Escherichia coli

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-type strain . Mutation in EmrB but not AcrB efflux protein slightly increased POA susceptibility . Two transposon mutants with increased susceptibility to POA were found to harbor mutations in acnA encoding aconitase-1 and ygiY encoding a putative two-component sensor protein . Complementation of the AcnA and YgiY mutants conferred resistance to POA, whereas the complemented TolC mutant became resistant to POA and other weak acids.

FEMS Microbiol Lett, 2002 Jun 4, 211(2), 161 - 7
Temperature-dependent translation of leaderless and canonical mRNAs in Escherichia coli; Grill S et al.; Leaderless mRNAs beginning with a 5'-terminal start codon occur in all biological systems . In this work, we have studied the comparative translational efficiency of leaderless and leadered mRNAs as a function of temperature by in vitro translation competition assays with Escherichia coli extracts . At low temperature (25 degrees C) leaderless mRNAs were found to be translated comparatively better than mRNAs containing an internal canonical ribosome binding site, whereas at high temperature (42 degrees C) the translational efficiency of canonical mRNAs is by far superior to that of leaderless mRNA . The inverse correlation between temperature and translational efficiency characteristic for the two mRNA classes was attributed to structural features of the mRNA(s) and to the reduced stability of the translation initiation complex formed at a 5'-terminal start codon at elevated temperature.

FEMS Microbiol Lett, 2002 Jun 4, 211(2), 133 - 41
Cloning, expression, and functional characterization of the Mycobacterium tuberculosis secA gene; Owens MU et al.; To better understand the protein secretion mechanisms involved in the growth and pathogenesis of Mycobacterium tuberculosis, we examined the secA gene from M . tuberculosis (tbsecA; cosmid sequence accession No . z95121.gb_ba) . We generated plasmids containing the full-length tbsecA gene or a fusion containing the 5' sequence from the M . tuberculosis secA gene and the remainder from the Escherichia coli secA gene and evaluated the ability of each construct to complement the defective SecA protein in E . coli MM52ts when grown at the non-permissive temperature . The full-length tbsecA gene was unable to compensate for the temperature-sensitive defect, whereas E . coli MM52ts that has been transformed with plasmid pMF8TB226 containing a chimeric secA gene was able to grow at 42 degrees C . This work confirms that the topography of SecA and its ATP binding sites are highly conserved, whereas its membrane insertion domains are species specific.

FEMS Microbiol Lett, 2002 Jun 18, 212(1), 55 - 8
Role of fibronectin in curli-mediated internalization; Gophna U et al.; Curli fibers of Escherichia coli mediate internalization of bacteria by eukaryotic cells . As curli fibers bind fibronectin with high affinity, the role of fibronectin in the uptake process was studied . The experiments presented here support the involvement of fibronectin in internalization of bacteria . Furthermore, a peptide containing the RGD motif, responsible for interaction of fibronectin with cellular integrins, can strongly inhibit curli-mediated internalization . The ability of curli fibers to bind fibronectin can therefore be linked to virulence.

Mol Biochem Parasitol, 2002 Jun, 122(1), 45 - 54
Amino terminus of Plasmodium falciparum acidic basic repeat antigen interacts with the erythrocyte membrane through band 3 protein; Kushwaha A et al.; The acidic basic repeat antigen (ABRA) of Plasmodium falciparum is localised in the parasitophorous vacuole, and associates with the merozoite surface at the time of schizont rupture . By virtue of its protease-like activity, it is implicated in the process of merozoite invasion and schizont rupture, and therefore, possibly interacts with erythrocyte membrane proteins to execute its function during these events . In this study, using Escherichia coli expressed recombinant fragments of ABRA, we have demonstrated that ABRA interacts with red blood cells through its N-terminus . Out of the four human erythrocyte proteins tested, namely, band 3, glycophorin A and B and spectrin, ABRA showed dose-dependent and saturable binding with the band 3 protein . This binding was lost on chymotrypsin treatment of erythrocytes or their membrane extract . Studies with the deletion constructs of the N-terminus revealed that the binding domain lies in the cysteine-rich N-proximal region of ABRA . In addition to the recombinant fragments, native ABRA derived from the P . falciparum-infected erythrocytes also showed binding to band 3 protein . Sequencing of the cysteine-rich 528 bp region, amplified from fifteen field isolates of P . falciparum, showed that not only the five cysteines of mature ABRA but also the whole sequence is fully conserved, even at the nucleotide level . This sequence conservation of the N-terminus and its role in RBC binding suggests that this region may be crucial for any putative function of ABRA, therefore emphasising its importance as a vaccine/drug target.

J Org Chem, 2002 Jun 28, 67(13), 4590 - 4
Biosynthesis of terpenes . Preparation of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate, an intermediate of the deoxyxylulose phosphate pathway; Amslinger S et al.; (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (E-6) was synthesized in six reaction steps from hydroxyacetone (9) and (ethoxycarbonylmethenyl)-triphenylphosphorane (11) with an overall yield of 38% . The compound was shown to be identical with the product of IspG protein, which serves as an intermediate in the nonmevalonate terpene biosynthetic pathway.

Res Vet Sci, 2002 Jun, 72(3), 229 - 34
Anti-enteropathogenic Escherichia coli immunoglobulin Y isolated from eggs laid by immunised Leghorn chickens; Amaral JA et al.; IgY, the egg yolk immunoglobulin, equivalent to the IgG from mammals, has been used in veterinary practice for passive immunisation against bacterial or viral infectious diseases . Enteropathogenic Escherichia coli (EPEC) is the main etiological agent of infantile diarrhoea in Brazil and other developing countries . Our aims were to isolate immunoglobulin IgY from egg yolk laid by EPEC -immunised Leghorn chickens and to study its reactivity to the antigens from this pathogen, including some virulence factors . Leghorn chickens were immunised with a bacterial suspension intramuscularly (three hens) or intravenously (three hens) or with PBS (two hens) . Eggs were collected over a period of 17 weeks . IgY isolation procedures were carried out by salt precipitation (ammonium sulphate, in solid form) followed by centrifugations and dialysis . Final preparations were submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS - PAGE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting . All immunised animals developed good levels of antibodies reactive to whole bacteria or lipopolysaccharide (LPS), in contrast to the control ones . Immunoblottings allowed the recognition of several antigenic fractions of bacterial antigens, some of which had a molecular weight compatible with bacterial virulence factors, confirming the efficacy of the immunisation and the adequacy of the method .

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(3), 312 - 315
In Vitro Amidating Processing of Products Expressed by Gene Engineering; Yang YH et al.; To set up an in vitro amidating system, a recombinant human calcitonin with a glycine at C termial (mhCT-Gly) was used as the amidating substrate of recombinant rat peptidylglycine alpha-amidating monooxygenase (rPAM) . First, the mhCT-Gly gene was synthesized and cloned into a fusion expression vector to get an expression plasmid pGEXCT . The GST-fused mhCT-Gly was highly expressed in E.coli BL21(DE3) harboring the pGEXCT, and was purified rapidly by affinity chromatography . Second, using the method of ultrafiltration, the rPAM was prepared from the supernatant of cultured transfectant CHO cells which express rPAM stably . Finally, the in vitro amidating experiments were carried out using GST-mhCT-Gly as substrate and the prepared rPAM . The results of dot blot with the specific antibody and of mass spectrum assay indicated that amidating product GST-hCT-NH(2) could be easily detected . This study provides a useful method for the amidation of recombinant products in vitro.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(3), 243 - 247
A Structure-function Analysis of Human GDNF; Chen ZY et al.; The glial cell line-derived neurotrophic factor(GDNF) plays a very important role in the regeneration of the nervous system . Based on the results of the X-ray structure analysis of rat GDNF, human GDNF gene was modified with deletion and insertion mutagenesis by using PCR methods . The various mutants were all highly expressed in E.coli . The recombinant proteins were purified and their survival-promoting activities were determined by using cultures of the spinal cord neurons of embryonic mouse . The results showed that the "cystine knot motif" was critical for the maintenance of GDNF structure the alpha-helix, finger 1 and finger 2 region were critical for GDNF neurotrophic activity and the N-terminus of human GDNF was not essential for its biological functions.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(3), 235 - 238
Purification of Recombinant GM-CSF/IL-3 Fusion Protein; Zhang Y et al.; As a new artificial haemopoietic growth factor, GM-CSF/IL-3 fusion protein appears very promi-sing to be developed as a drug in the treatment of many diseases including cancer . The purification process of the recombinant GM-CSF/IL-3 fusion protein was studied . Similar to majority of the eukaryotic proteins, GM-CSF/IL-3 was expressed as inclusion bodies in E.coli . A series of purification steps, including cell breakage, inclusion body washing, inclusion body solubilization, protein renaturation and ion-exchange chromatography, have been set up to purify the recombinant fusion protein in an active form . Experimental results showed that the inclusion body solubility increased with increasing urea concentration . During the protein renaturation process, dialysis method was chosen to remove the denaturant urea . Stepwise decrease of urea concentration in the buffer could effectively improve the protein renaturation efficiency by reducing protein aggregation . At the same time, reduced and oxidized glutathionine were added to optimize the correct disulfide bonds formation . The recombinant protein was then purified by DEAE ion-exchange chromatography . The final protein recovery was over 30% . SDS-PAGE and reverse HPLC analysis revealed over 95% purity of the final purified recombinant protein . Therefore, a laboratory-scale purification procedure of the recombinant GM-CSF/IL-3 fusion protein was basically well established.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(4), 417 - 420
Deinococcus radiodurans CatB Gene Cloning and Expression in Escherichia coli; Zheng H et al.; A 1 611 bp length complete coding sequence of the catalase (Cat) was obtained through bioimformatical analysis of the database of D.radiodurans genome, and then was amplified from D . radiodurans genomic DNA by polymerase chain reaction . The amplified gene was cloned into pKK223-3 vector and transformed into E.coli UM2, a Cat-deficient mutant . Staining of non-denaturing polyacrylamide gels for Cat activity demonstrated that pKK223-3 insert encoded a Cat that co-migrated with the CatB found in D.radiodurans cell lysates . Expression of CatB gene from D.radiodurans in E.coli UM2 restored the resistance to H(2)O(2) at low concentrations.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(4), 401 - 405
Detection of Lis1 Gene Frame Shift Mutation in Human Hepatocarcinoma; Xia SL et al.; GST fusion protein expression system combined with protein truncation test(PTT) protocol was used to detect gene frame shift mutation . The RT-PCR products of Lis1 genes from hepatocarcinoma samples were respectively cloned into a GST fusion protein expression vector pGEX-1, then expressed in E.coli . The results showed a truncated 33 kD fusion protein in SDS-PAGE, although the full-translated product of Lis1 gene should be of 71 kD . Sequencing revealed insertion of an A residue, causing the premature termination of translation, between the 163th and 164th nucleotide of Lis1 gene . This improved PTT assay was proved to be a fast and effective way in detecting gene frame shift mutation.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(4), 333 - 336
Cloning, Expression and Tumor Suppression of Human Endostatin; He ZY et al.; Human endostatin cDNA was cloned from total RNA of normal Chinese liver cell line L02 by RT-PCR . Endostatin DNA sequence encoded 184 amino acid residues . Five base pairs and 3 amino acid residues are different from that reported, it may be due to interspecies difference . The endostatin cDNA was inserted into the pET-28a(+) containing T7 promoter . The recombinant plasmid was transformed the E.coli BL21(DE3) . Recombinant human endostatin was highly expressed as inclusion body when the expression strain BL-ENDO was induced with 1 mmol/L IPTG . Result of SDS-PAGE analysis revealed that recombinant human endostatin was accounted for up to 25% of soluble protein in E.coli . Purified and refolded recombinant human endostatin was active in inhibiting tumor growth and metastasis.

J Gen Virol, 2002 Jul, 83(Pt 7), 1601 - 12
Activity and intracellular localization of the human cytomegalovirus protein pp71; Marshall KR et al.; The human cytomegalovirus (HCMV) tegument phosphoprotein pp71 activates viral immediate early (IE) transcription and thus has a role in initiating lytic infection . Protein pp71 stimulates expression from a range of promoters in a sequence-independent manner, and in this respect behaves similarly to the herpes simplex virus type 1 (HSV-1) IE protein ICP0 . The intracellular localization of pp71 was investigated after its expression from transfected plasmids or from HSV-1 mutants constructed to produce pp71 transiently . The protein colocalized with the cell promyelocytic leukaemia (PML) protein at nuclear domain 10 (ND10) structures but, unlike ICP0, pp71 did not induce disruption of ND10 . The activity of pp71 in mouse sensory neurons in vivo was investigated after co-inoculation of animals with pairs of HSV-1 mutants, one expressing pp71 and the second containing the E . coli lacZ gene controlled by various promoters . In this system, pp71 stimulated beta-galactosidase expression from a range of viral IE promoters when mice were analysed at 4 days postinoculation . At later times, expression of pp71 resulted in a reduction in numbers of neurons containing beta-galactosidase, indicating cytotoxicity or promoter shutoff . The HSV-1 latency-active promoter was not responsive to pp71, demonstrating specificity in the activity of the protein . Pp71 was as active in mice lacking both copies of the PML gene (PML-/-) as in control animals, and in PML-/- fibroblasts pp71 stimulated gene expression as effectively as in other cell types . Therefore, neither the PML protein nor the normal ND10 structure is necessary for pp71 to stimulate gene expression.

Ann N Y Acad Sci, 2002 May, 957, 295 - 301
Oxidative stress and inflammatory reaction modulation by white wine; Bertelli AA et al.; Wine and olive oil, essential components of the Mediterranean diet, are considered important factors for a healthy life style . Tyrosol (T) and caffeic acid (CA) are found in both extra virgin olive oil and in white wine . Three white wines from the northeast Italy and four white wines from Germany were analyzed for their content of T and CA . These compounds were tested for their antioxidant activity and their capacity to modulate three different cytokines: IL-1 beta, IL-6, and TNF-alpha, which are currently considered to be the major cytokines influencing the acute phase of the inflammatory response . Furthermore, the antioxidant activity of T and CA was analyzed by monitoring the oxidation of a redox-sensitive probe by using laser scanning confocal microscopy . T and CA, applied at nanomolar range, were found to significantly reduce the generation of oxidants induced by azobis-amidinopropanedihydrochloride . Peripheral blood mononuclear cells (PBMC) from healthy volunteers were incubated at 37 degrees C for 12 hours with 100 ng LPS (E . coli and P . maltofilia) . Increasing doses of T and CA (150 nM to 300 microM) were added and cell-associated IL-1 beta and TNF-alpha were determined by immunoreactive tests after three freeze-thaw cycles . IL-6 release was also determined in cell surnatants . LPS-stimulated PBMC showed a significant increase in cytokine release, while T and CA, used at nanomolar concentrations, were able to modulate their expression . Taken together, these results suggest a remarkable effect of white wine non-alcoholic compounds on oxidative stress and inflammatory reaction.

Biotechnol Appl Biochem, 2002 Jun 1, 35(3), 199 - 203
Expression, purification and characterization of a recombinant levan fructotransferase; Yang SJ et al.; A 1.6 kb DNA fragment including the lftM gene, encoding a levan fructotransferase (LFTase) of Microbacterium sp . AL-210, was subcloned into a high-expression vector, pET-29b, and the recombinant enzyme was overexpressed in Escherichia coli . Most of the LFTase activity was detected in the cytoplasmic fraction after induction with isopropyl beta-d-thiogalactoside . The recombinant enzyme with a tag of six histidine residues at the C-terminus was purified 132-fold by affinity and gel-filtration chromatography . Analysis of the N-terminal amino acid sequence revealed that the first 42 amino acids were post-translationally cleaved off . The molecular mass of the purified LftM was approx . 54 kDa as determined by SDS/PAGE, which corresponded well with a predicted size from the nucleotide sequence of the lftM gene lacking 42 amino acids . The enzyme converted levan into difructose anhydride IV (DFA IV) with a K(m) of 2 mg/ml and a V(max) of 40.6 &mgr;mol/min at pH 7.0 and 40 degrees C . The pH-dependence study of the enzyme for DFA IV production showed that LftM had a broad pH optimum (5.0-8.0) and the pK(a) values obtained were 4.5 and 8.9 at 40 degrees C . These results suggest that the acidic residues at the active site may play important roles for the catalytic mechanism of the LFTase.

Biochem Biophys Res Commun, 2002 Jun 28, 294(5), 949 - 55
The P1' specificity of tobacco etch virus protease; Kapust RB et al.; Affinity tags have become indispensable tools for protein expression and purification . Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein . The stringent sequence specificity of the tobacco etch virus (TEV) protease has made it a useful reagent for this purpose . However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency . Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity . To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position . The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro . Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position . The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.

Dev Biol, 2002 Jul 1, 247(1), 47 - 61
Exploring myriapod segmentation: the expression patterns of even-skipped, engrailed, and wingless in a centipede; Hughes CL et al.; Segment formation is critical to arthropod development, yet there is still relatively little known about this process in most arthropods . Here, we present the expression patterns of the genes even-skipped (eve), engrailed, and wingless in a centipede, Lithobius atkinsoni . Despite some differences when compared with the patterns in insects and crustaceans, the expression of these genes in the centipede suggests that their basic roles are conserved across the mandibulate arthropods . For example, unlike the seven pair-rule stripes of eve expression in the Drosophila embryonic germband, the centipede eve gene is expressed strongly in the posterior of the embryo, and in only a few stripes between newly formed segments . Nonetheless, this pattern likely reflects a conserved role for eve in the process of segment formation, within the different context of a short-germband mode of embryonic development . In the centipede, the genes wingless and engrailed are expressed in stripes along the middle and posterior of each segment, respectively, similar to their expression in Drosophila . The adjacent expression of the engrailed and wingless stripes suggests that the regulatory relationship between the two genes may be conserved in the centipede, and thus this pathway may be a fundamental mechanism of segmental development in most arthropods . (c) 2002 Elsevier Science (USA).

Biotechniques, 2002 Jun, 32(6), 1266 - 8, 1270
One-step, highly efficient site-directed mutagenesis by toxic protein selection; Xu W et al.; A fast and efficient site-directed mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E . coli F plasmid . Once the target gene is cloned into pTPS19, desired mutations can be introduced with two primers . The first contains the desired mutation, and the second is designed to create a +1 frame shift in the ccdB gene to inactivate the CcdB protein . The mutants can be directly selected on LB plates containing IPTG, through which the toxic CcdB protein is induced, thereby eliminating cells carrying wild-type parental plasmids . Based on stringent selection through the toxic CcdB protein, mutagenesis efficiency of 90%-100% was reached even after one round of transformation.

Biopolymers, 2002, 67(2), 121 - 8
Dynamics of supercoiled and linear pTZ18U plasmids observed with a long-lifetime metal-ligand complex; Kang JS et al.; The metal-ligand complex, {Ru(bpy)2(dppz)}2+ (bpy = 2,2'-bipyridine, dppz = dipyrido{3,2-a:2',3'-c}phenazine) (Ru-BD), was used as a spectroscopic probe for studying nucleic acid dynamics . The Ru-BD complex displays a long lifetime of over 100 ns and a molecular light switch property upon DNA binding due to shielding of its dppz ligand from water . To further show the usefulness of this luminophore (Ru-BD) for probing DNA dynamics, we examined its intensity and anisotropy decays when intercalated into supercoiled and linear pTZ18U plasmids using frequency-domain fluorometry with a light-emitting diode (LED) as the modulated light source . Compared to the supercoiled plasmids with an average intensity decay time of 120.8 ns at 25 degrees C, we obtained somewhat longer lifetimes for the linear plasmids ((tau) = 141.4 ns at 25 degrees C), suggesting a more efficient shielding from water by the linear plasmids . The anisotropy decay data also showed longer rotational correlation times for the linear plasmids (495 and 35 ns at 25 degrees C) as compared to the supercoiled plasmids (412 and 27 ns at 25 degrees C) . The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the plasmids, respectively . The anisotropy values were quite similar, although the values of the supercoiled plasmids were slightly higher in both the steady-state and anisotropy decay measurements . These results indicate that Ru-BD can be applied in the study of both bending and torsional dynamics of nucleic acids.

Mol Genet Genomics, 2002 May, 267(3), 380 - 90 Epub 2002 May 09.
Cloning and characterization of a cell cycle-regulated gene encoding topoisomerase I from Nicotiana tabacum that is inducible by light, low temperature and abscisic acid; Mudgil Y et al.; We have cloned a full-length 2874-bp cDNA coding for tobacco topoisomerase I, with an ORF of 2559 bp encoding a protein of 852 amino acids with a calculated molecular mass of 95 kDa and an estimated pI of 9.51 . The deduced amino acid sequence shows homology to other eukaryotic topoisomerases I . Tobacco topoisomerase I was over-expressed in Escherichia coli, and the purified recombinant protein was found to relax both positively and negatively super-coiled DNA in the absence of the divalent cation Mg(2+)and ATP . These characteristic features indicate that the tobacco enzyme is a type I topoisomerase . The recombinant protein could be phosphorylated at (a) threonine residue(s) by protein kinase C . However, phosphorylation did not cause any change in its enzymatic activity . The genomic organization of the topoisomerase I gene revealed the presence of 8 exons and 7 introns in the region corresponding to the ORF and one intron in the 3' UTR region . Transcript analysis using RT-PCR showed basal constitutive expression in all organs examined, and the gene was expressed at all stages of the cell cycle--but the level of expression increased during the G1-S phase . The transcript level also increased following exposure to light, low-temperature stress and abscisic acid, a stress hormone.

Mol Genet Genomics, 2002 May, 267(3), 370 - 9 Epub 2002 Apr 26.
Efficient expression of the alpha-haemolysin determinant in the uropathogenic Escherichia coli strain 536 requires the leuX-encoded tRNA(5)(Leu); Dobrindt U et al.; The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries two alpha-haemolysin determinants which are located on different pathogenicity islands (PAI I(536) and PAI II(536)) . PAI II(536) is associated with the tRNA gene leuX . The leuX-encoded tRNA(5)(Leu) is required for the efficient expression of the hly determinants in strain 536 . HlyA levels were reduced and secretion of the protein was delayed in the leuX-negative mutant strain 536Delta102 . The lack of a functional tRNA(5)(Leu) resulted in a decrease in hly transcript levels in comparison to the wild-type strain . Analysis of several genes whose products are involved in the regulation of hly expression revealed that levels of RfaH and Hha, as well as the corresponding rfaH and hha transcripts, were higher in the leuX-negative background, whereas the expression of tolC and hns was not influenced by the leuX genotype . The analysis of hly transcript levels in hha deletion mutants of the E . coli strains 536 and 536Delta102 demonstrated that the increase in hha expression is partially responsible for the reduction in hly transcript levels in the leuX-negative background . These results demonstrate that the tRNA(5)(Leu) affects the expression of the alpha-haemolysin determinant at different levels in a regulatory cascade, and imply that, in addition to Hha, at least one further, as yet unidentified, regulatory factor must be involved in the regulation of hly transcription in the uropathogenic E . coli strain 536.

Genetics, 2002 Jun, 161(2), 623 - 32
Mutational analysis of the gephyrin-related molybdenum cofactor biosynthetic gene cnxE from the lower eukaryote Aspergillus nidulans; Heck IS et al.; We report the identification of a number of mutations that result in amino acid replacements (and their phenotypic characterization) in either the MogA-like domain or domains 2 and 3 of the MoeA-like region of the Aspergillus nidulans cnxE gene . These domains are functionally required since mutations that result in amino acid substitutions in any one domain lead to the loss or to a substantial reduction in all three identified molybdoenzyme activities (i.e., nitrate reductase, xanthine dehydrogenase, and nicotinate hydroxylase) . Certain cnxE mutants that show partial growth with nitrate as the nitrogen source in contrast do not grow on hypoxanthine or nicotinate . Complementation between mutants carrying lesions in the MogA-like domain or the MoeA-like region, respectively, most likely occurs at the protein level . A homology model of CnxE based on the dimeric structure of E . coli MoeA is presented and the position of inactivating mutations (due to amino acid replacements) in the MoeA-like functional region of the CnxE protein is mapped to this model . Finally, the activity of nicotinate hydroxylase, unlike that of nitrate reductase and xanthine dehydrogenase, is not restored in cnxE mutants grown in the presence of excess molybdate.

Genetics, 2002 Jun, 161(2), 483 - 92
A domain of RecC required for assembly of the regulatory RecD subunit into the Escherichia coli RecBCD holoenzyme; Amundsen SK et al.; The heterotrimeric RecBCD enzyme of Escherichia coli is required for the major pathway of double-strand DNA break repair and genetic exchange . Assembled as a heterotrimer, the enzyme has potent nuclease and helicase activity . Analysis of recC nonsense and deletion mutations revealed that the C terminus of RecC is required for assembly of the RecD subunit into RecBCD holoenzyme but not for recombination proficiency; the phenotype of these mutations mimics that of recD deletion mutations . Partial proteolysis of purified RecC polypeptide yielded a C-terminal fragment that corresponds to the RecD-interaction domain . RecD is essential for nuclease activity, regulation by the recombination hotspot Chi, and high affinity for DNA ends . The RecC-RecD interface thus appears critical for the regulation of RecBCD enzyme via the assembly and, we propose, disassembly or conformational change of the RecD subunit.

J Biol Chem, 2002 Sep 6, 277(36), 32706 - 13 Epub 2002 Jun 18.
DsbB catalyzes disulfide bond formation de novo; Regeimbal J et al.; DsbA and DsbB are responsible for disulfide bond formation . DsbA is the direct donor of disulfides, and DsbB oxidizes DsbA . DsbB has the unique ability to generate disulfides by quinone reduction . It is thought that DsbB oxidizes DsbA via thiol disulfide exchange . In this mechanism, a disulfide is formed across the N-terminal pair of cysteines (Cys-41/Cys-44) in DsbB by quinone reduction . This disulfide is then transferred on to the second pair of cysteine residues in DsbB (Cys-104/Cys-130) and then finally transferred to DsbA . We have shown here the redox potential of the two disulfides in DsbB are -271 and -284 mV, respectively, and considerably less oxidizing than the disulfide of DsbA at -120 mV . In addition, we have found the Cys-104/Cys-130 disulfide of DsbB to actually be a substrate for DsbA in vitro . These findings indicate that the disulfides in DsbB are unsuitable to function as the oxidant of DsbA . Furthermore, we have shown that mutants in DsbB that lack either pair or all of its cysteines are also capable of oxidizing DsbA . These unexpected findings raise the possibility that the oxidation of DsbA by DsbB does not occur via thiol disulfide exchange as is widely assumed but rather, directly via quinone reduction.

J Biol Chem, 2002 Sep 6, 277(36), 32606 - 15 Epub 2002 Jun 18.
5'-adenosinephosphosulfate lies at a metabolic branch point in mycobacteria; Williams SJ et al.; Bacterial sulfate assimilation pathways provide for activation of inorganic sulfur for the biosynthesis of cysteine and methionine, through either adenosine 5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as intermediates . PAPS is also the substrate for sulfotransferases that produce sulfolipids, putative virulence factors, in Mycobacterium tuberculosis such as SL-1 . In this report, genetic complementation using Escherichia coli mutant strains deficient in APS kinase and PAPS reductase was used to define the M . tuberculosis and Mycobacterium smegmatis CysH enzymes as APS reductases . Consequently, the sulfate assimilation pathway of M . tuberculosis proceeds from sulfate through APS, which is acted on by APS reductase in the first committed step toward cysteine and methionine . Thus, M . tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases . Deletion of CysH from M . smegmatis afforded a cysteine and methionine auxotroph consistent with a metabolic branch point centered on APS . In addition, we have redefined the substrate specificity of the B . subtilis CysH, formerly designated a PAPS reductase, as an APS reductase, based on its ability to complement a mutant E . coli strain deficient in APS kinase . Together, these studies show that two conserved sequence motifs, CCXXRKXXPL and SXGCXXCT, found in the C termini of all APS reductases, but not in PAPS reductases, may be used to predict the substrate specificity of these enzymes . A functional domain of the M . tuberculosis CysC protein was cloned and expressed in E . coli, confirming the ability of this organism to make PAPS . The expression of recombinant M . tuberculosis APS kinase provides a means for the discovery of inhibitors of this enzyme and thus of the biosynthesis of SL-1.

Vet Immunol Immunopathol, 2002 Sep 10, 87(3-4), 161 - 8
In vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding; Van Reeth K et al.; The early cytokines interferon-alpha (IFN-alpha), tumour necrosis factor-alpha (TNF-alpha), interleukin-1, -6 and -8 (IL-1, -6, -8) are produced during the most early stage of an infection . The activities of these cytokines have been studied extensively in vitro and in rodents, but in vivo studies on the role of these cytokines in infectious diseases of food animals are few . This review concentrates on in vivo studies of cytokine involvement in infectious respiratory diseases of swine, with an emphasis on viral infections . First evidence for the role of early cytokines in pneumonia in swine came from experimental infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae . The role of TNF-alpha and IL-1 in the symptoms and pathology of porcine pleuropneumonia has recently been proven by use of an adenovirus vector expressing the anti-inflammatory IL-10 . In the authors' laboratory, studies were undertaken to investigate the relationship between viral respiratory disease and bioactive lung lavage levels of IFN-alpha, TNF-alpha, IL-1 and IL-6 . Out of three respiratory viruses-porcine respiratory coronavirus (PRCV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV)-only SIV induced acute respiratory disease and severe lung damage by itself . Disease and lung pathology were tightly associated with the simultaneous production of IFN-alpha, TNF-alpha, IL-1 and IL-6 . In challenge studies of SIV-vaccinated pigs, levels of IFN-alpha, TNF-alpha and IL-6, but not IL-1 were correlated with clinical and virological protection . Multifactorial respiratory disease was reproduced by combined inoculations with PRCV or PRRSV followed by LPS from Escherichia coli . In comparison with the respective single inoculations, which were subclinical, there was a true potentiation of disease and production of TNF-alpha, IL-1 and IL-6 . TNF-alpha and IL-6 were best correlated with disease . In further studies, we will use more specific strategies to dissect the role of cytokines during viral infections.

Vet Immunol Immunopathol, 2002 Sep 10, 87(3-4), 147 - 60
Induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model; Yuan L et al.; Enteric viruses are a major cause of diarrhea in animals and humans . Among them, rotaviruses are one of the most important causes of diarrhea in young animals and human infants . A lack of understanding of mechanisms to induce intestinal immunity and the correlates of protective immunity in neonates has impaired development of safe and effective vaccines against enteric viruses . Studies of candidate vaccines using an adult mouse model of subclinical enteric viral infections often do not predict vaccine efficacy against disease evaluated in neonatal large animals . A series of studies have been conducted using a neonatal gnotobiotic pig model of rotavirus infection and diarrhea to identify correlates of protective immunity and to evaluate traditional and novel vaccine approaches for the induction of mucosal immune responses and protection to enteric viruses . Gnotobiotic pigs recovered from infection with virulent Wa human rotavirus (HRV) (mimic natural infection) had high numbers of intestinal IgA rotavirus-specific primary antibody-secreting cells (ASCs) and memory B-cells (to recall antigen) measured by ELISPOT assay, which correlated with complete protection against rotavirus challenge . Most short-term IgA memory B-cells were resident in the ileum, the major site of rotavirus replication . Spleen, not the bone marrow, was the major resident site for longer-term IgG memory B-cells . Candidate rotavirus vaccines evaluated in pigs for their ability to induce intestinal or systemic ASC and protection against rotavirus infection and diarrhea included attenuated live virus, inactivated virus, and baculovirus-expressed double-layered rotavirus-like particles (2/6-VLPs) . In combination with those candidate vaccines, various adjuvants, delivery systems, and immunization routes were tested, including incomplete Freund's adjuvant for i.m . immunization, and a mutant Escherichia coli heat labile enterotoxin R192G (mLT) for i.n . immunization . It was shown that orally administered replicating vaccines were most effective for priming for intestinal IgA ASC and memory B-cell responses, but i.n . administered non-replicating 2/6-VLPs plus mLT were effective as booster vaccines . We conclude that protective immunity depends on the magnitude, location, viral protein-specificity, and isotype of the antibody responses induced by vaccination . Therefore highly effective enteric viral vaccines should: (i) induce sufficient levels of intestinal IgA antibodies; (ii) include viral antigens that induce neutralizing antibodies; and (iii) require the use of effective mucosal adjuvants or antigen delivery systems for non-replicating oral or i.n . vaccines.

Eur J Biochem, 2002 Jun, 269(12), 3032 - 40
Identification and characterization of the Escherichia coli stress protein UP12, a putative in vivo substrate of GroEL; Bochkareva ES et al.; Many groups of proteins play important roles in the cell's response to various stresses . The molecular chaperone GroEL of Escherichia coli represents one such highly conserved family of stress proteins . We have observed that isolated GroEL complexes from stationary cultures contain various polypeptides that can be released from the chaperonin by GroES and/or ATP, and identified two such polypeptides as the proteins GatY and UP12 . Whereas GatY had been isolated previously, as an in vivo substrate of GroEL, the isolation of UP12 in a complex with GroEL was intriguing, because based on sequence similarity it was suggested that UP12 might also be a functional stress protein . UP12 belongs to a family of universal stress proteins (UspA family), of which UspA itself, and three additional paralogues, have been characterized previously . Here we show that UP12 accumulates under various growth inhibitory conditions and induced by heat shock . Furthermore, unlike wild-type cells, a UP12 deletion mutant recovers slowly from late stationary growth conditions, and has a marked sensitivity to the toxic agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) . Finally, coimmunoprecipitation experiments confirmed the initial observation that UP12 interacts with GroEL . Therefore, we suggest that UP12 may function as a universal stress protein, interaction of which with GroEL possibly ensures its proper folding state.

Eur J Biochem, 2002 Jun, 269(12), 2907 - 17
Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants; Kitagawa M et al.; To examine functions of two small heat shock proteins of Escherichia coli, IbpA and IbpB, we constructed His-IbpA and His-IbpB, in which a polyhistidine tag was fused to the N-terminals . Both purified His-IbpA and His-IbpB formed multimers, which have molecular masses of about 2.0-3.0 MDa and consist of about 100-150 subunits . They suppressed the inactivation of several enzymes including citrate synthase and 6-phosphogluconate dehydrogenase by heat, potassium superoxide, hydrogen peroxide and freeze-thawing, but not the inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide . Both His-IbpA and His-IbpB suppressed enzyme inactivation by various treatments and were also found to be associated with their non-native forms . However, both His-IbpA and His-IbpB were not able to reactivate enzymes inactivated by heat, oxidants or guanidine hydrochloride . When heated to 50 degrees C, each multimeric form of His-IbpA or His-IbpB was dissociated to form a monomer for His-IbpA, and an oligomer of about one-quarter size for His-IbpB . These structural changes were reversible, as both heated proteins regained the multimeric structures after incubation at 25 degrees C . However, when exposed to hydrogen peroxide or potassium superoxide, the large multimeric forms of His-IbpA and His-IbpB were maintained . The results suggest that His-IbpA and His-IbpB suppress the inactivation of enzymes and bind non-native proteins to protect their structures from heat and oxidants.

Eur J Biochem, 2002 Jun, 269(12), 2889 - 96
Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis; Mattern-Dogru E et al.; In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton . The PNAE cDNA was previously heterologously expressed in E . coli . Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily . The catalytic triad, which is typical for this family, is conserved . By site-directed mutagenesis, the members of the catalytic triad were identified . For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase . The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action . The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.

Eur J Biochem, 2002 Jun, 269(12), 2842 - 50
Cloning and expression of two novel aldo-keto reductases from Digitalis purpurea leaves; Gavidia I et al.; The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon-carbon double bonds of a wide variety of substrates including steroids . Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants . Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were isolated from a D . purpurea cDNA library using the rat Delta4-3-ketosteroid 5beta-reductase clone . Both cDNAs encode 315 amino acid proteins showing 98.4% identity . DpAR proteins present high identities (68-80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medicago sativa . A molecular phylogenetic tree suggests that these seven proteins belong to a new subfamily of the AKR superfamily . Southern analysis indicated that DpARs are encoded by a family of at most five genes . RNA-blot analyses demonstrated that the expression of DpAR genes is developmentally regulated and is restricted to leaves . The expression of DpAR genes has also been induced by wounding, elevated salt concentrations, drought stress and heat-shock treatment . The isolated cDNAs were expressed in Escherichia coli and the recombinant proteins purified . The expressed enzymes present reductase activity not only for various sugars but also for steroids, preferring NADH as a cofactor . These studies indicate the presence of plant AKR proteins with ketosteroid reductase activity . The function of the enzymes in cardenolide biosynthesis is discussed.

Biochem J, 2002 Jul 1, 365(Pt 1), 193 - 204
An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp . strain E2; Steenbakkers PJ et al.; The cellulosome produced by Piromyces sp . strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water . Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90 kDa . Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested . Incubation with 0.5% (w/v) SDS or 8 M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170 kDa components . To investigate the major 90 kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing . Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi . The 2800 bp cDNA clone contained an open reading frame of 2334 bp encoding a 757-residue extracellular protein . Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family . Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase . The catalytic domain is succeeded by a putative beta-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains . Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges . The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.

Biochem J, 2002 Jul 1, 365(Pt 1), 165 - 72
Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids; Wadum MC et al.; Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression . Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations . No such method is presently available . In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold . Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53 . FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm) . Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively . FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters . Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM) . FACI-53 showed a high fluorescence yield for C8-C12 acyl chains . It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.

J Am Chem Soc, 2002 Jun 26, 124(25), 7353 - 62
Gas-phase concentration, purification, and identification of whole proteins from complex mixtures; Reid GE et al.; Five proteins present in a relatively complex mixture derived from a whole cell lysate fraction of E . coli have been concentrated, purified, and dissociated in the gas phase, using a quadrupole ion trap mass spectrometer . Concentration of intact protein ions was effected using gas-phase ion/ion proton-transfer reactions in conjunction with mass-to-charge dependent ion "parking" to accumulate protein ions initially dispersed over a range of charge states into a single lower charge state . Sequential ion isolation events interspersed with additional ion parking ion/ion reaction periods were used to "charge-state purify" the protein ion of interest . Five of the most abundant protein components present in the mixture were subjected to this concentration/purification procedure and then dissociated by collisional activation of their intact multiply charged precursor ions . Four of the five proteins were subsequently identified by matching the uninterpreted product ion spectra against a partially annotated protein sequence database, coupled with a novel scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential cleavage sites . The identification of these proteins illustrates the potential of this "top-down" protein identification approach to reduce the reliance on condensed-phase chemistries and extensive separations for complex protein mixture analysis.

Protein Expr Purif, 2002 Jun, 25(1), 203 - 8
Cloning, overexpression, purification, and matrix-assisted refolding of DevS (Rv 3132c) histidine protein kinase of Mycobacterium tuberculosis; Saini DK et al.; The devR-devS (Rv 3133c-Rv 3132c) two-component system of Mycobacterium tuberculosis was identified in our laboratory by RNA subtractive hybridization . This genetic system was predicted to encode a response regulator and histidine protein kinase, respectively . The putative histidine kinase protein DevS was overexpressed to high levels in Escherichia coli as a fusion protein with a hexahistidine tag, His(6)-DevS201, in the form of inclusion bodies . Here we report a "redox-based" method of matrix-bound renaturation of DevS protein . The refolded protein was biochemically active in an autophosphorylation reaction characteristic of histidine kinases and was suitable for the generation of polyclonal antibodies and as an antigen in ELISA .

Protein Expr Purif, 2002 Jun, 25(1), 174 - 9
Chromatographic methods for the isolation of, and refolding of proteins from, Escherichia coli inclusion bodies; Gu Z et al.; New methods for the chromatographic isolation of inclusion bodies directly from crude Escherichia coli homogenates and for the refolding of denatured protein are presented . The traditional method of differential centrifugation for the isolation of purified inclusion bodies is replaced by a single gel-filtration step . The principle is that the exclusion limit of the gel particles is chosen such that only the inclusion bodies are excluded, i.e., all other components of the crude homogenate penetrate the gel under the conditions selected . In the novel column refolding process, a decreasing gradient of denaturant (urea or Gu-HCl), combined with an increasing pH gradient, is introduced into a gel-filtration column packed with a gel medium that has an exclusion limit lower than the molecular mass of the protein to be refolded . A limited sample volume of the protein, dissolved in the highest denaturant concentration at the lowest pH of the selected gradient combination, is applied to the column . During the course of elution, the zone of denatured protein moves down the column at a speed approximately threefold higher than that of the denaturant . This means that the protein sample will gradually pass through areas of increasingly lower denaturant concentrations and higher pH, which promotes refolding into the native conformation . The shape and slope of the gradients, as well as the flow rate, will influence the refolding rate and can be adjusted for different protein samples . The principle is illustrated using a denatured recombinant scFv fusion protein obtained from E . coli inclusion bodies .

Protein Expr Purif, 2002 Jun, 25(1), 138 - 48
Refolding and purification of recombinant human PDE7A expressed in Escherichia coli as inclusion bodies; Richter W et al.; We have investigated the refolding and purification of the catalytic domain of human 3',5'-cyclic nucleotide phosphodiesterase 7A1 (PDE7A1) expressed in Escherichia coli . A cDNA encoding an N-terminal-truncated PDE7A1(147-482-His) was amplified by RT-PCR from human peripheral blood cells and inserted into the vector pET21-C for bacterial expression of the enzyme fused to a C-terminal His-tag . The PDE was found to be expressed in the form of inclusion bodies which could be refolded to an active enzyme in buffer containing high concentrations of arginine hydrochloride, ethylene glycol, and magnesium chloride at pH 8.5 . The PDE7A1(147-482-His) construct could be purified after dialysis and concentration steps by either Zn2+-IDA-Sepharose chromatography or ResourceQ ion-exchange chromatography to homogeneity . In comparison to the metal-chelate column, the ResourceQ purification resulted in a distinctly better yield and enrichment of the protein . Both the Vmax (0.46 micromol . min(-1) . mg(-1) ) and the K(m) (0.1 microM) of the purified enzyme were found to be comparable with published data for native or recombinant catalytically active expressed PDE7A1 . Using SDS/PAGE, a molecular mass of 39 kDa was determined (theoretical value 38.783 kDa) . As known from several other mammalian PDEs, size-exclusion chromatography using refolded PDE7A1(147-482-His) indicated the formation of dimers . The purified enzyme was soluble at concentrations up to 100 microg/ml . A further increase of protein concentration resulted, however, in precipitation of the enzyme .

Protein Expr Purif, 2002 Jun, 25(1), 105 - 13
Expression, refolding, purification, molecular characterization, crystallization, and preliminary X-ray analysis of the receptor binding domain of human B7-2; Zhang X et al.; The cell-mediated immune response involves a series of specific molecular interactions between cell surface molecules on T cells and antigen-presenting cells . Of particular importance for the regulation of T cell activity is the interaction of the B7 isoforms, B7-1 and B7-2, with the T cell surface costimulatory receptors, CD28 and CTLA-4 . The binding of CD28 by B7-1/B7-2 results in an enhancement of T cell responses initiated by the interaction between a clonotypic T cell receptor and its specific, antigenic MHC-peptide complex, whereas the subsequent engagement of CTLA-4 by B7-1/B7-2 leads to a down-regulation of the response . Here we report the expression, refolding, purification, characterization, and crystallization of the receptor-binding domain of human B7-2 . The receptor-binding domain of human B7-2 was overexpressed in Escherichia coli as inclusion bodies, solubilized in 6 M guanidine-hydrochloride, and then refolded in vitro by rapid dilution into a renaturing buffer . Refolded B7-2 was subsequently purified to homogeneity by anion-exchange chromatography . Gel-filtration chromatography and native PAGE analysis showed that the receptor-binding domain of B7-2 is exclusively monomeric in solution . Purified B7-2 binds tightly to bacterially expressed monomeric and disulfide-linked homodimeric human CTLA-4 as shown by gel-filtration chromatography and native PAGE . This suggests that glycosylation is not important for the proper folding of the receptor-binding domain of B7-2 nor for its binding to CTLA-4 . In addition, these results suggest that refolded B7-2 is biologically active and may be a useful therapeutic and experimental reagent for regulating T cell activity . Refolded and purified B7-2 was crystallized by the hanging-drop vapor diffusion method, allowing for the initiation of an X-ray crystallographic study .

Protein Expr Purif, 2002 Jun, 25(1), 81 - 6
Expression and purification of two hydrophobic double-spanning membrane proteins derived from the cystic fibrosis transmembrane conductance regulator; Therien AG et al.; We describe a rapid method for the expression and purification of two hydrophobic protein constructs derived from the membrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein associated with cystic fibrosis . The proteins have no sequence homology but are both predicted to contain two membrane-spanning segments . The protocol involves the expression of CFTR constructs as thioredoxin fusion proteins in Escherichia coli, followed by partial purification by affinity chromatography, removal of the thioredoxin moiety by proteolytic cleavage in the presence of detergent, and final purification by reversed-phase high-performance liquid chromatography . The method yields milligram amounts of purified constructs that spontaneously insert into detergent micelles in alpha-helical conformation . We predict that this protocol will be applicable to a variety of proteins of similar size and hydrophobicity .

Protein Expr Purif, 2002 Jun, 25(1), 73 - 80
Expression of recombinant human betaine: homocysteine S-methyltransferase for x-ray crystallographic studies and further characterization of interaction with S-adenosylmethionine; Bose N et al.; Elevated homocysteine as a result of dysfunctional metabolic enzymes is an independent risk factor for arteriosclerosis . Betaine:homocysteine S-methyltransferase (BHMT) (EC 2.1.1.5) is an important enzyme in the pathway of homocysteine metabolism in that it recycles methionine from homocysteine and nonfolate methyl donors . To initiate X-ray crystallographic structural studies, we created a BHMT expression construct for use in Escherichia coli that has a polyhistidine purification tag with no extraneous protein, usually found in commercial vectors, between the tag and protein sequence . The extra amino acids can hinder the crystallization process . A modified pET28b vector was designed to produce N-terminal polyhistidine-tagged proteins with a simple construction scheme having broad applicability because of the use of rare SapI cloning sites . BHMT expressed using this vector could be rapidly purified using metal chelate chromatography . Gel exclusion chromatography analysis showed that recombinant polyhistidine-tagged human BHMT is a tetramer . S-Adenosylmethionine (SAMe) has no effect on the recombinant BHMT's ability to methylate homocysteine nor does the enzyme appear to bind SAMe when examined by microcalorimetry .

Protein Expr Purif, 2002 Jun, 25(1), 65 - 72
Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli; Jayalakshmi R et al.; Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements . Enzymes of the salvage pathway are, therefore, candidate drug targets . We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene . In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine . The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238 . This paper reports the conditions for hyperexpression of the recombinant protein in E . coli BL21(DE3) and purification of the protein to homogeneity . The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity . Glycerol and EDTA were found to stabilize enzyme activity during storage . The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg . The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively . The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength .

Protein Expr Purif, 2002 Jun, 25(1), 23 - 30
In vitro assembly, purification, and crystallization of the rab geranylgeranyl transferase:substrate complex; Rak A et al.; Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion . This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab proteins . The enzyme consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane . In order to understand the structural basis of Rab prenylation, we have investigated in vitro assembly and crystallization of the RabGGTase:REP-1:Rab complex . In order to ensure maximal stability of the ternary complex, we generated its monoprenylated form, which corresponds to a reaction intermediate and displays the highest affinity between the components . This was achieved by expressing the individual components in baculovirus and Escherichia coli systems with subsequent purification followed by in vitro monoprenylation of Rab7 with immobilized recombinant RabGGTase . Purified monoprenylated REP-1:Rab7 was complexed with recombinant RabGGTase and crystallized in hanging drops . The crystals obtained initially diffract to 8 A on an in-house X-ray source .

Protein Expr Purif, 2002 Jun, 25(1), 1 - 7
High throughput methods for gene cloning and expression; Dieckman L et al.; We outline a high throughput process for the production of bacterial expression clones using automated liquid handlers . The protocol consists of a series of interlinked methods representing liquid manipulations or incubations on various stations of the automation system . The methods employ the ligation-independent cloning approach that enables the simultaneous production of plasmids for different expression systems . The current cloning protocol spans 3 days with a linear throughput of 400 targets per production run . This automated approach enables the production of large numbers of bacterial expression clones and ultimately purified proteins . Although they were developed for structural genomics, these molecular protocols can also be applied in high throughput strategies such as those used for site-specific mutagenesis or protein interaction studies .

Prep Biochem Biotechnol, 2002 May, 32(2), 189 - 205
Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli; Ikura K et al.; Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena . Transglutaminase reactions are also applicable in applied enzymology . Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity . Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein . By the expression culture at lower temperatures (25 and 18 degrees C), however, a fraction of the soluble and active enzyme protein slightly increased . Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E . coli cells . The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme . These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.

Jpn J Antibiot, 2002 Apr, 55(2), 203 - 27
Preventive effect of TAK-751S on complications of hemorrhagic colitis (results of clinical study of TAK-751S); Ito H et al.; BACKGROUND: The effective therapy for hemolytic uremic syndrome and encephalopathy caused by enterohemorrhagic Escherichia coli have not been established . Great attention has been drawn to the results of the clinical study of TAK-751S, performed in Canada . In Japan, a nationwide clinical study of TAK-751S had been performed since 1997 to investigate the preventive effect on the onset of HUS and the safety . METHODS: TAK-751S was administered in daily doses of 500 mg/kg for one week to 128 pediatric patients with colitis who were suspected of enterohemorrhagic Escherichia coli (EHEC) infection . RESULTS: 1 . TAK-751S was confirmed to absorb Shiga toxin (Stx) existing inside the human intestine and to excrete Stx out of the body . 2 . The incidence of HUS is 5.9% (4/68) and a tendency to inhibit the onset of HUS was observed as compared with the historical control . The complications of central neuropathy such as encephalopathy were observed in 3 of these patients with HUS . 3 . Mild "sweating" and "nausea" were observed . There were 13 mild non-specific abnormalities of laboratory test values in 8 patients . CONCLUSIONS: From these results, it was clarified that TAK-751S absorbed and removed free Stx in the intestinal tract of pediatric patients with EHEC infection . The test drug could not inhibit the onset of HUS completely, but since HUS occurred within 48 hours after the start of administration in 3 of the 4 patients with onset of HUS, TAK-751S is a safe drug for pediatric patients with EHEC infection in which the preventive effect on HUS and encephalopathy are expected when it can be given from an early stage of the diseases . Furthermore, these results suggest that importance of rapid diagnosis of HUS.

Arch Microbiol, 2002 Jul, 178(1), 13 - 25 Epub 2002 Apr 13.
TfdD(II), one of the two chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4), cannot efficiently convert 2-chloro- cis, cis-muconate to trans-dienelactone to allow growth on 3-chlorobenzoate; Laemmli CM et al.; Ralstonia eutropha JMP134 (pJP4) harbors two functional gene clusters for the degradation of chlorocatechols, i.e . tfdCDEF (in short: tfd (I)) and tfdD (II) C (II) E (II) F (II) (in short: tfd (II)), which are both present on the catabolic plasmid pJP4 . In this study, we compared the function of both gene clusters for degradation of chlorocatechols by constructing isolated and hybrid tfd (I)- tfd (II) clusters on plasmids in R . eutropha, by activity assays of Tfd enzymes, and by HPLC/MS of individual enzymatic catalytic steps in chlorocatechol conversion . R . eutropha containing the tfd (II) cluster alone or hybrid tfd-clusters with tfdD (II) as sole gene for chloromuconate cycloisomerase were impaired in growth on 3-chlorobenzoate, in contrast to R . eutrophaharboring the complete tfd (I) cluster . Enzyme activities for TfdD(II) and for TfdE(II) were very low in R . eutropha when induced with 3-chlorobenzoate . By contrast, a relatively high enzyme activity was found for TfdF(II) . Spectral conversion assays with extracts from R . eutropha strains expressing tfdD (II) all showed accumulation of a compound with a similar UV spectrum as 2-chloro- cis,cis-muconate from 3-chlorocatechol . HPLC analysis of in vitro assays in which each individual step in 3-chlorocatechol conversion was reproduced by sequentially adding cell extracts of an Escherichia coli expressing one Tfd enzyme only demonstrated that TfdD(II) was unable to cause conversion of 2-chloro- cis,cis-muconate . No accumulation of intermediates was observed with 4-chlorocatechol . From these results, we conclude that at least TfdD(II) is a bottleneck in conversion of 3-chlorocatechol and, therefore, in efficient metabolism of 3-chlorobenzoate . This study showed the subtle functional and expression differences between similar enzymes of the tfd-encoded pathway and demonstrated that extreme care has to be taken when inferring functionality from sequence data alone.

Curr Microbiol, 2002 Aug, 45(2), 94 - 8
Biosynthesis of the branched-chain amino acids in the cyanobacterium Synechocystis PCC6803: existence of compensatory pathways; Kouhen OM et al.; Complementation of an E . coli mutant auxotrophic for the branched-chain amino acids (BCAA)--valine, leucine, and isoleucine--by the ilvG gene ( slr2088) of the cyanobacterium Synechocystis PCC6803 indicates that this gene encodes an active alpha-acetohydroxy acid synthase . Differences of response of the recombinants to the addition of the essential amino acids suggested a lower specificity for the initial reaction of the valine/leucine chain than for the isoleucine one . Inactivation of ilvG in Synechocystis led to a leaky phenotype, suggesting a capacity to compensate the auxotrophies by other processes . This observation is discussed in view of the general difficulty of obtaining auxotrophs in cyanobacteria.

Protein Sci, 2002 Jul, 11(7), 1850 - 3
Mapping the surface of Escherichia coli peptide deformylase by NMR with organic solvents; Byerly DW et al.; Identifying potential ligand binding sites on a protein surface is an important first step for targeted structure-based drug discovery . While performing control experiments with Escherichia coli peptide deformylase (PDF), we noted that the organic solvents used to solubilize some ligands perturbed many of the same resonances in PDF as the small molecule inhibitors . To further explore this observation, we recorded (15)N HSQC spectra of E . coli peptide deformylase (PDF) in the presence of trace quantities of several simple organic solvents (acetone, DMSO, ethanol, isopropanol) and identified their sites of interaction from local perturbation of amide chemical shifts . Analysis of the protein surface structure revealed that the ligand-induced shift perturbations map to the active site and one additional surface pocket . The correlation between sites of solvent and inhibitor binding highlights the utility of organic solvents to rapidly and effectively validate and characterize binding sites on proteins prior to designing a drug discovery screen . Further, the solvent-induced perturbations have implications for the use of organic solvents to dissolve candidate ligands in NMR-based screens.

Protein Sci, 2002 Jul, 11(7), 1714 - 9
High-throughput screening of soluble recombinant proteins; Shih YP et al.; The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies . Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format . Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products . All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%) . To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture ( approximately 1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE . Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs . The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis.

Protein Sci, 2002 Jul, 11(7), 1671 - 80
Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: implications for in vivo function; Hobart SA et al.; The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes . The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase . Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo . Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels . Its urea-induced equilibrium denaturation was monitored by far- and near-UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy . The denaturation transitions obtained were concentration-dependent and showed similar midpoints (C(m)) and m values, suggesting a two-state denaturation process involving the native dimer and unfolded monomers (N(2) <--> 2U) . The DeltaG(H(2)O) for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole . At concentrations <9 microM, the FIS dimer began to dissociate, as noted by the change in CD signal and size-exclusion high-pressure liquid chromatography retention times and peak width . The estimated dimer dissociation constant based on the CD and size-exclusion chromatography data is in the micromolar range, resulting in a DeltaG(H(2)O) of at least 5 kcal/mole less than that calculated from the urea denaturation data . This discrepancy suggests a deviation from a two-state denaturation model, perhaps due to a marginally stable monomeric intermediate . These observations have implications for the stability and function of FIS in vivo.

Protein Sci, 2002 Jul, 11(7), 1626 - 38
Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase; Guddat LW et al.; Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 A resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 A resolution) . Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E . coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 A . Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP . A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures . For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg(2+) in vivo . In the free structure, a Mg(2+) is coordinated to the side chains of Glu103 and Asp104 . This interaction may be important for stabilization of the enzyme before catalysis . E . coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine . The structures suggest that its substrate specificity is due to the modes of binding of the bases . In E . coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg(2+) complex) . However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

J Biol Chem, 2002 Aug 30, 277(35), 31484 - 90 Epub 2002 Jun 17.
Selective binding of synapse-associated protein 97 to GluR-A alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionate receptor subunit is determined by a novel sequence motif; Cai C et al.; A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses . To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97) . The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102 . Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain . Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position -9 to -11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the -2 position) . Analysis of the in vitro MAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.

J Biol Chem, 2002 Aug 30, 277(35), 31656 - 62 Epub 2002 Jun 17.
The unique pentagonal structure of an archaeal Rubisco is essential for its high thermostability; Maeda N et al.; We have previously determined the crystal structure of a novel pentagonal ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 . Here we have carried out biochemical studies to identify the necessities and/or advantages of this intriguing pentagonal structure . The structure indicated the presence of three neighboring residues (Glu-63, Arg-66, and Asp-69), participating in ionic interactions within unique dimer-dimer interfaces . We constructed three single mutant proteins (E63S, R66S, and D69S) and one triple mutant protein (E63S/R66S/D69S) by replacing the charged residues with serine . The wild type (WT) and all mutant proteins were purified and subjected to gel permeation chromatography at various temperatures . WT and D69S proteins were decameric at all temperatures examined between 30 and 90 degrees C . The majority of E63S and R66S were decamers at 30 degrees C but were found to gradually disassemble with the elevation in temperature . E63S/R66S/D69S was found in a dimeric form even at 30 degrees C . An interesting correlation was found between the subunit assembly and thermostability of the proteins . Circular dichroism and differential scanning calorimetry analyses indicated that the denaturation temperatures of dimeric enzymes (E63S, R66S, and E63S/R66S/D69S) were approximately 95 degrees C, whereas those of the enzymes retaining a decameric structure (WT and D69S) were approximately 110 degrees C . Disassembly into tetramer or dimer units did not alter the slopes of the Arrhenius plots, indicating that the decameric structure had no effect on catalytic performance per se . The results indicate that the decameric assembly of Tk-Rubisco contributes to enhance the thermostability of the enzyme . Taking into account the growth temperature of strain KOD1 (65-100 degrees C), the decameric structure of Tk-Rubisco can be considered essential for the stable presence of the enzyme in the host cells . This study provides an interesting example in which the thermostability of a protein can be enhanced by formation of a unique quaternary structure not found in mesophilic enzymes.

Blood, 2002 Jul 1, 100(1), 299 - 305
Intersubunit circular permutation of human hemoglobin; Sanders KE et al.; For many years, human hemoglobin (Hb) isolated from erythrocytes has been investigated as a potential oxygen delivery therapeutic . Advantages with respect to the need for blood typing were balanced with various undesirable properties of cell-free Hb, including cost, overall oxygen affinity, alterations in cooperativity, and ready dissociation into toxic dimeric species . The use of total gene synthesis has resulted in very high levels of functional human Hb expression in Escherichia coli, but there remains a desire for effecting the crosslinking of the hemoglobin tetramer and providing for ready means for increasing the globular molecular weight . In this communication, we report a novel method for linking alpha chains . By circularly permuting one alpha sequence, the second alpha chain in the Hb tetramer can be linked with glycine residues to form 2 bridges across the central cavity . The second alpha chain thus presents its amino and carboxyl termini on a solvent exposed surface, providing for additional polymerization of oxygen-carrying subunits or attachment of any other peptide-based therapeutic.

J Microbiol Methods, 2002 Sep, 51(1), 63 - 72
Design and development of a novel genetic probe for the analysis of repressor-operator interactions; Love JF et al.; While the native diphtheria tox promoter/operator (toxPO)-lacZ transcriptional fusion has allowed initial isolation and characterization of the diphtheria toxin repressor (DtxR), the low level of reporter gene expression has limited the detection and analysis of mutations affecting subtle changes in repressor-operator binding . In order to overcome this difficulty, we have constructed a novel hybrid promoter/operator-lacZ transcriptional fusion in which the "-35" and spacing of the tac promoter was fused to the "-10" and interrupted palindromic sequence of toxO . We show that the hybrid tacPtoxO is regulated by the transition metal ion-dependent DtxR and that lacZ expression is increased approximately 70-fold in the reporter strain Escherichia coli DH5alpha/lambdaRS45-tacPtoxO-lacZ relative to DH5alpha/lambdaRS45-toxPO-lacZ . In addition, we have constructed a transcriptional fusion between tacPtoxO and luc, pJL1 . We have used pJL1 to program S30 extracts of E . coli in order to direct in vitro the coupled transcription and translation of luciferase . We demonstrate the utility of this in vitro system in providing a direct functional link between in vivo and in vitro observations with DtxR and mutants of DtxR, which display subtle changes in activity in a manner not previously possible.

Biosystems, 2002 Mar-May, 65(2-3), 157 - 77
A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome; Chen S et al.; The recent explosion in available bacterial genome sequences has initiated the need to improve an ability to annotate important sequence and structural elements in a fast, efficient and accurate manner . In particular, small non-coding RNAs (sRNAs) have been difficult to predict . The sRNAs play an important number of structural, catalytic and regulatory roles in the cell . Although a few groups have recently published prediction methods for annotating sRNAs in bacterial genome, much remains to be done in this field . Toward the goal of developing an efficient method for predicting unknown sRNA genes in the completed Escherichia coli genome, we adopted a bioinformatics approach to search for DNA regions that contain a sigma70 promoter within a short distance of a rho-independent terminator . Among a total of 227 candidate sRNA genes initially identified, 32 were previously described sRNAs, orphan tRNAs, and partial tRNA and rRNA operons . Fifty-one are mRNAs genes encoding annotated extremely small open reading frames (ORFs) following an acceptable ribosome binding site . One hundred forty-four are potentially novel non-translatable sRNA genes . Using total RNA isolated from E . coli MG1655 cells grown under four different conditions, we verified transcripts of some of the genes by Northern hybridization . Here we summarize our data and discuss the rules and advantages/disadvantages of using this approach in annotating sRNA genes on bacterial genomes.

Biosystems, 2002 Mar-May, 65(2-3), 105 - 12
An observation of the initial stage towards a symbiotic relationship; Todoriki M et al.; Two well-characterized and phylogeneticaly different species, Escherichia coli and Dictyostelium discoideum, were used as the model organisms . When the two species were mixed and allowed to grow on minimal agar plates at 22 degrees C, remarkably, the two species achieved a state of coexistence at an average of 2-4 weeks . In addition, the emerged colonies housing the coexisting species had a mucoidal nature that was not observed from its origin . Moreover, the state of coexistence was confirmed to be stable, and so was the mucoidal nature of the emerged colonies . Comparing with the pure E . coli origin, the mucoidal colony showed a significant increase in higher molecular weight extracellular components, with polysaccharides as the major constituent . Qualitative analysis of the monosaccharide contents in the extracellular components of the mucoidal colony revealed not only a significant increase in the glucose content, but also significant amount of additional xylose and galactose . The system permits the initial stages of the development of relationship between two species be captured within a short period of time . This feature, together with being simple and reproducible in laboratory conditions, provides a new model system for the study of symbiosis, especially when initial stages are concerned.

Biochemistry, 2002 Jun 25, 41(25), 7969 - 78
A new electron transport mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin; Beilke D et al.; The adrenal ferredoxin (adrenodoxin, Adx) is an acidic 14.4-kDa {2Fe-2S} ferredoxin that belongs to the vertebrate ferredoxin family . It is involved in the electron transfer from the flavoenzyme NADPH-adrenodoxin-reductase to cytochromes P-450(scc) and P-450(11)(beta) . The interaction between the redox partners during electron transport has not yet been fully established . Determining the tertiary structure of an electron-transfer protein may be very helpful in understanding the transport mechanism . In the present work, we report a structural study on the oxidized and reduced forms of bovine adrenodoxin (bAdx) in solution using high-resolution NMR spectroscopy . The protein was produced in Escherichia coli and singly or doubly labeled with (15)N or (13)C/(15)N, respectively . Approximately 70 and 75% of the (15)N, (13)C, and (1)H resonances could be assigned for the reduced and the oxidized bAdx, respectively . The secondary and tertiary structures of the reduced and oxidized states were determined using NOE distance information . (1)H(N)-T(1) relaxation times of certain residues were used to obtain additional distance constraints to the {2Fe-2S} cluster . The results suggest that the solution structure of oxidized Adx is quite similar to the X-ray structure . However, structural changes occur upon reduction of the {2Fe-2S} cluster, as indicated by NMR measurements . It could be shown that these conformational changes, especially in the C-terminal region, cause the dissociation of the Adx dimer upon reduction . A new electron transport mechanism proceeding via a modified shuttle mechanism, with both monomers and dimers acting as electron carriers, is proposed.

Virology, 2002 May 10, 296(2), 321 - 9
RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block; Kalinina NO et al.; Cell-to-cell and long-distance transport of some plant viruses requires coordinated action of three movement proteins encoded by triple gene block (TGB) . The largest of TGB proteins, TGBp1, is a member of the superfamily I of DNA/RNA helicases and possesses a set of conserved helicase sequence motifs necessary for virus movement . A recombinant His-tagged form of TGBp1 of two hordeiviruses and potato virus X, a potexvirus, produced in Escherichia coli had unwinding activity on a partially duplexed RNA, but not DNA substrate . The helicase activity of these proteins was dependent on Mg2+ and ATP . The isolated C-terminal half of the PSLV TGBp1 retaining all helicase motifs was also able to unwind RNA duplex.

Anal Biochem, 2002 Jul 1, 306(1), 100 - 7
Fluorescence assay for the binding of ribonuclease A to the ribonuclease inhibitor protein; Abel RL et al.; Ribonuclease A (RNase A) and the ribonuclease inhibitor protein (RI) form one of the tightest known protein-protein complexes . RNase A variants and homologues, such as G88R RNase A, that retain ribonucleolytic activity in the presence of RI are toxic to cancer cells . Herein, a new and facile assay is described for measuring the equilibrium dissociation constant (K(d)) and dissociation rate constant (k(d)) for complexes of RI and RNase A . This assay is based on the decrease in fluorescence intensity that occurs when a fluorescein-labeled RNase A binds to RI . To allow time for equilibration, the assay is most readily applied to those complexes with K(d) values in the nanomolar range or higher . Using this assay, the value of K(d) for the complex of RI with fluorescein-labeled G88R RNase A was determined to be 0.55 +/- 0.03 nM . In addition, the value of K(d) was determined for the complex of RI with unlabeled G88R RNase A to be 0.57 +/- 0.05 nM by using a competition assay with fluorescein-labeled G88R RNase A . Finally, the value of k(d) for the complex of RI with fluorescein-labeled G88R RNase A was determined to be (7.5 +/- 0.4) x 10(-3) s(-1) by monitoring the increase in fluorescence intensity upon dissociation . This assay can be used to characterize complexes of RI with a wide variety of RNase A variants and homologues, including those with cytotoxic activity.

Anal Biochem, 2002 Jul 1, 306(1), 50 - 4
Protein microarrays to detect protein-protein interactions using red and green fluorescent proteins; Kukar T et al.; Proteomics, the study of protein function on a global scale, will play an important role in furthering our understanding of gene functions, complex biological pathways, and discovery of novel drug targets . A number of techniques have been developed for proteomic studies to identify and analyze proteins, compare protein expression levels, and study protein-protein interactions . Recent developments have applied a DNA array-type approach to immobilize proteins on a surface for high-throughput analysis . Here we report the development and construction of protein chips using derivatized glass and nitrocellulose-coated slides and the employment of recombinant proteins fused with green and red fluorescent proteins for detection . Fluorescent signals were found to be proportional to the amount of arrayed proteins and could be readily detected with a conventional fluorescence slide scanner . This technique allows the investigation of protein-protein interactions without the need for additional labeling steps of probe proteins.

Anal Chem, 2002 Jun 1, 74(11), 2500 - 4
A homogeneous noncompetitive immunoassay for the detection of small haptens; Yokozeki T et al.; We describe a noncompetitive homogeneous immunoassay for small haptens based on the antigen-dependent reassociation of antibody variable domains and beta-galactosidase (beta-gal) complementation (open sandwich enzymatic complementation immunoassay) . As a model system, the reassociation of two fusion proteins, an anti 4-hydroxy-3-nitrophenylacetyl (NP) antibody heavy-chain variable-region fragment fused to an N-terminal deletion mutant of beta-gal (V(H)delta alpha) and the light-chain variable-region fragment fused to a C-terminal deletion mutant of beta-gal (V(L)delta omega), was monitored by the enzymatic complementation between the two . Upon simple mixing of the reagents with the sample, an antigen (NP)-dependent increase in enzymatic activity was observed . When 5-iodo-NP was measured, a 10 times higher sensitivity was observed, probably due to its higher affinity . Compared with our corresponding heterogeneous open sandwich enzyme-linked immunosorbent assay, approximately 1000-fold improvement in the sensitivity was attained, probably due to lower background V(H)-V(L) association . In addition, the assay required less time, handling, sample volume, and assay reagents.

Anal Chem, 2002 Jun 1, 74(11), 2451 - 7
An integrated microfabricated cell sorter; Fu AY et al.; We have developed an integrated microfabricated cell sorter using multilayer soft lithography . This integrated cell sorter is incorporated with various microfluidic functionalities, including peristaltic pumps, dampers, switch valves, and input and output wells, to perform cell sorting in a coordinated and automated fashion . The active volume of an actuated valve on this integrated cell sorter can be as small as 1 pL, and the volume of optical interrogation is approximately 100 fL . Different algorithms of cell manipulation, including cell trapping, were implemented in these devices . We have also demonstrated sorting and recovery of Escherichia coli cells on the chip.

Mol Microbiol, 2002 Jun, 44(5), 1397 - 405
SecDFyajC forms a heterotetrameric complex with YidC; Nouwen N et al.; The Escherichia coli preprotein translocase is composed of a "preprotein conducting channel" domain that consists of the peripherally bound translocation ATPase SecA and the heterotrimeric SecYEG membrane protein complex . SecD, SecF, and YajC form another heterotrimeric complex that can associate with the SecYEG complex . YidC is an essential membrane protein that plays a role in the integration of newly synthesized membrane proteins, and has been shown to co-purify with SecYEG when all translocase components are overproduced . Here, we demonstrate that under conditions that YidC co-purifies with overproduced SecDFyajC it does not co-purify with overproduced SecYEG . Moreover, this interaction of YidC with the SecDFyajC complex is also found at chromosomal protein levels of SecD, SecF and YajC . Closer examination of the SecDFyajC-YidC complex showed that YidC binds to SecD and SecF, whereas YajC interacts only with SecF . As SecF and YajC have previously been shown to interact with SecY, we propose that these two proteins link the heterotetrameric SecDFyajC-YidC complex to the SecYEG complex.

Mol Microbiol, 2002 Jun, 44(5), 1387 - 96
Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli; Moll I et al.; Leaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life . It has been previously reported that translation of the leaderless cI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal protein S2 . Here, we have studied this phenomenon at the molecular level by making use of an E . coli rpsB(ts) mutant . The analysis of the ribosomes isolated under the non-permissive conditions revealed that in addition to ribosomal protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for binding of S1 to the 30S ribosomal subunit . In vitro translation assays and the selective translation of a leaderless mRNA in vivo at the non-permissive temperature corroborate and extend previous in vitro ribosome binding studies in that S1 is indeed dispensable for translation of leaderless mRNAs . The deaD/csdA gene, encoding the "DeaD/CsdA" DEAD-box helicase, has been isolated as a multicopy suppressor of rpsB(ts) mutations . Here, we show that expression of a plasmid-borne DeaD/CsdA gene restores both S1 and S2 on the ribosome at the non-permissive temperature in the rpsB(ts) strain, which in turn leads to suppression of the translational defect affecting canonical mRNSa . These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.

Mol Microbiol, 2002 Jun, 44(5), 1367 - 75
The datA locus predominantly contributes to the initiator titration mechanism in the control of replication initiation in Escherichia coli; Ogawa T et al.; Replication of the Escherichia coli chromosome is initiated synchronously from all origins (oriC) present in a cell at a fixed time in the cell cycle under given steady state culture conditions . A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which titrates exceptionally large amounts of the bacterial initiator protein, DnaA, to prevent overinitiation . Deletion of the datA locus results in extra initiations and altered temporal control of replication . There are many other sites on the E . coli chromosome that can bind DnaA protein, but the contribution of these sites to the control of replication initiation has not been investigated . In the present study, seven major DnaA binding sites other than datA have been examined for their influence on the timing of replication initiation . Disruption of these seven major binding sites, either individually or together, had no effect on the timing of initiation of replication . Thus, datA seems to be a unique site that adjusts the balance between free and bound DnaA to ensure that there is only a single initiation event in each bacterial cell cycle . Mutation either in the second or the third DnaA box (a 9 basepair DnaA-binding sequence) in datA was enough to induce asynchronous and extra initiations of replication to a similar extent as that observed with the datA-deleted strain . These DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.

Mol Microbiol, 2002 Jun, 44(5), 1323 - 9
Both metal binding sites in the homodimer are required for metalloregulation by the CadC repressor; Sun Y et al.; The cadCA operon of plasmid pI258, which confers resistance to the soft metals Cd(II), Pb(II) and Zn(II), is regulated by CadC, a metal-responsive transcriptional repressor . CadC is a 27.6 kDa homodimer composed of two 122-residue monomers . Three cysteine residues, Cys-7, Cys-58 and Cys-60, have been shown to be required for sensing soft metals . Thus, the repressor has two potential inducer binding sites, one on each monomer . However, it is not known whether both binding sites are required for derepression or whether binding of metal to a single site would result in transcript . In this study, heterodimers were purified in which one binding site was wild type and the other had substitutions of the cysteine residues . The wild type-mutant heterodimers retained the ability to bind to cad operator/promoter DNA but did not dissociate from the DNA upon addition of soft metal ions . The results indicate that both subunits in the dimer must have functional metal binding sites for metal sensing to lead to derepression

Mol Microbiol, 2002 Jun, 44(5), 1287 - 98
Expression of the Escherichia coli pcnB gene is translationally limited using an inefficient start codon: a second chromosomal example of translation initiated at AUU; Binns N et al.; Expression of the gene pcnB, encoding the dispensable Escherichia coli poly(A) polymerase (PAPI), which is toxic when overproduced, was investigated . Its promoter was identified and found to be moderately strong when used to express a beta-galactosidase reporter . Expression levels were not affected by increasing or decreasing PcnB concentration . Translation of pcnB was found to initiate from the non-canonical initiation codon AUU . The only other coli gene reported to use AUU as initiation codon is infC, which encodes the initiation factor IF-3 . AUU, in common with other rarely used initiation codons, is discriminated against by IF-3, resulting in the aborting of most AUU-promoted initiation events . This enables AUU to form part of an autoregulatory circuit controlling IF-3 production . We show that InfC discrimination reduces PcnB production fivefold . This is the first instance of this mechanism being used to limit severely the production of a potentially toxic product.

Mol Microbiol, 2002 Jun, 44(5), 1225 - 34
Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter; Furrer JL et al.; The enterobactin system for iron transport in Escherichia coli is well characterized with the exception of the mechanism of enterobactin secretion to the extracellular environment . Escherichia coli membrane protein P43, encoded by ybdA in the chromosomal region of genes involved in enterobactin synthesis, shows strong homology to the 12-transmembrane segment major facilitator superfamily of export pumps . A P43-null mutation was created and siderophore nutrition assays, performed with a panel of E . coli strains expressing one or more outer membrane receptors for enterobactin-related compounds, demonstrated that the P43 mutant was unable to secrete enterobactin efficiently . Products released from the mutant strain were identified with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that the P43 mutant secretes little, if any, enterobactin, but elevated levels of enterobactin breakdown products 2,3- dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer . These data establish that P43 is a critical component of the E . coli enterobactin secretion machinery and provides a rationale for the designation of the previous genetic locus ybdA as entS to reflect its relevant biological function.

Mol Microbiol, 2002 Jun, 44(5), 1141 - 51
A novel cyclic GMP-dependent protein kinase is expressed in the ring stage of the Plasmodium falciparum life cycle; Deng W et al.; Cyclic GMP-dependent protein kinases (PKGs) are the major mediators of the cGMP signal transduction pathway and regulate a variety of physiological effects . We report here the characterization of an unusual PKG from the human malaria parasite Plasmodium falciparum (designated PfPKG) . The 97.5 kDa protein contains some of the structural features of mammalian PKGs but, uniquely, contains a third predicted cGMP binding site and a degenerate fourth . Using both protein kinase activity assays and Western blotting with native P . falciparum proteins, we demonstrate here that PfPKG is expressed predominantly in the ring stage of the life cycle, suggesting a role in the development of asexual blood stage parasites . An Escherichia coli-derived recombinant protein (PfPKG2, Met115-Phe853) was purified and shown to have phosphotransferase activity in terms of both substrate phosphorylation and auto-phosphorylation . This activity was stimulated at least fivefold by 1.0 microM cyclic GMP, but was not stimulated by cAMP or by 8-pCPT-cGMP, which is a potent activator of mammalian PKGs . Several protein kinase inhibitors exhibited a range of inhibitory effects on PfPKG activity . Biochemical analysis therefore shows that PfPKG is distinct from mammalian PKGs with respect to both cyclic nucleotide analogue activation and inhibition profiles.

Mol Biol (Mosk), 2002 May-Jun, 36(3), 460 - 5
{Region of intermolecular complementarity in Escherichia coli 16S rRNA, mRNA, and tRNA molecules}; Shabalina SA; The results of computer analysis of complementarity regions in the sequences of E . coli 16S rRNA, mRNAs and tRNAs are reported in this article . The potential regions of intermolecular RNA-RNA hybridization, or clinger fragments, in 16S rRNA, which are complementary to the sites frequently occurring in mRNAs and tRNAs, were found . Major clinger fragments on 16S rRNA are universal for genes that belong to different functional groups . Our results show there are adaptations of the structural organization of the 16S rRNA molecule to messenger and transport RNA sequences . RNA interaction with clinger fragments may contribute to upregulation of translation process through increasing the local concentration of mRNAs and tRNAs in the vicinity of the ribosome and their proper positioning, as well as decrease the efficiency of translation through non-specific mRNA-16SrRNA interactions.

Genetika, 2002 May, 38(5), 613 - 21
{Formation of an additional promotor in the regulatory region of the Escherichia coli udp gene and its structural and functional characterization}; Evdokimova AA et al.; Structural and functional organization of the mutant udpP18 promoter generated after the spontaneous deletion of the G base in the -79 position relative to the start site of transcription from the main (P1) promoter within the regulatory region of the udp gene was studied . In this mutant, a new, functionally active promoter (P2) with the start site of transcription in the -64 position that contained the typical motif 5'-TG-3' located in front of the Pribnow sequence was formed . The data presented suggest that the expression of the P2 promoter, unlike that of P1, is not subjected to regulation with participation of the CytR protein and the cAMP-CRP complex . Results of mutational analysis of the P2 promoter showed that substitutions of the nucleotide G in the -14 position and nucleotide T in the -15 position significantly diminish the level of transcription from the P2 promoter . On the basis of these data, it is concluded that the P2 promoter could be assigned with respect to its characteristics to a group of promoters with an extended -10 region . The synergistic effect of P1 and P2 promoters on total expression of the udp gene in the mutant udpP18 was detected.

Plant Physiol, 2002 Jun, 129(2), 762 - 73
Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii; Kasahara M et al.; Phototropins (phot1 and phot2, formerly designated nph1 and npl1) are blue-light receptors that mediate phototropism, blue light-induced chloroplast relocation, and blue light-induced stomatal opening in Arabidopsis . Phototropins contain two light, oxygen, or voltage (LOV) domains at their N termini (LOV1 and LOV2), each a binding site for the chromophore flavin mononucleotide (FMN) . Their C termini contain a serine/threonine protein kinase domain . Here, we examine the kinetic properties of the LOV domains of Arabidopsis phot1 and phot2, rice (Oryza sativa) phot1 and phot2, and Chlamydomonas reinhardtii phot . When expressed in Escherichia coli, purified LOV domains from all phototropins examined bind FMN tightly and undergo a self-contained photocycle, characterized by fluorescence and absorption changes induced by blue light (T . Sakai, T . Kagawa, M . Kasahara, T.E . Swartz, J.M . Christie, W.R . Briggs, M . Wada, K . Okada {2001} Proc Natl Acad Sci USA 98: 6969-6974; M . Salomon, J.M . Christie, E . Knieb, U . Lempert, W.R . Briggs {2000} Biochemistry 39: 9401-9410) . The photocycle involves the light-induced formation of a cysteinyl adduct to the C(4a) carbon of the FMN chromophore, which subsequently breaks down in darkness . In each case, the relative quantum efficiencies for the photoreaction and the rate constants for dark recovery of LOV1, LOV2, and peptides containing both LOV domains are presented . Moreover, the data obtained from full-length Arabidopsis phot1 and phot2 expressed in insect cells closely resemble those obtained for the tandem LOV-domain fusion proteins expressed in E . coli . For both Arabidopsis and rice phototropins, the LOV domains of phot1 differ from those of phot2 in their reaction kinetic properties and relative quantum efficiencies . Thus, in addition to differing in amino acid sequence, the phototropins can be distinguished on the basis of the photochemical cycles of their LOV domains . The LOV domains of C . reinhardtii phot also undergo light-activated spectral changes consistent with cysteinyl adduct formation . Thus, the phototropin family extends over a wide evolutionary range from unicellular algae to higher plants.

Plant Physiol, 2002 Jun, 129(2), 594 - 604
The pgp1 mutant locus of Arabidopsis encodes a phosphatidylglycerolphosphate synthase with impaired activity; Xu C et al.; Phosphatidylglycerol is a ubiquitous phospholipid that is also present in the photosynthetic membranes of plants . Multiple independent lines of evidence suggest that this lipid plays a critical role for the proper function of photosynthetic membranes and cold acclimation . In eukaryotes, different subcellular compartments are competent for the biosynthesis of phosphatidylglycerol . Details on the plant-specific pathways in different organelles are scarce . Here, we describe a phosphatidylglycerol biosynthesis-deficient mutant of Arabidopsis, pgp1 . The overall content of phosphatidylglycerol is reduced by 30% . This mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerolphosphate synthase (EC 2.7.8.5) isoform encoded by a gene on chromosome 2 . The mutant shows an 80% reduction in plastidic phosphatidylglycerolphosphate synthase activity consistent with the plastidic location of this particular isoform . Mutant plants are pale green, and their photosynthesis is impaired . This mutant provides a promising new tool to elucidate the biosynthesis and function of plastidic phosphatidylglycerol in seed plants.

Plant Physiol, 2002 Jun, 129(2), 540 - 50
The branched-chain amino acid transaminase gene family in Arabidopsis encodes plastid and mitochondrial proteins; Diebold R et al.; Branched-chain amino acid transaminases (BCATs) play a crucial role in the metabolism of leucine, isoleucine, and valine . They catalyze the last step of the synthesis and/or the initial step of the degradation of this class of amino acids . In Arabidopsis, seven putative BCAT genes are identified by their similarity to their counterparts from other organisms . We have now cloned the respective cDNA sequences of six of these genes . The deduced amino acid sequences show between 47.5% and 84.1% identity to each other and about 30% to the homologous enzymes from yeast (Saccharomyces cerevisiae) and mammals . In addition, many amino acids in crucial positions as determined by crystallographic analyses of BCATs from Escherichia coli and human (Homo sapiens) are conserved in the AtBCATs . Complementation of a yeast Deltabat1/Deltabat2 double knockout strain revealed that five AtBCATs can function as BCATs in vivo . Transient expression of BCAT:green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) protoplasts shows that three isoenzymes are imported into chloroplasts (AtBCAT-2, -3, and -5), whereas a single enzyme is directed into mitochondria (AtBCAT-1).

J Biol Chem, 2002 Aug 30, 277(35), 32094 - 8 Epub 2002 Jun 14.
X-ray structure and ligand binding study of a moth chemosensory protein; Lartigue A et al.; Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception . Such proteins, of M(r) 13,000, have been isolated from several sensory organs of a wide range of insect species . Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae . One of them, CSPMbraA6, a 112-amino acid antennal protein, has been expressed in large quantities and is soluble in the Escherichia coli periplasm . X-ray structure determination has been performed in parallel with ligand binding assays using tryptophan fluorescence quenching . The protein has overall dimensions of 25 x 30 x 32 A and exhibits a novel type of alpha-helical fold with six helices connected by alpha-alpha loops . A narrow channel extends within the protein hydrophobic core . Fluorescence quenching with brominated alkyl alcohols or fatty acids and modeling studies indicates that CSPMbraA6 is able to bind such compounds with C12-18 alkyl chains . These ubiquitous proteins might have the role of extracting hydrophobic linear compounds (pheromones, odors, or fatty acids) dispersed in the phospholipid membrane and transporting them to their receptor.

J Biol Chem, 2002 Aug 30, 277(35), 31335 - 44 Epub 2002 Jun 14.
Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis . PimA is essential for growth of mycobacteria; Kordulakova J et al.; We examined the function of the pimA (Rv2610c) gene, located in the vicinity of the phosphatidylinositol synthase gene in the genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis, which encodes a putative mannosyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis . A cell-free assay was developed in which membranes from M . smegmatis overexpressing the pimA gene incorporate mannose from GDP-{(14)C}Man into di- and tri-acylated phosphatidylinositol mono-mannosides . Moreover, crude extracts from Escherichia coli producing a recombinant PimA protein synthesized diacylated phosphatidylinositol mono-mannoside from GDP-{(14)C}Man and bovine phosphatidylinositol . To determine whether PimA is an essential enzyme of mycobacteria, we constructed a pimA conditional mutant of M . smegmatis . The ability of this mutant to synthesize the PimA mannosyltransferase was dependent on the presence of a functional copy of the pimA gene carried on a temperature-sensitive rescue plasmid . We demonstrate here that the pimA mutant is unable to grow at the higher temperature at which the rescue plasmid is lost . Thus, the synthesis of phosphatidylinositol mono-mannosides and derived higher phosphatidylinositol mannosides in M . smegmatis appears to be dependent on PimA and essential for growth . This work provides the first direct evidence of the essentiality of phosphatidylinositol mannosides for the growth of mycobacteria.

FEBS Lett, 2002 Jun 19, 521(1-3), 127 - 32
2',3'-Dialdehydo-UDP-N-acetylglucosamine inhibits UDP-N-acetylglucosamine 2-epimerase, the key enzyme of sialic acid biosynthesis; Blume A et al.; Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems . UDP-N-acetylglucosamine 2-epimerase catalyzes the first step of their biosynthesis . Periodate-oxidized UDP-N-acetylglucosamine, namely 2',3'-dialdehydo-UDP-alpha-D-N-acetylglucosamine, was found to be an effective inhibitor of this enzyme, compared with the periodate oxidation products of compounds such as UDP, uridine or methyl riboside . It bound covalently to amino acids in the active site causing an irreversible inhibition . This compound may therefore represent a basis for the synthesis of potent inhibitors of UDP-N-acetylglucosamine 2-epimerase and, as a consequence, of the biosynthesis of sialic acids.

Mol Microbiol, 2002 Jun, 44(6), 1611 - 24
Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data; Martin RG et al.; Microarray analyses are providing a plethora of data concerning transcriptional responses to specific gene regulators and their inducers but do not distinguish between direct and indirect responses . Here, we identify directly activated promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by applying informatics, genomics and molecular genetics to microarray data obtained by others . Those studies found that overexpression of marA, or the treatment of cells with salicylate to derepress marA, or treatment with paraquat to induce soxS, resulted in elevated transcription of 153 genes . However, only 27 out of the promoters showed increased transcription under at least two of the aforementioned conditions and eight of those were previously known to be directly activated . A computer algorithm was used to identify potential activator binding sites located upstream of the remaining 19 promoters of this subset, and conventional genetic and biochemical approaches were applied to test whether these sites are critical for activation by the homologous MarA, SoxS and Rob transcriptional activators . Only seven out of the 19 promoters were found to be activated when fused to lacZ and tested as single lysogens . All seven contained an essential activator binding site . The remaining promoters were insensitive to stimulation by the inducers suggesting that the great majority of elevated microarray transcripts either were misidentified or resulted from indirect effects requiring sequences outside of the promoter region . We estimate that the total number of directly activated promoters in the regulon is less than 40.

Mol Microbiol, 2002 Jun, 44(6), 1599 - 610
CsrA regulates glycogen biosynthesis by preventing translation of glgC in Escherichia coli; Baker CS et al.; The carbon storage regulatory system of Escherichia coli controls the expression of genes involved in carbohydrate metabolism and cell motility . CsrA binding to glgCAP transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay . CsrB RNA functions as an antagonist of CsrA by sequestering this protein and preventing its action . In this paper, we elucidate further the mechanism of CsrA-mediated glgC regulation . Results from gel shift assays demonstrate that several molecules of CsrA can bind to each glgC transcript . RNA footprinting studies indicate that CsrA binds to the glgCAP leader transcript at two positions . One of these sites overlaps the glgC Shine-Dalgarno sequence, whereas the other CsrA target is located further upstream in an RNA hairpin . Results from toeprint and cell-free translation experiments indicate that bound CsrA prevents ribosome binding to the glgC Shine-Dalgarno sequence and that this reduces GlgC synthesis . The effect of two deletions in the upstream binding site was examined . Both of these deletions reduced, but did not eliminate, CsrA binding in vitro and CsrA-dependent regulation in vivo . Our findings establish that bound CsrA inhibits initiation of glgC translation, thereby reducing glycogen biosynthesis . This inhibition of translation probably contributes to destabilization of the glgC transcript that was observed previously.

Mol Microbiol, 2002 Jun, 44(6), 1533 - 50
Characterization and evidence of mobilization of the LEE pathogenicity island of rabbit-specific strains of enteropathogenic Escherichia coli; Tauschek M et al.; We have characterized the LEE pathogenicity islands (PAIs) of two rabbit-specific strains of enteropathogenic E . coli (REPEC), 83/39 (serotype O15:H-) and 84/110-1 (O103:H2), and have compared them to homologous loci from the human enteropathogenic and enterohaemorrhagic E . coli strains, E2348/69 and EDL933, and another REPEC strain, RDEC-1 . All five PAIs contain a 34 kb core region that is highly conserved in gene order and nucleotide sequence . However, the LEE of 83/39 is significantly larger (59 540 basepairs) than those of the human strains, which are less than 44 kb, and has inserted into pheU tRNA . The regions flanking the 34 kb core of 83/39 contain homologues of two putative virulence determinants, efa1/lifA and senA . The LEE of 84/110-1 is approximately 85 kb and is located at pheV tRNA . Its core is almost identical to those of 83/39 and RDEC-1, apart from a larger espF gene, but its flanking regions contain trcA, a putative virulence determinant of EPEC . All three REPEC LEE PAIs contain a gene for an integrase, Int-phe . The LEE PAI of 84/110-1 is also flanked by short direct repeats (representing the 3'-end of pheV tRNA), suggesting that it may be unstable . To investigate this possibility, we constructed a LEE::sacB derivative of 84/110-1 and showed that the PAI was capable of spontaneous deletion . We also showed that Int-phe can mediate site-specific integration of foreign DNA at the pheU tRNA locus of E . coli DH1 . Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI.

Mol Microbiol, 2002 Jun, 44(6), 1517 - 31
CpeR is an activator required for expression of the phycoerythrin operon (cpeBA) in the cyanobacterium Fremyella diplosiphon and is encoded in the phycoerythrin linker-polypeptide operon (cpeCDESTR); Cobley JG et al.; In the cyanobacteria, phycobilisomes are assembled from (alphabeta)(6) hexamers of the coloured phycobiliproteins, allophycocyanin, phycocyanin and phycoerythrin (PE) . The precise architecture of the phycobilisome is determined by the various colourless linker proteins that bind to the biliprotein hexamers . Genes for beta and alpha subunits of PE make up one operon (cpeBA), whereas genes for PE-associated linker polypeptides are in a second operon . In the chromatically adapting cyanobacterium Fremyella diplosiphon green light is required for the transcription of both cpeBA and the operon encoding the PE-associated linkers (cpeCDE) . From the genome of F . diplosiphon we have identified an open reading frame, cpeR, which, when expressed from a shuttle plasmid, is capable of suppressing various mutations that cause a decrease in PE synthesis . The introduction of a shuttle plasmid bearing cpeR+ into wild-type F . diplosiphon caused PE expression in red light . Fremyella diplosiphon cpeR-, created by in vitro mutagenesis and in vivo homologous recombination, is fully PE and, in this strain, cpeCDE is transcribed normally whereas the transcript from cpeBA is undetectable . Polymerase chain reaction (PCR) amplification of cDNA showed that cpeR is transcribed as part of the cpeCDE operon on an extended transcript . As CpeR is an activator required for expression of the cpeBA operon, we propose that at the onset of green light the operons cpeCDESTR and cpeBA are expressed in series as a genetic cascade.

Mol Microbiol, 2002 Jun, 44(6), 1483 - 92
Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface; Claessen D et al.; The filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle . After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores . The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer . We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes . The rdl genes are expressed in growing aerial hyphae but not in spores . Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer . Disruption of both rdlA and rdlB in S . coelicolor and S . lividans (DeltardlAB strains) did not affect the formation and differentiation of aerial hyphae . However, the characteristic rodlet layer was absent . Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid . Attachment to this substratum was greatly reduced in the DeltardlAB strains . Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.

Mol Microbiol, 2002 Jun, 44(6), 1441 - 54
Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp; Voncken F et al.; A mitochondrial-type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp . L2 . Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes . Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP--similar to isolated mitochondria of yeast and animals . Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp . L2 belongs to the family of veritable mitochondrial-type AACs . Hydrogenosome-bearing anaerobic ciliates possess clearly distinct mitochondrial-type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins . Also, phylogenetic analysis of genes encoding mitochondrial-type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.

Mol Microbiol, 2002 Jun, 44(6), 1429 - 40
Eclipse period without sequestration in Escherichia coli; Olsson J et al.; The classical Meselson-Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC . Populations of bacteria growing exponentially in heavy ((15)NH(4)+ and (13)C(6)-glucose) medium were shifted to light ((14)NH(4)+ and (12)C(6)-glucose) medium . The HH-, HL- and LL-DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene-specific probes . The eclipse period, estimated from the kinetics of conversion of HH-DNA to HL- and LL-DNA, turned out to be 0.60 generation times for the wild-type strain . This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured . For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively . Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised . The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.

Mol Microbiol, 2002 Jun, 44(6), 1421 - 8
Cooperation and competition in mismatch repair: very short-patch repair and methyl-directed mismatch repair in Escherichia coli; Bhagwat AS et al.; In Escherichia coli and related enteric bacteria, repair of base-base mismatches is performed by two overlapping biochemical processes, methyl-directed mismatch repair (MMR) and very short-patch (VSP) repair . While MMR repairs replication errors, VSP repair corrects to C*G mispairs created by 5-methylcytosine deamination to T . The efficiency of the two pathways changes during the bacterial life cycle; MMR is more efficient during exponential growth and VSP repair is more efficient during the stationary phase . VSP repair and MMR share two proteins, MutS and MutL, and although the two repair pathways are not equally dependent on these proteins, their dual use creates a competition within the cells between the repair processes . The structural and biochemical data on the endonuclease that initiates VSP repair, Vsr, suggest that this protein plays a role similar to MutH (also an endonuclease) in MMR . Biochemical and genetic studies of the two repair pathways have helped eliminate certain models for MMR and put restrictions on models that can be developed regarding either repair process . We review here recent information about the biochemistry of both repair processes and describe the balancing act performed by cells to optimize the competing processes during different phases of the bacterial life cycle.

Int Arch Allergy Immunol, 2002 Jun, 128(2), 105 - 14
IgE reactivity to profilin in pollen-sensitized subjects with adverse reactions to banana and pineapple; Reindl J et al.; BACKGROUND: The so-called 'latex-fruit syndrome' is a well-documented phenomenon in cross-reactive allergies . By contrast, there is a lack of information about allergy to exotic fruits in patients with a predominant pollen sensitization . Since the ubiquitous protein profilin has been identified as an allergen in natural rubber latex as well as in pollen-related foods, the aim of this study was to investigate the role of profilin in allergy to certain exotic fruits . METHODS: Recombinant profilins from banana and pineapple were cloned by a PCR technique after isolation of total RNA using degenerated profilin-specific primers . The unknown 5' ends of copy DNA (cDNA) were identified by rapid amplification of 5'cDNA ends (5'-RACE) and expression in Escherichia coli BL21(DE3) cells . The recombinant profilins were purified by affinity chromatography using poly-(L)-proline as the solid phase . IgE-binding capabilities were characterized by means of immunoblot and Enzyme Allergosorbent Test (EAST) . The cross-reactivity to birch pollen profilin and latex profilin was studied by EAST as well as by immunoblot inhibition experiments . RESULTS: Both banana and pineapple profilin were found to consist of 131 amino acid residues with high amino acid sequence identity to known allergenic pollen and food profilins (71-84%) . IgE binding to the recombinant profilins was observed in 7/16 sera from subjects with suspected banana allergy (44%) and in 8/19 sera from subjects with suspected pineapple allergy (42%) . Inhibition experiments indicated similar IgE reactivity of natural and recombinant allergens . In addition, high cross-reactivity to birch pollen profilin Bet v 2 and latex profilin Hev b 8 was demonstrated by immunoblot inhibition as well as EAST inhibition experiments . CONCLUSIONS: Since a high IgE-binding prevalence of about 40% was obtained in both banana and pineapple allergy, we conclude that profilin is an important mediator of IgE cross-reactivity between pollen and exotic fruits .

Int Arch Allergy Immunol, 2002 Jun, 128(2), 90 - 6
Cloning, isolation, and IgE-binding properties of Helix aspersa (brown garden snail) tropomyosin; Asturias JA et al.; BACKGROUND: Gastropod consumption is quite frequent in the Mediterranean countries and cross-reactivities with crustaceans have been described, but the mechanism of this allergenic cross-reactivity has not been studied in detail . This study aimed to produce recombinant Helix aspersa (brown garden snail) tropomyosin and investigate its implication for cross-reactivity among invertebrates . METHODS: A tropomyosin-specific cDNA encoding H . aspersa tropomyosin was synthetized, and recombinant allergen was overexpressed in Escherichia coli as nonfusion protein . IgE-binding reactivity was studied by immunoblotting and immunoblot inhibition experiments with sera from snail-allergic patients . RESULTS: Cloned brown garden snail tropomyosin shares high homology with other edible mollusk tropomyosins (84-69% identity) as well as with those from arthropods (65-62%), and less homology with vertebrate ones (56% identity) . Tropomyosin reacted with 18% of the sera from patients with snail allergy . Inhibition experiments, using natural and recombinant tropomyosins, showed different degrees of cross-reactivity between invertebrate tropomyosins . Sera from snail-allergic subjects recognized tropomyosins in both mollusks and crustacean extracts . CONCLUSIONS: Tropomyosin represents a minor allergen in snail extracts, but it is clearly involved in invertebrate cross-reactivity .

J Pharmacol Exp Ther, 2002 Jul, 302(1), 111 - 8
A novel cAMP-stimulated pathway in protein phosphatase 2A activation; Feschenko MS et al.; Elevated cAMP in NRK-52E and L6 cells causes a marked reduction in the phosphorylation of numerous phosphoproteins, as detected initially with phosphoserine-specific antibodies . Here, we show that elevation of cAMP in NRK cells by forskolin/3-isobutyl-1-methylxanthine (IBMX) treatment decreased phosphorylation of substrates for different protein kinases, pointing to a common protein phosphatase as a target for cAMP-dependent regulation . Forskolin/IBMX treatment completely dephosphorylated a selective protein phosphatase 2A (PP2A) substrate, elongation factor-2 (EF-2), at its Ca(2+) calmodulin-dependent kinase site, and decreased phosphorylation of substrates for cyclin-dependent kinases, including retinoblastoma (Rb) protein . As reported before, forskolin/IBMX also decreased phosphorylation of a protein kinase C substrate, the Na,K-ATPase . The cAMP-stimulated dephosphorylation was blocked by the protein phosphatases 1 (PP1) and PP2A inhibitor okadaic acid at concentrations selective for PP2A but was not blocked by tautomycin at concentrations selective for PP1 . The data implicate PP2A as a cAMP-activated phosphatase . Contrary to expectation, we found evidence that cAMP-dependent activation of PP2A did not depend on protein kinase A (PKA) . Pretreatment of cells with the PKA inhibitor H89 abolished PKA activity measured in cell extracts and significantly decreased cAMP-activated phosphorylation of a known PKA substrate, ARPP-19, in cells, but failed to block the cAMP-stimulated dephosphorylation of EF-2, Rb, and other proteins . This novel pathway of PP2A activation, acting on the time scale of minutes, represents yet another example of a cAMP-mediated, PKA-independent signaling mechanism . Because PP2A is active toward a variety of endogenous substrates, cAMP-stimulated dephosphorylation may have complicated the interpretation of many prior studies.

J Biol Chem, 2002 Sep 6, 277(36), 32438 - 44 Epub 2002 Jun 13.
Specificity of the stimulatory interaction between chromosomal HMGB proteins and the transcription factor Dof2 and its negative regulation by protein kinase CK2-mediated phosphorylation; Krohn NM et al.; The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that can contribute to transcriptional control by interaction with certain transcription factors . Using the transcription factor Dof2 and five different maize HMGB proteins, we have examined the specificity of the HMGB-transcription factor interaction . The HMG-box DNA binding domain of HMGB1 is sufficient for the interaction with Dof2 . Although all tested HMGB proteins can interact with Dof2, the various HMGB proteins stimulate the binding of Dof2 to its DNA target site with different efficiencies . The HMGB5 protein is clearly the most potent facilitator of Dof2 DNA binding . Maximal stimulation of the DNA binding by the HMGB proteins requires association of HMGB and Dof2 prior to DNA binding . HMGB5 and Dof2 form a ternary complex with the DNA, but within the protein-DNA complex the interaction of HMGB5 and Dof2 is different from that in solution, as in contrast to the proteins in solution, they cannot be cross-linked with glutaraldehyde when bound to DNA . Phosphorylation of HMGB1 by protein kinase CK2 abolishes the interaction with Dof2 and the stimulation of Dof2 DNA binding . These findings indicate that transcription factors may recruit certain members of the HMGB family as assistant factors.

J Biol Chem, 2002 Sep 6, 277(36), 33115 - 26 Epub 2002 Jun 13.
Structural plasticity and noncovalent substrate binding in the GroEL apical domain . A study using electrospay ionization mass spectrometry and fluorescence binding studies; Ashcroft AE et al.; Advances in understanding how GroEL binds to non-native proteins are reported . Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I . Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH . To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied . Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions . Fluorescence binding studies show that the apical domain can fully bind both peptides independently . No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites . Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.

J Biol Chem, 2002 Sep 6, 277(36), 33148 - 52 Epub 2002 Jun 13.
Genetic analysis of GalR tetramerization in DNA looping during repressosome assembly; Geanacopoulos M et al.; The Gal repressosome is a nucleoprotein complex consisting of 2 GalR dimers, 1 HU, and 1 DNA loop, which represses the transcription of the gal operon . We have adopted a structure-based genetic approach to complement ongoing physical studies of the complex . Homology-based and subsequent alanine-scanning mutageneses suggest that five residues in the DNA-distal subdomain of GalR dimer are important for repressosome formation . A further analysis of these and intragenic suppressors of looping-defective GalR mutants as well as gain-of-function mutants that permit repressosome assembly in the absence of HU show that GalR dimers contact each other in the repressosome in a partially stacked configuration.

Infect Immun, 2002 Jul, 70(7), 3930 - 4
Magnesium uptake by CorA is essential for viability of the gastric pathogen Helicobacter pylori; Pfeiffer J et al.; We show here that Mg(2+) acquisition by CorA is essential for Helicobacter pylori in vitro, as corA mutants did not grow in media without Mg(2+) supplementation . Complementation analysis performed with an Escherichia coli corA mutant revealed that H . pylori CorA transports nickel and cobalt in addition to Mg(2+) . However, Mg(2+) is the dominant CorA substrate, as the corA mutation affected neither cobalt and nickel resistance nor nickel induction of urease in H . pylori . The drastic Mg(2+) requirement (20 mM) of H . pylori corA mutants indicates that CorA plays a key role in the adaptation to the low-Mg(2+) conditions predominant in the gastric environment.

Infect Immun, 2002 Jul, 70(7), 3382 - 8
Borrelia burgdorferi population kinetics and selected gene expression at the host-vector interface; Hodzic E et al.; By using real-time quantitative PCR, the population dynamics and gene transcription of Borrelia burgdorferi were examined in ticks and skin of mice during acquisition of the infection from mice by ticks and during transmission of the infection from ticks to mice . Population dynamics were determined by using a flaB DNA target . A quantitative analysis of flaB, ospA, ospC, dbpA, and arp transcription was also performed . The results revealed that both uninfected larval and nymphal Ixodes scapularis ticks acquired B . burgdorferi as early as 1 day after attachment and that the sizes of spirochete populations within ticks increased during feeding . In addition, all gene targets revealed that there was RNA transcription during feeding . Similar events occurred within infected nymphal ticks feeding on uninfected hosts . Transmission from infected nymphal ticks to mice could be detected within 1 day after attachment . Analysis of skin during the first 3 days after attachment of infected ticks revealed rising numbers of spirochetes but minimal gene transcription . In contrast, the skin of mice with established infections revealed static populations of spirochetes and active but stable transcription of flaB, ospC, dbpA, and arp . There were consistent reductions in the number of spirochetes in the skin at the tick attachment sites compared to the number of spirochetes in the skin at nontick sites, but there were no differences in gene expression between tick and nontick skin sites . Evidence of ospA transcription in skin could be found 1 day after tick attachment but not thereafter.

EMBO J, 2002 Jun 17, 21(12), 3182 - 91
A novel uracil-DNA glycosylase with broad substrate specificity and an unusual active site; Sartori AA et al.; Uracil-DNA glycosylases (UDGs) catalyse the removal of uracil by flipping it out of the double helix into their binding pockets, where the glycosidic bond is hydrolysed by a water molecule activated by a polar amino acid . Interestingly, the four known UDG families differ in their active site make-up . The activating residues in UNG and SMUG enzymes are aspartates, thermostable UDGs resemble UNG-type enzymes, but carry glutamate rather than aspartate residues in their active sites, and the less active MUG/TDG enzymes contain an active site asparagine . We now describe the first member of a fifth UDG family, Pa-UDGb from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum, the active site of which lacks the polar residue that was hitherto thought to be essential for catalysis . Moreover, Pa-UDGb is the first member of the UDG family that efficiently catalyses the removal of an aberrant purine, hypoxanthine, from DNA . We postulate that this enzyme has evolved to counteract the mutagenic threat of cytosine and adenine deamination, which becomes particularly acute in organisms living at elevated temperatures.

EMBO J, 2002 Jun 17, 21(12), 3148 - 59
The DnaC helicase loader is a dual ATP/ADP switch protein; Davey MJ et al.; Helicases are transferred to replication origins by helicase loading factors . The Escherichia coli DnaC and eukaryotic Cdc6/18 helicase loaders contain ATP sites and are both members of the AAA+ family . One might expect that ATP is required for helicase loading; however, this study on DnaC illustrates that ATP is not actually needed for DnaC to load helicase onto single-strand DNA (ssDNA) . In fact, it seems to be a paradox that after transfer of helicase to DNA, DnaC-ATP inhibits helicase action . In addition, ATP is required for DnaC function at an early step in oriC replication in which ATP stimulates ssDNA binding by DnaC, leading to expansion of the ssDNA bubble at the origin . Two cofactors, ssDNA and DnaB, trigger hydrolysis of ATP, converting DnaC to the ADP form that no longer inhibits DnaB . These observations have led to the idea that DnaC is a 'dual' switch protein, where both the ATP and the ADP forms are sequentially required for replication . This dual switching process may underlie the sensitivity of DnaB to even small fluctuations in DnaC levels.

EMBO J, 2002 Jun 17, 21(12), 2958 - 67
A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes; Dudek J et al.; Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes . This pancreatic DnaJ-like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK-like proteins, such as BiP . The pancreatic endoplasmic reticulum contains a second DnaJ-like membrane protein, which had been termed Mtj1p in mouse . Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP . In addition to a lumenal J-domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation . The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p . We propose that Mtj1p represents a novel type of co-chaperone, mediating transmembrane recruitment of DnaK-like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK-like chaperones of the endoplasmic reticulum.

EMBO J, 2002 Jun 17, 21(12), 2866 - 76
Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain; Kalodimos CG et al.; The lac repressor-operator system is a model system for understanding protein-DNA interactions and allosteric mechanisms in gene regulation . Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive . Here we present the first high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator 01 . The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites . Specific recognition is accomplished by a combination of elongation and twist by 48 degrees of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA . The set of recognition mechanisms involved in the lac repressor-operator system is unique among other protein-DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.

Food Chem Toxicol, 2002 Jul, 40(7), 919 - 23
Effect of an extract of cauliflower (leaf) on the labeling of blood elements with technetium-99m and on the survival of Escherichia coli AB1157 submitted to the treatment with stannous chloride; Lima EA et al.; The labeling of red blood cells (RBC) with technetium-99m (99mTc) depends on a reducing agent and stannous chloride (SnCl(2)) and is widely utilized . This labeling may also be altered by drugs, and SnCl(2) reduces the survival of Escherichia coli cultures . Cauliflower (Brassica oleracea L . var . botrytis) is used in folk medicine and we evaluated its influence on (i) the labeling of blood elements with 99mTc, and (ii) on the survival of an E . coli strain . Blood was withdrawn from rats that drank the extract of cauliflower (15 days) . Blood was incubated with SnCl(2) and with 99mTc, as sodium pertechnetate, centrifuged and plasma (P) and RBC were isolated . Samples of P and RBC were also precipitated, centrifuged and soluble and insoluble fractions isolated . E . coli culture was treated with SnCl(2) in the presence of cauliflower . The extract of cauliflower did not alter the fixation of 99mTc on blood fractions; however, it abolished the lethal effect of SnCl(2) on the E . coli culture . We suggest that the substances present in the extract of cauliflower probably, would have redox property with different mechanisms of action . The oxidant action of the substances of the extract would not be strong enough to oxidise the stannous ions altering the 99mTc-labeling . However, the referred substances could oxidise these ions sufficiently to protect the E . coli culture against the lethal effect of the stannous ion.

Cancer Lett, 2002 Sep 26, 183(2), 155 - 62
Recombinant arginine deiminase as a potential anti-angiogenic agent; Beloussow K et al.; Arginine deiminase (ADI), isolated from Mycoplasma cell extracts, has been suggested to inhibit endothelial cell growth in vitro . However, anti-angiogenic activity by ADI has not yet been demonstrated . In this study, we investigated the in vitro effect of recombinant ADI (rADI) on the growth, migration, and tube formation of human umbilical vein endothelial (HUVE) cells . Mycoplasma arginine deiminase was cloned by PCR and the rADI was expressed in Escherichia coli . and purified to near homogeneity . The purified recombinant protein was found to have characteristics similar to those of the native enzyme: molecular weight (48 kDa) and specific enzymatic activity of converting L-arginine into citrulline (32.7 U/mg) . This recombinant enzyme also exhibited an inhibitory effect on the growth of HUVE cells . The anti-angiogenic activity was demonstrated by in vitro inhibition of migration into the scratch wounded area in HUVE cell monolayers and the inhibition of microvessel tube-like formation of HUVE cells on Matrigel-coated surfaces . These results suggest that arginine deiminase is a potential inhibitor for angiogenesis, and that arginine concentrations may play an important role in regulating neovascularization.

Gene Expr, 2002, 10(3), 109 - 14
Genetic analysis of the basis of translation in the -1 frame of an unusual non-ORF sequence isolated from phage display; Zemsky J et al.; An unusual peptide-encoding sequence, called H10, and several derivatives of this sequence were previously isolated from a random peptide library screened by phage display during drug discovery protocols . The H10 family of sequences had the unusual property of being expressed despite the absence of an open reading frame . When these sequences were fused to a reporter lacZ gene in all three frames, beta-galactosidase was expressed not only from the parental non-open reading frame, consistent with the original isolations, but also from the frame -1 to the parental . This unexpected translation in a second reading frame could result from either a recoding event or from an internal translation initiation event . In order to elucidate which type of event, a genetic approach was selected to eliminate a potential downstream initiator site within the H10 sequence . This report provides strong evidence that translation in the -1 frame in this family of sequences is indeed originating from a downstream translation initiation event . Unexpectedly, the mutation eliminating the downstream initiation event in the -1 frame simultaneously elevated expression in the original non-open reading frame.

Gene, 2002 May 15, 290(1-2), 181 - 91
The African trypanosome cyclophilin A homologue contains unusual conserved central and N-terminal domains and is developmentally regulated; Pelle R et al.; We have cloned and characterized the homologue of cyclophilin A (CypA) from Trypanosoma brucei brucei, Trypanosoma congolense, Trypanosoma evansi and Trypanosoma vivax . The 1-kilobase African trypanosome CypA complementary DNA contains an open reading frame of 531 base pairs, corresponding to 177 amino acids with a calculated molecular weight of 18,700 . The CypA gene is present at one copy/haploid genome in T . brucei, T . congolense and T . vivax and is located on large chromosomes (>3 Mb) in T . brucei . CypA is differentially transcribed in African trypanosomes and is localized in the cytosol as well as in the flagellum . It is also detected in the supernatant of in vitro cultivated parasites . The African trypanosome CypA is unique due to a ten amino acid residue N-terminus extension and a block that includes a three amino acid insertion around position 100 that might result in a differently structured surface . Wild-type recombinant CypA and several mutants were over-expressed in Escherichia coli and purified to >98% homogeneity . Antisera from cattle immunized with a trypanosome fraction containing immunosuppressive activity react strongly against CypA . These data indicate that trypanosome CypA might play an important role in the establishment and maintenance of infections in susceptible animals.

Int J Parasitol, 2002 Jun 15, 32(7), 877 - 87
Characterisation of a novel transporter from Cryptosporidium parvum; LaGier MJ et al.; P-ATPases are transmembrane proteins that hydrolyse ATP to drive cations or other substances across biomembranes . In this study we present the characterisation of a novel P-ATPase from the apicomplexan parasite Cryptosporidium parvum (CpATPase3), an opportunistic pathogen in autoimmune deficiency syndrome patients, for which no treatment is available . The single copy gene encodes 1488 amino acids, predicting a protein of 169.7 kDa . Primary sequence analysis, as well as an extensive phylogenetic reconstruction, indicated CpATPase3 belongs to a novel class of eukaryotic-specific P-ATPases (Type V) with undefined substrate preferences . Transcription and translation of the gene were confirmed by reverse-transcriptase polymerase chain reaction, and Western blot analysis of sporozoite protein extracts . Immunofluorescent microscopy of C . parvum sporozoites using rabbit antiserum raised against a glutathione-S-transferase-CpATPase3 (GST-ATP3) fusion protein showed that the parasite transporter was located within the apical complex associated with the parasite host-invasion machinery . Overall, these data demonstrate the diversity of C . parvum transporters, and raise the potential of Type V P-ATPases as apicomplexan-specific drug targets.

Int J Parasitol, 2002 Jun 15, 32(7), 833 - 42
A cement protein of the tick Rhipicephalus appendiculatus, located in the secretory e cell granules of the type III salivary gland acini, induces strong antibody responses in cattle; Bishop R et al.; Protein components of the cement cone of ixodid ticks are candidates for inclusion in vaccines against tick infestation, since they are essential for tick attachment and feeding . We describe here the cloning of a cDNA encoding a 36 kDa protein, designated Rhipicephalus Immuno-dominant Molecule 36 (RIM36), present in salivary glands and the cement cone material secreted by Rhipicephalus appendiculatus . The 334-amino-acid sequence of RIM36 has a high content of glycine, serine and proline . The protein contains a predicted N-terminal signal peptide and two classes of glycine-rich amino acid repeats, a GL{G/Y/S/F/L} tripeptide and a GSPLSGF septapeptide . Comparison of genomic and cDNA sequences reveals a 597 bp intron within the 3' end of the RIM36 gene . Immuno-electron microscopy demonstrates that RIM36 is predominantly located in the e cell granules of the type III salivary gland acini . An Escherichia coli recombinant form of the proline-rich C-terminal domain of RIM36 reacts with antisera from Bos indicus cattle, either experimentally infested with R . appendiculatus, or exposed to ticks in the field . The 36 kDa protein is strongly recognised on Western blots of salivary gland lysates and soluble extracts of purified R . appendiculatus cement cones by polyclonal antibodies generated against recombinant RIM36, and by antisera from cattle experimentally infested with ticks . The data indicate that this tick cement component is a target of strong antibody responses in cattle exposed to feeding ticks.

FEBS Lett, 2002 Apr 24, 517(1-3), 239 - 44
Methionine oxidation inhibits fibrillation of human alpha-synuclein in vitro; Uversky VN et al.; We examined the effect of methionine oxidation of human recombinant alpha-synuclein on its structural properties and propensity to fibrillate . Both oxidized and non-oxidized alpha-synucleins were natively unfolded under conditions of neutral pH, with the oxidized protein being slightly more disordered . Both proteins adopted identical partially folded conformations under conditions of acidic pH . The fibrillation of alpha-synuclein at neutral pH was completely inhibited by methionine oxidation . This inhibitory effect was eliminated at low pH . The addition of oxidized alpha-synuclein to the unoxidized form led to a substantial inhibition of alpha-synuclein fibrillation.

FEBS Lett, 2002 Apr 24, 517(1-3), 211 - 4
A cell-free protein synthesis system as an investigational tool for the translation stop processes; Kang TJ et al.; Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin . Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE . Optimal conditions were established for correct stop and programmed suppressions . Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.

FEBS Lett, 2002 Apr 24, 517(1-3), 110 - 4
The biosynthetic pathway for lipoic acid is present in plastids and mitochondria in Arabidopsis thaliana; Yasuno R et al.; In eukaryotes, the biosynthetic pathway for lipoic acid is present in mitochondria . However, it has been hypothesized that, in plants, the biosynthetic pathway is present in plastids in addition to mitochondria . In this study, Arabidopsis thaliana LIP1p cDNA for a plastidial form of lipoic acid synthase has been identified . We show that it encodes a lipoic acid synthase by demonstrating its ability to complement an Escherichia coli mutant lacking lipoic acid synthase activity . We also show that LIP1p is targeted to chloroplasts . These findings suggest that the biosynthetic pathway for lipoic acid is present not only in mitochondria but also in plastids.

FEBS Lett, 2002 Apr 24, 517(1-3), 79 - 82
Cold stress decreases the capacity for respiratory NADH oxidation in potato leaves; Svensson AS et al.; Cold stress effects on the expression of genes for respiratory chain enzymes were investigated in potato (Solanum tuberosum L., cv . Desiree) leaves . The nda1 and ndb1 genes, homologues to genes encoding the non-proton-pumping respiratory chain NADH dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton-pumping NADH dehydrogenase (complex I) . Using a real-time PCR system, we demonstrate a specific and gradual decrease of the NDA1 transcript after exposing the plants to 5 degrees C . After 6 days of cold treatment the NDA1 transcript abundance is 10% of the original level . This decrease is accompanied by specific decreases of immunodetected NDA protein and internal rotenone-insensitive NADH oxidation in mitochondria isolated from cold-treated plants . The alternative oxidase is not cold-induced neither at the protein nor at the activity level . The results are discussed in relation to the recent finding that the nda1 gene expression is completely light-dependent.

FEBS Lett, 2002 Apr 24, 517(1-3), 50 - 4
A comparative structural and functional analysis of cytochrome cM cytochrome c6 and plastocyanin from the cyanobacterium Synechocystis sp . PCC 6803; Molina-Heredia FP et al.; Cytochrome cM is a new c-class photosynthetic haem protein whose physiological role is still unknown . It has been proposed previously that cytochrome cM can replace cytochrome c6 and plastocyanin in transferring electrons between the two membrane complexes cytochrome b6-f and photosystem I in organisms growing under stress conditions . The experimental evidence herein provided allows us to discard such a hypothesis . We report a procedure to overexpress cytochrome cM from the cyanobacterium Synechocystis sp . PCC 6803 in Escherichia coli cells in mg quantities . This has allowed us to perform a comparative laser flash-induced kinetic analysis of photosystem I reduction by the three metalloproteins from Synechocystis . The bimolecular rate constant for the overall reaction is up to 100 times lower with cytochrome cM than with cytochrome c6 or plastocyanin . In addition, the redox potential value and surface electrostatic potential distribution of cytochrome cM are quite different from those of cytochrome c6 and plastocyanin . These findings strongly indicate that cytochrome cM cannot be recognised by and interact with the same redox partners as the other two metalloproteins.

Antiviral Res, 2002 Jun, 54(3), 163 - 74
Establishment and use of a cell line expressing HSV-1 thymidine kinase to characterize viral thymidine kinase-dependent drug-resistance; Kim JH et al.; To understand the mechanisms of antiviral drug resistance and to have a system to examine the cytotoxicity of herpes simplex virus type 1 (HSV-1) inhibitors that are thymidine kinase (TK)-dependent, we have constructed a plasmid pFTK1 by inserting a DNA fragment containing the TK gene of HSV-1 strain F into the eukaryotic expression vector pcDNA3.1/His A . TK-deficient 143B cells were transfected with this vector and neomycin-resistant cells were selected . Cell survival in HAT medium and TK activity of the cell lysates were examined to ascertain HSV-1 TK expression . A cell line expressing the viral TK gene, FTK143B (FTK), was established and used for characterization of two laboratory-derived TK-deficient drug-resistant HSV-1 mutants of strain F . The antiviral activities of several drugs, mostly nucleoside analogues, were compared in the Vero, 143B and FTK cell culture systems . We showed that both mutant viruses lost their resistance to acyclovir and to other HSV-1 TK-dependent compounds in FTK cells but not in Vero and 143B cells . Significantly increased cytotoxicity of ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine was also observed in the FTK cells . This HSV-1 TK gene-transfected cell model is a useful tool to rapidly determine HSV-1 drug resistance at the viral TK level.

Biophys Chem, 2002 Mar 28, 95(3), 253 - 72
Structural basis for the unusual properties of 2',5' nucleic acids and their complexes with RNA and DNA; Premraj BJ et al.; To provide insights into the unusual properties of 2',5' nucleic acids (iso nucleic acids), that includes their rejection by Nature as information molecules, modeling studies have been carried out to examine if they indeed possess the stereochemical ability to form helical duplexes and triplexes, just as their 3',5' linked constitutional isomers . The results show that the formation of helical duplexes with 2',5' linkages demands a mandatory displacement of the Watson and Crick base pairs from the helical axis, as a direct consequence of the lateral shift of the sugar-phosphate backbone from the periphery towards the interior of the helix . Thus, both duplexes and triplexes formed with a 2',5'-sugar-phosphate backbone possess this intrinsic trait, manifested normally only in A type duplexes of DNA and RNA . It was found that only a 10-fold symmetric parallel triplex with isomorphous T.AT triplets is stereochemically favorable for isoDNA with 'extended' nucleotide repeats, unlike the 12-fold symmetric triplex favored by DNA . The wider nature of a 12-fold triplex, concomitant with mandatory slide requirement for helix formation in isoDNA, demands even larger displacement, especially with 'extended' nucleotide structural repeats, thereby violating symmetry . However, a symmetric triplex possessing higher twist, can be naturally formed for isoDNA with a 'compact' nucleotide repeat . Two nanosecond molecular dynamics simulation of a 2',5'-B DNA duplex, formed with an intrinsic base pair displacement of -3.3 A, does not seem to favor a total transition to a typical A type duplex, although enhanced slide, X-displacement, decrease in helical rise and narrowing of the major groove during simulation seem to indicate a trend . Modeling of the interaction between the chimeric isoDNA.RNA duplex and E . coli RNase H has provided a structural basis for the inhibitory action of the enzyme . Interaction of residues Gln 80, Trp 81, Asn 16 and Lys 99, of E . coli RNase H with DNA of the DNA.RNA hybrid, are lost when the DNA backbone is replaced by isoDNA . Based on modeling and experimental observations, it is argued that 2',5' nucleic acids possess restricted conformational flexibility for helical polymorphism . The inability of isoDNA to favor the biologically relevant B form duplex and the associated topological inadequacies related to nucleic acid compaction and interactions with regulatory proteins may be some of the factors that might have led to the rejection of 2',5' links.

J Biochem Biophys Methods, 2002 Apr 18, 51(2), 195 - 201
Addition of Polybrene improves stability of organophosphate hydrolase immobilized in poly(vinyl alcohol) cryogel carrier; Efremenko EN et al.; Organophosphate hydrolase, covalently attached to the beads of poly(vinyl alcohol) cryogel in the presence of Polybrene, was fivefold more stable in 15% (v/v) ethanol solution than the free enzyme . Immobilized biocatalyst, prepared with an addition of Polybrene, retained a half of its initial activity in 50% (v/v) aqueous ethanol solution, 90% of activity during 10 working cycles of Paraoxon hydrolysis and 85% of activity after storage in the 50 mM CHES buffer (pH 9.0) at room temperature for 2 months.

Curr Biol, 2002 Jun 4, 12(11), R399 - 401
Signal transduction: receptor clusters as information processing arrays; Thomason PA et al.; The organization of transmembrane receptors into higher-order arrays occurs in cells as different as bacteria, lymphocytes and neurons . What are the implications of receptor clustering for short-term and long-term signaling processes that occur in response to ligand binding?

Ann Trop Med Parasitol, 2002 Apr, 96(3), 317 - 24
The multiplex-PCR-based detection and genotyping of diarrhoeagenic Escherichia coli in diarrhoeal stools; Matar GM et al.; In several hospitals in Beirut, Lebanon, 77 isolates of Escherichia coli were successfully derived from the stools of patients with diarrhoeal diseases, by culture on MacConkey or MacConkey-sorbitol agar . When the isolates were screened, using a multiplex PCR, 14 (from 14 different patients) were each found positive for one of the various genes defining the enterotoxigenic (five), enteroinvasive (four), enteroaggregative (three) or enteropathogenic (two) groups . Genotyping of these 14 diarrhoeagenic isolates, by pulsed-field gel electrophoresis, indicated that all were genomically distinct with the exception of two of the enteroaggregative isolates (which were of the same genotype) . The E . coli apparently involved in diarrhoeal disease in Beirut therefore belong to at least four different diarrhoeagenic groups and show strain variation within each group . Diarrhoea in the absence of diarrhoeagenic E . coli may be the result of infection with bacteria other than E . coli or viral or parasitic enteropathogens.

Plant J, 2002 Jun, 30(6), 733 - 44
Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast; Franklin S et al.; Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported . Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low . We hypothesized that the inability to achieve high levels of recombinant protein expression in the C . reinhardtii chloroplast was due to the codon bias seen in the C . reinhardtii chloroplast genome . To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C . reinhardtii chloroplast-encoded proteins . We monitored the accumulation of GFP in C . reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C . reinhardtii rbcL 5'- and 3'-UTRs . We compared this expression with the accumulation of GFP in C . reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs . We demonstrate that C . reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains . We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C . reinhardtii chloroplast gene expression.

Plant J, 2002 Jun, 30(6), 613 - 23
Male gametophyte development in bread wheat (Triticum aestivum L.): molecular, cellular, and biochemical analyses of a sporophytic contribution to pollen wall ontogeny; Wang A et al.; Bread wheat (hexaploid AABBDD genome; 16 billion basepairs) is a genetically complex, self-pollinating plant with bisexual flowers that produce short-lived pollen . Very little is known about the molecular biology of its gametophyte development despite a longstanding interest in hybrid seeds . We present here a comprehensive characterization of three apparently homeologous genes (TAA1a, TAA1b and TAA1c) and demonstrate their anther-specific biochemical function . These eight-exon genes, found at only one copy per haploid complement in this large genome, express specifically within the sporophytic tapetum cells . The presence of TAA1 mRNA and protein was evident only at specific stages of pollen development as the microspore wall thickened during the progression of free microspores into vacuolated-microspores . This temporal regulation matched the assembly of wall-impregnated sporopollenin, a phenylpropanoid-lipid polymer containing very long chain fatty alcohols (VLCFAlc), described in the literature . Our results establish that sporophytic genes contribute to the production of fatty alcohols: Transgenic expression of TAA1 afforded production of long/VLCFAlc in tobacco seeds (18 : 1; 20 : 1; 22 : 1; 24 : 0; 26 : 0) and in Escherichia coli (14 : 0; 16 : 0; 18 : 1), suggesting biochemical versatility of TAA1 with respect to cellular milieu and substrate spectrum . Pollen walls additionally contain fatty alcohols in the form of wax esters and other lipids, and some of these lipids are known to play a role in the highly specific sexual interactions at the pollen-pistil interface . This study provides a handle to study these and to manipulate pollen traits, and, furthermore, to understand the molecular biology of fatty alcohol metabolism in general.

Arch Biochem Biophys, 2002 Jul 1, 403(1), 63 - 70
The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor; Kramer G et al.; The molecular chaperone DnaK and trigger factor (TF), a ribosome-associated protein with folding activity, have been implicated in assisting nascent polypeptides to acquire a three-dimensional structure on Escherichia coli ribosomes . We asked whether ribosomes that lack trigger factor would recruit DnaK for synthesis and folding of nascent peptides . For these analyses, translating ribosomes with a homogeneous population of nascent peptides were isolated . Truncated forms of rhodanese and E . coli translation initiation factor 3 (IF3) were generated with tandem rare arginine codons in the coding sequence . These codons cause strong translational pausing during coupled transcription/translation in E . coli extracts, generating nascent polypeptides on ribosomes . Protein synthesis in the TF(-) extract was initiated with biotin-Met-tRNA(f) . Ribosomes with nascent polypeptides were isolated by interaction of the N-terminal biotin with streptavidin on magnetobeads . These translating ribosomes that lack TF contain the molecular chaperone DnaK in considerably less than stoichiometric amounts.

Arch Biochem Biophys, 2002 Jul 1, 403(1), 25 - 34
Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases; Salas JJ et al.; The specificity of plant acyl-acyl carrier protein (ACP) thioesterases is the major determinant of the chain length and level of saturated fatty acids found in most plant tissues . Although these enzymes have been previously characterized from a number of sources, information on kinetic parameters for a wide range of substrates with cloned enzymes is lacking . In the present study the substrate specificity of recombinant FatA thioesterase isoforms from Arabidopsis (AtFatA) and coriander (CsFatA) and FatB from Arabidopsis (AtFatB) have been re-examined with a comprehensive range of substrates including 14:1-ACP and 16:1-ACP . AtFatA displayed the highest catalytic efficiencies (kcat/Km) towards oleoyl-ACP with activities at least 20-fold lower for all other tested substrates and 75-fold lower with palmitoyl-ACP . Both chain length and double bond presence strongly influenced kcat of FatA with minor influence on Km . Arabidopsis FatB substrate specificity was found to differ from previous reports and this difference could be attributed to the influence of ACP structure . FatB activity with palmitoyl-ACP was 2.5-fold higher and the ratio of 16:0-ACP/14:0-ACP hydrolysis was 6.4-fold higher with spinach ACP compared to E . coli ACP . Additionally, the influence of amino acid domains from both AtFatA and AtFatB on their substrate specificity was studied by utilizing a domain-swapping approach . The characterization of the resulting chimeric enzymes pointed to the N-terminus as a determinant of the substrate specificity for both FatA and FatB acyl-ACP thioesterases.

Biochem Biophys Res Commun, 2002 Jun 21, 294(4), 893 - 9
GroEL interacts transiently with oxidatively inactivated rhodanese facilitating its reactivation; Melkani GC et al.; When the enzyme rhodanese was inactivated with hydrogen peroxide (H(2)O(2)), it underwent significant conformational changes, leading to an increased exposure of hydrophobic surfaces . Thus, this protein seemed to be an ideal substrate for GroEL, since GroEL uses hydrophobic interactions to bind to its substrate polypeptides . Here, we report on the facilitated reactivation (86%) of H(2)O(2)-inactivated rhodanese by GroEL alone . Reactivation by GroEL required a reductant and the enzyme substrate, but not GroES or ATP . Further, we found that GroEL interacted weakly and/or transiently with H(2)O(2)-inactivated rhodanese . A strong interaction with rhodanese was obtained when the enzyme was pre-incubated with urea, indicating that exposure of hydrophobic surfaces alone on oxidized rhodanese was not sufficient for the formation of a strong complex and that a more unfolded structure of rhodanese was required to interact strongly with GroEL . Unlike prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that GroEL can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein . Additionally, the mechanism for the GroEL-facilitated reactivation of rhodanese shown here appears to be different than that for the chaperonin-assisted folding of chemically unfolded polypeptides in which a nucleotide and sometimes GroES is required . (c) 2002 Elsevier Science (USA).

Avian Dis, 2002 Apr-Jun, 46(2), 412 - 22
Experimental infection of turkeys with avian pneumovirus and either Newcastle disease virus or Escherichia coli; Turpin EA et al.; Avian pneumoviruses (APVs) are RNA viruses responsible for upper respiratory disease in poultry . Experimental infections are typically less severe than those observed in field cases . Previous studies with APV and Escherichia coli suggest this discrepancy is due to secondary agents . Field observations indicate APV infections are more severe with concurrent infection by Newcastle disease virus (NDV) . In the current study, we examined the role of lentogenic NDV in the APV disease process . Two-week-old commercial turkey poults were infected with the Colorado strain of APV . Three days later, these poults received an additional inoculation of either NDV or E . coli . Dual infection of APV with either NDV or E . coli resulted in increased morbidity rates, with poults receiving APV/NDV having the highest morbidity rates and displaying lesions of swollen infraorbital sinuses . These lesions were not present in the single APV, NDV, or E coli groups . These results demonstrate that coinfection with APV and NDV can result in clinical signs and lesions similar to those in field outbreaks of APV.

Avian Dis, 2002 Apr-Jun, 46(2), 360 - 9
Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome; Pakpinyo S et al.; Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease . In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998 . Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC . Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E . coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures . EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys . AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks . Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria . Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy . EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes . EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus . In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates . These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.

Avian Dis, 2002 Apr-Jun, 46(2), 287 - 97
Safety, immunogenicity, and efficacy of two Escherichia coli cya crp mutants as vaccines for broilers; Peighambari SM et al.; Attenuated derivatives (delta cya delta crp mutants) of an O2 and an O78 avian septicemic Escherichia coli strain were used to immunize broiler chickens by spray to determine the safety, immunogenicity, and efficacy of the derivatives in single- and double-dose regimens . In the safety and immunogenicity studies, groups of 10 chickens were vaccinated by spray (droplet size approximately 20 microm) with the parent E . coli, the mutant organisms, or phosphate-buffered saline (PBS) at 14 days of age and euthanatised 21 days later . There was no deaths or gross pathologic finding in any of the chickens immunized with the vaccine strains . Compared with the levels in chickens exposed to PBS, there were significantly higher levels of immunoglobulin (Ig) G antibody in serum and air sac washings and of IgA antibody in air sac washings in response to the virulent parent strains than to the vaccine strains . In efficacy studies, chickens were immunized with the O2 or the O78 vaccine strain or PBS at day 14 and with the O2 vaccine strain or PBS at days 10 and 14 and challenged with the parent strain 10 days after the last vaccination . There was no significant difference in local IgA and IgG and serum IgG responses between vaccinated and control groups . Chickens vaccinated with the O2 strain, but not the O78 strain, had significantly lower air sac lesion scores compared with those of the unvaccinated groups in both single- and double-dose regimens . We conclude that the mutant O2 strain provided moderate protection against airsacculitis.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 7962 - 7
Regulatory interaction of phosducin-like protein with the cytosolic chaperonin complex; McLaughlin JN et al.; Phosducin and phosducin-like protein (PhLP) bind G protein betagamma subunits and regulate their activity . This report describes a previously uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identification . Chaperonin containing tailless complex polypeptide 1 (CCT), a cytosolic chaperone responsible for the folding of many cellular proteins, binds PhLP with a stoichiometry of one PhLP per CCT complex . Unlike protein-folding substrates of CCT, which interact only in their nonnative conformations, PhLP binds in its native state . Native PhLP competes directly for binding of protein substrates of CCT and thereby inhibits CCT activity . Overexpression of PhLP inhibited the ability of CCT to fold newly synthesized beta-actin by 80% . These results suggest that the interaction between PhLP and CCT may be a means to regulate CCT-dependent protein folding or alternatively, to control the availability of PhLP to modulate G protein signaling.

Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8602 - 7 Epub 2002 Jun 11.
NTP-sensing by rRNA promoters in Escherichia coli is direct; Schneider DA et al.; We showed previously that rrn P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription in vitro . We proposed that this requirement for high initiating NTP concentrations contributes to control of the rrn P1 promoters from the seven Escherichia coli rRNA operons . However, the previous studies did not prove that variation in NTP concentration affects rrn P1 promoter activity directly in vivo . Here, we create conditions in vivo in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from rrn P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter . These results demonstrate that the effect of initiating NTP concentration on rrn P1 promoter activity in vivo is direct . As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by rrn P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from rrnB P1 . We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by rrn P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.

Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8737 - 41 Epub 2002 Jun 11.
SOS-induced DNA polymerases enhance long-term survival and evolutionary fitness; Yeiser B et al.; Escherichia coli encodes three SOS-induced DNA polymerases: pol II, pol IV, and pol V . We show here that each of these polymerases confers a competitive fitness advantage during the stationary phase of the bacterial life cycle, in the absence of external DNA-damaging agents known to induce the SOS response . When grown individually, wild-type and SOS pol mutants exhibit indistinguishable temporal growth and death patterns . In contrast, when grown in competition with wild-type E . coli, mutants lacking one or more SOS polymerase suffer a severe reduction in fitness . These mutants also fail to express the "growth advantage in stationary phase" phenotype as do wild-type strains, instead expressing two additional new types of "growth advantage in stationary phase" phenotype . These polymerases contribute to survival by providing essential functions to ensure replication of the chromosome and by generating genetic diversity.

Nucleic Acids Res, 2002 Jun 15, 30(12), 2692 - 700
DNA binding of the transcription activator protein MelR from Escherichia coli and its C-terminal domain; Howard VJ et al.; MelR is an Escherichia coli transcription factor belonging to the AraC family . It activates expression of the melAB operon in response to melibiose . Full-length MelR (MelR303) binds to two pairs of sites upstream of the melAB transcription start site, denoted sites 1' and 1 and sites 2 and 2', and to a fifth site, R, which overlaps the divergent melR promoter . The C-terminal domain of MelR (MelR173) does not activate transcription . Here we show that, like MelR303, when MelR173 binds to sites 1 and 2 it recruits CRP to bind between these sites . Hence, the C-terminal domain is involved in heterologous interactions . MelR173 binds to the R site, which has 11 of 18 bp identical to sites 1 and 2 but, surprisingly, does not bind to site 1', which has 12 of 18 bp identical, nor to site 2' . Using electrophoretic mobility shift assays, we show that the binding of MelR303 to sites 1' and 2' is due to cooperative binding with the adjacent site . This homologous cooperativity requires the N-terminal domain of the protein . Activation of the melAB promoter requires MelR to occupy site 2', which overlaps the -35 hexamer . Hence, both domains of MelR are required for transcription activation.

Nucleic Acids Res, 2002 Jun 15, 30(12), 2620 - 7
Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA; Shimizu T et al.; Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030-1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro . Mixing of P1 and P2 formed the P1-P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking . This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1-P2)2, assumed from its molecular weight derived from sedimentation analysis . Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain . The protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli equivalent RNA domain, were tested for their function by replacing with the E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits . Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity . The results suggest that formation of the P1-P2 heterodimer is required for subsequent formation of the P0(P1-P2)2 complex and its functional rRNA binding in silkworm ribosomes.

J Biol Chem, 2002 Sep 6, 277(36), 32430 - 7 Epub 2002 Jun 11.
A second UDP-glucose pyrophosphorylase is required for differentiation and development in Dictyostelium discoideum; Bishop JD et al.; Uridine diphosphoglucose pyrophosphorylase (UDPGP) is a developmentally regulated enzyme in Dictyostelium discoideum, which is involved in trehalose, cellulose, and glycogen synthesis . Two independent UDPGP proteins are believed to be responsible for this activity . To determine the relative contributions of each protein, the genes encoding them were disrupted individually . Cells lacking the udpgp1 gene exhibit normal growth and development and make normal levels of cellulose . In agreement with these phenotypes, udpgp1(-) cells still have UDPGP activity, although at a reduced level . This supports the importance of the second UDPGP gene . This newly identified gene, ugpB, encodes an active UDPGP as determined by complementation in Escherichia coli . When this gene is disrupted, cells undergo aberrant differentiation and development ending with small, gnarled fruiting bodies . These cells also have decreased spore viability and decreased levels of glycogen, whose production requires UDPGP activity . These phenotypes suggest that UgpB constitutes the major UDPGP activity produced during development . Sequence analysis of the two UDPGP genes shows that UgpB has higher homology to other eukaryotic UDPGPs than does UDPGP1 . This includes the presence of 5 conserved lysine residues . Udpgp1 only has 1 of these lysines.

Physiol Plant, 2001 Oct, 113(2), 176 - 184
Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli; Marillia EF et al.; A genomic clone encoding a thiohydroximate S-glucosyltransferase (S-GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers . Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening . The genomic clone was 5 896 bp long and contained a 173-bp intron . At least two copies of the S-GT gene were present in B . napus . The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide . The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain . The recombinant protein was expressed in Escherichia coli, and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation . The high thiohydroximate S-GT activity detected from the recombinant protein confirmed that this clone was indeed a S-glucosyltransferase.

Physiol Plant, 2002 Mar, 114(3), 482 - 490
Molecular cloning of a functional protein phosphatase 2C (FsPP2C2) with unusual features and synergistically up-regulated by ABA and calcium in dormant seeds of Fagus sylvatica; Lorenzo O et al.; Phosphorylation/dephosphorylation of proteins is a general mechanism of hormonal signal transduction, including ABA, and serine/threonine protein phosphatases 2C (PP2C, EC 3.1.3.16) have been suggested to play an important role in this process . By means of differential reverse transcriptase-polymerase chain reaction (RT-PCR) and further screening of a cDNA library made from mRNA of ABA-treated Fagus sylvatica L . seeds, a full-length cDNA clone (FsPP2C2) encoding a putative PP2C was obtained . Comparison to the databases revealed high homology to plant PP2C and most features of these enzymes, but unusual characteristics were found within the catalytic domain and the N-terminal region of the amino acid sequence . The coding region of FsPP2C2 was expressed in Escherichia coli as histidine tag fusion protein and shows Mg2+-dependent in vitro phosphatase activity . Transcription of the FsPP2C2 gene is low during seeds stratification at 4 degrees C or under gibberellic acid (GA3) treatment and clearly increases when seeds are treated with ABA and calcium (Ca2+) together, while the addition of calcium chelators (EGTA or TMB-8) decreases its expression . Furthermore, FsPP2C2 is only expressed in ABA-treated tissues, preferentially in seeds, which suggests that this PP2C is specifically induced by ABA in dormant seeds, in a Ca2+-dependent manner, and also in other ABA-treated tissues.

Br J Haematol, 2002 Jun, 117(4), 980 - 7
Congenital erythropoietic porphyria: identification and expression of eight novel mutations in the uroporphyrinogen III synthase gene; Shady AA et al.; Mutations in the uroporphyrinogen III synthase (URO-synthase) gene cause congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of haem biosynthesis . Molecular analysis of the URO-synthase gene in seven unrelated CEP patients revealed eight novel mutations . These included four missense mutations (A69T, E81D, G188W and I219S), a deletion (21delG), two insertions (398insG and 672ins28) and one complex mutation (627del6ins39), as well as three previously reported mutations, C73R, T228M, and -86C-->A . When the four novel missense mutations were expressed in Escherichia coli, only E81D expressed significant enzymatic activity (30% of expressed wild-type activity), which was thermolabile . In addition, reverse transcription polymerase chain reaction studies demonstrated that E81D, which altered the penultimate nucleotide in exon 4, impaired splicing and caused about 85% exon 4 skipping . The identification and expression of these mutations provided genotype-phenotype correlations and further evidence of the molecular heterogeneity underlying this erythropoietic porphyria.

Genes Cells, 2002 Jun, 7(6), 553 - 66
Characterization of HscC (Hsc62), homologue of Hsp70 in Escherichia coli: over-expression of HscC modulates the activity of house keeping sigma factor sigma70; Arifuzzaman M et al.; BACKGROUND: HscC, the third member of the Hsp70 family in Escherichia coli, shares 33% identity with the other two homologues, DnaK and HscA, and displays ATPase activity . Genetic and biochemical evidence indicates that the DnaK-DnaJ chaperone system interacts with sigma32 and is involved in the negative regulation of the heat shock response . Although HscC is a highly conserved protein in the Hsp70 family, its function is still unknown . RESULTS: We observed that the over-expression of HscC caused severe growth inhibition . To explore this effect, we performed primer extension analysis and a beta-galactosidase assay and found that HscC reduced the sigma70-dependent promoter activity . An in vitro transcription assay revealed that HscC inhibited sigma70-dependent transcription . In addition, the co-purification analysis showed that sigma70 co-eluted with HscC . CONCLUSION: These results indicate that HscC forms a complex with sigma70 and may function as its negative modulator.

Biochemistry (Mosc), 2002 May, 67(5), 547 - 52
Effect of hydrogen peroxide on the activity and structure of Escherichia coli chaperone GroEL; Wang F et al.; Chaperone GroEL was treated with different concentrations of hydrogen peroxide . The conformational states of GroEL were monitored by protein intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate fluorescence, and far-UV CD measurements . The results show that GroEL has unusual ability to resist oxidative stress . GroEL kept its quaternary structure and activity even when treated with 10 mM hydrogen peroxide . Two fragments were formed when GroEL was treated with high concentrations of hydrogen peroxide (more than 20 mM) . It is suggested that GroEL, as a molecular chaperone, is related to oxidative process in vivo.

J Agric Food Chem, 2002 Jun 19, 50(13), 3731 - 7
Enhanced expression of chicken cystatin as a thioredoxin fusion form in Escherichia coli AD494(DE3)pLysS and its effect on the prevention of surimi gel softening; Jiang ST et al.; The DNA encoding chicken lung cystatin was ligated into a thioredoxin-pET 23a+ expression vector and transformed into Escherichia coli AD494(DE3)pLysS . A high level of soluble recombinant thioredoxin-cystatin (trx-cystatin) was expressed in the cytoplasm of the E . coli transformant . As compared with recombinant cystatin (trx-free), a 38.7% increase of inhibitory activity in the soluble fraction was achieved by introducing the trx fusion protein . Trx-cystatin was purified to electrophoretical homogeneity by 3 min of heating at 90 degrees C and Sephacryl S-100 chromatography . The molecular mass of trx-cystatin was 29 kDa, which was the expected size based on its composition of recombinant trx (16 kDa) and chicken cystatin (13 kDa) . The purified trx-cystatin behaved as a thermally stable and papain-like proteinase inhibitor comparable to either recombinant or natural chicken cystatins . The inhibitor could inhibit the gel softening of mackerel surimi.

J Agric Food Chem, 2002 Jun 19, 50(13), 3723 - 30
Immobilization and utilization of the recombinant fusion proteins trypsin-streptavidin and streptavidin-transglutaminase for modification of whey protein isolate functionality; Wilcox CP et al.; A method was developed for the production of a hydrolyzed/polymerized whey protein derivative with altered solution and gelation properties using a combination of recombinant DNA and immobilized enzyme technologies . The recombinant fusion proteins trypsin-streptavidin (TrypSA) and streptavidin-transglutaminase (cSAcTG) were produced in Escherichia coli, extracted, and then immobilized by selective adsorption on biotinylated controlled-pore glass . Recirculation through a TrypSA reactor induced limited proteolysis of whey proteins . Hydrolysates were then recirculated through a cSAcTG reactor for incremental periods of time to arrive at increasing degrees of polymerization . The polymers were subsequently analyzed for viscosity/flow behavior, gelation properties, and fracture properties using shear rate ramps/intrinsic viscosity, small-strain oscillatory rheology, and vane viscometry, respectively . By combining limited proteolysis with controlled cross-linking, it was possible to create derivatives of whey proteins with enhanced functional properties . Increases in the degree of whey protein modification were correlated with greater apparent viscosity and intrinsic viscosity, lowered gel point temperatures, and stronger, more brittle gels . This method allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of modification . Utilization of a similar process may allow for the production of designer proteins engineered with specific functionalities.

J Chromatogr A, 2002 Apr 12, 953(1-2), 111 - 21
Comparison of histidine-tag capture chemistries for purification following chemical extraction; Choe WS et al.; The purification of a 6x-histidine tagged viral coat protein (L1) in expanded mode directly following chemical extraction from the cytoplasm of Escherichia coli HMS174(DE3) is investigated . Chelating adsorbents based on the ligands iminodiacetic acid (IDA) and nitrilotriacetic acid, using chelated metal ions Ni2+ and Cu2+, were compared . The use of Ni2+-IDA resulted in a high purification factor (9.7) and moderate recovery yield (58%) . However, the eluted fractions had an overall L1 purity less than 50% and were therefore significantly contaminated with other host proteins . In batch tests, Cu2+-IDA was found to be superior to all other combinations as it was characterised by higher binding capacities and faster adsorption kinetics . A subsequent immobilised metal affinity chromatography-expanded bed adsorption experiment using Cu2+-IDA resulted in a higher L1 purification factor (20), recovery yield (71%) and purity (89%) . The process presented here combines direct chemical extraction with expanded bed recovery . It is simpler than traditional methods, and should find more widespread application in the recovery of inclusion body proteins . Robust pseudo-affinity ligands such as metal chelates show potential for selective primary recovery of unfolded proteins, and could be used for further processing such as on-column refolding.

Farmaco, 2002 May, 57(5), 363 - 7
Synthesis and pharmacological properties of benzisothiazole/benzimidazole derivatives with acidic groups; Vicini P et al.; The synthesis and the pharmacological evaluation of benzisothiazole and benzimidazole tetrazolyl- and carboxyl- derivatives 1-6 are described . Structural modification was aimed at investigating the influence of two isosteric substituents (tetrazolyl- and carboxyl-) on the title benzofused heterocycles . The antiphlogistic, antipyretic and analgesic activities have been investigated in in vivo experimental models . Additional investigations have been performed in vitro to study the antiplatelet and spasmolytic activity of the compounds synthetized . All the compounds produced peripheral analgesic effects, but were less effective in hot plate test . The tetrazole and the carboxylic benzisothiazole derivatives 2 and 3 proved to be the most effective drugs within the series, exhibiting maximal inhibition of writhes with a potency 3-fold higher than that of acetaminophen . Only compound 5 provided indication for a central analgesic activity since it was active in hot plate test although with a low potency . The findings obtained in these in vivo and in vitro studies indicate that these compounds do not share the same mechanism of action of acetaminophen . All of the compounds under study present lower acute toxicity than acetaminophen when orally administered in mice.

Folia Microbiol (Praha), 2002, 47(2), 185 - 8
Detection of Shiga toxins, intimin and enterohemolysin in Escherichia coli strains isolated from children in eastern Slovakia; Liptakova A et al.; Fifty Escherichia coli strains isolated from stool samples of 51 healthy children, 143 strains isolated from stool samples of 327 children with diarrhea and 24 strains isolated from stool samples of 21 children with suspected hemolytic uremic syndrome were examined for the presence of Shiga toxin-producing E . coli virulence factors (shiga toxin 1 and 2, intimin and enterohemolysin) and their genes . Vero-cell assay and latex agglutination were used for detection of Shiga toxin 1 and 2, TSB agar with washed erythrocytes was used for detection of enterohemolysin; genes encoding shiga toxin 1 and 2, intimin and enterohemolysin were detected using multiplex PCR . The presence of E . coli strains harboring genes encoding shiga toxin 1 and 2 (12 strains), intimin (34 strains) and enterohemolysin (12 strains) was demonstrated.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(5), 524 - 528
Cloning and Secretion Expression of Heat-shock Protein 70 Gene of Helicobacter pylori; Liu CJ et al.; Heat-shock protein 70 gene (hsp70)was obtained by PCR method from Helicobacter pylori chromosomal DNA . Sequencing analysis exhibited that the hsp70 gene isolated from Hp Y(2) was highly homologous with the gene encoded in Helicobacter pylori 26695 and J99, which had been sequenced for complete genome . The hsp70 gene was recombined in vitro with fusion secretion expression vector pMAL-p2 and was transformed into E.coli cells . The E.coli strains, containing hsp70 recombinant plasmid, expressed a 113 kD fusion protein which accounted for 19.4% of the total bacterial periplasm protein after the induction with IPTG for 5 h at 30 degrees . The expressed fusion protein could react specifically with anti-Helicobacter pylori rabbit IgG, as proved by Western blot method.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(5), 485 - 489
Cloning, Expression and Biological Activity of VEGI(151), a Novel Vascular Endothelial Cell Growth Inhibitor; Wang L et al.; VEGI(vascular endothelial cell growth inhibitor) is a novel cytokine which belongs to the TNF super-family . In this study, the VEGI gene from ECV304 cells was cloned . A truncated form of VEGI, where 23 amino acids from N-terminal were deleted and named VEGI(151), was expressed in E.coli with 25.5% of expression rate . The purity of VEGI(151) reached 92.5% after purification . VEGI(151) showed significant inhibitory effect on endothelial cells . IC(50) of VEGI151 was 10 mg/L at 24 h . At the concentration of 0.613 mg/L, VEGI(151) induced apoptosis of endothelial cells within 36 h . However, neither stimulatory effect nor inhibitory effect of VEGI(151) was detected on tumor cells(A549 HepG2 Hela)cultured in vitro . These results suggest that endothelial cells was the main target cells of VEGI(151) . Our findings indicate that VEGI(151) is a potential therapeutic drug on angiogenic disease and cancer.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(5), 441 - 444
Effect of F209S Mutation of Escherichia coli AroG on Resistance to Phenylalanine Feedback Inhibition; Jiang PH et al.; In Escherichia coli, 80% of the 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP) synthase was encoded by aroG gene . The aroG gene was amplified by polymerase chain reaction(PCR) from strain K-12 and a mutant strain resistant to phenylalanine analogues . The PCR products were cloned and subject to DNA sequence analysis . A single base mutation of Tright curved arrow C was detected at nucleotide 625, which causes a substitution of Phe(209) by Ser in the gene product . The gene was expressed on pTrc99A in E.coli strain JM105 . Under the induction of IPTG, distinct band with the expected molecule weight was detected on SDS-polyacrylamide gel electrophoresis and the specific activity of DAHP of the crude extract of the transformed cells increased by 1.8-fold . Enzyme activity inhibition analysis revealed the high resistance of mutant AroG to feedback inhibition by phenylalanine . JM105 cells harboring with mutant aroG gene showed were able to grow on medium containing higher concentration of analogues than that carrying normal aroG gene . Discussion was focused on the varieties of mutations contributing to desensitization of feedback inhibition.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(5), 435 - 440
A Strong Promoter Provided with the Gene Encoding Arginyl-tRNA Synthetase(argS) from Escherichia coli; Liu MF et al.; Previous studies showed that the gene argS encoding the arginyl-tRNA synthetase(ArgRS) from Escherichia coli(E.coli), was overexpressed 1 000 folds in the E.coli transformant TG1/pUC-argS, while the gene leuS, encoding the leucyl-tRNA synthetase(LeuRS) from E.coli, was only overproduced 35-fold in the same case . To investigate why the expression of these two aminoacyl-tRNA synthetase genes is so different, a fused gene (termed parg-leuS) was constructed by replacement of the 5' flanking region of leuS to 5' flanking region of argS . In the E.coli transformant TG1/pUC-parg-leuS, the activity of LeuRS was only improved 8.5-fold, which was much lower than that of the transformant harboring the recombinant plasmid pUC18-leuS or pKK-leuS . However, by RNA dot hybridization the amount of mRNA produced in the transcription of parg-leuS was about 5 times than that of the wild type leuS, and was similar to that of pKK-leuS, suggesting that the promoter of argS is very strong . Analysis of the secondary structure around the initiation codon among three mRNAs showed that the secondary structure of the mRNA from parg-leuS was the strongest of the three mRNAs . From the results, it could be deduced that expression of the fused gene parg-leuS might be controlled at the translational level and the strong secondary structure of this mRNA may hinder translation initiation and result in a low translation efficiency.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(6), 627 - 632
Cloning and Expression of Insulin Receptor Ligand-binding Domains; Zhang H et al.; Insulin receptor is a transmembrane protein consisting of four subunits, that form a heterotetramer(alpha(2)beta(2))with molecular weight of 350 kD . Because the extracellular subunit(alpha)consists of 731 residues and a cysteine-rich domain, it is difficult to express and crystallize such a large ligand-binding subunit, thus hampering further study on "insulin-receptor" complex . Based on the fact that the domains L1 and L2 of the alpha subunit, consisted of 119 and 118 residues, contained the high and low affinity insulin binding sites, respectively, the cDNAs of L1 and L2 were obtained from a human placental cDNA library by PCR . The cDNAs of L1, L2 and L1-(Ala)(10)-L2(designed ten-alanine-connected L1 and L2)were cloned, respectively, into an expression plasmid pET-3a, and E.coli BL21(DE3)transformants with such plasmids were successfully induced to express the goal proteins . The expression products were isolated and purified by the washing and solubilization of inclusion body, gel filtration chromatography and ion exchange chromatography . Each final product displayed a single band, corresponding the purity above 99%, in SDS-PAGE . These products have also been confirmed respectively as the L1, L2 and L1-(Ala)(10)-L2 by DNA sequencing, amino acid composition analysis and N-terminal amino acid sequencing.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(6), 609 - 614
Difference between Biological Activities and Secondary Structures of Expression Product of atpE Gene from Broad Bean and Maize Chloroplast; Wang DF et al.; The chloroplast atpE genes from broad bean and maize were overexpressed in E.coli, respectively . After proper refolding and purification, two types of epsilon-subunit proteins with biological activity were obtained . When reconstituted with CF(1)(-epsilon)from different chloroplast ATPase, the effects of the reconstructed epsilon-subunit protein of maize CF(1) to the Ca(2+)-ATPase activity of epsilon-deficient CF(1) and to the proton leakage through CF(0) were markedly stronger than that of broad bean CF(1) . It was also observed that the enhancing effect of maize epsilon-subunit protein to the chloroplast photophosphorylation activity was greater than that of broad bean epsilon-subunit protein . The results indicate that (1)the inhibition of epsilon-subunit is closely related to the its affinity with other parts of CF(1) (2)both functions of epsilon-subunit as inhibitor of ATPase and as proton pathway are closely linked . The circular dichroism was performed to show the differences in their secondary structure of these epsilon-subunit proteins based on the deduced primary sequences . Unconstrained analysis of the CD spectrum for the maize epsilon-subunit protein gave 22.6% alpha-helix, 30.6% beta-sheet, 9.3% beta-turn, and 37.7% unordered structure and 31.4%alpha-helix, 22.3% beta-sheet, 13.8% beta-turn, and 32.4% unordered structure for the epsilon-subunit from broad bean.

J Biol Chem, 2002 Aug 9, 277(32), 28823 - 9 Epub 2002 Jun 10.
Mechanism of Galpha i-mediated inhibition of type V adenylyl cyclase; Dessauer CW et al.; The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2) . The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain . The mechanism of these two regulators also appears to be in opposition . Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another . We show herein that Galpha(i) reduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site . Mutants that increase the affinity of C1 for C2 decrease the ability of Galpha(i) to inhibit the enzyme . In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface . Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP . Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs . Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.

J Bacteriol, 2002 Jul, 184(13), 3759 - 64
The enigmatic Escherichia coli fadE gene is yafH; Campbell JW et al.; The identity of the gene encoding acyl coenzyme A dehydrogenase is a major remaining mystery of the Escherichia coli fatty acid degradation (fad) regulon . Our prior genome array analyses showed that transcription of the yafH gene is controlled by the FadR regulatory protein . We now report direct experimental proof that yafH and fadE are the same gene.

J Bacteriol, 2002 Jul, 184(13), 3756 - 8
Pyridoxal 5-phosphate inhibition of substrate selectivity mutants of UhpT, the sugar 6-phosphate carrier of Escherichia coli; Hall JA et al.; In the sugar phosphate transporter UhpT, gain-of-function derivatives that prefer phosphoenolpyruvate (PEP) as substrate have an uncompensated lysine residue on transmembrane segment 11 . We show here that these variants are also highly susceptible to substrate-protectable inhibition by covalent modification of lysine with pyridoxal 5-phosphate . The chemical requirements of this interaction provide evidence that the gain-of-function phenotype results from the pairing of the uncompensated lysines in these mutants with the anionic carboxyl group of PEP.

J Bacteriol, 2002 Jul, 184(13), 3746 - 8
PII T-loop mutations affecting signal transduction to NtrB also abolish yeast two-hybrid interactions; Martinez-Argudo I et al.; Mutations A49P and Delta47-53 at the T loop of the Escherichia coli GlnB (PII) protein impair regulatory interactions with the two-component sensor regulator NtrB (P . Jiang, P . Zucker, M . R . Atkinson, E . S . Kamberov, W . Tirasophon, P . Chandran, B . R . Schepke, and A . J . Ninfa, J . Bacteriol . 179: 4342-4353, 1997) . We show here that these mutations also impair interactions between PII and NtrB in the yeast two-hybrid system, indicating that defects in NtrB regulation closely reflect binding impairment . The reported results underline the strength of two-hybrid assays for analysis of interactions involving the T loop of PII proteins.

J Bacteriol, 2002 Jul, 184(13), 3723 - 33
Assembly of colicin A in the outer membrane of producing Escherichia coli cells requires both phospholipase A and one porin, but phospholipase A is sufficient for secretion; Cavard D; Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of Escherichia coli . These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling . Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the cal gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium . Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process . Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins . In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed . Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA . Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.

J Bacteriol, 2002 Jul, 184(13), 3699 - 703
Toxic waste disposal in Escherichia coli; Helling RB et al.; About 10% of the nalidixic acid-resistant (Nal(r)) mutants in a transposition-induced library exhibited a growth factor requirement as the result of cysH, icdA, metE, or purB mutation . Resistance in all of these mutants required a functional AcrAB-TolC efflux pump, but the EmrAB-TolC pump played no obvious role . Transcription of acrAB was increased in each type of Nal(r) mutant . In the icdA and purB mutants, each of the known signaling pathways appeared to be used in activating the AcrAB-TolC pump . The metabolites that accumulate upstream of the blocks caused by the mutations are hypothesized to increase the levels of the AcrAB-TolC pump, thereby removing nalidixic acid from the organism.

J Bacteriol, 2002 Jul, 184(13), 3689 - 98
A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins; Fukui T et al.; In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain, Archae . We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon . Lon from Thermococcus kodakaraensis KOD1 (Lon(Tk)) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA(+) superfamily and a C-terminal protease domain including a putative catalytic triad . Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that Lon(Tk) was actually a membrane-bound protein . The recombinant Lon(Tk) possessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70 degrees C, respectively . Unlike the enzyme from Escherichia coli, we found that Lon(Tk) showed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP . When three kinds of proteins with different thermostabilities were examined as substrates, it was found that Lon(Tk) required ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis . In contrast, Lon(Tk) degraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in Lon(Tk) different from that of its bacterial counterpart.

J Bacteriol, 2002 Jul, 184(13), 3501 - 7
Nitric oxide-induced homologous recombination in Escherichia coli is promoted by DNA glycosylases; Spek EJ et al.; Nitric oxide (NO*) is involved in neurotransmission, inflammation, and many other biological processes . Exposure of cells to NO* leads to DNA damage, including formation of deaminated and oxidized bases . Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO* toxicity, which indicates that base excision repair (BER) intermediates are being generated . Here, we show that AP endonuclease-deficient cells can be protected from NO* toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase . These results suggest that Ung and Fpg remove nontoxic NO*-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases . Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination . The RecBCD complex is critical for repair of double-strand breaks via homologous recombination . When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced . We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO*-induced recombination . Taken together, a picture emerges in which the action of DNA glycosylases on NO*-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination . These studies shed light on the underlying mechanism of NO*-induced homologous recombination.

J Bacteriol, 2002 Jul, 184(13), 3492 - 500
Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134; Louie TM et al.; Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP) . Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized . In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate . We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts . Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH2)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ) . The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases . Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively . The three genes were individually inactivated in JMP134 . The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ . Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ . For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH2 was supplied by an Escherichia coli flavin reductase . TcpC produced in E . coli oxidized 6-CHQ to 2-chloromaleylacetate . Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC . Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis . The function of TcpB remains unknown.

J Bacteriol, 2002 Jul, 184(13), 3457 - 65
The type IV pilus assembly complex: biogenic interactions among the bundle-forming pilus proteins of enteropathogenic Escherichia coli; Ramer SW et al.; Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon . Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins . This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex . These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts . The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU . Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.

J Bacteriol, 2002 Jul, 184(13), 3419 - 25
Molecular characterization of the PceA reductive dehalogenase of desulfitobacterium sp . strain Y51; Suyama A et al.; The tetrachloroethene (PCE) reductive dehalogenase (encoded by the pceA gene and designated PceA dehalogenase) of Desulfitobacterium sp . strain Y51 was purified and characterized . The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE) . The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to cis-1,2-dichloroethene at a specific activity of 113.6 nmol x min(-1) x mg of protein(-1) . The apparent K(m) values for PCE and TCE were 105.7 and 535.3 microM, respectively . Chlorinated ethenes other than PCE and TCE were not dehalogenated . However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane . The pceA gene of Desulfitobacterium sp . strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in Escherichia coli for the preparation of antibodies . Immunoblot analyses located PceA in the periplasm of the cell.

Bioorg Med Chem, 2002 Aug, 10(8), 2739 - 49
10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway; Marsilje TH et al.; The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported . Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase . The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency {K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively} . Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition . The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation . Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E . coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase . On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca . 100-fold higher intracellular concentrations) . However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E . coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E . coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.

Structure (Camb), 2002 Jun, 10(6), 877 - 86
A hyperthermostable D-ribose-5-phosphate isomerase from Pyrococcus horikoshii characterization and three-dimensional structure; Ishikawa K et al.; A gene homologous to D-ribose-5-phosphate isomerase (EC 5.3.1.6) was found in the genome of Pyrococcus horikoshii . D-ribose-5-phosphate isomerase (PRI) is of particular metabolic importance since it catalyzes the interconversion between the ribose and ribulose forms involved in the pentose phosphate cycle and in the process of photosynthesis . The gene consisting of 687 bp was overexpressed in Escherichia coli, and the resulting enzyme showed activity at high temperatures with an optimum over 90 degrees C . The crystal structures of the enzyme, free and in complex with D-4-phosphoerythronic acid inhibitor, were determined . PRI is a tetramer in the crystal and in solution, and each monomer has a new fold consisting of two alpha/beta domains . The 3D structures and the characterization of different mutants indicate a direct or indirect catalytic role for the residues E107, D85, and K98.

Structure (Camb), 2002 Jun, 10(6), 825 - 35
X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site; Roujeinikova A et al.; Acyl carrier protein (ACP) is an essential cofactor in biosynthesis of fatty acids and many other reactions that require acyl transfer steps . We have determined the first crystal structures of an acylated form of ACP from E . coli, that of butyryl-ACP . Our analysis of the molecular surface of ACP reveals a plastic hydrophobic cavity in the vicinity of the phosphopantethylated Ser36 residue that is expanded and occupied by the butyryl and beta-mercaptoethylamine moieties of the acylated 4'-phosphopantetheine group in one of our crystal forms . In the other form, the cavity is contracted, and we propose that the protein has adopted the conformation after delivery of substrate into the active site of a partner enzyme.

Structure (Camb), 2002 Jun, 10(6), 737 - 9
HtrA--a renaissance protein; Day CL et al.; Initially identified as proteases, members of the HtrA/DegP family of proteins have also been shown to act as chaperones in bacteria, and more recently implicated, as regulators of apoptosis in mammals . Two new structures of mammalian HtrA2 and E . coli DegP provide insights into the origin of this plurality of function.

Biochem J, 2002 Oct 1, 367(Pt 1), 279 - 85
Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis; Choudhuri BS et al.; The genes encoding ATP-binding cassette (ABC) transporters occupy 2.5% of the genome of Mycobacterium tuberculosis . However, none of these putative ABC transporters has been characterized so far . We describe the development of expression systems for simultaneous expression of the ATP-binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump . Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters . The localization of DrrA to the membrane depended on the simultaneous expression of DrrB . ATP binding was positively regulated by doxorubicin and daunorubicin . At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA . Simultaneous expression of the two polypeptides was essential to obtain a functional doxorubicin efflux pump . Expression of DrrAB in E . coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound . 2',7'-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a neutral compound, also behaved as a substrate of the reconstituted efflux pump . When expressed in M . smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics . The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters.

Biochemistry, 2002 Jun 18, 41(24), 7695 - 706
Thermodynamics of aminoglycoside-rRNA recognition: the binding of neomycin-class aminoglycosides to the A site of 16S rRNA; Kaul M et al.; We use spectroscopic and calorimetric techniques to characterize the binding of the aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin to a RNA oligonucleotide that models the A-site of Escherichia coli 16S rRNA . Our results reveal the following significant features: (i) Aminoglycoside binding enhances the thermal stability of the A-site RNA duplex, with the extent of this thermal enhancement decreasing with increasing pH and/or Na(+) concentration . (ii) The RNA binding enthalpies of the aminoglycosides become more exothermic (favorable) with increasing pH, an observation consistent with binding-linked protonation of one or more drug amino groups . (iii) Isothermal titration calorimetry (ITC) studies conducted as a function of buffer reveal that aminoglycoside binding to the host RNA is linked to the uptake of protons, with the number of linked protons being dependent on pH . Specifically, increasing the pH results in a corresponding increase in the number of linked protons . (iv) ITC studies conducted at 25 and 37 degrees C reveal that aminoglycoside-RNA complexation is associated with a negative heat capacity change (Delta C(p)), the magnitude of which becomes greater with increasing pH . (v) The observed RNA binding affinities of the aminoglycosides decrease with increasing pH and/or Na(+) concentration . In addition, the thermodynamic forces underlying these RNA binding affinities also change as a function of pH . Specifically, with increasing pH, the enthalpic contribution to the observed RNA binding affinity increases, while the corresponding entropic contribution to binding decreases . (vi) The affinities of the aminoglycosides for the host RNA follow the hierarchy neomycin > paromomycin > ribostamycin . The enhanced affinity of neomycin relative to either paromomycin or ribostamycin is primarily, if not entirely, enthalpic in origin . (vii) The salt dependencies of the RNA binding affinities of neomycin and paromomycin are consistent with at least three drug NH(3)(+) groups participating in electrostatic interactions with the host RNA . In the aggregate, our results reveal the impact of specific alterations in aminoglycoside structure on the thermodynamics of binding to an A-site model RNA oligonucleotide . Such systematic comparative studies are critical first steps toward establishing the thermodynamic database required for enhancing our understanding of the molecular forces that dictate and control aminoglycoside recognition of RNA.

Biochemistry, 2002 Jun 18, 41(24), 7610 - 6
Novel phenalenone derivatives from a marine-derived fungus exhibit distinct inhibition spectra against eukaryotic DNA polymerases; Perpelescu M et al.; A number of compounds used for cancer chemotherapy exert their effects by inhibiting DNA replication . New inhibitors of DNA polymerases, therefore, could be potential candidates for new anti-cancer drugs . This study tested the effects of two phenalenone-skeleton-based compounds, which were isolated from a marine-derived fungus Penicillium sp., sculezonone-B (SCUL-B) and sculezonone-A (SCUL-A), upon DNA polymerase activity . Both compounds inhibited bovine DNA polymerases alpha and gamma, moderately affected the activity of DNA polymerase epsilon, and had almost no effect on HIV-reverse transcriptase and an E . coli DNA polymerase I Klenow fragment . Most notably, whereas SCUL-A inhibited pol beta (IC(50) = 17 microM), SCUL-B has only a weak influence upon this polymerase (IC(50) = 90 microM) . Kinetic studies showed that inhibition of both DNA polymerases alpha and beta by either SCUL-A or SCUL-B was competitive with respect to dTTP substrate and noncompetitive with the template-primer . Whereas pol alpha inhibition by SCUL-B is competitive with respect to dATP, the inhibition by SCUL-A was found to be a mixed type with dATP substrate . The K(i) values of SCUL-B were calculated to be 1.8 and 6.8 microM for DNA polymerases alpha and gamma, respectively . The K(i) of DNA polymerase beta for SCUL-A was 12 microM and that for DNA polymerase alpha, 16 microM . Therefore, deletion of the OH-group at C12 enhanced inhibition of DNA polymerase beta . Since computational analyses of these two inhibitors revealed a remarkable difference in the distribution of negative electrostatic charge on the surface of molecules, we infer that different electrostatic charges might elicit different inhibition spectra from these two compounds.

Biochemistry, 2002 Jun 18, 41(24), 7540 - 8
Thermodynamic, kinetic, and structural basis for recognition and repair of 8-oxoguanine in DNA by Fpg protein from Escherichia coli; Ishchenko AA et al.; X-ray analysis does not provide quantitative estimates of the relative importance of the molecular contacts it reveals or of the relative contributions of specific and nonspecific interactions to the total affinity of specific DNA to enzymes . Stepwise increase of DNA ligand complexity has been used to estimate the relative contributions of virtually every nucleotide unit of 8-oxoguanine-containing DNA to its total affinity for Escherichia coli 8-oxoguanine DNA glycosylase (Fpg protein) . Fpg protein can interact with up to 13 nucleotide units or base pairs of single- and double-stranded ribo- and deoxyribo-oligonucleotides of different lengths and sequences through weak additive contacts with their internucleotide phosphate groups . Bindings of both single-stranded and double-stranded oligonucleotides follow similar algorithms, with additive contributions to the free energy of binding of the structural components (phosphate, sugar, and base) . Thermodynamic models are provided for both specific and nonspecific DNA sequences with Fpg protein . Fpg protein interacts nonspecifically with virtually all of the base-pair units within its DNA-binding cleft: this provides approximately 7 orders of magnitude of affinity (Delta G degrees approximately equal to -9.8 kcal/mol) for DNA . In contrast, the relative contribution of the 8-oxoguanine unit of the substrate (Delta G degrees approximately equal to -0.90 kcal/mol) together with other specific interactions is <2 orders of magnitude (Delta G degrees approximately equal to -2.8 kcal/mol) . Michaelis complex formation of Fpg protein with DNA containing 8-oxoguanine cannot of itself provide the major part of the enzyme specificity, which lies in the k(cat) term; the rate is increased by 6-8 orders of magnitude on going from nonspecific to specific oligodeoxynucleotides.

Biochem Biophys Res Commun, 2002 Jun 14, 294(3), 650 - 4
Identification of human L-fucose kinase amino acid sequence; Hinderlich S et al.; Fucose is a major component of complex carbohydrates . L-Fucose kinase (fucokinase) takes part in the salvage pathway for reutilization of fucose from the degradation of oligosaccharides . The amino acid sequence of human fucokinase was derived from a cDNA encoding a protein of hitherto unidentified function . Human fucokinase polypeptide chain consists of 990 amino acids with a predicted molecular mass of 107 kDa . The C-terminal part of its amino acid sequence showed sequence motifs typical for sugar kinases . Fucokinase full-length protein and a deletion mutant lacking the first 363 amino acids of the N-terminus were expressed in Escherichia coli BL21 cells . Both proteins displayed fucokinase activity . These results reveal that the discovered cDNA encodes the fucokinase protein and they confirm that a functional kinase domain is located in the C-terminal part of the enzyme.

Folia Histochem Cytobiol, 2002, 40(2), 133 - 4
Efficiency of lipofection of adherent cells is limited by apoptosis; Bednarek I et al.; Stability of gene expression and transfection efficiency plays the main role in the application of gene transfer method . In somatic cell gene delivery, expression of the gene product is limited by the function of the cell to which it is delivered . In the present study analyzing the lipofected adherent cells, we have shown that lower level of transgene: beta-galactosidase activity at later time period correlated with decrease in cell viability, which was shown to be due to apoptosis . Apoptosis following DNA uptake occurred only when DNA was present during lipofection.

Vet Res, 2002 May-Jun, 33(3), 313 - 26
Protection evaluation against Chlamydophila abortus challenge by DNA vaccination with a dnaK-encoding plasmid in pregnant and non-pregnant mice; Hechard C et al.; Mice were intramuscularly immunized with a dnaK-encoding DNA plasmid . The protective effect of DNA immunization against Chlamydophila abortus infection was studied in pregnant and non-pregnant mice models . In non-pregnant mice, the dnaK vaccine induced a specific humoral response with the predominant IgG2a isotype, which failed to have in vitro neutralizing properties . No delayed-type hypersensitivity reaction was observed and the spleens of dnaK vaccinated-mice were not protected against C . abortus challenge . In pregnant mice, the dnaK vaccine induced a non-specific partial protection from abortion . This may be due to the immunogenic properties of the CpG motifs of bacterial DNA present in the vaccinal plasmid backbone . Nevertheless, spleens of dnaK vaccinated-pregnant mice were not protected.

Methods, 2002 Apr, 26(4), 298 - 306
Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial processing peptidase; Cavadini P et al.; Most mitochondrial proteins are synthesized in the cytoplasm as larger precursors carrying N-terminal matrix-targeting presequences, and are subsequently transported to the mitochondria . The presequence mediates the interaction between the precursor polypeptide and components of the mitochondrial protein import machinery, a complex apparatus that is responsible for translocation of the precursor across the two mitochondrial membranes . Once the precursor has reached the mitochondrial matrix, the presequence is removed by the general mitochondrial processing peptidase (MPP) . Some precursors undergo additional processing steps carried out by specialized processing peptidases . For most mitochondrial proteins, however, cleavage by MPP is the step that precedes folding and assembly into the native form . We describe methods to isolate import-competent mitochondria from rat liver and to perform import reactions with precursor proteins synthesized in vitro by coupled transcription-translation . We also describe methods to perform in vitro processing reactions of mitochondrial precursors by recombinant MPP and to identify the cleavage sites used by this enzyme.

J Mol Biol, 2002 May 3, 318(3), 911 - 32
Homology, pathway distance and chromosomal localization of the small molecule metabolism enzymes in Escherichia coli; Rison SC et al.; Here, we analyse Escherichia coli enzymes involved in small molecule metabolism (SMM) . We introduce the concept of pathway distance as a measure of the number of distinct metabolic steps separating two SMM enzymes, and we consider protein homology (as determined by assigning enzymes to structural and sequence families) and gene interval (the number of genes separating two genes on the E . coli chromosome) . The relationships between these three contexts (pathway distance, homology and chromosomal localisation) is investigated extensively . We make use of these relationships to suggest possible SMM evolution mechanisms . Homology between enzyme pairs close in the SMM was higher than expected by chance but was still rare . When observed, homologues usually conserved their reaction mechanism and/or co-factor binding rather than shared substrate binding . The correlation between pathway distance and gene intervals was clear . Enzymes catalysing nearby SMM reactions were usually encoded by genes close by on the E . coli chromosome . We found many co-regulated blocks of three to four genes (usually non-homologous) encoding enzymes occurring within four metabolic steps of one another; nearly all of these blocks formed part of known or predicted operons . The "inline reuse" of enzymes (i.e . the use of the same enzyme to catalyse two or more different steps of a metabolic pathway) is also discussed: of these enzymes, four were multifunctional (i.e . catalysed a different reaction in each instance), nine had multiple substrate specificity (i.e . catalysed the same reaction on different substrates in each instance) and one catalysed the same reaction on the same substrate but as part of two different complexes . We also identified 59 sets of isozymic proteins most commonly duplicated to function under different conditions, or with a different preferred substrate or minor substrate . In addition to transcriptional units, isozymes and inline reuse of enzymes provide mechanisms for controlling the SMM network . Our data suggest that several pathway evolution mechanisms may occur in concert, although chemistry-driven duplication/recruitment is favoured . SMM exploits regulatory strategies involving chromosomal location, isozymes and the reuse of enzymes . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 3, 318(3), 877 - 88
Core side-chain packing and backbone conformation in Lpp-56 coiled-coil mutants; Liu J et al.; Native proteins exhibit precise geometric packing of atoms in their hydrophobic interiors . Nonetheless, controversy remains about the role of core side-chain packing in specifying and stabilizing the folded structures of proteins . Here we investigate the role of core packing in determining the conformation and stability of the Lpp-56 trimerization domain . The X-ray crystal structures of Lpp-56 mutants with alanine substitutions at two and four interior core positions reveal trimeric coiled coils in which the twist of individual helices and the helix-helix spacing vary significantly to achieve the most favored superhelical packing arrangement . Introduction of each alanine "layer" into the hydrophobic core destabilizes the superhelix by 1.4 kcal mol(-1) . Although the methyl groups of the alanine residues pack at their optimum van der Waals contacts in the coiled-coil trimer, they provide a smaller component of hydrophobic interactions than bulky hydrophobic side-chains to the thermodynamic stability . Thus, specific side-chain packing in the hydrophobic core of coiled coils are important determinants of protein main-chain conformation and stability . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 3, 318(3), 787 - 804
Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB; Collins ES et al.; In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells . The translocation function is located in the N-terminal domain of the colicin molecule . (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible . Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically . The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms) . The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances . Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus . This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains . Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues . Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region . The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself . c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 3, 318(3), 651 - 63
FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter; Pemberton IK et al.; We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli . FIS photo-footprints strongly to three binding sites upstream of the core promoter . The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter . The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter . In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling . These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase . We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator . We further suggest that FIS plays a similar role and may facilitate polymerase escape . c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 10, 318(4), 1117 - 26
Crystal structure of the Homer 1 family conserved region reveals the interaction between the EVH1 domain and own proline-rich motif; Irie K et al.; PSD-Zip45 (also named Homer 1c/Vesl-1L) is a synaptic scaffolding protein, which interacts with neurotransmitter receptors and other scaffolding proteins to target them into post-synaptic density (PSD), a specialized protein complex at the synaptic junction . Binding of the PSD-Zip45 to the receptors and scaffolding proteins results in colocalization and clustering of its binding partners in PSD . It has an Ena/VASP homology 1 (EVH1) domain in the N terminus for receptor binding, two leucine zipper motifs in the C terminus for clustering, and a linking region whose function is unclear despite the high level of conservation within the Homer 1 family . The X-ray crystallographic analysis of the largest fragment of residues 1-163, including an EVH1 domain reported here, demonstrates that the EVH1 domain contains an alpha-helix longer than that of the previous models, and that the linking part included in the conserved region of Homer 1 (CRH1) of the PSD-Zip45 interacts with the EVH1 domain of the neighbour CRH1 molecule in the crystal . The results suggest that the EVH1 domain recognizes the PPXXF motif found in the binding partners, and the SPLTP sequence (P-motif) in the linking region of the CRH1 . The two types of binding are partly overlapped in the EVH1 domain, implying a mechanism to regulate multimerization of Homer 1 family proteins . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 10, 318(4), 1097 - 115
NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein; Vogtherr M et al.; We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy . Our results lead to a well-defined structure with a backbone rmsd of 0.23 A . As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold . Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar . Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet . Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor . Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet . Our data indicate, however, that this strand may be partially structured as "nascent" beta strand . This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 10, 318(4), 1057 - 69
Conformational changes during the catalytic cycle of gluconate kinase as revealed by X-ray crystallography; Kraft L et al.; The crystal structure of gluconate kinase from Escherichia coli has been determined to 2.0 A resolution by X-ray crystallography . The three-dimensional structure was solved by multi-wavelength anomalous dispersion, using a crystal of selenomethionine-substituted enzyme . Gluconate kinase is an alpha/beta structure consisting of a twisted parallel beta-sheet surrounded by alpha-helices with overall topology similar to nucleoside monophosphate (NMP) kinases, such as adenylate kinase . In order to identify residues involved in substrate binding and catalysis, structures of binary complexes with ATP, the ATP analogue adenosine 5'-(beta,gamma-methylene) triphosphate and the product, gluconate-6-phosphate have been determined . Significant conformational changes are induced upon binding of ATP to the enzyme . The largest changes involve a hinge-bending motion of the NMP(bind) part and a motion of the LID with adjacent helices, which opens the cavity to the second substrate, gluconate . Opening of the active site cleft upon ATP binding is the opposite of what has been observed in the NMP kinase family so far, which usually close their active site to prevent fortuitous hydrolysis of ATP . The conformational change positions the side-chain of Arg120 to stack with the purine ring of ATP and the side-chain of Arg124 is shifted to interact with the alpha-phosphate in ATP, at the same time protecting ATP from solvent water . The beta and gamma-phosphate groups of ATP bind in the predicted P-loop . A conserved lysine side-chain interacts with the gamma-phosphate group, and might promote phosphoryl transfer . Gluconate-6-phosphate binds with its phosphate group in a similar position as the gamma-phosphate of ATP, consistent with inline phosphoryl transfer . The gluconate binding-pocket in GntK is located in a different position than the nucleoside binding-site usually found in NMP kinases . (c) 2002 Elsevier Science Ltd.

J Mol Biol, 2002 May 10, 318(4), 1031 - 42
Leucine-induced dissociation of Escherichia coli Lrp hexadecamers to octamers; Chen S et al.; Lrp is a global regulator of metabolism in Escherichia coli that helps cells respond to changes in environmental conditions . The action of Lrp as a transcriptional activator or repressor is sometimes affected when the medium contains exogenous leucine . In this study, we examined the thermodynamics of leucine binding to Lrp and to a leucine response mutant, Lrp-1, and leucine-induced dissociation of Lrp hexadecamer to leucine-bound octamer . The results of dynamic light-scattering and fluorescence measurements suggest that Lrp has two leucine-binding sites, one a high-affinity site and the other a low-affinity site that is coupled to the dissociation reaction . The Gibbs free energy change for leucine binding to the high-affinity site is about -7.0 kcal/mol . Binding of two leucine molecules to low-affinity sites on the hexadecamer or one leucine molecule to one octamer induces the dissociation of hexadecamer to leucine-bound octamer . The Gibbs free energy change for leucine binding to the low-affinity site was estimated to be in the range -4.66 to -5.03 kcal/mol for leucine binding to an octamer or -6.01 to -6.75 kcal/mol for leucine binding to a hexadecamer . The thermodynamic parameters derived from this study were used together with other data to estimate the distribution of free Lrp hexadecamer, octamer, leucine-bound hexadecamer, and leucine-bound octamer in cells . Mathematical modeling, employed to simulate modulation of Lrp action in response to growth conditions, gave results that are consistent with known patterns of Lrp action on different operons . (c) 2002 Elsevier Science Ltd.

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