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