Escherichia coli

Bioinformatics, 2001, 17 Suppl 1, S296 - 305
Protein-protein interaction map inference using interacting domain profile pairs; Wojcik J et al.; A number of predictive methods have been designed to predict protein interaction from sequence or expression data . On the experimental front, however, high-throughput proteomics technologies are starting to yield large volumes of protein-protein interaction data . High-quality experimental protein interaction maps constitute the natural dataset upon which to build interaction predictions . Thus the motivation to develop the first interaction-based protein interaction map prediction algorithm . A technique to predict protein-protein interaction maps across organisms is introduced, the 'interaction-domain pair profile' method . The method uses a high-quality protein interaction map with interaction domain information as input to predict an interaction map in another organism . It combines sequence similarity searches with clustering based on interaction patterns and interaction domain information . We apply this approach to the prediction of an interaction map of Escherichia coli from the recently published interaction map of the human gastric pathogen Helicobacter pylori . Results are compared with predictions of a second inference method based only on full-length protein sequence similarity - the "naive" method . The domain-based method is shown to i) eliminate a significant amount of false-positives of the naive method that are the consequences of multi-domain proteins; ii) increase the sensitivity compared to the naive method by identifying new potential interactions . AVAILABILITY: Contact the authors.

Pediatr Int, 2001 Aug, 43(4), 343 - 9
Nitric oxide inhalation and nitric oxide synthase inhibitor supplement for endotoxin-induced hypotension; Suzuki S et al.; BACKGROUND: This study was performed to determine whether a combined therapy of nitric oxide (NO) inhalation and nitric oxide synthase (NOS) inhibitor is effective in experimental animals with endotoxin-induced refractive hypotension accompanied by pulmonary hypertension . METHODS: Escherichia coli lipopolysaccharide (1 mg/kg) was administered to 10 newborn piglets to induce endotoxemia . The experiment then began 60 min later, when the systemic arterial pressure dropped . The inhalation of 20 p.p.m . NO at 60 and 120 min of endotoxemia created a control group . Another group was also administered N w-nitro-L-arginine (L-NNA; 5 mg) after the first NO inhalation at 60 min of endotoxemia (the L-NNA group) . Pulmonary arterial pressure, systemic arterial pressure and cardiac output were measured and compared among the groups . RESULTS: Three of the 5 piglets in the control group died of hypotensive shock, while in the L-NNA group the systemic arterial pressure recovered to pre-endotoxin administration levels . The L-NNA group produced a further increase in pulmonary arterial pressure against which NO inhalation was effective . CONCLUSION: Nitric oxide inhalation alone carries a potential risk of further lowering systemic arterial pressure in a piglet with hypotension induced by endotoxin, whereas the combined therapy resulted in the recovery of the blood pressure to pre-endotoxin levels . The combined therapy was simultaneously effective against pulmonary hypertension.

Clin Exp Immunol, 2001 Jun, 124(3), 423 - 8
Escherichia coli-induced expression of IL-1 alpha, IL-1 beta, IL-6 and IL-8 in normal human renal tubular epithelial cells; Brauner A et al.; The aim of the present study was to investigate whether the IL-1 family cytokines, in addition to IL-6 and IL-8, could be induced in normal human cortical epithelial cells in response to bacterial stimuli . Human renal tissue was obtained from 9 patients undergoing elective tumour nephrectomy . Renal cortical epithelial cells of tubular origin were prepared from the unaffected tissue . The proximal tubular cells were stimulated for 2, 6 and 24 h with a heat-inactivated pyelonephritogenic Escherichia coli strain DS-17 . Cultured unstimulated tubular cells served as controls . IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, IL-6, IL-8, IL-10, TNF-alpha, G-CSF and GM-CSF were analysed using immunohistochemistry at the single cell level . The nonstimulated cells were found to express low levels of IL-6 and IL-8 (mean value < 3% of total cells) . In contrast, E . coli exposure resulted in significantly increased incidences of IL-6 and IL-8 expressing cells (mean values approximately 18% of total cells) peaking within two hours of stimulation (P < 0.008 and P < 0.02 versus non-stimulated cells, respectively) . A gradual decrease was thereafter observed at 6 and 24 h, respectively, although persistently higher compared to controls . A different kinetic response was found for IL-1 alpha, IL-1 beta and IL-1 receptor antagonist-expressing cells, which peaked 24 h after E . coli stimulation (mean values 3--10%) (P < 0.008, P < 0.02, P < 0.02 versus non-stimulated cells, respectively) . Low levels of TNF-alpha and GM-CSF were found in 3 of the 9 donated epithelial cells, peaking at 2 h, and IL-10 and G-CSF producing cells in 1 patient each . In conclusion we found that heat-inactivated pyelonephritic E . coli induced a proinflammatory cytokine response in the normal human proximal tubular cells including the IL-1 family, IL-6 and IL-8.

J Med Chem, 2001 Aug 2, 44(16), 2667 - 70
Reduced amide bond peptidomimetics . (4S)-N-(4-amino-5-{aminoakyl}aminopentyl)-N'-nitroguanidines, potent and highly selective inhibitors of neuronal nitric oxide synthase; Hah JM et al.; Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically useful in the treatment of certain disease states arising from the overproduction of nitric oxide . Recently, we reported nitroarginine-containing dipeptide amides (Huang, H; Martasek, P.; Roman, L . J.; Masters, B . S . S.; Silverman, R . B . J . Med . Chem . 1999, 42, 3147.) and some peptidomimetic analogues (Huang, H; Martasek, P.; Roman, L . J.; Silverman, R.B . J . Med Chem . 2000, 43, 2938.) as potent and selective inhibitors of neuronal NOS (nNOS) . Here, reduced amide bond pseudodipeptide analogues are synthesized and evaluated for their activity . The deletion of the carbonyl group from the amide bond either preserves or improves the potency for nNOS . Significantly, the selectivities for nNOS over eNOS (endothelial NOS), and iNOS (inducible NOS) are greatly increased in these series . The most potent nNOS inhibitor among these compounds is (4S)-N-(4-amino-5-{aminoethyl}aminopentyl)-N'-nitroguanidine (7) (K(i) = 120 nM), which also shows the highest selectivity over eNOS (greater than 2500-fold) and 320-fold selectivity over iNOS . The reduced amide bond is an excellent surrogate of the amide bond, and it will facilitate the design of new potent and selective inhibitors of nNOS.

J Am Chem Soc, 2001 Apr 18, 123(15), 3569 - 76
High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy: the tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast; Bar G et al.; High-frequency pulsed EPR and ENDOR have been employed to characterize the tyrosyl radical (Y*)-diiron cofactor in the Y2-containing R2 subunit of ribonucleotide reductase (RNR) from yeast . The present work represents the first use of 140-GHz time domain EPR and ENDOR to examine this system and demonstrates the capabilities of the method to elucidate the electronic structure and the chemical environment of protein radicals . Low-temperature spin-echo-detected EPR spectra of yeast Y* reveal an EPR line shape typical of a tyrosyl radical; however, when compared with the EPR spectra of Y* from E . coli RNR, a substantial upfield shift of the g(1)-value is observed . The origin of the shift in g(1) was investigated by 140-GHz (1)H and (2)H pulsed ENDOR experiments of the Y2-containing subunit in protonated and D(2)O-exchanged buffer . (2)H ENDOR spectra and simulations provide unambiguous evidence for one strongly coupled (2)H arising from a bond between the radical and an exchangeable proton of an adjacent residue or a water molecule . Orientation-selective 140-GHz ENDOR spectra indicate the direction of the hydrogen bond with respect to the molecular symmetry axes and the bond length (1.81 A) . Finally, we have performed saturation recovery experiments and observed enhanced spin lattice relaxation rates of the Y* above 10 K . At temperatures higher than 20 K, the relaxation rates are isotropic across the EPR line, a phenomenon that we attribute to isotropic exchange interaction between Y* and the first excited paramagnetic state of the diiron cluster adjacent to it . From the activation energy of the rates, we determine the exchange interaction between the two irons of the cluster, J(exc) = -85 cm(-)(1) . The relaxation mechanism and the presence of the hydrogen bond are discussed in terms of the differences in the structure of the Y*-diiron cofactor in yeast Y2 and other class I R2s.

J Am Chem Soc, 2001 Apr 18, 123(15), 3418 - 28
Energetically most likely substrate and active-site protonation sites and pathways in the catalytic mechanism of dihydrofolate reductase; Cummins PL et al.; Despite much experimental and computational study, key aspects of the mechanism of reduction of dihydrofolate (DHF) by dihydrofolate reductase (DHFR) remain unresolved, while the secondary DHFR-catalyzed reduction of folate has been little studied . Major differences between proposed DHF mechanisms are whether the carboxylate group of the conserved active-site Asp or Glu residue is protonated or ionized during the reaction, and whether there is direct protonation of N5 or a proton shuttle from an initially protonated carboxylate group via O4 . We have addressed these questions for both reduction steps with a comprehensive set of ab initio quantum chemical calculations on active-site fragment complexes, including the carboxyl side chain and, progressively, all other polar active-site residue groups including conserved water molecules . Addition of two protons in two steps was considered . The polarization effects of the remainder of the enzyme system were approximated by a dielectric continuum self-consistent reaction field (SCRF) model using an effective dielectric constant (epsilon) of 2 . Optimized geometries were calculated using the density functional (B3LYP) method and Onsager SCRF model with the 6-31G basis . Single-point energy calculations were then carried out at the B3LYP/6-311+G level with either the Onsager or dielectric polarizable continuum model . Additional checking calculations at MP2 and HF levels, or with other basis sets or values of epsilon, were also done . From the results, the conserved water molecule, corresponding to W206 in the E . coli DHFR complexes, that is H-bonded to both the OD2 oxygen atom of the carboxyl (Asp) side chain and O4 of the pterin/dihydropterin ring, appears critically important and may determine the protonation site for the enzyme-bound substrates . In the absence of W206, the most stable monoprotonated species are the neutral-pair 4-enol forms of substrates with the carboxyl group OD2 oxygen protonated and H-bonded to N3 . If W206 is included, then the most stable forms are still the neutral-pair complexes but now for the N3-H keto forms with the protonated OD2 atom H-bonding with W206 . A second proton addition to these complexes gives protonations at N8 (folate) or N5 (DHF) . Calculated H-bond distances correlate well with those for the conserved W206 observed in many X-ray structures . For all structures with occluded M20 loop conformations (closed active site), OD2-N3 distances are less than OD2-NA2 distances, which is consistent with those calculated for protonated OD2 complexes . Thus, the results (B3LYP; epsilon = 2 calculations) support a mechanism for both folate and DHF reduction in which the OD2 carboxyl oxygen is first protonated, followed by a direct protonation at N8 (folate) and N5 (DHF) to obtain the active cation complexes, i.e., doubly protonated . The results do not support a proposed protonated carboxyl with DHF in the enol form for the Michaelis complex, nor an ionized carboxyl with protonated enol-DHF as a catalytic intermediate . However, as additional calculations for the monoprotonated complete complexes show a reduction in the energy differences between the neutral-pair keto and ion-pair keto (N8- or N5-protonated) forms, we are extending the treatment using combined quantum mechanics and molecular mechanics (QM/MM) and molecular dynamics simulation methods to refine the description of the protein/solvent environment and prediction of the relative stabilization free energies of the various (OD2, O4, N5, and N8) protonation sites.

J Struct Biol, 2001 Feb-Mar, 133(2-3), 203 - 13
Classification and reconstruction of a heterogeneous set of electron microscopic images: a case study of GroEL-substrate complexes; Falke S et al.; Image analysis methods were used to separate images of a large macromolecular complex, the chaperonin GroEL, in a preparation in which it is partially liganded to a nonnative protein substrate, glutamine synthetase . The relatively small difference ( approximately 6%) in size between the chaperonin in its free and complexed forms, and the absence of gross changes in overall conformation, made separation of the two types of particles challenging . Different approaches were evaluated and used for alignment and classification of images, both in two common projections and in three dimensions, yielding 2D averages and a 3D reconstruction . The results of 3D analysis describe the conformational changes effected by binding of this particular protein substrate and demonstrate the utility of 2D analysis as an indicator of structural change in this system .

Microb Comp Genomics, 2000, 5(4), 205 - 22
MultiFun, a multifunctional classification scheme for Escherichia coli K-12 gene products; Serres MH et al.; An enriched classification system for cellular functions of gene products of Escherichia coli K-12 was developed based on the initial classification by Riley . In the new classification scheme, MultiFun, cellular functions are divided into 10 major categories: Metabolism, Information Transfer, Regulation, Transport, Cell Processes, Cell Structure, Location, Extra-chromosomal Origin, DNA Site, and Cryptic Gene . These major categories are further sub-divided into a hierarchical scheme . Two thousand nine hundred twenty-two gene products of E . coli K-12 were assigned to one or more functions depending on the role they play in the cell . Functional assignments were made to 66% of E . coli gene products, ranging from 1 to 16 assignments per gene product . The expansion of cellular function categories and the assignment to more than one category (multifunction) provides a more complete description of the gene products and their roles and hence better reflects the functional complexity of organisms . We believe this classification system will be useful in the field of genome analysis, both for annotation purposes and for comparative studies . The functional classification scheme and the cellular function assignments made to E . coli gene products can be accessed from the web at the databases GenProtEC and EcoCyc .

Microbiol Immunol, 2001, 45(5), 349 - 55
Region of heat-stable enterotoxin II of Escherichia coli involved in translocation across the outer membrane; Okamoto K et al.; Heat-stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre- and mature regions . The pre-region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm . The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys-10 and Cys-48 and between Cys-21 and Cys-36 . STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC . However, it remains unknown which regions of STII are involved in interaction with TolC . In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant . A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant . On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low . These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu . The experiment using a dsbA-deficient strain of E . coli showed that the disulfide bond between Cys-21 and Cys-36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.

Biosci Biotechnol Biochem, 2001 Jun, 65(6), 1310 - 4
Characterization of recombinant yeast exo-beta-1,3-glucanase (Exg 1p) expressed in Escherichia coli cells; Suzuki K et al.; Yeast exo-beta-1,3-glucanase gene (EXG1) was expressed in Escherichia coli and the recombinant enzyme (Exg1p) was characterized . The recombinant Exglp had an apparent molecular mass of 45 kDa by SDS-PAGE and the enzyme has a broad specificity for beta-1,3-linkages as well as beta-1,6-linkages, and also for other beta-glucosidic linked substrates, such as cellobiose and pNPG . Kinetic analyses indicate that the enzyme prefers small substrates such as laminaribiose, gentiobiose, and pNPG rather than polysaccharide substrates, such as laminaran or pustulan . With a high concentration of laminaribiose, the enzyme catalyzed transglucosidation forming laminarioligosaccharides . The enzyme was strongly inhibited with high concentrations of laminaran.

Curr Issues Mol Biol, 2000 Jul, 2(3), 71 - 85
Introductory experiments in recombinant DNA; Tait RC; Nine practical exercises demonstrate the basic principles in recombinant DNA . The exercises explain the principles that DNA equals genes and that changes in DNA cause changes in genetic properties . The aim is to provide a teaching resource that can be used to illustrate the theory and applications of molecular biology to highschool students, undergraduate students, medics, dentists, doctors, nurses, life scientists, and anyone learning the basics of DNA technology.

Mol Biotechnol, 2001 Jun, 18(2), 155 - 67
Evaluating phenotype and genotype of drug-resistant strains in herpesviruses; Andrei G et al.; The isolation of drug-resistant strains of herpesviruses, including Herpes Simplex Virus type I (HSV-1) and type 2 (HSV-2), Varicella-Zoster Virus (VZV), and cytomegalovirus (CMV), has been reported with increasing frequency in immunocompromised patients and is a matter of major concern . Determination of antiviral drug susceptibilities is a prerequisite for the management of drug-resistant herpesvirus infections . Phenotyping studies should be correlated with genotyping, i.e., characterization of the mutations in the target genes . The isolation of drug-resistant virus in the laboratory and the determination of their phenotype and genotype may be useful to clarify the mechanisms of selective drug action . We describe here the procedures used for in vitro selection of drug-resistant herpesvirus mutants and the determination of their patterns of drug-susceptibility . The subcloning of the HSV-1 DNA polymerase gene is described as an example of the methodology followed to determine the mutation(s) in the drug-target viral gene that are associated with the resistant phenotype . To avoid the introduction of mutations by PCR amplification, all subcloning experiments were executed directly on viral DNA . Viral DNA was prepared from each plaque-purified viral strain and a 3.4 kb BamHI fragment containing 87% of the HSV-1 DNA polymerase gene coding region was purified and further digested with SacI; the two resulting fragments were subcloned into pU18 and propagated in Escherichia coli . Plasmid DNA was isolated and the inserts were sequenced using dideoxynucleotide chain termination method with T7 DNA polymerase and Taq DNA polymerase in an automated laser fluorescent DNA sequencer . pUC/M13 reverse, universal primers and oligonucleodite primers based on the wild-type virus sequence were used . The nucleotide sequences of the DNA polymerase genes of the different mutants was then compared with the nucleotide sequence of the wild-type HSV-1 KOS strain.

In Silico Biol, 1999, 1(2), 93 - 108
Use of contiguity on the chromosome to predict functional coupling; Overbeek R et al.; The availability of a growing number of completely sequenced genomes opens new opportunities for understanding of complex biological systems . Success of genome-based biology will, to a large extent, depend on the development of new approaches and tools for efficient comparative analysis of the genomes and their organization . We have developed a technique for detecting possible functional coupling between genes based on detection of potential operons . The approach involves computation of "pairs of close bidirectional best hits", which are pairs of genes that apparently occur within operons in multiple genomes . Using these pairs, one can compose evidence (based on the number of distinct genomes and the phylogenetic distance between the orthologous pairs) that a pair of genes is potentially functionally coupled . The technique has revealed a surprisingly rich and apparently accurate set of functionally coupled genes . The approach depends on the use of a relatively large number of genomes, and the amount of detected coupling grows dramatically as the number of genomes increases.

Proc Natl Acad Sci U S A, 2001 Jul 31, 98(16), 8997 - 9001 Epub 2001 Jul 24.
The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro); Gong F et al.; Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination . In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system . We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNA(Pro)) is not cleaved; it accumulates in the S-30 reaction mixture . In this paper, we examine the requirements for TnaC-peptidyl-tRNA(Pro) accumulation and cleavage, in vitro . We show that this peptidyl-tRNA remains bound to the translating ribosome . Removal of free tryptophan and addition of release factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNA(Pro) and release of TnaC from the ribosome-mRNA complex . Release factor-mediated cleavage is prevented by the addition of tryptophan . TnaC of the ribosome-bound TnaC-peptidyl-tRNA(Pro) was transferable to puromycin . This transfer was also blocked by tryptophan . Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action . Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNA(Pro) inhibits both TnaC peptidyl-tRNA(Pro) hydrolysis and TnaC peptidyl transfer . The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho factor binding and subsequent transcription termination.

Nucleic Acids Res, 2001 Aug 1, 29(15), 3188 - 94
Role of DNA minor groove interactions in substrate recognition by the M.SinI and M.EcoRII DNA (cytosine-5) methyltransferases; Kiss A et al.; The SinI and EcoRII DNA methyltransferases recognize sequences (GG(A)/(T)CC and CC(A)/(T)GG, respectively), which are characterized by an (A)/(T) ambiguity . Recognition of the A.T and T.A base pair was studied by in vitro methyltransferase assays using oligonucleotide substrates containing a hypoxanthine.C base pair in the central position of the recognition sequence . Both enzymes methylated the substituted oligonucleotide with an efficiency that was comparable to methylation of the canonical substrate . These observations indicate that M.SinI and M.EcoRII discriminate between their canonical recognition site and the site containing a G.C or a C.G base pair in the center of the recognition sequence (GG(G)/(C)CC and CC(G)/(C)GG, respectively) by interaction(s) in the DNA minor groove . M.SinI mutants displaying a decreased capacity to discriminate between the GG(A)/(T)CC and GG(G)/(C)CC sequences were isolated by random mutagenesis and selection for the relaxed specificity phenotype . These mutations led to amino acid substitutions outside the variable region, previously thought to be the sole determinant of sequence specificity . These observations indicate that (A)/(T) versus (G)/(C) discrimination is mediated by interactions between the large domain of the methyltransferase and the minor groove surface of the DNA.

Nucleic Acids Res, 2001 Aug 1, 29(15), 3145 - 53
Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex; Kolb FA et al.; In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis . These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops . In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment . This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control . The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity . This study addresses the question of why removal of bulged nucleotides blocks stable complex formation . Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA-CopT complexes suggests that, subsequent to loop-loop contact, helix propagation is prevented . Instead, a fully base paired loop-loop interaction is formed, inducing a continuous stacking of three helices . Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked . In contrast to the four-way junction topology, the loop-loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.

Nucleic Acids Res, 2001 Aug 1, 29(15), 3137 - 44
Changing the target base specificity of the EcoRV DNA methyltransferase by rational de novo protein-design; Roth M et al.; The EcoRV DNA-(adenine-N(6))-methyltransferase (M.EcoRV) specifically modifies the first adenine residue within GATATC sequences . During catalysis, the enzyme flips its target base out of the DNA helix and binds it into a target base binding pocket which is formed in part by Lys16 and Tyr196 . A cytosine residue is accepted by wild-type M.EcoRV as a substrate at a 31-fold reduced efficiency with respect to the k(cat)/K(M) values if it is located in a CT mismatch substrate (GCTATC/GATATC) . Cytosine residues positioned in a CG base pair (GCTATC/GATAGC) are modified at much more reduced rates, because flipping out the target base is much more difficult in this case . We intended to change the target base specificity of M.EcoRV from adenine-N(6) to cytosine-N(4) . To this end we generated, purified and characterized 15 variants of the enzyme, containing single, double and triple amino acid exchanges following different design approaches . One concept was to reduce the size of the target base binding pocket by site-directed mutagenesis . The K16R variant showed an altered specificity, with a 22-fold preference for cytosine as the target base in a mismatch substrate . This corresponds to a 680-fold change in specificity, which was accompanied by only a small loss in catalytic activity with the cytosine substrate . The K16R/Y196W variant no longer methylated adenine residues at all and its activity towards cytosine was reduced only 17-fold . Therefore, we have changed the target base specificity of M.EcoRV from adenine to cytosine by rational protein design . Because there are no natural paragons for the variants described here, a change of the target base specificity of a DNA interacting enzyme was possible by rational de novo design of its active site.

Vet Microbiol, 2001 Sep 28, 82(3), 275 - 83
Detection and characterization of Shiga toxin-producing Escherichia coli in feral pigeons; Morabito S et al.; Escherichia coli strains producing a variant of Shiga toxin 2 (Stx2), designated Stx2f, have been recently described in the stools of feral pigeons . During 1997-1998, 649 pigeons were trapped and examined in three different squares of Rome . Stool samples were collected from each bird and enrichment cultures were examined for the presence of Stx by the vero cell assay . Stx-producing E . coli (STEC) were isolated from the positive cultures and characterized by serotyping and PCR analysis of stx and other virulence-related genes . Stx was detected in 10.8% of the stool enrichment cultures . The percentage of positive birds did not differ significantly for the three flocks considered and the season of sample collection . Conversely, STEC carriage was significantly more frequent in young than in adult birds (17.9 versus 8.2%) . None of the birds examined showed signs of disease . STEC strains were isolated from 30 of 42 Stx-positive cultures examined . All the strains produced Stx2f, and most of them possessed genes encoding for intimin and the cytolethal distending toxin (CLDT) . Six serogroups were identified, but most of the isolates belonged to O45, O18ab, and O75 . Molecular typing indicated that most of the isolates within a flock were clonally-related . This work confirms that pigeons represent a natural reservoir of STEC strains characterized by the production of the toxin variant Stx2f, and by the frequent presence of eae and cldt genes . Further work is needed to clarify whether these STEC may represent a cause of avian disease or even a potential health hazard for humans.

Gene, 2001 Jul 11, 272(1-2), 267 - 74
In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor; Danielsen S et al.; The 'detergent lipase' Lipolase, from Thermomyces lanuginosa was subjected to a combinatorial protein engineering/phage display approach with the aim of identifying new enzyme variants with improved characteristics in the presence of detergents . First it was demonstrated that wild-type Lipolase could be produced in Escherichia coli retaining full activity and be displayed as an active enzyme fused to coat protein 3 on E . coli phage M13 . A phagemid library designed to result in approximately two to three mutations per lipase gene was then constructed . Nine amino acids located in two regions close to the active site were targeted for randomization . Selections using a mechanism-based biotinylated inhibitor showed that phages displaying Lipolase could be specifically enriched from a population of control phages . Selections on a library phage stock in the presence of inhibitor and a commercial powder detergent resulted in a step-wise increase in the proportion of active clones . Analysis of 84 active clones revealed that they all expressed lipase activity, but with lower activities than that of a wild-type Lipolase-producing clone . In six of the seven most active clones a wild-type serine at position 83 had been replaced by threonine, a substitution known to alter the substrate chain length preference of Lipolase variants . Furthermore, the selection had enriched enzyme variants with a high degree of conservatism in one of the variegated regions, suggesting that this region is important for enzymatic activity and that the designed selection procedure was relevant . The selected variants contained primarily basic amino acid residues within the other variegated region . Taken together, the described results show that selection protocols based on enzymatic activity can be designed for this enzyme class which should be of importance for future protein engineering attempts.

Mutat Res, 2001 Aug 8, 479(1-2), 37 - 52
Stationary phase deletions in Escherichia coli . II . Mutations which stimulate stationary phase deletions in plasmid pMC874; Balbinder E; Deletions in the plasmid pMC874 take place in resting cells incubating on McConkey's or minimal lactose agar and are time rather than generation dependent . These deletions join the km(r) promoter to a promoterless lac operon giving rise to Lac(+) papillae on McConkey's lactose agar, and can occur in the absence of sequence homologies such as direct or inverted repeats . Using this as a selective screen we isolated 31 mutants designated dli (for deletion increase), which enhanced to different extents the frequency of this unusual class of deletions . Six of these were characterized by phenotypic tests and their ability to stimulate other deletion events such as the excision of Tn10 from various chromosomal sites and the loss of cloned fragments between two EcoR1 sites in the gene for chloramphenicol resistance (cat) of plasmid pBR325 . Two of them showed contrasting phenotypes and were studied further: one (dli1) stimulated Lac(+) deletions in pMC874 in resting cells but not Tn10 excision from chromosomal locations in log phase cells, and the other one (dli2) did exactly the reverse, i.e . it enhanced Tn10 excision but not Lac(+) deletion incidence . Mapping and complementation tests showed that dli1 is a null mutation in recC and was renamed recC2251 . This is strong evidence that resting phase deletions in pMC874 are stimulated by the absence of a functional RecBCD enzyme . The dli2 mutation was identified by mapping and phenotypic tests as a mutation in uvrD, the gene for helicase II, and it was tentatively designated uvrD(-)dli2 . These results show that (1) pMC874 is an excellent system to select mutants for genetic functions involved in the generation of resting phase deletions, and (2) there are at least two major deletion pathways in E . coli, one active in resting and the other in actively dividing cells.

Mutat Res, 2001 Aug 8, 479(1-2), 19 - 36
Stationary phase deletions in Escherichia coli . I--Evidence for a new deletion pathway; Balbinder E; Deletions in the plasmid pMC874 join the promoter of the km(r) (kanamycin resistance) gene coding for the enzyme aminoglycoside 3'-phosphotransferase to a promoterless lac operon downstream giving a phenotypic change from Lac(-)-->Lac(+) . They differ from most deletions studied in Escherichia coli, which occur in actively dividing cells, in several important respects, as follows . (1) They occur in "resting" cells incubating on McConkey's or minimal lactose agar and increase in number gradually over a period of 1-2 weeks . Thus, like "adaptive" mutations, they are time rather than generation dependent . (2) They are extremely rare events (frequency 1x10(-11)-5x10(-11)) in wild type cells, but their frequency is increased between 1 and 2 orders of magnitude by null recC(-) mutations . In these respects they differ from "adaptive" mutations which are equally frequent in recC(+) and recC(-) cells . (3) Their frequency is not increased by mutations which stimulate log phase deletions . (4) Based on a computer search for homologies and sequencing of one deletion, it appears that they differ from log phase deletions in that they can occur in the absence of major terminal homologies (direct repeats) or intervening homologies (inverted repeats) which could stabilize a transient secondary structure and determine the deletion endpoints . Thus, they are not explained by the misaligned mutagenesis model . In conclusion, resting phase deletions occur through a totally different pathway from deletions in actively dividing cells and probably originate from unrepaired double strand breaks.

Structure (Camb), 2001 Jul 3, 9(7), 597 - 604
Crystal structure of the alpha-actinin rod reveals an extensive torsional twist; Ylanne J et al.; BACKGROUND: Alpha-actinin is a ubiquitously expressed protein found in numerous actin structures . It consists of an N-terminal actin binding domain, a central rod domain, and a C-terminal domain and functions as a homodimer to cross-link actin filaments . The rod domain determines the distance between cross-linked actin filaments and also serves as an interaction site for several cytoskeletal and signaling proteins . RESULTS: We report here the crystal structure of the alpha-actinin rod . The structure is a twisted antiparallel dimer that contains a conserved acidic surface . CONCLUSIONS: The novel features revealed by the structure allow prediction of the orientation of parallel and antiparallel cross-linked actin filaments in relation to alpha-actinin . The conserved acidic surface is a possible interaction site for several cytoplasmic tails of transmembrane proteins involved in the recruitment of alpha-actinin to the plasma membrane.

Structure (Camb), 2001 Jul 3, 9(7), 559 - 69
Structure of the RGS-like domain from PDZ-RhoGEF: linking heterotrimeric g protein-coupled signaling to Rho GTPases; Longenecker KL et al.; BACKGROUND: The multidomain PDZ-RhoGEF is one of many known guanine nucleotide exchange factors that upregulate Rho GTPases . PDZ-RhoGEF and related family members play a critical role in a molecular signaling pathway from heterotrimeric G protein-coupled receptors to Rho proteins . A approximately 200 residue RGS-like (RGSL) domain in PDZ-RhoGEF and its homologs is responsible for the direct association with Galpha12/13 proteins . To better understand structure-function relationships, we initiated crystallographic studies of the RGSL domain from human PDZ-RhoGEF . RESULTS: A recombinant construct of the RGSL domain was expressed in Escherichia coli and purified, but it did not crystallize . Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic acid residues for mutagenesis to alanine . A triple-point mutant functionally identical to the wild-type protein was crystallized, and its structure was determined by the MAD method using Se-methionine (Se-Met) incorporation . A molecular model of the RGSL domain was refined at 2.2 A resolution, revealing an all-helical tertiary fold with the mutations located at intermolecular lattice contacts . CONCLUSIONS: The first nine helices adopt a fold similar to that observed for RGS proteins, although the sequence identity with other such known structures is below 20% . The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobic interactions . Comparison with RGS proteins suggests features that are likely relevant for interaction with G proteins . Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable to other proteins resistant to crystallization.

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 271 - 5
Differential activities of the SoxR protein of Escherichia coli: SoxS is not required for gene activation under iron deprivation; Fuentes AM et al.; When Escherichia coli cells are under superoxide stress, proteins SoxR and SoxS, acting sequentially, control the expression of a set of repair and defense genes . One of these genes, fumC, encoding fumarase C, was reported to be also activated by iron deprivation in a soxRS-dependent manner . However, the same condition failed to induce the expression of a soxS'::lacZ fusion . The expression of acnA (aconitase A) is also activated by SoxR alone when under iron deprivation, but not of sodA (Mn-superoxide-dismutase) . SoxR completely inhibited the migration of a DNA fragment containing the promoter region of fumC, in gel-shift experiments . SoxR might bind to a different region than SoxS within the fumC promoter, or an unknown intermediate other than SoxS might be acting . It is possible that the regulatory role of SoxR is more complex than previously considered.

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 193 - 8
The Aspergillus nidulans carnitine carrier encoded by the acuH gene is exclusively located in the mitochondria; Ramon De Lucas J et al.; The location of the Aspergillus nidulans carnitine/acyl-carnitine carrier (ACUH) was studied . ACUH with a His-tag at its N-terminus was over-expressed in Escherichia coli and purified by Ni(2+) affinity chromatography . The purified protein was utilised to raise polyclonal antibodies which were characterised by Western blotting . For localisation studies A . nidulans T1 strain, that contains the acuH gene under control of the strong promoter alcA(p), was derived . Results obtained demonstrate the exclusively mitochondrial localisation of ACUH and therefore exclude the targeting of the acuH gene product to the peroxisomal membrane.

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 187 - 91
On surrogate methods for detecting lateral gene transfer; Ragan MA; Surrogate methods for detecting lateral gene transfer are those that do not require inference of phylogenetic trees . Herein I apply four such methods to identify open reading frames (ORFs) in the genome of Escherichia coli K12 that may have arisen by lateral gene transfer . Only two of these methods detect the same ORFs more frequently than expected by chance, whereas several intersections contain many fewer ORFs than expected . Each of the four methods detects a different non-random set of ORFs . The methods may detect lateral ORFs of different relative ages; testing this hypothesis will require rigorous inference of trees.

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 163 - 8
Diversity of surface structures and virulence genetic markers among enteroaggregative Escherichia coli (EAEC) strains with and without the EAEC DNA probe sequence; Suzart S et al.; The expression of surface structures and the presence of DNA sequences related to putative virulence factors were investigated in 22 enteroaggregative Escherichia coli strains (EAEC) . Fimbria was the most frequent (72.7%) structure identified . Only strains hybridising with the EAEC DNA probe carried aggA, but one strain produced a similar but unrelated bundle-like structure . All probe-positive and 62.5% of the probe-negative strains carried the virulence genes tested; aspU and irp2 prevailed among the former strains . The EAEC probe-positive strains were more diverse, and some of these strains, which promoted cell detachment, also carried the hly and pap sequences, thus suggesting they might represent uropathogenic E . coli.

J Immunol Methods, 2001 Sep 1, 255(1-2), 135 - 48
In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation; Andersson C et al.; We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J . Immunol . Methods 222 (1999) 171; 238 (2000) 181) . Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction . For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E . coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography . Upon expression in E . coli, fatty acids would be linked to the produced gene products . To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid . A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study . The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments . The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid . Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association . Iscom formation was further supported by electron microscopy analysis . In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice . For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented . Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

FEBS Lett, 2001 Jul 20, 501(2-3), 115 - 20
Use of amphipathic polymers to deliver a membrane protein to lipid bilayers; Nagy JK et al.; Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes . The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols . Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers . For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency . Results were not completely clear for the other amphipol.

Vet Parasitol, 2001 Aug 1, 99(2), 147 - 54
Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen; Boldbaatar D et al.; The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector . The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H . lineatum in cattle . Western blotting with rHC as antigen clearly differentiated between H . lineatum-infested cattle sera and normal cattle sera . Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting . The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay . These data demonstrated that Western blotting, with rHC expressed in E . coli, might be a useful method for the diagnosis of cattle hypodermosis.

Biochim Biophys Acta, 2001 Jul 30, 1520(1), 54 - 62
Identification of Dof proteins with implication in the gibberellin-regulated expression of a peptidase gene following the germination of rice grains; Washio K; Type III carboxypeptidase (CPD3) is one of the hydrolytic enzymes whose expression is up-regulated by gibberellins (GA) in the aleurones of germinated cereal grains . A number of pyrimidine boxes and a sequence resembling the gibberellic acid response element (GARE) are observed in the region upstream of the transcription initiation site of the CPD3 gene, showing a characteristic of cereal GA-responsive genes . Transient gene expression assays in germinated rice aleurone demonstrated that the CPD3 promoter was able to confer hormonally responses on the expression of the reporter gene . By southwestern screening, several cDNAs encoding the Dof class proteins were isolated from a rice aleurone library . Each mRNA accumulation for five novel members of Dof proteins (OsDof1--5) occurs with a different time course and in a tissue-specific manner following the germination of grains . Of these, the expression of the OsDof3 gene is abundant in aleurones where it precedes that of the CPD3 gene, implying that this is an early response gene of GA . The OsDof3 protein, expressed in Escherichia coli, selectively bound AAAG motifs of the pyrimidine boxes through the DNA-binding activity of its Dof domain . Co-expression experiments in aleurones suggested that the OsDof3 protein should play a regulatory role in the expression of the CPD3 gene under the control of GA.

Biochim Biophys Acta, 2001 Jul 30, 1520(1), 7 - 20
Mapping proteins of the 50S subunit from Escherichia coli ribosomes; Willumeit R et al.; Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago . Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins . Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction . Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes . The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.

Biochim Biophys Acta, 2001 Aug 6, 1513(2), 223 - 31
Critical parameters for functional reconstitution of glucose transport in Trypanosoma brucei membrane vesicles; Bayele HK; The glucose transporter of Trypanosoma brucei was reconstituted by incorporating Escherichia coli phospholipid liposomes into detergent-solubilised trypanosome membranes . Proteoliposome vesicles were formed by detergent dilution and used in glucose-uptake assays . The minima for functional reconstitution of the glucose transporter were established and used to probe the mechanism of glucose transport . The uptake pattern of radiolabelled glucose showed a counterflow transient at about 3 s, after which the sugar equilibrated across the proteoliposomal membrane . This observation is consistent with a facilitated transporter . There was a six-fold increase in the initial rate of glucose uptake compared to non-reconstituted or native membranes . In addition, the transporter exhibited stereospecificity to D-glucose but poorly transported L-glucose . Directionality, stereoselectivity or substrate specificity and cis-inhibition by phloridzin were therefore the main criteria for validation of glucose transport . The observed counterflow transient also provided further evidence for a facilitated glucose transporter within the trypanosome plasma membrane, and was the single most important criterion for this assertion . A stoichiometry of 0.78 mol of glucose per mol of transporter was estimated.

J Surg Res, 2001 Aug, 99(2), 245 - 52
Metalloproteinase inhibition prevents acute respiratory distress syndrome; Carney DE et al.; BACKGROUND: The acute respiratory distress syndrome (ARDS) occurs in patients with clearly identifiable risk factors, and its treatment remains merely supportive . We postulated that patients at risk for ARDS can be protected against lung injury by a prophylactic treatment strategy that targets neutrophil-derived proteases . We hypothesized that a chemically modified tetracycline 3 (COL-3), a potent inhibitor of neutrophil matrix metalloproteinases (MMPs) and neutrophil elastase (NE) with minimal toxicity, would prevent ARDS in our porcine endotoxin-induced ARDS model . METHODS: Yorkshire pigs were anesthetized, intubated, surgically instrumented for hemodynamic monitoring, and randomized into three groups: (1) control (n = 4), surgical instrumentation only; (2) lipopolysaccharide (LPS) (n = 4), infusion of Escherichia coli lipopolysaccharide at 100 microg/kg; and (3) COL-3 + LPS (n = 5), ingestion of COL-3 (100 mg/kg) 12 h before LPS infusion . All animals were monitored for 6 h following LPS or sham LPS infusion . Serial bronchoalveolar lavage (BAL) samples were analyzed for MMP concentration by gelatin zymography . Lung tissue was fixed for morphometric assessment at necropsy . RESULTS: LPS infusion was marked by significant (P < 0.05) physiological deterioration as compared with the control group, including increased plateau airway pressure (P(plat)) (control = 15.7 +/- 0.4 mm Hg, LPS = 23.0 +/- 1.5 mm Hg) and a decrement in arterial oxygen partial pressure (P(a)O(2)) (LPS = 66 +/- 15 mm Hg, Control = 263 +/- 25 mm Hg) 6 h following LPS or sham LPS infusion, respectively . Pretreatment with COL-3 reduced the above pathophysiological changes 6 h following LPS infusion (P(plat) = 18.5 +/- 1.7 mm Hg, P(a)O(2) = 199 +/- 35 mm Hg; P = NS vs control) . MMP-9 and MMP-2 concentration in BAL fluid was significantly increased between 2 and 4 h post-LPS infusion; COL-3 reduced the increase in MMP-9 and MMP-2 concentration at all time periods . Morphometrically LPS caused a significant sequestration of neutrophils and monocytes into pulmonary tissue . Pretreatment with COL-3 ameliorated this response . The wet/dry lung weight ratio was significantly greater (P < 0.05) in the LPS group (10.1 +/- 1.0 ratio) than in either the control (6.4 +/- 0.5 ratio) or LPS+COL-3 (7.4 +/- 0.6 ratio) group . CONCLUSIONS: A single prophylactic treatment with COL-3 prevented lung injury in our model of endotoxin-induced ARDS . The proposed mechanism of COL-3 is a synergistic inhibition of the terminal neutrophil effectors MMPs and NE . Similar to the universal practice of prophylaxis against gastric stress ulceration and deep venous thromboses in trauma patients, chemically modified tetracyclines may likewise be administered to prevent acute lung injury in critically injured patients at risk of developing ARDS .

J Surg Res, 2001 Aug, 99(2), 187 - 93
Suppression of tumor necrosis factor alpha production by cAMP in human monocytes: dissociation with mRNA level and independent of interleukin-10; Shames BD et al.; BACKGROUND: Elevation of cellular cAMP inhibits lipopolysaccharide(LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) production and increases the expression of interleukin (IL)-10 in mononuclear cells . TNF-alpha gene expression obligates activation of the transcription factor nuclear factor kappaB (NF-kappaB) . Exogenous IL-10 inhibits NF-kappaB in monocytes and thus attenuates TNF-alpha production . We examined the role of endogenous IL-10 in the regulation of NF-kappaB activation and TNF-alpha production in human monocytes by cAMP . METHODS: Human monocytes were stimulated with Escherichia coli LPS (100 ng/ml) with and without forskolin (FSK, 50 microM) or dibutyryl cyclic AMP (dbcAMP, 100 microM) . Cytokine (TNF-alpha and IL-10) release was measured by immunoassay . TNF-alpha mRNA was measured by reverse transcription polymerase chain reaction, and NF-kappaB DNA binding activity was assessed by gel mobility shift assay . RESULTS: cAMP-elevating agents inhibited LPS-stimulated TNF-alpha release (0.77 +/- 0.13 ng/10(6) cells in LPS + dbcAMP and 0.68 +/- 0.19 ng/10(6) cells in LPS + FSK, both P < 0.05 vs 1.61 +/- 0.34 ng/10(6) cells in LPS alone) . Conversely, cAMP enhanced LPS-stimulated IL-10 release (100 +/- 21.5 pg/10(6) cells in LPS + dbcAMP and 110 +/- 25.2 pg/10(6) cells in LPS + FSK, both P < 0.05 vs 53.3 +/- 12.8 pg/10(6) cells in LPS alone) . Neither TNF-alpha mRNA expression nor NF-kappaB activation stimulated by LPS was inhibited by the cAMP-elevating agents . Neutralization of IL-10 with a specific antibody did not attenuate the effect of cAMP-elevating agents on TNF-alpha production . CONCLUSION: The results indicate that cAMP inhibits LPS-stimulated TNF-alpha production through a posttranscriptional mechanism that is independent of endogenous IL-10 .

J Mol Biol, 2001 Aug 3, 311(1), 217 - 28
Functional determinants of the Epstein-Barr virus protease; Buisson M et al.; Herpesvirus proteases are essential for the production of progeny virus . They cleave the assembly protein that fills the immature capsid in order to make place for the viral DNA . The recombinant protease of the human gamma-herpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and purified . Circular dichroism indicated that the protein was properly folded with a secondary structure content similar to that of other herpesvirus proteases . Gel filtration and sedimentation analysis indicated a fast monomer-dimer equilibrium of the protease with a K(d) of about 60 microM . This value was not influenced by glycerol but was lowered to 1.7 microM in the presence of 0.5 M sodium citrate . We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease . We found that conditions that stabilised the dimer also led to a higher enzymatic activity . Through sequential deletion of amino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2' . This minimal sequence is shorter than that for other herpesvirus proteases . The implications of our findings are discussed with reference to the viral life-cycle . These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors .

Poult Sci, 2001 Jul, 80(7), 879 - 84
Immune response and resistance to infectious bursal disease virus of chicken lines selected for high or low antibody response to Escherichia coli; Pitcovski J et al.; Two experimental broiler lines were developed by divergent selection for high (HH) and low (LL) antibody response to Escherichia coli . Antibody response of these lines to immunization with a commercial vaccine (whole inactivated virus, WIV) against infectious bursal disease virus (IBDV) or with proteins VP2 and VP3 of that virus, and their resistance to challenge with a virulent IBDV, were tested . The study was performed with 213 male and female chicks from the tenth generation of the HH and LL lines . At 15 d of age, after disappearance of maternal antibodies, chicks from each line were randomly divided into four groups and injected with WIV, VP2, VP3, or adjuvant alone as a negative control . Chicks were bled 18 d postinjection, and antibody titers were determined by ELISA . Ten days later, the chicks were challenged with a virulent strain of the virus and killed after 10 d; the ratio of bursa of Fabricius to 100 g BW was determined for each bird . Significant differences in antibody titers were found among immunized and control chicks . Chicks from the HH line exhibited significantly higher antibody titers than LL chicks in response to WIV and VP2 vaccines but not to VP3 vaccine . Following challenge, bursa weight (relative to BW) of HH and LL chicks vaccinated with WIV and VP2 was significantly higher (P < 0.01) than that of chicks vaccinated with VP3 or the challenged unvaccinated control . No difference was found in this parameter between the latter two groups . Possible explanations for the differences in the line response to VP2 and VP3 are discussed.

Planta, 2001 Jun, 213(2), 258 - 64
Copy-DNA cloning and characterisation of a potato alpha-glucosidase: expression in Escherichia coli and effects of down-regulation in transgenic potato; Taylor MA et al.; Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristics of an alpha-glucosidase . Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were alpha-glucosidases and alpha-xylosidases of plant origin . The MAL2 cDNA was expressed in Escherichia coli and the recombinant MAL2 protein was affinity-purified . MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nitrophenyl-alpha-D-glucopyranoside with a pH optimum of 5.5-5.7 . The substrate with the lowest Km value was maltotetraose (3.7 mM) . The MAL2 expression product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p-nitrophenyl-alpha-D-xylopyranoside or gelatinised potato starch . MAL2 was down-regulated in transgenic potato plants using an antisense approach . In several independent transgenic antisense lines, MAL2 expression was severely down-regulated . Despite this, no decrease in total extractable alpha-glucosidase and alpha-xylosidase activity could be detected in tissues from the transgenic plants . In glasshouse trials, no visible phenotype, change in tuber yield or carbohydrate content was associated with MAL2 down-regulation . The implications of these results are discussed.

Tissue Cell, 2001 Jun, 33(3), 233 - 40
Interleukin-1beta-induced type IIA secreted phospholipase A2 gene expression and extracellular activity in rat vascular endothelial cells; Schwemmer M et al.; Two phospholipase A2 (PLA2) isoforms, secretory and cytosolic, have been implicated in inflammation . Secretory type IIA PLA2 (sPLA2-IIA), which hydrolyzes fatty acids bound at the sn-2 position of glycerophospholipids, has been detected universally in a variety of mammalian tissues and cells . The expression of the sPLA2-IIA gene and its extracellular activity were shown to be regulated by different factors such as hypoxia, cytokines and phorbol esters . In the present study, we examined the effects of interleukin-1beta (IL-1beta) on the expression of the 14kDa sPLA2-IIA, determined using reverse transcription polymerase chain reaction and radiometric Escherichia coli enzyme assay in primary cultures of rat endothelial cells and in two different rat endothelial cell lines (SVAREC and RBE4) . These experiments revealed that IL-1beta induces sPLA2-IIa gene expression and secretion of the enzyme in endothelial cells in a dose- and time-dependent manner . The cAMP-elevator forskolin did not augment the cytokine-induced elevation of sPLA2-IIa enzyme activity but significantly increased the IL-1beta-stimulated sPLA2-IIa mRNA contents in endothelial cells.

J Rheumatol, 2001 Jul, 28(7), 1492 - 5
Autoantibodies to osteopontin in patients with osteoarthritis and rheumatoid arthritis; Sakata M et al.; OBJECTIVE: Osteopontin (OPN), secreted mainly from chondrocytes, is suggested to be involved in the ossification and remodeling of bone and also in regulation of cytokine profiles . We investigated whether patients with osteoarthritis (OA) and rheumatoid arthritis (RA) display autoimmunity against OPN . METHODS: Recombinant human OPN (rhOPN) was prepared as a fusion protein with beta-galactosidase using E . coli . Serum samples from patients with OA or RA and from age matched healthy donors were tested for autoantibodies to rhOPN using ELISA and Western blotting . Reactivity of the same samples to purified native human OPN (nhOPN) was investigated by ELISA separately, to evaluate conformational epitopes . RESULTS: By ELISA, autoantibodies to rhOPN were found in one (0.95%) of 105 patients with OA and 2 (2.3%) of 88 patients with RA . These autoantibodies to rhOPN were confirmed by Western blotting . In contrast, 11 (9.5%) of 105 OA serum and 13 (15%) of 88 RA serum samples reacted to nhOPN . The anti-OPN positive RA patients showed high serum levels of rheumatoid factor and C-reactive protein and accelerated erythrocyte sedimentation rate compared to the anti-OPN negative group, although the differences did not achieve statistical significance . CONCLUSION: Our data showed that OPN is one of the autoantigens in OA and RA . Preferential recognition of nhOPN to rhOPN indicates that major epitope(s) of OPN would be conformational . Clinically, existence of the anti-OPN antibodies may be linked to disease severity in RA.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1189 - 91 Epub 2001 Jul 23.
Crystallization and preliminary X-ray diffraction studies of recombinant Escherichia coli 4-diphosphocytidyl-2-C-methyl-D-erythritol synthetase; Kemp LE et al.; Diphosphocytidyl-methylerythritol (DPCME) synthetase is involved in the mevalonate-independent pathway of isoprenoid biosynthesis, where it catalyses the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol 4-phosphate and CTP . The Escherichia coli enzyme has been cloned, expressed in high yield, purified and crystallized . Elongated tetragonal prismatic crystals grown by the hanging-drop vapour-diffusion method using polyethylene glycol (PEG) 4000 as the precipitant belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 73.60, c = 175.56 A . Diffraction data have been recorded to 2.4 A resolution using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1167 - 70 Epub 2001 Jul 23.
Coexpression, copurification, crystallization and preliminary X-ray analysis of a complex of ARL2-GTP and PDE delta; Renault L et al.; The small GTPase ARL2 (from Mus musculus) and an effector protein, the delta subunit of human cGMP phosphodiesterase (hPDE delta), were coexpressed and copurified from Escherichia coli as a stable complex . Coexpression significantly increased the otherwise low yield of PDE delta production in E . coli . The complex, which contains ARL2 in the activated GTP-bound form, was crystallized in two forms . The first belongs to the monoclinic space group P2(1), with unit-cell parameters a = 48.1, b = 45.7, c = 74.7 A, beta = 94.0 degrees and one complex (39 kDa) in the asymmetric unit . Cryocooled crystals diffract to 2.3 A using synchrotron radiation . The micro-focused X-ray beam at beamline ID13 (ESRF) allowed the use of very small crystals, which helped to overcome twinning and enabled the identification of a molecular-replacement solution . The second form recrystallized from the first one after several months . These crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.5, b = 65.4, c = 104.4 A and one complex in the asymmetric unit . They diffracted to 1.8 A using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1159 - 61 Epub 2001 Jul 23.
Crystallization and preliminary X-ray crystallographic studies of recombinant thermoresistant gluconate kinase GntK from Escherichia coli; Kraft L et al.; The thermoresistant gluconate kinase GntK from Escherichia coli, an essential enzyme in gluconate metabolism, has been expressed, purified and crystallized . For crystallization, the hanging-drop vapour-diffusion method was used with polyethylene glycol (PEG) 6000 and lithium chloride as precipitants . Three crystal forms belonging to the monoclinic space group C2 or the orthorhombic space groups P2(1)2(1)2(1) and P2(1)2(1)2 were obtained . The unit-cell parameters are a = 75.0, b = 79.3, c = 70.2 A, beta = 105.3 degrees (C2), a = 52.0, b = 79.3, c = 89.8 A (P2(1)2(1)2(1)) and a = 70.1, b = 74.1, c = 78.9 A (P2(1)2(1)2) . In all three crystal forms, there are two molecules in the asymmetric unit; the different forms occur in the same crystallization drop . The crystals diffract to at least 2.0 A using synchrotron radiation.

Protein Sci, 2001 Aug, 10(8), 1635 - 44
Reversible formation of on-pathway macroscopic aggregates during the folding of maltose binding protein; Ganesh C et al.; Maltose binding protein (MBP) is widely used as a model for protein folding and export studies . We show here that macroscopic aggregates form transiently during the refolding of MBP at micromolar protein concentrations . Disaggregation occurs spontaneously without any aid, and the refolded material has structure and activity identical to those of the native, nondenatured protein . A considerable fraction of protein undergoing folding partitions into the aggregate phase and can be manually separated from the soluble phase by centrifugation . The separated MBP precipitate can be resolubilized and yields active, refolded protein . This demonstrates that both the soluble and aggregate phases contribute to the final yield of refolded protein . SecB, the cognate Escherichia coli cytosolic chaperone in vivo for MBP, reduces but does not entirely prevent aggregation, whereas GroEL and a variety of other control proteins have no effect . Kinetic studies using a variety of spectroscopic probes show that aggregation occurs through a collapsed intermediate with some secondary structure . The aggregate formed during refolding can convert directly to a near native state without going through the unfolded state . Further, optical and electron microscopic studies indicate that the MBP precipitate is not an amyloid.

Protein Sci, 2001 Aug, 10(8), 1549 - 62
Novel inter-protein cross-link identified in the GGH-ecotin D137Y dimer; Person MD et al.; In the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl-glycyl-histidine (GGH) mediates efficient and specific oxidative protein cross-linking . The fusion of GGH to the N terminus of a protein allows for the cross-linking reagent to be delivered in a site-specific fashion, making this system extremely useful for analyzing protein-protein contacts in complicated mixtures of biomolecules . Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross-linking efficiency . Cross-linking increased four-fold for GGH-ecotin D137Y in comparison to wild-type GGH-ecotin, presumably through bityrosine formation at the dimer interface . Here we report the first complete structural analysis of the cross-linked GGH-ecotin D137Y dimer . Using a combination of mass spectrometric and chemical derivatization methods, a sole novel cross-link between the N-terminal glycine residues and the engineered tyrosine at position 137 has been characterized . The dimer cross-link is localized to a single site without other protein modifications, but different reaction pathways produce structurally related products . We propose a mechanism that involves covalent bond formation between the protein backbone and a dopaquinone moiety derived from a specific tyrosine residue . This finding establishes that it is not necessary to have two tyrosine residues within close proximity in the protein interface to obtain high protein cross-linking yields, and suggests that the cross-linking reagent may be of more general utility than previously thought.

Biochemistry, 2001 Jul 31, 40(30), 9040 - 8
Regulation of the plant-type 5'-adenylyl sulfate reductase by oxidative stress; Bick JA et al.; 5'-Adenylyl sulfate (APS) reductase (EC 1.8.4.9) catalyzes a key reaction in the plant sulfate assimilation pathway leading to the synthesis of cysteine and the antioxidant glutathione . In Arabidopsis thaliana APS reductase is encoded by a family of three genes . In vitro biochemical studies revealed that the enzyme product derived from one of them (APR1) is activated by oxidation, probably through the formation of a disulfide bond . The APR1 enzyme is 45-fold more active when expressed in a trxB strain of Escherichia coli than in a trxB(+) wild type . The enzyme is inactivated in vitro by treatment with disulfide reductants and is reactivated with thiol oxidants . Redox titrations show that the regulation site has a midpoint potential of -330 mV at pH 8.5 and involves a two-electron redox reaction . Exposure of a variety of plants to ozone induces a rapid increase in APS reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione . During the response to ozone, the level of immunodetectable APS reductase enzyme does not increase . Treatment of A . thaliana seedlings with oxidized glutathione or paraquat induces APS reductase activity even when transcription or translation is blocked with inhibitors . The results suggest that a posttranslational mechanism controls APS reductase . A model is proposed whereby redox regulation of APS reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress.

Biochemistry, 2001 Jul 31, 40(30), 8971 - 80
Kinetic study of folding and misfolding of diacylglycerol kinase in model membranes; Nagy JK et al.; Despite the relevance of membrane protein misfolding to a number of common diseases, our understanding of the folding and misfolding of membrane proteins lags well behind soluble proteins . Here, the overall kinetics of membrane insertion and folding of the homotrimeric integral membrane protein diacylglycerol kinase (DAGK) is addressed . DAGK was purified into lipid/detergent-free urea and guanidinium solutions and subjected to general structural characterization . In urea, the enzyme was observed to be monomeric but maintained considerable tertiary structure . In guanidinium, it was also monomeric but exhibited much less tertiary structure . Aliquots of these DAGK stock solutions were diluted 200-fold into lipid vesicles or into detergent/lipid mixed micelles, and the rates and efficiencies of folding/insertion were monitored . Reactions were also carried out in which micellar DAGK solutions were diluted into vesicular solutions . Productive insertion of DAGK from denaturant solutions into mixed micelles occurred much more rapidly than into lipid vesicles, suggesting that bilayer transversal represents the rate-limiting step for DAGK assembly in vesicles . The efficiency of productive folding/insertion into vesicles was highest in reactions initiated with micellar DAGK stock solutions (where DAGK maintains a nativelike fold and oligomeric state) and lowest in reactions starting with guanidinium stocks (where DAGK is an unfolded monomer) . Moreover, the final ratio of irreversibly misfolded DAGK to reversibly misfolded enzyme was highest following reactions initiated with guanidinium stock solutions and lowest when micellar stocks were used . Finally, it was also observed that very low concentrations of detergents were able to both enhance the bilayer insertion rate and suppress misfolding.

Biochemistry, 2001 Jul 31, 40(30), 8808 - 14
Ca2+ binding site 2 in calcineurin-B modulates calmodulin-dependent calcineurin phosphatase activity; Feng B et al.; Calcineurin is the Ca(2+)- and calmodulin-dependent Ser/Thr phosphatase . Human calcineurin-Aalpha and wild-type or mutated calcineurin-Bs were coexpressed in Escherichia coli and purified by calmodulin-Sepharose affinity chromatography . Four calcineurin-B mutants were studied . Each had a single conserved Glu in the 12th position of one EF-hand Ca(2+) binding site replaced by a Lys, resulting in the loss of Ca(2+) binding to that site . Phosphatase activities of the enzymes toward a (32)P-labeled phosphopeptide substrate were measured . Inactivating Ca(2+) binding sites 1, 2, or 3 in calcineurin-B reduced Ca(2+)-dependent phosphatase activity of the enzymes in the absence of calmodulin with the site 2 mutation being most effective . Inactivating Ca(2+) binding site 4 did not change enzyme activity or sensitivity to Ca(2+) in either the absence or presence of calmodulin . The calmodulin-dependent phosphatase activity of the enzymes containing site 1, 2, or 3 mutations in calcineurin-B was also decreased compared to enzyme with wild-type calcineurin-B . Of these enzymes, the one with the site 2 mutation was most profoundly affected as determined by the magnitude of the shift in Ca(2+) concentration dependence . Binding of a fluorescein-labeled calmodulin to the wild-type and the site 2 mutant enzymes was examined using fluorescence polarization measurements . The decrease in Ca(2+) sensitivity for the enzyme with calcineurin-B site 2 inactivated is apparently due to a decrease in the affinity of that enzyme for calmodulin at low Ca(2+) concentrations . These data support a role for Ca(2+) binding site 3 in the carboxyl half of calcineurin-B in transmitting the Ca(2+) signal to calcineurin-A and indicate that site 2 in the amino half of calcineurin-B is critical for enzyme activation.

Biochemistry, 2001 Jul 31, 40(30), 8773 - 82
Tryptophan residues at subunit interfaces used as fluorescence probes to investigate homotropic and heterotropic regulation of aspartate transcarbamylase; Fetler L et al.; The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications . The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes . These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation . To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues . The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes . Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states . The bisubstrate analogue N-phosphonacetyl-L-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively . The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r . These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.

Parasitology, 2001 Jul, 123(Pt 1), 77 - 83
Molecular cloning and characterization of a serine proteinase inhibitor from Trichinella spiralis; Nagano I et al.; We produced a recombinant protein from a cDNA library from muscle larvae of Trichinella spiralis which had proteinase inhibitory activity . The predicted amino acid sequence of the clone had an identity of only 30% to the serine proteinase inhibitors (serpins) from Caenorhabditis elegans or Brugia malayi . At the putative reactive region, however, the identity was about 50% . The recombinant protein expressed in Escherichia coli inhibited 82% of the activity of the serine proteinase (trypsin) . Stage-specific expression of this protein was suggested from the following experiments . Antibody against the recombinant protein could stain proteins migrating at about 42 kDa (which is the expected size from the sequence) in crude extracts from newborn larvae and 18-day post-infection (p.i.) muscle larvae, but it failed to stain any proteins in crude extracts from 30-day p.i . muscle larvae . Production of mRNA transcript for the serpin gene was restricted largely to the newborn larvae and to 18-day p.i . muscle larvae . The antibody reacted with the stichocytes of the larvae at 18 days p.i., but did not react with the muscle larvae at 24 days and 30 days p.i.

Anal Chem, 2001 Jul 1, 73(13), 2968 - 75
Two-layer sample preparation method for MALDI mass spectrometric analysis of protein and peptide samples containing sodium dodecyl sulfate; Zhang N et al.; Sodium dodecyl sulfate (SDS) is widely used in protein sample workup . However, many mass spectrometric methods cannot tolerate the presence of this strong surfactant in a protein sample . We present a practical and robust technique based on a two-layer matrix/sample deposition method for the analysis of protein and peptide samples containing SDS by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) . The two-layer method involves the deposition of a mixture of sample and matrix on top of a thin layer of matrix crystals . It was found that for SDS-containing samples, the intensity of the MALDI signals can be affected by the conditions of sample preparation: on-probe washing, choice of matrix, deposition method, solvent system, and protein-to-SDS ratio . However, we found that, under appropriate conditions, the two-layer method gave reliable MALDI signals for samples with levels of SDS up to approximately 1% . The applications of this method are demonstrated for MALDI analysis of hydrophobic membrane proteins as well as bacterial extracts . We envision that this two-layer method capable of handling impure samples including those containing SDS will play an important role in protein molecular weight analysis as well as in proteome identification by MALDI-MS and MS/MS.

J Immunol, 2001 Aug 1, 167(3), 1274 - 82
Processing of exogenous antigens for presentation by class I MHC molecules involves post-Golgi peptide exchange influenced by peptide-MHC complex stability and acidic pH; Chefalo PJ et al.; Vacuolar alternate class I MHC (MHC-I) Ag processing allows presentation of exogenous Ag by MHC-I molecules with binding of antigenic peptides to post-Golgi MHC-I molecules . We investigated the role of previously bound peptides and their dissociation in generating peptide-receptive MHC-I molecules . TAP1-knockout macrophages were incubated overnight with an initial exogenous peptide, producing a large cohort of peptide-K(b) complexes that could influence subsequent peptide dissociation/exchange . Initial incubation with FAPGNYPAL, KVVRFDKL, or RGYVYQGL enhanced rather than reduced subsequent binding and presentation of a readout peptide (SIINFEKL or FAPGNYPAL) to T cells . Thus, K(b) molecules may be stabilized by an initial (stabilizing) peptide, enhancing their ability to bind readout peptide and implicating peptide dissociation/exchange . In contrast, incubation with SIINFEKL as stabilizing peptide reduced presentation of readout peptide . SIINFEKL-K(b) complexes were more stable than other peptide-K(b) complexes, which may limit their contribution to peptide exchange . Stabilizing peptides (FAPGNYPAL, KVVRFDKL, or RGYVYQGL) enhanced alternate MHC-I processing of HB101.Crl-OVA (Escherichia coli expressing an OVA fusion protein), indicating that alternate MHC-I Ag processing involves peptide dissociation/exchange . Stabilizing peptide enhanced processing of HB101.Crl-OVA more than presentation of exogenous OVA peptide (SIINFEKL), suggesting that peptide dissociation/exchange may be enhanced in the acidic phagosomal processing environment . Furthermore, exposure of cells to acidic pH increased subsequent binding and presentation of readout peptide . Thus, peptide dissociation/exchange contributes to alternate MHC-I Ag processing and may be influenced by both stability of peptide-MHC-I complexes and pH.

J Bacteriol, 2001 Aug, 183(16), 4918 - 26
Isolation and characterization of a Shewanella putrefaciens MR-1 electron transport regulator etrA mutant: reassessment of the role of EtrA; Maier TM et al.; Shewanella putrefaciens MR-1 has emerged as a good model to study anaerobic respiration and electron transport-linked metal reduction . Its remarkable respiratory plasticity suggests the potential for a complex regulatory system to coordinate electron acceptor use in the absence of O(2) . It had previously been suggested that EtrA (electron transport regulator A), an analog of Fnr (fumarate nitrate regulator) from Escherichia coli, may regulate gene expression for anaerobic electron transport . An etrA knockout strain (ETRA-153) was isolated from MR-1 using a gene replacement strategy . Reverse transcription-PCR analysis of total RNA demonstrated the loss of the etrA mRNA in ETRA-153 . ETRA-153 cells retained the ability to grow on all electron acceptors tested, including fumarate, trimethylamine N-oxide (TMAO), thiosulfate, dimethyl sulfoxide, ferric citrate, nitrate, and O(2), as well as the ability to reduce ferric citrate, manganese(IV), nitrate, and nitrite . EtrA is therefore not necessary for growth on, or the reduction of, these electron acceptors . However, ETRA-153 had reduced initial growth rates on fumarate and nitrate but not on TMAO . The activities for fumarate and nitrate reductase were lower in ETRA-153, as were the levels of fumarate reductase protein and transcript . ETRA-153 was also deficient in one type of ubiquinone . These results are in contrast to those previously reported for the putative etrA mutant METR-1 . Molecular analysis of METR-1 indicated that its etrA gene is not interrupted; its reported phenotype was likely due to the use of inappropriate anaerobic growth conditions.

J Bacteriol, 2001 Aug, 183(16), 4914 - 7
Autoamplification of a two-component regulatory system results in "learning" behavior; Hoffer SM et al.; We have tested the hypothesis that the autoamplification of two-component regulatory systems results in "learning" behavior, i.e., that bacteria respond faster or more extensively to a signal when a similar signal has been perceived in the past . Indeed, the induction of alkaline phosphatase activity upon phosphate limitation was faster if the cultures had been limited for phosphate previously, and this faster response correlated with the autoamplification of the cognate two-component system.

J Bacteriol, 2001 Aug, 183(16), 4900 - 4
Specificity and topology of the Escherichia coli xanthosine permease, a representative of the NHS subfamily of the major facilitator superfamily; Norholm MH et al.; The specificity of XapB permease was compared with that of the known nucleoside transporters NupG and NupC . XapB-mediated xanthosine uptake is abolished by 2,4-dinitrophenol and exhibits saturation kinetics with an apparent K(m) of 136 microM . A 12-transmembrane-segment model was confirmed by translational fusions to alkaline phosphatase and the alpha fragment of beta-galactosidase.

J Bacteriol, 2001 Aug, 183(16), 4806 - 13
Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli; Patzer SI et al.; The uptake of Mn(2+), a cofactor for several enzymes in Escherichia coli, is mediated by MntH, a proton-dependent metal transporter, which also recognizes Fe(2+) with lower affinity . MntH belongs to the NRAMP family of eukaryotic Fe(2+) and Mn(2+) transporters . In E . coli strains with chromosomal mntH-lacZ fusions, mntH was partially repressed by both Mn(2+) and Fe(2+) . Inactivation of fur resulted in the loss of Fe(2+)-dependent repression of mntH transcription, demonstrating that Fe(2+) repression depends on the global iron regulator Fur . However, these fur mutants still showed Mn(2+)-dependent repression of mntH . The Mn(2+)-responsive transcriptional regulator of mntH was identified as the gene product of o155 (renamed MntR) . mntR mutants were impaired in Mn(2+) but not Fe(2+) repression of mntH transcription . Binding of purified MntR to the mntH operator was manganese dependent . The binding region was localized by DNase I footprinting analysis and covers a nearly perfect palindrome . The Fur binding site, localized within 22 nucleotides of the mntH operator by in vivo operator titration assays, resembles the Fur-box consensus sequence.

J Bacteriol, 2001 Aug, 183(16), 4727 - 36
matB, a common fimbrillin gene of Escherichia coli, expressed in a genetically conserved, virulent clonal group; Pouttu R et al.; A novel fimbrial type in Escherichia coli was identified and characterized . The expression of the fimbria was associated with the O18acK1H7 clonal group of E . coli, which cause newborn meningitis and septicemia when grown at low temperature; hence, it was named the Mat (meningitis associated and temperature regulated) fimbria . The fimbriae were purified from a fimA::cat sfaA::Gm fliC::St derivative of the O18K1H7 isolate E . coli IHE 3034 . The purified Mat fimbrillin had an apparent molecular mass of 18 kDa and did not serologically cross-react with the type 1 or S fimbria of the same strain . The matB gene encoding the major fimbrillin was cloned from the genomic DNA of the fimA::cat sfaA::Gm fliC::St derivative of IHE 3034 . The predicted MatB sequence was of 195 amino acids, contained a signal sequence of 22 residues, and did not show significant homology to any of the previously characterized fimbrial proteins . The DNA sequence of matB was 97.8% identical to a region from nucleotides 17882 to 18469 in the 6- to 8-min region of the E . coli K-12 chromosome, reported to encode a hypothetical protein . The 7-kb DNA fragment containing matB of IHE 3034 was found by restriction mapping and partial DNA sequencing to be highly similar to the corresponding region in the K-12 chromosome . Trans complementation of the matB::cat mutation in the IHE 3034 chromosome showed that matB in combination with matA or matC restored surface expression of the Mat fimbria . A total of 27 isolates representing K-12 strains and the major pathogroups of E . coli were analyzed for the presence of a matB homolog as well as for expression of the Mat fimbria . A conserved matB homolog was found in 25 isolates; however, expression of the Mat fimbriae was detected only in the O18acK1H7 isolates . Expression of the Mat fimbria was temperature regulated, with no or a very small amount of fimbriae or intracellular MatB fimbrillin being detected in cells cultivated at 37(o)C . Reverse transcriptase PCR and complementation assays with mat genes controlled by the inducible trc promoter indicated that regulation of Mat fimbria expression involved both transcriptional and posttranscriptional events.

J Bacteriol, 2001 Aug, 183(16), 4709 - 17
Fructose uptake in Sinorhizobium meliloti is mediated by a high-affinity ATP-binding cassette transport system; Lambert A et al.; By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar . The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently . The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (K(m) of 6 microM) fructose uptake system in S . meliloti . The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family . The expression of S . meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter . The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein . However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S . meliloti . The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose . Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is expressed in bacteroids isolated from alfalfa nodulated by S . meliloti wild-type strains.

Clin Immunol, 2001 Aug, 100(2), 157 - 63
The role of cpg sequences in the induction of anti-DNA antibodies; Pisetsky DS et al.; To investigate the role of CpG sequences in anti-DNA induction, immunization experiments were performed in mice to assess the immunogenicity of native Escherichia coli (EC) and calf thymus (CT) in incomplete Freund's adjuvant . The effects of CpG sequences were further tested by comparing the adjuvant properties of a synthetic phosphorothioate oligonucleotide with a CpG motif to one with a GpC sequence . Both EC and CT DNA alone induced a limited anti-DNA response . For CT DNA, the addition of a CpG ODN significantly enhanced responses whereas for EC DNA, the presence of a CpG oligonucleotide (ODN) or control GpC ODN did not increase responses compared to EC DNA alone . Specificity analysis by ELISA indicated that these immunizations led to the generation of cross-reactive anti-DNA autoantibodies . These results thus extend the adjuvant effects of CpG sequences to self antigens and suggest mechanisms by which self and foreign antigens can interact in the generation of autoimmunity .

Pediatr Infect Dis J, 2001 Jul, 20(7), 672 - 8
Inhibition of enteroaggregative Escherichia coli adhesion to HEp-2 cells by secretory immunoglobulin A from human colostrum; Fernandes RM et al.; BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an important agent of the persistent diarrhea among low socioeconomic level children in developing countries that may be associated with chronic undernourishment . Breast-feeding is effective in protecting infants against diarrhea and other infectious diseases . The aim of the study is to verify the ability of human colostrum to inhibit aggregative adhesion of EAEC to HEp-2 cells and the presence of antibodies reactive to antigenic fractions of EAEC in colostrum samples . METHODS: Enzyme-linked immunosorbent assay, immunoblotting and adhesion assays of EAEC to HEp-2 cells were done with pooled or individual colostrum samples (n = 35) . Assays were performed with a well-known EAEC strain, 044:H18 E . coli (strain 042) . Colostral IgA was isolated by affinity chromatography in Sepharose anti-human alpha chain column . RESULTS: Total colostrum and isolated IgA inhibited EAEC adhesion, and this ability was associated with the presence of IgA antibodies against a 15-kDa band, compatible with the subunits of aggregative adherence fimbrial adhesin II, characteristic of the 042 strain, absent in its plasmid-cured isogenic strain, that was used as control . Individual colostrum samples also inhibited adhesion, showed variable antibody titles against EAEC antigens in enzyme-linked immunosorbent assay and recognized many antigenic fractions in immunoblotting assays, including the 15-kDa band . CONCLUSIONS: These results confirm that IgA from human colostrum inhibits adhesion of EAEC to HEp-2 cells and suggest that colostrum IgA antibodies reactive to EAEC antigens may play a role in protection of infants against diarrhea caused by these bacteria.

Electrophoresis, 2001 Jun, 22(10), 2110 - 9
Cell immobilization induces changes in the protein response of Escherichia coli K-12 to a cold shock; Perrot F et al.; We have compared the protein expression of gel-entrapped Escherichia coli cells submitted to a cold shock at 4 degrees C with those of exponential- and stationary-phase free-floating counterparts . Autoradiograms of two-dimensional gel electrophoresis patterns of proteins radiolabeled with L-{35S}methionine were compared using computing scanning densitometry . The levels of 203 proteins synthesized during the temperature shift were significantly and reproducibly higher than those corresponding to synthesis at 37 degrees C . A principal component analysis (PCA) was performed on the synthesis levels of these 203 proteins in the different incubation conditions tested . This study showed that the protein response of immobilized cells after the cold shock was significantly different from those of exponential- and stationary-phase free-floating organisms . For instance, protein SSB was specifically overexpressed by shocked immobilized organisms . Such induction of specific molecular mechanisms in immobilized bacteria might explain the high resistance of sessile-like organisms to stresses.

Xenobiotica, 2001 Mar, 31(3), 163 - 76
Specificity of 17beta-oestradiol and benzo{a}pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432; Shimada T et al.; 1 . Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg48 Ala119 Leu432, Arg48 Ala119 Val432 Gly48 Ala119 Leu432, Gly48 Ala119 Val432, Arg48 Ser119 Leu432, Arg48 Ser119 Val432, Gly48 Ser119 Leu432 and Gly48 Ser119 Va1432 (all with Asn453), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17beta-oestradiol and benzo{a}pyrene were determined . 2 . All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+.CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots . The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5 . 3 . CYP1B1 Arg48 variants tended to have higher activities for 17beta-oestradiol 4-hydroxylation than Gly48 variants, although there were no significant variations in 17beta-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants . Interestingly, ratios of formation of 17beta-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val432 forms than the corresponding Leu432 forms . 4 . In contrast, Leu432 forms of CYP1B1 showed higher rates of oxidation of benzo{a}pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val432 forms . 5 . These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17beta-oestradiol and benzo{a}pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

Scand J Clin Lab Invest, 2001 Jul, 61(4), 293 - 9
Surgical organ perfusion method for gene transfer into cells of the perifollicular area of the spleen: an experimental trial on farm pigs; Parpala-Sparman T et al.; In an attempt to develop a gene therapy method for splenic and systemic diseases, an evaluation was made of surgical methods for gene transfer into porcine spleen . We have developed a continuous closed-circuit organ perfusion method for gene transfer into porcine spleen . For gene transfer, we used a type-5 replication defective adenovirus vector expressing the E . coli beta-galactosidase gene as a reporter gene . In eight young 22-35 kg farm pigs, the spleen was perfused in vivo with the viral solution via laparotomy, for 30 or 60 min . Gene transfer was determined visually on histological cryosections after X-gal and PAS staining . Infusion of the viral solution through the splenic artery did not result in gene expression . Perfusion of spleen in vivo resulted in beta-galactosidase reporter gene expression in the macrophages located around capillaries terminating in the perifollicular zone and in the red pulp examined after four days . The present results suggest that the surgically performed spleen perfusion method can be used for gene transfer in the treatment of diseases having splenic manifestations and in systemic diseases.

Ann Thorac Surg, 2001 Jul, 72(1), 278 - 9; discussion 280
Massive hemoptysis from a lung abscess due to retained gallstones; Werber YB et al.; This case report describes a subhepatic abscess from spilled gallstones which eroded through the diaphragm causing a right lower lobe pulmonary abscess and presenting as massive hemoptysis.

Eur J Immunol, 2001 May, 31(5), 1513 - 22
Identification of Chlamydia trachomatis antigens recognized by human CD4+ T lymphocytes by screening an expression library; Goodall JC et al.; Identification of the immunogenic proteins that induce Chlamydia trachomatis (CT)-specific T cell responses is crucial to the development of protective vaccines and understanding the mechanisms of chlamydia-induced pathology . To characterize the targets of the human T cell response we have used chlamydia-reactive human T cell clones as cellular probes to screen a CT genomic library expressed in Escherichia coli using peripheral blood mononuclear cells to present antigens . The library was screened with three chlamydia-reactive T cell clones of unknown specificity and three novel stimulatory chlamydia antigens were identified . These E . coli recombinants were shown to express the chlamydia proteins, enolase, pmpD and CT579 . Enolase and pmpD proteins were purified and shown to induce the proliferation of synovial fluid mononuclear cells isolated from the knee joints of patients suffering from chlamydia-associated reactive arthritis . We suggest that these stimulatory antigens are common targets of the T cell response in this group of patients . A greater understanding of T cell-mediated immunity in uncomplicated CT infection, and in patients with CT-induced chronic inflammatory disease (trachoma, salpingitis, arthritis) may identify the principal immune responses associated with immunopathology.

Mol Cell Biol, 2001 Aug, 21(16), 5520 - 30
The oncoprotein Tax binds the SRC-1-interacting domain of CBP/p300 to mediate transcriptional activation; Scoggin KE et al.; Oncogenesis associated with human T-cell leukemia virus (HTLV) infection is directly linked to the virally encoded transcription factor Tax . To activate HTLV-1 transcription Tax interacts with the cellular protein CREB and the pleiotropic coactivators CBP and p300 . While extensively studied, the molecular mechanisms of Tax transcription function and coactivator utilization are not fully understood . Previous studies have focused on Tax binding to the KIX domain of CBP, as this was believed to be the key step in recruiting the coactivator to the HTLV-1 promoter . In this study, we identify a carboxy-terminal region of CBP (and p300) that strongly interacts with Tax and mediates Tax transcription function . Through deletion mutagenesis, we identify amino acids 2003 to 2212 of CBP, which we call carboxy-terminal region 2 (CR2), as the minimal region for Tax interaction . Interestingly, this domain corresponds to the steroid receptor coactivator 1 (SRC-1)-interacting domain of CBP . We show that a double point mutant targeted to one of the putative alpha-helical motifs in this domain significantly compromises the interaction with Tax . We also characterize the region of Tax responsible for interaction with CR2 and show that the previously identified transactivation domain of Tax (amino acids 312 to 319) participates in CR2 binding . This region of Tax corresponds to a consensus amphipathic helix, and single point mutations targeted to amino acids on the face of this helix abolish interaction with CR2 and dramatically reduce Tax transcription function . Finally, we demonstrate that Tax and SRC-1 bind to CR2 in a mutually exclusive fashion . Together, these studies identify a novel Tax-interacting site on CBP/p300 and extend our understanding of the molecular mechanism of Tax transactivation.

Mol Cell Biol, 2001 Aug, 21(16), 5408 - 16
Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts; Yehudai-Resheff S et al.; The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase) . In Escherichia coli, polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence . While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it . Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts . Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures . The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of P(i) and ADP . As expected of a phosphorylase, P(i) enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization . In addition, searching the complete Arabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide . These results suggest that there is no enzyme similar to E . coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.

J Biol Chem, 2001 Sep 21, 276(38), 35842 - 6 Epub 2001 Jul 19.
tau binds and organizes Escherichia coli replication proteins through distinct domains: domain III, shared by gamma and tau, oligomerizes DnaX; Glover BP et al.; The tau and gamma proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with gamma being a truncated version of tau arising from ribosomal frameshifting . tau is comprised of five structural domains, the first three of which are shared by gamma (Gao, D., and McHenry, C . (2001) J . Biol . Chem . 276, 4433-4453) . In the absence of the other holoenzyme subunits, DnaX exists as a tetramer . Association of delta, delta', chi, and psi with domain III of DnaX(4) results in a DnaX complex with a stoichiometry of DnaX(3)deltadelta'chipsi . To identify which domain facilitates DnaX self-association, we examined the properties of purified biotin-tagged DnaX fusion proteins containing domains I-II or III-V . Unlike domain I-II, treatment of domain III-V, gamma, and tau with the chemical cross-linking reagent BS3 resulted in the appearance of high molecular weight intramolecular cross-linked protein . Gel filtration of domains I-II and III-V demonstrated that domain I-II was monomeric, and domain III-V was an oligomer . Biotin-tagged domain III-V, and not domain I-II, was able to form a mixed DnaX complex by recruiting tau, delta, delta', chi, and psi onto streptavidin-agarose beads . Thus, domain III not only contains the delta, delta', chi, and psi binding interface, but also the region that enables DnaX to oligomerize.

J Biol Chem, 2001 Sep 14, 276(37), 34695 - 701 Epub 2001 Jul 19.
Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor; Leimkuhler S et al.; Amino acid sequence comparisons of Escherichia coli MoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of molybdopterin synthase might resemble the ubiquitin-activating step in the ubiquitin-targeted degradation of proteins in eukaryotes . To determine the exact role of MoeB in molybdopterin biosynthesis, the protein was purified after homologous overexpression . Using purified proteins, we have demonstrated the ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to gel filtration . Mass spectrometry of the complex revealed a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adenylate . However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed . There was also no evidence for a MoeB-bound sulfur during the sulfuration of MoaD . Amino acid substitutions were generated in every cysteine residue in MoeB . All of these exhibited activity comparable to the wild type, with the exception of mutations in cysteine residues located in putative Zn-binding motifs . For these cysteines, loss of activity correlated with loss of metal binding.

J Biol Chem, 2001 Sep 14, 276(37), 35217 - 22 Epub 2001 Jul 19.
Assembly of DNA polymerase III holoenzyme: co-assembly of gamma and tau is inhibited by DnaX complex accessory proteins but stimulated by DNA polymerase III core; Pritchard AE et al.; Although the two alternative Escherichia coli dnaX gene products, tau and gamma, are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood . When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing tau but not gamma . We developed a new assay based on the binding of complexes containing biotin-tagged tau to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III holoenzyme subunits on the kinetics of co-assembly of gamma and tau into the same complex . Auxiliary proteins in combination with delta' almost completely blocked co-assembly, whereas chipsi or delta' alone slowed the association only moderately compared with the interaction of tau with gamma alone . In contrast, DNA polymerase III core, in the absence of deltadelta' and chipsi, accelerated the co-assembly of tau and gamma, suggesting a role for DNA polymerase III' {tau(2)(pol III core)(2)} in the assembly pathway of holoenzyme.

EMBO Rep, 2001 Aug, 2(8), 709 - 14 Epub 2001 Jul 19.
YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids; Beck K et al.; Like its mitochondrial homolog Oxa1p, the inner membrane protein YidC of Escherichia coli is involved in the integration of membrane proteins . We have analyzed individual insertion steps of the polytopic E . coli membrane protein MtlA targeted as ribosome-nascent chain complexes to inner membrane vesicles . YidC can accommodate at least the first two transmembrane segments of MtlA at the protein lipid interface and retain them even though the length of the nascent chain would amply allow insertion into membrane lipids . An even longer insertion intermediate of MtlA is described that still has the first transmembrane helix bound to YidC while the third contacts SecE and YidC during integration . Our findings suggest that YidC forms a contiguous integration unit with the SecYE translocon and functions as an assembly site for polytopic membrane proteins mediating the formation of helix bundles prior to their release into the membrane lipids.

Biophys J, 2001 Aug, 81(2), 1195 - 204
Substrate binding to DNA photolyase studied by electron paramagnetic resonance spectroscopy; Weber S et al.; Structural changes in Escherichia coli DNA photolyase induced by binding of a (cis,syn)-cyclobutane pyrimidine dimer (CPD) are studied by continuous-wave electron paramagnetic resonance and electron-nuclear double resonance spectroscopies, using the flavin adenine dinucleotide (FAD) cofactor in its neutral radical form as a naturally occurring electron spin probe . The electron paramagnetic resonance/electron-nuclear double resonance spectral changes are consistent with a large distance (> or =0.6 nm) between the CPD lesion and the 7,8-dimethyl isoalloxazine ring of FAD, as was predicted by recent model calculations on photolyase enzyme-substrate complexes . Small shifts of the isotropic proton hyperfine coupling constants within the FAD's isoalloxazine moiety can be understood in terms of the cofactor binding site becoming more nonpolar because of the displacement of water molecules upon CPD docking to the enzyme . Molecular orbital calculations of hyperfine couplings using density functional theory, in conjunction with an isodensity polarized continuum model, are presented to rationalize these shifts in terms of the changed polarity of the medium surrounding the FAD cofactor.

Biophys J, 2001 Aug, 81(2), 917 - 36
Structural models of the MscL gating mechanism; Sukharev S et al.; Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations . The modeling began with the crystal structure of M . tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit . The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1 . A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate . As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore . Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart . During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex . The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s . These models are consistent with numerous experimental results and modeling criteria.

Int J Antimicrob Agents, 2001 Jul, 18(1), 89 - 91
Effect of cyclosporin on uropathogenic Escherichia coli adherence to human endothelial cells; Szkaradkiewicz A et al.; The effect of cyclosporin A on the adherence of Escherichia coli strains to human umbilical vein endothelial cells was studied . Compared with the controls, bacterial adherence significantly increased following preincubation of the cells with cyclosporin A at 50 mg/l and more significantly, to cells preincubated with cyclosporin A at 100-200 mg/l . The results showed that cyclosporin A use may be accompanied by an increased bacterial adherence to host cells in vivo . This effect was achieved using a wide range of therapeutic doses.

Cancer Lett, 2001 Sep 20, 170(2), 147 - 52
Identification and characterization of human deoxyguanosine kinase cDNA fragments; Mansson E et al.; Mitochondria require deoxyribonucleoside triphosphates for the synthesis of their DNA and one of the enzymes responsible for the initial phosphorylation of purine deoxyribonucleoside is deoxyguanosine kinase (dGK; EC 2.7.1.113) . Recent studies have suggested that dGK in addition to deoxycytidine kinase phosphorylates several anti-cancer agents, such as 9-beta-D-arabinofuranosylguanine (Ara-G), cladribine (CdA), and fludarabine . There appear to coexist several mRNA fragments of dGK . In the present study we found 10 fragments, the longest fragment had 834bp, and represented the entire open reading frame of dGK (780bp) . The nine additional fragments detected ranged from 807 to 269bp . All the fragments were found to contain the specific mitochondria translocation signal sequence . Expression of these fragments in Escherichia coli demonstrated that only the full-length dGK resulted in a protein that could phosphorylate CdA and Ara-G . Given the difficulty to measure the full-length dGK, these data are of value for studying the mRNA gene expression of dGK in cell lines and in leukemic cells from patients.

Mol Biochem Parasitol, 2001 Aug, 116(1), 73 - 9
Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum; Kawazu S et al.; We have identified the 2-Cys peroxiredoxin (PfPrx-1) from the human malaria parasite Plasmodium falciparum . The PfPrx-1 showed the highest identity at amino acid level to the type II Prx among the currently known six subfamilies of mammalian Prx . The sequence identity between the PfPrx-1 and the previously reported 1-Cys Prx of P . falciparum (PfPrx-2), which corresponded to mammalian type VI Prx, was 25% . This suggests that the parasite possesses two Prx subfamilies . The PfPrx-1 showed significant sequence similarities with those of 2-Cys peroxiredoxins of plants in the BLASTX search . This may reflect the consequences of a genetic transfer from an algal endosymbiont to the parasite nucleus during evolution . The recombinant PfPrx-1 protein (rPfPrx-1) was expressed as a histidine fusion protein in Escherichia coli and purified with Ni chromatography . The rPfPrx-1 existed as dimers under non-reducing conditions and dissociated into monomers in the presence of dithiothreitol . The PfPrx-1 protein also exists as a dimer in the parasites themselves . The reduction of the oxidized enzyme by the donation of electrons from E . coli thioredoxin (Trx)/Trx reductase system was demonstrated in its reaction with H(2)O(2), using the rPfPrx-1 protein . These results suggested that the PfPrx-1 can act as a terminal peroxidase of the parasite Trx system . An elevated expression of the PfPrx-1 protein seen in the trophozoite, the stage with active metabolism, suggests an association of the parasite Trx system with its intracellular redox control.

Mol Cell, 2001 Mar, 7(3), 603 - 14
Structural model for the cooperative assembly of HIV-1 Rev multimers on the RRE as deduced from analysis of assembly-defective mutants; Jain C et al.; The functional efficacy of the HIV-1 Rev protein is highly dependent on its ability to assemble onto its HIV-1 RNA target (the RRE) as a multimeric complex . To elucidate the mechanism of multimeric assembly, we have devised two rapid and broadly applicable strategies for examining cooperative interactions between proteins bound to RNA, one based on cooperative translational repression of a two-site reporter and the other on gel shift analysis with crude E . coli extracts . Using these strategies, we have identified two distinct surfaces of Rev (head and tail) that are critical for different steps in multimeric assembly . Our data indicate that Rev assembles cooperatively on the RRE via a series of symmetrical tail-to-tail and head-to-head protein-protein interactions . The insights into molecular architecture suggested by these findings have enabled us to derive a structural model for Rev and its multimerization on the RRE.

Mol Cell, 2001 Mar, 7(3), 571 - 9
SOS mutator DNA polymerase IV functions in adaptive mutation and not adaptive amplification; McKenzie GJ et al.; Adaptive point mutation and amplification are induced responses to environmental stress, promoting genetic changes that can enhance survival . A specialized adaptive mutation mechanism has been documented in one Escherichia coli assay, but its enzymatic basis remained unclear . We report that the SOS-inducible, error-prone DNA polymerase (pol) IV, encoded by dinB, is required for adaptive point mutation in the E . coli lac operon . A nonpolar dinB mutation reduces adaptive mutation frequencies by 85% but does not affect adaptive amplification, growth-dependent mutation, or survival after oxidative or UV damage . We show that pol IV, together with the major replicase, pol III, can account for all adaptive point mutations at lac . The results identify a role for pol IV in inducible genetic change.

Biochem J, 2001 Aug 1, 357(Pt 3), 749 - 57
Regulation of yeast acetohydroxyacid synthase by valine and ATP; Pang SS et al.; The first step in the common pathway for the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (AHAS; EC 4.1.3.18) . The enzyme is found in plants, fungi and bacteria, and is regulated by controls on transcription and translation, and by allosteric modulation of catalytic activity . It has long been known that the bacterial enzyme is composed of two types of subunit, and a similar arrangement has been found recently for the yeast and plant enzymes . One type of subunit contains the catalytic machinery, whereas the other has a regulatory function . Previously, we have shown {Pang and Duggleby (1999) Biochemistry 38, 5222--5231} that yeast AHAS can be reconstituted from its separately purified subunits . The reconstituted enzyme is inhibited by valine, and ATP reverses this inhibition . In the present work, we further characterize the structure and the regulatory properties of reconstituted yeast AHAS . High phosphate concentrations are required for reconstitution and it is shown that these conditions are necessary for physical association between the catalytic and regulatory subunits . It is demonstrated by CD spectral changes that ATP binds to the regulatory subunit alone, most probably as MgATP . Neither valine nor MgATP causes dissociation of the regulatory subunit from the catalytic subunit . The specificity of valine inhibition and MgATP activation are examined and it is found that the only effective analogue of either regulator of those tested is the non-hydrolysable ATP mimic, adenosine 5'-{beta,gamma-imido}triphosphate . The kinetics of regulation are studied in detail and it is shown that the activation by MgATP depends on the valine concentration in a complex manner that is consistent with a proposed quantitative model.

Biochem J, 2001 Aug 1, 357(Pt 3), 625 - 34
Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting; Washbourne P et al.; The release of neurotransmitter at a synapse occurs via the regulated fusion of synaptic vesicles with the plasma membrane . The fusion of the two lipid bilayers is mediated by a protein complex that includes the plasma membrane target soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (t-SNAREs), syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25), and the vesicle SNARE (v-SNARE), vesicle-associated membrane protein (VAMP) . Whereas syntaxin 1A and VAMP are tethered to the membrane by a C-terminal transmembrane domain, SNAP-25 has been suggested to be anchored to the membrane via four palmitoylated cysteine residues . We demonstrate that the cysteine residues of SNAP-25 are not required for membrane localization when syntaxin 1A is present . Analysis of the 7 S and 20 S complexes formed by mutants that lack cysteine residues demonstrates that the cysteines are required for efficient SNARE complex dissociation . Furthermore, these mutants are unable to support exocytosis, as demonstrated by a PC12 cell secretion assay . We hypothesize that syntaxin 1A serves to direct newly synthesized SNAP-25 through the Golgi transport pathway to the axons and synapses, and that palmitoylation of cysteine residues is not required for targeting, but to optimize interactions required for SNARE complex dissociation.

J Virol, 2001 Aug, 75(16), 7410 - 9
Reconstitution of a functional duck hepatitis B virus replication initiation complex from separate reverse transcriptase domains expressed in Escherichia coli; Beck J et al.; Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA) . Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (epsilon) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors . Functional complexes catalyze the synthesis of a short DNA primer that is templated by epsilon and covalently linked to the terminal protein (TP) domain of P protein . Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors . However, its limited translation capacity precludes a closer analysis of the complex . To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL . Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other . Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of epsilon-dependent DNA primer synthesis, demonstrating posttranslational activation . This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.

J Biol Chem, 2001 Sep 21, 276(38), 35523 - 9 Epub 2001 Jul 18.
Plants synthesize ethanolamine by direct decarboxylation of serine using a pyridoxal phosphate enzyme; Rontein D et al.; The established pathways from serine to ethanolamine are indirect and involve decarboxylation of phosphatidylserine . Here we show that plants can decarboxylate serine directly . Using a radioassay based on ethanolamine (Etn) formation, pyridoxal 5'-phosphate-dependent l-serine decarboxylase (SDC) activity was readily detected in soluble extracts from leaves of diverse species, including spinach, Arabidopsis, and rapeseed . A putative Arabidopsis SDC cDNA was identified by searching GenBank for sequences homologous to other amino acid decarboxylases and shown by expression in Escherichia coli to encode a soluble protein with SDC activity . This cDNA was further authenticated by complementing the Etn requirement of a yeast psd1 psd2 mutant . In a parallel approach, a cDNA was isolated from a rapeseed library by its ability to complement the Etn requirement of a yeast cho1 mutant and shown by expression in E . coli to specify SDC . The deduced Arabidopsis and rapeseed SDC polypeptides are 90% identical, lack obvious targeting signals, and belong to amino acid decarboxylase group II . Recombinant Arabidopsis SDC was shown to exist as a tetramer and to contain pyridoxal 5'-phosphate . It does not attack d-serine, l-phosphoserine, other l-amino acids, or phosphatidylserine and is not inhibited by Etn, choline, or their phosphoesters . As a soluble, pyridoxal 5'-phosphate enzyme, SDC contrasts sharply with phosphatidylserine decarboxylases, which are membrane proteins that have a pyruvoyl cofactor.

J Biol Chem, 2001 Sep 21, 276(38), 35707 - 13 Epub 2001 Jul 18.
Temporal translational control by a metastable RNA structure; Moller-Jensen J et al.; Programmed cell death by the hok/sok locus of plasmid R1 relies on a complex translational control mechanism . The highly stable hok mRNA is activated by 3'-end exonucleolytical processing . Removal of the mRNA 3' end releases a 5'-end sequence that triggers refolding of the mRNA . The refolded hok mRNA is translatable but can also bind the inhibitory Sok antisense RNA . Binding of Sok RNA leads to irreversible mRNA inactivation by an RNase III-dependent mechanism . A coherent model predicts that during transcription hok mRNA must be refractory to translation and antisense RNA binding . Here we provide genetic evidence for the existence of a 5' metastable structure in hok mRNA that locks the nascent transcript in an inactive configuration in vivo . Consistently, the metastable structure reduces the rate of Sok RNA binding and completely blocks hok translation in vitro . Structural analyses of native RNAs strongly support that the 5' metastable structure exists in the nascent transcript . Further structural analyses reveal that the mRNA 3' end triggers refolding of the mRNA 5' end into the more stable tac-stem conformation . These results provide a profound understanding of an unusual and intricate post-transcriptional control mechanism.

Hybridoma, 2001 Jun, 20(3), 175 - 81
Generation and characterization of recombinant ScFv antibodies detecting Eimeria acervulina surface antigens; Kim JK et al.; In our previous attempt to generate monoclonal antibodies (MAbs) against coccidia parasites that more accurately reflect the natural avian humoral immune response, we produced two chicken B-cell hybridomas, 5D11 and 2-1 . While both cell lines secreted antibodies reactive with sporozoites of Eimeria acervulina, they were produced in yields too low to conduct meaningful in vivo studies . To circumvent this problem, we produced four single chain variable fragment (scFv) antibodies from the V(H) and V(L) genes of hybridomas 5D11 and 2-1 . The concentration of these recombinant antibodies expressed in E . coli and purified to homogeneity was 5-6 mg/L . Three of the antibodies exhibited antigen binding specificity to Eimeria surface antigens equivalent to that of the native MAbs . Nucleotide sequence analysis of the V(L) genes from hybridomas 5D11 and 2-1 and genomic DNA revealed vestiges of gene conversion with V(lambda) pseudogenes . These recombinant scFv antibodies will prove useful for further characterization of natural Eimeria surface antigens as potential vaccine candidates.

Phys Rev E Stat Nonlin Soft Matter Phys . 2001 Jul;64(1 Pt 1):011913 . Epub 2001 Jun 21.
Self-consistent simulations of electroporation dynamics in biological cells subjected to ultrashort electrical pulses; Joshi RP et al.; The temporal dynamics of electroporation of cells subjected to ultrashort voltage pulses are studied based on a coupled scheme involving the Laplace, Nernst-Plank, and Smoluchowski equations . A pore radius dependent energy barrier for ionic transport, accounts for cellular variations . It is shown that a finite time delay exists in pore formation, and leads to a transient overshoot of the transmembrane potential V(mem) beyond 1.0 V . Pore resealing is shown to consist of an initial fast process, a 10(-4) s delay, followed by a much slower closing at a time constant of about 10(-1) s . This establishes a time-window during which the pores are mostly open, and hence, the system is most vulnerable to destruction by a second electric pulse . The existence of such a time window for effective killing by a second pulse is amply supported by our experimental data for E . coli cells . The time constant for the longer process also matches experiments . The study suggests that controlled manipulation of the pore "open times" can be achieved through multiple, ultrashort pulses.

Mol Genet Metab, 2001 Jul, 73(3), 280 - 4
A phenylalanine hydroxylase amino acid polymorphism with implications for molecular diagnostics; Gjetting T et al.; Mutations in the gene encoding phenylalanine hydroxylase (PAH, EC 1.14.16.1) are associated with various degrees of hyperphenylalaninemia, including classical phenylketonuria (PKU) . We examined the PAH gene in a Brazilian PKU family of African origin and identified three missense variants, R252W (c.754C --> T), K274E (c.820A --> G), and I318T (c.953T --> C), the two latter of which were transmitted in cis . Expression analyses in two different in vitro systems showed that I318T is associated with profoundly decreased enzyme activity, whereas the enzyme activity of K274E is indistinguishable from that of the wild-type protein . Detailed kinetic analyses of PAH expressed in E . coli showed that the K274E mutant protein has kinetic properties similar to that of the wild-type protein . Population studies have suggested that the K274E variant occurs on approximately 4% of African-American PAH alleles, whereas the neonatal screening incidence of PKU among African Americans is only 1:100,000 . This is to our knowledge the first demonstration of a PAH missense variant with no apparent association to PAH deficiency . Awareness of this common variant may be helpful to laboratories that perform molecular diagnosis of PAH deficiency in populations of African origin .

Methods Enzymol, 2001, 338, 283 - 304
Uniform 13C/15N-labeling of DNA by tandem repeat amplification; Werner MH et al.; An optimized procedure has been described for the large-scale production of stable isotopeenriched duplex oligonucleotides of designed sequence . Large-scale production of labeled nucleotide triphosphates can be produced in this procedure simultaneously with labeled proteins, thereby providing synthetic dNMP precursors at no additional cost . The procedure is robust, with a minimum product:template yield of 800:1 overall, and produces > 99% single-length product . Tandem repeat PCR amplification is a general approach to large scale synthesis of duplex oligonucleotides and may have applications to both NMR and X-ray methods, particularly for product lengths in excess of 25 base pairs where failed sequences from solid-phase synthesis can be difficult to remove chromatographically . A drawback of the present approach is that the product is a duplex of two equal-length strands, making single-stranded products more difficult to prepare . For this reason, it could be preferable to produce single-stranded products by the {figure: see text} method of Zimmer and Crothers . Although a single base type can be selectively enriched in this approach, chemical synthesis will provide greater flexibility for labeled DNAs requiring site-specific labels at only one or a small number of nucleotide positions in the sequence . Therefore, maximum flexibility in labeling patterns can be realized by judicious choice of labeling method appropriate to the type of DNA product and extent of isotopic enrichment desired.

Photochem Photobiol, 2001 Jul, 74(1), 46 - 54
Resistance of a lizard (the green anole, Anolis carolinensis; Polychridae) to ultraviolet radiation-induced immunosuppression; Cope RB et al.; The green anole (Anolis carolinensis) is the most northerly distributed of its Neotropical genus . This lizard avoids a winter hibernation phase by the use of sun basking behaviors . Inevitably, this species is exposed to high doses of ambient solar ultraviolet radiation (UVR) . Increases in terrestrial ultraviolet-B (UV-B) radiation secondary to stratospheric ozone depletion and habitat perturbation potentially place this species at risk of UVR-induced immunosuppression . Daily exposure to subinflammatory UVR (8 kJ/m2/day UV-B, 85 kJ/m2/day ultraviolet A {UV-A}), 6 days per week for 4 weeks (total cumulative doses of 192 kJ/m2 UV-B, 2.04 x 10(3) kJ/m2 UV-A) did not suppress the anole's acute or delayed type hypersensitivity (DTH) response to horseshoe crab hemocyanin . In comparison with the available literature UV-B doses as low as 0.1 and 15.9 kJ/m2 induced suppression of DTH responses in mice and humans, respectively . Exposure of anoles to UVR did not result in the inhibition of ex vivo splenocyte phagocytosis of fluorescein labeled Escherichia coli or ex vivo splenocyte nitric oxide production . Doses of UV-B ranging from 0.35 to 45 kJ/m2 have been reported to suppress murine splenic/peritoneal macrophage phagocytosis and nitric oxide production . These preliminary studies demonstrate the resistance of green anoles to UVR-induced immunosuppression . Methanol extracts of anole skin contained two peaks in the ultraviolet wavelength range that could be indicative of photoprotective substances . However, the resistance of green anoles to UVR is probably not completely attributable to absorption by UVR photoprotective substances in the skin but more likely results from a combination of other factors including absorption by the cutis and absorption and reflectance by various components of the dermis.

Biotechnol Bioeng, 2001 Mar 5, 72(5), 573 - 6
Characterization of an oxygen-dependent inducible promoter, the nar promoter of Escherichia coli, to utilize in metabolic engineering; Han SJ et al.; The nar promoters, whose transcription is maximally induced under microaerobic conditions in the presence of nitrate ion, were characterized in fed-batch culture to determine whether they can be used for metabolic engineering, by which overall production of valuable chemicals can be increased . For this purpose, we tested whether the expression level of a reporter gene, the lacZ gene from the nar promoter, could be maintained constant throughout the induction period by manipulation of dissolved oxygen (DO) levels at a given nitrate ion concentration . First, E . coli was grown under aerobic conditions (DO 80%) to absorbance at 600 nm (OD(600)) of 35, then the nar promoter was induced by reduction of DO to different levels, combined with different frequencies and duration of alternating microaerobic and aerobic conditions throughout the entire induction period . For a wild-type nar promoter (pMW61) in a mutant host E . coli with a mutation in the narG gene on the chromosome of the host (RK5265), it was possible to maintain production of beta-galactosidase activity per cell (specific beta-galactosidase activity) at a constant rate at 5000, 10,000, 15,000, and 20,000 Miller units, using different combinations of nitrate ion concentrations (0.1%, 0.5%, and 1%) and DO levels . In addition, it was possible to maintain production of specific beta-galactosidase activity at a constant rate at about 10,000 Miller units in the absence of nitrate ion when a nitrate-independent nar promoter (pMW618) in the narL(-) mutant of the W3110 E . coli strain (W3110narL(-)) was used . Based on these results, we conclude that the nar promoter system provides a convenient expression system for metabolic engineering as well as for maximal production of recombinant proteins under conditions of fed-batch culture .

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8433 - 9
The synaptic activity of HsDmc1, a human recombination protein specific to meiosis; Gupta RC et al.; Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51 . Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein . However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings . These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology . Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET) . Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate . The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A small middle dotT base pairs for recognition of homology . We conclude that Dmc1, like human Rad51 and E . coli RecA protein, promotes homologous pairing and strand exchange by a "synaptic pathway" involving a three-stranded nucleoprotein intermediate, rather than by a "helicase pathway" involving the separation and reannealing of DNA strands.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8425 - 32
Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: a possible advantage of DNA over RNA as genomic material; Shibata T et al.; Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes . A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins . In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology . Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure . This structure contains a hydrophobic interaction between the 2' methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers . This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints . The pivotal role of the 2' methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes . Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival . The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling) . These properties may give an extensive evolutionary advantage to DNA.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8350 - 4
Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli; Pham P et al.; DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD(2)(') proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), beta sliding clamp, and gamma clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS) . DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner . Replication-restart takes place almost immediately after the DNA is damaged (approximately 2 min post-UV irradiation), whereas TLS occurs after pol V is induced approximately 50 min later . We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart . Specific roles during TLS for pol V and each of its accessory factors have been recently determined . Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8319 - 25
Instability of repetitive DNA sequences: the role of replication in multiple mechanisms; Bzymek M et al.; Rearrangements between tandem sequence homologies of various lengths are a major source of genomic change and can be deleterious to the organism . These rearrangements can result in either deletion or duplication of genetic material flanked by direct sequence repeats . Molecular genetic analysis of repetitive sequence instability in Escherichia coli has provided several clues to the underlying mechanisms of these rearrangements . We present evidence for three mechanisms of RecA-independent sequence rearrangements: simple replication slippage, sister-chromosome exchange-associated slippage, and single-strand annealing . We discuss the constraints of these mechanisms and contrast their properties with RecA-dependent homologous recombination . Replication plays a critical role in the two slipped misalignment mechanisms, and difficulties in replication appear to trigger rearrangements via all these mechanisms.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8227 - 34
Rescue of stalled replication forks by RecG: simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation; McGlynn P et al.; Modification of damaged replication forks is emerging as a crucial factor for efficient chromosomal duplication and the avoidance of genetic instability . The RecG helicase of Escherichia coli, which is involved in recombination and DNA repair, has been postulated to act on stalled replication forks to promote replication restart via the formation of a four-stranded (Holliday) junction . Here we show that RecG can actively unwind the leading and lagging strand arms of model replication fork structures in vitro . Unwinding is achieved in each case by simultaneous interaction with and translocation along both the leading and lagging strand templates at a fork . Disruption of either of these interactions dramatically inhibits unwinding of the opposing duplex arm . Thus, RecG translocates simultaneously along two DNA strands, one with 5'-3' and the other with 3'-5' polarity . The unwinding of both nascent strands at a damaged fork, and their subsequent annealing to form a Holliday junction, may explain the ability of RecG to promote replication restart . Moreover, the preferential binding of partial forks lacking a leading strand suggests that RecG may have the ability to target stalled replication intermediates in vivo in which lagging strand synthesis has continued beyond the leading strand.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8211 - 8
RecA protein promotes the regression of stalled replication forks in vitro; Robu ME et al.; Replication forks are halted by many types of DNA damage . At the site of a leading-strand DNA lesion, forks may stall and leave the lesion in a single-strand gap . Fork regression is the first step in several proposed pathways that permit repair without generating a double-strand break . Using model DNA substrates designed to mimic one of the known structures of a fork stalled at a leading-strand lesion, we show here that RecA protein of Escherichia coli will promote a fork regression reaction in vitro . The regression process exhibits an absolute requirement for ATP hydrolysis and is enhanced when dATP replaces ATP . The reaction is not affected by the inclusion of the RecO and R proteins . We present this reaction as one of several potential RecA protein roles in the repair of stalled and/or collapsed replication forks in bacteria.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8203 - 10
Effects of mutations involving cell division, recombination, and chromosome dimer resolution on a priA2::kan mutant; McCool JD et al.; Recombinational repair of replication forks can occur either to a crossover (XO) or noncrossover (non-XO) depending on Holliday junction resolution . Once the fork is repaired by recombination, PriA is important for restarting these forks in Escherichia coli . PriA mutants are Rec(-) and UV sensitive and have poor viability and 10-fold elevated basal levels of SOS expression . PriA sulB mutant cells and their nucleoids were studied by differential interference contrast and fluorescence microscopy of 4',6-diamidino-2-phenylindole-stained log phase cells . Two populations of cells were seen . Eighty four percent appeared like wild type, and 16% of the cells were filamented and had poorly partitioned chromosomes (Par(-)) . To probe potential mechanisms leading to the two populations of cells, mutations were added to the priA sulB mutant . Mutating sulA or introducing lexA3 decreased, but did not eliminate filamentation or defects in partitioning . Mutating either recA or recB virtually eliminated the Par(-) phenotype . Filamentation in the recB mutant decreased to 3%, but increased to 28% in the recA mutant . The ability to resolve and/or branch migrate Holliday junctions also appeared crucial in the priA mutant because removing either recG or ruvC was lethal . Lastly, it was tested whether the ability to resolve chromosome dimers caused by XOs was important in a priA mutant by mutating dif and the C-terminal portion of ftsK . Mutation of dif showed no change in phenotype whereas ftsK1cat was lethal with priA2kan . A model is proposed where the PriA-independent pathway of replication restart functions at forks that have been repaired to non-XOs.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8196 - 202
Participation of recombination proteins in rescue of arrested replication forks in UV-irradiated Escherichia coli need not involve recombination; Courcelle J et al.; Alternative reproductive cycles make use of different strategies to generate different reproductive products . In Escherichia coli, recA and several other rec genes are required for the generation of recombinant genomes during Hfr conjugation . During normal asexual reproduction, many of these same genes are needed to generate clonal products from UV-irradiated cells . However, unlike conjugation, this latter process also requires the function of the nucleotide excision repair genes . Following UV irradiation, the recovery of DNA replication requires uvrA and uvrC, as well as recA, recF, and recR . The rec genes appear to be required to protect and maintain replication forks that are arrested at DNA lesions, based on the extensive degradation of the nascent DNA that occurs in their absence . The products of the recJ and recQ genes process the blocked replication forks before the resumption of replication and may affect the fidelity of the recovery process . We discuss a model in which several rec gene products process replication forks arrested by DNA damage to facilitate the repair of the blocking DNA lesions by nucleotide excision repair, thereby allowing processive replication to resume with no need for strand exchanges or recombination . The poor survival of cellular populations that depend on recombinational pathways (compared with that in their excision repair proficient counterparts) suggests that at least some of the rec genes may be designed to function together with nucleotide excision repair in a common and predominant pathway by which cells faithfully recover replication and survive following UV-induced DNA damage.

Proc Natl Acad Sci U S A, 2001 Jul 31, 98(16), 9359 - 64 Epub 2001 Jul 17.
Assembling filamentous phage occlude pIV channels; Marciano DK et al.; Filamentous phage f1 is exported from its Escherichia coli host without killing the bacterial cell . Phage-encoded protein pIV, which is required for phage assembly and secretion, forms large highly conductive channels in the outer membrane of E . coli . It has been proposed that the phage are extruded across the bacterial outer membrane through pIV channels . To test this prediction, we developed an in vivo assay by using a mutant pIV that functions in phage export but whose channel opens in the absence of phage extrusion . In E . coli lacking its native maltooligosacharride transporter LamB, this pIV variant allowed oligosaccharide transport across the outer membrane . This entry of oligosaccharide was decreased by phage production and still further decreased by production of phage that cannot be released from the cell surface . Thus, exiting phage block the pIV-dependent entry of oligosaccharide, suggesting that phage occupy the lumen of pIV channels . This study provides the first evidence, to our knowledge, for viral exit through a large aqueous channel.

J Biol Chem, 2001 Sep 28, 276(39), 36268 - 74 Epub 2001 Jul 17.
Thiocarboxylation of molybdopterin synthase provides evidence for the mechanism of dithiolene formation in metal-binding pterins; Gutzke G et al.; Molybdopterin (MPT) is a pyranopterin with a unique dithiolene group coordinating molybdenum (Mo) or tungsten (W) in all Mo- and W-enzymes except nitrogenase . In Escherichia coli, MPT is formed by incorporation of two sulfur atoms into precursor Z, which is catalyzed by MPT synthase . The recently solved crystal structure of MPT synthase (Rudolph, M . J., Wuebbens, M . M., Rajagopalan, K . V., and Schindelin, H . (2000) Nat . Struct . Biol . 8, 42-46) shows the heterotetrameric nature of the enzyme that is composed of two small (MoaD) and two large subunits (MoaE) . According to sequence and structural similarities among MoaD, ubiquitin, and ThiS, a thiocarboxylation of the C terminus of MoaD is proposed that would serve as the source of sulfur that is transferred to precursor Z . Here, we describe the in vitro generation of carboxylated and thiocarboxylated MoaD . Both forms of MoaD are monomeric and are able to form a heterotetrameric complex after coincubation in equimolar ratios with MoaE . Only the thiocarboxylated MPT synthase complex was found to be able to convert precursor Z in vitro to MPT . Slight but significant differences between the carboxylated and the thiocarboxylated MPT synthase can be seen using size exclusion chromatography . A two-step reaction of MPT synthesis is proposed where the dithiolene is generated by two thiocarboxylates derived from a single tetrameric MPT synthase.

J Biol Chem, 2001 Sep 28, 276(39), 36831 - 8 Epub 2001 Jul 17.
Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis; Hoffmann L et al.; Animal catechol O-methyltransferases and plant caffeoyl-coenzyme A O-methyltransferases share about 20% sequence identity and display common structural features . The crystallographic structure of rat liver catechol O-methyltransferase was used as a template to construct a homology model for tobacco caffeoyl-coenzyme A O-methyltransferase . Integrating substrate specificity data, the three-dimensional model identified several amino acid residues putatively involved in substrate binding . These residues were mutated by a polymerase chain reaction method and wild-type and mutant enzymes were each expressed in Escherichia coli and purified . Substitution of Arg-220 with Thr resulted in the total loss of enzyme activity, thus indicating that Arg-220 is involved in the electrostatic interaction with the coenzyme A moiety of the substrate . Changes of Asp-58 to Ala and Gln-61 to Ser were shown to increase K(m) values for caffeoyl coenzyme A and to decrease catalytic activity . Deletions of two amino acid sequences specific for plant enzymes abolished activity . The secondary structures of the mutants, as measured by circular dichroism, were essentially unperturbed as compared with the wild type . Similar changes in circular dichroism spectra were observed after addition of caffeoyl coenzyme A to the wild-type enzyme and the substitution mutants but not in the case of deletion mutants, thus revealing the importance of these sequences in substrate-enzyme interactions.

J Biol Chem, 2001 Sep 28, 276(39), 36261 - 7 Epub 2001 Jul 17.
Elucidation of the differences between the 430- and 455-nm absorbing forms of P450-isocyanide adducts by resonance Raman spectroscopy; Tomita T et al.; Alkylisocyanide adducts of microsomal P450 exist in two interconvertible forms, each giving the Soret maximum around 430 or 455 nm . This is demonstrated with a rabbit liver P450 2B4 . Resonance Raman spectra of the 430- and 455-nm forms were examined for typical P450s of the two types as well as for P450 2B4 because the 430-nm form of P450 2B4 is liable to change into P420 . P450cam and P450nor were selected as a model of the 430- and 455-nm forms, respectively . For the n-butyl isocyanide (CNBu) adduct, the Fe(II)-CNBu stretching band was observed for the first time at 480/467 cm(-1) for P450cam and at 471/459 cm(-1) for P450nor with their (12)CNBu/(13)CNBu derivatives . For P450cam, but not P450nor, other (13)C isotope-sensitive bands were observed at 412/402, 844/835, and 940/926 cm(-1) . The C-N stretching mode was identified by Fourier transform IR spectroscopy at 2116/2080 cm(-1) for P450cam and at 2148/2108 cm(-1) for P450nor for the (12)C/(13)C derivatives . These findings suggest that the binding geometry of isocyanide differs between the two forms-bent and linear structures for P450cam-CNBu and P450nor-CNBu, respectively . In contrast, in the ferric state, the Raman (13)C isotopic frequency shifts, and the IR C-N stretching frequencies (2213/2170 and 2215/2172 cm(-1)) were similar between P450cam and P450nor, suggesting similar bent structures for both.

J Biol Chem, 2001 Sep 28, 276(39), 36225 - 32 Epub 2001 Jul 17.
The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ) . Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin; Schiffler B et al.; The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail . Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented . The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1 . According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants) . The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion . This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system . Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.

BMC Biochem . 2001;2(1):6 . Epub 2001 Jul 10.
Color transitions in coral's fluorescent proteins by site-directed mutagenesis; Gurskaya NG et al.; BACKGROUND: Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression . Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors . Fluorescent proteins can be subdivided into cyan (approximately 485 nm), green (approximately 505 nm), yellow (approximately 540 nm), and red (>580 nm) emitters . RESULTS: Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence . First, a blue-shifted mutant of cyan amFP486 was generated . Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence . Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins . The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow . Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence . CONCLUSIONS: Several amino acid substitutions were found to strongly affect fluorescence maxima . Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color . These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals.

J Am Chem Soc, 2001 Jul 25, 123(29), 7017 - 30
Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase; Baldwin J et al.; The outcome of O2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122*) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)2-(carboxylate)4 ligand set characteristic of the diiron-carboxylate family . Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH) . We previously showed that this substitution allows accumulation of a mu-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H(peroxo)) in O2 activation by MMOH . In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X . Decay of the mu-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122* and which converts very slowly (t1/2 approximately 7 h) upon incubation at 0 degrees C to an intensely purple final product . X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster . Resonance Raman (RR) spectra of the purple product generated with 16O2 or 18O2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O2 . Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mossbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product . The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which mu-O2- is lost and the F208-derived phenolate coordinates . The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon-hydroxyphenylalanine is formed and pathways leading to Y122* formation predominate in both R2-D84E and R2-W48F.

Comput Chem, 2001 Jul, 25(4), 401 - 10
Sequence alignment: an approximation law for the Z-value with applications to databank scanning; Bacro JN et al.; The Z-value is an attempt to estimate the statistical significance of a Smith and Waterman dynamic programming alignment score (H-score) through the use of a Monte-Carlo procedure . In this paper, we give an approximation for the Z-value law deduced from the Poisson clumping heuristic developed by Waterman and Vingron (Stat . Sci . 9 (1994) 367) in the case of independent and identically distributed sequences comparison . As for non-gapped alignment scores, our approximation is of Gumbel type but with parameters that are sequence independent . This result makes clear the related experimental results mentioned by Comet et al . (Comput . Chem . 23 (1999) 317) . Using 'quasi-real' sequences (i.e . randomly shuffled sequences of the same length and amino acid composition as the real ones) we investigate the relevance of our approximation result . Since the Monte-Carlo approach we use generates a bias for the Gumbel decay parameter estimation, a correction procedure is proposed . Applications to real sequences are considered and we show how our results can be used to detect the potential biological relationships between real sequences.

Mol Genet Genomics, 2001 Jun, 265(4), 748 - 54
DNA methylation inhibits expression and transposition of the Neurospora Tad retrotransposon; Zhou Y et al.; Tad is a LINE-like retrotransposon of the filamentous fungus Neurospora crassa . We have analyzed both expression and transposition of this element using strains with a single copy of Tad located in the 5' noncoding sequences of the am (glutamate dehydrogenase) gene . Tad in this position has been shown to carry a de novo cytosine methylation signal which causes reversible methylation of both Tad and am upstream sequences . Here we find that methylation of the Tad sequences inhibits both Tad expression and transposition . This inhibition can be relieved by the use of 5-azacytidine, a drug which reduces cytosine methylation, or by placing the Tad/am sequences in a dim-2 genetic background.

Mol Genet Genomics, 2001 Jun, 265(4), 615 - 21
Temperature-sensitive mutations in various genes of Escherichia coli K12 can be suppressed by the ssrA gene for 10Sa RNA (tmRNA); Nakano H et al.; An Escherichia coli strain with a deletion in the ssrA gene that encodes 10Sa RNA (tmRNA) was used to screen for temperature-sensitive (ts) mutants whose ts phenotypes were suppressible by introduction of the wild-type ssrA gene . Mutants in four different genes were isolated . Ts mutants of this type were also obtained in a screen for mutations in thyA, the structural gene for thymidylate synthase . The ThyA activity in crude extracts prepared from the ts mutants was temperature-sensitive . The presence of the ssrA gene caused an increase in the total amount of the temperature-sensitive enzyme expressed, rather than suppressing the ts activity of the enzyme itself . SsrA-DD, a mutant form of 10Sa RNA, suppressed the ts phenotype of a thyA mutant, suggesting that degradation of a tagged peptide was not required for suppression of the ts phenotype . Considering the fact that ssrA-suppressible mutants could be isolated as temperature-sensitive mutants with mutations in different genes, it seems evident that trans-translation can occur on mRNA that is not lacking its stop codon.

Nature, 2001 Jun 14, 411(6839), 820 - 4
Structure of a human gammadelta T-cell antigen receptor; Allison TJ et al.; T-cell antigen receptors composed of gamma and delta polypeptide chains (gammadelta TCRs) can directly recognize antigens in the form of intact proteins or non-peptide compounds, unlike alphabeta TCRs, which recognize antigens bound to major histocompatibility complex molecules (MHC) . About 5% of peripheral blood T cells bear gammadelta TCRs, most of which recognize non-peptide phosphorylated antigens . Here we describe the 3.1 A resolution structure of a human gammadelta TCR from a T-cell clone that is phosphoantigen-reactive . The orientation of the variable (V) and constant (C) regions of the gammadelta TCR is unique when compared with alphabeta TCRs or antibodies, and results from an unusually small angle between the Vgamma and Cgamma domains . The complementarity-determining regions (CDRs) of the V domains exhibit a chemically reasonable binding site for phosphorylated antigens, providing a possible explanation for the canonical usage of the Vgamma9 and Vdelta2 gene segments by phosphoantigen-reactive receptors . Although the gammadelta TCR V domains are similar in overall structure to those of alphabeta TCRs, gammadelta TCR C domains are markedly different . Structural differences in Cgamma and Cdelta, and in the location of the disulphide bond between them, may enable gammadelta TCRs to form different recognition/signalling complexes than alphabeta TCRs.

J Vet Med Sci, 2001 Jun, 63(6), 609 - 18
Identification of the porcine cytomegalovirus major capsid protein gene; Rupasinghe V et al.; A major capsid protein (MCP) gene homologue of porcine cytomegalovirus (PCMV) was identified . Sequence analysis indicated that the PCMV MCP gene is 4,026 nucleotides in length encoding a protein of 1,341 amino acid residues . The predicted molecular weight of the PCMV MCP is 151,456 Da, equivalent to those of other herpesvirus MCP counterparts . Phylogenetic analysis using herpesviral MCP gene sequences confirmed that PCMV is a betaherpesvirus with higher homology with human herpesvirus-6 and -7 than human and mouse cytomegaloviruses . The serum of pig experimentally infected with PCMV did not react with bacterially expressed MCP, suggesting that the PCMV MCP may not be related to the humoral immune response in the course of PCMV infection . Also, we established polymerase chain reaction (PCR) protocols using primers corresponding to MCP gene sequences for detection of PCMV infection . The PCR protocol would be effective for the diagnosis of slow-growing PCMV infection, for which traditional methods involving virus-isolation are not useful.

J Neural Transm, 2000, 107(12), 1393 - 401
Lipopolysaccharide administration produces time-dependent and region-specific alterations in tryptophan and tyrosine hydroxylase activities in rat brain; Nolan Y et al.; The present study examined the effect of systemic administration of lipopolysaccharide (LPS; 100 and 250 microg/kg, i.p.) on tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities in frontal cortex, striatum and midbrain of the rat . Enzyme activities were determined by measuring accumulation of the transient intermediates 5-hydroxytrptophan (5-HTP) and L-dihydroxyphenylalanine (L-DOPA) following in vivo administration of the decarboxylase inhibitor, NSD 1015 . TPH activity was increased 2 hours after administration of LPS (100 and 250 microg/kg) in both frontal cortex and midbrain, and a secondary increase was seen in the midbrain 12 hours after challenge . LPS provoked an increase in TH activity in the midbrain only, and this was evident for up to 24 hours after LPS administration . Thus in addition to previous studies demonstrating that LPS increases in vivo NA, DA and 5-HT release, this study shows that LPS increases the activity of the rate-limiting enzymes responsible for their synthesis.

Res Commun Mol Pathol Pharmacol, 2001 Jul, 109(1-2), 53 - 63
Kinetics of testosterone 6beta-hydroxylation in the reconstituted system with similar ratios of purified CYP3A4, NADPH-cytochrome p450 oxidoreductase and cytochrome B5 to human liver microsomes; Taguchi M et al.; Kinetics of testosterone 6beta-hydroxylation were determined using a reconstituted system that consisted of CYP3A4, cytochrome b5 and NADPH-cytochrome P450 oxidoreductase (OR) with similar ratios as those seen in human liver microsomes and compared with those determined using human liver microsomes . Two reconstituted systems were constructed in accordance with two human liver microsomal samples that showed extremely high and low ratios of OR/CYP3A4 . The Km values of testosterone 6beta-hydroxylation obtained from the reconstituted systems with high and low OR/CYP3A4 ratios were 29.3 and 35.2 microM, respectively, which were similar to that of the corresponding human liver microsomal samples (23.2 and 40.0 microM, respectively) . However, Vmax values obtained from the reconstituted systems (3.7 and 0.8 pmol/min/pmol CYP3A4) were much lower than those from the human liver microsomes (44.2 and 31.1 pmol/min/pmol CYP3A4) . The results suggest that the interaction between substrate and CYP3A4 in the reconstituted systems appear to be similar to human liver microsomes but that the velocity of the substrate metabolism in the reconstituted systems is different from that in human liver microsomes . In conclusion, our reconstituted systems could be used for the determination of affinity but not for the determination of the maximum velocity of substrate metabolism . Further studies on the protein-protein interactions between CYP3A4, OR, cytochrome b5 and/or a specific lipid environment are required to establish a reconstituted system showing similar kinetic properties to those of human liver microsomes.

J Gen Virol, 2001 Aug, 82(Pt 8), 1959 - 63
Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not; Drew PD et al.; Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells . We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting . MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains . Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli . These scAb retained the binding properties of the whole antibody . However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206 . Shortening the peptide linker joining the V(H) to the V(kappa) domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy . The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.

J Biol Chem, 2001 Sep 14, 276(37), 34847 - 52 Epub 2001 Jul 16.
Function of YidC for the insertion of M13 procoat protein in Escherichia coli: translocation of mutants that show differences in their membrane potential dependence and Sec requirement; Samuelson JC et al.; The membrane insertion of the Sec-independent M13 Procoat protein in bacteria requires the membrane electrochemical potential and the integral membrane protein YidC . We show here that YidC is involved in the translocation but not in the targeting of the Procoat protein, because we found the protein was partitioned into the membrane in the absence of YidC . YidC can function also to promote membrane insertion of Procoat mutants that insert independently of the membrane potential, proving that the effect of YidC depletion is not due to a dissipation of the membrane potential . We also found that YidC is absolutely required for Sec-dependent translocation of a long periplasmic loop of a mutant Procoat in which the periplasmic region has been extended from 20 to 194 residues . Furthermore, when Sec-dependent membrane proteins with large periplasmic domains were overproduced under YidC-limited conditions, we found that the exported proteins pro-OmpA and pre-peptidoglycan-associated lipoprotein accumulated in the cytoplasm . This suggests for Sec-dependent proteins that YidC functions at a late stage in membrane insertion, after the Sec translocase interacts with the translocating membrane protein . These studies are consistent with the understanding that YidC cooperates with the Sec translocase for membrane translocation and that YidC is required for clearing the protein-conducting channel.

J Biol Chem, 2001 Sep 28, 276(39), 36508 - 13 Epub 2001 Jul 16.
Oxyluciferin, a luminescence product of firefly luciferase, is enzymatically regenerated into luciferin; Gomi K et al.; The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis . The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW(XL) . This enzyme was a single polypeptide with a molecular mass of 38 kDa . LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid . In the presence of d-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin . The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis . We have cloned a cDNA encoding LRE from poly(A)+ RNA of the lantern of P . pyralis using reverse transcription-polymerase chain reaction, 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE . The primary structure of LRE from P . pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619 . The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.

J Biol Chem, 2001 Sep 7, 276(36), 34348 - 54 Epub 2001 Jul 16.
Motor function and regulation of myosin X; Homma K et al.; Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues . Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level . We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells . The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP) . The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain . Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end . The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility . Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V . Consistently nearly all actin dissociated from myosin X in the presence of ATP . ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting . These results suggest that myosin X is a nonprocessive motor . Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.

J Biol Chem, 2001 Sep 21, 276(38), 35581 - 8 Epub 2001 Jul 16.
Nonsense mutations in cspA cause ribosome trapping leading to complete growth inhibition and cell death at low temperature in Escherichia coli; Xia B et al.; CspA, the major cold shock protein of Escherichia coli, is dramatically induced immediately after cold shock . CspA production is transient and reduces to a low basal level when cells become adapted . Here we show that expression from multicopy plasmids of mutant cspA mRNAs bearing nonsense mutations in the coding region caused sustained high levels of the mutant mRNAs at low temperature, resulting in complete inhibition of cell growth ultimately leading to cell death . We demonstrate that the observed growth inhibition was caused by largely exclusive occupation of cellular ribosomes by the mutant cspA mRNAs . Such sequestration of ribosomes even occurs without a single peptide bond formation, implying that the robust translatability of the cspA mRNA is determined at the step of initiation . Further analysis demonstrated that the downstream box of the cspA mRNA was dispensable for the effect, whereas the upstream box of the mRNA was essential . Our system may offer a novel means to study sequence or structural elements involved in the translation of the cspA mRNA and may also be utilized to regulate bacterial growth at low temperature.

J Biol Chem, 2001 Sep 14, 276(37), 34600 - 6 Epub 2001 Jul 16.
SeqA protein aggregation is necessary for SeqA function; Lee H et al.; The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation . Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer . SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner . Using a bacterial two-hybrid system, we demonstrate that the N-terminal region of SeqA, especifically the 9th amino acid residue, glutamic acid, is required for functional SeqA-SeqA interaction . Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated DNA with altered binding patterns as compared with wild-type SeqA . Aggregates of SeqA(E9K) are defective in hemimethylated DNA binding . Here we demonstrate that proper interaction between SeqA tetramers is required for both hemimethylated DNA binding and formation of active aggregates . SeqA tetramers and aggregates might be involved in the formation of SeqA foci required for the segregation of chromosomal DNA as well as the regulation of chromosomal initiation.

J Steroid Biochem Mol Biol, 2001 Jun, 77(4-5), 261 - 9
Expression and characterization of the human 3 beta-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b); Meloche CA et al.; The human hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST), is highly expressed in liver and adrenal cortex and displays reactivity towards a broad range of hydroxysteroids including 3 beta-hydroxysteroids, 3 alpha-hydroxysteroids, estrogens with a 3-phenolic moiety, and 17-hydroxyl group of androgens . In contrast, characterization of the newly described human hydroxysteroid sulfotransferase SULT2B1 isoforms shows that these enzymes are selective for the sulfation of 3 beta-hydroxysteroids, such as pregnenolone, epiandrosterone, DHEA, and androstenediol . There was no activity detected towards testosterone, dexamethasone, beta-estradiol, androsterone, or p-nitrophenol . The SULT2B1 gene encodes two isoforms, SULT2B1a and SULT2B1b, which are generated by alternate splicing of the first exon; therefore the SULT2B1 isoforms differ at their N-terminals . Northern Blot analysis detected a SULT2B1 message in RNA isolated from the human prostate and placenta . No SULT2B1 message was observed in RNA isolated from human liver, colon, lung, kidney, brain, or testis tissue . Purified SULT2B1a was used to generate a specific rabbit polyclonal anti-SULT2B1 antibody . The anti-SULT2B1 antibody did not react with expressed human EST, P-PST-1, M-PST, DHEA-ST, or ST1B2, during immunoblot analysis . The substrate specificity of the expressed SULT2B1 isoforms suggests that these enzymes are capable of regulating the activity of adrenal androgens in human tissues via their inactivation by sulfation.

Eur J Pharm Sci, 2001 Aug, 14(1), 63 - 7
Topically applied liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation; Vorauer-Uhl K et al.; The overproduction of biochemical mediators, and activation of leukocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects . The formation of short-lived, highly reactive metabolites, such as oxygen free radicals, increases with increasing tissue ischemia, and causes further cell damage . Human recombinant Cu/Zn-superoxide dismutase (rh-Cu/Zn-SOD), an enzyme which captures these radicals, may have a beneficial effect on the postburn inflammation processes . In this study, the influence of rh-Cu/Zn-SOD application to thermally injured tissue of rabbit backskin was examined . Three different delivery strategies were compared, pure or liposomally encapsulated enzyme, or intralesionally injected rh-Cu/Zn-SOD . For control, one animal group was treated with plain gel and another group was kept untreated . At 24 h following trauma a statistically significant difference in lesion sizes between the enzyme treated and control groups was observed . After 72 h tissue swelling had diminished significantly more in the rh-Cu/Zn-SOD treated groups as compared to the control animals . The best results were achieved by spreading liposomes encapsulating the enzyme onto the wounds . Our results suggest that local treatment of burn wounds with enzymatic radical scavengers such as rh-Cu/Zn-SOD has a beneficial effect on the extent of the postburn damage.

Vaccine, 2001 Jul 20, 19(30), 4287 - 96
cDNA cloning, characterization and vaccine effect analysis of Haemaphysalis longicornis tick saliva proteins; Tsuda A et al.; Immunological control of ticks is currently the only sustainable and practical alternative method to the current use of acaricides which has serious limitations . The success of this method is dependent upon identification and cloning of potential tick vaccine antigens . We used a combination of immuno-screening of an adult tick cDNA library as well as the 3 and 5 rapid amplification of cDNA ends to clone two cDNAs, encoding tick saliva proteins from Haemaphysalis longicornis . The two cDNAs herein named HL 34 and 35 are 1000 bp each and encode polypeptides with 292 and 321 amino acid residues respectively . Northern blotting analysis of total RNA from ticks at different feeding stages revealed that expression of both HL 34 and HL35 mRNAs is induced during the slow feeding phase . We speculate that the functions of both genes are closely associated with blood feeding . Expression analysis by RT-PCR showed that both genes are expressed in other tick organs in addition to salivary glands . Recombinant HL 34 was successfully expressed in Escherichia coli and its suitability as a tick vaccine antigen was analyzed in rabbits . We propose that rHL34 could be a useful component of a cocktail tick vaccine.

Vaccine, 2001 Jul 20, 19(30), 4135 - 42
Identification of vaccine candidate antigens from a genomic analysis of Porphyromonas gingivalis; Ross BC et al.; Porphyromonas gingivalis is a key periodontal pathogen which has been implicated in the etiology of chronic adult periodontitis . Our aim was to develop a protein based vaccine for the prevention and or treatment of this disease . We used a whole genome sequencing approach to identify potential vaccine candidates . From a genomic sequence, we selected 120 genes using a series of bioinformatics methods . The selected genes were cloned for expression in Escherichia coli and screened with P . gingivalis antisera before purification and testing in an animal model . Two of these recombinant proteins (PG32 and PG33) demonstrated significant protection in the animal model, while a number were reactive with various antisera . This process allows the rapid identification of vaccine candidates from genomic data.

Vet Immunol Immunopathol, 2001 Aug 10, 80(3-4), 245 - 57
Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay; Miyamoto T et al.; Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays . These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection . Anti-IL-2 mAbs bound specifically to E . coli-derived rchIL-2 in ELISA and identified a 16kDa IL-2 polypeptide band in Western blot . Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay . Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity . To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody . Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed . These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2 . These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.

FEBS Lett, 2001 Jul 13, 501(1), 1 - 5
YidC/Oxa1p/Alb3: evolutionarily conserved mediators of membrane protein assembly; Luirink J et al.; This review focuses on a novel, evolutionarily conserved mediator of membrane protein assembly in bacteria, mitochondria and chloroplasts . This factor is designated YidC in Escherichia coli, and is localized in the inner membrane . YidC is homologous to Oxa1p in the mitochondrial inner membrane and Alb3 in the chloroplast thylakoid membrane, but does not seem to have a homologue in the endoplasmic reticulum membrane . It has been suggested that YidC operates both as a separate unit and in connection with the SecYEG-translocon depending on the substrate membrane protein that is integrated into the membrane . Mitochondria do not possess a SecYEG-like complex and Oxa1p is thought to form, or to contribute to the formation of, a novel translocon in the mitochondrial inner membrane . Alb3 in the chloroplast thylakoid membrane is, just like YidC and Oxa1p, involved in membrane protein assembly, but only few details are known.

J Am Chem Soc, 2001 May 16, 123(19), 4373 - 81
A photoactivatable prenylated cysteine designed to study isoprenoid recognition; Kale TA et al.; Protein prenylation, involving the alkylation of a specific C-terminal cysteine with a C(15) or C(20) isoprenoid unit, is an essential posttranslational modification required by most GTP-binding proteins for normal biological activity . Despite the ubiquitous nature of this modification and numerous efforts aimed at inhibiting prenylating enzymes for therapeutic purposes, the function of prenylation remains unclear . To explore the role the isoprenoid plays in mediating protein-protein recognition, we have synthesized a photoactivatable, isoprenoid-containing cysteine analogue (2) designed to act as a mimic of the C-terminus of prenylated proteins . Photolysis experiments with 2 and RhoGDI (GDI), a protein which interacts with prenylated Rho proteins, suggest that the GDI is in direct contact with the isoprenoid moiety . These results, obtained using purified GDI as well as Escherichia coli (E . coli) crude extract containing GDI, suggest that this analogue will be an effective and versatile tool for the investigation of putative isoprenoid binding sites in a variety of systems . Incorporation of this analogue into peptides or proteins should allow for even more specific interactions between the photoactivatable isoprenoid and any number of isoprenoid binding proteins.

J Am Chem Soc, 2001 Apr 25, 123(16), 3790 - 8
The electronic structure of the flavin cofactor in DNA photolyase; Weber S et al.; Density functional theory is used to calculate the electronic structure of the neutral flavin radical, FADH(*), formed in the light-induced electron-transfer reaction of DNA repair in cis,syn-cyclobutane pyrimidine dimer photolyases . Using the hybrid B3LYP functional together with the double-zeta basis set EPR-II, (1)H, (13)C, (15)N, and (17)O isotropic and anisotropic hyperfine couplings are calculated and explained by reference to the electron densities of the highest occupied molecular orbital and of the unpaired spin distribution on the radical . Comparison of calculated and experimental hyperfine couplings obtained from EPR and ENDOR/TRIPLE resonance leads to a refined structure for the FAD cofactor in Escherichia coli DNA photolyase . Hydrogen bonding at N3H, O4, and N5H results in significant changes in the unpaired spin density distribution and hyperfine coupling constants . The calculated electronic structure of FADH(*) provides evidence for a superexchange-mediated electron transfer between the cyclobutane pyrimidine dimer lesion and the 7,8-dimethyl isoalloxazine moiety of the flavin cofactor via the adenine moiety.

J Am Chem Soc, 2001 Apr 4, 123(13), 3055 - 63
A structural mode-coupling approach to 15N NMR relaxation in proteins; Tugarinov V et al.; The two-body Slowly Relaxing Local Structure (SRLS) model was applied to (15)N NMR spin relaxation in proteins and compared with the commonly used original and extended model-free (MF) approaches . In MF, the dynamic modes are assumed to be decoupled, local ordering at the N-H sites is represented by generalized order parameters, and internal motions are described by effective correlation times . SRLS accounts for dynamical coupling between the global diffusion of the protein and the internal motion of the N-H bond vector . The local ordering associated with the coupling potential and the internal N-H diffusion are tensors with orientations that may be tilted relative to the global diffusion and magnetic frames . SRLS generates spectral density functions that differ from the MF formulas . The MF spectral densities can be regarded as limiting cases of the SRLS spectral density . SRLS-based model-fitting and model-selection schemes similar to the currently used MF-based ones were devised, and a correspondence between analogous SRLS and model-free parameters was established . It was found that experimental NMR data are sensitive to the presence of mixed modes . Our results showed that MF can significantly overestimate order parameters and underestimate local motion correlation times in proteins . The extent of these digressions in the derived microdynamic parameters is estimated in the various parameter ranges, and correlated with the time scale separation between local and global motions . The SRLS-based analysis was tested extensively on (15)N relaxation data from several isotropically tumbling proteins . The results of SRLS-based fitting are illustrated with RNase H from E . coli, a protein extensively studied previously with MF.

J Am Chem Soc, 2001 Apr 4, 123(13), 3048 - 54
Sensitivity of tyrosyl radical g-values to changes in protein structure: a high-field EPR study of mutants of ribonucleotide reductase; Un S et al.; The local electrostatic environment plays a critical role in determining the physicochemical properties of reactive radicals in proteins . High-field electron paramagnetic resonance (HF-EPR) spectroscopy has been used to determine the sensitivity of the tyrosyl radical g-values to local electrostatic environment . Site-specific mutants of ribonucleotide reductase from Escherichia coli were used to study the effect of introducing a charge group on the HF-EPR spectrum of the stable tyrosyl (Y122) radical . The changes affected by the mutations were small, but measurable . Mutation of isoleucine-74 to an arginine (I74R) or lysine (I74K) induced disorder in the hyperfine interactions . Similar effects were observed for the mutation of valine-136 to an arginine (V136R) or asparagine (V136N) . For five or six mutants studied, the g(x)() component of the g-tensor was distributed . For the isoleucine-74 to lysine (I74K) and leucine-77 to phenylalanine (L77F) mutants, a shift of 1 x 10(-)(4) in g(x)() value was also detected . For the I74K mutant, it is shown that the shift is consistent with the introduction of a charged residue, but cannot be distinguished from changes in the electrostatic effect of the nearby diiron center . For the L77F mutant, the shift is induced by the diiron center . Using existing tyrosyl radical g-tensor measurements, we have developed a simple effective charge model that allows us to rationalize the effect of the local electrostatic environments in a number of proteins.

J Am Chem Soc, 2001 Feb 14, 123(6), 1047 - 58
Reactivity of zinc finger cores: analysis of protein packing and electrostatic screening; Maynard AT et al.; The chemical stability of 207 zinc fingers, derived from 92 experimental protein structures, is evaluated according to the protein packing and electrostatic screening of their zinc cores . These properties are used as measures of the protein protection of zinc cores, to predictively rank relative zinc finger reactivities and assess differences in function . On average, there is a substantial and concomitant increase in the screening of increasingly anionic core motifs, suggesting zinc fingers have evolved in a manner that promotes shielding of their potentially reactive core thiolates . In contrast, enzymatic zinc cores are functionally differentiated by negative electrostatic screening . Zinc finger cores are predominantly screened by networks of backbone:core NH-S hydrogen bonds that electronically stabilize core thiolates and enhance backbone packing . Stabilizing protein:core interactions can be mapped to conserved residues, including {Arg,Lys}:core salt-bridges in some protein families . Labile zinc fingers are identified by poorly screened cores, possibly indicating redox or metallothionein (MT) regulated function . Consistent with experiment, the cores of the C-terminal finger of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 (NCp7) and Escherichia coli Ada protein (Ada) "finger" are identified as reactive . The C-terminal zinc fingers of nuclear receptors are predicted to be the most labile in this study, particularly the human estrogen receptor (hER), which contains a triad of reactive thiolates . We propose that hER DNA binding is redox and MT regulated through the C-terminal finger and that weak electrophilic agents may inhibit hER-mediated transcription, implicated in breast cancer progression.

Biochemistry, 2001 Jul 24, 40(29), 8531 - 5
Elongation factor Ts can act as a steric chaperone by increasing the solubility of nucleotide binding-impaired elongation factor-Tu; Krab IM et al.; Several elongation factor (EF) Tu mutants (T25A, H22Y/T25S, D80N, D138N) that have impaired nucleotide binding show decreased solubility on overexpression in the E . coli cell, an indication that they do not fold correctly . Moreover, EF-Tu{T25A} and EF-Tu{D80N} were shown to inhibit cell growth on expression, an effect attributed to their sequestration of EF-Ts {Krab, I . M., and Parmeggiani, A . (1999) J . Biol . Chem . 274, 11132--11138; Krab, I . M., and Parmeggiani, A . (1999) Biochemistry 38, 13035--13041} . We present here results showing that the co-overexpression of EF-Ts at a 1:1 ratio dramatically improves the solubility of mutant EF-Tu, although in the case of EF-Tu{D138N}--which cannot at all bind the nucleotides available in the cell--this is a slow process . Moreover, with co-overexpression of EF-Ts, the mentioned growth inhibition is relieved . We conclude that for the formation of a correct EF-Tu structure the nucleotide plays an important role as a "folding nucleus", and also that in its absence EF-Ts can act as a folding template or steric chaperone for the correct folding of EF-Tu.

Biol Pharm Bull, 2001 Jul, 24(7), 760 - 6
Amino acid sequence and characterization of a rice bran ribonuclease; Iwama M et al.; A base-nonspecific and acid ribonuclease (RNase Os) belonging to the RNase T2 family was purified from rice bran to a homogeneous state by SDS-PAGE . The primary structure of RNase Os was determined by protein chemistry and molecular cloning . The RNase Os was a simple protein and consisted of 205 amino acid residues . Its molecular weight was 22578 and its amino acid sequence showed that it was most similar to barley RNase among the known RNase T2 family enzymes having 157 amino acid residues identical with barley RNase . However, its N-terminus was blocked by a gamma-pyroglutamyl residue . The optimal pH of RNase Os was around 5.5 . The base preference at the B1 and B2 site of RNase Os was estimated from the rates of hydrolysis of 16 dinucleoside phosphates, to be guanine as the case of RNase LE from tomato . RNase Os was successfully expressed from yeast cells using the E . coli yeast expression vector pYE-RNAP.

Ann Acad Med Singapore, 2001 May, 30(3), 270 - 3
Nitric oxide production by human peripheral blood mononuclear cells; Toomtong P et al.; INTRODUCTION: There are conflicting data on the ability of human mononuclear cells to produce nitric oxide (NO) . We investigated nitric oxide production from peripheral blood mononuclear cells (PBMs) by using a new sensitive fluorescent indicator . MATERIALS AND METHODS: PBMs from healthy volunteers were collected, plated in 96-well microplates, and loaded with the fluorescent nitric oxide probe, 4,5-diaminofluorescein diacetate (DAF-2DA) . Experiments were performed in normal control and endotoxin-stimulated PBMs, with and without exogenous L-arginine . The exogenous nitric oxide donor S-nitroso-N-acetyl-penicillamine (SNAP) was used as a positive control . Fluorescence intensity was measured with a fluorescence microplate reader . RESULTS: Nitric oxide production by human PBMs can be demonstrated by the use of the fluorescent indicator, DAF-2DA, in both control and endotoxin-stimulated conditions . Nitric oxide production was independent of the concentration of exogenous L-arginine . The addition of endotoxin did not change nitric oxide production . PBMs treated with SNAP showed a concentration dependent increase in fluorescence . Nitric oxide production over 5 hours was constant and identical in both control and stimulated groups . CONCLUSION: This fluorescent indicator technique is useful for the study of NO production by human PBMs . Nitric oxide production by PBMs was independent of exogenous L-arginine concentration and was not affected by endotoxin.

Ann Acad Med Singapore, 2001 May, 30(3), 265 - 9
Retinol palmitate counteracts oxidative injury during experimental septic shock; Basu S et al.; INTRODUCTION: Retinols seem to be of clinical importance in ameliorating the clinical consequences of septic shock . These beneficial effects of retinols are suggested to be due to an antioxidant property . The present study was undertaken in order to confirm or rule out such an effect of retinol palmitate (RP) in experimental septic shock by measuring F2-isoprostanes and a major prostaglandin F2 alpha metabolite as indicators of oxidative injury and inflammatory response, respectively . MATERIALS AND METHODS: Fourteen anaesthetised pigs were randomly given an injection of RP (2.300 IU x kg-1) or the corresponding volume of vehicle . All pigs received a continuous infusion of E . coli endotoxin (10 micrograms x kg-1 x h-1) . Blood samples were analysed for lipid peroxidation products (8-iso-PGF2 alpha), indicating free radical induced oxidative injury and 15-keto-dihydro-PGF2 alpha indicating cyclooxygenase-mediated inflammatory response) . RESULTS: Significantly elevated levels of 8-iso-PGF2 alpha were seen at 3, 5 and 6 hours of endotoxaemia in the vehicle + endotoxin group as compared to RP + endotoxin group . Endotoxin induced cyclooxygenase-mediated inflammatory response was not affected by RP . CONCLUSIONS: This study is the first one to show that RP counteracts oxidative injury rather than inflammatory response in experimental septic shock . These results may be of importance for the understanding of some beneficial effects of RP during endotoxaemia (i.e . improved systemic haemodynamics and reduced serum levels of endotoxin) . Our results may explain the therapeutic effects of nutrients rich in caroten/retinols used in some clinical studies.

Pest Manag Sci, 2001 Jan, 57(1), 33 - 40
Mode of bleaching phytotoxicity of herbicidal diphenylpyrrolidinones; Ogawa H et al.; A mode of action study of herbicidal diphenylpyrrolidinones was carried out through carotenoid analyses in intact Scenedesmus cells and by a cell-free plant-type phytoene desaturase assay using Escherichia coli transformants . A series of forty-eight diphenylpyrrolidinones decreased the carotenoid content of Scenedesmus cells in the light and inhibited phytoene desaturase . The relationship between substituents at various positions and inhibition of phytoene desaturase is discussed . Using very active bleaching diphenylpyrrolidinones, a 10(-5) M concentration affected neither the zeta-carotene desaturase nor the protoporphyrinogen-IX oxidase . Although some differences in their inhibitory activity were found between the in vivo and cell-free assays, it is concluded that the compounds are essentially bleachers affecting carotenoid biosynthesis in plants . Enzyme kinetics studies with recombinant phytoene desaturase revealed a non-competitive inhibition with respect to the substrate phytoene . A competition against the inhibitor was shown by the cofactor NADP+, suggesting an interaction of pyrrolidinones at the cofactor-binding site of phytoene desaturase.

Proteins, 2001 Aug 15, 44(3), 304 - 11
The crystal structure of chorismate lyase shows a new fold and a tightly retained product; Gallagher DT et al.; The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4-hydroxy benzoate (4HB) for the ubiquinone pathway . In Escherichia coli, CL is monomeric, with 164 residues . We have determined the structure of the CL product complex by crystallographic heavy-atom methods and report the structure at 1.4-A resolution for a fully active double Cys-to-Ser mutant and at 2.0-A resolution for the wild-type . The fold involves a 6-stranded antiparallel beta-sheet with no spanning helices and novel connectivity . The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main-chain amides and by the buried side-chains of Arg 76 and Glu 155 . The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix-turn-helix loops . The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10-fold stronger binding of product than substrate . Substrate binding and kinetically rate-limiting product release apparently require the rearrangement of these active-site-covering loops . Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane-bound enzyme 4HB octaprenyltransferase.

Proteins, 2001 Aug 15, 44(3), 270 - 81
Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli; Melik-Adamyan W et al.; The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer . Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited . To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution . The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket . In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel . The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure . It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide . The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel . Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme . An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism . The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.

J Vasc Res, 2001 Jul-Aug, 38(4), 315 - 23
Enhanced gene transfer to rabbit jugular veins by an adenovirus containing a cyclic RGD motif in the HI loop of the fiber knob; Hay CM et al.; Gene therapy using recombinant adenoviral vectors represents a promising therapeutic tool to prevent vein graft stenosis, the main complication of coronary artery bypass grafting . However, the low transduction efficiency of vascular smooth muscle cells and endothelial cells (EC) is a potential limitation, presumably due to the low levels of functional adenovirus receptor (coxsackie:adenovirus receptor; CAR) . Designing vectors specifically targeted to alpha(v) integrins is a strategy that might overcome the poor expression of CAR in vascular smooth muscle cells and EC . RGD, a receptor-binding motif that can interact with alpha(v) integrins, was inserted into the HI loop and at the C-terminus of the adenoviral fiber protein in two separate adenovirus vectors encoding a beta-galactosidase reporter gene . Av1nBgCRGD (C-terminus) and Av1nBgHIRGD (HI loop) were evaluated in EC in culture and in jugular vein organ culture . Transduction of primary rat and rabbit EC with Av1nBgHIRGD was significantly more efficient when compared to Av1nBgCRGD or Av1nBg . Transduction of mouse, rat and rabbit jugular veins in organ culture using Av1nBg showed that adenovirus-mediated gene expression was greatest in rabbit jugular veins compared to rat and mouse veins . Av1nBgHIRGD augmented gene expression approximately four-fold in rabbit jugular veins when compared to Av1nBg . Histochemical analysis showed that numerous EC but few smooth muscle cells were transduced at all vector concentrations . A substantial number of adventitial fibroblasts were transduced only at the highest vector concentrations of Av1nBgHIRGD . These findings demonstrate that integrin-targeted vectors allow for enhanced gene delivery to veins and strengthen the viability of adenoviral-mediated gene transfer of therapeutic transgenes to human veins prior to vein grafting .

J Biol Chem, 2001 Sep 14, 276(37), 35201 - 8 Epub 2001 Jul 13.
Homologous pairing promoted by the human Rad52 protein; Kagawa W et al.; The Rad52 protein, which is unique to eukaryotes, plays important roles in the Rad51-dependent and the Rad51-independent pathways of DNA recombination . In the present study, we have biochemically characterized the homologous pairing activity of the HsRad52 protein (Homo sapiens Rad52) and found that the presynaptic complex formation with ssDNA is essential in its catalysis of homologous pairing . We have identified an N-terminal fragment (amino acid residues 1-237, HsRad52(1-237)) that is defective in binding to the human Rad51 protein, which catalyzed homologous pairing as efficiently as the wild type HsRad52 . Electron microscopic visualization revealed that HsRad52 and HsRad52(1-237) both formed nucleoprotein filaments with single-stranded DNA . These lines of evidence suggest the role of HsRad52 in the homologous pairing step of the Rad51-independent recombination pathway . Our results reveal the striking similarity between HsRad52 and the Escherichia coli RecT protein, which functions in a RecA-independent recombination pathway.

J Biol Chem, 2001 Sep 7, 276(36), 34339 - 47 Epub 2001 Jul 13.
Distinct MutS DNA-binding modes that are differentially modulated by ATP binding and hydrolysis; Blackwell LJ et al.; The role of MutS ATPase in mismatch repair is controversial . To clarify further the function of this activity, we have examined adenine nucleotide effects on interactions of Escherichia coli MutS with homoduplex and heteroduplex DNAs . In contrast to previous results with human MutS alpha, we find that a physical block at one end of a linear heteroduplex is sufficient to support stable MutS complex formation in the presence of ATP.Mg(2+) . Surface plasmon resonance analysis at low ionic strength indicates that the lifetime of MutS complexes with heteroduplex DNA depends on the nature of the nucleotide present when MutS binds . Whereas complexes prepared in the absence of nucleotide or in the presence of ADP undergo rapid dissociation upon challenge with ATP x Mg(2+), complexes produced in the presence of ATP x Mg(2+), adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) x Mg(2+), or ATP (no Mg(2+)) are resistant to dissociation upon ATP challenge . AMPPNP x Mg(2+) and ATP (no Mg(2+)) reduce MutS affinity for heteroduplex but have little effect on homoduplex affinity, resulting in abolition of specificity for mispaired DNA at physiological salt concentrations . Conversely, the highest mismatch specificity is observed in the absence of nucleotide or in the presence of ADP . ADP has only a limited effect on heteroduplex affinity but reduces MutS affinity for homoduplex DNA.

J Biol Chem, 2001 Sep 7, 276(36), 33833 - 9 Epub 2001 Jul 13.
Strong RNA splicing enhancers identified by a modified method of cycled selection interact with SR protein; Tian H et al.; A modified method of cycled selection was used to characterize splicing enhancers for exon inclusion from a pool of beta-globin-based three exon/two intron pre-mRNAs with a variable number of random nucleotides incorporated in the internal exon . The pre-mRNAs generated by this method contained random sequences ranging from 0 to 18 nucleotides in length . This method was used to isolate particular splicing enhancer motifs from a previously enriched pool of extremely diverse enhancers . After four cycles of selection for mRNA containing the internal exon, a distinct enhancer motif (GACGAC...CAGCAG) was highly enriched . This motif served as strong splicing enhancers in a heterogeneous exon . We have shown here that the selected enhancer motif promotes exon inclusion through specific interaction with SRp30 . We have also shown that although present in many of our selected splicing enhancers conforming to this motif, a typical purine-rich enhancer sequence is dispensable for either enhancer activity or binding with SRp30.

J Comput Biol, 2001, 8(2), 151 - 75
Determination of bias in the relative abundance of oligonucleotides in DNA sequences; Elhai J; Different statistical measures of bias of oligonucleotide sequences in DNA sequences were compared, both by theoretical analysis and according to their abilities to predict the relative abundances of oligonucleotides in the genome of Escherichia coli . The expected frequency of an oligonucleotide calculated from a maximal order Markov model was shown to be a degenerate case of the expected frequency calculated from biases of all subwords arising when noncontiguous subwords exhibit no bias . Since (at least in E . coli) noncontiguous sequences exhibit significant bias, the total compositional bias approach is expected to represent biases in genomic sequences more faithfully than Markov approaches . In fact, the efficacy of statistics based on Markov analysis even at the highest order were inferior in predicting actual frequencies of oligonucleotides to methods that factored out biases of internal subwords with gaps . Using total compositional bias as a measure of relative abundance, tetranucleotide and hexanucleotide palindromes were found to be distributed differently from nonpalindromic sequences, with their means shifted somewhat towards underrepresentation . A subpopulation of palindromic hexanucleotides, however, was highly underrepresented, and this group consisted almost entirely of targets for Type II restriction enzymes found within strains of E . coli . Sites recognized by Type I endonucleases from related strains were not markedly biased, and with pentanucleotides, palindromic and nonpalindromic sequences had nearly identical distributions . The loss of restriction sites may be explained by the free transfer of plasmids encoding restriction enzymes and episodic selection for the presence of the enzymes.

Mol Microbiol, 2001 Jul, 41(1), 207 - 16
Codon-usage based regulation of colicin K synthesis by the stress alarmone ppGpp; Kuhar I et al.; The molecular mechanism of the upregulation of Escherichia coli colicin K (Cka) synthesis during stress conditions was studied . Nutrient starvation experiments and the use of relA spoT mutant strains, IPTG-regulated overproduction of ppGpp and lacZ fusions revealed that the stringent response alarmone guanosine 3',5'-bispyrophosphate (ppGpp) is the main positive effector of Cka synthesis . Comparison of the amounts of protein produced (Western blotting) and specific mRNA (Northern blotting) before and after nutrient starvation demonstrated increases in Cka protein with unaltered specific mRNA levels, suggesting a post-transcriptional regulatory mechanism . Reporter (beta-galactosidase) assays using truncated cka of variable length fused to lacZ located the key regulatory region close to the 5' end of the cka mRNA . Closer analysis of this region indicated the presence of several rare codons, including the leucine-encoding codon CUA . Synonymous exchange of the rare codons with more frequently used ones abolished the regulatory effect of ppGpp . Supplementation of the strain with the plasmid CodonPlus carrying several rare tRNA genes yielded similar results, indicating that codon usage (in particular, the fifth codon for the amino acid leucine) and tRNA availability (i.e . tRNAleu) are the key elements of the regulatory function of ppGpp . We conclude that ppGpp regulates Cka synthesis via a novel post-transcriptional mechanism that is based on rare codon usage and variable cognate tRNA availability.

Mol Microbiol, 2001 Jul, 41(1), 83 - 91
Escherichia coli FtsZ polymers contain mostly GTP and have a high nucleotide turnover; Mingorance J et al.; The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures . To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter-binding assay, whereas polymerization of the protein was followed in parallel by light scattering . Unpolymerized FtsZ bound 1 mol of GTP mol(-1) protein monomer . At pH 7.5 and in the presence of Mg(2+) and K(+), there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant . These results show that the long FtsZ polymers formed in solution contain mostly GTP . Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization . Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium . These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.

Mol Microbiol, 2001 Jul, 41(1), 73 - 82
The ratio between CcdA and CcdB modulates the transcriptional repression of the ccd poison-antidote system; Afif H et al.; The ccd operon of the F plasmid encodes CcdB, a toxin targeting the essential gyrase of Escherichia coli, and CcdA, the unstable antidote that interacts with CcdB to neutralize its toxicity . Although work from our group and others has established that CcdA and CcdB are required for transcriptional repression of the operon, the underlying mechanism remains unclear . The results presented here indicate that, although CcdA is the DNA-binding element of the CcdA-CcdB complex, the stoichiometry of the two proteins determines whether or not the complex binds to the ccd operator-promoter region . Using electrophoretic mobility shift assays, we show that a (CcdA)2-(CcdB)2 complex binds DNA . The addition of extra CcdB to that protein-DNA complex completely abolishes DNA retardation . Based on these results, we propose a model in which the ratio between CcdA and CcdB regulates the repression state of the ccd operon . When the level of CcdA is superior or equal to that of CcdB, repression results . In contrast, derepression occurs when CcdB is in excess of CcdA . By ensuring an antidote-toxin ratio greater than one, this mechanism could prevent the harmful effect of CcdB in plasmid-containing bacteria.

Mol Microbiol, 2001 Jul, 41(1), 9 - 17
Feedback controls restrain the initiation of Escherichia coli chromosomal replication; Katayama T; In Escherichia coli, initiation of chromosomal replication is activated by a nucleoprotein complex formed primarily between the DnaA protein and oriC (replication origin) DNA . After replicational initiation, this complex has to be inactivated in order to repress the appearance of initiation events until the next scheduled round of initiation . Studies of the mechanisms responsible for this repression have recently revealed direct coupling between these mechanisms and key elements of the replication process, suggesting that feedback-type regulatory loops exist between the factors implicated in initiation and the elements yielded by the replication process . The loading of the ring-shaped beta-subunit of DNA polymerase III onto DNA plays a key role in the inactivation of the DnaA protein . Duplication of oriC DNA results in hemimethylated DNA, which is inert for reinitiation . Titration of large amounts of DnaA protein to a non-oriC locus can repress untimely initiations, and timely duplication of this locus is required for this repression in rapidly growing cells . All these systems functionally complement one another to ensure the maintenance of the interinitiation interval between two normal DNA replication cycles . The mechanisms that link the replication cycle to the progression of the cell cycle are also discussed.

Eur J Biochem, 2001 Jul, 268(14), 4086 - 94
Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin; Taguchi G et al.; Scopoletin is one of the phytoalexins in tobacco . Cells of the T-13 cell line (Nicotiana tabacum L . Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles . We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin . Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe . The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, approximately 20-50% identities with other reported glucosyltransferases . Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins . They also strongly reacted with 2-naphthol as a substrate . These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor . RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different . This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators . These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.

Eur J Biochem, 2001 Jul, 268(14), 4036 - 43
The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts; Inoue K et al.; Toc75 is an outer envelope membrane protein of chloroplasts . It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide . The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts . We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC . Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry . In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine . However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine . Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase . The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.

Eur J Biochem, 2001 Jul, 268(14), 4011 - 8
Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralis; Greller G et al.; We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis . The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins . One hundred grams of cells yielded 3 mg of pure complex . The final product showed ATPase activity at 70 degrees C and was soluble at low detergent concentration . ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T . litoralis or the soluble engineered protein . The purified native TMBP was identified as a glycoprotein.

Eur J Biochem, 2001 Jul, 268(14), 4001 - 10
Dichloromethane mediated in vivo selection and functional characterization of rat glutathione S-transferase theta 1-1 variants; Gisi D et al.; Methylobacterium dichloromethanicum DM4 is able to grow with dichloromethane as the sole carbon and energy source by using a dichloromethane dehalogenase/glutathione S-transferase (GST) for the conversion of dichloromethane to formaldehyde . Mammalian homologs of this bacterial enzyme are also known to catalyze this reaction . However, the dehalogenation of dichloromethane by GST T1-1 from rat was highly mutagenic and toxic to methylotrophic bacteria . Plasmid-driven expression of rat GST T1-1 in strain DM4-2cr, a mutant of strain DM4 lacking dichloromethane dehalogenase, reduced cell viability 10(5)-fold in the presence of dichloromethane . This effect was exploited to select dichloromethane-resistant transconjugants of strain DM4-2cr carrying a plasmid-encoded rGSTT1 gene . Transconjugants that still expressed the GST T1 protein after dichloromethane treatment included rGSTT1 mutants encoding protein variants with sequence changes from the wild-type ranging from single residue exchanges to large insertions and deletions . A structural model of rat GST T1-1 suggested that sequence variation was clustered around the glutathione activation site and at the protein C-terminus believed to cap the active site . The enzymatic activity of purified His-tagged GST T1-1 variants expressed in Escherichia coli was markedly reduced with both dichloromethane and the alternative substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane . These results provide the first experimental evidence for the involvement of Gln102 and Arg107 in catalysis, and illustrate the potential of in vivo approaches to identify catalytic residues in GSTs whose activity leads to toxic effects.

Chem Res Toxicol, 2001 Jul, 14(7), 901 - 11
Regioselective differences in C(8)- and N-oxidation of 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline by human and rat liver microsomes and cytochromes P450 1A2; Turesky RJ et al.; The metabolism of the mutagen 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) was investigated with human and rat liver microsomes, recombinant human cytochrome P450 1A2 (P450 1A2) expressed in Escherichia coli cells, and rat P450 1A2 . Human liver microsomes and human P450 1A2 catalyzed the oxidation of the exocyclic amine group of MeIQx to form the genotoxic product 2-(hydroxyamino)-3,8-dimethylimidazo{4,5-f}quinoxaline (HONH-MeIQx) . Human P450 1A2 also catalyzed the oxidation of C(8)-methyl group of MeIQx to form 2-amino-(8-hydroxymethyl)-3-methylimidazo{4,5-f}quinoxaline (8-CH(2)OH-IQx), 2-amino-3-methylimidazo{4,5-f}quinoxaline-8-carbaldehyde (IQx-8-CHO), and 2-amino-3-methylimidazo{4,5-f}quinoxaline-8-carboxylic acid (IQx-8-COOH) . Thus, chemically stable C(8)-oxidation products of MeIQx may be useful biomarkers of P450 1A2 activity in humans . Rat liver microsomes were 10-15-fold less active than the human counterpart at both N-oxidation and C(8)-oxidation of MeIQx when expressed as nanomoles of product formed per minute per nanomoles of P450 1A2 . Differences in regioselective oxidation of MeIQx were also observed with human and rat liver microsomes and the respective P450 1A2 orthologs . In contrast to human liver microsomes and P450 1A2, rat liver microsomes and purified rat P4501A2 were unable to catalyze the oxidation of MeIQx to the carboxylic derivative IQx-8-COOH, an important detoxication product formed in humans . However, rat liver microsomes and rat P4501A2, but not human liver microsomes or human P450 1A2, extensively catalyzed ring oxidation at the C-5 position of MeIQx to form the detoxication product 2-amino-3,8-dimethyl-5-hydroxyimidazo{4,5-f}quinoxaline (5-HO-MeIQx) . There are important differences between human and rat P450 1A2, both in catalytic activities and oxidation pathways of MeIQx, that may affect the biological activity of this carcinogen and must be considered when assessing human health risk.

Chem Res Toxicol, 2001 Jul, 14(7), 894 - 900
Mutagenesis by O(6)-methyl-, O(6)-ethyl-, and O(6)-benzylguanine and O(4)-methylthymine in human cells: effects of O(6)-alkylguanine-DNA alkyltransferase and mismatch repair; Pauly GT et al.; Double-stranded and gapped shuttle vectors were used to study mutagenesis in human cells by O(6)-methyl (m(6)G)-, O(6)-ethyl (e(6)G)-, and O(6)-benzylguanine (b(6)G), and O(4)-methylthymine (m(4)T) when these bases were incorporated site-specifically in the ATG initiation codon of a lacZ' gene . Vectors were transfected into either human kidney cells (293) or colon tumor cells (SO) or into mismatch repair defective human colon tumor cells (H6 and LoVo) . Cellular O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) was optionally inactivated by treating cells with O(6)-benzylguanine prior to transfection . In alkyltransferase competent cells, the mutagenicity of all the modified bases was substantially higher in gapped plasmids than in double-stranded plasmids . Alkyltransferase inactivation increased mutagenesis by the three O(6)-substituted guanines in both double-stranded and gapped plasmids but did not affect m(4)T mutagenesis . In the absence of alkyltransferase, mutagenesis by m(6)G and to a lesser extent e(6)G in double-stranded vectors was higher in the mismatch repair defective H6 and LoVo cells than in SO or 293 cells indicating that e(6)G as well as m(6)G were subject to mismatch repair processing in these cells . The level of mutagenesis by m(4)T and b(6)G was not affected by mismatch repair status . When incorporated in gapped plasmids and in the absence of alkyltransferase, the order of mutagenicity for the modified bases was m(4)T > e(6)G congruent with m(6)G > b(6)G . The O(6)-substituted guanines primarily produced G-->A transitions while m(4)T primarily produced T-->C transitions . However, m(4)T also produced a significant number of T-->A transversion mutations in addition to T-->C transitions in mismatch repair deficient LoVo cells.

Microb Pathog, 2001 Aug, 31(2), 103 - 7
Escherichia coli S fimbriae do not contribute to intestinal colonization or translocation in the gnotobiotic rat; Herias VM et al.; Escherichia coli S fimbriae, which bind to sialic acid residues, are a virulence factor for extraintestinal infection, but also promote binding to intestinal epithelial cells . In this study, we investigated whether S fimbriae would enhance intestinal colonization by E . coli or promote translocation to extraintestinal sites . A mixture of two E . coli isogenic strains both expressing type-1 fimbriae but differing in the carriage of S fimbriae (Sfim+ and Sfim-) were given perorally to germfree neonatal, infant or adult rats . The Sfim+ bound better to rat intestinal mucus and epithelial cells . However, both strains colonized equally well in both the small and large intestine and their rate of translocation to the mesenteric lymph nodes was similar . Infant rats had higher E . coli levels in the small intestine than adult rats, but their translocation rates were lower . This was at least partly due to their milk diet, since weaned infant rats had more translocating bacteria than infant rats that continued suckling their mother . The results suggest that S fimbriae, despite binding to intestinal epithelial cells and mucus, do not contribute to either colonization or translocation in the gnotobiotic rat .

Microb Pathog, 2001 Aug, 31(2), 69 - 79
Escherichia coli K1 purA and sorC are preferentially expressed upon association with human brain microvascular endothelial cells; Hoffman JA et al.; In order to better understand the events that allow Escherichia coli K1 to cross the blood-brain barrier we used differential fluorescence induction to identify bacterial genes that are preferentially expressed when associated with human brain microvascular endothelial cells (HBMEC), which comprise the blood-brain barrier . Random gene fusions of E . coli K1 DNA were created in a promoterless gfp vector and gene fusion libraries were incubated with and without HBMEC . The cells were subjected to a series of fluorescence-activated cell sorting screens to identify promoter fusions which lead to fluorescence when bacteria were associated with HBMEC, yet not fluorescent when grown in media alone . Two genes were identified, purA (encodes adenylosuccinate synthetase) and a sorC homologue (encodes a member of the sorC family of transcriptional regulators), whose expression were preferentially induced when bacteria were associated with eukaryotic cells . Individual gene disruption mutants of E . coli K1 purA and sorC demonstrated significantly decreased HBMEC invasion phenotype in vitro, when compared to the wild-type strain, and could be complemented when the respective wild-type sequences were supplied in trans . The purA and sorC mutants were deficient in their ability to grow in defined minimal media, without adenine, and with sorbose as sole carbon source, respectively, yet capable of normal growth in complex media . We have identified novel phenotypes associated with E . coli K1 purA and sorC, which provide evidence that these genes contribute to the invasion of HBMEC .

J Mol Biol, 2001 Jul 20, 310(4), 907 - 18
Solution structure of Escherichia coli glutaredoxin-2 shows similarity to mammalian glutathione-S-transferases; Xia B et al.; Glutaredoxin 2 (Grx2) from Escherichia coli is distinguished from other glutaredoxins by its larger size, low overall sequence identity and lack of electron donor activity with ribonucleotide reductase . However, catalysis of glutathione (GSH)-dependent general disulfide reduction by Grx2 is extremely efficient . The high-resolution solution structure of E . coli Grx2 shows a two-domain protein, with residues 1 to 72 forming a classical "thioredoxin-fold" glutaredoxin domain, connected by an 11 residue linker to the highly helical C-terminal domain, residues 84 to 215 . The active site, Cys9-Pro10-Tyr11-Cys12, is buried in the interface between the two domains, but Cys9 is solvent-accessible, consistent with its role in catalysis . The structures reveal the hither to unknown fact that Grx2 is structurally similar to glutathione-S-transferases (GST), although there is no obvious sequence homology . The similarity of these structures gives important insights into the functional significance of a new class of mammalian GST-like proteins, the single-cysteine omega class, which have glutaredoxin oxidoreductase activity rather than GSH-S-transferase conjugating activity . E . coli Grx 2 is structurally and functionally a member of this new expanding family of large glutaredoxins . The primary function of Grx2 as a GST-like glutaredoxin is to catalyze reversible glutathionylation of proteins with GSH in cellular redox regulation including stress responses .

J Mol Biol, 2001 Jul 20, 310(4), 817 - 26
X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution; Safo MK et al.; Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2) . Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly . The crystal structure of pyridoxine 5'-phosphate oxidase from E . coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution . One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN . A protein conformational change has occurred that partially closes the active site . The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN . When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site . A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent .

J Mol Biol, 2001 Jul 20, 310(4), 801 - 16
Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: separating their roles in specific and non-specific binding; Sidorova NY et al.; We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding . For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein . We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature . The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area . In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies . We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay . The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action . The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.

J Mol Biol, 2001 Jul 20, 310(4), 735 - 53
AA.AG@helix.ends: A:A and A:G base-pairs at the ends of 16 S and 23 S rRNA helices; Elgavish T et al.; This study reveals that AA and AG oppositions occur frequently at the ends of helices in RNA crystal and NMR structures in the PDB database and in the 16 S and 23 S rRNA comparative structure models, with the G usually 3' to the helix for the AG oppositions . In addition, these oppositions are frequently base-paired and usually in the sheared conformation, although other conformations are present in NMR and crystal structures . These A:A and A:G base-pairs are present in a variety of structural environments, including GNRA tetraloops, E and E-like loops, interfaced between two helices that are coaxially stacked, tandem G:A base-pairs, U-turns, and adenosine platforms . Finally, given structural studies that reveal conformational rearrangements occurring in regions of the RNA with AA and AG oppositions at the ends of helices, we suggest that these conformationally unique helix extensions might be associated with functionally important structural rearrangements .

J Mol Biol, 2001 Jul 20, 310(4), 709 - 22
The relationship between translational control and mRNA degradation for the Escherichia coli threonyl-tRNA synthetase gene; Nogueira T et al.; Expression of thrS, the gene encoding Escherichia coli threonyl-tRNA synthetase, is negatively autoregulated at the translational level . Regulation is due to the binding of threonyl-tRNA synthetase to its own mRNA at a site called the operator, located immediately upstream of the initiation codon . The present work investigates the relationship between regulation and mRNA degradation . We show that two regulatory mutations, which increase thrS expression, cause an increase in the steady-state mRNA concentration . Unexpectedly, however, the half-life of thrS mRNA in the derepressed mutants is equal to that of the wild-type, indicating that mRNA stability is independent of the repression level . All our results can be explained if one assumes that thrS mRNA is either fully translated or immediately degraded . The immediately degraded RNAs are never detected due to their extremely short half-lives, while the fully translated messengers share the same half-lives, irrespective of the mutations . The increase in the steady-state level of thrS mRNA in the derepressed mutants is simply explained by an increase in the population of translated molecules, i.e . those never bound by the repressor, ThrRS . Despite this peculiarity, thrS mRNA degradation seems to follow the classical degradation pathway . Its stability is increased in a strain defective for RNase E, indicating that an endonucleolytic cleavage by this enzyme is the rate-limiting process in degradation . We also observe an accumulation of small fragments corresponding to the 5' end of the message in a strain defective for polynucleotide phosphorylase, indicating that, following the endonucleolytic cleavages, fragments are normally degraded by 3' to 5' exonucleolytic trimming . Although mRNA degradation was suspected to increase the efficiency of translational control based on several considerations, our results indicate that inhibition of mRNA degradation has no effect on the level of repression by ThrRS .

Tohoku J Exp Med, 2001 Apr, 193(4), 293 - 9
Normal tension glaucoma and primary open angle glaucoma associated with increased platelet aggregation; Matsumoto M et al.; On purpose of the present study was to evaluate platelet aggregation and fibrinolytic systems in patients with normal tension glaucoma (NTG) or primary open angle glaucoma (POAG) . For platelet aggregation, we photoelectrophotometrically investigated adenosine diphosphate (ADP) or collagen-induced platelet aggregation in consecutively selected patients with glaucoma (22 patients with NTG and 13 patients with POAG) and 42 glaucoma free control subjects with normal ocular findings . The aggregation patterns of the patients' platelets reacted abnormally to ADP 1 microM or collagen 0.5 microg/ml as evidenced by secondary aggregation were compared with those of control subjects . For blood coagulative and fibrinolytic systems, we measured prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin-antithrombin III complex (TAT), alpha2 plasmin inhibitor-plasmin complex . Seventeen of 22 patients (77%) with NTG and 5 of 13 patients (38%) with POAG showed abnormal secondary aggregation . A significant difference was observed between the two groups . No control subjects showed abnormal secondary aggregation . In the fibrinolytic test, all the parameters examined showed within normal ranges, although the log10(TAT) value was higher in NTG than in POAG . Results of the present study suggested that increased platelet aggregation as defined by ADP or collagen induced abnormal secondary aggregation in vitro is frequently associated with glaucoma patients and this tendency is more apparent in NTG than that in POAG.

J Periodontal Res, 2001 Jun, 36(3), 160 - 8
Elevated mRNA expression for supervillin and vascular endothelial growth factor in human neutrophils stimulated with lipopolysaccharide from Porphyromonas gingivalis; Morozumi T et al.; Differential gene expression was examined in human neutrophils stimulated with lipopolysaccharide from Porphyromonas gingivalis (P . gingivalis-LPS) using RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) . LPS from Escherichia coli (E . coli-LPS) was used as control . More than 200 differently expressed transcripts were found in 8 subjects showing differential regulation at the transcriptional level . Densitometric analyses revealed that 42-100 genes were upregulated, while 53-116 genes were downregulated by P . gingivalis-LPS compared with E . coli-LPS . The results of sequencing identification showed the presence of supervillin (SVIL) and vascular endothelial growth factor (VEGF) genes in the clones which were upregulated by P . gingivalis-LPS . Consequently, semiquantitative analyses proved that the level of mRNA for SVIL and VEGF were significantly higher in P . gingivalis-LPS-stimulated neutrophils than in other bacterial (E . coli, Actinobacillus actinomycetemcomitans, Prevotella intermedia) LPS- or synthetic lipid A-stimulated neutrophils . Our findings suggest that an elevated mRNA expression for SVIL could be associated with impaired function of neutrophils when stimulated by P . gingivalis-LPS . Further, the VEGF mRNA over-expression might be related to the pathogenesis of P . gingivalis-associated periodontitis . The RAP-PCR technique used in this study enabled us to identify a number of P . gingivalis-LPS regulated genes which hitherto have not been reported.

RNA, 2001 Jul, 7(7), 999 - 1012
Determinants on tmRNA for initiating efficient and precise trans-translation: some mutations upstream of the tag-encoding sequence of Escherichia coli tmRNA shift the initiation point of trans-translation in vitro; Lee S et al.; tmRNA facilitates a novel translation, trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence . The mechanism underlying resumption of translation at a definite position is not known . In the present study, the effects of mutations around the initiation point of the tag-encoding sequence of Escherichia coli tmRNA on the efficiency and the frame of tag translation were assessed by measuring the incorporations of several amino acids into in vitro poly (U)-dependent tag-peptide synthesis . One-nucleotide insertions within the tag-encoding region did not shift the frame of tag translation . Any 1-nt deletion within the span of -5 to -1, but not at -6, made the frame of tag translation heterologous . Positions at which a single base substitution caused a decrease of trans-translation efficiency were concentrated within the span of -4 to -2 . In particular, an A-4 to C-4 mutation seriously damaged the trans-translation, although this mutant retained normal aminoacylation and ribosome-binding abilities . A possible stem and loop structure around this region was not required for transtranslation . It was concluded that the tag translation requires the primary sequence encompassing -6 to +11, in which the central 3 nt, A-4, G-3, and U-2, play an essential role . It was also found that several base substitutions within the span of -6 to -1 extensively shifted the tag-initiation point by -1.

RNA, 2001 Jul, 7(7), 990 - 8
A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain; Gutgsell NS et al.; This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth . We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated . In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive . In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine . Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines . Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.

RNA, 2001 Jul, 7(7), 958 - 68
Purine N7 groups that are crucial to the interaction of Escherichia coli rnase P RNA with tRNA; Heide C et al.; We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+) . The majority of identified positions represent highly or universally conserved nucleotides . Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306) . Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA-RNase P RNA conjugate in the presence of 3 M NH4+ or Na+ . This indicates that these N7 functional groups play a key role in the structural organization of ribozyme-substrate and -product complexes . We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E . coli RNase P RNA . This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E . coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76).

Nucleic Acids Res, 2001 Jul 15, 29(14), 3099 - 107
Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii; Chung JH et al.; A novel dNTP pyrophosphatase, Mj0226 from Methanococcus jannaschii, which catalyzes the hydrolysis of nucleoside triphosphates to the monophosphate and PPi, has been characterized . Mj0226 protein catalyzes hydrolysis of two major substrates, dITP and XTP, suggesting that the 6-keto group of hypoxanthine and xanthine is critical for interaction with the protein . Under optimal reaction conditions the k(ca)(t) /K(m) value for these substrates was approximately 10 000 times that with dATP . Neither endonuclease nor 3'-exonuclease activities were detected in this protein . Interestingly, dITP was efficiently inserted opposite a dC residue in a DNA template and four dNTPs were also incorporated opposite a hypoxanthine residue in template DNA by DNA polymerase I . Two protein homologs of Mj0226 from Escherichia coli and Archaeoglobus fulgidus were also cloned and purified . These have catalytic activities similar to Mj0226 protein under optimal conditions . The implications of these results have significance in understanding how homologous proteins, including Mj0226, act biologically in many organisms . It seems likely that Mj0226 and its homologs have a major role in preventing mutations caused by incorporation of dITP and XTP formed spontaneously in the nucleotide pool into DNA . This report is the first identification and functional characterization of an enzyme hydrolyzing non-canonical nucleotides, dITP and XTP.

Nucleic Acids Res, 2001 Jul 15, 29(14), 3020 - 9
Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity; Jammi NV et al.; The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), inhibiting the function of the eIF2 complex and continued initiation of translation . When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues . This autophosphorylation reaction stimulates the eIF2alpha kinase activity of PKR . The binding of certain viral RNAs inhibits the activation of PKR . Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli . We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme . The in vitro autophosphorylation and eIF2alpha kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA . Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism . Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA.Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho- and dephosphoPKR . We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs . These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2alpha.

Nucleic Acids Res, 2001 Jul 15, 29(14), 2986 - 93
Biochemical characterization of the small isoform of Drosophila melanogaster RECQ5 helicase; Ozsoy AZ et al.; Recently the gene encoding a member of the RecQ helicase family, RecQ5, was cloned from the fruit fly, Drosophila melanogaster {J.J.Sekelsky, M.H.Brodsky, G.M.Rubin and R.S.Hawley (1999) Nucleic Acids Res., 27, 3762-3769} . The Drosophila RecQ5 transcript is alternatively spliced, like its human counterpart, to yield three protein isoforms . Two of these isoforms are almost identical and have a predicted molecular weight of 54 kDa . The third isoform is larger and contains, in addition to the helicase domain shared by all three isoforms, a long highly charged C-terminal region . A small isoform of the Drosophila RecQ5 protein (RECQ5) has been expressed in Escherichia coli and purified . The purified protein is a single-stranded DNA-stimulated ATPase (dATPase) and a 3'-->5' DNA helicase . Hydrolysis of the nucleotide cofactor is required for unwinding activity and dATP supported the unwinding reaction better than other NTPs . The turnover number for the single-stranded DNA-stimulated dATPase activity was 1380 min(-1), approximately 1.5-fold higher than that observed for the ATPase activity (900 min(-1)) . The purified protein catalyzed unwinding of partial duplex substrates up to at least 93 bp, however, unwinding of an 89 bp blunt duplex substrate was not detected.

Mol Biol Cell, 2001 Jul, 12(7), 2061 - 73
Activation of rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells; Fiorentini C et al.; Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function . It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors . In this case, the internalized material recycles back to the cell surface . We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes . In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation . Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes . The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.

J Biol Chem, 2001 Sep 28, 276(39), 36200 - 6 Epub 2001 Jul 12.
Insertion of PsaK into the thylakoid membrane in a "Horseshoe" conformation occurs in the absence of signal recognition particle, nucleoside triphosphates, or functional albino3; Mant A et al.; The photosystem I subunit PsaK spans the thylakoid membrane twice, with the N and C termini both located in the lumen . The insertion mechanism of a thylakoid membrane protein adopting this type of topology has not been studied before, and we have used in vitro assays to determine the requirements for PsaK insertion into thylakoids . PsaK inserts with high efficiency and we show that one transmembrane span (the C-terminal region) can insert independently of the other, indicating that a "hairpin"-type mechanism is not essential . Insertion of PsaK does not require stromal extract, indicating that signal recognition particle (SRP) is not involved . Removal of nucleoside triphosphates inhibits insertion only slightly, both in the presence and absence of stroma, suggesting a mild stimulatory effect of a factor in the translation system and again ruling out an involvement of SRP or its partner protein, FtsY . We, furthermore, find no evidence for the involvement of known membrane-bound translocation apparatus; proteolysis of thylakoids destroys the Sec and Tat translocons but does not block PsaK insertion, and antibodies against the Oxa1/YidC homolog, Alb3, block the SRP-dependent insertion of Lhcb1 but again have no effect on PsaK insertion . Because YidC is required for the efficient insertion of every membrane protein tested in Escherichia coli (whether SRP-dependent or -independent), PsaK is the first protein identified as being independent of YidC/Alb3-type factors in either thylakoids or bacteria . The data raise the possibility of a wholly spontaneous insertion pathway.

Antimicrob Agents Chemother, 2001 Aug, 45(8), 2378 - 80
Mutation in the DNA gyrase A Gene of Escherichia coli that expands the quinolone resistance-determining region; Friedman SM et al.; In three Escherichia coli mutants, a change (Ala-51 to Val) in the gyrase A protein outside the standard quinolone resistance-determining region (QRDR) lowered the level of quinolone susceptibility more than changes at amino acids 67, 82, 84, and 106 did . Revision of the QRDR to include amino acid 51 is indicated.

Chem Biol, 2001 Jul, 8(7), 725 - 38
Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin; Sanchez C et al.; BACKGROUND: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides . PPTases have been classified according to their carrier protein specificity . In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity . However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene . RESULTS: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S . verticillus . The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters . The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution . Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S . verticillus or other Streptomyces species . As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP . CONCLUSIONS: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type . The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.

J Neurooncol, 2001 Mar, 52(1), 11 - 21
Role of ceramide during cisplatin-induced apoptosis in C6 glioma cells; Noda S et al.; Cisplatin is commonly used for the treatment of malignant brain tumors . However, the mechanisms of cell death by cisplatin are not fully understood . Therefore, the present study was designed to elucidate the apoptotic signaling pathway(s) activated by cisplatin in a C6 rat glioma cell line . C6 cells were treated with various concentrations of cisplatin (0.2-10 microg/ml) for 24-72 h . At 10 microg/ml cisplatin, over 90% of the cells became dead at 72 h . Apoptotic death was confirmed by condensation and fragmentation of nuclei, and DNA laddering . Even in cells treated with 1.5 microg/ml cisplatin, typical apoptotic cells were observed at 72 h . The intracellular level of ceramide, measured Escherichia coli diacylglycerol kinase markedly increased during 24-72 h after the addition of 10 microg/ml cisplatin . The activity of caspase-3(-like) proteases increased and reached a peak at 48 h . Inhibitors of caspases reduced the number of apoptotic cells . Pretreatment of C6 cells with glutathione or N-acetyl-cysteine, which are known to block the activation of neutral magnesium-dependent sphingomyelinase, inhibited ceramide formation, leading to suppression of both activation of caspase-3(-like) proteases and apoptosis by cisplatin . In contrast, pretreatment of the cells with N-oleoylethanolamine (OE), a ceramidase inhibitor, potentiated apoptosis induced by cisplatin . Furthermore, OE enhanced sensitivity of the cisplatin-resistant cells to cisplatin . These results suggest that ceramide is closely implicated in apoptosis of glioma cells by cisplatin through activation of caspase-3(-like) proteases.

Virus Genes, 2001 Jun, 22(3), 363 - 72
Generation of a recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus expressing a foreign gene under the control of a very late promoter; Arana EI et al.; A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) expressing beta-galactosidase under the control of the polyhedrin promoter was generated in our laboratory . To this end, we cloned the AgMNPV-2D genomic DNA fragment containing the polh gene and subcloned and sequenced the polyhedrin gene and its flanking regions . Based on this sequence information, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites . The transfer vector (pAgPHZ) was constructed by the consecutive cloning of these PCR fragments flanking the Escherichia coli LacZ gene, in place of the polh gene . pAgPHZ was used for cotransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the required recombinant, generated by homologous recombination with the polh locus, was identified by its polh(-)/LacZ+ plaque phenotype . Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses . The kinetics and levels of expression of beta-galactosidase in UFL-AG-286 cells infected with the recombinant were tested by SDS-PAGE and enzymatic activity assays.

Adv Microb Physiol, 2001, 45, 1 - 49
The regulation of pap and type 1 fimbriation in Escherichia coli; Blomfield IC; The ability of bacterial pathogens to bind to the host mucosa is a critical step in the pathogenesis of many bacterial infections and, for Escherichia coli, a large number of different fimbrial adhesins have been implicated as virulence factors . In this chapter, our current understanding of the regulatory mechanisms that control the expression of two of the best characterized fimbrial adhesins, pyelonephritis-associated pilus (encoded by pap) and the type 1 fimbria (encoded by fim), will be described . The expression of both fimbrial adhesins is controlled by phase variation (the reversible and apparently random switching between expressing ('on') and non-expressing ('off') states), and is regulated in response to environmental conditions . The phase variation of pap (and of some other fimbriae in Escherichia coli) is determined by the formation of alternative nucleoprotein complexes that either activate (phase 'on') or suppress (phase 'off') transcription of the fimbria genes . Formation of each complex protects a single Dam methylation site (5' GATC) from modification (GATCdist in phase 'on' cells and GATCprox in phase 'off' cells) . Furthermore, complex formation is inhibited by methylation of the two 5' GATC sites . Both the phase variation of pap and the transcription of the pap genes in phase 'on' cells, are regulated and expression is subject to both positive and negative feedback control . In contrast to pap, the phase variation of fim is determined by the site-specific inversion of a short element of DNA (the fim switch) . In phase 'on' cells, a promoter within the invertible element directs the transcription of the fim structural genes, whereas in phase 'off' cells transcription of the fimbrial genes is silenced . Despite the very different molecular mechanisms controlling the expression of pap and fim, the two systems share many features in common and have probably evolved to fulfill the same function . In addition to details about the molecular mechanisms that control pap and fim, the possible physiological significance of the observed regulation will be discussed.

J Mol Graph Model, 2001, 19(3-4), 304 - 6, 379
H-BloX: visualizing alignment block entropies; Zuegge J et al.; H-BloX is a web-based JavaScript application that allows the calculation and visualization of Shannon information content or relative entropy (Kullback-Leibler 'distance') within sequence alignment blocks . The application was designed for use in both teaching and research . Amino acid, nucleic acid sequences, or any other type of aligned chemical structures may serve as the input . Various interpretations of the meaning of 'entropy' or 'information content' are possible, including treatment as a chemical diversity measure or the degree of feature conservation . For analysis of numerical data by H-BloX, values must be converted to a user-defined character alphabet before computation of entropy or information content . H-BloX was successfully applied to feature identification in Escherichia coli signal peptides and their cleavage sites . Characteristics known features became visible, e.g., the hydrophobic core region and the well-known '-3,-1' cleavage site pattern . Based on the H-BloX analysis, the hydrophobic core is centered at amino acid residue position 13, counting from the N-terminal end of the protein precursor sequence . This result was obtained by using a built-in feature of H-BloX that enables conversion of amino acid sequences to a different alphabet that is based on hydrophobicity assignments . H-BloX can be accessed online or downloaded as HTML/JavaScript at http://bopwww.biologie.uni-freiburg.de/~bioinfo/HBloX/html/index.html.

Braz J Med Biol Res, 2001 Jul, 34(7), 913 - 7
Role of Tamm-Horsfall protein in the binding and in vivo phagocytosis of type 1 fimbriated Escherichia coli by mouse peritoneal macrophages; Bastos AC et al.; Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli . In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP . At low THP concentrations (12.5 microg/ml and 50 microg/ml) no significant difference was observed in the phagocytosis of E . coli F1+ . However, at high THP concentrations (500 microg/ml and 1250 microg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages.

J Biol Chem, 2001 Sep 7, 276(36), 34189 - 98 Epub 2001 Jul 11.
Biophysical characterization of interactions involving importin-alpha during nuclear import; Catimel B et al.; Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer . Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore . We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism) . Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor) . Association with importin-beta (stoichiometry, 1:1; K(D) = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K(D) = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K(D) = 1.7 x 10(-8) m; nucleoplasmin NLS, K(D) = 1.4 x 10(-8) m) . The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha . Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import.

J Biol Chem, 2001 Sep 7, 276(36), 34318 - 22 Epub 2001 Jul 11.
Recombinant small subunit of smooth muscle myosin light chain phosphatase . Molecular properties and interactions with the targeting subunit; Langsetmo K et al.; We expressed the small subunit of smooth muscle myosin light chain phosphatase (MPs) in Escherichia coli, and have studied its molecular properties as well as its interaction with the targeting subunit (MPt) . MPs (M(r) = 18,500) has an anomalously low electrophoretic mobility, running with an apparent M(r) of approximately 21,000 in sodium dodecyl sulfate-gel electrophoresis . CD spectroscopy shows that it is approximately 45% alpha-helix and undergoes a cooperative temperature-induced unfolding with a transition midpoint of 73 degrees C . Limited proteolysis rapidly degrades MPs to a stable C-terminal fragment (M(r) = 10,000) that retains most of the helical content . Rotary shadowing electron microscopy reveals that it is an elongated protein with two domains . Sedimentation velocity measurements show that recombinant MPt (M(r) = 107,000), intact MPs, and the 10-kDa MPs fragment are all dimeric, and that MPs and MPt form a complex with a molar mass consistent with a 1:1 heterodimer . Sequence analysis predicts that regions in the C-terminal portions of both MPs and MPt have high probabilities for coiled coil formation . A synthetic peptide from a region of MPs encompassing residues 77-116 was found to be 100% alpha-helical, dimeric, and formed a complex with MPt with a molecular mass corresponding to a heterodimer . Based on these results, we propose that MPs is an elongated molecule with an N-terminal head and a C-terminal stalk domain . It dimerizes via a coiled coil interaction in the stalk domain, and interacts with MPt via heterodimeric coiled coil formation . Since other proteins with known regulatory function toward MP also have predicted coiled coil regions, our results suggest that these regulatory proteins target MP via the same coiled coil strand exchange mechanism with MPt.

Bioinformatics, 2001 Jul, 17(7), 608 - 21
Identifying target sites for cooperatively binding factors; GuhaThakurta D et al.; MOTIVATION: Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors . Though several methods exist for identification of individual protein binding site patterns in DNA sequences, there are few methods for discovery of binding site patterns for cooperatively acting factors . Here we present an algorithm, Co-Bind (for COperative BINDing), for discovering DNA target sites for cooperatively acting transcription factors . The method utilizes a Gibbs sampling strategy to model the cooperativity between two transcription factors and defines position weight matrices for the binding sites . Sequences from both the training set and the entire genome are taken into account, in order to discriminate against commonly occurring patterns in the genome, and produce patterns which are significant only in the training set . RESULTS: We have tested Co-Bind on semi-synthetic and real data sets to show it can efficiently identify DNA target site patterns for cooperatively binding transcription factors . In cases where binding site patterns are weak and cannot be identified by other available methods, Co-Bind, by virtue of modeling the cooperativity between factors, can identify those sites efficiently . Though developed to model protein-DNA interactions, the scope of Co-Bind may be extended to combinatorial, sequence specific, interactions in other macromolecules . AVAILABILITY: The program is available upon request from the authors or may be downloaded from http://ural.wustl.edu.

Curr Biol, 2001 Jun 26, 11(12), 941 - 50
Novel small RNA-encoding genes in the intergenic regions of Escherichia coli; Argaman L et al.; Background: Small, untranslated RNA molecules were identified initially in bacteria, but examples can be found in all kingdoms of life . These RNAs carry out diverse functions, and many of them are regulators of gene expression . Genes encoding small, untranslated RNAs are difficult to detect experimentally or to predict by traditional sequence analysis approaches . Thus, in spite of the rising recognition that such RNAs may play key roles in bacterial physiology, many of the small RNAs known to date were discovered fortuitously.Results: To search the Escherichia coli genome sequence for genes encoding small RNAs, we developed a computational strategy employing transcription signals and genomic features of the known small RNA-encoding genes . The search, for which we used rather restrictive criteria, has led to the prediction of 24 putative sRNA-encoding genes, of which 23 were tested experimentally . Here we report on the discovery of 14 genes encoding novel small RNAs in E . coli and their expression patterns under a variety of physiological conditions . Most of the newly discovered RNAs are abundant . Interestingly, the expression level of a significant number of these RNAs increases upon entry into stationary phase.Conclusions: Based on our results, we conclude that small RNAs are much more widespread than previously imagined and that these versatile molecules may play important roles in the fine-tuning of cell responses to changing environments.

Carbohydr Res, 2001 Jul 3, 333(2), 179 - 83
Structural studies of the O-polysaccharide from the Escherichia coli O77 lipopolysaccharide; Yildirim H et al.; The structure of the O-antigen polysaccharide (PS) from Escherichia coli O77 has been determined . Sugar and methylation analysis together with 1H and 13C NMR spectroscopy were the main methods used . The PS is composed of tetrasaccharide repeating units with the following structure:-->2)-alpha-D-Manp-(1-->2)-beta-D-Manp-(1-->3)-alpha-D-GlcpNAc-(1-->6)-alpha-D-Manp-(1-->

J Am Chem Soc, 2001 Jul 18, 123(28), 6892 - 903
Chi1 torsion angle dynamics in proteins from dipolar couplings; Mittermaier A et al.; Experiments are presented for the measurement of one-bond carbon-proton dipolar coupling values at CH and CH2 ositions in 13C-labeled, approximately 50% fractionally deuterated proteins . 13Cbeta-1Hbeta dipolar couplings have been measured for 38 of 49 possible residues in the 63-amino-acid B1 domain of peptostreptococcal protein L in two aligning media and interpreted in the context of side-chain chi1 torsion angle dynamics . The beta protons for 18 of the 25 beta-methylene-containing amino acids for which dipolar data are available can be unambiguously stereoassigned, and for those residues which are best fit to a single rotamer model the chi(1) angles obtained deviate from crystal structure values by only 5.2 degrees (rmsd) . The results for 11 other residues are significantly better fit by a model that assumes jumps between the three canonical (chi1 approximately -60 degrees, 60 degrees, 180 degrees ) rotamers . Relative populations of the rotamers are determined to within +/-6% uncertainty on average and correlate with dihedral angles observed for the three molecules in the crystal asymmetric unit . Entropic penalties for quenching chi1 jumps are considered for six mobile residues thought to be involved in binding to human immunoglobulins . This study demonstrates that dipolar couplings may be used to characterize both the conformation of static residues and side-chain motion with high precision.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8566 - 71 Epub 2001 Jul 10.
Mechanism of DNA polymerase II-mediated frameshift mutagenesis; Becherel OJ et al.; Escherichia coli possesses three SOS-inducible DNA polymerases (Pol II, IV, and V) that were recently found to participate in translesion synthesis and mutagenesis . Involvement of these polymerases appears to depend on the nature of the lesion and its local sequence context, as illustrated by the bypass of a single N-2-acetylaminofluorene adduct within the NarI mutation hot spot . Indeed, error-free bypass requires Pol V (umuDC), whereas mutagenic (-2 frameshift) bypass depends on Pol II (polB) . In this paper, we show that purified DNA Pol II is able in vitro to generate the -2 frameshift bypass product observed in vivo at the NarI sites . Although the Delta polB strain is completely defective in this mutation pathway, introduction of the polB gene on a low copy number plasmid restores the -2 frameshift pathway . In fact, modification of the relative copy number of polB versus umuDC genes results in a corresponding modification in the use of the frameshift versus error-free translesion pathways, suggesting a direct competition between Pol II and V for the bypass of the same lesion . Whether such a polymerase competition model for translesion synthesis will prove to be generally applicable remains to be confirmed.

Infect Immun, 2001 Aug, 69(8), 5182 - 5
Concurrent upregulation of urokinase plasminogen activator receptor and CD11b during tuberculosis and experimental endotoxemia; Juffermans NP et al.; Patients with tuberculosis had higher expression of monocyte urokinase receptor (uPAR) and CD11b than controls . In vitro, lipoarabinomannan and lipopolysaccharide (LPS) from Escherichia coli shared the ability to enhance uPAR and CD11b expression on monocytes and granulocytes . In healthy volunteers, LPS induced increases in monocyte and granulocyte uPAR and CD11b.

Infect Immun, 2001 Aug, 69(8), 5080 - 7
Vacuolating cytotoxin of Helicobacter pylori induces apoptosis in the human gastric epithelial cell line AGS; Kuck D et al.; Helicobacter pylori induces cell death by apoptosis . However, the apoptosis-inducing factor is still unknown . The virulence factor vacuolating cytotoxin A (VacA) is a potential candidate, and thus its role in apoptosis induction was investigated in the human gastric epithelial cell line AGS . The supernatant from the vacA wild-type strain P12 was able to induce apoptotic cell death, whereas the supernatant from its isogenic mutant strain P14 could not . That VacA was indeed the apoptosis-inducing factor was demonstrated further by substantial reduction of apoptosis upon treatment of AGS cells with a supernatant specifically depleted of native VacA . Furthermore, a recombinant VacA produced in Escherichia coli was also able to induce apoptosis in AGS cells but failed to induce cellular vacuolation . These findings demonstrate that the vacuolating cytototoxin of H . pylori is a bacterial factor capable of inducing apoptosis in gastric epithelial cells.

Infect Immun, 2001 Aug, 69(8), 4969 - 79
Construction and characterization of genetically defined aro omp mutants of enterotoxigenic Escherichia coli and preliminary studies of safety and immunogenicity in humans; Turner AK et al.; Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in travelers to countries where the disease is endemic and causes a major disease burden in the indigenous population, particularly children . We describe here the generation and preclinical characterization of candidate strains of ETEC which are intended to provide the basis of a live attenuated oral vaccine to prevent this disease . It has been shown previously that a spontaneously arising toxin-negative variant ETEC strain, E1392/75-2A, could confer 75% protection against challenge when administered to volunteers . Unfortunately this strain induced mild diarrhea in 15% of recipients . To eliminate the unacceptable reactogenicity of strain E1392/75-2A, it was further attenuated by introducing three different combinations of defined deletion mutations into the chromosome . A mouse intranasal model of immunization was developed and used to show that all of the strains were immunogenic . Immune responses against colonization factor antigens (CFAs) were particularly strong when the bacterial inocula were grown on "CFA agar," which induces strong expression of these antigens . Two of the strains were selected for a phase I dose escalation safety study with healthy adult volunteers . Freshly grown organisms were harvested from CFA agar plates and administered to volunteers as a suspension containing from 5 x 10(7) to 5 x 10(9) CFU . The vaccine was well tolerated at all doses and induced significant immune responses in all recipients at the highest dose of either strain . The results provide the basis for further clinical evaluation of these vaccine candidates.

Infect Immun, 2001 Aug, 69(8), 4923 - 30
Escherichia coli enterotoxin B subunit triggers apoptosis of CD8(+) T cells by activating transcription factor c-myc; Soriani M et al.; Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties . Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8(+) T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways . Here we show that EtxB treatment of CD8(+) T cells but not of CD4(+) T cells triggers the specific up-regulation of the transcription factor c-myc, implicated in the control of cell proliferation, differentiation, and death . A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment . Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis . Myc activation was also associated with an increase of IkappaBalpha turnover, suggesting that elevated Myc expression may be dependent on NF-kappaB . When CD8(+) T cells were pretreated with inhibitors of IkappaBalpha turnover and NF-kappaB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression . Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription . These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

Infect Immun, 2001 Aug, 69(8), 4759 - 66
Assessment of Helicobacter pylori gene expression within mouse and human gastric mucosae by real-time reverse transcriptase PCR; Rokbi B et al.; Despite increasing knowledge on the biology of Helicobacter pylori, little is known about the expression pattern of its genome during infection . While mouse models of infection have been widely used for the screening of protective antigens, the reliability of the mouse model for gene expression analysis has not been assessed . In an attempt to address this question, we have developed a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa . The expression of four genes, 16S rRNA, ureA (encoding urease A subunit), katA (catalase), and alpA (an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans . We found that the selected genes were all expressed within both mouse and human infected mucosae . Moreover, the relative abundance of transcripts was the same (16S rRNA > ureA > katA > alpA), in the two models . Finally, results obtained with the mouse model suggest a negative effect of bacterial burden on the number of transcripts of each gene expressed per CFU (P < 0.05 for 16S rRNA, alpA, and katA) . Overall, this study demonstrates that real-time RT-PCR is a powerful tool for the detection and quantification of H . pylori gene expression within the gastric mucosa and strongly indicates that mice experimentally infected with H . pylori provide a valuable model for the analysis of bacterial gene expression during infection.

Glycobiology, 2001 Jul, 11(7), 605 - 11
The observation of multivalent complexes of Shiga-like toxin with globotriaoside and the determination of their stoichiometry by nanoelectrospray Fourier-transform ion cyclotron resonance mass spectrometry; Kitova EN et al.; We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the millimolar range, such as P(k) trisaccharide (globotriaoside) complexed with the Shiga-like toxin (SLT) of pathogenic E . coli . It is further demonstrated that nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as the SLTs . At increasing trisaccharide:protein ratios it was shown that the B(5 )toxin subunit complexes with 5 P(k) trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied . From the distribution of bound P(k)'s at the five binding sites, it was possible to establish association constants for each of the five sites and to confirm that binding occurs noncooperatively, the association constants for each site are identical and that compared to site 1, site 2 exhibits a tenfold higher affinity for the globotriaoside synthetic ligand 1 . The facile identification of the occupancy of binding sites represents information that is not readily available by other techniques . This sensitive and rapid estimation of association constants for protein-ligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application.

EMBO J, 2001 Jul 16, 20(14), 3831 - 9
The exosome of Trypanosoma brucei; Estevez AM et al.; The yeast exosome is a complex of at least 10 essential 3'-5' riboexonucleases which is involved in 3'-processing of many RNA species . An exosome-like complex has been found or predicted to exist in other eukaryotes but not in Escherichia coli . The unicellular parasite Trypanosoma brucei diverged very early in eukaryotic evolution . We show here that T.brucei contains at least eight exosome subunit homologs, but only a subset of these associate in a complex . Accordingly, the T.brucei exosome is smaller than that of yeast . Both free and complex-associated homologs are essential for cell viability and are involved in 5.8S rRNA maturation . We suggest that the exosome was present in primitive eukaryotes, and became increasingly complex during subsequent evolution.

EMBO J, 2001 Jul 16, 20(14), 3811 - 20
Ribosomal protein S4 is a transcription factor with properties remarkably similar to NusA, a protein involved in both non-ribosomal and ribosomal RNA antitermination; Torres M et al.; Escherichia coli ribosomal RNA (rRNA) operons contain antitermination motifs necessary for forming terminator-resistant transcription complexes . In preliminary work, we isolated 'antiterminating' transcription complexes and identified four new proteins potentially involved in rRNA transcription antitermination: ribosomal (r-) proteins S4, L3, L4 and L13 . We show here that these r-proteins and Nus factors lead to an 11-fold increase in terminator read-through in in vitro transcription reactions . A significant portion of the effect was a result of r-protein S4 . We show that S4 acted as a general antitermination factor, with properties very similar to NusA . It retarded termination and increased read-through at Rho-dependent terminators, even in the absence of the rRNA antiterminator motif . High concentrations of NusG showed reduced antitermination by S4 . Like rrn antitermination, S4 selectively antiterminated at Rho-dependent terminators . Lastly, S4 tightly bound RNA polymerase in vivo . Our results suggest that, like NusA, S4 is a general transcription antitermination factor that associates with RNA polymerase during normal transcription and is also involved in rRNA operon antitermination . A model for key r-proteins playing a regulatory role in rRNA synthesis is presented.

EMBO J, 2001 Jul 16, 20(14), 3667 - 75
Adenylyl cyclase Rv1625c of Mycobacterium tuberculosis: a progenitor of mammalian adenylyl cyclases; Guo YL et al.; The gene Rv1625c from Mycobacterium tuberculosis encodes a membrane-anchored adenylyl cyclase corresponding to exactly one-half of a mammalian adenylyl cyclase . An engineered, soluble form of Rv1625c was expressed in Escherichia coli . It formed a homodimeric cyclase with two catalytic centers . Amino acid mutations predicted to affect catalysis resulted in inactive monomers . A single catalytic center with wild-type activity could be reconstituted from mutated monomers in stringent analogy to the mammalian heterodimeric cyclase structure . The proposed existence of supramolecular adenylyl cyclase complexes was established by reconstitution from peptide-linked, mutation-inactivated homodimers resulting in pseudo-trimeric and -tetrameric complexes . The mycobacterial holoenzyme was expressed successfully in E.coli and mammalian HEK293 cells, i.e . its membrane targeting sequence was compatible with the bacterial and eukaryotic machinery for processing and membrane insertion . The membrane-anchored mycobacterial cyclase expressed in E.coli was purified to homogeneity as a first step toward the complete structural elucidation of this important protein . As the closest progenitor of the mammalian adenylyl cyclase family to date, the mycobacterial cyclase probably was spread by horizontal gene transfer.

Am J Physiol Gastrointest Liver Physiol, 2001 Aug, 281(2), G569 - 76
Enteral feeding decreases gut apoptosis, permeability, and lung inflammation during murine endotoxemia; Alscher KT et al.; We tested the hypothesis that endotoxemia and fasting are associated with increased gut apoptotic activity, gut permeability, and inflammation in a distant organ . Fed or fasted CD-1 mice were studied 6 h after intraperitoneal injection of either saline (sham) or endotoxin (4 mg/kg of 0111:B4 Escherichia coli lipopolysaccharide) . We found that endotoxin increased gut caspase-3 and -6 activity by 4.9 +/- 0.6- and 4.5 +/- 0.5-fold, respectively (P < 0.001), and increased terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) staining of mucosal cells (P < 0.05) . Feeding decreased caspase-3 activity by 40% (P < 0.05) and decreased endotoxin-induced TUNEL staining (P < 0.05) . Endotoxin increased gut poly(ADP-ribose) polymerase activity by 15% (P < 0.05) . Endotoxin increased gut permeability by 44% (P < 0.05), an effect reduced 36% by feeding (P < 0.05) . Similarly, endotoxin increased pulmonary neutrophil infiltration (6.0 +/- 1.0-fold, P < 0.001) and increased lung interleukin (IL)-6 (5.9 +/- 0.1-fold, P < 0.001) and macrophage inflammatory protein (MIP)-2 expression (290 +/- 40-fold, P < 0.001), whereas feeding decreased this effect by 43% for neutrophils, 40% for IL-6 (P < 0.05), and 35% for MIP-2 (P < 0.05) . Thus endotoxin increases gut apoptotic activity, gut permeability, and pulmonary inflammation . Enteral feeding may decrease the distant organ inflammation by reducing gut apoptosis, thereby maintaining gut mucosal function during endotoxemia.

Nutr Res, 2001 Jul, 21(7), 1035 - 1044
Arginine-genotype interactions and immune status; Kwak H et al.; The effects of arginine on selective immune responses were investigated in a high arginine-requiring (HA) and low arginine-requiring (LA) strain of chickens . Female chickens from these strains were fed diet containing a nutritionally inadequate level of arginine (0.53% arginine diet) or a surfeit level of arginine (1.53% arginine diet) for 2 weeks . Compared to LA chickens, HA chickens showed a higher feed efficiency, body weight gain, and relative thymus and spleen weights with L-arginine supplementation (p < 0.05) . In both HA and LA chickens, a deficiency of arginine significantly decreased the delayed-type hypersensitivity response (p < 0.05) and nitric oxide (NO) production from macrophages . Chickens of the HA strain had higher NO production than those of LA strain with E . coli lipopolysaccharide (LPS) activation . This study indicates that dietary arginine concentration influences the immune status of chickens and that strains that differ in arginine requirements for growth may differ in their arginine needs for immune function.

Exp Eye Res, 2001 Aug, 73(2), 257 - 64
The functional unit of interphotoreceptor retinoid-binding protein (IRBP)--purification, characterization and preliminary crystallographic analysis; Loew A et al.; To define the relationship between the structure and function of interphotoreceptor retinoid-binding protein (IRBP) we seek to prepare crystals of IRBP suitable for X-ray crystallographic analysis . As recent studies suggest that each of IRBPs four homologous regions or modules possess ligand-binding activity, we here explore the feasibility of preparing crystals from an individual module . Xenopus laevis IRBP, which has a similar four-module structure as that of mammalian and avian IRBPs, was selected for these studies in view of the advantage of the Xenopus retina for cellular and transgenic approaches . In the present study we focused on the second module of Xenopus IRBP . This module was expressed as a thioredoxin/histidine-patch fusion protein to promote its soluble expression in Escherichia coli and subsequent purification . The ligand-binding properties of the fusion protein were determined by fluorescence spectroscopy . For the preparation of crystals, the module was enzymatically separated from the fusion tag . Crystals of the native and selenomethionine derivatized module were prepared by vapor diffusion in hanging drops . Module II of IRBP binds 1.57 +/- 0.041 and 1.49 +/- 0.15 equivalents of at all- trans retinol and 9-(9-anthroyloxy) stearic acid, respectively, with KDs in the 0.1 microM range . Crystals of this module had an elongated rectangular beam-like morphology . A complete dataset of a frozen selenomethionine crystal extending to 1.85 A resolution was collected . Focusing on the individual modules will likely provide an effective strategy to correlate biochemical and structural data to define the functional domains of IRBP . The quality and resolution of the data obtained suggests that it will be possible in the near future to solve the X-ray crystal structure of the IRBP modules .

Semin Thromb Hemost, 2001 Jun, 27(3), 287 - 92
Function of von Willebrand factor in children with diarrhea-associated hemolytic-uremic syndrome (D+ HUS); Sutor AH et al.; Reports on von Willebrand factor (vWF) in hemolytic-uremic syndrome (HUS) are not unequivocal . Because of potential pathogenic implications, we examined the ability of vWF to bind to collagen in vitro, which reflects its function . Plasma vWF antigen (vWF:Ag) and collagen-binding activity (vWF:CBA) were measured by enzyme-linked immunosorbent assay in children with (1) diarrhea-associated (D+) HUS (n = 27), (2) chronic renal insufficiency (CRI) (n = 8), (3) gastroenteritis (GE) not associated with HUS (n = 15), (4) immune thrombocytopenia (ITP) (n = 40) and from controls (n = 35) . Structural vWF was evaluated by multimer analysis . Children with D+ HUS had vWF:Ag of 2.53 and vWF:CBA of 1.98 U/mL . The corresponding values for patients with ITP were 1.35 and 1.82 U/mL, with CRI 1.55 and 1.55 U/mL, and with GE 1.68 and 2.10 U/mL; all values were higher than in controls (1.04 and 1.16 U/mL) . The mean ratio of vWF:CBA to vWF:Ag ratio in controls was 1.13; only children with HUS had a dysfunctional vWF, as indicated by a low ratio of 0.78; the ratio was elevated in children with ITP (1.36) and GE (1.27) and was normal in those with CRI (1.06) . No ultralarge molecular multimers of vWF were detected in any group, including HUS . The very high concentration of plasma vWF:Ag in HUS probably reflects endothelial cell damage or irritation . In contrast to all other groups, only children with HUS had a dysfunctional vWF, caused either by a primary (due to enterohemorrhagic Escherichia coli) or secondary (due to consumption of functionally active vWF) process . This abnormality was not obvious as structural anomaly by multimer analysis.

Semin Thromb Hemost, 2001 Jun, 27(3), 201 - 5
The role of coagulation and fibrinolysis in the pathogenesis of diarrhea-associated hemolytic uremic syndrome; Proesmans W; The hemolytic-uremic syndrome (HUS) is a frequent cause of acute renal failure in childhood . It comprises acute acquired hemolysis, thrombocytopenia, and renal dysfunction . Very many disease processes can lead to this constellation, the most frequent one in childhood being an infection by bacteria that produce Shiga toxin or Shiga-like toxins (SLTs) . In industrialized countries, the first identified human pathogen to cause HUS was Escherichia coli O157H7, and this organism remains the most common one . The mechanisms by which these bacteria cause hemorrhagic colitis and HUS are incompletely understood . The bacteria are able to adhere to the mucosa of the colon . The local and systemic effects that follow the intestinal invasion are responsible for the bloody diarrhea . In a further step, the SLTs reach the blood stream and attach to the endothelium of the small arterioles mainly in the kidney but also in other organs . The endothelial cells express a toxin-specific receptor that enables the contact between toxin and cells . Damage to the endothelium causes platelet aggregation and activation, which trigger fibrin deposition . Although thrombotic changes in the microcirculation have been recognized in many histological studies, it is only recently that coagulation studies have been able to demonstrate clearly localized intravascular coagulation . The finding that the fibrinolytic system is inhibited in HUS has been challenged . By using specific and sensitive tests to measure the active moiety of plasminogen activator inhibitor type 1 (PAI-1) and comparing the findings in HUS patients and appropriate controls, it has become clear that in diarrhea-associated HUS the fibrinolytic system is rather stimulated . In this contribution the pathophysiology of diarrhea-associated HUS is discussed with special emphasis on coagulation and fibrinolysis.

Chemistry, 2001 Jun 1, 7(11), 2398 - 406
Effects of dimerization on protein electron transfer; van Amsterdam IM et al.; In order to investigate the relationship between the rate of protein-protein electron transfer and the structure of the association complex, a dimer of the blue copper protein azurin was constructed and its electron exchange properties were determined . For this purpose, a site for covalent cross-linking was engineered by replacing the surface-exposed asparagine 42 with a cysteine . This mutation enabled the formation of disulfide-linked homo-dimers of azurin . Based on NMR line-broadening experiments, the electron self-exchange (e.s.e.) rate constant for this dimer was determined to be 4.2(+/-0.7) x 10(5)M(-1)s(-1), which is a seven-fold decrease relative to wild-type azurin . This difference is ascribed to a less accessible hydrophobic patch in the dimer . To discriminate between intramolecular electron transfer within a dimer and intermolecular electron transfer between two dimers, the e.s.e . rate constant of (Cu-Cu)-N42C dimers was compared with that of (Zn-Cu)- and (Ag-Cu)-N42C dimers . As Zn and Ag are redox inactive, the intramolecular electron transfer reaction in these latter dimers can be eliminated . The e.s.e . rate constants of the three dimers are the same and an upper limit for the intramolecular electron transfer rate of 10 s(-1) could be determined . This rate is compatible with a Cu-Cu distance of 18 A or more, which is larger than the Cu - Cu distance of 15 A observed in the wild-type crystal structure that shows two monomers that face each other with opposing hydrophobic patches . Modelling of the dimer shows that the Cu-Cu distance should be in the range of 17 A < rCu-Cu < 28 A, which is in agreement with the experimental findings . For efficient electron transfer, it appears crucial that the two molecules interact in the proper orientation . Direct cross-linking may disturb the formation of such an optimal electron transfer complex.

Res Microbiol, 2001 Jun, 152(5), 487 - 92
A novel gene from Myxococcus xanthus that facilitates membrane translocation of an extracellular endoglucanase in Escherichia coli?
Bensmail L, Monnier C, Quillet L, Guespin-Michel JF, Barray S.
Expression in Escherichia coli of the Myxococcus xanthus gene celA, which encodes an extracellular endoglucanase, resulted in CelA being distributed between cytoplasm, periplasm and membrane . The presence of an adjacent open reading frame downstream from the full celA gene, or the absence of a putative lipoprotein signal sequence, confined CelA distribution to the periplasm and membrane, or to the cytoplasm and periplasm, respectively.

Res Microbiol, 2001 Jun, 152(5), 469 - 79
Regulation of microcin C51 operon expression: the role of global regulators of transcription; Fomenko D et al.; Expression of the microcin C51 operon in Escherichia coli cells is regulated as a function of the phase of growth; it is stimulated during the decelerating phase of growth . Using single-copy P(mcc)-lac transcriptional fusion (the promoter region of the microcin C51 operon fused to a promoterless lac operon in lambda phage), we showed that transcription from the microcin operon promoter is dependent on sigma(s) (RpoS) factor . However, some level of P(mcc)-lac expression is possible in rpoS null mutants, indicating that another sigma factor might be involved in transcription of the microcin C51 operon . Overproduction of sigma70 decreased Pmcc-directed transcription, presumably as a result of competition of sigma factors for the limited amount of core RNA polymerase . The cyclic AMP-CRP complex was shown to stimulate transcription from Pmcc: the absence of CRP or cAMP in crp or cya mutant cells strongly decreased the level of P(mcc)-lac expression . The production of C51 microcin decreased or was absent in rpoS, crp and cya mutant cells . Leucine-responsive protein Lrp and histone-like protein H-NS repressed P(mcc)-lac expression in the exponential and decelerating phases of growth . In studies of P(mcc)-lac expression in double mutant cells, we showed that proteins CRP, Lrp and H-NS acted in rpoS-dependent and rpoS-independent ways in transcription of the microcin C51 operon . Mutation hns(-) resulted in an increase in P(mcc)-lac expression in crp, rpoS and lrp mutant cells, as in wild-type cells.

Rapid Commun Mass Spectrom, 2001, 15(14), 1166 - 71
Screening combinatorial libraries for optimal enzyme substrates by mass spectrometry; Wang P et al.; A method has been developed for the rapid identification of optimal enzyme substrates from combinatorial libraries . This methodology was validated by screening a 361-member N-terminally formylated tripeptide library, f-XXR (X = 19 different amino acids), for optimal substrates of Escherichia coli peptide deformylase (PDF) . The library was synthesized on a solid phase via the split-pool synthesis method . The N-terminal formyl group was added by treating the resin with a 1:1 (mol/mol) mixture of HCO(2)H and DCO(2)D in the presence of dicyclohexylcarbodiimide . In a mass spectrum, each member of the library produced a doublet peak (separated by 1.0063 Da) . Limited treatment of this library with E . coli PDF resulted in the deformylation of those peptides that are the most efficient substrates of the enzyme . The deformylated products, due to loss of the mass-degenerate formyl group, each generated a singlet peak in the mass spectrum . Thus, the PDF product peaks were readily identified and sequenced via tandem mass spectrometry . The results showed that PDF strongly prefers a norleucine and, to a lesser extent, a phenylalanine as the N-terminal residue, whereas it has little selectivity at the penultimate position . This result is in excellent agreement with the literature data and therefore demonstrates the methodology as an effective approach to the identification of optimal enzyme substrates . This method should be generally applicable to other enzymes as well as synthetic catalysts .

Crit Care Med, 2001 Jul, 29(7 Suppl), S78 - 89
Staging of the pathophysiologic responses of the primate microvasculature to Escherichia coli and endotoxin: examination of the elements of the compensated response and their links to the corresponding uncompensated lethal variants; Taylor FB Jr; OBJECTIVE: Review of primate studies of Escherichia coli sepsis and endotoxemia with a reexamination of the rationale for diagnosis and treatment of these multistage disorders . SETTING: Animal research and intensive care units in a university medical school . SUBJECTS: Cyanocephalus baboons (E . coli) and normal human subjects (endotoxin) . INTERVENTIONS: Baboon studies: anti-tissue factor, protein C, endothelial protein C receptor, and anti-tumor necrosis factor antibodies, and active site inhibited factor recombinant VIIa and factor Xa . RESULTS AND CONCLUSIONS: This review concerns the primate microvascular endothelial response to inflammatory and hemostatic stress . Studies of the impact of inflammatory and hemostatic stress on this microvasculature have fallen into four categories . First, studies of pure hemostatic stress using factor Xa phospholipid vesicles showed that blockade of protein C as well as protein C plus tissue plasminogen activator produced a severe but transient consumptive and a lethal thrombotic coagulopathy, respectively . These studies showed that the protein C and fibrinolytic systems can work in tandem to regulate even a severe response if the endothelium is not rendered dysfunctional by metabolic or inflammatory factors . Second, studies of compensated (nonlethal) inflammatory stress using E . coli or endotoxin in baboon and human subjects showed that even under minimal stress in which there is no evidence of overt disseminated intravascular coagulation, injury of the endothelium and activation of neutrophils and hemostatic factors are closely associated . This showed that molecular markers of hemostatic activity could be used to detect microvascular endothelial stress (nonovert disseminated intravascular coagulation) in patients who are compensated but at risk . These studies also showed that the compensated response to inflammatory stress could exhibit two stages, each with its unique inflammatory and hemostatic response signature . The first is driven by vasoactive peptides, cytokines, and thrombin, followed 12 to 14 hrs later by a second stage driven by C-reactive protein/complement complexes, tissue factor, and plasminogen activator inhibitor 1 secondary to oxidative stress after reperfusion . Third, studies of uncompensated (lethal) inflammatory stress using E . coli showed that irreversible thrombosis of the microvasculature was not a link in the lethal chain of events even though inhibition of components of the protein C network (protein C and endothelial protein C receptor) converted compensated responses to sublethal E . coli into uncompensated lethal responses . Fourth, these studies also showed that there were variants of the lethal response ranging from capillary leak and shock to recurrent sustained inflammatory disorders . We believe that each of these variants arises from their sublethal counterparts, depending on underlying or modulating host factors operating at the time of challenge . Such underlying conditions range from preexisting microvascular ischemia, reperfusion, and oxidative stress to alteration or reprogramming of monocyte/macrophage responses (tolerance to hyperresponsiveness) . Characterization of these underlying conditions in patients who are at risk should aid in identifying and optimizing management of these variants.

Crit Care Med, 2001 Jul, 29(7), 1412 - 6
Effects of iloprost, a stable prostacyclin analog, on intestinal leukocyte adherence and microvascular blood flow in rat experimental endotoxemia; Lehmann C et al.; OBJECTIVE: To investigate the effects of iloprost, a stable prostacyclin analog, on leukocyte adherence in intestinal venules and intestinal microvascular blood flow in experimental endotoxemia . DESIGN: Prospective, randomized, controlled animal study . SETTING: Experimental laboratory . SUBJECTS: Twenty-one male Wistar rats weighing 190 +/- 40 g . INTERVENTIONS: The rats were divided equally into three groups: the first was a control group; the second received endotoxin (20 mg/kg lipopolysaccharide from Escherichia coli O55:B5 intravenously); and the third received endotoxin and intravenous iloprost infusion (2 ng.kg-1.min-1) . MEASUREMENTS AND MAIN RESULTS: The distal small intestine of the animals was examined by using intravital fluorescence videomicroscopy 2 hrs after endotoxin challenge . Leukocytes were stained in vivo by means of rhodamine 6G . Intestinal microvascular blood flow was measured by laser Doppler flowmetry in the terminal ileum . Iloprost treatment significantly attenuated the count of adherent leukocytes in collecting venules (control, 61 +/- 10 n/mm2; lipopolysaccharide, 364 +/- 60 n/mm2; iloprost, 232 +/- 29 n/mm2; p <.05) and in postcapillary venules (control, 96 +/- 14 n/mm2; lipopolysaccharide, 470 +/- 21 n/mm2; iloprost 390 +/- 41 n/mm2; p <.05) . Intestinal microvascular blood flow was decreased significantly in the lipopolysaccharide group (-49%), whereas iloprost-treated animals showed no significant difference compared with the control group . CONCLUSIONS: The study demonstrated that administration of iloprost attenuated leukocyte adherence in postcapillary and collecting intestinal venules and improved intestinal microvascular blood flow . Thus, iloprost treatment may impact endotoxin-induced intestinal injury.

J Gerontol A Biol Sci Med Sci, 2001 Jul, 56(7), B281 - 7
Heat shock protein accumulation is upregulated in a long-lived mutant of Caenorhabditis elegans; Walker GA et al.; We present evidence for elevated levels of heat shock protein 16 (HSP16) in an intrinsically thermotolerant, long-lived strain of Caenorhabditis elegans during and after heat stress . Mutation of the age-1 gene, encoding a phosphatidylinositol 3-kinase catalytic subunit, results in both extended life span (Age) and increased intrinsic thermotolerance (Itt) in adult hermaphrodites . We subjected age-synchronous cohorts of worms to lethal and nonlethal thermal stress and observed the accumulation of a small (16-18 kd) heat-shock-specific polypeptide detected by an antibody raised against C . elegans HSP16 . Strains carrying the mutation hx546 consistently accumulated HSP16 to higher levels than a wild-type strain . Significantly, overaccumulation of HSP16 in the age-1(hx546) strain following heat was observed throughout the adult life span . A chimeric transgene containing the Escherichia coli beta-galactosidase gene fused to a C . elegans HSP16-41 transcriptional promoter was introduced into wild-type and age-1(hx546) backgrounds . Heat-inducible expression of the transgene was elevated in the age-1(hx546) strain compared with the wild-type strain under a wide variety of heat shock and recovery conditions . These observations are consistent with a model in which Age mutations exhibit thermotolerance and extended life span as a result of elevated levels of molecular chaperones.

J Biol Chem, 2001 Sep 28, 276(39), 36543 - 9 Epub 2001 Jul 09.
Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p; Bill RM et al.; Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae . In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved . To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo . In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p . We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function . In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type . We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p . Our model predicts that the pore of Fps1p is more flexible than that of GlpF . We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.

J Biol Chem, 2001 Aug 31, 276(35), 32559 - 66 Epub 2001 Jul 09.
Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis; Veenendaal AK et al.; In Escherichia coli, the SecYEG complex mediates the translocation and membrane integration of proteins . Both genetic and biochemical data indicate interactions of several transmembrane segments (TMSs) of SecY with SecE . By means of cysteine scanning mutagenesis, we have identified intermolecular sites of contact between TMS7 of SecY and TMS3 of SecE . The cross-linking of SecY to SecE demonstrates that these subunits are present in a one-to-one stoichiometry within the SecYEG complex . Sites in TMS3 of SecE involved in SecE dimerization are confined to a specific alpha-helical interface and occur in an oligomeric SecYEG complex . Although cross-linking reversibly inactivates translocation, the contact between TMS7 of SecY and TMS3 of SecE remains unaltered upon insertion of the preprotein into the translocation channel . These data support a model for an oligomeric translocation channel in which pairs of SecYEG complexes contact each other via SecE.

FEMS Microbiol Lett, 2001 Jul 10, 201(1), 73 - 7
Purification and properties of Aquifex aeolicus DNA polymerase expressed in Escherichia coli; Chang JR et al.; The gene encoding Aquifex aeolicus (Aae) DNA polymerase was expressed under the control of the trp promoter on a high-copy plasmid, pTRPNS, in Escherichia coli . The expressed enzyme was purified 11-fold with a 13.8% yield and a specific activity of 2268.3 U mg(-1) . The optimum pH of the enzyme was 6.8-7.2 . The optimal concentrations of KCl and Mg(2+) were 20-30 mM and 4-5 mM, respectively . Aae DNA polymerase contained a double-strand-dependent 3'-->5' proofreading exonuclease activity but lacked any detectable 5'-->3' exonuclease activity.

DNA Cell Biol, 2001 Jun, 20(6), 349 - 57
Molecular cloning, expression, localization, and gene organization of PTX1, a human nuclear protein that is downregulated in prostate cancer; Kwok SC et al.; A cDNA, designated PTX1, has been isolated by subtractive hybridization on the basis that it is expressed in normal prostate but not in prostate carcinoma . The full-length cDNA was subsequently established by 5' and 3' RACE . Nucleotide sequence analysis of the 5'- and 3'-RACE clones yielded a composite cDNA of 1327 bp, which predicted a protein of 377 amino acid residues with a putative nuclear import signal (RRLNRKK) at its N terminus . The PTX1 gene was localized to human chromosome 12 and was found to be ubiquitously expressed . A segment of the cDNA was expressed in E . coli to produce a fragment of the PTX1 protein for the generation of specific antibodies . The resulting antibodies detected a 73-kDa protein in both nuclear and cytoplasmic extracts of prostate, although the level in the cytoplasmic extract was much lower . Using immunohistochemical analysis, the PTX1 protein was localized mainly in the nuclei of glandular epithelia of normal prostate . The nuclear staining was greatly reduced in prostate carcinoma . The gene organization of PTX1 was established by comparing the cDNA sequence with the published human genomic sequence.

Biochemistry, 2001 Jul 27, 40(28), 8387 - 96
New insights into the metal center of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; Jordan PA et al.; Metal binding properties for a series of metal-substituted forms of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, DAHPS(Tyr), have been followed by UV-vis and EPR spectroscopy . The results show that there are two metal species present at pH = 7.0 and these are coordinated in a distorted metal binding site with a mixed nitrogen and oxygen donor atom coordination set . There is no spectroscopic evidence for strong M-S interactions in this system at any pH . Metal saturation occurs at a substoichiometric ratio of 0.8-0.85 metal/monomer, and the binding trends mirror previously published enzyme activity profiles . There is a conformational change for CuDAHPS under basic conditions, and equivalent protein handling for apoDAHPS leads to apparent loss of metal binding ability . Addition of the substrate PEP does not alter the UV-vis spectra, but there are small changes in the EPR spectra of CuDAHPS(Tyr) . Further addition of the substrate analogue A5P has no effect on either spectra . Taken together, these results serve to link previous studies on enzyme activity with the recently determined X-ray crystal structure for DAHPS(Phe) and represent the first detailed spectroscopic characterization of the metal binding properties of DAHPS(Tyr).

Biochemistry, 2001 Jul 27, 40(28), 8343 - 51
Biotin synthase contains two distinct iron-sulfur cluster binding sites: chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions; Ugulava NB et al.; Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin . Biotin synthase (BioB) is aerobically purified as a dimer that contains {2Fe-2S}(2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing {4Fe-4S}(2+) and/or {4Fe-4S}(+) clusters . To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy . In contrast to previous reports, we find that aerobically purified BioB contains ca . 1.2-1.5 {2Fe-2S}(2+) clusters per monomer with epsilon(452) = 8400 M(-)(1) cm(-)(1) per monomer . Upon reduction, the {2Fe-2S}(2+) clusters are converted to {4Fe-4S} clusters with two widely separate reduction potentials of -140 and -430 mV . BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two {4Fe-4S}(2+) clusters per monomer with epsilon(400) = 30 000 M(-)(1) cm(-)(1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively . Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one {2Fe-2S}(2+) cluster and one {4Fe-4S}(2+) cluster per monomer . These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two {2Fe-2S}(2+) and two {4Fe-4S}(2+) clusters.

Biochemistry, 2001 Jul 27, 40(28), 8204 - 15
Structures of purine nucleoside phosphorylase from Mycobacterium tuberculosis in complexes with immucillin-H and its pieces; Shi W et al.; A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs . The crystal structure of M . tuberculosis PNP in complex with the transition-state analogue immucillin-H (ImmH) and inorganic phosphate was solved at 1.75 A resolution and confirms the trimeric structure . Binding of the inhibitor occurs independently at the three catalytic sites, unlike mammalian PNPs which demonstrate negative cooperativity in ImmH binding . Reduced subunit interface contacts for TB-PNP, compared to the mammalian enzymes, correlate with the loss of the cooperative inhibitor binding . Mammalian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociation constants for ImmH . The structure supports a catalytic mechanism of reactant destabilization by neighboring group electrostatic interactions, transition-state stabilization, and leaving group activation . Despite an overall amino acid sequence identity of 33% between bovine and TB-PNPs and almost complete conservation in active site residues, one catalytic site difference suggests a strategy for the design of transition-state analogues with specificity for TB-PNP . The structure of TB-PNP was also solved to 2.0 A with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO(4) to reconstruct the ImmH complex with its separate components . One subunit of the trimer has 9dHX, IR, and PO(4) bound, while the remaining two subunits contain only 9dHX . In the filled subunit, 9dHX retains the contacts found in the ImmH complex . However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucleophilic phosphate rotates away from the IR group . Loose packing of the pieces of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.

Biochemistry, 2001 Jul 27, 40(28), 8196 - 203
Purine nucleoside phosphorylase from Mycobacterium tuberculosis . Analysis of inhibition by a transition-state analogue and dissection by parts; Basso LA et al.; Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis . The M . tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP) . The gene was cloned, expressed, purified, and found to exhibit PNP activity . Purified M . tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme . Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's . This inhibitor also exhibits slow-onset inhibition of M . tuberculosis PNP with a rapid, reversible inhibitor binding (K(i) of 2.2 nM) followed by an overall dissociation constant (K(i)) of 28 pM, yielding a K(m)/K(i) value of 10(6) . Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s(-)(1), while relaxation of the complex is slower at 1.4 x 10(-)(3) s(-)(1) . The pH dependence of the K(i) value of immucillin-H to the M . tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species . The M . tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP . Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K(is) of 0.39 microM) while the 1,4-dideoxy-1,4-iminoribitol binds weakly (K(is) of 2.9 mM) . Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol (beta = 13) . However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 microM) compared to 9-deazahypoxanthine alone, and by a factor of >10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements . The structural basis for M . tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper {Shi, W., Basso, L . A., Santos, D . S., Tyler, P . C., Furneaux, R . H., Blanchard, J . S., Almo, S . C., and Schramm, V . L . (2001) Biochemistry 40, 8204-8215}.

Biochemistry, 2001 Jul 27, 40(28), 8188 - 95
Effects of bilayer thickness on the activity of diacylglycerol kinase of Escherichia coli; Pilot JD et al.; We have developed a procedure for the reconstitution of Escherichia coli diacylglycerol kinase (DGK) into phospholipid bilayers containing diacylglycerol substrate . When DGK is reconstituted into a series of phosphatidylcholines containing monounsaturated fatty acyl chains, activity against dihexanoylglycerol (DHG) as a substrate was found to be markedly dependent on the fatty acyl chain length with the highest activity in dioleoylphosphatidylcholine {di(C18:1)PC} and a lower activity in bilayers with shorter or longer fatty acyl chains . Low activities in the short chain phospholipid dimyristoleoylphosphatidylcholine {di(C14:1)PC} followed from an increase in the K(m) value for DHG and ATP, with no effect on v(max) . In contrast, in the long chain lipid dierucoylphosphatidylcholine {di(C24:1)PC}, the low activity followed from a decrease in v(max) with no effect on K(m) . In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture . Cholesterol increased the activity in di(C14:1)PC but slightly decreased it in di(C18:1)PC or di(C24:1)PC, effects that could follow from changes in bilayer thickness caused by cholesterol.

Biochemistry, 2001 Jul 27, 40(28), 8181 - 7
Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase; Biswas EE; Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner . The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD) . Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains . We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus . The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography . The purified protein migrated as a 66 kDa protein on SDS-PAGE . Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 {Biswas, E . E., and Biswas, S . B . (2000) Biochemistry 39, 15879-15886} . NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding . Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP . In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP) . NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)) . The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm) . Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP >> UTP . These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.

Biochem Biophys Res Commun, 2001 Jul 13, 285(2), 540 - 5
A novel human homologue of the SH3BGR gene encodes a small protein similar to Glutaredoxin 1 of Escherichia coli; Mazzocco M et al.; Glutaredoxins (GRXs) are ubiquitous GSH-dependent oxidoreductases, which catalyze the reduction of protein-glutathionyl-mixed disulfides and are considered to play an important role in the enzymatic regulation of redox-sensitive proteins . In this paper, we describe the identification and characterization of a new human homologue of the SH3BGR gene, named SH3BGRL3 (SH3 domain binding glutamic acid-rich protein like 3) . SH3BGRL3 is widely expressed and codes for a highly conserved small protein, which shows a significant similarity to Glutaredoxin 1 (GRX1) of Escherichia coli and is predicted to belong to the Thioredoxin Superfamily . However, the SH3BGRL3 protein lacks both the conserved cysteine residues, which characterize the enzymatic active site of GRX . This structural feature raises the possibility that SH3BGRL3 could function as an endogenous modulator of GRX biological activity . EGFP-SH3BGRL3 fusion protein expressed in COS-7 cells localizes both to the nucleus and to the cytoplasm . The SH3BGRL3 gene was mapped to chromosome 1p34.3-35 .

Biochem Biophys Res Commun, 2001 Jul 13, 285(2), 530 - 6
Differential virulence-gene mRNA expression in coccoid forms of Helicobacter pylori; Monstein HJ et al.; Controversy exists whether coccoid forms of Helicobacter pylori maintain transcriptional and translational processes . The aim of the present study was to investigate mRNA levels in coccoid H . pylori and, if possible, to establish a correlation with the state of nonrandom fragmentation of rRNA in those cells . For that purpose, UreA, UreI, CagA, VacA, SodB, and Hsp60 mRNA levels in bacillary and coccoid forms of H . pylori CCUG 17874(T), H . pylori 26695, and H . pylori J99, respectively, were studied by means of a multiplex reverse-transcription PCR assay and Southern blot analysis of the RT-PCR-amplified products . Nonrandom fragmentation of 23S rRNA was assessed by a recently described assay . Virulence-gene-derived mRNA transcripts were visualized in DNase I-treated RNA preparations . All three strains revealed the presence of different mRNA patterns in bacillary and coccoid forms . Putative promoter sequences similar to the consensus Escherichia coli -10 hexamer TATAAA box were present in all six virulence genes analyzed . Moreover, the decrease seen in mRNA levels during conversion into the coccoid form appeared to correlate with the 23S rRNA nonrandom fragmentation pattern . The present data indicate that modulation of virulence-gene expression is differently regulated in bacillary and coccoid H . pylori .

Biochem Biophys Res Commun, 2001 Jul 13, 285(2), 364 - 71
Identification and characterization of GDP-d-mannose 4,6-dehydratase and GDP-l-fucose snthetase in a GDP-l-fucose biosynthetic gene cluster from Helicobacter pylori; Wu B et al.; In this study two open reading frames, namely HP0044 and HP0045 from H . pylori, were cloned and overexpressed in E . coli . The two recombinant proteins were demonstrated to have GDP-d-mannose 4,6-dehydratase (GMD) and GDP-l-fucose synthetase (GFS) activities, respectively . The recombinant GMD was a tetramer and had an optimum pH of 6.5 . Exogenous NADP(+) was essential for its activity . The K(m) and K(cat) for GDP-d-mannose were 117.3 microM and 0.27 s(-1), respectively . The recombinant GFS was a homodimer with an optimum pH of 8.0 . The K(m) and K(cat) for GDP-4-keto-6-deoxy-d-mannose were 64.08 microM and 0.75 s(-1), respectively . It can use both NADPH and NADH, but less efficient with the latter . Amino acid sequence alignment and phylogenetic analysis showed that H . pylori GFS was highly homologous to the GFS of E . coli O111 and both of them were located on a separate phylogenetic branch from other GFS . The unique clustering and origin of the two genes were also discussed .

Biochem Biophys Res Commun, 2001 Jul 13, 285(2), 348 - 54
The effect of modifications of the charged residues in the transmembrane helices on the transport activity of the melibiose carrier of Escherichia coli; Ding PZ et al.; The melibiose transport carrier of Escherichia coli (coded by melB gene) is a cotransport system which couples the transport of a-galactosides to protons, sodium, or lithium ions . The charged amino acid residues in membrane-spanning helices are of considerable interest because many of them have important function in substrate recognition . In most cases changing these charged residue to an uncharged residue (cysteine) results in total loss of activity . In this communication we describe experiments in which the cysteine substitution for a charged residue was chemically changed by sulfhydryl reagents (MTSEA and MTSET to restore a positive charge and MTSES a negative charge) or by iodoacetic acid or through oxidation by hydrogen peroxide so as to regain the original negative charge . In two cases (D55C and D124C) the reconstructed negative charges via the oxidation of the thiol to the sulfinic and/or sulfonic acid resulted in partial recovery of transport: D55C up to 27% of the normal and D124C up to 4% of the normal in melibiose accumulation; D55C up to 36% of the normal and D124 up to 4.5% of the normal in downhill transport . Sulfhydryl reagents and iodoacetic acid failed to recover transport in all cases . We infer that the configurations of the charges as well as the structure of the side chains that carry them are critical in the maintenance of the transport .

Biochem Biophys Res Commun, 2001 Jul 13, 285(2), 201 - 6
Efficient accommodation of recombinant, foot-and-mouth disease virus RGD peptides to cell-surface integrins; Alcala P et al.; The engineering of either complete virus cell-binding proteins or derived ligand peptides generates promising nonviral vectors for cell targeting and gene therapy . In this work, we have explored the molecular interaction between a recombinant, integrin-binding foot-and-mouth disease virus RGD peptide displayed on the surface of a carrier protein and its receptors on the cell surface . By increasing the number of viral segments, cell binding to recombinant proteins was significantly improved . This fact resulted in a dramatic growth stimulation of virus-sensitive BHK(21) cells but not virus-resistant HeLa cells in protein-coated wells . Surprisingly, growth stimulation was not observed in vitronectin-coated plates, suggesting that integrins other than alpha(v)beta(3) could be involved in binding of the recombinant peptide, maybe as coreceptors . On the other hand, both free and cell-linked integrins did not modify the enzymatic activity of RGD-based enzymatic sensors that contrarily, were activated by the induced fit of anti-RGD antibodies . Those findings are discussed in the context of a proper mimicry of the unusually complex architecture of this cell-binding site as engineered in multifunctional proteins .

Am J Forensic Med Pathol, 2001 Mar, 22(1), 88 - 91
Hepatic abscess formation and unexpected death: a delayed complication of occult intraabdominal foreign body; Byard RW et al.; A case of unexpected death in a 65-year-old woman, caused by a migrating foreign body that resulted in multifocal hepatic abscesses, is reported . The foreign body was subsequently identified as a portion of chicken fibula . The prolonged time course of the illness, with relatively nonspecific symptoms and signs, resulted in establishment of the diagnosis only at autopsy.

Parasitology, 2001 Jun, 122(Pt 6), 683 - 7
Assessment of benzimidazole binding to individual recombinant tubulin isotypes from Haemonchus contortus; Oxberry ME et al.; One a- and 2 beta-tubulin isotypes (isotypes 1 and 2) from the parasitic nematode Haemonchus contortus were artificially expressed in E . coli and purified to obtain tubulin that was capable of polymerizing into microtubules . Binding of {14C} mebendazole (MBZ), a benzimidazole compound, to each individual unpolymerized isotype and to microtubules polymerized from recombinant alpha- and beta-tubulin was assessed and Kd and Bmax values determined . Mebendazole bound to the individual tubulin isotypes with a stoichiometry of 1:1 . Binding occurred with highest affinity to alpha-tubulin followed by beta-tubulin isotype 2 and beta-tubulin isotype 1 indicating that alpha-tubulin may play a role in benzimidazole binding to microtubules . Upon polymerization of alpha- and beta-tubulin isotype 2 into microtubules the stoichiometry of binding increased to 2:1 (mebendazole : tubulin) while binding affinity remained the same . Mebendazole binding to alpha/beta-isotype 1 microtubules remained unchanged following polymerization . The increase in the number of benzimidazole receptors on alpha/beta-isotype 2 microtubules suggests the formation of a new benzimidazole receptor upon polymerization.

Chemosphere, 2001 Jul, 44(2), 281 - 7
Combined treatment of textile effluent using the sequence Phanerochaete chrysosporium-ozone; Kunz A et al.; Textile effluents cause a high environmental impact when released into the environment without correct treatment . In this work, we have evaluated the capacity of treatment of a textile effluent using a biological and a chemical method using the sequence Phanerochaete chrysosporium-ozone . The fungal treatment was performed by direct incubation of a fungus spore suspension in textile effluent for nine days . Then, the effluent was ozonized at pH 11 and room temperature . Color, total organic carbon, molecular mass distribution and total phenols were determined . In biological experiments, enzymatic activity (lignin peroxidase, manganese peroxidase and laccase) were also monitored . Toxicity tests were carried out with Scenedesmus subspicatus and with Escherichia coli . Good decoloration, total phenols reduction and textile effluent molecular mass reduction were obtained during the process . No significant total organic carbon reduction was observed . The toxicity of the textile effluent was reduced with both test organisms showing no inhibition at the end of the treatment.

Biochem Genet, 2001 Feb, 39(1-2), 15 - 31
Complementation cloning and characterization of the pyrroline 5-carboxylate reductase gene from Drosophila melanogaster; Misener SR et al.; The first insect cDNA and genomic sequences encoding pyrroline 5-carboxylate reductase (EC 1.5.1.2) have been isolated from Drosophila melanogaster . The cDNA sequence was identified by interspecies complementation of an E . coli proline auxotroph and encodes a protein 280 amino acids in length with 25-41% identity to pyrroline 5-carboxylate reductases isolated from other organisms . The corresponding gene is single copy and is located at cytological position 91E-F, and in one of the P1 clones in that region . With a single 61-bp intron, and an impressively small 135- to 200-bp region that presumably acts as a bidirectional promoter, the gene itself shows remarkable economy . The calculated molecular weight of 29,700 predicts that the native enzyme is likely an octomer . Sequencing of the promoter region and expression studies, as well as the known function of the enzyme in redox regulation and the high levels of free proline in insects, suggest that this housekeeping gene encodes an enzyme with a crucial role in intermediary metabolism.

J Infect Dis, 2001 Aug 1, 184(3), 373 - 6 Epub 2001 Jun 26.
Effects of anti-inflammatory agents on serum levels of calcitonin precursors during human experimental endotoxemia; Preas HL 2nd et al.; Calcitonin precursor (CTpr) levels are both markers and mediators of inflammation . The duration of their elevation after intravenous endotoxin challenge and the effects of anti-inflammatory therapies were studied in 52 subjects . CTpr levels maximized at 24 h in all subjects . At 7 days (n=4), after levels of acute-phase cytokines and C-reactive protein had normalized, CTpr levels remained 2-4-fold above baseline levels . The elimination half-life of CTpr levels ranged from 26.9 to 45.7 h . At 24 h, endotoxin and ibuprofen (compared with endotoxin alone) increased CTpr levels approximately 2-fold (P=.03), whereas soluble tumor necrosis factor receptor blunted the increase in CTpr levels by 2-3-fold (P=.0015) . However, soluble interleukin-1 receptor failed to alter the increase in CTpr levels . Thus, the fact that anti-inflammatory agents may alter CTpr levels resulting from a single stimulus must be considered when CTpr is used as a clinical marker . Of importance, this study reveals that anti-inflammatory agents may modulate the CTpr level, which is a potential toxic mediator of inflammation.

J Mol Evol, 2001 Jun, 52(6), 502 - 9
Plasticity of fitness and diversification process during an experimental molecular evolution; Kashiwagi A et al.; A simplified experimental evolution encompassing the essence of natural one was designed in an attempt to understand the involved mechanism . In our system, molecular evolution was observed through three serial cycles of consecutive random mutagenesis of the glutamine synthetase gene and chemostat culture of the transformed Escherichia coli cells containing the mutated genes . Selection pressure was imposed solely on the glutamine synthetase gene when varieties of mutant genes compete in an unstructured environment of the chemostat . The molecular phylogeny and population dynamics were deduced from the nucleotide sequences of the genes isolated from each of the chemostat runs . An initial mutant population in each cycle, comprised of diversified closely-related genes, ended up with several varieties of mutants in a state of coexistence . Competition between two mutant genes in the final population of the first cycle ascertained that the observed coexisting state is not an incidental event and that cellular interaction via environmental nutrients is a possible mechanism of coexistence . In addition, the mutant gene once extinct in the previous passage was found to have the capacity to reinvade and constitute the gene pool of the later cycle of molecular evolution . These results, including the kinetic characteristics of the purified wild-type and mutant glutamine synthetases in the phylogenetic tree, revealed that the enzyme activity had diverged, rather than optimized, to a fittest value during the course of evolution . Here, we proposed that the plasticity of gene fitness in consequence of cellular interaction via the environment is an essential mechanism governing molecular evolution.

J Mol Evol, 2001 May, 52(5), 391 - 404
A phylogenetic framework for the aquaporin family in eukaryotes; Zardoya R et al.; A comprehensive evolutionary analysis of aquaporins, a family of intrinsic membrane proteins that function as water channels, was conducted to establish groups of homology (i.e., to identify orthologues and paralogues) within the family and to gain insights into the functional constraints acting on the structure of the aquaporin molecule structure . Aquaporins are present in all living organisms, and therefore, they provide an excellent opportunity to further our understanding of the broader biological significance of molecular evolution by gene duplication followed by functional and structural specialization . Based on the resulting phylogeny, the 153 channel proteins analyzed were classified into six major paralogous groups: (1) GLPs, or glycerol-transporting channel proteins, which include mammalian AQP3, AQP7, and AQP9, several nematode paralogues, a yeast paralogue, and Escherichia coli GLP; (2) AQPs, or aquaporins, which include metazoan AQP0, AQP1, AQP2, AQP4, AQP5, and AQP6; (3) PIPs, or plasma membrane intrinsic proteins of plants, which include PIP1 and PIP2; (4) TIPs, or tonoplast intrinsic proteins of plants, which include alphaTIP, gammaTIP, and deltaTIP; (5) NODs, or nodulins of plants; and (6) AQP8s, or metazoan aquaporin 8 proteins . Of these groups, AQPs, PIPs, and TIPs cluster together . According to the results, the capacity to transport glycerol shown by several members of the family was acquired only early in the history of the family . The new phylogeny reveals that several water channel proteins are misclassified and require reassignment, whereas several previously undetermined ones can now be classified with confidence . The deduced phylogenetic framework was used to characterize the molecular features of water channel proteins . Three motifs are common to all family members: AEF (Ala-Glu-Phe), which is located in the N-terminal domain; and two NPA (Asp-Pro-Ala) boxes, which are located in the center and C-terminal domains, respectively . Other residues are found to be conserved within the major groups but not among them . Overall, the PIP subfamily showed the least variation . In general, no radical amino acid replacements affecting tertiary structure were identified, with the exception of Ala-->Ser in the TIP subfamily . Constancy of rates of evolution was demonstrated within the different paralogues but rejected among several of them (GLP and NOD).

News Physiol Sci, 2001 Jun, 16, 130 - 4
Small is mighty: EmrE, a multidrug transporter as an experimental paradigm; Schuldiner S et al.; EmrE is a multidrug transporter from Escherichia coli that functions as a homooligomer and is unique in its small size . In each monomer there are four tightly packed transmembrane segments and one membrane-embedded charged residue . This residue provides the basis for the coupling mechanism as part of a binding site "time shared" by substrates and protons.

J Biol Chem, 2001 Aug 24, 276(34), 31891 - 6 Epub 2001 Jul 06.
Different roles for basic and aromatic amino acids in conserved region 2 of Escherichia coli sigma(70) in the nucleation and maintenance of the single-stranded DNA bubble in open RNA polymerase-promoter complexes; Tomsic M et al.; Amino acid residues in region 2 of final sigma(70) have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex . Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2 . We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with single-stranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the -10 region . We concluded that two basic amino acids (Lys(414) and Lys(418)) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr(430) and Trp(433)) . It is likely that these four amino acids, which are close to each other in the structure of final sigma(70), together are involved in the nucleation of the strand separation process.

J Biol Chem, 2001 Sep 7, 276(36), 33588 - 95 Epub 2001 Jul 06.
The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA; Mueller EG et al.; The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA . We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity (Palenchar, P . M., Buck, C . J., Cheng, H., Larson, T . J., and Mueller, E . G . (2000) J . Biol . Chem . 275, 8283-8286) . In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, R., and Lauhon, C . T . (1999) Biochemistry 38, 16561-16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI . We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro . Cys-108 and Cys-202 were converted to alanine with no significant effect on ThiI activity, and C207A ThiI was only mildly impaired . Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro . We also examined the possibility that ThiI contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species . We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native ThiI and that disulfide bonds do form in ThiI during turnover . We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters.

J Biol Chem, 2001 Sep 28, 276(39), 36493 - 500 Epub 2001 Jul 06.
The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin; Swartz TE et al.; The plant blue light receptor, phot1, a member of the phototropin family, is a plasma membrane-associated flavoprotein that contains two ( approximately 110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus . The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins . In response to blue light, phototropins undergo autophosphorylation . E . coli-expressed LOV domains bind riboflavin-5'-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm . These spectral features correspond to the action spectrum for phototropism in higher plants . Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient approximately 660 nm-absorbing species that spectroscopically resembles a flavin triplet state . This putative triplet state subsequently decays with a 4-micros time constant into a 390 nm-absorbing metastable form . The LOV2 domain (450 nm) recovers spontaneously with half-times of approximately 50 s . It has been shown that the metastable species is likely a flavin-cysteine (Cys(39) thiol) adduct at the flavin C(4a) position . A LOV2C39A mutant generates the early photoproduct but not the adduct . Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys(39) exists as a thiolate.

J Biol Chem, 2001 Sep 14, 276(37), 34388 - 95 Epub 2001 Jul 06.
Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase; Pham AS et al.; Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate . Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme . Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step . Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J . P . (1965) J . Mol . Biol . 12, 88-118) . The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex . Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.

J Biol Chem, 2001 Sep 7, 276(36), 33645 - 51 Epub 2001 Jul 06.
Identification and characterization of SppA, a novel light-inducible chloroplast protease complex associated with thylakoid membranes; Lensch M et al.; A new component of the chloroplast proteolytic machinery from Arabidopsis thaliana was identified as a SppA-type protease . The sequence of the mature protein, deduced from a full-length cDNA, displays 22% identity to the serine-type protease IV (SppA) from Escherichia coli and 27% identity to Synechocystis SppA1 (sll1703) but lacks the putative transmembrane spanning segments predicted from the E . coli sequence . The N-terminal sequence exhibits typical features of a cleavable chloroplast stroma-targeting sequence . The chloroplast localization of SppA was confirmed by in organello import experiments using an in vitro expression system and by immunodetection with antigen-specific antisera . Subfractionation of intact chloroplasts demonstrated that SppA is associated exclusively with thylakoid membranes, predominantly stroma lamellae, and is a part of some high molecular mass complex of about 270 kDa that exhibits proteolytic activity . Treatments with chaotropic salts and proteases showed that SppA is largely exposed to the stroma but that it behaves as an intrinsic membrane protein that may have an unusual monotopic arrangement in the thylakoids . We demonstrate that SppA is a light-inducible protease and discuss its possible involvement in the light-dependent degradation of antenna and photosystem II complexes that both involve serine-type proteases.

J Bacteriol, 2001 Aug, 183(15), 4664 - 7
ZitB (YbgR), a member of the cation diffusion facilitator family, is an additional zinc transporter in Escherichia coli; Grass G et al.; The Escherichia coli zitB gene encodes a Zn(II) transporter belonging to the cation diffusion facilitator family . ZitB is specifically induced by zinc . ZitB expression on a plasmid rendered zntA-disrupted E . coli cells more resistant to zinc, and the cells exhibited reduced accumulation of (65)Zn, suggesting ZitB-mediated efflux of zinc.

J Bacteriol, 2001 Aug, 183(15), 4659 - 63
Activation by gene amplification of pitB, encoding a third phosphate transporter of Escherichia coli K-12; Hoffer SM et al.; Two systems for the uptake of inorganic phosphate (P(i)) in Escherichia coli, PitA and Pst, have been described . A revertant of a pitA pstS double mutant that could grow on P(i) was isolated . We demonstrate that the expression of a new P(i) transporter, PitB, is activated in this strain by a gene amplification event.

J Bacteriol, 2001 Aug, 183(15), 4643 - 7
Roles for the Rhodobacter sphaeroides CcmA and CcmG proteins; Cox RL et al.; Rhodobacter sphaeroides cells containing an in-frame deletion within ccmA lack detectable soluble and membrane-bound c-type cytochromes and are unable to grow under conditions where these proteins are required . Only strains merodiploid for ccmABCDG were found after attempting to generate cells containing either a ccmG null mutation or a ccmA allele that should be polar on to expression of ccmBCDG, suggesting that CcmG has another important role in R . sphaeroides.

J Bacteriol, 2001 Aug, 183(15), 4571 - 9
Computation-directed identification of OxyR DNA binding sites in Escherichia coli; Zheng M et al.; A computational search was carried out to identify additional targets for the Escherichia coli OxyR transcription factor . This approach predicted OxyR binding sites upstream of dsbG, encoding a periplasmic disulfide bond chaperone-isomerase; upstream of fhuF, encoding a protein required for iron uptake; and within yfdI . DNase I footprinting assays confirmed that oxidized OxyR bound to the predicted site centered 54 bp upstream of the dsbG gene and 238 bp upstream of a known OxyR binding site in the promoter region of the divergently transcribed ahpC gene . Although the new binding site was near dsbG, Northern blotting and primer extension assays showed that OxyR binding to the dsbG-proximal site led to the induction of a second ahpCF transcript, while OxyR binding to the ahpCF-proximal site leads to the induction of both dsbG and ahpC transcripts . Oxidized OxyR binding to the predicted site centered 40 bp upstream of the fhuF gene was confirmed by DNase I footprinting, but these assays further revealed a second higher-affinity site in the fhuF promoter . Interestingly, the two OxyR sites in the fhuF promoter overlapped with two regions bound by the Fur repressor . Expression analysis revealed that fhuF was repressed by hydrogen peroxide in an OxyR-dependent manner . Finally, DNase I footprinting experiments showed OxyR binding to the site predicted to be within the coding sequence of yfdI . These results demonstrate the versatile modes of regulation by OxyR and illustrate the need to learn more about the ensembles of binding sites and transcripts in the E . coli genome.

J Bacteriol, 2001 Aug, 183(15), 4562 - 70
DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide; Zheng M et al.; The genome-wide transcription profile of Escherichia coli cells treated with hydrogen peroxide was examined with a DNA microarray composed of 4,169 E . coli open reading frames . By measuring gene expression in isogenic wild-type and oxyR deletion strains, we confirmed that the peroxide response regulator OxyR activates most of the highly hydrogen peroxide-inducible genes . The DNA microarray measurements allowed the identification of several new OxyR-activated genes, including the hemH heme biosynthetic gene; the six-gene suf operon, which may participate in Fe-S cluster assembly or repair; and four genes of unknown function . We also identified several genes, including uxuA, encoding mannonate hydrolase, whose expression might be repressed by OxyR, since their expression was elevated in the DeltaoxyR mutant strain . In addition, the induction of some genes was found to be OxyR independent, indicating the existence of other peroxide sensors and regulators in E . coli . For example, the isc operon, which specifies Fe-S cluster formation and repair activities, was induced by hydrogen peroxide in strains lacking either OxyR or the superoxide response regulators SoxRS . These results expand our understanding of the oxidative stress response and raise interesting questions regarding the nature of other regulators that modulate gene expression in response to hydrogen peroxide.

J Bacteriol, 2001 Aug, 183(15), 4493 - 8
Properties of a revertant of Escherichia coli viable in the presence of spermidine accumulation: increase in L-glycerol 3-phosphate; Raj VS et al.; Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase . When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine . When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained . We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase sigma(38) subunit, factors important for cell viability, was increased in the revertant . Levels of L-glycerol 3-phosphate also increased in the revertant . Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E . coli CAG2242 partially prevented the cell death caused by accumulation of spermidine . It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine . These results indicate that L-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.

J Bacteriol, 2001 Aug, 183(15), 4459 - 67
Identification of some DNA damage-inducible genes of Mycobacterium tuberculosis: apparent lack of correlation with LexA binding; Brooks PC et al.; The repair of DNA damage is expected to be particularly important to intracellular pathogens such as Mycobacterium tuberculosis, and so it is of interest to examine the response of M . tuberculosis to DNA damage . The expression of recA, a key component in DNA repair and recombination, is induced by DNA damage in M . tuberculosis . In this study, we have analyzed the expression following DNA damage in M . tuberculosis of a number of other genes which are DNA damage inducible in Escherichia coli . While many of these genes were also induced by DNA damage in M . tuberculosis, some were not . In addition, one gene (ruvC) which is not induced by DNA damage in E . coli was induced in M . tuberculosis, a result likely linked to its different transcriptional arrangement in M . tuberculosis . We also searched the sequences upstream of the genes being studied for the mycobacterial SOS box (the binding site for LexA) and assessed LexA binding to potential sites identified . LexA is the repressor protein responsible for regulating expression of these SOS genes in E . coli . However, two of the genes which were DNA damage inducible in M . tuberculosis did not have identifiable sites to which LexA bound . The absence of binding sites for LexA upstream of these genes was confirmed by analysis of LexA binding to overlapping DNA fragments covering a region from 500 bp upstream of the coding sequence to 100 bp within it . Therefore, it appears most likely that an alternative mechanism of gene regulation in response to DNA damage exists in M . tuberculosis.

J Bacteriol, 2001 Aug, 183(15), 4435 - 50
Novel topology of BfpE, a cytoplasmic membrane protein required for type IV fimbrial biogenesis in enteropathogenic Escherichia coli; Blank TE et al.; Enteropathogenic Escherichia coli (EPEC) produces the bundle-forming pilus (BFP), a type IV fimbria that has been implicated in virulence, autoaggregation, and localized adherence to epithelial cells . The bfpE gene is one of a cluster of bfp genes previously shown to encode functions that direct BFP biosynthesis . Here, we show that an EPEC strain carrying a nonpolar mutation in bfpE fails to autoaggregate, adhere to HEp-2 cells, or form BFP, thereby demonstrating that BfpE is required for BFP biogenesis . BfpE is a cytoplasmic membrane protein of the GspF family . To determine the membrane topology of BfpE, we fused bfpE derivatives containing 3' truncations and/or internal deletions to alkaline phosphatase and/or beta-galactosidase reporter genes, whose products are active only when localized to the periplasm or cytoplasm, respectively . In addition, we constructed BfpE sandwich fusions using a dual alkaline phosphatase/beta-galactosidase reporter cassette and analyzed BfpE deletion derivatives by sucrose density flotation gradient fractionation . The data from these analyses support a topology in which BfpE contains four hydrophobic transmembrane (TM) segments, a large cytoplasmic segment at its N terminus, and a large periplasmic segment near its C terminus . This topology is dramatically different from that of OutF, another member of the GspF family, which has three TM segments and is predominantly cytoplasmic . These findings provide a structural basis for predicting protein-protein interactions required for assembly of the BFP biogenesis machinery.

Mol Microbiol, 2001 Jun, 40(6), 1415 - 26
Disruptions of the Ustilago maydis REC2 gene identify a protein domain important in directing recombinational repair of DNA; Kojic M et al.; The REC2 gene of Ustilago maydis encodes a homologue of the Escherichia coli RecA protein and was first identified in a screen for UV-sensitive mutants . The original isolate, rec2-1, was found to be deficient in repair of DNA damage, genetic recombination and meiosis . We report here that the rec2-197 allele, which was constructed by gene disruption, retains some biological activity and is partially dominant with respect to REC2 . The basis for the residual activity is probably as a result of expression of a diffusible product from the rec2-197 allele that augments or interferes with REC2 functions . This product appears to be a polypeptide expressed from a remnant of the 5' end of the open reading frame that was not removed in creating the gene disruption . The mutator activity and disturbed meiosis of rec2-197 suggest that the Rec2 protein functions in a process that avoids spontaneous mutation and insures faithful meiotic chromosome segregation . A prediction based on the phenotype of rec2-197 is that Rec2 protein interacts with one or more other proteins in directing these functions . To identify interacting proteins we performed a yeast two-hybrid screen and found Rad51 as a candidate . Rec2-197 and Rad51 appear to interact to a similar degree.

Mol Microbiol, 2001 Jun, 40(6), 1381 - 90
Role of the response regulator RssB in sigma recognition and initiation of sigma proteolysis in Escherichia coli; Klauck E et al.; In growing Escherichia coli cells, the master regulator of the general stress response, sigmaS (RpoS), is subject to rapid proteolysis . In response to stresses such as sudden carbon starvation, osmotic upshift or shift to acidic pH, sigmaS degradation is inhibited, sigmaS accumulates and numerous sigmaS-dependent genes with stress-protective functions are activated . sigmaS proteolysis is dependent on ClpXP protease and the response regulator RssB, whose phosphorylated form binds directly to sigmaS in vitro . Here, we show that substitutions of aspartate 58 (D58) in RssB, which result in higher sigmaS levels in vivo, produce RssB variants unable to bind sigmaS in vitro . Thus, RssB is the direct substrate recognition factor in sigmaS proteolysis, whose affinity for sigmaS depends on phosphorylation of its D58 residue . RssB does not dimerize or oligomerize upon this phosphorylation and sigmaS binding, and RssB and sigmaS exhibit a 1:1 stoichiometry in the complex . The receiver as well as the output domain of RssB are required for sigmaS binding (as shown in vivo and in vitro) and for complementation of an rssB null mutation . Thus, the N-terminal receiver domain plays an active and positive role in RssB function . Finally, we demonstrate that RssB is not co-degraded with sigmaS, i.e . RssB has a catalytic role in the initiation of sigmaS turnover . A model is presented that integrates the details of RssB-sigmaS interaction, the RssB catalytic cycle and potential stress signal input in the control of sigmaS proteolysis.

Mol Microbiol, 2001 Jun, 40(6), 1323 - 33
degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide sigma (E) activity; Alba BM et al.; DegS (HhoB), a putative serine protease related to DegP/HtrA, regulates the basal and induced activity of the essential Escherichia coli sigma factor sigma (E), which is involved in the cellular response to extracytoplasmic stress . DegS promotes the destabilization of the sigma (E)-specific anti-sigma factor RseA, thereby releasing sigma (E) to direct gene expression . We demonstrate that degS is an essential E . coli gene and show that the essential function of DegS is to provide the cell with sigma (E) activity . We also show that the putative active site of DegS is periplasmic and that DegS requires its N-terminal transmembrane domain for its sigma (E)-related function.

Mol Microbiol, 2001 Jun, 40(6), 1261 - 72
The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides; Martin AC et al.; Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci . These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2) . We have deleted each of these cheA and cheW homologues in-frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays . In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG . In E . coli, deletion of either cheA or cheW results in a non-chemotactic phenotype, and these strains also show no receptor clustering . Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions . Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates . Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay . These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl-accepting chemotaxis protein (MCP) clustering . Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis . We propose a model in which the multiple different MCPs in R . sphaeroides are contained within a polar chemoreceptor cluster . Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.

J Appl Microbiol, 2001 Jul, 91(1), 104 - 9
Surface hygiene monitored using a reporter of fis in Escherichia coli; Goulsbra AM et al.; AIMS: To examine the value of the fis promoter in monitoring regrowth of a surface-attached bacterial population following exposure to chemical stress using several candidate reporters, beta-galactosidase (lacZYA), bacterial luciferase (luxAB) and enhanced green fluorescent protein (EGFP) . METHODS AND RESULTS: The pattern of expression for the reporters within Escherichia coli cells attached to surfaces was determined . Both the bacterial luciferase reporter and EGFP were readily detected, but EGFP was found to overcome problems associated with luciferase and beta-galactosidase . The effect of surface pretreatment, using polymer systems, on bacterial attachment and growth confirmed the usefulness of this approach . CONCLUSION: The fis promoter, combined with EGFP, can be used successfully to study adhesion, biocidal damage and recovery . The stability of the EGFP enabled the magnitude of the total recovery response to be monitored as cells remained fluorescent after the decline in fis expression . SIGNIFICANCE AND IMPACT OF THE STUDY: The E . coli Pfis-egfp reporter system provides a new, versatile and sensitive tool to investigate bacterial adhesion both quantitatively and qualitatively.

Int J Urol, 2001 Jul, 8(7), S5 - 8
Suicide gene therapy on LNCaP human prostate cancer cells; Yoshimura I et al.; The efficacy of combination suicide gene therapy was evaluated using a Herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system and an Escherichia coli cytosine deaminase/5-fluorocytosine (CD/5-FC) system on the LNCaP human prostate cancer cell model . Two types of plasmid vectors with the HSV-TK gene were constructed . A constitutive chicken beta-actin promoter drove one and a prostate-specific antigen (PSA) promoter drove the other . Similarly, a pair of plasmids with the CD gene under a cytomegalovirus (CMV) promoter and the PSA promoter was also constructed . LNCaP cells were transfected in vitro with either or both of those plasmids using a cationic lipid reagent . Transfected cells were treated with GCV and/or 5-FC . The percentage of viable LNCaP cells 7 days after treatment with HSV-TK/GCV or CD/5-FC under a constitutive promoter was 40% and 41% of controls, respectively . The cell viability when two suicide genes were combined was 23% . The cell viabilities after four days with PSA promoter-HSV-TK vectors, CD vectors and a combination of both were 79%, 88% and 88%, respectively . Suicide gene therapy using either HSV-TK/GCV, CD/5-FC, or both, was effective in the LNCaP model . An additive effect was observed when the two suicide genes were used together . The PSA promoter did not seem to be effective enough to elicit cytotoxicity under the experimental conditions used here.

Abdom Imaging, 2001 Jul-Aug, 26(4), 411 - 3
Acute mesenteric and retroperitoneal lymphadenitis in systemic lupus erythematosus: case report; Radin R; In a young woman with clinical evidence of acute cutaneous, musculoskeletal, and neurologic manifestations of systemic lupus erythematosus, computed tomography (CT) showed enlarged, centrally hypoattenuating mesenteric and retroperitoneal lymph nodes . After treatment with steroids, the CT appearance of the lymph nodes returned to normal . The differential diagnosis of lymph nodes with central hypoattenuation includes Mycobacterium tuberculosis infection, metastatic disease (especially squamous cell carcinoma and germ cell tumor), Whipple's disease, and celiac disease in addition to lupus lymphadenitis.

Korean J Parasitol, 2001 Jun, 39(2), 171 - 6
Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite; Son ES et al.; Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens . Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax . Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG . The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination . When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera . In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively . We obtained higher detection rates when using 5 antigens (91.9%) rather than using each antigen solely (55.6-80%), a combination of 2 (76.3-87.5%), 3 (85.6-90.6%), or 4 antigens (89.4-91.3%) . This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.

J Basic Microbiol, 2001, 41(2), 75 - 83
Involvement of gntS in the control of GntI, the main system for gluconate metabolism in Escherichia coli; Isturiz T et al.; The initial steps of gluconate metabolism in E . coli, transport and phosphorylation, occur through duplicate activities . These activities have been included in two systems designated as GntI (main) and GntII (subsidiary), encoded by differently regulated operons located at the 76.4-77 and 95.3-96.9 regions on the map respectively . Despite recent molecular advances related to genetics and physiology of these systems, there is no information about the coordination of their expression when E . coli grows on gluconate . Under these conditions, the subsidiary gluconokinase (gntV gene, min 96.8) as well as the GntI activities are expressed in inducible form . Therefore it was of interest to find out if GntS, the positive regulator of gntV has a similar effect on GntI activities expression . Our results agree with this hypothesis . GntS, in addition to its regulatory action on the gntV gene, seems to assist, direct or indirectly, the expression of the GntI activities . A gntS E . coli mutant does not grow on gluconate but spontaneously pseudoreverts to a gluconate growing phenotype at high rate per cell generation when cultivated in rich media with or without gluconate or mineral medium containing any other suitable carbon source . In the pseudorevertants, the thermosensitive gluconokinase remains repressed while the GntI activities are inducibly expressed . At present, the location and nature of the gntS suppressor mutation are not known . Phage P1Kc mediated transductions have ruled out that it alters the gntT gene . This is the first report on GntI activities alteration due to a lesion located out of the bioH-asd region.

J Immunol, 2001 Jul 15, 167(2), 797 - 804
Engagement of Fc epsilon RI on human monocytes induces the production of IL-10 and prevents their differentiation in dendritic cells; Novak N et al.; The local cytokine environment and the presence of stimulatory signals determine whether circulating monocytes will finally acquire characteristics of dendritic cells (DCs) or macrophages . Because FcepsilonRI expressed on professional APCs, e.g., monocytes and DCs, has been suggested to play a key role in the pathophysiology of atopic diseases, we evaluated the effect of receptor ligation on the generation of monocyte-derived DCs (MoDCs) . Aggregation of FcepsilonRI at the initiation of the IL-4-GM-CSF-driven differentiation resulted in the emergence of macrophage-like cells with a strong expression of the mannose receptor and a low level of CD1a and the DC-specific markers CD83 and the actin-bundling protein (p55) . These cells sustained the ability to take up FITC-labeled Escherichia coli by phagocytosis and were significantly less efficient in stimulating purified allogeneic T cells . In addition, receptor ligation of FcepsilonRI at the beginning of the culture prevented the generation of MoDCs, mainly due to a dramatic increase in the IL-10 production . These results suggest that FcepsilonRI aggregation prevents the generation of CD1a(+) MoDCs and imply a novel pivotal function of this receptor in modulating the differentiation of monocytes.

J Biol Chem, 2001 Sep 21, 276(38), 35836 - 41 Epub 2001 Jul 05.
Structural basis for the thioredoxin-like activity profile of the glutaredoxin-like NrdH-redoxin from Escherichia coli; Stehr M et al.; NrdH-redoxin is a representative of a class of small redox proteins that contain a conserved CXXC motif and are characterized by a glutaredoxin-like amino acid sequence and thioredoxin-like activity profile . The crystal structure of recombinant Escherichia coli NrdH-redoxin in the oxidized state has been determined at 1.7 A resolution by multiwavelength anomalous diffraction . NrdH-redoxin belongs to the thioredoxin superfamily and is structurally most similar to E . coli glutaredoxin 3 and phage T4 glutaredoxin . The angle between the C-terminal helix alpha3 and strand beta4, which differs between thioredoxin and glutaredoxin, has an intermediate value in NrdH-redoxin . The orientation of this helix is to a large extent determined by an extended hydrogen-bond network involving the highly conserved sequence motif (61)WSGFRP(D/E)(67), which is unique to this subclass of the thioredoxin superfamily . Residues that bind glutathione in glutaredoxins are in general not conserved in NrdH-redoxin, and no glutathione-binding cleft is present . Instead, NrdH-redoxin contains a wide hydrophobic pocket at the surface, similar to thioredoxin . Modeling studies suggest that NrdH-redoxin can interact with E . coli thioredoxin reductase at this pocket and also via a loop that is complementary to a crevice in the reductase in a similar manner as observed in the E . coli thioredoxin-thioredoxin reductase complex.

J Biol Chem, 2001 Sep 7, 276(36), 34122 - 30 Epub 2001 Jul 05.
Expression and localization of the mouse homologue of the yeast V-ATPase 21-kDa Subunit c" (Vma16p); Nishi T et al.; We have identified a cDNA encoding the mouse homologue of the yeast V-ATPase 21-kDa subunit c" (Vma16p) . The encoded protein contains 205 amino acid residues with five putative membrane spanning segments and shows 48% identity and 64% similarity to the yeast protein . Despite this homology, however, the mouse cDNA does not complement the phenotype of a yeast strain in which the VMA16 gene has been disrupted . Northern blot analysis demonstrated that the 21-kDa subunit is expressed in most tissues examined and showed an expression pattern almost identical to that of the 16-kDa proteolipid subunit (subunit c) . The presence of multiple mRNA species suggests the existence of alternatively spliced forms of the 21-kDa subunit which, from Southern blot analysis, are derived from a single gene . Promoter analysis using the luciferase reporter gene revealed that a region 186 bases upstream of the initiation site is sufficient to show a low level of transcriptional activity but that transcription is significantly enhanced by inclusion of the region -186 to -706 . The 21-kDa protein was Myc-tagged and the 16-kDa protein was HA-tagged and the tagged proteins were co-expressed in COS-1 cells in order to study their intracellular localization by immunofluorescence microscopy . Both proteins showed significant punctate and perinuclear staining and were predominantly co-localized throughout the cell, consistent with their presence in the same V(0) complexes . Selective permeabilization of cells with digitonin (to permeabilize the plasma membrane) or Triton X-100 (to permeabilize both intracellular and plasma membranes) followed by immunofluorescence microscopy revealed that the carboxyl terminus of the 21-kDa subunit is exposed on the cytoplasmic side of the membrane whereas the carboxyl terminus of the 16-kDa subunit is located on the lumenal side of the membrane.

J Biol Chem, 2001 Sep 7, 276(36), 34213 - 20 Epub 2001 Jul 05.
Role of DNA end distortion in catalysis by avian sarcoma virus integrase; Katz RA et al.; Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction) . In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction) . Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN . Here we used potassium permanganate modification to show that the avian sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro . This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA . Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion . End-distortion activity was also observed with mutant or heterologous DNA substrates . However, further analyses showed that using Mn(2+) as a cofactor, processing site specificity of these substrates was also maintained . Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction . Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.

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