Biochem Biophys Res Commun, 2002 Jul 5, 295(1), 167 - 73 Structural characterization of Escherichia coli sialic acid synthase; Hwang TS et al.; Sialic acid synthase encoded by the neuB gene of Escherichia coli catalyzes the condensation of N-acetylmannosamine and phosphoenolpyruvate to form N-acetylneuraminic acid . This report demonstrates the first structural information on sialic acid synthase by CD, MALDI-TOF, and chemical cross-linking studies . Also, a specific cleavage by endogenous protease(s) has been identified at Lys(280) of the enzyme (40 kDa) by LC-MS and N-terminal sequencing analyses . The cleavage results in the formation of two inactive fragments of 33 and 7 kDa . The structural analysis indicates that the fragmentation is associated with a significant change of the enzyme from a tetrameric to trimeric form, and alterations in both secondary and native quaternary structures . (c) 2002 Elsevier Science (USA). Biochem Biophys Res Commun, 2002 Jul 5, 295(1), 163 - 6 Secretory production of recombinant human C-reactive protein in Escherichia coli, capable of binding with phosphorylcholine, and its characterization; Tanaka T et al.; Recombinant human CRP (rhCRP) was secreted into culture supernatant of Escherichia coli by co-expressing kil gene that has a function to secrete colicin E1 outside the cell . Highly purified 5 g rhCRP was produced from 180 L culture supernatant by affinity chromatography . The purified rhCRP was indistinguishable from the native one with respect to Ca(2+)-dependent binding ability to phosphorylcholine, electrophoretic behavior, N-terminal amino acid analysis, and immunochemical properties . The molecular weight of rhCRP monomer was determined to be 23059.7 Da by TOF/MS analysis . These results indicate that rhCRP has the same protein structure as native one and that rhCRP has the potential as a reference material and/or calibrator of high-sensitivity CRP assay to predict the risk of cardiovascular disease . (c) 2002 Elsevier Science (USA). Biochem Biophys Res Commun, 2002 Jul 5, 295(1), 92 - 7 Extensive overproduction of the AdhE protein by rng mutations depends on mutations in the cra gene or in the Cra-box of the adhE promoter; Kaga N et al.; Escherichia coli RNase G encoded by the rng gene is involved in degradation of adhE mRNA . Overproduction of the AdhE protein by rng mutants was found to depend on the genetic background of strains derived from DC272 (adhC81) or MC1061 . We found that DC272 carried a point mutation in the Cra-binding site of the adhE promoter . The Cra protein encoded by the cra gene is known to act as a repressor of adhE . P1-phage-mediated transduction and lacZ fusion analysis with the mutant adhE promoter confirmed that this mutation is responsible for overproduction . On the other hand, Southern hybridization revealed that MC1061 had a 0.85-kb deletion of the cra gene . Overproduction of AdhE in the MC1061 background was reversed to the wild-type levels by introduction of a plasmid carrying the cra(+) gene . These results indicated that expression of the adhE gene was regulated transcriptionally by Cra and posttranscriptionally by RNase G . (c) 2002 Elsevier Science (USA). Biochem Biophys Res Commun, 2002 Jul 5, 295(1), 67 - 73 The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain; Grillo C et al.; ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain . ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins . However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction . In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one . A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57 . (c) 2002 Elsevier Science (USA). Biochem Biophys Res Commun, 2002 Jul 5, 295(1), 31 - 6 Selection of human antibody fragments on the basis of stabilization of the variable domain in the presence of target antigens; Watanabe H et al.; Here we report a novel method for selecting human antibody fragments from nonimmunized variable domain libraries . The antibody fragments are selected on the basis of stabilization of the variable domain fragment (F(v)) in the presence of target antigens ("open sandwich selection") . One variable domain is displayed on phages and another is prepared as soluble molecules . These two reagents are mixed with the biotinylated target molecule and ternary complexes are captured by using streptavidin-conjugated magnet beads . After extensive washing, enriched clones are eluted by using target antigen . Some of the clones selected after 3 rounds are prepared as soluble domains, which then undergo another selection process . We obtained several human antibody fragments specific for human soluble erythropoietin receptor by using this method . Our method minimizes several of the disadvantages associated with human antibody selection through a phage-display system, such as construction of a large-scale library, deletion of genes during selection, and nonspecific binding . (c) 2002 Elsevier Science (USA). JBR-BTR, 2002 Apr-May, 85(2), 100 - 3 Diffusion-weighted MR imaging in the evaluation of renal infection: preliminary results; Verswijvel G et al.; Diffusion-weighted (DW) magnetic resonance imaging (MRI) has already gained status in the neuroradiological MRI approach of a patient suffering from a variety of neurological diseases . The clinical application of DW MRI in the evaluation of renal disease is not standard . This manuscript describes preliminary results of the application of DW imaging in renal infection to aid differential diagnosis and/or lesion detection in clinical MRI . Three patients with acute pyelonephritis and two patients with renal abscess (one with xanthogranulomatous pyelonephritis, the other with a solitary pyogenic renal abscess) were examined with MRI . Areas of acute pyelonephritis and abscedation have restricted proton diffusion and are demonstrated on the DW images . Refinement of the technique, in-depth investigation of the pathological background of the MR findings and evaluation of its true clinical value need further investigation . This manuscripts shows preliminary findings of DW imaging in patients with renal infectious disease. J Mol Biol, 2002 May 17, 318(5), 1265 - 74 The 1.9 A crystal structure of heat-labile shrimp alkaline phosphatase; de Backer M et al.; Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man . This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP) . Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g . to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature . Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9 A . Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer . SAP shares the core, an extended beta-sheet flanked by alpha-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure) . Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms . Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzyme's net charge of -80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site . The negatively charged substrate must therefore be directed strongly towards the active site . It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation . SAP demonstrates this principle very clearly. J Mol Biol, 2002 May 17, 318(5), 1251 - 64 Biochemical properties of single-stranded DNA-binding protein from Mycobacterium smegmatis, a fast-growing mycobacterium and its physical and functional interaction with uracil DNA glycosylases; Acharya N et al.; The single-stranded DNA-binding proteins (SSBs) are vital to virtually all DNA functions . Here, we report on the biochemical properties of SSB from a fast-growing mycobacteria, Mycobacterium smegmatis, and the interaction of the homotetrameric SSBs with uracil DNA glycosylases (UDGs) from M . smegmatis (Msm), Mycobacterium tuberculosis (Mtu) and Escherichia coli (Eco) . UDG is a crucial DNA repair enzyme, which removes the promutagenic uracil residues . MsmSSB stimulates activity of the homologous Msm UDG and of the heterologous Mtu-, and Eco-UDGs . On the contrary, while the MtuSSB stimulates the Mtu UDG, it inhibits the other two UDGs . Although the MsmSSB shares 84% identity with MtuSSB, the two are strikingly different, in that MsmSSB contains a glycine-rich segment (11 out of 13 residues) in the spacer connecting the N-terminal DNA-binding domain with the C-terminal acidic tail . While the DNA-binding properties of MsmSSB, such as its affinity to oligomeric DNA, requirement of minimum size DNA and the modes of interaction are indistinguishable from those of Eco-, and Mtu-SSBs, it is unclear if the glycine-rich segment confers structural advantage to MsmSSB, responsible for its stimulatory effect on all UDGs tested . More importantly, by using a small polypeptide inhibitor of UDGs, and the deletion mutants of SSBs, we suggest that the C-terminal acidic tail of the SSBs interacts within the DNA-binding groove of the UDGs, and propose a role for SSBs in the recruitment of UDGs to the damaged DNA. J Mol Biol, 2002 May 17, 318(5), 1207 - 20 Recognition of tRNA backbone for aminoacylation with cysteine: evolution from Escherichia coli to human; Ming X et al.; The underlying basis of the genetic code is specific aminoacylation of tRNAs by aminoacyl-tRNA synthetases . Although the code is conserved, bases in tRNA that establish aminoacylation are not necessarily conserved . Even when the bases are conserved, positions of backbone groups that contribute to aminoacylation may vary . We show here that, although the Escherichia coli and human cysteinyl-tRNA synthetases both recognize the same bases (U73 and the GCA anticodon) of tRNA for aminoacylation, they have different emphasis on the tRNA backbone . The E . coli enzyme recognizes two clusters of phosphate groups . One is at A36 in the anticodon and the other is in the core of the tRNA structure and includes phosphate groups at positions 9, 12, 14, and 60 . Metal-ion rescue experiments show that those at positions 9, 12, and 60 are involved with binding divalent metal ions that are important for aminoacylation . The E . coli enzyme also recognizes 2'-hydroxyl groups within the same two clusters: at positions 33, 35, and 36 in the anticodon loop, and at positions 49, 55, and 61 in the core . The human enzyme, by contrast, recognizes few phosphate or 2'-hydroxy groups for aminoacylation . The evolution from the backbone-dependent recognition by the E . coli enzyme to the backbone-independent recognition by the human enzyme demonstrates a previously unrecognized shift that nonetheless has preserved the specificity for aminoacylation with cysteine. J Mol Biol, 2002 May 17, 318(5), 1189 - 206 Identification of new poly(A) polymerase-inhibitory proteins capable of regulating pre-mRNA polyadenylation; Ko B et al.; The 3' ends of nearly all eukaryotic pre-mRNAs undergo cleavage and polyadenylation, thereby acquiring a poly(A) tail added by the enzyme poly(A) polymerase (PAP) . Two well-characterized examples of regulated poly(A) tail addition in the nucleus consist of spliceosomal proteins, either the U1A or U170K proteins, binding to the pre-mRNA and inhibiting PAP via their PAP regulatory domains (PRDs) . These two proteins are the only known examples of this type of gene regulation . On the basis of sequence comparisons, it was predicted that many other proteins, including some members of the SR family of splicing proteins, contain functional PRDs . Here we demonstrate that the putative PRDs found in the SR domains of the SR proteins SRP75 and U2AF65, via fusion to a heterologous MS2 RNA binding protein, specifically and efficiently inhibit PAP in vitro and pre-mRNA polyadenylation in vitro and in vivo . A similar region from the SR domain of SRP40 does not exhibit these activities, indicating that this is not a general property of SR domains . We find that the polyadenylation- and PAP-inhibitory activity of a given polypeptide can be accurately predicted based on sequence similarity to known PRDs and can be measured even if the polypeptides' RNA target is unknown . Our results also indicate that PRDs function as part of a network of interactions within the pre-mRNA processing complex and suggest that this type of regulation will be more widespread than previously thought. Biotechniques, 2002 Jun, Suppl, 44 - 7 TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing; Nelson JR et al.; We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions . The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism . This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification . Large amounts of product (1-3 microg) can be obtained in as little as 4 hours . Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture . Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification . Once completed, the product DNA can be used directly in sequencing reactions . Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed. Acta Virol, 2001, 45(5-6), 311 - 7 The UL9 ori-binding protein of herpes simplex virus 1: its expression and localization in vero cells; Durmanova V et al.; The ori-binding protein (OBP), an early protein which is encoded by the herpes simples virus 1 (HSV-1) UL9 gene and initiates the replication of viral DNA, was expressed in Escherichia coli, purified on an avidin resin and used for preparation of a mouse antiserum to OBP (OBP antiserum) . Expression and localization of OBP in HSV-1-infected Vero cells was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence test . RT-PCR revealed the presence of abundant UL9 transcripts from 3 to 12 hrs post infection (p.i) . Traces of UL9 mRNA were detected already at 1.5 hr p.i . The OBP antiserum detected clumps of irregularly shaped structures in the nuclei of infected Vero cells first at 4 hrs p.i . These nuclear structures peaked at 5-6 hrs p.i . and later on (at 8-12 hrs p.i.) they changed into fine granules filling the whole nucleus. Neoplasia, 2002 Jul-Aug, 4(4), 304 - 11 The treatment of malignant meningioma with verotoxin; Salhia B et al.; Malignant meningiomas (MMs) are aggressive intracranial neoplasms with a 75% 5-year recurrence rate . Verotoxin 1 (VT1) is an Escherichia coli toxin, which has recently been shown to have anti-neoplastic action by targeting the globotriosylceramide (Gb(3)) glycolipid on tumor cells and tumor neovasculature . To investigate the potential use of VT1 as a clinical agent for MM, we initially tested 16 meningiomas for Gb(3) expression . Nine of 11 MMs (82%), but only one of five benign meningiomas (20%), were positive for Gb(3) . An orthotopic xenograft model was used to test the efficacy of VT1 treatment for MM . We first demonstrated that Gb(3) was highly expressed by the MM cell line, IOMM-Lee, and that this cell line was highly sensitive to VT1 treatment in vitro . A single intratumoral injection of VT1 significantly improved survival in nude mice harboring intracranial tumours (P<.0001) . Factor-eight immunostaining of tumours harvested from VT1-treated animals revealed a marked reduction in the tumour microvascular density . In addition, the tumors of VT1-treated animals displayed increased apoptosis by TUNEL analysis and showed a significant decrease in cell proliferation, as determined by MIB-5 immunostaining . VT1 treatment of MM is effective in our orthotopic xenograft model, and warrants further exploration as a potential treatment for these highly anaplastic and aggressive neoplasms. Protein Eng, 2002 Jun, 15(6), 485 - 92 Rational design of 'water-soluble' bacteriorhodopsin variants; Mitra K et al.; We have explored the interchangeability of soluble and membrane proteins by attempting to render a helical membrane protein 'water soluble' through mutation of its lipid-exposed residues . Using an atomic resolution structure of bacteriorhodopsin (bR), two different strategies were developed to identify lipid-exposed residues for mutation . In the first strategy all residues in trimeric bR with solvent accessibility >35% were marked for replacement . Replacement residues were chosen so as to map an average surface of helical soluble proteins onto the bR surface, resulting in the mutagenesis of 14.9% of surface residues . The second strategy took into account the observation that accessible residues can be categorized as fully or partially accessible . Consequently, three mutants were designed based on monomeric bR, all with their accessible residues changed and with varying extents of mutagenesis of partially accessible residues . 13.5-24.3% of the wild-type surface was altered in these designs . The construct for the first design was cloned into Escherichia coli . Trace amounts of the mutant protein were expressed with the concurrent overexpression of an endogenous prolyl isomerase . In contrast, all three mutant proteins of the second design expressed well and could be purified to homogeneity . Systematic refolding trials were undertaken with limited success at solubilization in aqueous media . We have discussed the feasibility of applying the 'solubilization strategy' outlined here to membrane proteins. Mol Biol Evol, 2002 Jul, 19(7), 1162 - 70 The evolution of the heat-shock protein GroEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection; Fares MA et al.; The heat-shock protein GroEL is a double-ring-structured chaperonin that assists the folding of many newly synthesized proteins in Escherichia coli and the refolding in vitro, with the cochaperonin GroES, of conformationally damaged proteins . This protein is constitutively overexpressed in the primary symbiotic bacteria of many insects, constituting approximately 10% of the total protein in Buchnera, the primary endosymbiont of aphids . In the present study, we perform a maximum likelihood (ML) analysis to unveil the selective constraints in GroEL . In addition, we apply a new statistical approach to determine the patterns of evolution in this highly interesting protein . The main conclusion derived from our analysis is that GroEL has suffered an accelerated rate of amino acid substitution upon the symbiotic integration of Buchnera into the aphids . It is most interesting that the ML analysis of codon substitutions in the different branches of the phylogenetic tree strongly supports the action of positive selection in the different lineages of BUCHNERA: Additionally, the new sliding window analysis of the complete groEL sequence reveals different regions of the molecule under the action of positive selection, mainly located in the apical domain, that are important for both peptide and GroES binding. J Biol Chem, 2002 Sep 6, 277(36), 33267 - 74 Epub 2002 Jun 24. Complex role of the beta 2-beta 3 loop in the interaction of U1A with U1 hairpin II RNA; Katsamba PS et al.; RNA recognition motifs (RRMs) are characterized by highly conserved regions located centrally on a beta-sheet, which forms the RNA binding surface . Variable flanking regions, such as the loop connecting beta-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs . The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA . In this complex, the beta(2)-beta(3) loop protrudes through the 10-nucleotide RNA loop . Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal "fit" of the beta(2)-beta(3) loop into the RNA loop is an important factor in complexation . To understand this interaction further, we mutated or deleted loop residues Lys(50) and Met(51), which protrude centrally into the RNA loop but do not make any direct contacts to the bases . Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops . Alanine replacement mutations only modestly affected binding to wild type U1hpII . Interestingly, simultaneous replacement of Lys(50) and Met(51) with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop . Deletion of Lys(50) or Met(51) caused a dramatic loss in stability of the U1A.U1hpII complex . However, deletion of both residues simultaneously was much less deleterious . Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues . The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop . Our results indicate that one of the roles of the beta(2)-beta(3) loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA.protein complex. J Bacteriol, 2002 Jul, 184(14), 4044 - 7 The fixA and fixB genes are necessary for anaerobic carnitine reduction in Escherichia coli; Walt A et al.; In Escherichia coli, the use of carnitine as a terminal electron acceptor depends on a functional caiTABCDE operon . It had been suggested that the adjacent but divergent fixABCX operon is also required for carnitine metabolism, perhaps to provide electrons for carnitine reduction . We have constructed E . coli fixA and fixB mutants and find that they are unable to reduce carnitine to gamma-butyrobetaine under anaerobic conditions. J Bacteriol, 2002 Jul, 184(14), 4033 - 8 Differential spectrum of mutations that activate the Escherichia coli bgl operon in an rpoS genetic background; Moorthy S et al.; The bgl promoter is silent in wild-type Escherichia coli under standard laboratory conditions, and as a result, cells exhibit a beta-glucoside-negative (Bgl-) phenotype . Silencing is brought about by negative elements that flank the promoter and include DNA structural elements and sequences that interact with the nucleoid-associated protein H-NS . Mutations that confer a Bgl+ phenotype arise spontaneously at a detectable frequency . Transposition of DNA insertion elements within the regulatory locus, bglR, constitutes the major class of activating mutations identified in laboratory cultures . The rpoS-encoded sigmaS, the stationary-phase sigma factor, is involved in both physiological as well as genetic changes that occur in the cell under stationary-state conditions . In an attempt to see if the rpoS status of the cell influences the nature of the mutations that activate the bgl promoter, we analyzed spontaneously arising Bgl+ mutants in rpoS+ and rpoS genetic backgrounds . We show that the spectrum of activating mutations in rpoS cells is different from that in rpoS+ cells . Unlike rpoS+ cells, where insertions in bglR are the predominant activating mutations, mutations in hns make up the majority in rpoS cells . The physiological significance of these differences is discussed in the context of survival of natural populations of E . coli. J Bacteriol, 2002 Jul, 184(14), 3864 - 70 Cleavage of Treponema denticola PrcA polypeptide to yield protease complex-associated proteins Prca1 and Prca2 is dependent on PrtP; Lee SY et al.; Analysis of potential virulence factors of oral spirochetes focuses on surface and secreted proteins . The Treponema denticola chymotrypsin-like protease (CTLP) is implicated in degradation of host cell molecules and contributes to tissue invasion . The CTLP complex, composed of the 72-kDa PrtP protein and two auxiliary proteins with molecular masses of approximately 40 and 30 kDa, is also involved in localization and oligomerization of the T . denticola major surface protein (Msp) . The larger auxiliary protein was reported to be encoded by an open reading frame (ORF2) directly upstream of prtP . The deduced 39-kDa translation product of ORF2 contains a sequence matching the N-terminal sequence determined from one of the CTLP complex proteins . No proteins with significant homology are known, nor was information available on the third protein of the complex . DNA sequence analysis showed that ORF2 extended an additional 852 bp upstream of the reported sequence . The complete gene, designated prcA, encodes a predicted N-terminally-acylated polypeptide of approximately 70 kDa . Isogenic mutants with mutations in prtP, prcA, and prcA-prtP all lacked CTLP protease activity . The prcA mutant lacked all three CTLP proteins . The prcA-prtP mutant produced only a C-terminally-truncated 62-kDa PrcA protein . The prtP mutant produced a full-length 70-kDa PrcA . Immunoblot analysis of recombinant PrcA constructs confirmed that PrcA is cleaved to yield the two smaller proteins of the CTLP complex, designated PrcA1 and PrcA2 . These data indicate that PrtP is required for cleavage of PrcA and suggest that this cleavage may be required for formation or stability of outer membrane complexes. J Bacteriol, 2002 Jul, 184(14), 3823 - 33 Analysis of DNA regulatory elements required for expression of the Legionella pneumophila icm and dot virulence genes; Gal-Mor O et al.; To investigate the regulation of the Legionella pneumophila icm and dot genes required for intracellular growth, a series of nine icm::lacZ fusions were constructed . These icm::lacZ fusions were found to have different levels of expression in L . pneumophila, and five of them were more highly expressed at stationary phase than at exponential phase . When the expression of these fusions in Escherichia coli was tested, all of them were found to be expressed but three of them had dramatic changes in their levels of expression in comparison to those in L . pneumophila . Site-directed and PCR random mutagenesis with these icm::lacZ fusions was used to identify DNA regulatory elements of icm genes . Four icm genes (icmT, icmP, icmQ, and icmM) that had low levels of expression in L . pneumophila were found to contain a 6-bp sequence (TATACT) essential for their expression . This sequence was shown by primer extension to serve as their -10 promoter elements . A similar sequence, which constitutes the -10 promoter elements of the icmV, icmW, and icmR genes which had high levels of expression in L . pneumophila, was also identified . In addition, regulatory elements that probably serve as binding sites for transcription regulators were found in these genes . Altogether, 12 regulatory elements, 7 of which constitute the -10 promoter elements of the icm genes, were found . Even though all the icm and dot genes are part of one system required for L . pneumophila intracellular growth and even though their promoters are probably recognized by the vegetative sigma factor, it seems that they are subjected to different regulation mediated by several regulatory factors. J Bacteriol, 2002 Jul, 184(14), 3801 - 7 Evidence from terminal recombination gradients that FtsK uses replichore polarity to control chromosome terminus positioning at division in Escherichia coli; Corre J et al.; Chromosome dimers in Escherichia coli are resolved at the dif locus by two recombinases, XerC and XerD, and the septum-anchored FtsK protein . Chromosome dimer resolution (CDR) is subject to strong spatiotemporal control: it takes place at the time of cell division, and it requires the dif resolution site to be located at the junction between the two polarized chromosome arms or replichores . Failure of CDR results in trapping of DNA by the septum and RecABCD recombination (terminal recombination) . We had proposed that dif sites of a dimer are first moved to the septum by mechanisms based on local polarity and that normally CDR then occurs as the septum closes . To determine whether FtsK plays a role in the mobilization process, as well as in the recombination reaction, we characterized terminal recombination in an ftsK mutant . The frequency of recombination at various points in the terminus region of the chromosome was measured and compared with the recombination frequency on a xerC mutant chromosome with respect to intensity, the region affected, and response to polarity distortion . The use of a prophage excision assay, which allows variation of the site of recombination and interference with local polarity, allowed us to find that cooperating FtsK-dependent and -independent processes localize dif at the septum and that DNA mobilization by FtsK is oriented by the polarity probably due to skewed sequence motifs of the mobilized material. Am J Clin Nutr, 2002 Jul, 76(1), 232 - 8 Acute ingestion of a meal rich in n-3 polyunsaturated fatty acids results in rapid gastric emptying in humans; Robertson MD et al.; BACKGROUND: n-3 Polyunsaturated fatty acids (PUFAs) have proven benefits for both the development of atherosclerosis and inflammatory conditions . The effects on atherosclerosis may be partly mediated by the observed reduction in fasting and postprandial triacylglycerol concentrations after both acute and chronic n-3 PUFA ingestion . OBJECTIVE: The aim of this study was to assess gastric emptying and gastrointestinal hormone release after the consumption of mixed meals rich in n-3 PUFAs or other classes of fatty acids . DESIGN: Ten healthy women (aged 50-62 y) completed 4 separate study visits in a single-blind, randomized design . On each occasion, subjects consumed 40 g oil rich in either saturated fatty acids, monounsaturated fatty acids, n-6 PUFAs, or n-3 PUFAs as part of a mixed meal . {1-(13)C}Octanoic acid (100 mg) was added to each oil . Gastric emptying was assessed by a labeled octanoic acid breath test, and concentrations of gastrointestinal hormones and plasma lipids were measured . RESULTS: Recovery of (13)C in breath was enhanced after n-3 PUFA ingestion (P < 0.005) . The cholecystokinin response after the n-3 PUFA meal was significantly delayed (P < 0.001), and the glucagon-like peptide 1 response was significantly reduced (P < 0.05) . CONCLUSION: The inclusion of n-3 PUFAs in a meal alters the gastric emptying rate, potentially as the result of changes in the pattern of cholecystokinin and glucagon-like peptide 1 release. Genes Cells, 2002 Jul, 7(7), 653 - 62 The Cpx stress response system of Escherichia coli senses plasma membrane proteins and controls HtpX, a membrane protease with a cytosolic active site; Shimohata N et al.; BACKGROUND: The abnormal accumulation of misfolded proteins outside the plasma (cytoplasmic or inner) membrane up-regulates the synthesis of a class of envelope-localized catalysts of protein folding and degradation . The pathway for this transmembrane signalling is mediated by the CpxR-CpxA two-component phospho-relay mechanism . RESULTS: We now show that an abnormality in the plasma membrane proteins, due either to the impairment of FtsH, a protease acting against integral membrane proteins, or to the overproduction of a substrate membrane protein of FtsH, activates this stress response pathway . Under such conditions, the cpxR gene function becomes essential for cell growth . We further show that the expression of a putative protease, HtpX, in the plasma membrane, is under the control of CpxR . Synthetic growth inhibition was observed when the ftsH and htpX disruption mutations had been combined, suggesting that these gene products have some complementary or overlapping proteolytic functions . Topology analyses indicated that the metalloproteinase active site of HtpX is located on the cytosolic side of the membrane . CONCLUSIONS: Taken together, these results suggest that the Cpx "extracytoplasmic" stress response system controls the quality of the plasma membrane, even on its cytoplasmic side. Biochemistry, 2002 Jul 2, 41(26), 8499 - 507 Characterization of two partially unfolded intermediates of the molecular chaperone DnaK at low pH; Sehorn MG et al.; In this study, the effect of pH on the conformation and the reactivity of the Escherichia coli Hsp70 molecular chaperone DnaK was investigated using spectroscopic and chemical assays . DnaK exhibits negligible binding of the hydrophobic dye 1-anilino-naphthalene-8-sulfonate (ANS) between pH 7 to 5.0, whereas appreciable binding occurs between pH 4.5 to 4.0 . The binding of ANS to a protein is diagnostic of the presence of accessible ordered hydrophobic surfaces . Such hydrophobic surfaces are often displayed by partially folded protein intermediates such as molten globules . Nucleotide inhibits 70% of the ANS binding at pH 4.5 but none of the ANS binding at pH 4.0 . Proteolysis of nucleotide-free DnaK at pH 4.5 with cathepsin D yields detectable fragments (masses > 20 kDa) of the C-terminal peptide-binding domain but none of the N-terminal ATPase domain, thus the ATPase domain is preferentially targeted for proteolysis . In contrast, proteolysis of nucleotide-free DnaK at pH 4.0 with cathepsin D cuts near the linker region, yielding both functional domains . Our interpretation of these data is that incubation of DnaK at pH 4.5 produces a partially unfolded form of the ATPase domain, in which secondary structure is mainly intact, but tertiary structure is reduced . Incubation of the protein at pH 4.0 produces an intermediate in which both functional domains have collapsed and possibly separated . Nucleotide inhibits the conformational change that occurs at pH 4.5 but not at 4.0. Biochemistry, 2002 Jul 2, 41(26), 8493 - 8 Cysteine biosynthetic enzymes are the pieces of a metabolic energy pump; Wei J et al.; Understanding the mechanisms of free energy transfer in metabolism is fundamental to understanding how the chemical forces that sustain the molecular organization of the cell are distributed . Recent studies of molecular motors (1-3) and ATP-driven proton transport (4-6) describe how chemical potential is transferred at the molecular level . These systems catalyze energy transfer through structural change and appear to be dedicated exclusively to their coupling tasks (7, 8) . Here we report the discovery of a new class of energy-transfer system . It is a biosynthetic pump composed of cysteine biosynthesis enzymes, ATP sulfurylase and O-acetylserine sulfhydrylase, each with its own catalytic function and from whose interactions emerge new function: the hydrolysis of ATP . The hydrolysis is kinetically and energetically linked to the chemistry catalyzed by ATP sulfurylase, the first enzyme in the cysteine biosynthetic pathway, in such a way that each molecule of ATP hydrolyzed, each stroke of the pump, produces 1 equivalent of that enzyme's product . These findings integrate cysteine metabolism and broaden our understanding of the ways in which higher order allostery is used to effect free energy transfer. Biochemistry, 2002 Jul 2, 41(26), 8464 - 70 DNA-mediated charge transport as a probe of MutY/DNA interaction; Boon EM et al.; MutY is an Escherichia coli DNA repair enzyme that binds to 8-oxo-G:A and G:A mismatches and catalyzes the deglycosylation of the mismatched 2'-deoxyadenosine . We have applied DNA-mediated charge transport to probe the interaction of MutY with its DNA substrate . Oligonucleotides synthesized with a tethered rhodium intercalator and guanine doublets placed before and after the MutY binding site are used to assay for base flipping activity by MutY . On the basis of this assay, we find no evidence that MutY uses progressive base flipping as a means to find its binding site; protein binding does not perturb long-range DNA charge transport . DNA-mediated charge transport can be utilized to promote protein-DNA cross-linking from a distance . Long-range oxidation of 8-oxo-G within the MutY binding site using tethered rhodium intercalators promoted cross-linking and yielded information on MutY side chains that interact with this base . On the basis of photooxidative cross-linking of the wild type but not K142A mutant, it is evident that, within the protein complex, lysine 142 makes important contacts with 8-oxo-G. Biochemistry, 2002 Jul 2, 41(26), 8414 - 21 Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli; Gonzalez G et al.; The EcDos protein belongs to a group of heme-based sensors that detect their ligands with a heme-binding PAS domain . Among these various heme-PAS proteins, EcDos is unique in having its heme iron coordinated at both axial positions to residues of the protein . To achieve its high affinities for ligands, one of the axial heme-iron residues in EcDos must be readily displaceable . Here we present evidence from mutagenesis, ligand-binding measurements, and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopies about the nature of the displaceable residue in the heme-PAS domain of EcDos, i.e., EcDosH . The magnetic circular dichroism spectra in the near-infrared region establish histidine-methionine coordination in met-EcDos . To determine whether in deoxy-EcDos coordination of the sixth axial position is also to methionine, methionine 95 was substituted with isoleucine . This substitution caused the ferrous heme iron to change from an exclusively hexacoordinate low-spin form (EcDosH) to an exclusively pentacoordinate high-spin form (M95I EcDosH) . This was accompanied by a modest acceleration of the dissociation rates of ligands but a dramatic increase (60-1300-fold) in the association rate constants for binding of O(2), CO, and NO . As a result, the affinity for O(2) was enhanced 10-fold in M95I EcDosH, but the partition constant M = {K(d)(O(2))/K(d)(CO)} between CO and O(2) was raised to about 30 from the extraordinarily low EcDosH value of 1 . Thus a major consequence of the increased O(2) affinity of this sensor was the loss of its unusually strong ligand discrimination. Biochemistry, 2002 Jul 2, 41(26), 8377 - 84 Neuronal nitric oxide synthase ligand and protein vibrations at the substrate binding site . A study by FTIR; Ingledew WJ et al.; Improvements in sensitivity and data processing of Fourier transform infrared (FTIR) spectroscopy enable it to be used to detect changes in protein structure at the atomic level . This paper reports a study of neuronal nitric oxide synthase (nNOS) by FTIR difference spectroscopy in the 1000-2500 cm(-1) range where vibrational bands of ligands, prosthetic groups, and protein and amino acid side chains are found . We have exploited the photolyzable CO compound of the ferrous heme of nNOS to produce light-induced CO photolysis difference spectra and to compare spectra after hydrogen/deuterium exchange . In (reduced) minus (reduced plus CO) difference spectra, negative bands at 1931 and 1907 cm(-1) are observed due to photolysis of multiple forms of ferrous heme-ligated CO, similar to those observed by resonance Raman spectroscopy {Wang et al . (1997) Biochemistry 36, 4595-4606} . Photolysis of the ferrous heme CO compound is accompanied by hitherto unreported changes in the 1000-2000 cm(-1) region that arise from changes of protein backbone, substrate, amino acid side chain, and cofactor vibrations . Preliminary assignments of vibrations are made on the basis of frequencies and the effects of hydrogen/deuterium exchange, and in the light of known atomic structures. Biochemistry, 2002 Jul 2, 41(26), 8310 - 20 Probing the interaction of bovine cytochrome P450scc (CYP11A1) with adrenodoxin: evaluating site-directed mutations by molecular modeling; Usanov SA et al.; The present study was undertaken to evaluate the role of positively charged amino acid residues proposed to reside on the proximal surface of bovine cytochrome P450 cholesterol side chain cleavage (P450scc, CYP11A1) and to determine which residues may be involved in protein-protein interactions with the electron carrier adrenodoxin (Adx) . In previous studies, nine different lysine residues were identified by chemical and immunological cross-linking experiments as potentially interacting with Adx, while in the present study, two arginine residues have been identified from sequence alignments . From these 11 residues, 13 different P450scc mutants were made of which only seven were able to be expressed and characterized . Each of the seven mutants were evaluated for their ability to bind Adx, to be reduced, and for their enzymatic activity . Among these, K403Q and K405Q showed a consistent decrease in Adx binding, the ability to be reduced by Adx, and enzymatic activity, with K405Q being affected to a much greater extent . More dramatic was the complete loss of Adx binding by R426Q, while still retaining its ability to be chemically reduced and bind carbon monoxide . Independently, a homology model of P450scc was constructed and docked with the structure of Adx . Four potential sites of interaction were identified: P450scc:K403 with Adx:D76, P450scc:K405 with Adx:D72; P450scc:R426 with Adx:E73, and P450scc:K267 with Adx:E47 . Thus, the biochemical and molecular modeling studies together support the hypothesis that K267, K403, K405, and R426 participate in the electrostatic interaction of P450scc with Adx. Biochemistry, 2002 Jul 2, 41(26), 8263 - 76 Determination of active concentrations and association and dissociation rate constants of interacting biomolecules: an analytical solution to the theory for kinetic and mass transport limitations in biosensor technology and its experimental verification; Sigmundsson K et al.; Accurate determination of kinetic rate constants for interacting biomolecules requires knowledge of the active concentrations of the participating molecules . Also, in other biomedical and clinical applications, sensitive, precise and accurate methods are needed to determine the concentration of biologically active molecules, which frequently constitute only a fraction of the total molecular pool . Here we report a novel development of the approach to determining active concentrations based on surface plasmon resonance (SPR) technology . The method relies on changes in binding rates with varying flow rates under conditions of partial mass transport, and does not require standards of known concentrations, given that the molecular mass of the molecule of interest is known . We introduce an analytical solution to the differential equations describing the formation of a 1:1 bimolecular complex, taking into account both the association and dissociation reactions, under partial mass transport limitations . This solution can be used in global fitting to binding curves obtained at different flow rates . The accuracy, precision, and sensitivity of this approach were determined in experiments involving binding of tyrosine-phosphorylated recombinant proteins to anti-phosphotyrosine antibodies, where the active concentration could be determined independently by in vitro phosphorylation with (33)P . There was an excellent agreement between the active concentrations determined by the analytical SPR-based method and by determination of the level of radioactivity of the phosphorylated protein . The SPR-based method allows determination of protein concentrations at picomolar levels . A procedure for accurate determinations of association and dissociation rate constants, based on the analytical solution of the mass transport and binding theory, is outlined. J Magn Reson, 2002 May, 156(1), 155 - 9 Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment; Brutscher B; Two novel experiments, intra-HNCA and intra-COHNCA, are presented for sequential backbone resonance assignment of (13)C, (15)N labeled proteins . The advantage with respect to conventional pulse schemes is the suppression of the sequential (15)N-->(13)C(alpha) coherence transfer pathway, which can be separately obtained from a HNCOCA correlation experiment . This results in a two-fold reduction of the number of detected correlation peaks . Spectral simplification is especially important for efficient automated assignment protocols as required in the context of high-throughput protein studies by NMR . The performance of the new experiments is demonstrated on an 18-kDa protein fragment of the E . coli sulfite reductase and compared to conventional techniques in terms of sensitivity and resolution. Biol Pharm Bull, 2002 Jun, 25(6), 722 - 7 Effect of replacing the aspartic acid/glutamic acid residues of bullfrog sialic acid binding lectin with asparagine/glutamine and arginine on the inhibition of cell proliferation in murine leukemia P388 cells; Ogawa Y et al.; The sialic acid binding lectin from bullfrog oocytes (cSBL) is known to have anti-tumor activity . In a previous report, to elucidate the relationship between the net charge and anti-tumor activity of cSBL, we examined the effect of chemical modifications of cSBL with a water-soluble carbodiimide in the presence of various nucleophiles . The results suggested that the anti-tumor activity and internalization into tumor cells increased with an increase in the net charge of cSBL . However, in the chemically modified cSBL, a modification site was observed on average in two of the carboxyl groups of cSBL . To confirm these previous results and to determine which modified carboxyl group contributes to the increase in anti-tumor activity, we prepared mutants with substitutions of Asn/Gln and Arg at three acidic amino acid residues of cSBL and studied their anti-tumor activity and internalization efficiency . The results showed the enhancing effect of charge on anti-tumor activity and internalization, and suggested that the replacement of D24 and E88 of cSBL with arginine is more effective than that of E97 . The double mutant D24RE88R showed comparable anti-tumor activity to the ethylenediamine-modified cSBL reported previously . The mutant was well-characterized as a pure cSBL derivative suitable for studying the mechanism of the anti-tumor action of cSBL. Biol Pharm Bull, 2002 Jun, 25(6), 705 - 9 cDNA cloning of the Sry-related gene Sox6 from rat with tissue-specific expression; Narahara M et al.; A cDNA encoding rat homologue of the previously characterized mouse Sox6 was isolated by a polymerase chain reaction (PCR) cloning strategy . Comparison of this eDNA with homologous mouse, human and rainbow trout cDNA exhibited an overall amino acid sequence identity of 99.6, 89.3 and 76.3% respectively . The leucine-zipper and HMG-box motif were almost completely conserved between these homologues . The expression of Sox6 was determined in rat by Northern hybridization and Real-time quantitative reverse transcription (RT)-PCR . rSox6 (rat Sox6) was specifically expressed in the neonatal brain and adult testis with Northern blotting . Real-time quantitative RT-PCR for the determination of Sox6 mRNA was examined . The rSox6 was expressed in the neonatal brain and adult testis as well as by Northern blotting and also expressed in the adult eyeball and slightly in the ovary. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 1999 Jul, 13(4), 206 - 8 {Direct gene transfer into rabbit peripheral nerve in vivo}; Zhang SQ et al.; OBJECTIVE: To probe the possibility of direct transfer of exogenous gene into peripheral nerve and its following expression in vivo . METHODS: The PCMV beta plasmid containing cytomegalovirus (CMV) promoter and Escherichia Coli (E . Coli), beta-Galactosidease (beta-Gal) structural gene (lacZ gene) was constructed and injected into the rabbit sciatic nerve . The control group was injected PBS solution . The injected nerves were sampled and tested by beta-Gal enzyme activity assay of the 5-bromo-4-chloro-3-indolyl-beta-D-galactoside and beta-Gal histochemical stain . RESULTS: In the control group, no beta-Gal enzyme activity was detected in the different stages after operation, and beta-Gal histochemical stains showed positive . In the experimental group, enzyme activity could be detected from 2 days to 30 days after operation, and the histochemical stains showed negative . CONCLUSION: The exogenous gene can be transferred into peripheral nerve and expressed with bioactivity, thus the gene therapy to accelerate the recovery of nerve is practical. Biophys J, 2002 Jul, 83(1), 309 - 21 Site-directed mutagenesis of tyrosine 118 within the central constriction site of the LamB (maltoporin) channel of Escherichia coli . II . Effect on maltose and maltooligosaccharide binding kinetics; Orlik F et al.; The 3-D structure of the maltooligosaccharide-specific LamB channel of Escherichia coli (also called maltoporin) is known from x-ray crystallography . The central constriction of the channel formed by the external loop 3 is controlled by tyrosine 118 . Y118 was replaced by site-directed mutagenesis by 10 other amino acids (alanine (A), isoleucine (I), asparagine (N), serine (S), cysteine (C), aspartic acid (D), arginine (R), histidine (H), phenylalanine (F), and tryptophan (W)) including neutral ones, negatively and positively charged amino acids to study the effect of their size, their hydrophobicity index, and their charge on maltose and maltooligosaccharide binding to LamB . The mutants were reconstituted into lipid bilayer membranes and the stability constants for binding of maltose, maltotriose, maltopentaose, and maltoheptaose to the channel were measured using titration experiments . The mutation of Y118 to any other non-aromatic amino acid led to a substantial decrease of the stability constant of binding by factors between about two and six . The highest effect was observed for the mutant Y118A . Replacement of Y118 by the two other aromatic amino acids, phenylalanine (F) and tryptophan (W), resulted in a substantial increase of the stability constant maximally by a factor of almost 400 for the Y118W mutant . The carbohydrate-induced block of the channel function was used for the study of current noise through the different mutant LamB channels . The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k(1) and k(-1)) of sugar binding . The results suggest that both rate constants were affected by the mutations . For most mutants, with the exception of Y118F and Y118W, k(1) decreased and k(-1) increased, whereas the opposite was found for the aromatic amino acid mutants . The results suggest that tyrosine 118 has a crucial effect on carbohydrate transport through LamB. Biophys J, 2002 Jul, 83(1), 290 - 8 Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes; Sukharev S; The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift . It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J . 18:1730) . Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer . MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of approximately 200 kDa . Consistent with that, the protein cross-linking patterns predict a hexamer . The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20-60 mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ . At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations . The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient . Therefore, the open probability curves were recorded with descending series of pressures . Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (gamma) . Po(gamma) curves had the midpoint at 5.5 +/- 0.1 dyne/cm and gave estimates for the energy of opening DeltaG = 11.4 +/- 0.5 kT, and the transition-related area change DeltaA = 8.4 +/- 0.4 nm(2) when fitted with a two-state Boltzmann model . The correspondence between channel properties in the native and reconstituted systems is discussed. Biophys J, 2002 Jul, 83(1), 154 - 60 Pressure-induced water transport in membrane channels studied by molecular dynamics; Zhu F et al.; A method is proposed to measure the water permeability of membrane channels by means of molecular dynamics simulations . By applying a constant force to the bulk water molecules and a counter force on the complementary system, a hydrostatic pressure difference across the membrane can be established, producing a net directional water flow . The hydraulic or osmotic permeability can then be determined by the ratio of the water flux and the pressure difference . The method is applied and tested on an aquaglyceroporin channel through a series of simulations totaling 5 ns in duration. Biophys J, 2002 Jul, 83(1), 79 - 86 Energy balance for analysis of complex metabolic networks; Beard DA et al.; Predicting behavior of large-scale biochemical networks represents one of the greatest challenges of bioinformatics and computational biology . Computational tools for predicting fluxes in biochemical networks are applied in the fields of integrated and systems biology, bioinformatics, and genomics, and to aid in drug discovery and identification of potential drug targets . Approaches, such as flux balance analysis (FBA), that account for the known stoichiometry of the reaction network while avoiding implementation of detailed reaction kinetics are promising tools for the analysis of large complex networks . Here we introduce energy balance analysis (EBA)--the theory and methodology for enforcing the laws of thermodynamics in such simulations--making the results more physically realistic and revealing greater insight into the regulatory and control mechanisms operating in complex large-scale systems . We show that EBA eliminates thermodynamically infeasible results associated with FBA. J Biol Chem, 2002 Sep 6, 277(36), 32650 - 8 Epub 2002 Jun 21. Stationary and time-resolved resonance Raman spectra of His77 and Met95 mutants of the isolated heme domain of a direct oxygen sensor from Escherichia coli; Sato A et al.; The heme environments of Met(95) and His(77) mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E . coli (E . coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme . The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10 . The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band (nu(Fe-His)) at 214 cm(-1), and the nu(Fe-CO) versus nu(CO) plot of CO-WT E . coli DOS PAS fell on the line of His-coordinated heme proteins . The photodissociated CO-H77A mutant complex did not yield the nu(Fe-His) band but gave a nu(Fe-Im) band in the presence of imidazole . The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e . the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a five-coordinate heme complex . Taken together, we suggest that the heme of the reduced WT enzyme is coordinated by His(77) and Met(95), and that Met(95) is displaced by CO and O(2) . Presumably, the protein conformational change that occurs upon exchange of an unknown ligand for Met(95) following heme reduction may lead to activation of the phosphodiesterase domain of E . coli DOS. J Biol Chem, 2002 Sep 13, 277(37), 34055 - 66 Epub 2002 Jun 21. Reduced flavins promote oxidative DNA damage in non-respiring Escherichia coli by delivering electrons to intracellular free iron; Woodmansee AN et al.; When cells are exposed to external H(2)O(2), the H(2)O(2) rapidly diffuses inside and oxidizes ferrous iron, thereby forming hydroxyl radicals that damage DNA . Thus the process of oxidative DNA damage requires only H(2)O(2), free iron, and an as-yet unidentified electron donor that reduces ferric iron to the ferrous state . Previous work showed that H(2)O(2) kills Escherichia coli especially rapidly when respiration is inhibited either by cyanide or by genetic defects in respiratory enzymes . In this study we established that these respiratory blocks accelerate the rate of DNA damage . The respiratory blocks did not substantially affect the amounts of intracellular free iron or H(2)O(2), indicating that that they accelerated damage because they increased the availability of the electron donor . The goal of this work was to identify that donor . As expected, the respiratory inhibitors caused a large increase in the amount of intracellular NADH . However, NADH itself was a poor reductant of free iron in vitro . This suggests that in non-respiring cells electrons are transferred from NADH to another carrier that directly reduces the iron . Genetic manipulations of the amounts of intracellular glutathione, NADPH, alpha-ketoacids, ferredoxin, and thioredoxin indicated that none of these was the direct electron donor . However, cells were protected from cyanide-stimulated DNA damage if they lacked flavin reductase, an enzyme that transfers electrons from NADH to free FAD . The K(m) value of this enzyme for NADH is much higher than the usual intracellular NADH concentration, which explains why its flux increased when NADH levels rose during respiratory inhibition . Flavins that were reduced by purified flavin reductase rapidly transferred electrons to free iron and drove a DNA-damaging Fenton system in vitro . Thus the rate of oxidative DNA damage can be limited by the rate at which electron donors reduce free iron, and reduced flavins become the predominant donors in E . coli when respiration is blocked . It remains unclear whether flavins or other reductants drive Fenton chemistry in respiring cells. Chem Biol, 2002 Jun, 9(6), 747 - 55 Crystal structure of a complex between the aminoglycoside tobramycin and an oligonucleotide containing the ribosomal decoding a site; Vicens Q et al.; Aminoglycoside antibiotics target the decoding aminoacyl site (A site) on the 16S ribosomal RNA and induce miscoding during translation . Here, we present the crystal structure, at 2.54 A resolution, of an RNA oligonucleotide containing the A site sequence complexed to the 4,6-disubstituted 2-deoxystreptamine aminoglycoside tobramycin . The three aminosugar rings making up tobramycin interact with the deep-groove atoms directly or via water molecules and stabilize a fully bulged-out conformation of adenines A(1492) and A(1493) . The comparison between this structure and the one previously solved in the presence of paromomycin confirms the importance of the functional groups on the common neamine part of these two antibiotics for binding to RNA . Furthermore, the analysis of the present structure provides a molecular explanation to some of the resistance mechanisms that have spread among bacteria and rendered aminoglycoside antibiotics inefficient. Int J Radiat Biol, 2002 Jul, 78(7), 585 - 92 Characterization of 2-hydroxyadenine DNA glycosylase activity of Escherichia coli MutY protein; Hashiguchi K et al.; PURPOSE: 2-Hydroxyadenine (2-ohA) is an oxidation product of adenine generated in DNA by ionizing radiation and various chemical oxidants . 2-ohA has mutational potential comparable to that of 8-oxoguanine in bacteria and mammalian cells . Recent studies have shown that 2-ohA is removed from DNA by a human MutY homolog, MYH protein, in vitro . On the other hand, the repair mechanisms for 2-ohA in Escherichia coli are not yet understood . MATERIALS AND METHODS: Gel shift assays were used to assess the binding activity of E . coli full-length MutY protein and its N-terminal (residues 1-226) domain (M25) to 2-ohA/G-, 2-ohA/A-, 2-ohA/C- and 2-ohA/T-containing 24-mer oligonucleotides . Furthermore, whether these proteins specifically cleave 2-ohA-containing duplex oligonucleotides was examined . RESULTS: The purified MutY and M25 proteins had similar binding affinities to 2-ohA/G-, 2-ohA/A- and 2-ohA/C-containing oligonucleotides . MutY protein removed 2-ohA preferentially from 2-ohA/G mispairs . M25 protein showed the reduced catalytic activity for 2-ohA/G-containing oligonucleotides . CONCLUSIONS: E . coli MutY protein has a DNA glycosylase activity that removes 2-ohA from 2-ohA/G mispairs in DNA . The C-terminal domain is required for the removal of 2-ohA from DNA, but is not crucial for binding to 2-ohA-containing oligonucleotides. Jpn J Cancer Res, 2002 Jun, 93(6), 706 - 15 Dose of adenoviral vectors expressing interleukin-2 plays an important role in combined gene therapy with cytosine deaminase/5-fluorocytosine: preclinical consideration; Nakamori M et al.; Using a syngeneic murine model, we investigated the therapeutic efficacy of combined gene therapy using adenoviral vectors expressing murine interleukin-2 (AdmIL-2) and Escherichia coli cytosine deaminase (AdCD) . In a subcutaneous tumor model, tumor-bearing mice were treated with an intratumoral injection of adenoviral vectors and received an intraperitoneal administration of 5-fluorocytosine (5-FC) . Only the mice treated with AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) survived significantly longer than mice treated with AdCD alone (P < 0.01) . Moreover, 40% of these treated mice obtained complete remission from tumor-bearing status . The cytotoxicity of splenocytes obtained from the treated mice was related to the survival period . Tumor-specific cytotoxic T lymphocyte assay showed that the cell-mediated cytotoxic response was specific for parental tumor cells . In a hepatic metastasis model, mice treated with an intravenous administration of both AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) demonstrated the most significant reduction of metastatic foci and the longest survival following a 5-FC administration . These results suggest that gene therapy combined with AdmIL-2 and AdCD may be a promising strategy for clinical application and, in addition, that translation of combined gene therapy from murine models into the clinical setting will require careful attention to the variables of cytokine expression levels in the design of clinical trials and in the evaluation of treatment efficacy. BMC Microbiol . 2002 Jun 12;2(1):13. Oxygen and nitrate-dependent regulation of dmsABC operon expression in Escherichia coli: sites for Fnr and NarL protein interactions; Bearson SM et al.; BACKGROUND: Escherichia coli, can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation . Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present . RESULTS: The regions of dmsA regulatory DNA required for Fnr and NarL interactions in response to anaerobiosis and nitrate, respectively, were examined . Mutations within the Fnr site that deviated from the wild type sequence, TTGATaccgAACAA, or that removed an entire half-site, either impaired or abolished the anaerobic activation of dmsA-lacZ expression . The region for phosphorylated NarL (NarL-phosphate) binding at the dmsA promoter was identified by DNase I and hydroxyl radical footprinting methods . A large 97 bp region that overlaps the Fnr and RNA polymerase recognition sites was protected by NarL-phosphate but not by the non-phosphorylated form of NarL . Hydroxyl radical footprinting analysis confirmed the NarL-phosphate DNase I protections of both dmsA strands and revealed 8-9 protected sites of 3-5 bp occurring at ten bp intervals that are offset by 3 bp in the 3' direction . CONCLUSION: These findings suggest that multiple molecules of phosphorylated NarL bind along one face of the DNA and may interfere with Fnr and/or RNA polymerase interactions at the dmsA regulatory region . The interplay of these transcription factors insures a hierarchical expression of the dmsABC genes when respiration of the preferred electron acceptors, oxygen and nitrate, is not possible. J Mol Biol, 2002 Jul 5, 320(2), 343 - 57 Thermodynamic consequences of burial of polar and non-polar amino acid residues in the protein interior; Loladze VV et al.; Effects of amino acid substitutions at four fully buried sites of the ubiquitin molecule on the thermodynamic parameters (enthalpy, Gibbs energy) of unfolding were evaluated experimentally using differential scanning calorimetry . The same set of substitutions has been incorporated at each of four sites . These substitutions have been designed to perturb packing (van der Waals) interactions, hydration, and/or hydrogen bonding . From the analysis of the thermodynamic parameters for these ubiquitin variants we conclude that: (i) packing of non-polar groups in the protein interior is favorable and is largely defined by a favorable enthalpy of van der Waals interactions . The removal of one methylene group from the protein interior will destabilize a protein by approximately 5 kJ/mol, and will decrease the enthalpy of a protein by 12 kJ/mol . (ii) Burial of polar groups in the non-polar interior of a protein is highly destabilizing, and the degree of destabilization depends on the relative polarity of this group . For example, burial of Thr side-chain in the non-polar interior will be less destabilizing than burial of Asn side-chain . This decrease in stability is defined by a large enthalpy of dehydration of polar groups upon burial . (iii) The destabilizing effect of dehydration of polar groups upon burial can be compensated if these buried polar groups form hydrogen bonding . The enthalpy of this hydrogen bonding will compensate for the unfavorable dehydration energy and as a result the effect will be energetically neutral or even slightly stabilizing. J Mol Biol, 2002 Jul 5, 320(2), 311 - 9 High pressure NMR reveals that apomyoglobin is an equilibrium mixture from the native to the unfolded; Kitahara R et al.; Pressure-induced reversible conformational changes of sperm whale apomyoglobin have been studied between 30 bar and 3000 bar on individual residue basis by utilizing 1H/15N hetero nuclear single-quantum coherence two-dimensional NMR spectroscopy at pH 6.0 and 35 degrees C . Apomyoglobin showed a series of pressure-dependent NMR spectra as a function of pressure, assignable to the native (N), intermediates (I), molten globule (MG) and unfolded (U) conformers . At 30 bar, the native fold (N) shows disorder only in the F helix . Between 500 bar and 1200 bar, a series of locally disordered conformers I are produced, in which local disorder occurs in the C helix, the CD loop, the G helix and part of the H helix . At 2000 bar, most cross-peaks exhibit severe line-broadening, suggesting the formation of a molten globule, but at 3000 bar all the cross-peaks reappear, showing that the molten globule turns into a well-hydrated, mobile unfolded conformation U . Since all the spectral changes were reversible with pressure, apomyoglobin is considered to exist as an equilibrium mixture of the N, I, MG and U conformers at all pressures . MG is situated at 2.4+/-(0.1) kcal/mol above N at 1 bar and the unfolding transition from the combined N-I state to MG is accompanied by a loss of partial molar volume by 75+/-(3) ml/mol . On the basis of these observations, we postulate a theorem that the partial molar volume of a protein decreases in parallel with the loss of its conformational order. J Mol Biol, 2002 Jul 5, 320(2), 249 - 61 Crystal structure of MabA from Mycobacterium tuberculosis, a reductase involved in long-chain fatty acid biosynthesis; Cohen-Gonsaud M et al.; The fatty acid elongation system FAS-II is involved in the biosynthesis of mycolic acids, which are major and specific long-chain fatty acids of the cell envelope of Mycobacterium tuberculosis and other mycobacteria, including Mycobacterium smegmatis . The protein MabA, also named FabG1, has been shown recently to be part of FAS-II and to catalyse the NADPH-specific reduction of long chain beta-ketoacyl derivatives . This activity corresponds to the second step of an FAS-II elongation round . FAS-II is inhibited by the antituberculous drug isoniazid through the inhibition of the 2-trans-enoyl-acyl carrier protein reductase InhA . Thus, the other enzymes making up this enzymatic complex represent potential targets for designing new antituberculous drugs . The crystal structure of the apo-form MabA was solved to 2.03 A resolution by molecular replacement . MabA is tetrameric and shares the conserved fold of the short-chain dehydrogenases/reductases (SDRs) . However, it exhibits some significant local rearrangements of the active-site loops in the absence of a cofactor, particularly the beta5-alpha5 region carrying the unique tryptophan residue, in agreement with previous fluorescence spectroscopy data . A similar conformation has been observed in the beta-ketoacyl reductase from Escherichia coli and the distantly related dehydratase . The distinctive enzymatic and structural properties of MabA are discussed in view of its crystal structure and that of related enzymes. J Mol Biol, 2002 Jul 5, 320(2), 201 - 13 Expression, purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides; Potter CA et al.; The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator . Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases . Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties . Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation . The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity . Phosphotransfer from RegB to RegA (overexpressed and purified from E . coli) by RegB is very rapid, as has been reported for the soluble domain . Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB. J Mol Biol, 2002 Jun 21, 319(5), 1279 - 90 Mutations that reduce aggregation of the Alzheimer's Abeta42 peptide: an unbiased search for the sequence determinants of Abeta amyloidogenesis; Wurth C et al.; The primary component of amyloid plaque in the brains of Alzheimer's patients is the 42 residue amyloid-beta-peptide (Abeta42) . Although the amino acid residue sequence of Abeta42 is known, the molecular determinants of Abeta amyloidogenesis have not been elucidated . To facilitate an unbiased search for the sequence determinants of Abeta aggregation, we developed a genetic screen that couples a readily observable phenotype in E . coli to the ability of a mutation in Abeta42 to reduce aggregation . The screen is based on our finding that fusions of the wild-type Abeta42 sequence to green fluorescent protein (GFP) form insoluble aggregates in which GFP is inactive . Cells expressing such fusions do not fluoresce . To isolate variants of Abeta42 with reduced tendencies to aggregate, we constructed and screened libraries of Abeta42-GFP fusions in which the sequence of Abeta42 was mutated randomly . Cells expressing GFP fusions to soluble (non-aggregating) variants of Abeta42 exhibit green fluorescence . Implementation of this screen enabled the isolation of 36 variants of Abeta42 with reduced tendencies to aggregate . The sequences of most of these variants are consistent with previous models implicating hydrophobic regions as determinants of Abeta42 aggregation . Some of the variants, however, contain amino acid substitutions not implicated in pre-existing models of Abeta amyloidogenesis . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 21, 319(5), 1177 - 97 Ions and counterions in a biological channel: a molecular dynamics simulation of OmpF porin from Escherichia coli in an explicit membrane with 1 M KCl aqueous salt solution; Im W et al.; A 5 ns all-atom molecular dynamics trajectory of Escherichia coli OmpF porin embedded in an explicit dimyristoyl-phosphatidylcholine (DMPC) bilayer bathed by a 1 M {KCl} aqueous salt solution is generated to explore the microscopic details of the mechanism of ion permeation . The atomic model includes the OmpF trimer, 124 DMPC, 13470 water molecules as well as 231 K+ and 201 Cl-, for a total of 70,693 atoms . The structural and dynamical results are in excellent agreement with the X-ray data . The global root-mean-square deviation of the backbone atoms relative to the X-ray structure is 1.4 A . A cluster of three fully charged arginine (Arg42, Arg82, and Arg132) facing two acidic residues (Asp113 and Glu117) on L3 in the narrowest part of the aqueous pore is observed to be very stable in the crystallographic conformation . In this region of the pore, the water molecules are markedly oriented perpendicular to the channel axis due to the strong transversal electrostatic field arising from those residues . On average the size of the pore is smaller during the simulation than in the X-ray structure, undergoing small fluctuations . No large movements of loop L3 leading to a gating of the pore are observed . Remarkably, it is observed that K+ and Cl- follow two well-separated average pathways spanning over nearly 40 A along the axis of the pore . In the center of the monomer, the two screw-like pathways have a left-handed twist, undergoing a counter-clockwise rotation of 180 degrees from the extracellular vestibule to the pore periplasmic side . In the pore, the dynamical diffusion constants of the ions are reduced by about 50% relative to their value in bulk solvent . Analysis of ion solvation across the channel reveals that the contributions from the water and the protein are complementary, keeping the total solvation number of both ions nearly constant . Unsurprisingly, K+ have a higher propensity to occupy the aqueous pore than Cl-, consistent with the cation selectivity of the channel . However, further analysis suggests that ion-ion pairs play an important role . In particular, it is observed that the passage of Cl- occurs only in the presence of K+ counterions, and isolated K+ can move through the channel and permeate on their own . The presence of K+ in the pore screens the negative electrostatic potential arising from OmpF to help the translocation of Cl- by formation of ion pairs . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 21, 319(5), 1085 - 96 The Escherichia coli dam DNA methyltransferase modifies DNA in a highly processive reaction; Urig S et al.; The Escherichia coli dam adenine-N6 methyltransferase modifies DNA at GATC sequences . It is involved in post-replicative mismatch repair, control of DNA replication and gene regulation . We show that E . coli dam acts as a functional monomer and methylates only one strand of the DNA in each binding event . The preferred way of ternary complex assembly is that the enzyme first binds to DNA and then to S-adenosylmethionine . The enzyme methylates an oligonucleotide containing two dam sites and a 879 bp PCR product with four sites in a fully processive reaction . On lambda-DNA comprising 48,502 bp and 116 dam sites, E . coli dam scans 3000 dam sites per binding event in a random walk, that on average leads to a processive methylation of 55 sites . Processive methylation of DNA considerably accelerates DNA methylation . The highly processive mechanism of E . coli dam could explain why small amounts of E . coli dam are able to maintain the methylation state of dam sites during DNA replication . Furthermore, our data support the general rule that solitary DNA methyltransferase modify DNA processively whereas methyltransferases belonging to a restriction-modification system show a distributive mechanism, because processive methylation of DNA would interfere with the biological function of restriction-modification systems . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 21, 319(5), 1067 - 83 Beta subunit residues 186-433 and 436-445 are commonly used by Esigma54 and Esigma70 RNA polymerase for open promoter complex formation; Wigneshweraraj SR et al.; During transcription initiation by DNA-dependent RNA polymerase (RNAP) promoter DNA has to be melted locally to allow the synthesis of RNA transcript . Localized melting of promoter DNA is a target for genetic regulation and is poorly understood at the molecular level . The Escherichia coli RNAP holoenzyme is a six-subunit (alpha(2)betabeta'omegasigma; Esigma) protein complex . The sigma subunit is directly responsible for promoter recognition and contributes to localized DNA melting . Mutations in the beta subunit have profound effects on promoter melting by Esigma70 . The sigma54 subunit is a representative of an unrelated class of the sigma subunits . Here, we determined whether mutations in the beta subunit that affect late stages of promoter complex formation by Esigma70 also influence promoter complex formation by the enhancer-dependent Esigma54 . Analyses of in vitro defects in promoter complex formation and transcription initiation exhibited by mutant Esigma54 suggest that during promoter complex formation by Esigma54 and Esigma70 a common set of beta subunit sequences is used . Late stages of promoter complex formation and localized melting of promoter DNA by Esigma70 and Esigma54 thus proceed through a common pathway . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 21, 319(5), 1059 - 66 Secondary structure prediction for aligned RNA sequences; Hofacker IL et al.; Most functional RNA molecules have characteristic secondary structures that are highly conserved in evolution . Here we present a method for computing the consensus structure of a set aligned RNA sequences taking into account both thermodynamic stability and sequence covariation . Comparison with phylogenetic structures of rRNAs shows that a reliability of prediction of more than 80% is achieved for only five related sequences . As an application we show that the Early Noduline mRNA contains significant secondary structure that is supported by sequence covariation . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 21, 319(5), 1015 - 34 Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe); Cabello-Villegas J et al.; The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells . tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications . The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis . The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species . Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group . The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop . This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe) . The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation . Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation . The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 28, 320(1), 97 - 106 The projection domain of MAP4 suppresses the microtubule-bundling activity of the microtubule-binding domain; Iida J et al.; Microtubule-associated protein 4 (MAP4), a major MAP expressed in proliferating non-neuronal cells, consists of an N-terminal projection (PJ) domain and a C-terminal microtubule-binding (MTB) domain . The PJ domain of MAP4 is divided into three regions; the N-terminal acidic region (the Na-region), the multiple KDM-repeated sequence region (the KDM-region), and the b-region followed by the MTB domain . To investigate roles of the PJ domain, we prepared three truncated forms of human MAP4 with different PJ domain lengths; PJ1, PJ2 and MTB with deletion of about one-third, two-third and all of the PJ domain, respectively, and examined their effects on bundle formation of microtubules (MTs) . MTs polymerized by full length MAP4 were singly distributed as observed by both negative staining electron microscopy and dark field microscopy . MTs with PJ1 were also separated in solution but became pairs when pelleted by centrifugation . PJ2 formed planar two-dimensional bundles consisting of several MTs (the 2D-bundle) . MTB induced large bundles of many MTs, tightly packed without space in between (termed the 3D-bundle) . To study how the PJ domain decreases the bundle-forming activity of the MTB domain of MAP4, we made three additional deletion-mutants of MAP4, called Na-MTB, KDM-MTB and Na-PJ2 . Na-MTB and KDM-MTB, in which the KDM/b-region and both of Na- and b-regions were deleted respectively, were prepared by fusing the Na-region or KDM-region to MTB . Both of Na-MTB and KDM-MTB suppressed the 3D-bundle formation as effectively as PJ2 . MTs polymerized with Na-PJ2, the KDM-deletion mutant made by adding the Na-region to PJ2, were singular and did not become bundles . These results indicated that the PJ domain kept individual MTs separated by suppressing the bundle-forming ability of the MTB domain . The suppressive activity of the PJ domain was correlated with the length, but not the amino acid sequence, of the PJ . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 28, 320(1), 73 - 84 The isomerization of the UvrB-DNA preincision complex couples the UvrB and UvrC activities; Delagoutte E et al.; In Escherichia coli nucleotide excision repair, the UvrB-DNA preincision complex plays a key role, linking adduct recognition to incision . We previously showed that the efficiency of the incision is inversely related to the stability of the preincision complex . We postulated that an isomerization reaction converts {UvrB-DNA}, stable but incompetent for incision, into the {UvrB-DNA}' complex, unstable and competent for incision . Here, we identify two parameters, negative supercoiling and presence of a nick at the fifth phosphodiester bond 3' to the lesion, that accelerate the isomerization leading to an increasing incision efficiency . We also show that the {UvrB-DNA} complex is more resistant to a salt concentration increase than the {UvrB-DNA}' complex . Finally, we report that the {UvrB-DNA}' is recognized by UvrC . These data suggest that the isomerization reaction leads to an exposure of single-stranded DNA around the lesion . This newly exposed single-stranded DNA serves as a binding site and substrate for the UvrC endonuclease . We propose that the isomerization reaction is responsible for coupling UvrB and UvrC activities and that this reaction corresponds to the binding of ATP . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 28, 320(1), 39 - 53 Structural and functional studies of an intermediate on the pathway to operator binding by Escherichia coli MetJ; He YY et al.; We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E . coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence . The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs . The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site . In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains . Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time . The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity . In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif . Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers . (c) 2002 Elsevier Science Ltd. J Mol Biol, 2002 Jun 28, 320(1), 1 - 10 Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli; Jurado P et al.; Production of intracellular antibodies in Escherichia coli has been thought unlikely owing to an inability to form stable disulfide bonds in the cytoplasm, a necessary step in the folding of most immunoglobulin (Ig) domains . This work investigates whether E . coli strains carrying mutations in the major intracellular disulfide bond-reduction systems (i.e . the thioredoxin and the glutathione/glutaredoxin pathways) allow the oxidation and folding of single chain variable fragment (scFv) antibodies in the cytoplasm . The effect of the co-expression of disulfide bond chaperones in these cells was also examined . An scFv that recognizes the alternative sigma factor sigma(54) was used as a model to investigate disulfide bond formation and the folding of Ig domains in E . coli . The results demonstrate that functional intrabodies, with oxidized disulfide bonds in their Ig domains, are produced efficiently in E . coli cells carrying mutations in the glutathione oxidoreductase (gor) and the thioredoxin reductase (trxB) genes and co-expressing a signal-sequence-less derivative of the disulfide-bond isomerase DsbC ((Delta)ssDsbC) . We obtained evidence indicating that (Delta)ssDsbC acts as a chaperone promoting the correct folding and oxidation of scFvs . (c) 2002 Elsevier Science Ltd. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1998, 16(6), 406 - 10 {Cloning and sequencing of the gene coding the sexual stage antigen Pfs48/45 of Plasmodium falciparum}; Luo S et al.; AIM: To express the antigen Pfs48/45 in vitro and provide an antigen for the development of the transmission-blocking vaccine . METHODS: According to the published nucleotide sequence of Pfs48/45 of Plasmodium falciparum isolate NF54, a pair of oligonucleotides was designed and used as primers(P1, P2) . The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of P . falciparum isolate FCC1/HN has been amplified by using polymerase chain reaction (PCR) technique . The PCR product was purified and directly sequenced by the dideoxynucleotide terminator method with 5'-end primer P1 . At the same time, the purified PCR product was digested with BamHI and EcoRI and cloned into the plasmid pcDNA3, then the recombinant clones were transformed into E . coli strain TG1 . The recombinant plasmid pcDNA3-Pfs48/45 was screened and identified by PCR amplification and restriction analysis . RESULTS: 1 . The gene fragment Pfs48/45 was specifically amplified from the genomic DNA of Plasmodium falciparum isolate FCC1/HN; 2 . The sequence demonstrated that the that the 5'-end nucleotide and predicted aminoacid sequence of Pfs48/45 from FCC1/HN isolate was basically identical with that from NF54 isolate . We found that the sequence of the Pfs48/45 gene from the isolate FCC1/HN differs from the published sequence(isolate NF54) only at positions 307 and 372(T-->C) . The substitution of T-->C at the position of 372 generates a new restriction site Taq I . The PCR product digested by Taq I generates DNA fragment of 984 bp and 379 bp, suggesting that the PCR product is the gene encoding the transmission blocking antigen Pfs48/45; 3 . The gene fragment of Pfs48/45 was directly inserted into the BamHI and EcoRI site of plasmid pcDNA3 . CONCLUSION: The nucleotide sequence of Pfs48/45 of Plasmodium falciparum isolate FCC1/HN from south China was similar to that of isolate NF54 . The recombinant plasmid pcDNA3-Pfs48/45 was successfully constructed, providing a means to evaluate the role and biological function of this sexual-stage-specific protein of Pfs48/45. Nippon Rinsho, 2002 Jun, 60(6), 1131 - 7 {New drugs that prevent cytotoxicity of Shiga toxins}; Natori Y; Shiga toxin(Stx) produced by enterohemorrhagic E . coli is the virulence factor that causes not only enterohemorrhagic colitis but also fatal complications, such as hemolytic uremic syndrome . To prevent the complications, new strategies targeted to Stx have been tested, mostly using mimics of the trisaccharide structure of neutral lipid Gb3, the receptor for Stx . One group of such new drugs are agents that can bind to Stx in gastrointestinal tract and prevent its spread to extraintestinal sites, and the other group are water-soluble neutralizers that suppress Stx cytotoxicity in the circulation . Although most of these are now under the laboratory investigations, one of these drugs may hopefully be utilized clinically to prevent hemolytic uremic syndrome in future. Nippon Rinsho, 2002 Jun, 60(6), 1126 - 30 {Therapy for children with Shiga toxin-E . coli-associated hemolytic uremic syndrome}; Ito K et al.; Hemolytic uremic syndrome(HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute nephropathy . Clinical features and outcome of children with HUS initiated by infections with Shiga toxin(Stx)-producing strains of Escherichia coli(E . coli) infection are different from those of patients with the other forms of HUS or thrombotic thrombocytopenic purpura(TTP) . Childhood Stx-E . coli-associated HUS usually recovers spontaneously and dose not require specific treatments including plasma therapy . In contrast, a general consensus has been achieved that plasma exchange or infusion should always be tried in adult HUS/TTP to minimize the risk of death or long-term sequelae . In this paper, we briefly reviewed therapy for patients with Stx-E . coli-associated HUS. Nippon Rinsho, 2002 Jun, 60(6), 1121 - 5 {Treatment in initial stage of VTEC infection}; Igarashi T; Verocytotoxin producing Escherichia coli(VTEC) causes gastrointestinal infections worldwide . In at most 8% of the children in Japan who are infected with VTEC, hemolytic-uremic syndrome(HUS) develops soon after the onset of diarrhea . Treatment with antibiotics does not ameliorate VTEC infections, and in some studies from western countries, it has been associated with worse clinical outcomes . It was recently indicated in Japan that early administration of fosfomycin to the patients with VTEC infection can decrease the risk of HUS . Moreover, early administration of Synsorb-Pk with high affinity to verocytotoxin to the patients with gastrointestinal VTEC infection was demonstrated to decrease the incidence of very mild and mild HUS, but it did not decrease the risk of moderate and severe HUS . In the developing stage of HUS, intravenous administration of fluid and electrolytes should be determined cautiously to prevent hyponatremia and systemic congestion. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1243 - 5 Epub 2002 Jun 20. Crystallization and preliminary analysis of Escherichia coli YodA; David G et al.; The Escherichia coli protein YodA was overexpressed, purified and crystallized in several crystal forms . The function of this protein is not known, although it has been identified under conditions of bacterial stress . Three of the four crystal forms were obtained in the presence of divalent cations (zinc, nickel and cadmium), suggesting that YodA may be a metal-binding protein. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1240 - 2 Epub 2002 Jun 20. Expression, purification, crystallization and preliminary X-ray analysis of a DNA-binding protein from Methanococcus jannaschii; Wang G et al.; A small DNA-binding protein of 87 amino-acid residues from the hyperthermophilic archaeon Methanococcus jannaschii (Mja10b) was cloned and overexpressed in Escherichia coli . The protein was crystallized and the crystals belong to the space group P6(1)22/P6(5)22, with unit-cell parameters a = b = 50.85, c = 124.02 A, alpha = beta = 90, gamma = 120 degrees . The crystals diffracted to a maximum resolution of 2.2 A at 100 K using Cu Kalpha radiation . The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 2.4 A(3) Da(-1) and a solvent content of 49% by volume . A full set of X-ray diffraction data was collected to 2.2 A from the native crystal. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1224 - 5 Epub 2002 Jun 20. Preliminary crystallographic analysis of the cysteine desulfurase IscS from Escherichia coli; Urbina HD et al.; IscS is a widely distributed cysteine desulfurase that catalyzes the pyridoxal phosphate dependent beta-elimination of sulfur from L-cysteine and plays a central role in the delivery of sulfur to a variety of metabolic pathways . Crystals of Escherichia coli IscS have been obtained by the hanging-drop vapor-diffusion method using polyethylene glycol (PEG) as a precipitant . Initial seed crystals were obtained using PEG 6000 and sodium acetate, and diffraction-quality crystals were grown using a mixture of PEG 2000 and PEG 10 000 in the presence of sodium citrate . A complete native X-ray diffraction data set was collected from a single crystal at 103 K to a resolution of 2.1 A . The crystals belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 73.7086, b = 101.9741, c = 108.617 A (alpha = beta = gamma = 90 degrees ) . Analysis of the Matthews equation and self-rotation function suggest two molecules per asymmetric unit, consistent with the presence of a single dimeric molecule. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1217 - 9 Epub 2002 Jun 20. Crystallization and preliminary X-ray crystallographic analysis of a yedU gene product from Escherichia coli; Kim OG et al.; A yedU gene product with a molecular mass of 31 kDa is a hypothetical protein with no known function . The protein was purified and crystallized at 296 K . X-ray diffraction data have been collected to 2.3 A using synchrotron radiation . The crystals belong to the primitive orthorhombic system, with unit-cell parameters a = 50.56, b = 63.45, c = 168.02 A . The asymmetric unit contains two monomers of the protein, with a corresponding V(M) of 2.25 A(3) Da(-1) and a solvent content of 44.84%. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1214 - 6 Epub 2002 Jun 20. Crystallization and preliminary crystallographic studies of human TGF-beta type II receptor ligand-binding domain; Boesen CC et al.; Three constructs (residues 15-136, 22-136 and 27-136) of the truncated extracellular domain of human transforming growth factor beta type II receptor (TBRII) were overexpressed in Escherichia coli . The constructs are referred to as TBRII(15-136), TBRII(22-136) and TBRII(27-136) . The refolded receptors were purified using a combination of ion-exchange and size-exclusion chromatography . The purified receptors have an apparent molecular weight of 14 kDa as judged by size-exclusion chromatography . In the crystallization trials, TBRII(15-136) and TBRII(22-136) formed mostly crystal-like spheres but failed to produce data-quality crystals . TBRII(27-136) yielded large single crystals from hanging drops using the vapor-diffusion procedure with PEG 2000 or 4000 at pH 5.0 . The crystals diffracted to 1.05 A {using the X9B beamline operated at lambda = 1.0092 A of the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory} and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.5, b = 40.7, c = 76.2 A . There was one molecule in the asymmetric unit, which corresponds to a solvent content of 42.1%. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1207 - 10 Epub 2002 Jun 20. Crystallization and preliminary X-ray analysis of the Escherichia coli adaptor protein ClpS, free and in complex with the N-terminal domain of ClpA; Zeth K et al.; Protein degradation in Escherichia coli is accomplished by a handful of large oligomeric complexes . In most cases, these proteolytic machines are comprised of a chaperone (e.g . ClpA) that is required to prepare the substrate for degradation by the peptidase (e.g . ClpP) . Recently, it was shown that the substrate recognition of the chaperone ClpA could be modified by the adaptor protein ClpS . To investigate the structural implications of this change in substrate specificity, ClpS was crystallized alone and in complex with the N-terminal domain of ClpA (ClpA(N)) . Crystals of ClpS diffract to 2.9 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 82.63, b = 145.67, c = 152.31 A . Two different crystal forms of the ClpA(N)-ClpS complex were characterized . Crystal form I (CFI) belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 91.63, b = 112.47, c = 38.47 A; data to 1.92 A resolution were collected . Crystals of form II (CFII) belong to space group P4(1/3)2(1)2, with unit-cell parameters a = b = 93.57, c = 78.77 A, and diffract to 1.85 A resolution . Data sets collected from heavy-atom derivatives of CFI indicated the incorporation of Pt and Hg atoms . Structure solution using MIR and MAD methods is currently under way. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1182 - 92 Epub 2002 Jun 20. Structure of the C-terminally truncated human ProMMP9, a gelatin-binding matrix metalloproteinase; Elkins PA et al.; The X-ray crystal structure of the proform of human matrix metalloproteinase MMP9 has been solved to 2.5 A resolution . The construct includes the prodomain, the catalytic domain and three FnII (fibronectin type II) domains . The prodomain is inserted into the active-site cleft, blocking access to the catalytic zinc . Comparison with the crystal structure of the most closely related MMP, MMP2, indicates that the conformations of residues in the active-site cleft and in the cysteine-switch peptide of the prodomain are highly conserved and that design of MMP9-specific inhibitors will be challenging . In common with MMP2, the MMP9 S1' inhibitor-binding pocket is large compared with that of other MMPs . One small point of difference in the S1' binding pockets of MMP9 and MMP2 may provide an opportunity to explore the design of specific inhibitors . The side chain of Arg424 in MMP9 is angled slightly away from the S1' pocket when compared with the corresponding residue in MMP2, Thr424 . The secondary structure of the FnII domains is conserved between the two closely related MMPs, although the second FnII domain makes no contact with the catalytic domain in MMP9, while the same domain in MMP2 has a substantial area of interaction with the catalytic domain. Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1111 - 7 Epub 2002 Jun 20. Thiol-reactive lanthanide chelates for phasing protein X-ray diffraction data; Purdy MD et al.; Lanthanides can contribute a large anomalous component to X-ray scattering when present and ordered in a target crystal . This large anomalous signal is a useful source of phase information in X-ray crystallographic studies of biological macromolecules . Thiol-reactive lanthanide chelates were tested as a means of incorporation of lanthanides into protein crystals . Two compounds, each capable of being loaded with a lanthanide of choice, were synthesized: diethylenetriaminepentaacetic 3-(2-pyridyldithio)propionyl hydrazide (DTPA-PDPH) and 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic 3-(2-pyridyldithio)propionyl hydrazide (DOTA-PDPH) . A cysteine mutant of the 34 kDa phosphate-binding protein (PBP-A197C) from Escherichia coli was used as a test case . PBP-A197C was labeled with DTPA-PDPH loaded with dysprosium . Characteristics of DTPA-PDPH enabled spectroscopic monitoring of the labeling reaction . Complete labeling of PBP-A197C was confirmed by mass spectrometry and SDS-PAGE analysis . Labeled PBP-A197C (PBP-A197C-DTPA-Dy) crystallized identically to unlabeled protein . X-ray diffraction data were collected from PBP-A197C-DTPA-Dy crystals in-house with a Cu Kalpha rotating-anode source and with a tuneable synchrotron source (ALS 5.0.2) . Synchrotron data were collected at energies corresponding to the Dy L(III) edge f" peak and a high-energy remote . Each data set was treated as an independent SAD experiment . A large anomalous signal was present in the data collected in-house and at the synchrotron . The Dy site was easily located in anomalous difference Patterson maps calculated from each of the data sets . In each case, SAD phasing resulted in high-quality electron-density maps, as evidenced by the success of automated model building . The generality of the method was analyzed with several other test proteins . Labeling of some of these proteins with thiol-reactive lanthanide chelates was deleterious to protein solubility or crystallization . In two of the cases the lanthanide chelate was disordered in the crystals . These results suggest that this method may not be well suited for high-throughput crystallography . However, for difficult cases requiring a large anomalous signal, thiol-reactive lanthanide chelates may prove to be a valuable tool. Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8597 - 601 Epub 2002 Jun 19. Employing Escherichia coli to functionally express, purify, and characterize a human transporter; Quick M et al.; Large-scale purification of recombinant human membrane proteins represents a rate-limiting step toward the understanding of their role in health and disease . There are only four mammalian membrane proteins of known structure, and these were isolated from natural sources (see In addition, genetic diseases of membrane proteins are frequently caused by trafficking defects, and it is enigmatic whether these mutants are functional . Here, we report the employment of Escherichia coli for the functional expression, purification, and reconstitution of a human membrane protein, the human Na+/glucose cotransporter (hSGLT1) . The use of an E . coli mutant defective in the outer membrane protease OmpT, incubation temperatures below 20 degrees C, and transcriptional regulation from the lac promoter/operator are crucial to reduce proteolytic degradation . Purification of a recombinant hSGLT1 through affinity chromatography yields about 1 mg of purified recombinant hSGLT1 per 3 liters of cultured bacterial cells . Kinetic analysis of hSGLT1 in proteoliposomes reveals that a purified recombinant transporter, which is missorted in eukaryotic cells, retains full catalytic activity . These results indicate the power of bacteria to manufacture and isolate human membrane proteins implicated in genetic diseases. J Biol Chem, 2002 Sep 6, 277(36), 32616 - 23 Epub 2002 Jun 19. Heparin/Heparan sulfate domains in binding and signaling of fibroblast growth factor 8b; Loo BM et al.; The role of heparin and heparan sulfate in the binding and signaling of fibroblast growth factors (FGFs) has been subject to intense investigation, but the studies have largely been confined to two species (FGF1 and FGF2) of the family with approximately 20 members . We have investigated the structural requirements for heparin/heparan sulfate in binding and activation of FGF8 (splice variant b) . We present evidence that the minimal FGF8b-binding saccharide domain encompasses 5-7 monosaccharide units . The N-, 2-O-, and 6-O-sulfate substituents of heparin/heparan sulfate (HS) are all involved in the interaction, preferentially in the form of trisulfated IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3)) disaccharide constituents . These structural characteristics resemble those described earlier for FGF1 . By contrast, the saccharide structures required for the biological activity of FGF8b differed significantly from those characteristic for FGF1 and FGF2 . Experiments with cells lacking active HS indicated that extended >/=14-mer heparin domains were needed to enhance cell proliferation and Erk phosphorylation by FGF8b, whereas in cells stimulated with FGF1 or FGF2 the corresponding responses were achieved by much shorter, 6-8-mer, oligosaccharides . Furthermore, still longer domains were needed to activate FGF8b in cells with "non-optimal" FGF receptor expression . Collectively, our data suggest that the heparin/HS structures enhancing the biological activity of FGFs were influenced by the FGF species involved as well as by the cellular composition of FGF receptors. J Biol Chem, 2002 Aug 30, 277(35), 31373 - 80 Epub 2002 Jun 19. The cryptic adenine deaminase gene of Escherichia coli . Silencing by the nucleoid-associated DNA-binding protein, H-NS, and activation by insertion elements; Petersen C et al.; In Escherichia coli there are two pathways for conversion of adenine into guanine nucleotides, both involving the intermediary formation of IMP . The major pathway involves conversion of adenine into hypoxanthine in three steps via adenosine and inosine, with subsequent phosphoribosylation of hypoxanthine to IMP . The minor pathway involves formation of ATP, which is converted via the histidine pathway to the purine intermediate 5-amino-4-imidazolecarboxamide ribonucleotide and, subsequently, to IMP . Here we describe E . coli mutants, in which a third pathway for conversion of adenine to IMP has been activated . This pathway was shown to involve direct deamination of adenine to hypoxanthine by a manganese-dependent adenine deaminase encoded by a cryptic gene, yicP, which we propose be renamed ade . Insertion elements, located from -145 to +13 bp relative to the transcription start site, activated the ade gene as did unlinked mutations in the hns gene, encoding the histone-like protein H-NS . Gene fusion analysis indicated that ade transcription is repressed more than 10-fold by H-NS and that a region of 231 bp including the ade promoter is sufficient for this regulation . The activating insertion elements essentially eliminated the H-NS-mediated silencing, and stimulated ade gene expression 2-3-fold independently of the H-NS protein. J Biol Chem, 2002 Sep 6, 277(36), 32714 - 21 Epub 2002 Jun 20. The linker region plays an important role in the interdomain communication of the response regulator OmpR; Mattison K et al.; OmpR is the response regulator of a two-component regulatory system that controls the expression of the porin genes ompF and ompC in Escherichia coli . This regulator consists of two domains joined by a flexible linker region . The amino-terminal domain is phosphorylated by the sensor kinase EnvZ, and the carboxyl-terminal domain binds DNA via a winged helix-turn-helix motif . In vitro studies have shown that amino-terminal phosphorylation enhances the DNA binding affinity of OmpR and, conversely, that DNA binding by the carboxyl terminus increases OmpR phosphorylation . In the present work, we demonstrate that the linker region contributes to this communication between the two domains of OmpR . Changing the specific amino acid composition of the linker alters OmpR function, as does increasing or decreasing its length . Three linker mutants give rise to an OmpF(+) OmpC(-) phenotype, but the defects are not due to a shared molecular mechanism . Currently, functional homology between response regulators is predicted based on similarities in the amino and carboxyl-terminal domains . The results presented here indicate that linker length and composition should also be considered . Furthermore, classification of response regulators in the same subfamily does not necessarily imply that they share a common response mechanism. Virus Res, 2002 Jun, 86(1-2), 133 - 41 Expression of Cardamom mosaic virus coat protein in Escherichia coli and its assembly into filamentous aggregates; Jacob T et al.; Cardamom mosaic virus (CdMV), a member of the genus Macluravirus of Potyviridae, causes a mosaic disease in cardamom . A polyclonal antiserum was raised against the purified virus and IgG was prepared . Electron microscopic studies on the purified virus showed flexuous filamentous particles of approximately 800 nm in length, typical of members of Potyviridae . The coat protein (CP) encoding sequence of the virus was expressed in Escherichia coli and the protein purified by affinity chromatography under denaturing conditions . The viral nature of the expressed CP was confirmed by positive reaction with anti CdMV IgG in a Western blot . The expressed CP aggregated irreversibly upon renaturation at concentrations above 0.07 mg/ml . The expression of the CP led to the formation of filamentous aggregates in E . coli as observed by immuno-gold electron microscopy . The filamentous aggregates were of 100-150 nm in length . Immuno-capture RT-PCR confirmed the absence of coat protein mRNA in the filamentous aggregates . Deletion mutations, which were expected to inhibit virus assembly, were introduced in the core region of the coat protein . However, these mutations did not improve the solubility of the CP in non-denaturing buffers. FEMS Microbiol Lett, 2002 Jun 4, 211(2), 265 - 70 Escherichia coli genes involved in resistance to pyrazinoic acid, the active component of the tuberculosis drug pyrazinamide; Schaller A et al.; The natural resistance of Escherichia coli to pyrazinoic acid (POA), the active derivative of pyrazinamide, was investigated . The TolC mutant was found to be more susceptible to POA and other weak acids than the wild