dimeric MutS proteins reveals only modest decreases in their ATPase activity compared to full-length protein . In contrast, disruption of the HuH region results in a greatly attenuated ATPase activity . In addition, only dimeric MutS proteins are proficient for mismatch binding . Finally, an analysis of the mismatch repair competency of truncated Escherichia coli MutS proteins in a rifampicin mutator assay confirms that the HuH region is critical for in vivo function . These findings indicate that dimerization is critical for both the ATPase and DNA mismatch binding activities of MutS, and corroborate several key features of the MutS structure recently deduced from X-ray crystallographic studies.

J Mol Biol, 2001 Jan 26, 305(4), 689 - 702
Mechanism of regulation of transcription initiation by ppGpp . II . Models for positive control based on properties of RNAP mutants and competition for RNAP; Barker MM et al.; Strains containing ppGpp, a nucleotide whose synthesis is dependent on the RelA and SpoT proteins of Escherichia coli, display slightly lower rRNA promoter activity and much higher amino acid biosynthesis/transport promoter activity than deltarelAdeltaspoT strains . In the accompanying paper, we show that ppGpp directly inhibits rRNA promoter activity in vitro by decreasing the lifetime of the rrn P1 open complex . However, ppGpp does not stimulate amino acid promoter activity in vitro . We show here that RNA polymerase (RNAP) mutants, selected to confer prototrophy to deltarelAdeltaspoT strains, mimic the effects of ppGpp on wild-type RNAP . Based on the positions of the mutant residues that confer prototrophy in the structure of core RNAP, we suggest molecular models for how the mutants, and by analogy ppGpp, generally decrease the lifetime of open complexes . We show that amino acid promoters require higher concentrations of RNAP for function in vitro and in vivo than control promoters, and are more sensitive to competition for RNAP in vivo than control promoters . Furthermore, we show that the requirement of an amino acid promoter for ppGpp in vivo can be alleviated by increasing its rate-limiting RNAP-binding step . Our data are consistent with a previously proposed passive model in which ppGpp inhibits stable RNA synthesis directly by reducing the lifetime of the rrn P1 open complex, liberating enough RNAP to stimulate transcription from amino acid promoters . Our data also place considerable constraints on models invoking hypothetical factors that might increase amino acid promoter activity in a ppGpp-dependent fashion .

J Mol Biol, 2001 Jan 26, 305(4), 673 - 88
Mechanism of regulation of transcription initiation by ppGpp . I . Effects of ppGpp on transcription initiation in vivo and in vitro; Barker MM et al.; To determine the role of ppGpp in both negative and positive regulation of transcription initiation during exponential growth in Escherichia coli, we examined transcription in vivo and in vitro from the growth-rate-dependent rRNA promoter rrnB P1 and from the inversely growth-rate-dependent amino acid biosynthesis/transport promoters PargI, PhisG, PlysC, PpheA, PthrABC, and PlivJ . rrnB P1 promoter activity was slightly higher at all growth-rates in strains unable to synthesize ppGpp (deltarelAdeltaspoT) than in wild-type strains . Consistent with this observation and with the large decrease in rRNA transcription during the stringent response (when ppGpp levels are much higher), ppGpp inhibited transcription from rrnB P1 in vitro . In contrast, amino acid promoter activity was considerably lower in deltarelAdeltaspoT strains than in wild-type strains, but ppGpp had no effect on amino acid promoter activity in vitro . Detailed kinetic analysis in vitro indicated that open complexes at amino acid promoters formed much more slowly and were much longer-lived than rrnB P1 open complexes . ppGpp did not increase the rates of association with, or escape from, amino acid promoters in vitro, consistent with its failure to stimulate transcription directly . In contrast, ppGpp decreased the half-lives of open complexes at all promoters, whether the half-life was seconds (rrnB P1) or hours (amino acid promoters) . The results described here and in the accompanying paper indicate that ppGpp directly inhibits transcription, but only from promoters like rrnB P1 that make short-lived open complexes . The results indicate that stimulation of amino acid promoters occurs indirectly . The accompanying paper evaluates potential models for positive control of amino acid promoters by ppGpp that might explain the requirement of ppGpp for amino acid prototrophy .

Growth Horm IGF Res, 2000 Dec, 10(6), 360 - 6
Characterization of the enzymatic specificity of the IGF-dependent insulin-like growth factor binding protein-4 (IGFBP-4) protease; Chelius D et al.; The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by cultured adult human fibroblasts was recently identified as pregnancy-associated plasma protein-A (PAPP-A) . In this study we showed that in addition to human IGFBP-4 the IGF-dependent IGFBP-4 protease also digests recombinant rat IGFBP-4 into two fragments by specifically cleaving at the carboxyl-terminal side of methionine at position 131 for rat IGFBP-4 . Thus the cleavage site is at the KMKV site, which is not represented in other IGFBPs . While kallikrein may cleave at this site, its action is not specific.

Mol Genet Metab, 2001 Feb, 72(2), 132 - 43
In vitro expression of 34 naturally occurring mutant variants of phenylalanine hydroxylase: correlation with metabolic phenotypes and susceptibility toward protein aggregation; Gjetting T et al.; Phenylalanine hydroxylase (PAH) is a homotetrameric enzyme that catalyzes the conversion of phenylalanine to tyrosine, the rate-limiting step of phenylalanine disposal in humans . Primary dysfunction of PAH caused by mutations in the PAH gene results in hyperphenylalaninemia, which may impair cognitive development unless corrected by dietary restriction of phenylalanine . The mechanism(s) by which PAH missense mutations cause enzyme impairment has been studied in detail only in a small number of cases, but existing evidence points to a major role of enhanced proteolytic degradation due to aberrant folding of mutant polypeptides . We have used two heterologous in vitro expression systems (a mammalian cell-free transcription-translation system and the pET system of Escherichia coli) to examine 34 mutations that have been associated with PAH deficiency in the Danish population . These mutations represent a broad range of amino acid substitutions, functional enzyme domains, and metabolic phenotypes . In both systems, residual in vitro activities correlated broadly with metabolic phenotypes, however, with significant discrepancies . Analysis of E . coli extracts by nondenaturing polyacrylamide gel electrophoresis and storage experiments showed that (i) in general, mutations in the N-terminal regulatory domain are associated with relatively stable proteins compared to most mutations in the central catalytic domain, and (ii) for mutations in the catalytic domain, high levels of protein aggregation do not always correspond with a severe phenotype . Our data support and extend previous evidence that PAH mutations exert their pathogenic effects by several distinct mechanisms that may operate individually or in concert .

Cryobiology, 2000 Nov, 41(3), 204 - 31
Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose; Koshimoto C et al.; Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice . However, successful and reproducible cryopreservation has proven to be a difficult problem . Furthermore, the underlying factors responsible for success or failure are mostly obscure . Several contributors to these difficulties have been identified . Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes . We have hypothesized two other contributors to the difficulties . One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells . The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals . That damage may reduce their ability to survive freezing . If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure . To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media . A previous report showed a protective effect . That is confirmed here under a broader range of conditions . The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C . Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits . Surprisingly, the best results were achieved in the total absence of glycerol . The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS . We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C . The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase . These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation . Oxyrase appears to lessen that damage substantially .

Protein Eng, 2000 Nov, 13(11), 791 - 9
Possible role of two phenylalanine residues in the active site of human cytidine deaminase; Vincenzetti S et al.; Site-directed mutagenesis on human cytidine deaminase (CDA) was employed to mutate specifically two highly conserved phenylalanine residues, F36 and F137, to tryptophan; at the same time, the unique tryptophan residue present in the sequence at position 113 was mutated to phenylalanine . These double mutations were performed in order to have for each protein a single tryptophan signal for fluorescence studies relative to position 36 or 137 . The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F, showed by circular dicroism and thermal stability an overall structure not greatly affected by the mutations . The titration of Trp residues by N-bromosuccinimide (NBS) suggested that residue W113 of the wild-type CDA and W36 of mutant F36W/W113F are buried in the tertiary structure of the enzyme, whereas the residue W137 of mutant F137W/W113F is located near the surface of the molecule . Kinetic experiments and equilibrium experiments with FZEB showed that the residue W113 seems not to be part of the active site of the enzyme whereas the Phe/Trp substitution in F36W/W113F and F137W/W113F mutant enzymes had a negative effect on substrate binding and catalysis, suggesting that F137 and F36 of the wild-type CDA are involved in a stabilizing interaction between ligand and enzyme.

Protein Eng, 2000 Nov, 13(11), 779 - 82
Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule; Chen H et al.; The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr and B27Thr, buried in the dimer of insulin, were identified by means of alanine-scanning mutagenesis . The receptor binding activity, in vivo biological potency and self-association properties of the seven single alanine human insulin mutants were determined . Four of the seven single alanine mutants, {B12Ala}human insulin, {B16Ala}human insulin, {B24Ala}human insulin and {B26Ala}human insulin, are monomeric insulin, which indicates that B12Val, B16Tyr, B24Phe and B26Tyr are crucial for the formation of insulin dimer . The monomeric {B16Ala}human insulin and {B26Ala}human insulin retain 27 and 54% receptor binding activity, respectively, and nearly the same in vivo biological potency compared with native insulin, so they could be developed as the fast-acting insulin.

Plant Physiol, 2001 Feb, 125(2), 943 - 54
Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis . Identification of multiple forms of TOM20; Werhahn W et al.; The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane . Here we report the purification of this protein complex from Arabidopsis . On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD . The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing . The 21- and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms . Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20 . All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria . Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced . The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus . Implications on the function of plant TOM complexes are discussed . Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.

Plant Physiol, 2001 Feb, 125(2), 595 - 603
Involvement of a nuclear-encoded basic helix-loop-helix protein in transcription of the light-responsive promoter of psbD; Baba K et al.; In the chloroplast psbD light-responsive promoter (LRP), a highly conserved sequence exists upstream from the bacterial -10/-35 elements . Multiple sequence-specific DNA binding proteins are predicted to bind to the conserved sequence as transcription factors . Using yeast one-hybrid screening of an Arabidopsis cDNA library, a possible DNA binding protein of the psbD LRP upstream sequence was identified . The protein, designated PTF1, is a novel protein of 355 amino acids (estimated molecular weight of 39.6) that contains a basic helix-loop-helix DNA binding motif in the predicted N-terminal region of the mature protein . Transient expression assay of PTF1-GFP fusion protein showed that PTF1 was localized in chloroplasts . Using the modified DNA sequence in the one-hybrid system, the ACC repeat was shown to be essential for PTF1 binding . The rate of psbD LRP mRNA accumulation was reduced in a T-DNA-inserted Arabidopsis ptf1 mutant . Compared with wild-type plants, the mutant had pale green cotyledons and its growth was inhibited under short-day conditions . These results suggest that PTF1 is a trans-acting factor of the psbD LRP.

Nucleic Acids Res . 2001 Feb 15;29(4):E23.
A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain; Akagi K et al.; A tetracycline-controlled gene expression system provides a powerful tool to dissect the functions of gene products . However, it often appears difficult to establish cell lines or transgenic animals stably expressing tetracycline-dependent transactivators, possibly as a result of toxicity of the transactivator domains used . In order to overcome this problem, we developed a novel tetracycline-dependent transactivator that works efficiently in mammalian cells . This transactivator is a fusion of the tet reverse repressor mutant and the transcriptional activating domain of human E2F4, which is ubiquitously expressed in vivo . We demonstrate here that this tetracycline-regulated gene expression system provides a two log transcriptional activation in mammalian cells as assessed by northern blot and luciferase analyses . Combining this system with green fluorescent protein reporter systems or microarray gene expression profiling will facilitate the study of gene function.

Nucleic Acids Res . 2001 Feb 15;29(4):E16.
Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides; Lutz S et al.; Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology . We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs . Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation . The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment . From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps . As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain . Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.

Nucleic Acids Res, 2001 Feb 15, 29(4), 976 - 85
An important 2'-OH group for an RNA-protein interaction; Hou YM et al.; We have investigated the role of 2'-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNA(Cys) and cysteine-tRNA synthetase . This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase . A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2'-OH by 2'-deoxy or 2'-O-methyl groups were tested . Except for position U73, all substitutions had little effect on aminoacylation . Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2'-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2'-O-propyl group decreased aminoacylation by another 2-fold . The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2'-OH at position U73 is unimportant for aminoacylation . The decrease in activity upon alkyl substitution suggests that the 2'-OH group instead provides a monitor of the steric environment during the RNA-synthetase interaction . The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase . A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix . This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2'-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

Nucleic Acids Res, 2001 Feb 15, 29(4), 970 - 5
Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs; Kuroda H et al.; Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine-Dalgarno (SD) and downstream box (DB) sequences, respectively . In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability . The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation . The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5'-untranslated region (5'-UTR) and DB region . Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts . We report here that the phage T7g10 5'-UTR and DB region promotes accumulation of NPTII up to approximately 16% of total soluble leaf protein (TSP) . Enhanced mRNA stability and an improved NPTII yield ( approximately 23% of TSP) was obtained from a construct in which the T7g10 5'-UTR was linked with the NPTII coding region via a NheI site . However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively . Reduced NPTII accumulation is in part due to accelerated mRNA turnover.

Nucleic Acids Res, 2001 Feb 15, 29(4), 872 - 9
Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair; Gaillard PH et al.; ERCC1-XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF . The enzyme cuts DNA at junctions where a single strand moves 5' to 3' away from a branch point with duplex DNA . This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination . To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex . We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins . Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts . XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells . A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional . This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case . Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF . The results show for the first time that functional ERCC1-XPF can be formed from separately produced subunits . Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1-XPF heterodimeric interaction . This suggests that the ERCC1 and XPF families are related via an ancient duplication.

Nucleic Acids Res . 2001 Feb 1;29(3):E12.
Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry; Dizdaroglu M et al.; Measurement of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied . A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase . The atmospheric pressure ionization-electrospray process was used for mass spectral measurements . A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS) . Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA . The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS) . It was found that approximately five lesions per 10(6) DNA bases can be detected using amounts of DNA as low as 2 microgram . The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10(6) DNA bases, or even lower, when more DNA is used . Up to 50 microgram of DNA per injection were used without adversely affecting the measurements . Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein . The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical . Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose . This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA . In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM . Again, similar results were obtained by the two techniques . The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo . Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.

Nucleic Acids Res, 2001 Feb 1, 29(3), 854 - 61
Topography of lacUV5 initiation complexes; Studitsky V et al.; Formation of a transcriptionally competent open complex is a highly regulated multistep process involving at least two intermediates . The rate of formation and stability of the intermediate complexes often determine promoter strength . However, the detailed mechanism of formation of the open complex and the high resolution structures of these intermediates are not known . In this study the structures of the open and intermediate complexes formed on the lacUV5 promoter by Escherichia coli RNA polymerase were analyzed using 'zero length' DNA-protein cross-linking . In both the open and the intermediate complexes the core subunits (ss' and ss) interact with lacUV5 DNA in a similar way, forming DNA-protein contacts flanking the initiation site . At the same time, the recognition (sigma(70)) subunit closely interacts with the promoter only in the open complex . In combination with our previous results, the data suggest that during promoter recognition contacts of the sigma subunit with core RNA polymerase and promoter DNA are rearranged in concert . These rearrangements constitute a landmark of transition from the intermediate to the open complex, identifying the sigma subunit as a key player directing formation of the open complex.

Nucleic Acids Res, 2001 Feb 1, 29(3), 774 - 82
Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes; McCue L et al.; Toward the goal of identifying complete sets of transcription factor (TF)-binding sites in the genomes of several gamma proteobacteria, and hence describing their transcription regulatory networks, we present a phylogenetic footprinting method for identifying these sites . Probable transcription regulatory sites upstream of Escherichia coli genes were identified by cross-species comparison using an extended Gibbs sampling algorithm . Close examination of a study set of 184 genes with documented transcription regulatory sites revealed that when orthologous data were available from at least two other gamma proteobacterial species, 81% of our predictions corresponded with the documented sites, and 67% corresponded when data from only one other species were available . That the remaining predictions included bona fide TF-binding sites was proven by affinity purification of a putative transcription factor (YijC) bound to such a site upstream of the fabA gene . Predicted regulatory sites for 2097 E.coli genes are available at http://www.wadsworth.org/resnres/bioinfo/.

Nucleic Acids Res, 2001 Feb 1, 29(3), 743 - 52
Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch; Yang H et al.; Adenine-DNA glycosylase MutY of Escherichia coli catalyzes the cleavage of adenine when mismatched with 7,8-dihydro-8-oxoguanine (GO), an oxidatively damaged base . The biological outcome is the prevention of C/G-->A/T transversions . The molecular mechanism of base excision repair (BER) of A/GO in mammals is not well understood . In this study we report stimulation of mammalian adenine-DNA glycosylase activity by apurinic/apyrimidinic (AP) endonuclease using murine homolog of MutY (Myh) and human AP endonuclease (Ape1), which shares 94% amino acid identity with its murine homolog Apex . After removal of adenine by the Myh glycosylase activity, intact AP DNA remains due to lack of an efficient Myh AP lyase activity . The study of wild-type Ape1 and its catalytic mutant H309N demonstrates that Ape1 catalytic activity is required for formation of cleaved AP DNA . It also appears that Ape1 stimulates Myh glycosylase activity by increasing formation of the Myh-DNA complex . This stimulation is independent of the catalytic activity of Ape1 . Consequently, Ape1 preserves the Myh preference for A/GO over A/G and improves overall glycosylase efficiency . Our study suggests that protein-protein interactions may occur in vivo to achieve efficient BER of A/GO.

Nucleic Acids Res, 2001 Feb 1, 29(3), 677 - 82
Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA; Robert F et al.; Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3' major domain of 16S rRNA and initiates its folding . It also binds to its own mRNA, the str mRNA, and represses its translation . Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA . This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures . Overexpression of S7 is known to inhibit bacterial growth . This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA . Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7 . This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.

Nucleic Acids Res, 2001 Feb 1, 29(3), 604 - 13
A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum; Yang H et al.; Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration . The biological outcome includes increased mutation rate and potential lethality . A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans . So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage . Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C . The predicted product of 223 amino acids shares significant sequence homology with several {4Fe-4S}-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth) . The histidine-tagged recombinant protein was expressed in E.coli and purified . Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT) . This activity is enhanced when DHT is paired with G . Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea . Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.

Mol Pharmacol, 2001 Feb, 59(2), 386 - 92
cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome P450; Domanski TL et al.; The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression . This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43 . Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion . CYP3A43 expression was detected in liver, kidney, pancreas, and prostate . The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7 . CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity . The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification . CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm . The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5 . Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5 . Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity . The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.

Microbiology, 2001 Jan, 147(Pt 1), 171 - 81
Dimensional regulation of cell-cycle events in Escherichia coli during steady-state growth; Grover N et al.; Two opposing models have been put forward in the literature to describe the changes in the shape of individual Escherichia coli cells in steady-state growth that take place during the cell cycle: the Length model, which maintains that the regulating dimension is cell length, and the Volume model, which asserts it to be cell volume . In addition, the former model envisages cell diameter as decreasing with length up to constriction whereas the latter sees it as being constrained by the rigid cell wall . These two models differ in the correlations they predict between the various cellular dimensions (diameter, length, volume) not only across the entire population of bacteria but also, and especially, within subpopulations that define specific cell-cycle events (division, for example, or onset of constriction); the coefficients of variation at these specific events are also expected to be very different . Observations from cells prepared for electron microscopy (air-dried) and for phase-contrast microscopy (hydrated) appeared qualitatively largely in accordance with the predictions of the Length model . To obtain a more quantitative comparison, simulations were carried out of populations defined by each of the models; again, the results favoured the Length model . Finally, in age-selected cells using membrane elution, the diameter-length and diameter-volume correlations were in complete agreement with the Length model, as were the coefficients of variation . It is concluded that, at least with respect to cell-cycle events such as onset of constriction and cell division, length rather than volume is the controlling dimension.

Microbiology, 2001 Jan, 147(Pt 1), 161 - 9
Antigen 43, the major phase-variable protein of the Escherichia coli outer membrane, can exist as a family of proteins encoded by multiple alleles; Roche A et al.; agn43 encodes a major phase-variable outer-membrane protein, antigen 43 (Ag43), involved in autoaggregation of Escherichia coli cells . The gene is present in single copy on the chromosome of E . coli K-12 . In contrast, Southern hybridization and gene inactivation studies demonstrate that control producer strain E . coli ML308-225 possesses duplicate copies of agn43 (agn43A and agn43B) . Construction and analyses of single and double knockout mutants clearly show that both alleles are capable of expressing antigen in a phase-variable manner, with observed differences in the ON<-->OFF switch frequencies appearing to favour expression of Ag43B under conditions of normal laboratory growth . Comparative analysis of agn43A and agn43B gene sequences revealed 98% identity at the nucleotide and predicted protein levels, with differences in the protein sequence of the surface-expressed alpha(43) subunit altering the surface probability of one of the predicted epitopes . Analysis of a panel of enteropathogenic E . coli strains by Southern hybridization using agn43-specific gene probes provided strong evidence for the presence of varying numbers of agn43 alleles within clinical isolates . Taken together, the results indicate the presence of a family of distinct Ag43 proteins encoded by multiple chromosomal alleles.

Microbiology, 2001 Jan, 147(Pt 1), 153 - 9
Anomalous DnaA protein binding to the regulatory region of the Escherichia coli aldA gene; Ozaki T et al.; A binding site for DnaA protein was identified in the regulatory region of the aldA gene of Escherichia coli . Specific binding was demonstrated by in vitro assays including filter binding as well as mobility shift in a polyacrylamide gel of the DnaA-DNA complex . In cells growing in minimal medium containing glucose, expression of ss-galactosidase activity from an aldA-lacZ fusion gene was suppressed by oversupply of DnaA protein and was enhanced by reducing the free DnaA level . These results suggest that DnaA protein negatively regulates expression of the aldA gene under these conditions . Despite fairly strong binding, the bound DNA fragment had no consensus 9 bp DnaA binding sequence (DnaA box), and anomalous binding to an AT-rich sequence located close to the transcription start site was revealed by a footprinting experiment.

J Virol, 2001 Mar, 75(5), 2314 - 23
Human GLI-2 is a tat activation response element-independent Tat cofactor; Browning CM et al.; Zinc finger-containing GLI proteins are involved in the development of Caenorhabditis elegans, Xenopus, Drosophila, zebrafish, mice, and humans . In this study, we show that an isoform of human GLI-2 strongly synergizes with the Tat transactivating proteins of human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and markedly stimulates viral replication . GLI-2 also synergizes with the previously described Tat cofactor cyclin T1 to stimulate Tat function . Surprisingly, GLI-2/Tat synergy is not dependent on either a typical GLI DNA binding site or an intact Tat activation response element but does require an intact TATA box . Thus, GLI-2/Tat synergy results from a mechanism of action which is novel both for a GLI protein and for a Tat cofactor . These findings link the GLI family of transcriptional and developmental regulatory proteins to Tat function and HIV replication.

J Virol, 2001 Feb, 75(4), 1870 - 8
Human cytomegalovirus with IE-2 (UL122) deleted fails to express early lytic genes; Marchini A et al.; Much evidence suggests that the major immediate-early (IE) transactivator of human cytomegalovirus (HCMV), IE-2, is likely to be critical for efficient viral replication; however, the lack of an IE-2 mutant HCMV has precluded an experimental test of this hypothesis . As an initial step toward characterizing an IE-2 mutant, we first cloned the HCMV Towne genome as a bacterial artificial chromosome (BAC) and analyzed the ability of transfected Towne-BAC DNA (T-BACwt) to produce plaques following introduction into permissive human fibroblasts . Like Towne viral DNA, transfected T-BACwt DNA was infectious in permissive cells, and the resulting virus stocks were indistinguishable from Towne virus . We then used homologous recombination in Escherichia coli to delete the majority of UL122, the open reading frame encoding the unique portion of IE-2, from T-BACwt . From this deleted BAC, a third BAC clone in which the deletion was repaired with wild-type UL122 was created . In numerous transfections of permissive human foreskin fibroblast cells with these three BAC DNA clones, the rescued BAC and T-BACwt consistently yielded plaques, while the UL122 mutant BAC never generated plaques, even after 4 weeks . Protein and mRNA of other IE genes were readily detected from transfected UL122 mutant BAC DNA; however, reverse transcription-PCR failed to detect mRNA expression from any of five early genes examined . The generalized failure of this mutant to express early genes is consistent with expectations from in vitro assays which have demonstrated that IE-2 transactivates most HCMV promoters . These experiments provide the first direct demonstration that IE-2 is required for successful HCMV infection and indicate that virus lacking IE-2 arrests early in the replication cycle.

J Virol, 2001 Feb, 75(4), 1681 - 8
Fowlpox virus encodes a novel DNA repair enzyme, CPD-photolyase, that restores infectivity of UV light-damaged virus; Srinivasan V et al.; Fowlpox virus (FPV), a pathogen of poultry, can persist in desiccated scabs shed from infected hosts . Although the mechanisms which ensure virus survival are unknown, it is likely that some type of remedial action against environmentally induced damage is required . In this regard, we have identified an open reading frame (ORF) coding for a putative class II cyclobutane pyrimidine dimer (CPD)-photolyase in the genome of FPV . This enzyme repairs the UV light-induced formation of CPDs in DNA by using blue light as an energy source and thus could enhance the viability of FPV during its exposure to sunlight . Based on transcriptional analyses, the photolyase gene was found to be expressed late during the FPV replicative cycle . That the resultant protein retained DNA repair activity was demonstrated by the ability of the corresponding FPV ORF to complement functionally a photolyase-deficient Escherichia coli strain . Interestingly, insertional inactivation of the FPV photolyase gene did not impair the replication of such a genetically altered virus in cultured cells . However, greater sensitivity of this mutant than of the parental virus to UV light irradiation was evident when both were subsequently photoreactivated in the absence of host participation . Therefore, FPV appears to incorporate its photolyase into mature virions where the enzyme can promote their survival in the environment . Although expression of a homologous protein has been predicted for some chordopoxviruses, this report is the first to demonstrate that a poxvirus can utilize light to repair damage to its genome.

J Virol, 2001 Feb, 75(4), 1664 - 71
Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant; Richards CM et al.; The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed . Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB) . By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses . As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells . The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice . Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis . In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice . The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.

J Immunol, 2001 Feb 1, 166(3), 1698 - 702
Molecular basis for paradoxical carriers of adenosine deaminase (ADA) deficiency that show extremely low levels of ADA activity in peripheral blood cells without immunodeficiency; Ariga T et al.; Adenosine deaminase (ADA) deficiency causes an autosomal recessive form of severe combined immunodeficiency and also less severe phenotypes, depending to a large degree on genotype . In general, ADA activity in cells of carriers is approximately half-normal . Unexpectedly, healthy first-degree relatives of two unrelated ADA-deficient severe combined immunodeficient patients (mother and brother in family I; mother in family II) had only 1-2% of normal ADA activity in PBMC, lower than has previously been found in PBMC of healthy individuals with so-called "partial ADA deficiency." The level of deoxyadenosine nucleotides in erythrocytes of these paradoxical carriers was slightly elevated, but much lower than levels found in immunodeficient patients with ADA deficiency . ADA activity in EBV-lymphoblastoid cell lines (LCL) and T cell lines established from these carriers was 10-20% of normal . Each of these carriers possessed two mutated ADA alleles . Expression of cloned mutant ADA cDNAs in an ADA-deletion strain of Escherichia coli indicated that the novel mutations G239S and M310T were responsible for the residual ADA activity . ADA activity in EBV-LCL extracts of the paradoxical carriers was much more labile than ADA from normal EBV-LCL . Immunoblotting suggested that this lability was due to denaturation rather than to degradation of the mutant protein . These results further define the threshold level of ADA activity necessary for sustaining immune function.

J Bacteriol, 2001 Mar, 183(5), 1810 - 2
Expression of individual copies of Methylococcus capsulatus bath particulate methane monooxygenase genes; Stolyar S et al.; The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions . The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth.

J Bacteriol, 2001 Mar, 183(5), 1801 - 4
Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth; Jack RL et al.; The transcription start sites for the tatABCD and tatE loci, encoding components of the Tat (twin-arginine translocase) protein export pathway, have been identified . Expression studies indicate that the tatABCD and tatE transcription units are expressed constitutively . Translational fusion experiments suggest that TatA is synthesized at a much higher level than the other Tat proteins.

J Bacteriol, 2001 Mar, 183(5), 1796 - 800
The miaA mutator phenotype of Escherichia coli K-12 requires recombination functions; Zhao J et al.; miaA mutants, which contain A-37 instead of the ms(2)i(6)A-37 hypermodification in their tRNA, show a moderate mutator phenotype leading to increased GC-->TA transversion . We show that the miaA mutator phenotype is dependent on recombination functions similar to, but not exactly the same as, those required for translation stress-induced mutagenesis.

J Bacteriol, 2001 Mar, 183(5), 1631 - 44
Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains; Brown EW et al.; mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination . These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains . While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear . The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events . To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E . coli strains in which mutS alleles have recombined . Comparison of mutS phylogeny against predicted E . coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains . We interpret these incongruences as signatures of horizontal exchange among mutS alleles . Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E . coli strain phylogeny . This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E . coli chromosome . Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.

J Bacteriol, 2001 Mar, 183(5), 1524 - 30
Introduction of a carboxyl group in the first transmembrane helix of Escherichia coli F1Fo ATPase subunit c and cytoplasmic pH regulation; Jones PC; The multicopy subunit c of the H(+)-transporting F1Fo ATP synthase of Escherichia coli folds across the membrane as a hairpin of two hydrophobic alpha helices . The subunits interact in a front-to-back fashion, forming an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery . A conserved carboxyl, Asp(61) in E . coli, centered in the second transmembrane helix is essential for H+ transport . A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28 in several bacteria, some the cause of serious infectious disease . This side chain has been predicted to pack proximal to the essential carboxyl in helix 2 . It appears that in some of these bacteria the primary function of the enzyme is H+ pumping for cytoplasmic pH regulation . In this study, Ile28 was changed to Asp and Glu . Both mutants were functional . However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled H+ pumping and passive H+ transport through Fo . The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic pH and that the ion binding pocket is aqueous accessible . The presence of a single carboxyl at position 28, in mutants I28D/D61G and I28E/D61G, did not support growth on a succinate carbon source . However, I28E/D61G was functional in ATPase-coupled H+ transport . This result indicates that the side chain at position 28 is part of the ion binding pocket.

J Bacteriol, 2001 Mar, 183(5), 1505 - 10
A eukaryotic-type protein kinase, SpkA, is required for normal motility of the unicellular Cyanobacterium synechocystis sp . strain PCC 6803; Kamei A et al.; The genome of the unicellular cyanobacterium Synechocystis sp . strain PCC 6803 comprises many open reading frames (ORFs) which putatively encode eukaryotic-type protein kinase and protein phosphatase . Based on gene disruption analysis, a region of the hypothetical ORF sll1575, which retained a part of the protein kinase motif, was found to be required for normal motility in the original isolate of strain PCC 6803 . Sequence determination revealed that in this strain sll1575 was part of a gene (designated spkA) which harbored an entire eukaryotic-type Ser/Thr protein kinase motif . Strain ATCC 27184 and a glucose-tolerant strain derived from the same isolate as the PCC strain had a frameshift mutation dividing spkA into ORFs sll1574 and sll1575 . The structural integrity of spkA agreed well with the motility phenotype, determined by colony morphology on agar plates . The spkA gene was expressed in Escherichia coli as a His-tagged protein, which was purified by Ni2+ affinity chromatography . With {gamma-32P}ATP, SpkA was autophosphorylated and transferred the phosphate group to casein, myelin basic protein, and histone . SpkA also phosphorylated several proteins in the membrane fraction of Synechocystis cells . These results suggest that SpkA is a eukaryotic-type Ser/Thr protein kinase and regulates cellular motility via phosphorylation of the membrane proteins in Synechocystis.

Infect Immun, 2001 Feb, 69(2), 1181 - 4
Amino-terminal hydrophobic region of Helicobacter pylori vacuolating cytotoxin (VacA) mediates transmembrane protein dimerization; McClain MS et al.; Helicobacter pylori VacA is a secreted protein toxin that forms channels in lipid bilayers and induces multiple structural and functional alterations in eukaryotic cells . A unique hydrophobic segment at the amino terminus of VacA contains three tandem repeats of a GxxxG motif that is characteristic of transmembrane dimerization sequences . To examine functional properties of this region, we expressed and analyzed ToxR-VacA-maltose binding protein fusions using the TOXCAT system, which was recently developed by W . P . Russ and D . M . Engelman (Proc . Natl . Acad . Sci . USA 96:863-868, 1999) to study transmembrane helix-helix associations in a natural membrane environment . A wild-type VacA hydrophobic region mediated insertion of the fusion protein into the inner membrane of Escherichia coli and mediated protein dimerization . A fusion protein containing a mutant VacA hydrophobic region (in which glycine 14 of VacA was replaced by alanine) also inserted into the inner membrane but dimerized significantly less efficiently than the fusion protein containing the wild-type VacA sequence . Based on these results, we speculate that the wild-type VacA amino-terminal hydrophobic region contributes to oligomerization of the toxin within membranes of eukaryotic cells.

Infect Immun, 2001 Feb, 69(2), 1053 - 60
Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells; Navarro-Garcia F et al.; We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregative Escherichia coli (EAEC) . Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa . Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily . Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects . Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin . We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion . Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet.

Infect Immun, 2001 Feb, 69(2), 1016 - 24
Microsporidian invasion apparatus: identification of a novel polar tube protein and evidence for clustering of ptp1 and ptp2 genes in three Encephalitozoon species; Delbac F et al.; Microsporidia are unicellular eukaryotes occurring as obligate intracellular parasites which produce resistant spores . A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell . The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for Encephalitozoon cuniculi and E . hellem . Our immunological investigations provided evidence for an additional PTP in E . cuniculi, termed PTP2 . The corresponding gene was sequenced and then expressed in Escherichia coli . As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube . The single copy ptp1 and ptp2 genes of E . cuniculi were tandemly arranged on chromosome VI . Polyadenylation of the mRNAs was demonstrated . Identification and sequencing of homologous genes in the two other human-infecting Encephalitozoon species (ptp2 in E . hellem and ptp1 and ptp2 in E . intestinalis) were facilitated by conserved gene clustering . PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail . Unlike PTP1, this protein is devoid of large tandem repeats . The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly . The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.

Infect Immun, 2001 Feb, 69(2), 706 - 11
Cloning of a beta-hemolysin gene of Brachyspira (Serpulina) hyodysenteriae and its expression in Escherichia coli; Hsu T et al.; Brachyspira (Serpulina) hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs . The production of a beta-hemolysin has been considered a major virulence attribute of this organism . Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein sequence . Thus, questions still remain concerning the structural gene sequence of the hemolysin . To answer this question unequivocally, the beta-hemolytic toxin was purified from extracts of log-phase spirochetes, and the N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K) and compared with the translated sequences of previously cloned genes, tlyA to tlyC . The lack of homology between tlyA to tlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here . A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen a B . hyodysenteriae genomic library . Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8.93-kDa polypeptide containing the N-terminal sequence of the purified beta-hemolysin . To distinguish this gene from the tlyA to tlyC genes, it has been designated hlyA . A hemolysis-negative Escherichia coli strains containing hlyA was beta-hemolytic on blood agar media . Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of native B . hyodysenteriae beta-hemolysin . Based on sequence analysis, the translated protein had a pI of 4.3, an alpha-helical structure, and a phosphopantetheine binding motif . Hybridization analysis of genomic DNA indicated that the hlyA gene was present in B . hyodysenteriae and B . intermedia but was not detected in B . innocens, B . pilosicoli, or B . murdochii under high-stringency conditions . The location of hlyA on the chromosomal map was distinct from the locations of tlyA, tlyB, and tlyC.

Infect Immun, 2001 Feb, 69(2), 640 - 9
Escherichia coli strain RDEC-1 AF/R1 endogenous fimbrial glycoconjugate receptor molecules in rabbit small intestine; Ryu H et al.; Escherichia coli strain RDEC-1 causes a diarrheagenic infection in rabbits with AF/R1 fimbriae, which have been identified as an important colonization factor in RDEC-1 adherence leading to disease . The AF/R1-mediated RDEC-1 adherence model has been used as a model systems for E . coli diarrheal diseases . In this study, RDEC-1 adhered specifically to small intestinal brush borders, with both sialic acid and beta-galactosyl residues apparently involved . The AF/R1-mediated adherence activity of {(14)C}-labeled RDEC-1 was analyzed quantitatively by using 24-well plates coated with purified brush borders and purified microvilli . Two microvillus membrane proteins (130 and 140 kDa) were individually isolated, and chicken antibody raised to each protein inhibited bacterial adherence . These same two proteins, previously shown to be recognized by AF/R1, were individually digested with trypsin, and the amino acid sequences of peptides were determined by reversed-phase capillary liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS) . This LC-MS analysis indicated that these proteins are subunits of the rabbit sucrase-isomaltase protein (SI) complex . Guinea pig serum raised to purified rabbit SI complex inhibited bacterial adherence to microvilli . Additionally, as determined by high-performance thin-layer chromatography and autoradiography, RDEC-1 adhered selectively, via AF/R1 fimbriae, to a glycolipid tentatively identified as galactosylceramide (Gal beta 1-1Cer) in the lipid extract of rabbit small intestinal brush borders . RDEC-1 adherence to Gal beta 1-1Cer was partially inhibited in the presence of galactose . These combined results indicate that the endogenous receptor molecule for AF/R1 fimbriae of RDEC-1 is each individual component of the SI complex, although binding to glycolipid may be responsible for an additional adherence mechanism.

Carcinogenesis, 2001 Jan, 22(1), 49 - 55
Differential effects of oltipraz on CYP1A and CYP2B in rat lung; Le Ferrec E et al.; Oltipraz (OPZ) is a potent chemopreventive agent against chemically-induced carcinogenesis in several animal models . It affects the expression and/or activity of xenobiotic-metabolizing enzymes and its effects are altered in the course of inflammation in liver . The present study was undertaken to analyse the effect of OPZ alone or in combination with Escherichia coli lipopolysaccharide (LPS) on the expression and activities of glutathione S-transferases (GSTs) and cytochrome P450 (CYPs) in rat lung and kidney . Male Wistar rats were fed a diet containing OPZ for 1-5 days . LPS was injected 24 h before the end of OPZ treatment (from 48 to 72 h) . Total GST activity, measured using 1-chloro-2,4-dinitrobenzene as a substrate, increased slightly in both lung and kidney during OPZ treatment . As previously demonstrated in the liver, OPZ induced rat GSTP1 in both kidney and lung and this effect was totally (kidney) or partially (lung) inhibited by co-treatment with LPS . CYP1A expression and activity were strongly increased in both tissues 24 h after starting OPZ treatment and maintained for 5 days . This increase was suppressed during the acute-phase response to endotoxin . OPZ has no effect on CYP2B1 mRNA expression in the lung, but it dramatically decreased the amount and activity of the corresponding apoprotein . The OPZ-dependent decrease in the CYP2B1 apoprotein was abolished and its corresponding activity partially reversed during LPS treatment . In reconstitution experiments using cytosol from OPZ-treated or control rat lungs and microsomal fractions, CYP2B1 apoprotein was rapidly degraded in the presence of cytosol from treated rats . This effect was partially reversed in the presence of MG132, a proteasome inhibitor . These observations support the conclusion that the decrease of CYP2B1 by OPZ involves proteasome-dependent degradation and represents a new mechanism of regulation by this compound.

Biophys J, 2001 Feb, 80(2), 916 - 22
Photo-induced proton transport of pharaonis phoborhodopsin (sensory rhodopsin II) is ceased by association with the transducer; Sudo Y et al.; Phoborhodopsin (pR; also sensory rhodopsin II, sRII) is a retinoid protein in Halobacterium salinarum and works as a receptor of negative phototaxis . Pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis . In bacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm . We expressed pHtrII-free ppR or ppR-pHtrII complex in H . salinarum Pho81/wr(-) cells . Flash-photolysis experiments showed no essential changes between pHtrII-free ppR and the complex . Using SnO2 electrode, which works as a sensitive pH electrode, and envelope membrane vesicles, we showed the photo-induced outward proton transport . This membranous proton transport was also shown using membrane vesicles from Escherichia coli in which ppR was functionally expressed . On the other hand, the proton transport was ceased when ppR formed a complex with pHtrII . Using membrane sheet, it was shown that the complex undergoes first proton uptake and then release during the photocycle, the same as pHtrII-free ppR, although the net proton transport ceases . Taking into consideration that the complex of sRII (pR) and its transducer undergoes extracellular proton circulation (J . Sasaki and J . L., Biophys . J . 77:2145-2152), we inferred that association with pHtrII closes a cytoplasmic channel of ppR, which lead to the extracellular proton circulation.

Am J Respir Cell Mol Biol, 2001 Feb, 24(2), 164 - 9
Differential induction of TNF-alpha and MnSOD by endotoxin: role of reactive oxygen species and NADPH oxidase; White JE et al.; Endotoxin (lipopolysaccharide {LPS}) is known to induce the production of tumor necrosis factor (TNF)-alpha and the induction of manganese superoxide dismutase (MnSOD) . We have recently demonstrated that induction of TNF-alpha and MnSOD by LPS is mediated through different signal transduction pathways . In the current study, we investigated the role of reactive oxygen species (ROS) in the induction of TNF-alpha and MnSOD messenger RNAs (mRNAs) in human monocytes . Hypoxia (1% O2) inhibited the production of superoxide (O2-) and the induction of MnSOD, but not TNF-alpha, mRNA . Diphenylene iodonium (DPI), a potent inhibitor of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, had no effect on LPS induction of MnSOD mRNA, whereas it markedly inhibited LPS-induced O2- production . Neither hypoxia nor DPI had any effect on LPS activation of nuclear factor (NF)-kappaB . These results suggest that (1) ROS is important in the induction of MnSOD, but not TNF-alpha, mRNA by LPS, (2) ROS from sources other than NADPH oxidase is involved in LPS induction of MnSOD mRNA, and (3) ROS-mediated LPS induction of MnSOD mRNA is independent of NF-kappaB activation.

Am J Physiol Lung Cell Mol Physiol, 2001 Feb, 280(2), L279 - 85
Intra-amniotic endotoxin increases pulmonary surfactant proteins and induces SP-B processing in fetal sheep; Bachurski CJ et al.; Intra-amniotic (IA) endotoxin induces lung maturation within 6 days in fetal sheep of 125 days gestational age . To determine the early fetal lung response to IA endotoxin, the timing and characteristics of changes in surfactant components were evaluated . Fetal sheep were exposed to 20 mg of Escherichia coli 055:B5 endotoxin by IA injection from 1 to 15 days before preterm delivery at 125 days gestational age . Surfactant protein (SP) A, SP-B, and SP-C mRNAs were maximally induced at 2 days . SP-D mRNA was increased fourfold at 1 day and remained at peak levels for up to 7 days . Bronchoalveolar lavage fluid from control animals contained very little SP-B protein, 75% of which was a partially processed intermediate . The alveolar pool of SP-B was significantly increased between 4 and 7 days in conjunction with conversion to the fully processed active airway peptide . All SPs were significantly elevated in the bronchoalveolar lavage fluid by 7 days . IA endotoxin caused rapid and sustained increases in SP mRNAs that preceded the increase in alveolar saturated phosphatidylcholine processing of SP-B and improved lung compliance in prematurely delivered lambs.

Am J Physiol Endocrinol Metab, 2001 Feb, 280(2), E221 - 8
Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells; Taylor WE et al.; Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass . To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells . After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC) . These proteins were identified by immunoblotting and were purified . Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ({3H}thymidine incorporation), and protein synthesis ({1-14C}leucine incorporation) in C2C12 cells . The inhibitory effects of both proteins were greater in myotubes than in myoblasts . Neither protein had any significant effects on protein degradation or apoptosis . In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 1113 - 7
Fitness effects of advantageous mutations in evolving Escherichia coli populations; Imhof M et al.; The central role of beneficial mutations for adaptive processes in natural populations is well established . Thus, there has been a long-standing interest to study the nature of beneficial mutations . Their low frequency, however, has made this class of mutations almost inaccessible for systematic studies . In the absence of experimental data, the distribution of the fitness effects of beneficial mutations was assumed to resemble that of deleterious mutations . For an experimental proof of this assumption, we used a novel marker system to trace adaptive events in an evolving Escherichia coli culture and to determine the selective advantage of those beneficial mutations . Ten parallel cultures were propagated for about 1,000 generations by serial transfer, and 66 adaptive events were identified . From this data set, we estimate the rate of beneficial mutations to be 4 x 10(-9) per cell and generation . Consistent with an exponential distribution of the fitness effects, we observed a large fraction of advantageous mutations with a small effect and only few with large effect . The mean selection coefficient of advantageous mutations in our experiment was 0.02.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 980 - 5 Epub 2001 Jan 23.
The MinE ring required for proper placement of the division site is a mobile structure that changes its cellular location during the Escherichia coli division cycle; Fu X et al.; Placement of the division site at midcell in Escherichia coli requires the MinE protein . MinE acts by imparting topological specificity to the MinCD division inhibitor, preventing the inhibitor from acting at the midcell site while permitting it to block division at other unwanted sites along the length of the cell . It was previously shown that MinE assembled into a ring structure that appeared to be localized near midcell, apparently explaining the ability of MinE to specifically counteract MinCD at midcell . We report here that the MinE ring is not fixed in position near midcell but is a dynamic structure that undergoes a repetitive cycle of movement first to one cell pole and then to the opposite pole . Taken together with studies of the dynamic behavior of the MinD protein, the results suggest that the topological specificity of division site placement may not involve a localized action of MinE to counteract the MinCD division inhibitor at midcell but rather the ability of MinE to move the division inhibitor away from midcell and to the cell poles.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 908 - 13 Epub 2001 Jan 23.
The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichia coli; Qin L et al.; Escherichia coli modulates its porin expression through a histidine kinase, EnvZ, and its cognate response regulator, OmpR . EnvZ is a bifunctional enzyme that possesses both OmpR kinase and phosphorylated OmpR (OmpR-P) phosphatase activities and thus controls the cellular level of OmpR-P . In an in vitro-assay system, the addition of OmpR to the reaction mixture consisting of the cytoplasmic domain of EnvZ (EnvZc) and ATP produces a barely detectable amount of OmpR-P because of the dual activities of EnvZ . Here we report that DNA fragments containing the upstream promoter regions of the porin genes (ompF and ompC) can shift the equilibrium between OmpR and OmpR-P dramatically toward OmpR-P . Among the four reactions occurring in the mixture, only the EnvZ phosphatase activity was inhibited severely by the specific DNA, in contrast to the previous report by Kenney and her associates that DNA stimulates OmpR phosphorylation by EnvZ {Ames, S . K., Frankema, N . & Kenney, L . J . (1999) Proc . Natl . Acad . Sci . USA 96, 11792-11797} . The autophosphorylation of EnvZc and the phosphotransfer from phosphorylated EnvZc to OmpR were not affected by DNA, whereas the autodephosphorylation of OmpR-P was inhibited slightly . We propose that the apparent inhibitory effect of DNA on the EnvZ phosphatase function is caused by sequestrating OmpR-P from the reaction as a result of OmpR-P binding to DNA.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 903 - 7 Epub 2001 Jan 23.
Directed evolution of ampicillin-resistant activity from a functionally unrelated DNA fragment: A laboratory model of molecular evolution; Yano T et al.; To establish an experimental system to directly observe molecular evolution, a DNA fragment that confers ampicillin resistance on Escherichia coli was cloned from an archaeal genomic DNA . The activity of this clone was enhanced by 50 rounds of directed evolution by using DNA shuffling . Analysis of the evolved DNA fragments shows that two genetic regions have coevolved: One region, which has no obvious ORF, is essential for the activity, whereas the other, which appears to encode a protein, is not essential but enhances the activity of the former region . Analysis of the evolutionary intermediates shows that negative mutations are effectively removed while beneficial mutations accumulate and illustrates how a protein has evolved over the course of the evolution experiments . Although the mechanism of the activity remains unclear, the evolved DNA fragments also confer resistance to other drugs that inhibit bacterial cell-wall synthesis . The present system would serve as an experimental model to study evolutionary dynamics in the laboratory and provide the concept of screening natural libraries to obtain starting materials for directed evolution.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 898 - 902 Epub 2001 Jan 23.
Rotation of the c subunit oligomer in fully functional F1Fo ATP synthase; Tsunoda SP et al.; The F(1)F(o)-type ATP synthase is the smallest motor enzyme known . Previous studies had established that the central gamma and epsilon subunits of the F(1) part rotate relative to a stator of alpha(3)beta(3) and delta subunits during catalysis . We now show that the ring of c subunits in the F(o) part moves along with the gamma and epsilon subunits . This was demonstrated by linking the three rotor subunits with disulfide bridges between cysteine residues introduced genetically at the interfaces between the gamma, epsilon, and c subunits . Essentially complete cross-linking of the gamma, epsilon, and c subunits was achieved by using CuCl(2) to induce oxidation . This fixing of the three subunits together had no significant effect on ATP hydrolysis, proton translocation, or ATP synthesis, and each of these functions retained inhibitor sensitivity . These results unequivocally place the c subunit oligomer in the rotor part of this molecular machine.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 852 - 7
Monolayers of derivatized poly(L-lysine)-grafted poly(ethylene glycol) on metal oxides as a class of biomolecular interfaces; Ruiz-Taylor LA et al.; We report on the design and characterization of a class of biomolecular interfaces based on derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers adsorbed on negatively charged surfaces . As a model system, we synthesized biotin-derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers, PLL-g-{(PEGm)((1-x)) (PEG-biotin)(x)}, where x varies from 0 to 1 . Monolayers were produced on titanium dioxide substrates and characterized by x-ray photoelectron spectroscopy . The specific biorecognition properties of these biotinylated surfaces were investigated with the use of radiolabeled streptavidin alone and within complex protein mixtures . The PLL-g-PEG-biotin monolayers specifically capture streptavidin, even from a complex protein mixture, while still preventing nonspecific adsorption of other proteins . This streptavidin layer can subsequently capture biotinylated proteins . Finally, with the use of microfluidic networks and protein arraying, we demonstrate the potential of this class of biomolecular interfaces for applications based on protein patterning.

Mol Endocrinol, 2001 Feb, 15(2), 241 - 54
Two distinct nuclear receptor-interaction domains and CREB-binding protein-dependent transactivation function of activating signal cointegrator-2; Lee SK et al.; ASC-2 is a recently isolated transcriptional cointegrator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors, AP-1, nuclear factor kappaB (NFkappaB), serum response factor (SRF), and numerous other transcription factors . ASC-2 contained two nuclear receptor-interaction domains, both of which are dependent on the integrity of their core LXXLL sequences . Surprisingly, the C-terminal LXXLL motif specifically interacted with oxysterol receptor LXRss, whereas the N-terminal motif bound a broad range of nuclear receptors . These interactions appeared to be essential because a specific subregion of ASC-2 including the N- or C-terminal LXXLL motif acted as a potent dominant negative mutant with transactivation by appropriate nuclear receptors . In addition, the autonomous transactivation domain (AD) of ASC-2 was found to consist of three separable subregions; i.e . AD1, AD2, and AD3 . In particular, AD2 and AD3 were binding sites for CREB binding protein (CBP), and CBP-neutralizing E1A repressed the autonomous transactivation function of ASC-2 . Furthermore, the receptor transactivation was not enhanced by ASC-2 in the presence of E1A and significantly impaired by overexpressed AD2 . From these results, we concluded that ASC-2 directly binds to nuclear receptors and recruits CBP to mediate the nuclear receptor transactivation in vivo.

Mol Cell Biol, 2001 Feb, 21(4), 979 - 89
The two RNA ligases of the Trypanosoma brucei RNA editing complex: cloning the essential band IV gene and identifying the band V gene; Rusche LN et al.; Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase . A complex of seven different polypeptides purified from Trypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of approximately 57 kDa (band IV) and approximately 50 kDa (band V) . From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex . It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining . Overexpression studies revealed that T . brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels . The protein's mitochondrial targeting was confirmed by its location, size when expressed in T . brucei and E . coli, and N-terminal sequence . Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes . The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions . We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

J Clin Microbiol, 2001 Feb, 39(2), 798 - 800
Relationship between susceptibility to hemolytic-uremic syndrome and levels of globotriaosylceramide in human sera; Watarai S et al.; The relationship between differential susceptibility to hemolytic-uremic syndrome (HUS) and levels of globotriaosylceramide (Gb3) in serum was studied in patients infected with verotoxin-producing Escherichia coli (VTEC) . The serum Gb3 levels in patients with HUS were lower than these in diarrheal patients without subsequent HUS or in patients without clinical symptoms, indicating that individuals with a lower content of serum Gb3 show a higher incidence of HUS following VTEC infection.

J Clin Microbiol, 2001 Feb, 39(2), 782 - 6
Identification of a cluster of strains bearing a new adhesin among genetically diverse enterotoxigenic Escherichia coli isolates of serogroup O20; Pichel M et al.; About one-third of the enterotoxigenic Escherichia coli isolates lack any of the known colonization factors . Among this group, those of serogroup O20 are the most frequently isolated in Argentina . By combining analysis of adhesion to Caco-2 cells, random amplified polymorphic DNA, and pulsed-field gel electrophoresis techniques, we were able to identify three sets of closely related strains with different binding properties . Further analysis of the most prevalent group revealed that all these isolates expressed the recently described adhesin CS22.

J Cell Biol, 2001 Feb 5, 152(3), 553 - 62
Functional differences in yeast protein disulfide isomerases; Norgaard P et al.; PDI1 is the essential gene encoding protein disulfide isomerase in yeast . The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1 . We have investigated the effects of simultaneous deletions of these genes . In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues . This shows that the homologues are not functionally interchangeable . In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p . Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs) . This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation . Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification . There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

J Bacteriol, 2001 Feb, 183(4), 1499 - 503
Inhibition of Escherichia coli acetyl coenzyme A carboxylase by acyl-acyl carrier protein; Davis MS et al.; Escherichia coli acetyl coenzyme A carboxylase (ACC), the first enzyme of the fatty acid biosynthetic pathway, is inhibited by acylated derivatives of acyl carrier protein (ACP) . ACP lacking an acyl moiety does not inhibit ACC . Acylated derivatives of ACP having chain lengths of 6 to 20 carbon atoms were similarly inhibitory at physiologically relevant concentrations . The observed feedback inhibition was specific to the protein moiety, as shown by the inability of the palmitoyl thioester of spinach ACP I to inhibit ACC.

J Bacteriol, 2001 Feb, 183(4), 1489 - 90
Aminopeptidases A, B, and N and dipeptidase D are the four cysteinylglycinases of Escherichia coli K-12; Suzuki H et al.; Aminopeptidases A, B, and N and dipeptidase D, with broad substrate specificity, are the four cysteinylglycinases of Escherichia coli K-12, and there is no peptidase specific for the cleavage of cysteinylglycine.

J Bacteriol, 2001 Feb, 183(4), 1459 - 61
Unexpected presence of defective glpR alleles in various strains of Escherichia coli; Holtman CK et al.; Alleles of glpR associated with the same GlpR(-) phenotype produce substitutions in different conserved portions of the glycerol 3-phosphate repressor which are not part of the helix-turn-helix motif . Analysis of the effects on growth and enzyme expression show that glucose repression of glycerol utilization is not dependent on a functional repressor.

J Bacteriol, 2001 Feb, 183(4), 1413 - 22
Influence of the nucleoid on placement of FtsZ and MinE rings in Escherichia coli; Sun Q et al.; We previously presented evidence that replicating but unsegregated nucleoids, along with the Min system, act as topological inhibitors to restrict assembly of the FtsZ ring (Z ring) to discrete sites in the cell . To test if nonreplicating nucleoids have similar exclusion effects, we examined Z rings in dnaA (temperature sensitive) mutants . Z rings were excluded from centrally localized nucleoids and were often observed at nucleoid edges . Cells with nonreplicating nucleoids formed filaments, some of which contained large nucleoid-free areas in which Z rings were positioned at regular intervals . Because MinE may protect FtsZ from the action of the MinC inhibitor in these nucleoid-free zones, we examined the localization of a MinE-green fluorescent protein (GFP) fusion with respect to Z rings and nucleoids . Like Z rings, MinE-GFP appeared to localize independently of nucleoid position, forming rings at regular intervals in nucleoid-free regions . Unlike FtsZ, however, MinE-GFP often localized on top of nucleoids, replicating or not, suggesting that MinE is relatively insensitive to the nucleoid inhibition effect . These data suggest that both replicating and nonreplicating nucleoids are capable of topologically excluding Z rings but not MinE.

J Bacteriol, 2001 Feb, 183(4), 1339 - 45
Expression characteristics of the transfer-related kilB gene product of Streptomyces plasmid pIJ101: implications for the plasmid spread function; Pettis GS et al.; Intermycelial transfer of Streptomyces plasmid pIJ101 occurs prior to cellular differentiation and is mediated by plasmid functions that are also required for production of zones of growth-inhibited recipient cells (i.e., pocks) that develop around individual donors during mating on agar medium . Several other pIJ101 functions, including that of the kilB gene, whose unregulated expression on pIJ101 is lethal, are required for normal pock size and so have been postulated to mediate intramycelial spread of the plasmid throughout recipient cells . Using antibodies raised against a KilB fusion protein expressed in Escherichia coli, native KilB protein was detected throughout development of pIJ101-containing Streptomyces lividans cells, with the concentration of KilB increasing dramatically and reaching a maximum during the final stages (i.e., sporulation and secondary metabolism) of cellular differentiation . Insertion of the kilB gene of pIJ101 into the S . lividans chromosome in cells lacking the pIJ101 KorB protein, which normally represses kilB gene transcription, resulted in elevated but still temporally increasing amounts of KilB . The increased expression or accumulation of the KilB spread protein throughout cellular differentiation of S . lividans, which leads to maximum KilB concentrations during developmental stages that occur far later than when intermycelial transfer of pIJ101 is mediated, supports the existence of a subsequent intramycelial component to the pIJ101 spread function . The results also suggest that intramycelial spread of pIJ101 molecules within the recipient extends beyond intercompartmental movements within the substrate mycelia and includes undetermined steps within the spore-yielding aerial hyphae as well.

J Bacteriol, 2001 Feb, 183(4), 1215 - 24
umuDC-mediated cold sensitivity is a manifestation of functions of the UmuD(2)C complex involved in a DNA damage checkpoint control; Sutton MD et al.; The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage . After induction, they help to promote cell survival via two temporally separate pathways . First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD'(2)C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA . Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis . Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show that umuDC-mediated cold sensitivity can be genetically separated from the role of UmuD'(2)C in SOS mutagenesis . Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that umuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC . Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD(2)C complex and not by the separate actions of the UmuD and UmuC proteins . Finally, we present evidence for structural differences between UmuD and UmuD' in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the umuDC gene products.

J Bacteriol, 2001 Feb, 183(4), 1205 - 14
Role of CspC and CspE in regulation of expression of RpoS and UspA, the stress response proteins in Escherichia coli; Phadtare S et al.; Nine homologous proteins, CspA to CspI, constitute the CspA family of Escherichia coli . Recent studies are aimed at elucidating the individual cellular functions of these proteins . Two members of this family, CspC and CspE, are constitutively produced at 37 degrees C . In the present study, these two proteins were evaluated for their cellular role(s) . The expression of three stress proteins, OsmY, Dps, and UspA, is significantly affected by the overexpression and deletion of CspC and CspE . RpoS is a regulatory element for osmY and dps . Further analysis showed a larger amount and greater stability of the rpoS mRNA as well as a higher level of RpoS itself with the overexpression of CspC and CspE . This suggests that CspC and CspE upregulate the expression of OsmY and Dps by regulating the expression of RpoS itself . Indeed, this upregulation is lost in the Delta rpoS strain . Other RpoS-controlled proteins such as ProP and KatG, are also upregulated by the overexpression of CspC . The present study suggests that CspC and CspE are the important elements involved in the regulation of the expression of RpoS, a global stress response regulator, and UspA, a protein responding to numerous stresses . In the light of these observations, it seems plausible that CspC and CspE function as regulatory elements for the expression of stress proteins in the complex stress response network of E . coli.

J Bacteriol, 2001 Feb, 183(4), 1147 - 58
Oversynthesis of a new Escherichia coli small RNA suppresses export toxicity of DsbA'-PhoA unfoldable periplasmic proteins; Guigueno A et al.; In Escherichia coli, the DsbA'-PhoA hybrid proteins carrying an unfoldable DsbA' fragment can be targeted to the envelope, where they exert their toxicity . Hybrid proteins stick to the periplasmic face of the inner membrane and paralyze the export mechanism, becoming lethal if sufficiently overproduced and if not degraded by the DegP protease (A . Guigueno, P . Belin, and P . L . Boquet, J . Bacteriol . 179:3260-3269, 1997) . We isolated a multicopy suppressor that restores viability to a degP strain without modifying the expression level of the toxic fusion . Suppression does not involve activation of the known envelope stress-combative pathways, the Cpx pathway and the sigma(E) regulon . Subclone analysis of the suppressor revealed a 195-bp DNA fragment that is responsible for toxicity suppression . The cloned gene, called uptR, is approximately 130 bp long (including the promoter and a transcription termination signal) and is transcribed into a small RNA (92 nucleotides) . Using site-directed mutagenesis, we found that UptR RNA does not require translation for toxicity suppression . UptR-mediated action reduces the amount of membrane-bound toxic hybrid protein . UptR RNA is the first example of a small RNA implicated in extracytoplasmic toxicity suppression . It appears to offer a new way of suppressing toxicity, and its possible modes of action are discussed.

EMBO J, 2001 Feb 1, 20(3), 619 - 29
Impairment of lagging strand synthesis triggers the formation of a RuvABC substrate at replication forks; Flores MJ et al.; The holD gene codes for the psi subunit of the Escherichia coli DNA polymerase III holoenzyme, a component of the gamma complex clamp loader . A holD mutant was isolated for the first time in a screen for mutations that increase the frequency of tandem repeat deletions . In contrast to tandem repeat deletions in wild-type strains, deletion events stimulated by the holD mutation require RecA . They do not require RecF, and hence do not result from the recombinational repair of gaps, arguing against uncoupling of the leading and lagging strand polymerases in the holD mutant . The holD recBC combination of mutations is lethal and holD recBts recCts strains suffer DNA double-strand breaks (DSBs) at restrictive temperature . DSBs require the presence of the Holliday junction-specific enzymes RuvABC and are prevented in the presence of RecBCD . We propose that impairment of replication due to the holD mutation causes the arrest of the entire replisome; consequently, Holliday junctions are formed by replication fork reversal, and unequal crossing over during RecA- and RecBCD-mediated re-incorporation of reversed forks causes the hyper-recombination phenotype.

EMBO J, 2001 Feb 1, 20(3), 601 - 11
Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition; Verhoeven EE et al.; Nucleotide excision repair (NER) is a major DNA repair mechanism that recognizes a broad range of DNA damages . In Escherichia coli, damage recognition in NER is accomplished by the UvrA and UvrB proteins . We have analysed the structural properties of the different protein-DNA complexes formed by UvrA, UvrB and (damaged) DNA using atomic force microscopy . Analysis of the UvrA(2)B complex in search of damage revealed the DNA to be wrapped around the UvrB protein, comprising a region of about seven helical turns . In the UvrB-DNA pre-incision complex the DNA is wrapped in a similar way and this DNA configuration is dependent on ATP binding . Based on these results, a role for DNA wrapping in damage recognition is proposed . Evidence is presented that DNA wrapping in the pre-incision complex also stimulates the rate of incision by UvrC.

EMBO J, 2001 Feb 1, 20(3), 579 - 88
t-loops at trypanosome telomeres; Munoz-Jordan JL et al.; Mammalian telomeres form large duplex loops (t-loops) that may sequester chromosome ends by invasion of the 3' TTAGGG overhang into the duplex TTAGGG repeat array . Here we document t-loops in Trypanosoma brucei, a kinetoplastid protozoan with abundant telomeres due to the presence of many minichromosomes . These telomeres contained 10-20 kb duplex TTAGGG repeats and a 3' TTAGGG overhang . Electron microscopy of psoralen/UV cross-linked DNA revealed t-loops in enriched telomeric restriction fragments and at the ends of isolated minichromosomes . In mammals, t-loops are large (up to 25 kb), often comprising most of the telomere . Despite similar telomere lengths, trypanosome t-loops were much smaller (approximately 1 kb), indicating that t-loop sizes are regulated . Coating of non-cross-linked minichromosomes with Escherichia coli single-strand binding protein (SSB) often revealed 3' overhangs at both telomeres and several cross-linked minichromosomes had t-loops at both ends . These results suggest that t-loops and their prerequisite 3' tails can be formed on the products of both leading and lagging strand synthesis . We conclude that t-loops are a conserved feature of eukaryotic telomeres.

EMBO J, 2001 Feb 1, 20(3), 491 - 8
The Escherichia coli glucose transporter enzyme IICB(Glc) recruits the global repressor Mlc; Nam TW et al.; In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes . Exposure of cells to glucose can result in repression or induction of gene expression . While the mechanism for carbon catabolite repression by glucose was well documented, that for glucose induction was not clearly understood in Escherichia coli . Recently, glucose induction of several E.coli genes has been shown to be mediated by the global repressor Mlc . Here, we elucidate a general mechanism for glucose induction of gene expression in E.coli, revealing a novel type of regulatory circuit for gene expression mediated by the phosphorylation state-dependent interaction of a membrane-bound protein with a repressor . The dephospho-form of enzyme IICB(Glc), but not its phospho-form, interacts directly with Mlc and induces transcription of Mlc-regulated genes by displacing Mlc from its target sequences . Therefore, the glucose induction of Mlc-regulated genes is caused by dephosphorylation of the membrane-bound transporter enzyme IICB(Glc), which directly recruits Mlc to derepress its regulon.

FASEB J, 2001 Feb, 15(2), 535 - 44
Delayed activation of PPARgamma by LPS and IFN-gamma attenuates the oxidative burst in macrophages; Von Knethen A A et al.; Desensitization of macrophages is important during the development of sepsis . It was our intention to identify mechanisms that promote macrophage deactivation upon contact with endotoxin (LPS) and interferon-gamma (IFN-gamma) in vitro . Macrophage activation was achieved with 12-O-tetradecanoylphorbol 13-acetate (TPA), and the oxidative burst (i.e., oxygen radical formation) was followed by oxidation of the redox-sensitive dyes hydroethidine and dichlorodihydrofluorescein diacetate . Prestimulation of macrophages for 15 h with a combination of LPS/IFN-gamma attenuated oxygen radical formation in response to TPA . Taking the anti-inflammatory properties of the peroxisome proliferator-activating receptorgamma (PPARgamma) into consideration, we established activation of PPARgamma in response to LPS/IFN-gamma by an electrophoretic mobility shift, supershift, and a reporter gene assay . The reporter contains a triple PPAR-responsive element (PPRE) in front of a thymidine kinase minimal promoter driving the luciferase gene . We demonstrated that PPRE decoy oligonucleotides, supplied in front of LPS/IFN-gamma, allowed a full oxidative burst to recover upon TPA addition . Furthermore, we suppressed the oxidative burst by using the PPARgamma agonists 15-deoxy-Delta12,14-prostaglandin J2, BRL 49653, or ciglitazone . No effect was observed with WY 14643, a PPARalpha agonist . We conclude that activation of PPARs, most likely PPARgamma, promotes macrophage desensitization, thus attenuating the oxidative burst . This process appears important during development of sepsis.

FASEB J, 2001 Feb, 15(2), 294 - 6 Epub 2000 Dec 08.
Cardiac contractile impairment associated with increased phosphorylation of troponin I in endotoxemic rats; Tavernier B et al.; The subcellular mechanisms underlying intrinsic myocardial depression during sepsis remain poorly defined, in particular the relative roles of altered intracellular Ca2+ transients versus changes in myofilament properties . We studied contractile function of cardiac myocytes isolated 12 h after induction of endotoxemia (5 mg/kg intravenous E . coli lipopolysaccharide {LPS}) in conscious rats . Cardiomyocytes from LPS-injected rats had depressed twitch shortening compared with control cells (4.10.2% versus 7.80.3%; P2+ transients (peak indo-1 ratio 1.130.02 versus 1.120.02; P = NS) . Contractile depression was unaffected by inhibitors of nitric oxide synthase . Steady-state myofilament response to Ca2+, assessed by tetanization of intact cells over a range of {Ca2+}, was reduced significantly in the LPS group (P2+ was unaffected by isoproterenol (3 nmol/L) in endotoxemic cells, whereas there was a rightward shift in control cells . A reduction in myofilament response to Ca2+ is the major determinant of intrinsic cardiac depression in systemic endotoxemia . This condition appears to be related to an increase in myocardial troponin I phosphorylation.

Bioorg Chem, 2000 Oct, 28(5), 293 - 305
Effects of Nucleoside Analogs on Native and Site-Directed Mutants of HTLV Type 1 Reverse Transcriptase; Anantharaman V V et al.; A bacterial assay was developed for testing HTLV-1 reverse transcriptase sensitivity to common nucleoside analog inhibitors in an Escherichia coli strain characterized by a temperature sensitive PolI/RecA deletion phenotype . This genetic complementation assay exploits the ability of HTLV-1 reverse transcriptase to functionally replace these missing activities at nonpermissive temperatures . The four inhibitors tested, dideoxyinosine, dideoxyadenosine, deoxythymidine, and didehydrodeoxythymidine are well-known inhibitors of HIV reverse transcriptase . All except dideoxyadenosine showed a strong activity against HTLV-1 reverse transcriptase with IC(50); in the nanomolar range . Sequence alignments were used to identify amino acid residues in HTLV-1 reverse transcriptase, which correspond to those identified as important for drug-resistance in HIV reverse transcriptase . Mutations of some of these HTLV-1 residues altered the IC(50) for the inhibitors as expected, which suggests that these amino acids have a function in HTLV-1 reverse transcriptase similar to that of their homologs in HIV reverse transcriptase .

Bioorg Chem, 2000 Aug, 28(4), 191 - 204
A Secondary beta Deuterium Kinetic Isotope Effect in the Chorismate Synthase Reaction; Bornemann S et al.; Chorismate synthase (EC 4.6.1.4) is the shikimate pathway enzyme that catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate . The enzyme reaction is unusual because it involves a trans-1,4 elimination of the C-3 phosphate and the C-6 proR hydrogen and it has an absolute requirement for reduced flavin . Several mechanisms have been proposed to account for the cofactor requirement and stereochemistry of the reaction, including a radical mechanism . This paper describes the synthesis of {4-(2)H}EPSP and the observation of kinetic isotope effects using this substrate with both Neurospora crassa and Escherichia coli chorismate synthases . The magnitude of the effects were (D)(V) = 1.08 +/- 0.01 for the N . crassa enzyme and 1.10 +/- 0.02 on phosphate release under single-turnover conditions for the E . coli enzyme . The effects are best rationalised as substantial secondary beta isotope effects . It is most likely that the C(3)-O bond is cleaved first in a nonconcerted E1 or radical reaction mechanism . Although this study alone cannot rule out a concerted E2-type mechanism, the C(3)-O bond would have to be substantially more broken than the proR C(6)-H bond in a transition state of such a mechanism . Importantly, although the E . coli and N . crassa enzymes have different rate limiting steps, their catalytic mechanisms are most likely to be chemically identical .

J Virol, 2000 May, 74(9), 4291 - 301
Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3; Butkiewicz N et al.; GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model . It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV . Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion . We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates . Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A . Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement . This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B . The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV . Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29 . A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity . Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered . Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.

J Gastroenterol, 2000, 35(3), 214 - 20
Induction of hepatic stellate cell proliferation by LPS-stimulated peripheral blood mononuclear cells from patients with liver cirrhosis; Toda K et al.; We studied hepatic stellate cell proliferation in vitro . Peripheral blood mononuclear cells (PBMC) from patients with chronic active hepatitis C (CAH) and liver cirrhosis (LC) were cultured for 24h in the presence or absence of Escherichia coli lipopolysaccharides (LPS) . Hepatic stellate cell proliferation induced by the culture supernatants was measured, and interleukin-1 (IL-1) and IL-6 levels in the culture supernatants were quantified . Culture supernatants of LPS-stimulated PBMC from LC patients induced rat hepatic stellate cell proliferation by almost 2.8-fold (stimulation index, 2.83 +/- 1.41) compared with when the cells were cultured without addition of PBMC culture supernatants . Production of IL-1beta was significantly higher in the culture supernatants of both CAH and LC patients than in those of ten healthy controls (P < 0.01 and P < 0.05, respectively) . But there was no significant correlation between IL-1 production and the induction of hepatic stellate cell proliferation by the culture supernatants . Although there were no significant differences in IL-6 production by LPS-stimulated PBMC among healthy controls and CAH and LC patients, we observed a significant correlation between IL-6 production and the induction of hepatic stellate cell proliferation in the culture supernatants of LC patients . Rat hepatic stellate cells themselves produced IL-6, and treatment with IL-6 antisense oligodeoxynucleotides suppressed the cell proliferation, suggesting that IL-6 is an autocrine growth factor for hepatic stellate cells . The addition of human recombinant IL-6 (hrIL-6) augmented rat hepatic stellate cell proliferation, indicating that excessive IL-6 may further facilitate cell proliferation . These findings suggest that a cytokine cascade including IL-6 may participate in hepatic stellate cell proliferation in LC patients when they are exposed to endotoxin.

J Med Microbiol, 2000 Apr, 49(4), 327 - 38
The role of fimbriae and flagella in the adherence of avian strains of Escherichia coli O78:K80 to tissue culture cells and tracheal and gut explants; La Ragione RM et al.; To investigate the role of fimbriae and flagella in the pathogenesis of avian colibacillosis, isogenic insertionally inactivated mutant strains of Escherichia coli O78:K80 strain EC34195 defective in the elaboration of type-1 and curli fimbriae and flagella were constructed by allelic exchange . Single and multiple non-fimbriate and non-flagellate mutant strains were compared to the wild-type in vitro in adherence assays with a HEp-2 cell line, a mucus-secreting cell line HT2916E, a non-mucus-secreting cell line HT2919A, tracheal explant and proximal gut explant . Mutant strains defective in the elaboration of type-1 fimbriae were significantly less adherent--in the order of 90% reduction--than the wild-type strain in all assays . Mutant strains defective in the elaboration of flagella were generally as adherent as the wild-type strain except when assayed with the mucus-secreting cell line HT2916E, for which a significant reduction of adherence--of the order of 90%--compared with the wild-type strain was observed . Mutant strains defective for the elaboration of curli fimbriae adhered as well as the wild-type strain in all assays, except when assayed in tests with gut explant tissue for which a significant reduction of adherence--of the order of 80%--compared with the wild-type strain was observed . Adherence to explants was to epithelial, not serous, surfaces and was 10-fold greater to tracheal than to gut explants . Together, these data support the hypothesis that type-1 fimbriae are significant factors in adherence, aided by flagella for penetration of mucus and curli fimbriae for adherence to the gut.

Trends Microbiol, 2000 Apr, 8(4), 172 - 9
PII signal transduction proteins; Ninfa AJ et al.; PII proteins, found in Bacteria, Archaea and plants, help coordinate carbon and nitrogen assimilation by regulating the activity of signal transduction enzymes in response to diverse signals . Recent studies of bacterial PII proteins have revealed a solution to the signal transduction problem of how to coordinate multiple receptors in response to diverse stimuli yet permit selective control of these receptors under various conditions and allow adaptation of the system as a whole to long-term stimulation.

J Biol Chem, 2000 Apr 14, 275(15), 11440 - 50
DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme; Yao N et al.; The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis . Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template . The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner . This study examines the DNA structure required for clamp loading action . We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex . Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength . Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA . On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA . The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring . However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation . A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta . Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta . The possible biological significance of these structural constraints is discussed.

J Biol Chem, 2000 Apr 14, 275(15), 11361 - 7
The yeast ARG7 gene product is autoproteolyzed to two subunit peptides, yielding active ornithine acetyltransferase; Abadjieva A et al.; Yeast ornithine acetyltransferase has been purified from total yeast extracts as a heterodimer of two subpeptides (Liu, Y., Van Heeswijck, R., Hoj, P., and Hoogenraad, N . (1995) Eur . J . Biochem . 228, 291-296), confirmed to derive from a single ARG7-encoded precursor (Crabeel, M., Abadjieva, A., Hilven, P., Desimpelaere, J., and Soetens, O . (1997) Eur . J . Biochem . 250, 232-241) . By Western immunoblotting, we show that Arg7p is also present as two subpeptides in isolated mitochondria, but that processing occurs before targeting to the mitochondria: deletion of the N-terminal leader peptide results in cytosolic accumulation of N-Arg7p, whereas C-Arg7p partially reaches the organelle by itself . When artificially co-expressed from separate genes, the two subpeptides can complement an arg7 mutation; ornithine acetyltransferase activity is measurable . Maturation of Arg7p occurs at threonine 215 (N-side), in the region most conserved among the 17 ornithine acetyltransferases characterized . Changing this conserved residue to alanine completely abolishes maturation . Furthermore, Arg7p is both processed and active in Escherichia coli, a heterologous background, and is also cleaved in vitro when produced by coupled transcription/translation in a reticulocyte lysate . Together, these data suggest classic autoproteolysis initiated by threonine 215 . Most importantly, maturation is required for the enzyme to be functional, since the T215A substitution mutant is catalytically inactive and incapable of genetic complementation, despite its correct targeting to the mitochondria.

J Biol Chem, 2000 Apr 14, 275(15), 11355 - 60
Mutations in single hairpin units of genetically fused subunit c provide support for a rotary catalytic mechanism in F(0)F(1) ATP synthase; Jones PC et al.; Previously, we generated genetically fused dimers and trimers of subunit c of the Escherichia coli ATP synthase based upon the precedent of naturally occurring dimers in V-type H(+)-transporting ATPases . The c(2) and c(3) oligomers have proven useful in testing hypothesis regarding the mechanism of energy coupling . In the first part of this paper, the uncoupling Q42E substitution has been introduced into the second loop of the c(2) dimer or the third loop of the c(3) trimer . Both mutant proteins proved to be as functional as the wild type c(2) dimer or wild type c(3) trimer . The results argue against an obligatory movement of the epsilon subunit between loops of monomeric subunit c in the c(12) oligomer during rotary catalysis . Rather, the results support the hypothesis that the c-epsilon connection remains fixed as the c-oligomer rotates . In the second section of this paper, we report on the effect of substitution of the proton translocating Asp(61) in every second helical hairpin of the c(2) dimer, or in every third hairpin of the c(3) trimer . Based upon the precedent of V-type ATPases, where the c(2) dimer occurs naturally with a single proton translocating carboxyl in every second hairpin, these modified versions of the E . coli c(2) and c(3) fused proteins were predicted to have a functional H(+)-transporting ATPase activity, with a reduced H(+)/ATP stoichiometry, but to be inactive as ATP synthases . A variety of Asp(61)-substituted proteins proved to lack either activity indicating that the switch in function in V-type ATPases is a consequence of more than a single substitution.

J Biol Chem, 2000 Apr 14, 275(15), 11257 - 63
RNase H overproduction corrects a defect at the level of transcription elongation during rRNA synthesis in the absence of DNA topoisomerase I in Escherichia coli; Hraiky C et al.; It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth . Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated . In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I . We found that this inhibition is due to transcriptional blocks . RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis . Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I . Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases . Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation.

J Biol Chem, 2000 Apr 14, 275(15), 11235 - 40
A guanylyl cyclase from Paramecium with 22 transmembrane spans . Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases; Linder JU et al.; Paramecium has a 280-kDa guanylyl cyclase . The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order . We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins . The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting . Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct . Adenylyl cyclase activity was minimal . The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp . Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase . All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.

J Biol Chem, 2000 Apr 14, 275(15), 10983 - 8
Functional expression of O-linked GlcNAc transferase . Domain structure and substrate specificity; Lubas WA et al.; O-GlcNAc transferase (OGT) modifies nuclear pore proteins and transcription factors . In Arabidopsis, the OGT homolog participates in the gibberellin signaling pathway . We and others have proposed that mammalian OGT is the terminal step in a glucose-sensitive signal transduction pathway that becomes disregulated in insulin resistance . To facilitate mutational analysis of OGT in the absence of competing endogenous activity, we expressed the 103-kDa human OGT in Escherichia coli . Kinetic parameters for the purified recombinant enzyme (K(m) = 1.2 microM for Nup 62; K(m) = 0.5 microM for UDP-GlcNAc) are nearly identical to purified mammalian OGT . Deletions in the highly conserved C terminus result in a complete loss of activity . The N-terminal tetratricopeptide repeat domain is required for optimal recognition of substrates . Removal of the first three tetratricopeptide repeats greatly reduces the O-GlcNAc addition to macromolecular substrates . However, this altered enzyme retains full activity against appropriate synthetic peptides . Autoglycosylation of OGT is augmented when the first six tetratricopeptide repeats are removed showing that these repeats are not required for catalysis . Given its proposed role in modulating insulin action, OGT may modify kinases involved in this signaling cascade . Among the many kinases tested, OGT glycosylates glycogen synthase kinase-3 and casein kinase II, two enzymes critical in the regulation of glycogen synthesis.

J Biol Chem, 2000 Apr 14, 275(15), 10899 - 904
Polymerase arrest at the lambdaP(R) promoter during transcription initiation; Sen R et al.; During transcription initiation by Escherichia coli RNA polymerase, a fraction of the homogeneous enzyme population has been kinetically shown to form two types of nonproductive complexes at some promoters: moribund complexes, which produce only abortive transcripts, and fully inactive ternary complexes (Kubori, T., and Shimamoto, N . (1996) J . Mol . Biol . 256, 449-457) . Here we report biochemical isolation of the complexes arrested at the lambdaP(R) promoter and an analysis of their structure by DNA and protein footprintings . We found that the isolated promoter-arrested complexes retain a stoichiometric amount of sigma(70) subunit . Exonuclease III footprints of the arrested complexes are backtracked compared with that of the binary complex, and KMnO(4) footprinting reveals a decrease in the melting of DNA in the promoter region . Protein footprints of the retained sigma(70) have shown a more exposed conformation in region 3, compared with binary complexes . This feature is similar to that of the complexes arrested in inactive state during transcription elongation, indicating the existence of a common inactivating mechanism during transcription initiation and elongation . The possible involvement of the promoter arrest in transcriptional regulation is discussed.

J Biol Chem, 2000 Apr 14, 275(15), 10727 - 30
Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA; Kambampati R et al.; IscS from Escherichia coli is a cysteine desulfurase that has been shown to be involved in Fe-S cluster formation . The enzyme converts L-cysteine to L-alanine and sulfane sulfur (S(0)) in the form of a cysteine persulfide in its active site . Recently, we reported that IscS can donate sulfur for the in vitro biosynthesis of 4-thiouridine (s(4)U), a modified nucleotide in tRNA . In addition to IscS, s(4)U synthesis in E . coli also requires the thiamin biosynthetic enzyme ThiI, Mg-ATP, and L-cysteine as the sulfur donor . We now report evidence that the sulfane sulfur generated by IscS is transferred sequentially to ThiI and then to tRNA during the in vitro synthesis of s(4)U . Treatment of ThiI with 5-((2-iodoacetamido)ethyl)-1-aminonapthalene sulfonic acid (I-AEDANS) results in irreversible inhibition, suggesting the presence of a reactive cysteine that is required for binding and/or catalysis . Both ATP and tRNA can protect ThiI from I-AEDANS inhibition . Finally, using gel shift and protease protection assays, we show that ThiI binds to unmodified E . coli tRNA(Phe) . Together, these results suggest that ThiI is a recipient of S(0) from IscS and catalyzes the ultimate sulfur transfer step in the biosynthesis of s(4)U.

Curr Biol, 2000 Apr 6, 10(7), 405 - 8
Conformational rearrangements of an archaeal chaperonin upon ATPase cycling; Gutsche I et al.; Chaperonins are double-ring protein assemblies with a central cavity that provides a sequestered environment for in vivo protein folding . Their reaction cycle is thought to consist of a nucleotide-regulated alternation between an open substrate-acceptor state and a closed folding-active state . The cavity of ATP-charged group I chaperonins, typified by Escherichia coli GroEL {1}, is sealed off by a co-chaperonin, whereas group II chaperonins--the archaeal thermosome and eukaryotic TRiC/CCT {2}--possess a built-in lid {3-5} . The mechanism of the lid's rearrangements requires clarification, as even in the absence of nucleotides, thermosomes of Thermoplama acidophilum appear open in vitrified ice {6} and closed in crystals {4} . Here we analyze the conformation of the thermosome at each step of the ATPase cycle by small-angle neutron scattering . The apo-chaperonin is open in solution, and ATP binding induces its further expansion . Closure seems to occur during ATP hydrolysis and before phosphate release, and represents the rate-limiting step of the cycle . The same closure can be triggered by the crystallization buffer . Thus, the allosteric regulation of group II chaperonins appears different from that of their group I counterparts.

Virology, 2000 Apr 10, 269(2), 471 - 80
Comparison of two single-chain antibodies that neutralize canine parvovirus: analysis of an antibody-combining site and mechanisms of neutralization; Yuan W et al.; We cloned the heavy- and light-chain variable domains of two monoclonal antibodies that recognize each of the two major neutralizing antigenic sites of the canine parvovirus (CPV) capsid . After expression in Escherichia coli as single-chain variable domains (scFv) with glycine-serine linker sequences, both scFv bound CPV capsids with the same specificity as the intact IgG, but with 10- to 20-fold lower avidity . Both scFvs neutralized CPV infectivity with efficiency similar to that of the IgG . Although both IgGs inhibited hemagglutination by CPV, only one scFv was inhibiting . The binding of one of the antibodies has previously been analyzed by cryoelectron microscopic reconstruction and the epitope-binding residues predicted . Mutagenesis of predicted contact residues in three heavy-chain complementarity-determining regions (CDR) showed that mutants of CDR1 or CDR3 reduced the binding of the scFv by about 10-fold compared with the wild-type scFv, while no effect was seen for one mutant of CDR2 . The levels of neutralization of CPV and of hemagglutination inhibition by the scFv mutants were proportional to their reduction in binding affinity compared with the wild type . Neither scFv blocked virus binding to host cells, but they both caused aggregation of the capsids and appeared to affect the process of infection after virus uptake into the cells .

Virology, 2000 Apr 10, 269(2), 462 - 70
The 23-kDa protein coded by the 3'-terminal gene of citrus tristeza virus is an RNA-binding protein; Lopez C et al.; The 23-kDa protein (p23), encoded by the 3'-proximal gene of the RNA of Citrus tristeza virus (CTV), was overexpressed in Escherichia coli fused to the maltose-binding protein and purified by affinity chromatography . Gel retardation and UV crosslinking assays demonstrated that p23 has the ability to cooperatively bind single-stranded RNA in a non-sequence-specific manner . Formation of the p23-RNA complex was dependent on the conformational state of p23 and on the presence of a basic region, but the complex was stable at high salt concentrations, suggesting that interactions other than those between the negatively charged RNA and the basic region of p23 are involved . Competition assays showed that the affinity of p23 for single-stranded and double-stranded RNA was similar but considerably higher than for single-stranded and double-stranded DNA . By use of a series of artificially generated mutants, the RNA-binding domain of p23 was mapped between positions 50-86, a region containing several basic amino acids and a putative zinc-finger domain . Additional p23-derivatives lacking the conserved residues presumably involved in coordinating the zinc ion showed RNA-binding activity, but with an apparent dissociation constant higher than the wild-type protein . These conserved residues might confer binding specificity or increase binding stability in vivo . Within the Closteroviridae family, p23 is the only protein characterized so far showing RNA-binding activity .

Biochem Biophys Res Commun, 2000 Apr 13, 270(2), 543 - 9
Mutation of the toxin binding site of PP-1c: comparison with PP-2B; Dawson JF et al.; The catalytic cores of PP-1c and PP-2B (calcineurin) are structurally conserved . However, PP-2B is resistant to inhibition by toxins of the okadaic acid and cyclic peptide classes, while PP-1c is potently inhibited . Molecular docking of the structure of microcystin-LR onto the catalytic core of PP-2B identified residues that may be responsible for blocking access of toxins to the catalytic site . Amino acids in PP-1c were substituted with these PP-2B residues to investigate their contribution to PP-2B toxin resistance . Mutants of PP-1c were also produced to test the importance of hydrophobic interactions to toxin binding . Our results suggest that different classes of toxin inhibitors interact with the same hydrophobic side chains of PP-1c through different mechanisms . Substitution of amino acids in PP-1c with PP-2B residues demonstrated no highly significant changes in toxin inhibition . We hypothesize that an interaction outside the catalytic core causing the L7 loop of PP-2B to block the catalytic site may be responsible for PP-2B resistance to toxins .

Dev Biol, 2000 Apr 15, 220(2), 343 - 57
Medial edge epithelial cell fate during palatal fusion; Martinez-Alvarez C et al.; To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial-mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed . However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance . Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both . To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy . Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed beta-galactosidase activity in cells after fusion to follow their fate . Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages . In addition, we have investigated the effects of the absence of transforming growth factor beta(3) (TGF-beta(3)) during palatal fusion . Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-beta(3) null and wild-type mice . We show that MEE cell death in TGF-beta(3) null palates is greatly reduced at the time of fusion, revealing that TGF-beta(3) has an important role as an inducer of apoptosis during palatal fusion . Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-beta(3) null mutants . We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration .

J Gene Med, 1999 Nov-Dec, 1(6), 407 - 14
Cationic polymeric gene delivery of beta-glucuronidase for doxorubicin prodrug therapy; Fonseca MJ et al.; BACKGROUND: An approach to improve current chemotherapy is the selective transduction of tumor cells with suicide genes to sensitize these cells to prodrugs of cytostatic agents . METHODS: In this study, gene transfer was accomplished with the cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), able to condense plasmid-DNA by electrostatic interaction . OVCAR-3 cells were transfected with plasmids encoding E . coli-derived or human beta-glucuronidase and the transfection efficiency and inhibition by serum was determined . Next, we measured the sensitivity of OVCAR-3 cells transiently expressing beta-glucuronidase to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin . RESULTS: OVCAR-3 cells were efficiently transfected with a plasmid encoding E . coli-derived beta-glucuronidase . The degree of transfection (30% of cells) was higher than that achieved with commercially available cationic lipids (DOTAP, Lipofectamine) without inhibition by serum . OVCAR-3 cells transiently expressing beta-glucuronidase were equally sensitive to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin itself, indicating complete conversion of prodrug to drug . Similar studies were performed with the plasmid encoding for human beta-glucuronidase, which is likely to be less immunogenic . Also in this case, OVCAR-3 cells showed an increased sensitivity to the prodrug DOX-GA3, although less pronounced than when the bacterial enzyme was used . A strong bystander effect was observed when OVCAR-3 cells transfected with beta-glucuronidase were mixed with non-transfected cells at different ratios . Complete tumor cell growth inhibition was already observed when only 15% of the cells expressed the activating enzyme . CONCLUSION: These studies suggest that beta-glucuronidase gene therapy using PDMAEMA as a carrier system and DOX-GA3 as the prodrug has a potential application in cancer gene therapy.

Med Microbiol Immunol (Berl), 1999 Nov, 188(2), 79 - 82
Isolation of monoclonal antibodies directed against the V protein of human parainfluenza virus type 2 and localization of the V protein in virus-infected cells; Nishio M et al.; Two monoclonal antibodies (mAbs) specific for the human parainfluenza virus type 2 (hPIV-2) V protein were obtained by immunizing mice with the V protein recombinantly expressed in Escherichia coli . Both mAbs were found to react with the V protein in ELISA and in Western blot analysis . Using these mAbs and previously obtained mAbs specific for hPIV-2 nucleoprotein (NP) or hPIV-2 phospho-(P) protein, we examined the intracellular distributions of the V, P and NP proteins in hPIV-2-infected cells by indirect immunofluorescence analyses . The P and NP proteins were organized in numerous granules in the cytoplasm of hPIV-2 infected cells . In contrast, the V protein showed diffuse nuclear and cytoplasm distributions.

Med Microbiol Immunol (Berl), 1999 Nov, 188(2), 73 - 8
Induction of apoptosis in human renal proximal tubular epithelial cells by Escherichia coli verocytotoxin 1 in vitro; Kodama T et al.; Verocytotoxin 1 and 2 (VT1 and 2) produced by verocytotoxin-producing Escherichia coli have been considered to play an important role in the pathogenesis of glomerular and tubular damage in the epidemic form of hemolytic uremic syndrome (HUS) . VTs are known to be cytotoxic to culture cells by inhibiting cellular protein synthesis . In this in vitro study, the mechanism(s) of tubular damage in HUS and the ability of VT1 to induce apoptosis in normal human renal proximal tubular epithelial cells (HRPTEC) were examined . VTI markedly reduced cell viability of HRPTEC and rapidly inhibited overall protein synthesis . VT1 directly induced apoptotic cell death in HRPTEC in a dose- and time-dependent fashion, and co-incubation with tumor necrosis factor-alpha enhanced the VT1-induced apoptosis . These results suggest that apoptosis induced by VT1, possibly in concert with host cytokines, in renal tubular cells may contribute to the tubular damage in HUS.

Med Microbiol Immunol (Berl), 1999 Nov, 188(2), 65 - 71
Influence of human anti-lipopolysaccharide immunoglobulins on tissue distribution and clearance of lipopolysaccharide in rats; Nys M et al.; To examine the influence of passive immunization on the biological fate of injected lipopolysaccharide (LPS), we used a human IgG preparation (anti-LPS IgG) rich in antibodies to a large panel of smooth and rough purified LPS extracts as well as a normal IgG preparation (standard IgG) . Our approach was to compare the uptake of 125I-labeled LPS by the tissues of saline or IgG-treated rats . After intravenous injection, one fraction of 125I-labeled Escherichia coli O55:B5 LPS is rapidly taken up by tissues, while another fraction remained in the blood . Uptake of 125I-labeled LPS was principally observed into the liver and spleen . In rats treated prophylactically with standard IgG, these tissues accumulated significantly larger amount of LPS than the tissues of rats treated with anti-LPS IgG . Nevertheless, both IgG preparations increased the specific binding of LPS by the liver and spleen . High levels of homologous unlabeled LPS decreased the uptake of LPS by the liver, presumably by occupying tissue receptors, whereas in the presence of E . coli O127:B8 LPS, an increase of the uptake of 125I-labeled LPS by the liver and lungs was observed . The pharmacokinetics and tissue distribution of LPS-IgG complexes pre-formed in vitro were compared . In the presence of standard IgG, a unexpected increase of the uptake of LPS by the tissues was recorded, whereas LPS-anti-LPS IgG complexes decreased the binding of 125I-labeled LPS to the tissues . On the other hand, the vascular effects induced by LPS did not appear to be modified in rats pretreated with either IgG preparation . In conclusion, although passive immunization against LPS slightly modified the uptake and clearance of LPS, neither in vitro nor in vivo formation of LPS-anti-LPS IgG complexes afforded a very significant protection against the toxic effects of LPS.

Crit Care Med, 2000 Mar, 28(3), 755 - 9
Effects of lidocaine administration on hemodynamics and cytokine responses to endotoxemia in rabbits; Taniguchi T et al.; OBJECTIVE: To investigate the effects of lidocaine administration on hemodynamics and cytokine concentrations in Escherichia coli endotoxemia in rabbits . DESIGN: Randomized, prospective laboratory study . SETTING: University laboratory . SUBJECTS: Thirty-two Japanese rabbits anesthetized with urethane and ventilated mechanically . INTERVENTIONS: Animals were randomly assigned to one of four groups: endotoxemic controls (n = 8), receiving intravenous E . coli endotoxin (0.5 mg/kg bolus) via the mesenteric vein; laparotomy controls (n = 8), treated identically to the endotoxemic controls except for the substitution of 0.9% saline for endotoxin; lidocaine controls (n = 8), treated identically to the laparotomy controls with the addition of intravenous lidocaine (3 mg/kg bolus followed by infusion at 2 mg/kg/hr) administered immediately after the injection of 0.9% saline; and lidocaine-treated rabbits (n = 8), treated identically to the endotoxemic controls with the addition of intravenous lidocaine (3 mg/kg bolus followed by infusion at 2 mg/kg/hr) administered immediately after the injection of endotoxin . MEASUREMENTS AND MAIN RESULTS: We compared the cardiac output, systemic vascular resistance, blood gases, and plasma cytokine concentrations (tumor necrosis factor, interleukin {IL}-6, and IL-8) for each group . After endotoxin injection, the mean arterial pressure, cardiac output, and systemic vascular resistance decreased progressively in the endotoxemic controls . At 4 hrs after injection, all of the variables except the heart rate and central venous pressure were lower in the endotoxemic controls than in the other groups . At 4 hrs after endotoxin injection, both IL-6 and IL-8 concentrations increased in all groups . However, the mean concentrations of IL-6 and IL-8 in the endotoxemic controls significantly exceeded those in the other groups . No significant differences existed between the laparotomy controls and lidocaine-treated rabbits . CONCLUSIONS: Lidocaine had a profound inhibitory effect on the hemodynamic and cytokine responses to endotoxemia when it was administered immediately after exposure to endotoxin . Our results demonstrate the potential usefulness of lidocaine as an anti-inflammatory agent in endotoxemia.

Pharmacol Toxicol, 2000 Mar, 86(3), 140 - 4
Nitric oxide protects murine embryonic liver cells (BNL CL.2) from cytotoxicity induced by glucose deprivation; Pae HO et al.; We investigated the protective effects of nitric oxide on cell death of murine embryonic liver cells (BNL CL.2) after glucose deprivation . Endogenous nitric oxide production by BNL CL.2 cells was induced by 6 hr pretreatment with interferon-gamma and lipopolysaccharide . We used sodium nitroprusside and S-nitroso-L-glutathione as exogenous nitric oxide-generating compounds . All agents were used at doses that did not show direct cytotoxicity as measured by crystal violet staining assay . In the BNL CL.2 cells, the viability dropped very steeply after 24 hr incubation with glucose-free media . Endogenous nitric oxide produced by treatment of the cells with interferon-gamma and lipopolysaccharide protected the cells from glucose deprivation-induced cytotoxicity, but did not protect them in the presence of the nitric oxide synthesis inhibitor, N(G)-monomethyl-L-arginine . Exogenous nitric oxide protected the cells from glucose deprivation-induced cytotoxicity in a concentration-dependent manner . Cytoprotection by nitric oxide donors was abolished by the use of nitric oxide scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazole, but not by the soluble guanosine cyclase inhibitor, 1H-{1,2,4}oxadiazole{4,3-a}quinoxalin-1-one . In addition, cytoprotective effects comparable to endogenous or exogenous nitric oxide were not observed when the cells were incubated with dibutyl guanosine 3',5'-cyclic monophosphate . Based upon these results, we suggest that nitric oxide may enhance the cell survival of BNL CL.2 cells after glucose deprivation via a guanosine 3',5'-cyclic monophosphate-independent pathway.

Mutat Res, 2000 Mar 14, 448(1), 97 - 104
Copper, zinc superoxide dismutase enhances DNA damage and mutagenicity induced by cysteine/iron; Yoon SJ et al.; Oxidative DNA damage caused by a cysteine metal-catalyzed oxidation system (Cys-MCO) comprised of Fe(3+), O(2), and a cysteine as an electron donor was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner, as reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks . Unlike CuZnSOD, manganese SOD (MnSOD) as well as iron SOD (FeSOD) did not enhance DNA damage . The capacity of CuZnSOD to enhance damage to DNA was inhibited by a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and a metal chelator, diethylenetriaminepentaacetic acid (DETAPAC) . The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with Cys-MCO . We found that the Cys-MCO system caused the release of free copper from CuZnSOD . CuZnSOD also caused the two-fold enhancement of a mutation in the pUC18 lacZ' gene in the presence of Cys-MCO when measured as a loss of alpha-complementation . Based on these results, we interpret the effects of CuZnSOD on Cys-MCO-induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed by the reaction of free Cu(2+), released from oxidatively damaged CuZnSOD, and H(2)O(2) produced by the Cys-MCO system.

Mutat Res, 2000 Mar 14, 448(1), 69 - 80
Subchronic administration of phenobarbital alters the mutation spectrum of lacI in the livers of Big Blue transgenic mice; Shane BS et al.; Phenobarbital (PHE) is a liver carcinogen in B6C3F1 mice and a weak mutagen that does not appear to form DNA adducts . To investigate PHE mutagenicity in vivo, B6C3F1 Big Blue(R) male transgenic mice harboring the lambdaLIZ shuttle vector containing the lacI target gene were fed PHE at 2500 ppm for 180 days . A modest increase in the mutant frequency (MF) from 5.02+/-2.4x10(-5) in the control group to 6.88+/-0.754x10(-5) in the PHE-treated group, which was marginally different (p<0.05), was obtained . To better assess the relevance of this increase in MF, a random collection of mutants from each PHE-exposed mouse was sequenced . After correcting for clonal expansion, which is the most conservative approach, the MF in the PHE-treated mice decreased to 6.39+/-1.02x10(-5), an insignificant difference (p=0.10) from that in control group . Despite this modest increase in MF, the mutation spectrum obtained from the PHE-exposed group was significantly different (pA:T transitions remained the same in the two spectra . It is postulated that the increase in transversions at G:C base pairs found in the PHE-derived spectrum is likely due to oxidative damage as a result of induction of CYP2B isozymes by the chronic administration of PHE . Results from this study demonstrate that PHE alters the spectrum of mutations, rather than inducing a significant global increase in the MF . The PHE-derived spectrum of lacI mutants from the liver of Big Blue(R) B6C3F1 male mice was remarkably similar (p=0.8) to that generated by oxazepam (OX), a compound which also induces CYP2B isozymes following chronic administration of the drug.

Mutat Res, 2000 Feb 14, 447(2), 275 - 80
Mutation induction by mechanical irritation caused by uracil-induced urolithiasis in Big Blue rats; Takahashi S et al.; Some chronic mechanical irritations induce cancers, and it is speculated that mutations are induced by increased rate of cell proliferation caused by the irritation . In this study, it was investigated using chronic mechanical irritation to urothelium caused by urolithiasis, whether mutations are really induced by such cell proliferation or not . Male rats transgenic for lacI (Big Blue(R) rats), in which lacI mutations accumulated in tissue can be measured, were fed 3% uracil, a component of RNA, to induce urolithiasis associated with papillomatosis, and eventually with bladder cancers . The frequency of independent mutations in the bladders of the treated rats showed 3-5 fold increases at weeks 10, 20, and 51 (P=0.01 at week 51) while the frequency was not elevated at week 2 . The mutation frequencies in the control bladders ranged from 3 to 9x10(-6) . In both groups, G to A transitions at CpG sites, indicative of spontaneous mutations, constituted the most prevalent mutations . Mechanical irritation caused by uracil was shown to induce a 3-5 fold increase of mutations, possibly through an elevation of spontaneous mutations by vigorous cell proliferation.

Mutat Res, 2000 Feb 14, 447(2), 187 - 98
Microsatellite instability induced by hydrogen peroxide in Escherichia coli; Jackson AL et al.; Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms . Using a selective assay for microsatellite instability in E . coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability . Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers . We demonstrate that exposure of E . coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences . Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats . All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences . We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.

J Agric Food Chem, 1998 Jan 19, 46(1), 344 - 348
Molecular Cloning of a cDNA Encoding Copper/Zinc Superoxide Dismutase from Papaya Fruit and Overexpression in Escherichia coli; Lin MT et al.; A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn SOD) of papaya fruit, Carica papaya L . cv . Tainong 2, was amplified according to the polymerase chain reaction technique from cDNA synthesized from fruit messenger RNA . Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 152 amino acid residues . The deduced amino acid sequence showed higher identity (81.6-84.9%) with the sequences of the cytosolic Cu/Zn SODs than those of the chloroplast from other plant species, and no recognizable plastid targeting peptide was found . These suggest that the papaya fruit cDNA clone encodes a cytosolic Cu/Zn SOD . The residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, are conserved as they are among all reported Cu/Zn SOD sequences . In addition, the coding region of Cu/Zn SOD cDNA from papaya was introduced into an expression vetor, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS . A predominant protein band was detected by Coomassie blue staining of native PAGE, and activity staining confirmed the result of Coomassie blue staining . These indicate that this Cu/Zn SOD cDNA clone can overexpress active Cu/Zn SOD enzyme in E . coli.

J Biol Chem, 2001 Mar 30, 276(13), 10432 - 6 Epub 2001 Jan 10.
Evidence that phenylalanine 69 in Escherichia coli RuvC resolvase forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate; Yoshikawa M et al.; Escherichia coli RuvC resolvase is a specific endonuclease that recognizes and cleaves Holliday junctions formed during homologous recombination and recombinational repair . This study examines the phenotype of RuvC mutants with amino acid substitutions at phenylalanine 69 (F69L, F69Y, F69W, and F69A), a catalytically important residue that faces the catalytic center of the enzyme . F69Y, but not the other three mutants, almost fully complements the UV sensitivity of a DeltaruvC strain and substantially resolves synthetic Holliday junctions in vitro . In the presence of 100 mm NaCl, RuvC F69A and F69L are defective in junction binding, but F69Y and F69W retain near wild-type binding activity during a gel shift binding assay . KMnO(4) was used to probe synthetic Holliday junction DNA in a complex with wild-type and mutant RuvC; F69A and F69L did not induce disruption of base pairing at the crossover to the same extent as wild-type RuvC . Thus, the aromatic ring of Phe-69 is involved in DNA binding, probably via a stacking interaction with a nucleotide base, and this interaction may induce a structural change in junction DNA that is required to form a catalytically competent complex.

J Biol Chem, 2001 Apr 6, 276(14), 11226 - 9 Epub 2001 Jan 10.
In vitro interaction of the Escherichia coli cyclic AMP receptor protein with the lactose repressor; Fried MG et al.; Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and lactose repressor associate to form a 2:1 complex in vitro . This is, to our knowledge, the first demonstration of a direct interaction of these proteins in the absence of DNA . No 1:1 complex was detected over a wide range of CAP concentrations, suggesting that binding is highly cooperative . Complex formation is stimulated by cAMP, with a net uptake of 1 equivalent of cAMP per molecule of CAP bound . Substitution of the dimeric lacI-18 mutant repressor for tetrameric wild-type repressor completely eliminates detectable binding . We therefore propose that CAP binds the cleft between dimeric units in the repressor tetramer . CAP-lac repressor interactions may play important roles in regulatory events that take place at overlapping CAP and repressor binding sites in the lactose promoter.

J Biol Chem, 2001 Apr 6, 276(14), 11393 - 401 Epub 2001 Jan 10.
Prmt5, which forms distinct homo-oligomers, is a member of the protein-arginine methyltransferase family; Rho J et al.; We found that JBP1, known as a human homolog (Skb1Hs) of Skb1 of fission yeast, interacts with NS3 of the hepatitis C virus in a yeast two-hybrid screen . Amino acid sequence analysis revealed that Skb1Hs/JBP1 contains conserved motifs of S-adenosyl-l-methionine-dependent protein-arginine methyltransferases (PRMTs) . Here, we demonstrate that Skb1Hs/JBP1, named PRMT5, is a distinct member of the PRMT family . Recombinant PRMT5 protein purified from human cells methylated myelin basic protein, histone, and the amino terminus of fibrillarin fused to glutathione S-transferase . Myelin basic protein methylated by PRMT5 contained monomethylated and dimethylated arginine residues . Recombinant glutathione S-transferase-PRMT5 protein expressed in Escherichia coli also contained the catalytic activity . Sedimentation analysis of purified PRMT5 on a sucrose density gradient indicated that PRMT5 formed distinct homo-oligomeric complexes, including a dimer and tetramer, that comigrated with the enzyme activity . The PRMT5 homo-oligomers were dissociated into a monomer in the presence of a reducing agent, whereas a monomer, dimer, and multimer were detected in the absence or at low concentrations of a reducing agent . The results indicate that both covalent linkage by a disulfide bond and noncovalent association are involved in the formation of PRMT5 homo-oligomers . Western blot analysis of sedimentation fractions suggests that endogenous PRMT5 is present as a homo-oligomer in a 293T cell extract . PRMT5 appears to have lower specific enzyme activity than PRMT1 . Although PRMT1 is known to be mainly located in the nucleus, human PRMT5 is predominantly localized in the cytoplasm.

J Biol Chem, 2001 Mar 30, 276(13), 10032 - 8 Epub 2001 Jan 04.
Identification of a novel thioredoxin-related transmembrane protein; Matsuo Y et al.; We recently identified a series of transforming growth factor-beta-responsive genes in A549 human adenocarcinoma cell line by a gene trap screening method . Here we report the molecular cloning and characterization of one of these genes, designated TMX, that encodes a novel protein of 280 amino acid residues . The TMX protein possesses an N-terminal signal peptide followed by one thioredoxin (Trx)-like domain with a unique active site sequence, Cys-Pro-Ala-Cys, and a potential transmembrane domain . There are putative TMX homologs with identical active site sequences in the Caenorhabditis elegans and Drosophila genomes . Using recombinant proteins expressed in Escherichia coli, we demonstrated the activity of the Trx domain of TMX to cleave the interchain disulfide bridges in insulin in vitro . The TMX transcript is widely expressed in normal human tissues, and subcellular fractionation and immunostaining for an epitope-tagged TMX protein suggest that TMX is predominantly localized in the endoplasmic reticulum (ER) . When TMX was expressed in HEK293 cells, it significantly suppressed the apoptosis induced by brefeldin A, an inhibitor of ER-Golgi transport . This activity was abolished when two Cys residues in the active site sequence were mutated to Ser, suggesting that the Trx-like activity of TMX may help relieve ER stress caused by brefeldin A.

J Biol Chem, 2001 Apr 13, 276(15), 11477 - 86 Epub 2001 Jan 04.
The unique cyanobacterial protein OpcA is an allosteric effector of glucose-6-phosphate dehydrogenase in Nostoc punctiforme ATCC 29133; Hagen KD et al.; Glucose-6-phosphate dehydrogenase (G6PD), encoded by zwf, is essential for nitrogen fixation and dark heterotrophic growth of the cyanobacterium Nostoc punctiforme ATCC 29133 . In N . punctiforme, zwf is part of a four-gene operon transcribed in the order fbp-tal-zwf-opcA . Genetic analyses indicated that opcA is required for G6PD activity . To define the role of opcA, the synthesis, aggregation state, and activity of G6PD in N . punctiforme strains expressing different amounts of G6PD and/or OpcA were examined . A single tetrameric form of G6PD was consistently observed for all strains, as well as for recombinant N . punctiforme His-G6PD purified from Escherichia coli, regardless of the quantity of OpcA present . However, His-G6PD and the G6PD of strain UCD 351, which lacks OpcA, had low affinities for glucose 6-phosphate (G6P) substrate (K(m)(app) = 65 and 85 mm, respectively) relative to wild-type N . punctiforme G6PD (K(m)(app) = 0.5 mm) . Near wild-type affinities for G6P were observed for these enzymes when saturating amounts of His-OpcA- or OpcA-containing extract were added . Kinetic studies were consistent with OpcA acting as an allosteric activator of G6PD . A role in redox modulation of G6PD activity was also indicated, because thioredoxin-mediated inactivation and reactivation of His-G6PD occurred only when His-OpcA was present.

J Biol Chem, 2001 Apr 13, 276(15), 11631 - 8 Epub 2001 Jan 04.
Reaction of peroxynitrite with Mn-superoxide dismutase . Role of the metal center in decomposition kinetics and nitration; Quijano C et al.; Manganese superoxide dismutase (Mn-SOD), a critical mitochondrial antioxidant enzyme, becomes inactivated and nitrated in vitro and potentially in vivo by peroxynitrite . Since peroxynitrite readily reacts with transition metal centers, we assessed the role of the manganese ion in the reaction between peroxynitrite and Mn-SOD . Peroxynitrite reacts with human recombinant and Escherichia coli Mn-SOD with a second order rate constant of 1.0 +/- 0.2 x 10(5) and 1.4 +/- 0.2 x 10(5) m(-)1 s(-)1 at pH 7.47 and 37 degrees C, respectively . The E . coli apoenzyme, obtained by removing the manganese ion from the active site, presents a rate constant <10(4) m(-)1 s(-)1 for the reaction with peroxynitrite, whereas that of the manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of the holoenzyme . Peroxynitrite-dependent nitration of 4-hydroxyphenylacetic acid was increased 21% by Mn-SOD . The apo/Mn also promoted nitration, but the apo and the zinc-substituted apoenzyme (apo/Zn) enzymes did not . The extent of tyrosine nitration in the enzyme was also affected by the presence and nature (i.e . manganese or zinc) of the metal center in the active site . For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap) . Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 +/- 0.1 x 10(4) m(-)1 s(-)1 and maximally increases nitration yields by 350% . Zn(tbap), on the other hand, affords protection against nitration . Our results indicate that the manganese ion in Mn-SOD plays an important role in the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of self and remote tyrosine residues.

J Biol Chem, 2001 Apr 13, 276(15), 12362 - 8 Epub 2001 Jan 09.
Demonstration of conformational changes associated with activation of the maltose transport complex; Mannering DE et al.; In Escherichia coli, interaction of a periplasmic maltose-binding protein with a membrane-associated ATP-binding cassette transporter stimulates ATP hydrolysis, resulting in translocation of maltose into the cell . The maltose transporter contains two transmembrane subunits, MalF and MalG, and two copies of a nucleotide-hydrolyzing subunit, MalK . Mutant transport complexes that function in the absence of binding protein are thought to be stabilized in an ATPase-active conformation . To probe the conformation of the nucleotide-binding site and to gain an understanding of the nature of the conformational changes that lead to activation, cysteine 40 within the Walker A motif of the MalK subunit was modified by the fluorophore 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid . Fluorescence differences indicated that residues involved in nucleotide binding were less accessible to aqueous solvent in the binding protein independent transporter than in the wild-type transporter . Similar differences in fluorescence were seen when a vanadate-trapped transition state conformation was compared with the ground state in the wild-type transporter . Our results and recent crystal structures are consistent with a model in which activation of ATPase activity is associated with conformational changes that bring the two MalK subunits closer together, completing the nucleotide-binding sites and burying ATP in the interface.

J Biol Chem, 2001 Apr 13, 276(15), 12385 - 94 Epub 2001 Jan 09.
The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex; Surtees JA et al.; Stable maintenance of P1 plasmids in Escherichia coli is mediated by a high affinity nucleoprotein complex called the partition complex, which consists of ParB and the E . coli integration host factor (IHF) bound specifically to the P1 parS site . IHF strongly stimulates ParB binding to parS, and the minimal partition complex contains a single dimer of ParB . To examine the architecture of the partition complex, we have investigated the DNA binding activity of various ParB fragments . Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for the formation of the minimal, high affinity partition complex . A fragment missing only the last 16 amino acids of ParB bound specifically to parS, but binding was weak and was no longer stimulated by IHF . The ability of IHF to stimulate ParB binding to parS correlated with the ability of ParB to dimerize via its C terminus . Using full and partial parS sites, we show that two regions of ParB, one in the center and the other near the C terminus of the protein, interact with distinct sequences within parS . Based on these data, we have proposed a model of how the ParB dimer binds parS to form the minimal partition complex.

J Biol Chem, 2001 Apr 6, 276(14), 11049 - 54 Epub 2001 Jan 08.
Binding of antigenic peptide to the endoplasmic reticulum-resident protein gp96/GRP94 heat shock chaperone occurs in higher order complexes . Essential role of some aromatic amino acid residues in the peptide-binding site; Linderoth NA et al.; Vaccination with heat shock protein gp96-antigenic peptide complexes produces a powerful specific immune response against cancers and infectious diseases in some experimental animal models, and gp96-peptide complexes are now being tested in human clinical trials . gp96 appears to serve as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways . A fundamental issue that needs to be addressed is the mechanism of binding of antigenic peptide to gp96 . Here, we show using scanning transmission electron microscopy that recombinant gp96 binds peptide in stable multimeric complexes, which may have biological significance . To open the possibility for genetically engineering gp96 for improved immunogenicity and to understand if molecular recognition plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket . Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for peptide whereas conversion of Trp-654 to Tyr increased peptide binding . Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively affected peptide binding whereas changing Trp-621 to Tyr or Val positively affected peptide binding . Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking interactions) may play a role in peptide loading by gp96.

J Biol Chem, 2001 Apr 6, 276(14), 11335 - 8 Epub 2001 Jan 03.
Synchronized domain-opening motion of GroEL is essential for communication between the two rings; Shiseki K et al.; Escherichia coli chaperonin GroEL consists of two stacked rings of seven identical subunits each . Accompanying binding of ATP and GroES to one ring of GroEL, that ring undergoes a large en bloc domain movement, in which the apical domain twists upward and outward . A mutant GroEL(AEX) (C138S,C458S,C519S,D83C,K327C) in the oxidized form is locked in a closed conformation by an interdomain disulfide cross-link and cannot hydrolyze ATP (Murai, N., Makino, Y., and Yoshida, M . (1996) J . Biol . Chem . 271, 28229-28234) . By reconstitution of GroEL complex from subunits of both wild-type GroEL and oxidized GroEL(AEX), hybrid GroEL complexes containing various numbers of oxidized GroEL(AEX) subunits were prepared . ATPase activity of the hybrid GroEL containing one or two oxidized GroEL(AEX) subunits per ring was about 70% higher than that of wild-type GroEL . Based on the detailed analysis of the ATPase activity, we concluded that inter-ring negative cooperativity was lost in the hybrid GroEL, indicating that synchronized opening of the subunits in one ring is necessary for the negative cooperativity . Indeed, hybrid GroEL complex reconstituted from subunits of wild-type and GroEL mutant (D398A), which is ATPase-deficient but can undergo domain opening motion, retained the negative cooperativity of ATPase . In contrast, the ability of GroEL to assist protein folding was impaired by the presence of a single oxidized GroEL(AEX) subunit in a ring . Taken together, cooperative conformational transitions in GroEL rings ensure the functional communication between the two rings of GroEL.

J Biol Chem, 2001 Apr 6, 276(14), 11339 - 46 Epub 2001 Jan 03.
Covalently linked heme in cytochrome p4504a fatty acid hydroxylases; Hoch U et al.; Three independent experimental methods, liquid chromatography, denaturing gel electrophoresis with heme staining, and mass spectrometry, establish that the CYP4A class of enzymes has a covalently bound heme group even though the heme is not cross-linked to the protein in other P450 enzymes . Covalent binding has been demonstrated for CYP4A1, -4A2, -4A3, -4A8, and -4A11 heterologously expressed in Escherichia coli . However, the covalent link is also present in CYP4A1 isolated from rat liver and is not an artifact of heterologous expression . The extent of heme covalent binding in the proteins as isolated varies and is substoichiometric . In CYP4A3, the heme is attached to the protein via an ester link to glutamic acid residue 318, which is on the I-helix, and is predicted to be within the active site . This is the first demonstration that a class of cytochrome P450 enzymes covalently binds their prosthetic heme group.

J Biol Chem, 2001 Mar 30, 276(13), 9846 - 54 Epub 2001 Jan 03.
The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation; Komatsu M et al.; Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p conjugation system and the Apg8p membrane targeting in autophagy and cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae . Similar to the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p, an E1-like enzyme . Apg12p is then transferred to Apg10p, an E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like enzyme, followed by conjugation with phosphatidylethanolamine . Evidence is presented here that Apg7p forms a homodimer with two active-site cysteine residues via the C-terminal region . The dimerization of Apg7p is independent of the other Apg proteins and facilitated by overexpressed Apg12p . The C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is neither an ATP binding domain nor an active-site cysteine essential for its E1 activity . The deletion of its carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine . Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p . These results suggest that Apg7p forms a homodimer via the C-terminal region and that the C-terminal region is essential for both the activity of the E1 enzyme for Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p.

J Biol Chem, 2001 Apr 13, 276(15), 11499 - 506 Epub 2001 Jan 02.
Expression of the mitochondrial ADP/ATP carrier in Escherichia coli . Renaturation, reconstitution, and the effect of mutations on 10 positive residues; Heimpel S et al.; Previously, the role of residues in the ADP/ATP carrier (AAC) from Saccharomyces cerevisiae has been studied by mutagenesis, but the dependence of mitochondrial biogenesis on functional AAC impedes segregation of the mutational effects on transport and biogenesis . Unlike other mitochondrial carriers, expression of the AAC from yeast or mammalians in Escherichia coli encountered difficulties because of disparate codon usage . Here we introduce the AAC from Neurospora crassa in E . coli, where it is accumulated in inclusion bodies and establish the reconstitution conditions . AAC expressed with heat shock vector gave higher activity than with pET-3a . Transport activity was absolutely dependent on cardiolipin . The 10 single mutations of intrahelical positive residues and of the matrix repeat (+X+) motif resulted in lower activity, except of R245A . R143A had decreased sensitivity toward carboxyatractylate . The ATP-linked exchange is generally more affected than ADP exchange . This reflects a charge network that propagates positive charge defects to ATP(4-) more strongly than to ADP(3-) transport . Comparison to the homologous mutants of yeast AAC2 permits attribution of the roles of these residues more to ADP/ATP transport or to AAC import into mitochondria.

J Biol Chem, 2001 Apr 13, 276(15), 11582 - 9 Epub 2001 Jan 02.
Isolation and characterization of skin-type, type I antifreeze polypeptides from the longhorn sculpin, Myoxocephalus octodecemspinosus; Low WK et al.; The antifreeze polypeptides (AFPs) are found in several marine fish and have been grouped into four distinct biochemical classes (type I-IV) . Recently, the new subclass of skin-type, type I AFPs that are produced intracellularly as mature polypeptides have been identified in the winter flounder (Pleuronectes americanus) and the shorthorn sculpin (Myoxocephalus scorpius) . This study demonstrates the presence of skin-type AFPs in the longhorn sculpin (Myoxocephalus octodecemspinosus), which produces type IV serum AFPs . Using polymerase chain reaction-based methods, a clone that encoded for a type I AFP was identified . The clone lacked a signal sequence, indicating that the mature polypeptide is produced in the cytosol . A recombinant protein was produced in Escherichia coli and antifreeze activity was characterized . Four individual Ala-rich polypeptides with antifreeze activity were isolated from the skin tissue . One polypeptide was completely sequenced by tandem MS . This study provides the first evidence of a fish species that produces two different biochemical classes of antifreeze proteins (type I and type IV), and enforces the notion that skin-type AFPs are a widespread biological phenomenon in fish.

J Biol Chem, 2001 Mar 30, 276(13), 9868 - 76 Epub 2001 Jan 02.
An ERG channel inhibitor from the scorpion Buthus eupeus; Korolkova YV et al.; The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A . K., Kozlov, S . A., Pluzhnikov, K . A., Grishin, E . V., and Brown, D . A . (1996) FEBS Lett . 384, 277-280) . Here we report the cloning, expression, and selectivity of BeKm-1 . A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons . Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues . The mature toxin consists of 36 amino acid residues . BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins . The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli . After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin . Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized . Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm . The effect of the recombinant BeKm-1 on different K(+) channels was also studied . BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1 . Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.

J Biol Chem, 2001 Apr 27, 276(17), 13685 - 8 Epub 2000 Dec 27.
An intrahelical salt bridge within the trigger site stabilizes the GCN4 leucine zipper; Kammerer RA et al.; We previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for the formation of its parallel two-stranded coiled coil . Here, we demonstrate that the intrinsic secondary structure of the trigger site is largely stabilized by an intrahelical salt bridge . Removal of this surface salt bridge by a single amino acid mutation induced only minor changes in the backbone structure of the GCN4 leucine zipper dimer as verified by nuclear magnetic resonance . The mutation, however, substantially destabilized the dimeric structure . These findings support the proposed hierarchic folding mechanism of the GCN4 coiled coil in which local helix formation within the trigger segment precedes dimerization.

J Biol Chem, 2001 Mar 30, 276(13), 10564 - 9 Epub 2000 Dec 27.
CyaG, a novel cyanobacterial adenylyl cyclase and a possible ancestor of mammalian guanylyl cyclases; Kasahara M et al.; A novel gene encoding an adenylyl cyclase, designated cyaG, was identified in the filamentous cyanobacterium Spirulina platensis . The predicted amino acid sequence of the C-terminal region of cyaG was similar to the catalytic domains of Class III adenylyl and guanylyl cyclases . The N-terminal region next to the catalytic domain of CyaG was similar to the dimerization domain, which is highly conserved among guanylyl cyclases . As a whole, CyaG is more closely related to guanylyl cyclases than to adenylyl cyclases in its primary structure . The catalytic domain of CyaG was expressed in Escherichia coli and partially purified . CyaG showed adenylyl cyclase (but not guanylyl cyclase) activity . By site-directed mutagenesis of three amino acid residues (Lys(533), Ile(603), and Asp(605)) within the purine ring recognition site of CyaG to Glu, Arg, and Cys, respectively, CyaG was transformed to a guanylyl cyclase that produced cGMP instead of cAMP . Thus having properties of both cyclases, CyaG may therefore represent a critical position in the evolution of Class III adenylyl and guanylyl cyclases.

J Biol Chem, 2001 Mar 30, 276(13), 9936 - 44 Epub 2000 Dec 27.
Functional characterization of and cooperation between the double-stranded RNA-binding motifs of the protein kinase PKR; Tian B et al.; The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR is regulated by dsRNAs that interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus . The dsRBM is a conserved protein motif found in many proteins from most organisms . In this study, we investigated the biochemical functions and cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and the cooperation between them . We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2 . In addition, dsRBM1 has RNA-annealing activity that is not displayed by dsRBM2 . Both dsRBMs have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1) . Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and strongly inhibits the dimerization of the intact PKR N terminus (p20) containing both dsRBMs . dsRBM1, like p20, activates reporter gene expression in transfection assays, and it plays a determinative role in localizing PKR to the nucleolus and cytoplasm of the cell . Thus, dsRBM2 has weak or no activity in dsRNA binding, stimulation of gene expression, and PKR localization, but it strongly enhances these functions of dsRBM1 when contained in p20 . However, dsRBM2 does not enhance the annealing activity of dsRBM1 . This study shows that the dsRBMs of PKR possess distinct properties and that some, but not all, of the functions of the enzyme depend on cooperation between the two motifs.

J Biol Chem, 2001 Apr 20, 276(16), 12513 - 9 Epub 2000 Dec 27.
Glycophorin as a receptor for Escherichia coli alpha-hemolysin in erythrocytes; Cortajarena AL et al.; Escherichia coli alpha-hemolysin (HlyA) can lyse both red blood cells (RBC) and liposomes . However, the cells are lysed at HlyA concentrations 1-2 orders of magnitude lower than liposomes (large unilamellar vesicles) . Treatment of RBC with trypsin, but not with chymotrypsin, reduces the sensitivity of RBC toward HlyA to the level of the liposomes . Since glycophorin, one of the main proteins in the RBC surface, can be hydrolyzed by trypsin much more readily than by chymotrypsin, the possibility was tested of a specific binding of HlyA to glycophorin . With this purpose, a number of experiments were performed . (a) HlyA was preincubated with purified glycophorin, after which it was found to be inactive against both RBC and liposomes . (b) Treatment of RBC with an anti-glycophorin antibody protected the cells against HlyA lysis . (c) Immobilized HlyA was able to bind glycophorin present in a detergent lysate of RBC ghosts . (d) Incorporation of glycophorin into pure phosphatidylcholine liposomes increased notoriously the sensitivity of the vesicles toward HlyA . (e) Treatment of the glycophorin-containing liposomes with trypsin reverted the vesicles to their original low sensitivity . The above results are interpreted in terms of glycophorin acting as a receptor for HlyA in RBC . The binding constant of HlyA for glycophorin was estimated, in RBC at sublytic HlyA concentrations, to be 1.5 x 10(-9) m.

J Biol Chem, 2001 Apr 6, 276(14), 11078 - 85 Epub 2000 Dec 22.
Cytochrome p450 CYP79F1 from arabidopsis catalyzes the conversion of dihomomethionine and trihomomethionine to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates; Hansen CH et al.; Glucosinolates are natural plant products that have received rising attention due to their role in interactions between pests and crop plants and as chemical protectors against cancer . Glucosinolates are derived from amino acids and have aldoximes as intermediates . We report that cytochrome P450 CYP79F1 catalyzes aldoxime formation in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana . Using recombinant CYP79F1 functionally expressed in Escherichia coli, we show that both dihomomethionine and trihomomethionine are metabolized by CYP79F1 resulting in the formation of 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively . 5-methylthiopentanaldoxime is the precursor of the major glucosinolates in leaves of A . thaliana, i.e . 4-methylthiobutylglucosinolate and 4-methylsulfinylbutylglucosinolate, and a variety of other glucosinolates in Brassica sp . Transgenic A . thaliana with cosuppression of CYP79F1 have a reduced content of aliphatic glucosinolates and a highly increased level of dihomomethionine and trihomomethionine . The transgenic plants have a morphological phenotype showing loss of apical dominance and formation of multiple axillary shoots . Our data provide the first evidence that a cytochrome P450 catalyzes the N-hydroxylation of chain-elongated methionine homologues to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates.

J Biol Chem, 2001 Mar 30, 276(13), 9917 - 23 Epub 2000 Dec 22.
The arc two-component signal transduction system inhibits in vitro Escherichia coli chromosomal initiation; Lee YS et al.; Under anaerobic growth conditions, Escherichia coli operates a two-component signal transduction system, termed Arc, that consists of ArcB protein, a transmembrane sensor kinase and ArcA protein, the cognate response regulator . In response to low oxygen levels, autophosphorylated ArcB phosphorylates ArcA, and the resulting phosphorylated ArcA (ArcA-P) functions as a transcriptional regulator of the genes necessary to maintain anaerobic growth . Under anaerobic conditions, cells maintain a slow growth rate, suggesting that the initiation of chromosomal replication is regulated to reduce the initiation frequency . DNase I footprinting experiments revealed that ArcA-P binds to the left region of the chromosomal origin, oriC . ArcA-P did not affect the in vitro replication of plasmid DNA containing the ColE1 origin nor the in vitro replication of viral DNAs; however, ArcA-P specifically inhibited in vitro E . coli chromosomal replication . This inhibition was caused by the prevention of open complex formation, a necessary step in the initiation of chromosomal replication . Our in vitro results suggest that the Arc two-component system participates in regulating chromosomal initiation under anaerobic growth conditions.

J Histochem Cytochem, 2001 Feb, 49(2), 247 - 58
A simple assay and histochemical localization of transglutaminase activity using a derivative of green fluorescent protein as substrate; Furutani Y et al.; Histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins . GFP alone did not serve as a substrate but its derivative His(6)-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His(6)-Xpress) . His(6)-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates . The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247-258, 2001)

J Appl Microbiol, 2001 Jan, 90(1), 7 - 26
A reconsideration of the evidence for Escherichia coli STa (heat stable) enterotoxin-driven fluid secretion: a new view of STa action and a new paradigm for fluid absorption; Lucas ML; A review of the evidence for Escherichia coli STa causing fluid secretion in vito leads to the conclusion that the concept of STa acting through enhanced chloride secretion in order to derange intestinal function is unproven . However, a consistent effect of STa in the small intestine is on Na+/H+ exchange, leading to interruption of luminal acidification . A model for the action of STa, involving inhibition of Na+/H+ exchange, is proposed which explains the ability of STa to reduce absorption in vito but its inability to cause secretion in vito in contrast to its apparent secretory effect in vitro . The apparent ability to demonstrate secretion in vitro is shown to derive from methodologies which do not involve measurement of mass transport of water but instead, infer it from in vitro and in vivo proxy measurements . The in vitro demonstration of notional secretion after STa exposure can be reconciled with the proposed new model for fluid absorption in that cell swelling is argued to arise as a transient consequence of STa challenge followed by regulatory volume decrease . Evidence for this derangement model is presented in the form of observations derived from acute in vivo physiological studies and clinical studies on patients without the exchanger . This process of appraisal of the evidence for the mechanism of action of STa has led to a new model for fluid absorption . This is based on the formation of hypotonicity at the brush border luminal surface rather than hypertonicity within the lateral spaces as required by the present standing gradient model of fluid absorption . Evidence from the literature is presented for this new paradigm of water absorption, which may only be relevant for small intestine and other tissues that have Na+/H+ exchangers in contact with HCO-3-containing solutions but which may also be generalizable to all mammalian absorbing epithelial membranes.

Plant Physiol, 2001 Jan, 125(1), 189 - 98
Molecular and biochemical analysis of a Madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransferase; Laflamme P et al.; The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G . Don) . Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical . Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coli were named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and horhammericine and the 4-O-acetylation of deacetylvindoline, respectively . Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT . Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems . The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells . The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances.

Mol Cell Biol, 2001 Feb, 21(3), 713 - 20
Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair; Wang X et al.; DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion . Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner . However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways . To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells . A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids . By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination . Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient . Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue . Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

Biol Chem, 2000 Nov, 381(11), 1089 - 101
The hyper-thermostable Fe-superoxide dismutase from the Archaeon Acidianus ambivalens: characterization, recombinant expression, crystallization and effects of metal exchange; Kardinahl S et al.; An iron-containing superoxide dismutase (SOD; EC 1.15.1.1) of the hyperthermophilic archaeon Acidianus ambivalens (Aa-SOD) has been purified and characterized and the gene has been cloned and sequenced . The SOD from the facultatively aerobic member of the crenarchaeota could be expressed in E . coli . Both, the native as well as the heterologously overproduced protein turned out to have extraordinarily high melting temperatures of 128 degrees C and 124.5 degrees C, respectively . To the best of our knowledge, this is the highest directly measured melting temperature of a native protein . Surprisingly, neither the native nor the recombinant superoxide dismutase displays 100% occupation of the metal coordination sites . Obviously it is not the incorporation of a metal ion that confers the extreme thermostability . Expression of the superoxide dismutase in the presence of different metals such as Fe, Co, Ni, Mn and Cu offered the possibility of studying the hitherto unknown cofactor preference of iron-superoxide dismutase . The recombinant enzyme displayed the highest preference for incorporation of cobalt although iron is used as the natural cofactor . Spectroscopic analysis by EPR, atomic absorption and UVNis spectroscopy as well as activity measurements and differential scanning calorimetry of the metal substituted superoxide dismutases were performed . However, the superoxide dismutase of A . ambivalens is active only with iron but may incorporate other metals equally well in the catalytic center without loss of conformational stability or heat tolerance . The co-form of the enzyme could be crystallized.

Biol Chem, 2000 Nov, 381(11), 1071 - 7
Cloning, heterologous expression and kinetic analysis of glycerol kinase (TbGLK1) from Trypanosoma brucei; Steinborn K et al.; We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR . The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms . It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme . Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages . Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other . Heterologous expression of the trypanosomal glycerol kinase in E . coli enabled us to perform a kinetic analysis of this enzyme . In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions . Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.

Eur J Oral Sci, 2000 Dec, 108(6), 530 - 7
One-step sandwich enzyme immunoassay using monoclonal antibodies for detection of human enamelysin (MMP-20); Wang T et al.; A one-step sandwich enzyme immunoassay (EIA) system for human matrix metalloproteinase 20 (MMP-20, enamelysin) was established by use of a solid-phase monoclonal antibody and a separate peroxidase-labeled monoclonal antibody . The EIA system was shown to be sensitive and quantitative for the detection of MMP-20 . As little as 1.0 ng/ml (50 pg/assay) of MMP-20 protein could be reliably detected . The EIA system was linear over a range of 2.5-160 ng/ml (125-8,000 pg/assay), and the EIA system was versatile in that it was capable of detecting with equal sensitivity proMMP-20, active MMP-20, and MMP-20 with COOH-terminal deletions . The EIA system was validated by the successful detection of MMP-20 in the culture medium of Chinese hamster ovary cells (CHO-K1) that were transfected with an MMP-20 expression vector . No MMP-20 was detected in normal human serum, normal saliva, or in selected tumors . However, when recombinant human MMP-20 was added to human saliva, the EIA system did detect quantifiable amounts of the MMP-20, indicating that the system will work within the framework of complex in biological fluids.

Vitam Horm, 2001, 61, 173 - 218
Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms; Meganathan R; The benzoquinone ubiquinone (coenzyme Q) and the naphthoquinones menaquinone (vitamin K2) and demethylmenaquinone are derived from the shikimate pathway, which has been described as a "metabolic tree with many branches." Menaquinone (MK) is considered a vitamin, but coenzyme (Q) is not; MK is an essential nutrient (it cannot be synthesized by mammals), whereas Q is not considered an essential nutrient since it can be synthesized from the amino acid tyrosine . The quinone nucleus of Q is derived directly from chorismate, whereas that of MK is derived from chorismate via isochorismate . The prenyl side chain of both quinones is derived from prenyl diphosphate, and the methyl groups are derived from S-adenosylmethionine . MK biosynthesis requires 2-ketoglutarate and the cofactors ATP, coenzyme A (CoASH), and thiamine pyrophosphate . In spite of the fact that both quinones originate from the shikimate pathway, there are important differences in their biosynthesis . In MK biosynthesis, the prenyl side chain is introduced in the next to last step, which is accompanied by loss of the carboxyl group, whereas in Q biosynthesis, the prenyl side chain is introduced at the second step, with retention of the carboxyl group . In MK biosynthesis, all the reactions of the pathway up to the prenylation (next to last step) are carried out by soluble enzymes, whereas all the enzymes involved in Q biosynthesis except the first are membrane bound . In MK biosynthesis the last step is a C-methylation; in Q biosynthesis, the last step is an O-methylation . In Q biosynthesis a second C-methylation and O-methylation take place in the middle part of the pathway . In spite of the fact that Q and MK biosynthesis diverges at chorismate, the C-methylations involved in both pathways are carried out by the same enzyme . Finally, Q biosynthesis under aerobic conditions requires molecular oxygen; anaerobic biosynthesis of Q and MK incorporates oxygen atoms derived from water . The current status of the pathways with particular emphasis on the reaction mechanisms, is discussed in this review.

Vitam Horm, 2001, 61, 121 - 55
Biosynthesis of vitamin B6 and structurally related derivatives; Drewke C et al.; In spite of the rather simple structure of pyridoxal 5'-phosphate (I), a member of the vitamin B6 group, the elucidation of its de novo biosynthesis remained largely unexplored until recently . Experiments designed to investigate the formation of the vitamin B6 pyridine nucleus mainly concentrated on Escherichia coli . The results of tracer experiments with radioactive and stable isotopes, feeding experiments, and molecular biological studies led to the prediction that 4-hydroxy-L-threonine (VIII, R = H) and 1-deoxy-D-xylulose (VII, R = H) are precursors which are assembled to yield the carbon-nitrogen skeleton of vitamin B6 . At this point, the involvement of the phosphorylated forms of these precursors in this assembly seems quite clear . However, vitamin B6 biosynthesis in organisms other than E . coli remains largely unknown . Toxic derivatives of vitamin B6, such as ginkgotoxin, occurring in higher plants may be suitable targets to gain further insight into this tricky problem.

Dtsch Tierarztl Wochenschr, 2000 Nov, 107(11), 444 - 54
{Epidemiology, pathogenesis, treatment and prevention of bovine acute Escherichia coli mastitis, a literature review}; Riemann T et al.; Comparisons of the rate of incidence of clinical mastitis caused by E . coli during the last 40 years demonstrate the increasing significance of this disease for modern milk production . Because of the acute nature of this disease it was not possible until now to establish by documentary evidence a spontaneous case from the beginning on--and thereby to give certain evidence to the pathogenesis . Nevertheless a multitude of reports about experimentally caused mastitis or observations of solitary aspects of this disease exists, which allow us to assume that pathogenesis of coliform mastitis as an environmental mastitis and contagious mastitis are basically distinguishable . The manyfold factors which provoke the formation of coliform infections pave the way for methods to reduce the rate of coliform mastitis--incidence by the preventive measures and show how to optimize the results of therapy by beginning earlier and including the whole organism into the concept of therapy.

Berl Munch Tierarztl Wochenschr, 2000 Nov-Dec, 113(11-12), 444 - 6
{Influence of the timing of an early postnatal booster vaccination on some fertility parameters in sows}; Karg H et al.; In a large pig production unit, with high prevalence of E . coli caused postpartal urogenital diseases of the breeding animals and diarrhoea of the piglets, the pregnant sows received during their late pregnancy a Porcovac plus E . coli vaccination . The vaccinated sows were assigned to two experimental groups and were treated postpartal (p . p.) as follows: Group 1 (421 sows): received an E . Coli booster on the first p . p . day Group 2 (299 sows): received a booster on the 7th p . p . day The following parameters were evaluated: A: Weaning-estrus-intervals B: Farrowing Rate The results showed a significant (p < 0.05) shorter weaning-estrus-interval (Parameter A) in group 1 when compared to group 2 (7.21 +/- 1.04 vs . 9.84 +/- 1.11) . Regarding parameter B (farrowing rate) the groups showed no significant difference (group 1: 89.9% vs . group 2: 91.8%) . It is the opinion of the authors that in large pig production units with high incidence of urogenital diseases an early p . p . E . coli Booster should be performed, because of its positive effect on the weaning-estrus-intervals.

J Mol Biol, 2001 Jan 19, 305(3), 633 - 42
Analysis of gain-of-function mutants of an ATP-dependent regulator of Tn7 transposition; Stellwagen AE et al.; The bacterial transposon Tn7 is distinguished by its unusual discrimination among targets, being particularly attracted to certain target DNA and actively avoiding other DNA . Tn7 transposition is mediated by the interaction of two alternative transposon-encoded target selection proteins, TnsD and TnsE, with a common core transposition machinery composed of the transposase (TnsAB) and an ATP-dependent DNA-binding protein TnsC . No transposition is observed with wild-type TnsABC . Here, we analyze the properties of two gain-of-function TnsC mutants that allow transposition in the absence of TnsD or TnsE . We find that these TnsC mutants have altered interactions with ATP and DNA that can account for their gain-of-function phenotype . We also show that TnsC is an ATPase and that it directly interacts with the TnsAB transposase . This work provides strong support to the view that TnsC and its ATP state are central to the control of Tn7 transposition .

J Mol Biol, 2001 Jan 19, 305(3), 603 - 18
How to untwist an alpha-helix: structural principles of an alpha-helical barrel; Calladine CR et al.; Recent crystallographic studies have revealed that 12 alpha-helices can pack in an anti-parallel fashion to form a hollow cylinder of nearly uniform radius . In this architecture, which we refer to as an alpha-barrel, the helices are inclined with respect to the cylindrical axis, and thus they curve and twist . As with conventional coiled-coils, the helices of the barrel associate via "knobs-into-holes" interactions; however, their packing is distinct in several important ways . First, the alpha-barrel helices untwist in comparison with the helices found in two-stranded coiled-coils and, as a consequence of this distortion, their knobs approach closely one end of the complementary holes . This effect defines a requirement for particular size and shape of the protruding residues, and it is associated with a relative axial translation of the paired helices . Second, as each helix packs laterally with two neighbours, the helices have two sequence patterns that are phased to match the two interfaces . The two types of interface are not equivalent and, as one travels around the circumference of the cylinder's interior, they alternate between one type where the knobs approach the holes straight-on, and a second type in which they are inclined . The choice of amino acid depends on the interface type, with small hydrophobic side-chains preferred for the direct contacts and larger aliphatic side-chains for the inclined contacts . Third, small residues are found preferentially on the inside of the tube, in order to make the "wedge" angle between helices compatible with a 12-member tube . Finally, hydrogen-bonding interactions of side-chains within and between helices support the assembly . Using these salient structural features, we present a sequence template that is compatible with some underlying rules for the packing of helices in the barrel, and which may have application to the design of higher-order assemblies from peptides, such as nano-tubes . We discuss the general implications of relative axial translation in coiled-coils and, in particular, the potential role that this movement could play in allosteric mechanisms .

J Mol Biol, 2001 Jan 19, 305(3), 523 - 33
Pro-sequence assisted folding and disulfide bond formation of human nerve growth factor; Rattenholl A et al.; Nerve growth factor (NGF) is a member of the neurotrophin family . These growth factors support neuronal survival and differentiation . Neurotrophins are synthesized as pre-pro-proteins . Whereas the pre-sequences mediate secretion, the function of the pro-peptides is largely unknown . To test the role of the pro-sequence as a folding enhancer, recombinant human pro-NGF (rh-pro-NGF) was produced in Escherichia coli . The oxidative refolding of rh-pro-NGF and rh-NGF was studied using electrospray mass spectrometry (ESIMS) time-course analysis . This analysis permitted both the identification and quantification of intermediates present during the process . The disulfide bonds formed at different times of the refolding processes were characterized by proteolytic digestion followed by matrix assisted laser desorption ionization mass spectrometry (MALDIMS) analysis . Folding yields and kinetics of rh-pro-NGF were significantly enhanced when compared to the in vitro refolding of mature rh-NGF . These results suggest that the pro-sequence of NGF promotes folding of the mature part .

J Mol Biol, 2001 Jan 19, 305(3), 389 - 99
Similarities in sunlight-induced mutational spectra of CpG-methylated transgenes and the p53 gene in skin cancer point to an important role of 5-methylcytosine residues in solar UV mutagenesis; You YH et al.; In the p53 gene of human sunlight-associated skin cancers, 35 % of the mutations involve trinucleotide sequences with the rare base 5-methylcytosine (5'PymCG) . In order to determine the involvement of 5-methylcytosine in sunlight-induced mutations, we have analyzed the cII transgene in mouse cells, a mutational target gene that we found is methylated at most CpG sequences . We report that the mutational spectra produced by irradiation with 254 nm UVC radiation and simulated sunlight, respectively, differ most dramatically by the much higher involvement of dipyrimidine structures containing 5-methylcytosine in the solar UV mutation spectrum (32 % versus 9 % of all mutations) . A distinct mutational hotspot induced by simulated sunlight occurs at a sequence 5'TmCG and is associated with high levels of cis-syn cyclobutane pyrimidine dimer formation . A comparison of sunlight-induced mutational spectra of the cII and lacI transgenes, as well as the p53 gene in skin tumors, shows that 5-methylcytosine is involved in 25 to 40 % of all mutations in all three systems . The combined data make a strong case that cyclobutane pyrimidine dimers forming preferentially at dipyrimidine sequences with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells .

J Mol Biol, 2001 Jan 19, 305(3), 377 - 88
Specific interaction between anticodon nuclease and the tRNA(Lys) wobble base; Jiang Y et al.; The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease cleaves its natural substrate 5' to the wobble base, yielding 2',3'-cyclic phosphate termini . Previous work has implicated the anticodon of tRNA(Lys) as a specificity element and a cluster of amino acid residues at the carboxy-proximal half of PrrC in its recognition . We further examined these assumptions by assaying unmodified and hypomodified derivatives of tRNA(Lys) as substrates of wild-type and mutant alleles of PrrC . The data show, first, that the anticodon sequence and wobble base modifications of tRNA(Lys) play major roles in the interaction with anticodon nuclease . Secondly, a specific contact between the substrate recognition site of PrrC and the tRNA(Lys) wobble base is revealed by PrrC missense mutations that suppress the inhibitory effects of wobble base modification mutations . Thirdly, the data distinguish between the anticodon recognition mechanisms of PrrC and lysyl-tRNA synthetase .

Environ Mol Mutagen, 2000, 36(4), 312 - 24
Second human protein with homology to the Escherichia coli abasic endonuclease exonuclease III; Hadi MZ et al.; There are two major apurinic/apyrimidinic (AP) endonuclease/3'-diesterase families designated after the Escherichia coli proteins exonuclease III (ExoIII) and endonuclease IV (EndoIV) . These repair proteins function to excise mutagenic and cytotoxic AP sites or 3'-phosphate/phosphoglycolate groups from DNA . In mammals, the predominant repair endonuclease is Ape1, a homolog of ExoIII, whereas a mammalian homolog to EndoIV has not been identified to date . We have identified a human protein termed Ape2 that represents a subclass of the ExoIII family (exhibiting highest similarity to the Saccharomyces cerevisiae ETH1/APN2 gene product) and maintains many of the essential functional residues of the ExoIII-like proteins . The human protein is 518 amino acids with a predicted molecular mass of 57.3 kDa and a pI of 8.65 . Unlike Ape1, this protein exhibited only weak ability to complement the repair defects of AP endonuclease/3'-repair-defective bacteria and yeast . Similarly, a weak, but specific, DNA-binding and incision activity for abasic site-containing substrates was observed with partially purified Ape2 protein . APE2 is located on the X chromosome at position p11.21 and consists of six exons . The transcript for APE2 is ubiquitously expressed, suggesting an important function for the encoded protein . An Ape2 green fluorescent fusion protein localized predominantly to the nucleus of HeLa cells, indicating a nuclear function; this localization was dependent on the C-terminal domain . We discuss our results in the context of the evolutionary conservation of the AP endonuclease families and their divergent activities and biological contributions.

Environ Mol Mutagen, 2000, 36(4), 266 - 73
Genotoxicity of 3-methylcholanthrene in liver of transgenic big Blue mice; Rihn BH et al.; Transgenic mice provide a unique tool for studying the tissue specificity and mutagenic potential of chemicals . Because 3-methylcholanthrene (3MC) was found mutagenic in bacteria, clastogenic in bone marrow, and induces DNA adducts in animals, we were interested to determinine whether this xenobiotic provokes (1) cell proliferation, (2) transcriptional activity changes, (3) DNA adducts, and (4) hepatic mutations in transgenic Big Blue mice carrying the lambdaLIZ phage shuttle vector . Big Blue C57/Bl male mice were treated with a single intraperitoneal dose of 80 mg/kg 3MC for 1, 3, 6, 14, or 30 days . Cell proliferation was checked by 5-bromo-2-deoxyuridine labeling and immunohistochemical detection . The maximal increase of the mitotic index was evidenced after 3 days (2.9 times the control value; P < 0.01) . The relative nucleus area, reflecting the transcriptional activity, was also the highest in the treated group after 3 days: 1.86 times the control value, on average (P < 0.01) . Four major DNA adducts, determined according to the {(32)P}-postlabeling method, were evidenced in liver DNA of treated mice, 6 days after the treatment: the spot intensities increased in a time-dependent manner . The mutant frequency of liver DNA was the highest after 14 days: 20.3 +/- 2.9 x 10(-5) in the treated vs . 7.6 +/- 2.7 x 10(-5) in the control mice (P < 0.01) . Sequencing of the lambda lacI mutant plaques showed mainly G:C --> T:A and C:G --> A:T transversions . In conclusion, 3MC at first induced nuclear enlargement and a slight increase of cell proliferation in liver, followed by parallel formation of DNA adducts and mutations . This study shows how transgenic models allow in vivo evaluation of mechanistically simultaneous endpoints .

Hybridoma, 2000 Dec, 19(6), 481 - 7
Production and characterization of an estrogen receptor beta subtype-specific mouse monoclonal antibody; Su JL et al.; An important step in differentiating the unique physiological roles of the alpha and beta forms of estrogen receptor is to determine the precise expression pattern of each of these receptors . We report the generation and characterization of a murine IgG1 monoclonal antibody (MAb), ER15.64A that is ERbeta subtype-specific and capable of recognizing full-length human ERbeta as well as all of its known protein isoforms . ER15.64A, raised against a ERbeta peptide (aa2-18)-keyhole limpet hemocyanine conjugate, reacted to the immunizing peptide and the full-length E . coli expressed ERbeta in ELISA and BIAcore assays . It also immunostained nuclei of Sf9 insect cells that were infected with an ERbeta-baculovirus . In Western analysis, ER15.64A recognized ERbeta1 and ERbeta2 proteins from a reticulocyte in vitro transcription/translation preparation . This antibody did not cross-react with recombinant ERalpha in ELISA, BIAcore, immunocytochemistry, or Western blot analysis . The specificity of ER15.64A should make this antibody a useful tool for monitoring expression of ERbeta and its isoforms at the protein level and should aid in distinguishing the pattern of ERbeta receptor expression from that of ERalpha.

Hybridoma, 2000 Dec, 19(6), 435 - 44
Production and characterization of monoclonal antibodies directed to the kringle V and protease domains of human apolipoprotein(a); Seo YK et al.; Production and use of anti-apolipoprotein(a) monoclonal antibodies (MAbs) specific to single copy regions in the polymorphic lipoprotein(a) (Lp(a)) has been emphasized to be important for the standardization of measurements of the coronary heart disease risk factor, Lp(a) . Here, mouse MAbs were prepared against the kringle V (V) and protease (P) domains of human apolipoprotein(a) (apo(a)), which domains are present in single copy in the apo(a) molecule . The cDNA for apo(a)VP was cloned from human liver cDNA library, and the V-P recombinant protein overexpressed in Escherichia coli was used as an antigen for the antibody production . Two antibodies named as MAb(a)20 and MAb(a)23 were finally produced, and they were characterized for their binding specificity and epitopes . The specificity of the antibodies was confirmed by an immunoblotting procedure and an enzyme-linked immunoassay (ELISA) . It was shown that the antibodies had little, if any, cross-reactivity with human plasminogen, which is relatively abundant in human serum and is highly homologous (85%) with apo(a) in amino acid (aa) sequence . For epitope analysis, 3'-deletional series of apo(a)VP cDNA were constructed, and expression products of them were analyzed for the binding MAb(a)20 and MAb(a)23 do . It has been revealed that distinct epitopes were recognized by the two MAbs: MAb(a)23 (gamma2b, kappa) bound to the V region about 60 aa downstream from the N-terminal, and MAb(a)20 (gamma1, kappa) bound to the P region close to the C-terminal . A one step-sandwich ELISA system for Lp(a) was developed using MAb(a)20 as a capturing antibody and horseradish peroxidase (HRP)-coupled MAb(a)23 as a detecting antibody . The assay was found to be sensitive and useful for detecting Lp(a) in the range of 4-150 microg/dL (80 pM-3 nM).

Farmaco, 2000 Sep-Oct, 55(9-10), 650 - 8
Photobiological properties of 1-(3'-hydroxypropyl)-4,6,8-trimethylfur; Marzano C et al.; A new furoquinolinone derivative, 1-(3'-hydroxypropyl)-4,6,8-trimethylfuro{2,3-h}quinolin-2(1H)-one (HPFQ, 4), was prepared, in which the nitrogen atom in position 1 carries a hydroxypropyl chain . The antiproliferative activity of HPFQ was studied in comparison with its analogue 1,4,6,8-tetramethylfuro{2,3-h}quinolin-2(1H)-one (FQ) and 8-methoxypsoralen (8-MOP) . By incubation in the dark, HPFQ, although retaining antitopoisomerase II activity, appeared less effective than FQ . Upon UVA irradiation, HPFQ produced little amounts of singlet oxygen, but detectable levels of superoxide anion; like FQ, HPFQ induced numbers of DNA-protein cross-links, but no interstrand cross-links in mammalian cells . The HPFQ phototoxicity was comparable to that of FQ and 8-MOP, while mutagenic activity, scored in two Escherichia coli strains, seemed much less remarkable.

Farmaco, 2000 Sep-Oct, 55(9-10), 641 - 9
Synthesis and biological studies of some benzopyran; Abd Allah OA; 3-Chlorobenzopyrano{2,3-c}pyrazole (2) was prepared and reacted with sodium azide to give compound 3, whereas its reaction with benzoyl hydrazide, ethyl glycinate, anthranilic acid or o-phenylenediamine afforded the products 4-7, respectively . Compounds 8 and 9 were synthesized by the reaction of compound 2 with 2-mercaptobenzothiazole or piperidine . 3-Hydrazinobenzopyrano{2,3-c}pyrazole (10) was obtained and subjected to cyclization with different reagents such as CS2, benzoic acid, acetyl acetone and diethyl malonate to give compounds 11-14, respectively . Compound 10 was cyclized also with phenacyl cyanide, ylidenemalononitriles to afford the products 15-20, respectively . On the other hand, compound 10 was cyclized with 3-{bis(methylthio)methylene}pentane-2,4-dione or 1,1-dicyano 2,2-dimethylthioethylene to give the corresponding compounds 22 and 23 which in turn were reacted with some compounds containing active methylene groups to afford the corresponding compounds 24-27, respectively . The biological activities of someselected compounds were given.

Protein Sci, 2000 Nov, 9(11), 2246 - 50
Enhanced internal dynamics of a membrane transport protein during substrate translocation; Doring K et al.; Conformational changes are essential for the activity of many proteins . If, or how fast, internal fluctuations are related to slow conformational changes that mediate protein function is not understood . In this study, we measure internal fluctuations of the transport protein lactose permease in the presence and absence of substrate by tryptophan fluorescence spectroscopy . We demonstrate that nanosecond fluctuations of alpha-helices are enhanced when the enzyme transports substrate . This correlates with previously published kinetic data from transport measurements showing that millisecond conformational transitions of the substrate-loaded carrier are faster than those in the absence of substrate . These findings corroborate the hypothesis of the hierarchical model of protein dynamics that predicts that slow conformational transitions are based on fast, thermally activated internal motions.

Protein Sci, 2000 Nov, 9(11), 2232 - 45
Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease; Ullrich B et al.; The Q7K/L331/L631 HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics . The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer . Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with k(q1) = 6.85 x 10(9) M(-1) s(-1) and k(q2) = 1.88 x 10(9) M(-1) s(-1), that merge into one in the complex with k(q) = 1.78 x 10(9) M(-1) s(-1) . 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence . In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms . In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur . A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59 . This could account for the short lifetime component . The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone . It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor . The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.

Protein Sci, 2000 Nov, 9(11), 2200 - 9
Three-dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights into the activation process; Bessis AS et al.; Metabotropic glutamate receptors (mGluRs) belong to the family 3 of G-protein-coupled receptors . On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7-transmembrane domains required for G-protein activation . To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4 mGluR (mGlu4R) . The fold recognition search allowed the identification of three hits with a low sequence identity, but with high secondary structure conservation: leucine isoleucine valine-binding protein (LIVBP) and leucine-binding protein (LBP) as already known, and acetamide-binding protein (AmiC) . These proteins are characterized by a bilobate structure in an open state for LIVBP/LBP and a closed state for AmiC, with ligand binding in the cleft . Models for both open and closed forms of mGlu4R ATD have been generated . ACPT-I (1-aminocyclopentane 1,3,4-tricarboxylic acid), a selective agonist, has been docked in the two models . In the open form, ACPT-I is only bound to lobe I through interactions with Lys74, Arg78, Ser159, and Thr182 . In the closed form, ACPT-I is trapped between both lobes with additional binding to Tyr230, Asp312, Ser313, and Lys317 from lobe II . These results support the hypothesis that mGluR agonists bind a closed form of the ATDs, suggesting that such a conformation of the binding domain corresponds to the active conformation.

Protein Sci, 2000 Nov, 9(11), 2161 - 9
Solution structure of DinI provides insight into its mode of RecA inactivation; Ramirez BE et al.; The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response . The 81-residue E . coli protein DinI inhibits activity of RecA in vivo . The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints . DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices . The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat . The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge . We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding . Biochemical data confirm that DinI is able to displace ssDNA from RecA.

Protein Sci, 2000 Nov, 9(11), 2109 - 17
Reversible denaturation of oligomeric human chaperonin 10: denatured state depends on chemical denaturant; Guidry JJ et al.; Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides . Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure . We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer . Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far-ultraviolet circular dichroism, and cross-linking; all denaturation reactions were reversible . GuHCl-induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition . In contrast, urea-induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation . There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant . The different cpn10 denatured states observed in high {GuHCl} and high {urea} were supported by cross-linking experiments . Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy-increase is significantly larger for the heptamer . A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol) . The thermodynamics for co-chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.

Protein Sci, 2000 Nov, 9(11), 2059 - 67
Solution structure of hpTX2, a toxin from Heteropoda venatoria spider that blocks Kv4.2 potassium channel; Bernard C et al.; HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind on Kv4.2 potassium channel . We have determined the solution structure of recombinant HpTX2 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics . The calculated structure belongs to the Inhibitory Cystin Knot structural family that consists in a compact disulfide-bonded core, from which four loops emerge . A poorly defined two-stranded antiparallel beta-sheet (residues 20-23 and 25-28) is detected . Analysis of the electrostatic charge anisotropy allows us to propose a functional map of HpTX2 different from the one described for kappa-conotoxin PVIIA, but strongly related to the one of charybdotoxin . The orientation of the dipole moment of HpTX2 emerges through K27 which could therefore be the critical lysine residue . Close to this lysine are a second basic residue, R23, an aromatic cluster (F7, W25, W30) and an hydrophobic side chain (L24) . The high density in aromatic side chains of the putative functional surface as well as the lack of an asparagine is proposed to be the structural basis of the specificity of HpTX2 toward Kv4.2 channel.

Acta Anaesthesiol Scand, 2001 Jan, 45(1), 112 - 8
Aminoguanidine does not influence tissue extravasation of albumin in endotoxaemic rats; Metcalf K et al.; BACKGROUND: It is generally maintained that protein and fluid are lost from the circulation under septic conditions . The role played by an increased production of nitric oxide, by the inducible nitric oxide synthase (iNOS), in this process is unclear . METHODS: Chloralose anaesthetised male Wistar rats received E . coli lipopolysaccharide (LPS), 3 mg kg(-1) i.v., and were studied for 5 h . Mean arterial pressure (MAP) and heart rate (HR) were monitored and haematocrit (Hct) was determined intermittently . Tissue plasma volume and tissue clearances of radiolabelled albumin over the last 2 h of the experiment were determined by a double-isotope method . In 8 rats, 2 h after LPS, aminoguanidine, an iNOS selective blocker, was given i.v . at a dose of 5 mg kg(-1) . This was followed by a continuous infusion for the duration of the experiment; altogether 20 mg kg(-1) was administered . In the control group (n=8), a corresponding volume of saline was infused . RESULTS: Aminoguanidine did not significantly influence Hct, MAP and HR, as evidenced by inter-group comparisons (Mann-Whitney test) . Tissue plasma clearances of albumin and tissue plasma volume were similar in both groups . CONCLUSION: Aminoguanidine at 20 mg kg(-1) did not reverse the haemodynamic changes induced by LPS . Neither did the drug affect the tissue plasma clearance of albumin or the tissue plasma volume.

J Math Biol, 2000 Nov, 41(5), 455 - 75
Fractional step methods applied to a chemotaxis model; Tyson R et al.; A fractional step numerical method is developed for the nonlinear partial differential equations arising in chemotaxis models, which include density-dependent diffusion terms for chemotaxis, as well as reaction and Fickian diffusion terms . We take the novel approach of viewing the chemotaxis term as an advection term which is possible in the context of fractional steps . This method is applied to pattern formation problems in bacterial growth and shown to give good results . High-resolution methods capable of capturing steep gradients (from CLAWPACK) are used for the advection step, while the A-stable and L-stable TR-BDF2 method is used for the diffusion step . A numerical instability that is seen with other diffusion methods is analyzed and eliminated.

FEMS Microbiol Lett, 2001 Jan 1, 194(1), 87 - 92
A Mycobacterium smegmatis gyrase B specific monoclonal antibody reveals association of gyrase A and B subunits in the cell; Manjunatha UH et al.; DNA gyrase is a unique topoisomerase, which plays important roles in macromolecular events like DNA replication, transcription and genetic recombination . In this study a high affinity monoclonal antibody to the gyrase B (GyrB) subunit of Mycobacterium smegmatis was characterized, which did not cross-react with either the Escherichia coli GyrB subunit or with GyrB subunits from other mycobacterial species . The antibody recognized an epitope in the N-terminus, novobiocin-binding domain of GyrB . Immunoprecipitation of gyrase from M . smegmatis cell lysate revealed an association, mediated by ionic interactions, of gyrase A and GyrB subunits in the cell . This antibody is a valuable tool for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase.

J Immunol Methods, 2001 Jan 1, 247(1-2), 141 - 51
A new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody; Matsuo R et al.; Signal transduction from cell surface receptors to the nucleus is regulated in most part by protein phosphorylation . For the purpose of identification of kinases which play an important role at a particular phosphorylation step in a series of signal transduction pathways, we have developed a new expression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide . We have applied this method for screening kinases which phosphorylate STAT3 at serine(727) . In this screening, antibody (PS727 antibody) specifically recognizing STAT3 in which serine(727) is phosphorylated was first prepared . Escherichia coli, bacteria expressing a serine(727)-containing fragment of STAT3 which was fused to glutathione-S-transferase (GST) (GST-STAT3-WT) were infected by lambda phage cDNA expression libraries . Phosphorylation of GST-STAT3-WT was effectively performed in E . coli as expected, and clones positive for PS727 antibody immunoreactivity were selected . Isolated 53 clones encode four serine/threonine kinases; extracellular signal regulated kinase 1 (ERK1/p44-MAPK), dual specificity Yak1 related kinase (DYRK), dual specificity Yak1 related kinase 2 (DYRK2) and homeodomain interacting protein kinase 2 (HIPK2) . These kinases have a potential to phosphorylate serine(727) in STAT3 protein also in mammalian cells . The present method is considered to be applicable in general to isolate kinases.

J Immunol Methods, 2001 Jan 1, 247(1-2), 83 - 94
Whole blood cultures to assess the immunostimulatory activities of CpG oligodeoxynucleotides; Pichyangkul S et al.; Specially designed oligodeoxynucleotide (ODN) sequences known as 'CpG' ODNs elicit innate and acquired immune responses . In general, screening of new CpG ODNs has been conducted by conventional lymphoproliferative assays or expression of activation markers in peripheral blood mononuclear cell (PBMC) cultures . Here, we compared conventional in vitro human PBMC assays with whole blood assays for screening the immunostimulatory properties of CpG ODNs . Commercially available DNA preparations and mycobacterial-based adjuvants were used as comparators . Activation was assessed by flow cytometry and cytokine production . CpG ODNs, identified by four-letter codes, consisted of 2006 (strong human cell stimulant), 1826 (strong murine cell stimulant), 1840 (weak immunostimulant), and 2041, a non-CpG ODN . In both test systems, and in accordance with previous reports, 2006 was an effective up-regulator of CD40 on human dendritic cells (DC1, DC2), monocytes, and B cells, and of CD69 on NK cells . In contrast to murine cells exposed to CpG ODNs, IL-12 (p40) and IFN-gamma production in human immune cells was negligible, but greatly enhanced by adding GM-CSF . Like 2006, two comparator mycobacterial adjuvant formulations activated DC1, DC2, monocytes and natural killer (NK) cells, but only 2006 had a strong effect on B cells . The usefulness of the whole blood assay was further demonstrated by studies in small volumes of umbilical cord mononuclear cells, that like adult blood cells, showed up-regulation of CD40 expression on B cells, DC, and monocytes, and CD69 on NK cells . The whole blood assay, in conjunction with flow cytometry, is useful for assessing the immunological properties of CpG ODN sequences.

FEBS Lett, 2000 Dec 29, 487(2), 272 - 6
The gene encoding the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of synechocystis PCC6803; Pieulle L et al.; The physiological function of the type 1 NAD(P)H dehydrogenase (Ndh-1) of Synechocystis sp . PCC6803 has been investigated by inactivating the gene ndhH encoding a subunit of the complex . Molecular analysis of independent transformants revealed that all clones were heteroploid, containing both wild-type and mutant ndhH copies, whatever the metabolic conditions used during genome segregation, including high CO(2) concentration . By replacing the chromosomal copy of the ndhH gene by a plasmidial copy under the control of a temperature-controlled promoter, we induce a conditional phenotype, growth being only possible at high temperature . This clearly shows for the first time that an ndh gene is indispensable to the survival of Synechocystis sp . PCC6803.

J Allergy Clin Immunol, 2001 Jan, 107(1), 31 - 5
CD14-dependent airway neutrophil response to inhaled LPS: role of atopy; Alexis N et al.; BACKGROUND: Inhaled endotoxin (LPS) is associated with airway neutrophilic (PMN) inflammation in both asthmatic and control subjects, with asthmatic subjects demonstrating possibly higher sensitivity . CD14 is the principal receptor mediating LPS responses in vivo . It is unknown whether constitutive CD14 can predict the magnitude of the PMN response after LPS inhalation and whether atopy plays a role in this response . OBJECTIVE: We sought to examine associations between constitutive airway CD14 expression and LPS-induced PMNs after 5 microg of LPS inhalation and to examine associations between markers of atopy (eosinophils and eosinophil cationic protein) and CD14 expression and LPS-induced PMNs . METHODS: Ten atopic asthmatic subjects and 8 healthy control subjects inhaled 0.9% saline and LPS (Escherichia coli 026:B6, 5 microg) separated by 3 weeks . Induced sputum was collected at 24 hours before and 6 hours after inhalation . Induced sputum was analyzed for total and differential cell counts and soluble markers (soluble {s}CD14, eosinophil cationic protein, IL8, and total protein) . Flow cytometry was used to analyze membrane-bound CD14 expression . RESULTS: Significant associations were found between the LPS-induced PMN response (PMNs per milligram of sputum) and both constitutive sCD14 (R = 0.7, P =.005) and membrane-bound CD14 (R = 0.9, P =.01) . Asthmatic subjects demonstrated significantly higher levels of constitutive sCD14 compared with control subjects, and baseline eosinophils were significantly associated with baseline sCD14 (R = 0.7, P =.01) and LPS-induced PMNs (R = 0.6, P =.03) . CONCLUSION: Constitutive airway CD14 expression can predict the magnitude of the PMN response after inhaled LPS . Atopy appears to play a role in the level of CD14 expression and may contribute to LPS sensitivity in asthmatic subjects.

Rheumatol Int, 2000 Dec, 20(1), 13 - 9
Synovial inflammation and hyperplasia induced by gliostatin/platelet-derived endothelial cell growth factor in rabbit knees; Waguri-Nagaya Y et al.; Neovascularization, proliferation of synovial cells, and mononuclear cell influx and activation are characteristic events observed in synovial joints in the pathohistology of rheumatoid arthritis (RA) . The objective of this study was to examine synovial inflammation in rabbit knees induced by intra-articular administration of human gliostatin/platelet-derived endothelial cell growth factor (GLS/PD-ECGF), which shares a high degree of chemical homology with thymidine phosphorylase (dThdPase) and is known to have angiogenic activity . Purified recombinant human gliostatin (rHuGLS) and its mutant protein, which was prepared by site-directed mutagenesis and which lacks dThdPase activity, were administered at various doses to rabbit knee joints . The effects of rHuGLS and the mutant were examined histologically . Intra-articular injection of rHuGLS resulted in the development of diffuse synovitis resembling RA . The mutant protein also brought about the same effect . These findings suggest that human GLS can cause RA-like synovitis in rabbit knee joints via a mechanism other than its dThdPase activity.

J Biomol Struct Dyn, 2000 Dec, 18(3), 325 - 34
RNA polymerase--promoter recognition . Specific features of electrostatic potential of "early" T4 phage DNA promoters; Kamzolova SG et al.; Comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken, along with calculation of topography of electrostatic potential around the native and ADP-ribosylated C-terminal domain of RNA polymerase alpha-subunit . The data obtained indicate that there is specific difference in the patterns of electrostatic potential distribution in far upstream regions of T4 promoters differing by their response to ADP-ribosylation of RNA polymerase . A specific change in profiles of electrostatic potential distribution for the native and ADP-ribosylated forms of RNA polymerase alpha-subunit was observed suggesting that this factor may be responsible for modulating T4 promoter activities in response to the enzyme modification.

Anesthesiology, 2000 Dec, 93(6), 1465 - 73
Impairment of cardiac beta-adrenoceptor cellular signaling by decreased expression of G(s alpha) in septic rabbits; Matsuda N et al.; BACKGROUND: Abnormalities in the beta-adrenergic control of cardiac function play a role in the pathogenesis of several disease states . Because circulatory failure in patients with septic shock is known to be less responsive to catecholamines, we investigated whether the beta-adrenoceptor-linked signal transduction mechanisms are altered in the heart of a septic animal model METHODS: Rabbits were rendered endotoxemic by an intravenous injection of 100 microg/kg Escherichia coli lipopolysaccharide . Three and 6 h later, the myocardial tissues were used for the experiments . RESULTS: The positive inotropic response to isoproterenol was significantly impaired in papillary muscles isolated from septic rabbits compared with those from controls . The impaired inotropic responsiveness to isoproterenol was not prevented by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine, indicating no involvement of nitric oxide overproduction . Adenylate cyclase activity stimulated with isoproterenol and 5'-guanylyl imidodiphosphate was markedly reduced in septic myocardium . The contractile and adenylate cyclase responses to colforsin daropate, a direct adenylate cyclase activator, were unaffected by sepsis . Radioligand binding experiments with (-){125I}iodocyanopindolol revealed no significant alteration in myocardial beta-adrenoceptor density or affinity in sepsis . Determination of cardiac G(s alpha) level by Western blotting showed a reduction of approximately 50% in sepsis . The relative content of G(s alpha) messenger RNA in septic myocardium also was reduced from the control level by about 50%, as determined by Northern blot analysis . Little change was found in protein and messenger RNA levels of G(s alpha) in septic myocardium . CONCLUSIONS: Impairment of myocardial functional responsiveness to beta-adrenoceptor stimulation appears in the early stage of sepsis . The impaired response to beta-adrenoceptor stimulation in the heart in this pathologic state may result in part from a decreased level of G(s alpha) protein which occurs at the level of gene expression.

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