Escherichia coli

Biomacromolecules, 2001 Fall, 2(3), 1023 - 8
Poly(amidoamine)s as potential nonviral vectors: ability to form interpolyelectrolyte complexes and to mediate transfection in vitro; Richardson SC et al.; Poly(amidoamine)s (PAAs) are water-soluble polymers that display pH-dependent membrane activity . PAAs have the potential to act as a synthetic alternative to fusogenic peptides and thus promote endosomal escape . The purpose of this study was to investigate for the first time whether PAA have the ability to complex DNA, protect it from nuclease degradation and to promote transfection in vitro . PAAs ISA 1 (Mn 6900) and ISA 23 (Mn 10,500) and their 2-phenylethylamine containing analogues ISA 4 and ISA 22 (Mn approximately 8000) were studied . All PAAs retarded the electrophoretic mobility of lambda Hind III DNA demonstrating interpolyelectrolyte complex (IPEC) formation and toroids of 80-150 nm in diameter (10:1 polymer excess) were visible using TEM . DNase II inhibition was observed . At a polymer:DNA ratio of 10:1, this was ISA 1(89.6 +/- 6.1%), ISA 4 (92.2 +/- 11.2%), ISA 22 (69.4 +/- 3.7%), and ISA 23 (58.0 +/- 10.0%) . PAAs demonstrated the ability to mediate pSV beta-galactosidase transfection of HepG2 cells . At a vector:DNA mass ratio of 5:1, ISA 23 showed equivalent transfection ability compared with polyethylenimine and LipofectIN and was more effective than LipofectACE . These properties suggest that PAAs warrant further development as endosomolytic vectors.

J Biol Chem, 2002 Jan 25, 277(4), 2992 - 6 Epub 2001 Nov 14.
Holliday junction resolution is modulated by archaeal chromatin components in vitro; Kvaratskhelia M et al.; The Holliday junction-resolving enzyme Hjc is conserved in the archaea and probably plays a role analogous to that of Escherichia coli RuvC in the pathway of homologous recombination . Hjc specifically recognizes four-way DNA junctions, cleaving them without sequence preference to generate recombinant DNA duplex products . Hjc imposes an X-shaped global conformation on the bound DNA junction and distorts base stacking around the point of cleavage, three nucleotides 3' of the junction center . We show that Hjc is autoinhibitory under single turnover assay conditions and that this can be relieved by the addition of either competitor duplex DNA or the architectural double-stranded DNA-binding protein Sso7d (i.e . by approximating in vivo conditions more closely) . Using a combination of isothermal titration calorimetry and fluorescent resonance energy transfer, we demonstrate that multiple Hjc dimers can bind to each synthetic four-way junction and provide evidence for significant distortion of the junction structure at high protein:DNA ratios . Analysis of crystal packing interactions in the crystal structure of Hjc suggests a molecular basis for this autoinhibition . The wider implications of these findings for the quantitative study of DNA-protein interactions is discussed.

J Biol Chem, 2002 Feb 22, 277(8), 5858 - 65 Epub 2001 Nov 14.
Why OrfY? Characterization of MMOD, a long overlooked component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath); Merkx M et al.; Soluble methane monooxygenase (sMMO) has been studied intensively to understand the mechanism by which it catalyzes the remarkable oxidation of methane to methanol . The cluster of genes that encode for the three characterized protein components of sMMO (MMOH, MMOB, and MMOR) contains an additional open reading frame (orfY) of unknown function . In the present study, MMOD, the protein encoded by orfY, was overexpressed as a fusion protein in Escherichia coli . Pure MMOD was obtained in high yields after proteolytic cleavage and a two-step purification procedure . Western blot analysis of Methylococcus capsulatus (Bath) soluble cell extracts showed that MMOD is expressed in the native organism although at significantly lower levels than the other sMMO proteins . The cofactorless MMOD protein is a potent inhibitor of sMMO activity and binds to the hydroxylase protein (MMOH) with an affinity similar to that of MMOB and MMOR . The addition of up to 2 MMOD per MMOH results in changes in the optical spectrum of the hydroxylase that suggest the formation of a (micro-oxo)diiron(III) center in a fraction of the MMOH-MMOD complexes . Possible functions for MMOD are discussed, including a role in the assembly of the MMOH diiron center similar to that suggested for DmpK, a protein that shares some properties with MMOD.

Antimicrob Agents Chemother, 2001 Dec, 45(12), 3660 - 2
Mechanism of action of the des-F(6) quinolone BMS-284756 measured by supercoiling inhibition and cleavable complex assays; Wu P et al.; BMS-284756 (T-3811ME), a novel des-F(6) quinolone, was tested in the supercoiling inhibition and cleavable complex assays against Escherichia coli DNA gyrase, a target of quinolones . The results suggest that BMS-284756 has the same mechanism of action against DNA gyrase as other quinolones and a similar level of potency.

Antimicrob Agents Chemother, 2001 Dec, 45(12), 3293 - 303
Dicyclic and tricyclic diaminopyrimidine derivatives as potent inhibitors of Cryptosporidium parvum dihydrofolate reductase: structure-activity and structure-selectivity correlations; Nelson RG et al.; A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J . R . Vasquez et al., Mol . Biochem . Parasitol . 79:153-165, 1996) . In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli . Fifty percent inhibitory concentrations (IC(50)s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates . Of the compounds tested, 25 had IC(50)s in the 1 to 10 microM range against one or both C . parvum enzymes and thus were not substantially different from trimethoprim (IC(50)s, ca . 4 microM) . Another 25 compounds had IC(50)s of <1.0 microM, and 9 of these had IC(50)s of <0.1 microM and thus were at least 40 times more potent than trimethoprim . The remaining 42 compounds were weak inhibitors (IC(50)s, >10 microM) and thus were not considered to be of interest as drugs useful against this organism . A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V . H . Brophy et al., Antimicrob . Agents Chemother . 44:1019-1028, 2000; H . Lau et al., Antimicrob . Agents Chemother . 45:187-195, 2001) . Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for C . parvum versus human DHFR . Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents . The compounds in the library were also tested as inhibitors of the proliferation of intracellular C . parvum oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 microM) and hypoxanthine (100 microM) . After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy . Sixteen compounds had IC(50)s of <3 microM, and five of these had IC(50)s of <0.3 microM and thus were comparable in potency to trimetrexate . The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of C . parvum in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable C . parvum infections.

Microbes Infect, 2001 Nov, 3(13), 1101 - 9
Priming of human neutrophils by mycobacterial lipoarabinomannans: role of granule mobilisation; Faldt J et al.; Lipoarabinomannans (LAMs) from mycobacteria were investigated concerning their effect on human neutrophils . Two types of LAM, the mannose-capped ManLAM from the virulent Mycobacterium tuberculosis H37Rv and the mannose-lacking AraLAM from a rapidly growing mycobacterial strain were used . Neither AraLAM nor ManLAM induced any significant direct activation of the NADPH-oxidase . Both LAMs, however, primed the neutrophils so that subsequent stimulation with the peptide chemoattractants fMet-Leu-Phe (fMLF), Trp-Lys-Tyr-Met-Val-DMet (WKYMVm) and the mammalian lactose-binding lectin galectin-3 resulted in a markedly enhanced oxidative response . The LAM-induced priming was accompanied by an increased exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the neutrophil surface, suggesting that the enhanced oxidative response could be due to upregulation of receptors on the cell surface as a result of granule mobilisation . Since LAM-primed neutrophils released 65% of the cell content of gelatinase but showed no increased release of vitamin B(12)-binding protein, mobilisation of the gelatinase granules rather than the specific granules is concluded to be responsible for the priming effects . This is in agreement with the subcellular localisation of receptors for fMLF, WKYMVm, as well as galectin-3, which are stored in the secretory vesicles and gelatinase granules . The priming effect appeared very similar to that of Escherichia coli lipopolysaccharide, and since no differences in activity could be detected between AraLAM and ManLAM, we hypothesize that the lipid anchor of the LAM is responsible for the priming effects.

Structure (Camb), 2001 Nov, 9(11), 1117 - 25
Escherichia coli GlpE is a prototype sulfurtransferase for the single-domain rhodanese homology superfamily; Spallarossa A et al.; BACKGROUND: Rhodanese domains are structural modules occurring in the three major evolutionary phyla . They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins . Although in vitro assays show sulfurtransferase or phosphatase activity associated with rhodanese or rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established . RESULTS: Eight ORFs coding for proteins consisting of (or containing) a rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome . One of these codes for the 12-kDa protein GlpE, a member of the sn-glycerol 3-phosphate (glp) regulon . The crystal structure of GlpE, reported here at 1.06 A resolution, displays alpha/beta topology based on five beta strands and five alpha helices . The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field . CONCLUSIONS: Relative to the two-domain rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting alpha helices and beta sheets, resulting in radical conformational changes surrounding the active site . As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii rhodaneses . Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain rhodanese module.

Structure (Camb), 2001 Nov, 9(11), 1095 - 106
Structure of Thermotoga maritima stationary phase survival protein SurE: a novel acid phosphatase; Zhang RG et al.; BACKGROUND: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth . rpoS codes for the stationary-phase RNA polymerase sigma subunit, and nlpD codes for a lipoprotein . The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls . The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E . coli subjected to high-temperature growth . The pcm and surE genes are highly conserved in bacteria, archaea, and plants . RESULTS: The structure of SurE from Thermotoga maritima was determined at 2.0 A . The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold . Monomers form a dimer that assembles into a tetramer . Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2 . The active site was identified in the N-terminal domain through analysis of conserved residues . Structure-based site-directed point mutations abolished phosphatase activity . T . maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability . CONCLUSIONS: The structure of SurE provided information about the protein's fold, oligomeric state, and active site . The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified . The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

Structure (Camb), 2001 Nov, 9(11), 1083 - 93
The mechanism of glycerol conduction in aquaglyceroporins; Jensen MO et al.; BACKGROUND: The E . coli glycerol facilitator, GlpF, selectively conducts glycerol and water, excluding ions and charged solutes . The detailed mechanism of the glycerol conduction and its relationship to the characteristic secondary structure of aquaporins and to the NPA motifs in the center of the channel are unknown . RESULTS: Molecular dynamics simulations of GlpF reveal spontaneous glycerol and water conduction driven, on a nanosecond timescale, by thermal fluctuations . The bidirectional conduction, guided and facilitated by the secondary structure, is characterized by breakage and formation of hydrogen bonds for which water and glycerol compete . The conduction involves only very minor changes in the protein structure, and cooperativity between the GlpF monomers is not evident . The two conserved NPA motifs are strictly linked together by several stable hydrogen bonds and their asparagine side chains form hydrogen bonds with the substrates passing the channel in single file . CONCLUSIONS: A complete conduction of glycerol through the GlpF was deduced from molecular dynamics simulations, and key residues facilitating the conduction were identified . The nonhelical parts of the two half-membrane-spanning segments expose carbonyl groups towards the channel interior, establishing a curve-linear pathway . The conformational stability of the NPA motifs is important in the conduction and critical for selectivity . Water and glycerol compete in a random manner for hydrogen bonding sites in the protein, and their translocations in single file are correlated . The suggested conduction mechanism should apply to the whole family.

Structure (Camb), 2001 Nov, 9(11), 1071 - 81
Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme; Martin P et al.; BACKGROUND: In Escherichia coli bearing the plasmid R773, resistance to arsenite, arsenate, antimonite, and tellurite is conferred by the arsRDABC plasmid operon that codes for an ATP-dependent anion pump . The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze the reduction of arsenate to arsenite prior to extrusion . RESULTS: Here, we report the first X-ray crystal structures of ArsC at 1.65 A and of ArsC complexed with arsenate and arsenite at 1.26 A resolution . The overall fold is unique . The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and arsenite . The covalent adduct of arsenate with Cys-12 in the active site of ArsC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 A . However, the corresponding adduct with arsenite binds as a hitherto unseen thiarsahydroxy adduct . Finally, the number of bound waters (385) in this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues . CONCLUSIONS: Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction . The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank.

Structure (Camb), 2001 Nov, 9(11), 1051 - 60
Crystal structure of transcription factor MalT domain III: a novel helix repeat fold implicated in regulated oligomerization; Steegborn C et al.; BACKGROUND: MalT from Escherichia coli, the best-studied member of the MalT family of ATP-dependent transcriptional activators, regulates the genes for malto-oligosaccharide utilization . The active form of this 4 domain protein is a homooligomer, and its multimerization is induced by the binding of maltotriose . Domains II and III of MalT were suggested to mediate the oligomerization process, but its molecular mechanism and the specific functions of these domains remain to be identified . RESULTS: We solved the crystal structure of MalT domain III at 1.45 A resolution by multiple isomorphous replacement phasing . The structure reveals eight copies of a two-helix bundle motif arranged in a novel, right-handed superhelix fold with closed walls, followed by a small C-terminal subdomain . The MalT superhelix contains a potential maltotriose binding site and forms a large hydrophobic protein-protein interaction interface that mediates the contact between two MalT domain III molecules . Structure-based analysis of the two-helix bundle motifs revealed a novel degenerated sequence pattern, and repeats of this pattern could be identified in other regulator proteins . CONCLUSIONS: MalT domain III contains a novel superhelix fold . Its protein-protein interaction interface, however, resembles protein binding sites of other superhelical proteins, suggesting a model with domain III mediating MalT oligomerization . Maltotriose seems to modulate the interaction interface and MalT oligomerization by occupying the ligand binding site inside the superhelix . Similar structural and mechanistic features in other MalT protein-family members and unrelated regulator proteins are indicated by the reappearance of a novel sequence motif derived from the MalT domain III structure.

Structure (Camb), 2001 Nov, 9(11), 999 - 1004
Intricacies in ATP-dependent clamp loading: variations across replication systems; Trakselis MA et al.; DNA replication requires the coordinated effort of many proteins to create a highly processive biomachine able to replicate entire genomes in a single process . The clamp proteins confer on replisomes this property of processivity but in turn require clamp loaders for their functional assembly onto DNA . A more detailed view of the mechanisms for holoenzyme assembly in replication systems has been obtained from the advent of novel solution experiments and the appearance of low- and high-resolution structures for the clamp loaders.

Biochem Soc Trans, 2001 Nov, 29(Pt 6), 806 - 11
Uncoupling protein, H+ transport and regulation; Klingenberg M et al.; The biochemical functions of uncoupling proteins (UCPs) are discussed with the view of UCP1 as a paradigm . In contrast with UCP1, the heterologous expression of UCP3 in yeast is found to result primarily in extra-mitochondrial deposits and thus is unsuitable for studying UCP3 function . On expression in Escherichia coli inclusion bodies, UCPs extracted and incorporated into vesicles showed no H(+) transport, only Cl(-) transport . Only after addition of coenzyme Q was fully nucleotide-sensitive high-H(+) transport reconstituted, with UCP1 as well as with UCP2 and UCP3 . The newly discovered cofactor role of coenzyme Q in H(+) transport is proposed to imply co-operation with fatty acids for the injection of H(+) into the UCP channel.

J Theor Biol, 2001 Nov 7, 213(1), 9 - 19
Gene arrangements and phylogeny in the class Proteobacteria; Kunisawa T; A simple method is presented for reconstructing phylogenetic trees on the basis of gene transposition . It is shown that differences in gene arrangements among genomes could allow us to determine whether a gene transposition event has occurred before or after species divergence from parsimonious considerations . The method is applied to evolutionary relationships among the bacterial class Proteobacteria, for which complete genomic sequences most densely accumulate and comprehensive gene order comparisons are possible . We were able to infer the emergence order of proteobacterial subclasses as epsilon-->beta-->gamma . This order is consistent with sequence-based inferences, which conversely confirms the usefulness of the approach presented here .

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 299 - 304
Design of an artificial light-harvesting unit by protein engineering: cytochrome b(562)-green fluorescent Protein chimera; Takeda S et al.; We have generated a novel model protein for an artificial light-harvesting complex composed of two proteins, cytochrome b(562) (cytb(562)) and enhanced green fluorescent protein (EGFP), in which two chromophores are fixed in each protein matrix . Cytb(562) was appended to the N-terminus of EGFP via a Gly-Ser linker and the resultant fusion protein was successfully expressed in Escherichia coli as a mixture of the apo- and the holo-forms as to the cytb(562) moiety . The fluorescence of EGFP was substantially quenched when the apo-form was reconstituted with hemin . Based on the fluorescence lifetime measurements, it appeared that light energy entrapped by EGFP is transferred to the heme of cytb(562) by resonance energy transfer (energy transfer yield: 65%) . Spatial organization of two chromophores using small and stable protein matrices will be promising toward the construction of an artificial light-harvesting complex by protein engineering .

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 264 - 8
NC1 domain of human type VIII collagen (alpha 1) inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis; Xu R et al.; Endostatin, a natural angiogenesis inhibitor, had been identified for years . It opened a new approach for cancer therapy . Sequence analysis revealed that endostatin is the NC1 domain (non-triple-helical domain) of collagen XVIII . In this report, the cDNA of NC1 domain of type VIII collagen (alpha 1) was cloned and expressed as soluble form in Escherichia coli . The recombinant protein was purified with Ni-NTA agarose column and named as vastatin . It inhibited the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner . The ED(50) of vastatin was 0.6 microg/ml, while the ED(50) of endostatin was 0.5 microg/ml . Treatment of BAE cell with vastatin caused G(0)-G(1) arrest and cell apoptosis . It is interesting that sequence analysis showed that there was only about 12% amino acid sequence homology between vastatin and endostatin . The structure-function relationship of these angiogenesis molecules remains to be elucidated .

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 252 - 6
A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages; Nakamura M et al.; Direct visualization of filamentous phage infection in Escherichia coli (E . coli) was attempted using biotinylated phages (BIO-phages) . The biotinylation of the phages did not influence their infectivity into E . coli . E . coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E . coli could be directly observed by using the avidin-biotin-peroxidase complex method with electron microscopy . This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin-avidin interaction . This simple and powerful method is applicable to the study of infection by various viruses .

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 161 - 6
Production and characterization of the recombinant Sphingomonas chlorophenolica pentachlorophenol 4-monooxygenase; Wang H et al.; Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen . In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein . Moreover, a tobacco etch virus (TEV) protease cleavage site (EKLYFQG) was introduced into GST-PCP4MO and a his-tagged TEV protease was employed . Hence, a two-step purification protocol was developed which allowed obtaining 15-20 mg of rePCP4MO from 1 L culture . The rePCP4MO revealed identity with native enzyme by SDS-PAGE and N-terminal sequence analyses . Furthermore, a polyclonal PCP4MO antibody was produced with GST-PCP4MO and purified by immunoaffinity chromatography, where both the native and recombinant forms of PCP4MO showed interaction . However, rePCP4MO was identified as apoprotein with no evidence for a typical flavoprotein spectrum . The catalytic activity could be detected in the presence of FAD . The K(m) and V(max) values for PCP were 50 microM and 30 nmol/min/mg, respectively .

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 125 - 9
Thermal stability of human ferritin: concentration dependence and enhanced stability of an N-terminal fusion mutant; Kim SW et al.; Though human L-chain ferritin has been known to be more resistant to physical denaturation than H-type ferritin, its stability characteristics and kinetic information have not been reported in detail . Overexpressed recombinant ferritin (FTN) in Escherichia coli formed inclusion bodies through noncovalent molecular interaction and easily dissolved with regaining the iron-uptake activity by a simple pH-shift process at high protein concentration (>600 mg l(-1)) . FTN was relatively thermostable at low protein concentration (0.2 g l(-1)), but it became extremely thermolabile at high protein concentration (1.3 g l(-1)), i.e., more than 80% of FTN was coprecipitated within 5 min under the same heat-induced denaturation condition . Aggregation rate constant for initial 5 min at high protein concentration was 6.04 x 10(-3) s(-1) for FTN . Surprisingly, glucagon . ferritin mutant (GFTN), consisting of an N-terminus fusion partner, human glucagon (29-residue alpha-helical peptide), showed significantly enhanced thermal stability even at high protein concentration . That is, in spite of 40-min heat treatment, more than 50% of GFTN the still remained soluble with maintaining the same functional properties . The aggregation rate constants were 2.75 x 10(-4) and 2.80 x 10(-4) s(-1) at low and high concentration, respectively, for GFTN . These results suggest a critical participation of the N-terminal domain of ferritin in the temperature-induced aggregation pathway . Presumably, partially denatured amino terminus of FTN is involved in nonspecific molecular interaction resulting in the off-pathway aggregation . It is notable that the purified GFTN showed the same molar capacity of iron (Fe(+3)) storage as standard ferritin . From the analysis of fluorescence emission spectrum, the physical stability of GFTN was also very comparable to that of standard ferritin under the various denaturation conditions induced by GdnHCl .

Transgenic Res, 2001 Oct, 10(5), 409 - 22
Selection and orientation of adjacent genes influences DAM-mediated male sterility in transformed maize; Unger E et al.; Anther-targeted expression of E . coli DNA (Adenosine-N6-)-Methyltransferase (DAM) in maize was tested as a means to produce male-sterile plants . A high frequency of male-sterile plants with reduced anther size was observed when DAM was regulated by the maize anther-specific promoter 5126 (5126:DAM) and placed upstream of the herbicide resistance gene, pat, regulated by the cauliflower mosaic virus (CaMV) 35S promoter (35S:PAT) . In contrast, placement of 5126:DAM upstream of a pat gene regulated by either the maize ubiquitin (UBI:PAT) or rice actin (rACTIN:PAT) promoters resulted in male-fertile plants . Based on these observed differences, DAM-mediated sterility was used as a phenotypic marker to assess the contribution of factors affecting gene expression such as orientation of the transcription units, choice of regulatory sequences mediating expression of adjacent genes, and effects of varying the anther-specific promoter regulating DAM . Constructs that place a portion of the CaMV 35S promoter, including the native AS-1 sequences, between 5126:DAM and UBI:PAT yielded a high frequency of male-sterile plants with reduced anther size . Significant differences in the frequency of male-sterile events and the associated anther size were also observed when the position of 35S:PAT was changed relative to 5126:DAM . These data provide evidence that gene expression in transformed maize plants can be impacted by simply altering the order, orientation or regulatory sequences of adjacent genes.

Protein Eng, 2001 Sep, 14(9), 699 - 704
Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution; Sun L et al.; We have used directed evolution methods to express a fungal enzyme, galactose oxidase (GOase), in functional form in Escherichia coli . The evolved enzymes retain the activity and substrate specificity of the native fungal oxidase, but are more thermostable, are expressed at a much higher level (up to 10.8 mg/l of purified GOase), and have reduced negative charge compared to wild type, all properties which are expected to facilitate applications and further evolution of the enzyme . Spectroscopic characterization of the recombinant enzymes reveals a tyrosyl radical of comparable stability to the native GOase from Fusarium.

Protein Eng, 2001 Sep, 14(9), 669 - 74
N-terminal portion acts as an initiator of the inactivation of pepsin at neutral pH; Tanaka T et al.; Porcine pepsin, an aspartic protease, is unstable at neutral pHs where it rapidly loses activity, however, its zymogen, pepsinogen, is stable at neutral pHs . The difference between the two is the presence of the prosegment in pepsinogen . In this study, possible factors responsible for instability were investigated and included: (i) the distribution of positively charged residues on the surface, (ii) an insertion of a peptide in the C-terminal domain and (iii) the dissociation of the N-terminal fragment of pepsin . Mutations to change the number and the distribution of positive charges on the surface had a minor effect on stability . No effect on stability was observed for the deletion of a peptide from the C-terminal domain . However, mutations on the N-terminal fragment had a major impact on stability . At pH 7.0, the N-fragment mutant was inactivated 5.8 times slower than the wild-type . The introduction of a disulfide bond between the N-terminal fragment and the enzyme body prevented the enzyme from denaturing . The above results showed that the inactivation of pepsin was initiated by the dissociation of the N-fragment and that the sequence of this portion was a major determinant for enzyme stability . Through this study, we have created porcine pepsin with increased pH stability at neutral pHs.

Protein Eng, 2001 Sep, 14(9), 647 - 54
Generation of protein lineages with new sequence spaces by functional salvage screen; Kim GJ et al.; A variety of different methods to generate diverse proteins, including random mutagenesis and recombination, are currently available and most of them accumulate the mutations on the target gene of a protein, whose sequence space remains unchanged . On the other hand, a pool of diverse genes, which is generated by random insertions, deletions and exchange of the homologous domains with different lengths in the target gene, would present the protein lineages resulting in new fitness landscapes . Here we report a method to generate a pool of protein variants with different sequence spaces by employing green fluorescent protein (GFP) as a model protein . This process, designated functional salvage screen (FSS), comprises the following procedures: a defective GFP template expressing no fluorescence is first constructed by genetically disrupting a predetermined region(s) of the protein and a library of GFP variants is generated from the defective template by incorporating the randomly fragmented genomic DNA from Escherichia coli into the defined region(s) of the target gene, followed by screening of the functionally salvaged, fluorescence-emitting GFPs . Two approaches, sequence-directed and PCR-coupled methods, were attempted to generate the library of GFP variants with new sequences derived from the genomic segments of E.coli . The functionally salvaged GFPs were selected and analyzed in terms of the sequence space and functional properties . The results demonstrate that the functional salvage process not only can be a simple and effective method to create protein lineages with new sequence spaces, but also can be useful in elucidating the involvement of a specific region(s) or domain(s) in the structure and function of protein.

Protein Eng, 2001 Sep, 14(9), 633 - 8
A cross-section of the fitness landscape of dihydrofolate reductase; Aita T et al.; In vitro molecular evolution is regarded as a hill-climbing on a fitness landscape in sequence space, where the 'fitness' is a quantitative measure of a certain physicochemical property of a biopolymer . We analyzed a 'cross-section' of the enzymatic activity landscape of dihydrofolate reductase (DHFR) by using a method of analysis of a fitness landscape . We limited the sequence space of interest to the five-dimensional sequence space, where the coordinate corresponds to the 1st, 16th, 20th, 42nd and 92nd site in the DHFR sequence . Thirty six mutants mapped into the limited sequence space were taken in the analysis . As a result, the cross-section is of the rough Mt Fuji type based on the mutational additivity . The ratio of the mean slope to the roughness is 2.8 and the Z-score of the original ratio against a distribution of random references is 7.0, which indicates a large statistical significance . The existence of such a cross-section was discussed in terms of the occurrence probability of sets of five sites distantly separated from each other on the DHFR 3D structure . Our results support the effectiveness of the evolution strategy which exploits the accumulation of advantageous single point mutations in such a cross-section.

Protein Eng, 2001 Sep, 14(9), 609 - 14
Similarity of phylogenetic trees as indicator of protein-protein interaction; Pazos F et al.; Deciphering the network of protein interactions that underlines cellular operations has become one of the main tasks of proteomics and computational biology . Recently, a set of bioinformatics approaches has emerged for the prediction of possible interactions by combining sequence and genomic information . Even though the initial results are very promising, the current methods are still far from perfect . We propose here a new way of discovering possible protein-protein interactions based on the comparison of the evolutionary distances between the sequences of the associated protein families, an idea based on previous observations of correspondence between the phylogenetic trees of associated proteins in systems such as ligands and receptors . Here, we extend the approach to different test sets, including the statistical evaluation of their capacity to predict protein interactions . To demonstrate the possibilities of the system to perform large-scale predictions of interactions, we present the application to a collection of more than 67 000 pairs of E.coli proteins, of which 2742 are predicted to correspond to interacting proteins.

J Cell Sci, 2001 Oct, 114(Pt 20), 3727 - 36
Nuclear localization of neutral sphingomyelinase 1: biochemical and immunocytochemical analyses; Mizutani Y et al.; To examine the intracellular localization of neutral sphingomyelinase 1 (nSMase 1), a rabbit polyclonal antibody was raised against a recombinant form of the enzyme expressed in E . coli . It has been reported that, in rat liver or in ascites hepatoma AH7974, high activity of neutral sphingomyelinase (SMase) is found at the plasma membrane, with a lesser but significant amount in nucleus and cytoplasm . The biochemical properties, dithiothreitol requirement and high salt concentration dependency, of cloned and expressed nSMase 1 resemble those of previously described nuclear neutral SMase of AH7974 . The present study was therefore focused on the nuclear localization of this enzyme . Western blotting of subcellular fractions using anti-rat nSMase 1 antibody revealed most nSMase 1 to be associated with the nuclei and some with microsomes, but not with plasma membranes . Consistently, neutral SMase activity in nuclear extract was immunoprecipitated by the antibody, while that of plasma membranes was not . The results indicate that nSMase 1 mainly resides in the nucleus and may thus differ from neutral SMase in plasma membrane . On gel-filtration column chromatography of nuclear extract, the profile of neutral SMase activity corresponded well with immunoreactive protein bands on western blotting, suggesting that a large part of nuclear neutral SMase may be nSMase 1 . Removal of the nuclear envelope by treatment with Triton X-100 did not significantly decrease the amount of nuclear nSMase 1, and western blotting of subnuclear fractions (i.e . nuclear envelope, chromatin, and nuclear matrix) revealed nSMase 1 signal exclusively in the nuclear matrix . Immunocytochemistry with AH7974, as well as rat fibroblast cell line 3Y1, demonstrated nSMase 1 to be localized mainly in the nucleus, with some in the cytoplasm . Moreover, immuno-electron microscopy clearly showed the signal of nSMase 1 to be more dense in the nucleus than in the cytoplasm of AH7974.

EMBO J, 2001 Nov 15, 20(22), 6443 - 52
Regulation of nuclear poly(A) addition controls the expression of immunoglobulin M secretory mRNA; Phillips C et al.; B-cell differentiation is accompanied by a dramatic increase in cytoplasmic accumulation and stability of the IgM heavy chain (mu) secretory mRNA . Despite considerable effort, the mechanism is unknown . We have identified three short motifs upstream of the secretory poly(A) site, which, when mutated in the mu heavy chain gene, significantly increase the accumulation of the secretory form of poly(A)(+) mRNA relative to the membrane form and regulate the expression of the secretory poly(A) site in a developmental manner . We show that these motifs bind U1A and inhibit polyadenylation in vitro and in vivo . Overexpression of U1A in vivo results in the selective inhibition of the secretory form . Thus, this novel mechanism selectively controls post-cleavage expression of the mu secretory mRNA . We present evidence that this mechanism is used to regulate alternative expression of other genes.

EMBO J, 2001 Nov 15, 20(22), 6371 - 82
Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint; Sironi L et al.; Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis . The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos . Here we show that Mad2 forms incompatible complexes with Mad1 and Cdc20, neither of which requires Mad2 oligomerization . A monomeric point mutant of Mad2 can sustain a cell cycle arrest of comparable strength to that of the wild-type protein . We show that the interaction of Mad2 with Mad1 is crucial for the localization of Mad2 to kinetochores, where Mad2 interacts with Cdc20 . We propose a model that features the kinetochore as a 'folding factory' for the formation of a Mad2-Cdc20 complex endowed with inhibitory activity on the anaphase promoting complex.

EMBO J, 2001 Nov 15, 20(22), 6297 - 305
Co-chaperones Bag-1, Hop and Hsp40 regulate Hsc70 and Hsp90 interactions with wild-type or mutant p53; King FW et al.; Using highly purified proteins, we have identified intermediate reactions that lead to the assembly of molecular chaperone complexes with wild-type or mutant p53R175H protein . Hsp90 possesses higher affinity for wild-type p53 than for the conformational mutant p53R175H . The presence of Hsp90 in a complex with wild-type p53 inhibits the binding of Hsp40 and Hsc70 to p53, consequently preventing the formation of wild-type p53-multiple chaperone complexes . The conformational mutant p53R175H can form a stable heterocomplex with Hsp90 only in the presence of Hsc70, Hsp40, Hop and ATP . The anti-apoptotic factor Bag-1 can dissociate Hsp90 from a pre- assembled complex wild-type p53 protein, but it cannot dissociate a pre-assembled p53R175H-Hsp40- Hsc70-Hop-Hsp90 heterocomplex . The results presented here provide possible molecular mechanisms that can help to explain the observed in vivo role of molecular chaperones in the stabilization and cellular localization of wild-type and mutant p53 protein.

EMBO J, 2001 Nov 15, 20(22), 6246 - 55
Apparent genetic redundancy facilitates ecological plasticity for nitrate transport; Unkles SE et al.; Aspergillus nidulans possesses two high-affinity nitrate transporters, encoded by the nrtA and the nrtB genes . Mutants expressing either gene grew normally on 1-10 mM nitrate as sole nitrogen source, whereas the double mutant failed to grow on nitrate concentrations up to 200 mM . These genes appear to be regulated coordinately in all growth conditions, growth stages and regulatory genetic backgrounds studied . Flux analysis of single gene mutants using 13NO3(-) revealed that K(m) values for the NrtA and NrtB transporters were approximately 100 and approximately 10 microM, respectively, while V(max) values, though variable according to age, were approximately 600 and approximately 100 nmol/mg dry weight/h, respectively, in young mycelia . This kinetic differentiation may provide the necessary physiological and ecological plasticity to acquire sufficient nitrate despite highly variable external concentrations . Our results suggest that genes involved in nitrate assimilation may be induced by extracellular sensing of ambient nitrate without obligatory entry into the cell.

Gene, 2001 Oct 31, 278(1-2), 115 - 24
Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis; Kaps I et al.; The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis . Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP) . A 160-fold variation of GFP expression levels in M . smegmatis was achieved by combining three promoters with different copy numbers of the vectors . An efficient energy transfer between BFP and GFP in M . smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors . Thus, these vectors will be valuable for all strategies where co-expression of proteins in M . smegmatis is needed, e.g . for constructing a two-hybrid system or for deleting essential genes.

FEBS Lett, 2001 Nov 9, 508(1), 103 - 6
SecDFyajC is not required for the maintenance of the proton motive force; Nouwen N et al.; SecDFyajC of Escherichia coli is required for efficient export of proteins in vivo . However, the functional role of SecDFyajC in protein translocation is unclear . We evaluated the postulated function of SecDFyajC in the maintenance of the proton motive force . As previously reported, inner membrane vesicles (IMVs) lacking SecDFyajC are defective in the generation of a stable proton motive force when energized with succinate . This phenomenon is, however, not observed when NADH is used as an electron donor . Moreover, the proton motive force generated in SecDFyajC-depleted vesicles stimulated translocation to the same extent as seen with IMVs containing SecDFyajC . Further analysis demonstrates that the reduced proton motive force with succinate in IMVs lacking SecDFyajC is due to a lower amount of the enzyme succinate dehydrogenase . The expression of this enzyme complex is repressed by growth on glucose media, the condition used to deplete SecDFyajC . These results demonstrate that SecDFyajC is not required for proton motive force-driven protein translocation.

J Am Chem Soc, 2001 Nov 21, 123(46), 11353 - 9
Biosynthesis of vitamin B(6) in yeast: incorporation pattern of glucose; Gupta RN et al.; Two yeasts, Saccharomyces cerevisiae ATCC 7752 and Candida utilis ATCC 9256, were incubated in the presence of variously multiply (13)C-labeled samples of D-glucose . The (13)C incorporation pattern within pyridoxamine dihydrochloride, established by (13)C NMR spectroscopy, differed from that which had previously been found within pyridoxine, isolated from Escherichia coli . Thus, the origin of the carbon skeleton of vitamin B(6) in yeast differs substantially from its origin in E . coli . In particular, in yeast the distribution of (13)C within the C(5) chain C-2',2,3,4,4' of pyridoxamine corresponds to the distribution of (13)C within the C(5) chain C-1,2,3,4,5 of the ribose component of cytidine . It follows that the C(5) chains of pyridoxamine and of ribose originate from a common glucose-derived pentulose or pentose intermediate . By contrast, in E . coli the C(5) chain of pyridoxine is derived from 1-deoxy-D-xylulose 5-phosphate which, in turn, originates by condensation of pyruvic acid with glyceraldehyde 3-phosphate.

Plant Physiol, 2001 Nov, 127(3), 1299 - 309
Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as a proton donor; Rouhier N et al.; A sequence coding for a peroxiredoxin (Prx) was isolated from a xylem/phloem cDNA library from Populus trichocarpa and subsequently inserted into an expression plasmid yielding the construction pET-Prx . The recombinant protein was produced in Escherichia coli cells and purified to homogeneity with a high yield . The poplar Prx is composed of 162 residues, a property that makes it the shortest plant Prx sequence isolated so far . It was shown that the protein is monomeric and possesses two conserved cysteines (Cys) . The Prx degrades hydrogen peroxide and alkyl hydroperoxides in the presence of an exogenous proton donor that can be either thioredoxin or glutaredoxin (Grx) . Based on this finding, we propose that the poplar protein represents a new type of Prx that differs from the so-called 2-Cys and 1-Cys Prx, a suggestion supported by the existence of natural fusion sequences constituted of a Prx motif coupled to a Grx motif . The protein was shown to be highly expressed in sieve tubes where thioredoxin h and Grx are also major proteins.

Plant Physiol, 2001 Nov, 127(3), 1243 - 55
Arabidopsis dynamin-like 2 that binds specifically to phosphatidylinositol 4-phosphate assembles into a high-molecular weight complex in vivo and in vitro; Kim YW et al.; Arabadopsis dynamin-like (ADL) 2, a member of the high-molecular weight (M(r)) dynamin family found in Arabidopsis, has been shown to be targeted to the plastid . In the chloroplast, most of the ADL2 was present in the fraction containing the envelope membranes when analyzed by suborganellar fractionation . Sucrose gradient and gel filtration experiments showed that when associated with membranes, ADL2 existed as a high-M(r) complex, whereas the soluble form existed as a monomer . The recombinant ADL2 expressed in Escherichia coli was present as a high-M(r) form and showed higher GTPase activity at a low NaCl concentration, whereas ADL2 existed as a low-M(r) form with a low level of GTPase activity at a high NaCl concentration . Electron microscopy studies revealed that the purified recombinant ADL2 formed spiral-coiled structures or rings . In the presence of guanosine-5'-O-(3-thio)triphosphate, these structures were transformed into a long rod structure . In contrast, in the presence of GDP, these structures disassembled into oligomers that were shown to be tetramer with 4-fold symmetry . Finally, a lipid-binding assay revealed that recombinant ADL2 purified from E . coli bound specifically to phosphatidylinositol 4-phosphate . Together, these results demonstrated that the biochemical properties of ADL2 were very similar to those of dynamin and other related proteins . Based on this similarity, we propose that ADL2 may be involved in vesicle formation at the chloroplast envelope membrane.

Plant Physiol, 2001 Nov, 127(3), 1224 - 33
Biochemical characterization of the Arabidopsis biotin synthase reaction . The importance of mitochondria in biotin synthesis; Picciocchi A et al.; Biotin synthase, encoded by the bio2 gene in Arabidopsis, catalyzes the final step in the biotin biosynthetic pathway . The development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein . Under optimized conditions, the turnover number of the reaction was >2 h(-1) with this in vitro system . Purified Bio2 protein was not efficient by itself in supporting biotin synthesis . However, heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yielded a functional biotin synthase complex . Biotin synthase in this heterologous system obeyed Michaelis-Menten kinetics with respect to dethiobiotin (K(m) = 30 microM) and exhibited a kinetic cooperativity with respect to S-adenosyl-methionine (Hill coefficient = 1.9; K(0.5) = 39 microM), an obligatory cofactor of the reaction . In vitro inhibition of biotin synthase activity by acidomycin, a structural analog of biotin, showed that biotin synthase reaction was the specific target of this inhibitor of biotin synthesis . It is important that combination experiments using purified Bio2 protein and extracts from pea (Pisum sativum) leaf or potato (Solanum tuberosum) organelles showed that only mitochondrial fractions could elicit biotin formation in the plant-reconstituted system . Our data demonstrated that one or more unidentified factors from mitochondrial matrix (pea and potato) and from mitochondrial membranes (pea), in addition to the Bio2 protein, are obligatory for the conversion of dethiobiotin to biotin, highlighting the importance of mitochondria in plant biotin synthesis.

Plant Physiol, 2001 Nov, 127(3), 1102 - 12
A novel phospholipase D of Arabidopsis that is activated by oleic acid and associated with the plasma membrane; Wang C et al.; Oleate-dependent phospholipase D (PLD; EC 3.1.4.4) has been reported in animal systems, but its molecular nature is unkown . Multiple PLDs have been characterized in plants, but none of the previously cloned PLDs exhibits the oleate-activated activity . Here, we describe the biochemical and molecular identification and characterization of an oleate-activated PLD in Arabidopsis . This PLD, designated PLDdelta, was associated tightly with the plasma membrane, and its level of expression was higher in old leaves, stems, flowers, and roots than in young leaves and siliques . A cDNA encoding the oleate-activated PLD was identified, and catalytically active PLDdelta was expressed from its cDNA in Escherichia coli . PLDdelta was activated by free oleic acid in a dose-dependent manner, with the optimal concentration being 0.5 mM . Other unsaturated fatty acids, linoleic and linolenic acids, were less effective than oleic acid, whereas the saturated fatty acids, stearic and palmitic acids, were totally ineffective . Phosphatidylinositol 4,5-bisphosphate stimulated PLDdelta to a lesser extent than oleate . Mutation at arginine (Arg)-611 led to a differential loss of the phosphatidylinositol 4,5-bisphosphate-stimulated activity of PLDdelta, indicating that separate sites mediate the oleate regulation of PLDdelta . Oleate stimulated PLDdelta's binding to phosphatidylcholine . Mutation at Arg-399 resulted in a decrease in oleate binding by PLDdelta and a loss of PLDdelta activity . However, this mutation bound similar levels of phosphatidylcholine as wild type, suggesting that Arg-399 is not required for PC binding . These results provide the molecular information on oleate-activated PLD and also suggest a mechanism for the oleate stimulation of this enzyme.

J Biol Chem, 2002 Feb 22, 277(8), 6073 - 9 Epub 2001 Nov 12.
Phosphatidylinositol 3-phosphate-interacting domains in PIKfyve . Binding specificity and role in PIKfyve . Endomenbrane localization; Sbrissa D et al.; PIKfyve is a phosphatidylinositol (PtdIns) 3-phosphate (P)-metabolizing enzyme, which, in addition to a C-terminally positioned catalytic domain, harbors several evolutionarily conserved domains, including a FYVE finger . The FYVE finger domains are thought to direct the protein localization to intracellular membrane PtdIns 3-P . Recent studies with several FYVE domain proteins challenge this general concept . Here we have examined the binding of PIKfyve's FYVE domain to PtdIns 3-P in vitro and in vivo and a plausible contribution of this binding mechanism for the intracellular localization of the full-length protein . We document now a specific and high affinity interaction of a recombinantly produced PIKfyve FYVE domain peptide fragment with PtdIns 3-P-containing liposomes that requires the presence of the conservative core of basic residues within the FYVE domain . PIKfyve localization to membranes of the late endocytic pathway was found to be absolutely dependent on the presence of an intact FYVE finger . Cell treatment with PI 3-kinase inhibitor wortmannin dissociated endosome-bound PIKfyve, indicating that the protein targeted the membrane PtdIns 3-P . An enzymatically inactive peptide fragment of the PIKfyve catalytic domain was found to also specifically bind to PtdIns 3-P-containing liposomes, with residue Lys-1999 being critical in the interaction . This binding, however, was of relatively low affinity and, in the cellular context, was found ineffective in directing the molecule to PtdIns 3-P-enriched endosomes . Collectively, these results demonstrate that interaction of the FYVE domain with PtdIns 3-P is absolutely necessary for PIKfyve targeting to the membranes of the late endocytic pathway and determine PIKfyve as a downstream effector of PtdIns 3-P.

J Biol Chem, 2002 Jan 25, 277(4), 2547 - 53 Epub 2001 Nov 12.
Complex formation of cytochrome P450cam with Putidaredoxin . Evidence for protein-specific interactions involving the proximal thiolate ligand; Unno M et al.; We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin . We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component . Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond . In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation . On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds . We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam) . These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation . These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.

J Biol Chem, 2002 Feb 15, 277(7), 4747 - 54 Epub 2001 Nov 12.
Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max: relation to phytochelatin synthase; Oven M et al.; The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes . To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals . The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana . When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation . GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not . Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture . The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1 . In addition to bis(glutathionato)-metal complexes, various other metal-thiolates were shown to contribute to the activation of phytochelatin synthase . These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O . K., Mari, S., Lu, Y . P., and Rea, P . A . (2000) J . Biol . Chem . 275, 31451-31459).

J Biol Chem, 2002 Feb 1, 277(5), 3210 - 8 Epub 2001 Nov 12.
Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell death via a caspase-1-dependent mechanism, independently of cytokine production; Le Feuvre RA et al.; ATP stimulation of cell surface P2X7 receptors results in cytolysis and cell death of macrophages . Activation of this receptor in bacterial lipopolysaccharide (LPS)-activated macrophages or monocytes also stimulates processing and release of the cytokine interleukin-1beta(IL-1beta) through activation of caspase-1 . The cytokine interleukin 18 (IL-18) is also cleaved by caspase-1 and shares pro-inflammatory characteristics with IL-1beta . The objective of the present study was to test the hypothesis that IL-1beta, IL-18, and/or caspase-1 activation contribute directly to macrophage cell death induced by LPS and ATP . Macrophages were cultured from normal mice or those in which genes for the P2X7 receptor, IL-1beta, IL-1alpha, IL-18, or caspase-1 had been deleted . Our data confirm the importance of the P2X7 receptor in ATP-stimulated cell death and IL-1beta release from LPS-primed macrophages . We demonstrate that prolonged stimulation with ATP leads to cell death, which is partly dependent on LPS priming and caspase-1, but independent of cytokine processing and release . We also provide evidence that LPS priming of macrophages makes them highly susceptible to the toxic effects of brief exposure to ATP, which leads to rapid cell death by a mechanism that is dependent on caspase-1 but, again, independent of cytokine processing and release.

J Biol Chem, 2002 Feb 1, 277(5), 3202 - 9 Epub 2001 Nov 08.
Neither Reb1p nor poly(dA*T) elements are responsible for the highly specific chromatin organization at the ILV1 promoter; Moreira JM et al.; Analysis of the chromatin structure at the yeast ILV1 locus revealed highly positioned nucleosomes covering the entire locus except for a hypersensitive site in the promoter region . All previously identified cis-acting elements required for GCN4-independent ILV1 basal level transcription, including a binding site for the REB1 protein (Reb1p), and a poly(dA*dT) element (26 As out of 32 nucleotides) situated 15 base pairs downstream of the Reb1p-binding site, reside within this hypersensitive site . The existence of a second A*T-rich element (25 As out of 33 nucleotides) present six base pairs upstream of the Reb1p-binding site, suggested that nucleosome exclusion from the hypersensitive site in the ILV1 promoter region might be dictated by synergistic action of the two poly(dA*dT) elements . Replacing one or both of them had, however, no effect on the chromatin structure of the ILV1 promoter, although drastically reduced basal transcription . Similarly, deletion of the Reb1p-binding site, albeit affecting ILV1 expression, had no detectable effect on chromatin at the ILV1 promoter . The absence of a good correlation between effects of these elements on gene activity and on chromatin structure at the ILV1 promoter indicates that the chromatin organization present at the ILV1 promoter is independent of the known regulatory elements and most likely dictated directly by the DNA sequence.

J Biol Chem, 2002 Jan 11, 277(2), 1255 - 60 Epub 2001 Nov 08.
MutS preferentially recognizes cisplatin- over oxaliplatin-modified DNA; Zdraveski ZZ et al.; Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin . Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents . This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts . MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA . ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts . In parallel cytotoxicity experiments, methylation-deficient E . coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds . A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks . The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.

Infect Immun, 2001 Dec, 69(12), 7941 - 5
Characterization of the adhesin of Escherichia coli F18 fimbriae; Smeds A et al.; Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in Escherichia coli is either the FedE or FedF protein . In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit E . coli adhesion to porcine enterocytes . Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein.

Infect Immun, 2001 Dec, 69(12), 7793 - 9
Dynamic changes in neutrophil defensins during endotoxemia; Klut ME et al.; Bacterial endotoxin or lipopolysaccharide (LPS) is an important causative agent of sepsis . This study determines the expression of defensins NP-2 and NP-5 and the function of polymorphonuclear leukocytes (PMN) in rabbits treated with LPS . PMN functional activity was assessed by measuring CD18 expression and H(2)O(2) production and by examining the lungs . NP-2 and, to a minor extent, NP-5 of circulating PMN increase during endotoxemia . This early increase is concomitant with neutrophilia and elevated CD18 expression and H(2)O(2) production, as well as with enhanced NP-2 immunoreactivity in pulmonary microvessels . A decline in defensins, shortly after the last LPS treatment, is associated with a decrease in the circulating activated PMN and enhanced immunoreactivity in the inflammatory cells, as well as with lung tissue damage . This study shows that LPS-induced changes in the defensins of circulating PMN correlate with the number and activated condition of these cells and suggests that PMN-derived products implement the inflammatory reaction that leads to lung injury and sepsis.

Infect Immun, 2001 Dec, 69(12), 7616 - 24
Molecular characterization of thermoinduced immunogenic proteins Q1p42 and Hsp15 of Leptospira interrogans; Nally JE et al.; Leptospira interrogans is a mammalian pathogen which must adapt to a range of new environmental conditions including temperature change when it infects new hosts . In vitro studies of organisms cultured at 30 degrees C and shifted to 37 degrees C for 5 to 7 days have confirmed that synthesis of several proteins involved in equine infection is regulated in response to temperature change (J . E . Nally, J . F . Timoney, and B . Stevenson, Infect . Immun . 69:400-404, 2001) . In order to specifically identify antigenic proteins upregulated at 37 degrees C, groups of three ponies were immunized with organisms shifted to 37 degrees C for 5 to 7 days or with organisms maintained at 30 degrees C . A lambda ZAP II genomic DNA library was screened with the pool of antisera to organisms shifted to 37 degrees C . Clones reactive with this pool but unreactive with the pool of pony antisera to organisms cultured at 30 degrees C were selected for further analysis . Sequence analysis of the first two clones identified open reading frames for proteins designated Qlp42 and Hsp15 . Qlp42 is predicted to be an outer membrane lipoprotein . Its synthesis was upregulated when cultures were shifted from 30 to 37 degrees C and downregulated when cultures were shifted from 37 to 30 degrees C . Although the predicted molecular mass of Qlp42 is 39.8 kDa for the mature protein, Qlp42-specific equine antiserum was reactive with two bands of 30 and 29.5 kDa . Hsp15 is a stress response protein and a member of the Hsp20/alpha-crystallin family . PCR detected homologues of qlp42 and hsp15 in pathogenic serovars of L . interrogans but not in the nonpathogenic Leptospira biflexa . Enzyme-linked immunosorbent assays of antibody in convalescent sera from mares naturally infected with L . interrogans suggest that Qlp42 is expressed during leptospiral infection.

Infect Immun, 2001 Dec, 69(12), 7356 - 64
Induction of epithelial cell death including apoptosis by enteropathogenic Escherichia coli expressing bundle-forming pili; Abul-Milh M et al.; Infection with enteropathogenic Escherichia coli (EPEC) is a major cause of severe infantile diarrhea, particularly in parts of the developing world . The bundle-forming pilus (BFP) of EPEC is an established virulence factor encoded on the EPEC adherence factor plasmid (EAF) and has been implicated in both localized adherence to host cells and bacterial autoaggregation . We investigated the role of BFP in the ability of EPEC binding to kill host epithelial cells . BFP-expressing strains killed all three cell lines tested, comprising HEp-2 (laryngeal), HeLa (cervical), and Caco-2 (colonic) cells . Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA, and morphological changes detected by electron microscopy indicated evidence of apoptosis . The extent of cell death was significantly greater for BFP-expressing strains, including E2348/69, a wild-type clinical isolate, as well as for a laboratory strain, HB101, transformed with a bfp-carrying plasmid . Strains which did not express BFP induced significantly less cell death, including a bfpA disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, HB101, and HB101 complemented with the locus of enterocyte effacement pathogenicity island . These results indicate a direct correlation between BFP expression and induction of cell death, including apoptosis, an event which may involve the targeting of host cell membrane phosphatidylethanolamine.

Infect Immun, 2001 Dec, 69(12), 7205 - 12
Characterization of receptor-mediated signal transduction by Escherichia coli type IIa heat-labile enterotoxin in the polarized human intestinal cell line T84; Wimer-Mackin S et al.; Escherichia coli type IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b . It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT) . In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium . Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl(-) secretory response . LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT . Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl(-) secretory response . These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa . The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase . Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b . These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells . The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT . However, the extent of association with lipid rafts and the magnitude of the Cl(-) secretory response in T84 cells were less for LTIIa than for CT . These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl(-) secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates of E . coli.

Am J Physiol Gastrointest Liver Physiol, 2001 Dec, 281(6), G1423 - 31
Hepatic and extrahepatic factors critical for liver injury during lipopolysaccharide exposure; Moulin F et al.; Bacterial endotoxin {lipopolysaccharide (LPS)} causes liver injury in vivo that is dependent on platelets, neutrophils {polymorphonuclear leukocytes (PMNs)}, and several inflammatory mediators, including thrombin . We tested the hypothesis that thrombin contributes to LPS-induced hepatocellular injury through direct interactions with platelets and/or PMNs in vitro . Perfusion of isolated livers from LPS-treated rats with buffer containing thrombin resulted in a significant increase in alanine aminotransferase (ALT) activity in the perfusion medium, indicating hepatocellular damage . This effect was completely abolished by prior depletion of PMNs from the LPS-treated donor rats but not by depletion of platelets, suggesting interaction between thrombin and PMNs in the pathogenesis . Thrombin did not, however, enhance degranulation of rat PMNs in vitro, and it was not directly toxic to isolated rat hepatocytes in the presence of PMNs even after LPS exposure, suggesting that hepatocellular killing by the PMN-thrombin combination requires the intervention of an additional factor(s) within the liver . In livers from naive donors perfused with buffer containing PMNs and LPS, no injury occurred in the absence of thrombin . Addition of thrombin (10 nM) to the medium caused pronounced ALT release . These results indicate that thrombin and PMNs are sufficient extrahepatic requirements for LPS-induced hepatocellular damage in intact liver.

Free Radic Biol Med, 2001 Nov 15, 31(10), 1170 - 8
Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex; Arner ES et al.; Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH . Conversely, increased cellular activity of the Trx system confers resistance to CDDP . In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism . The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site . Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1) . Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions . Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin . However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx . Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E . coli Trx system nor glutathione reductase were inhibited . Formation of GS-Pt is a major route for cellular elimination of CDDP . The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.

Vet Parasitol, 2001 Dec 3, 102(1-2), 35 - 44
Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1; Kimbita EN et al.; The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T . gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E . coli) as a glutathione-S-transferase (GST) fusion protein . The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis . The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein . The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T . gondii and sera from normal cats or mice . The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N . caninum) . Some 193 cat sera were tested for antibodies to T . gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay . Both positive and negative sera were confirmed by Western blot analysis . The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.

Biochemistry, 2001 Nov 20, 40(46), 14098 - 105
Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog; Steiger MA et al.; The final step of tRNA splicing in Saccharomyces cerevisiae requires 2'-phosphotransferase (Tpt1) to transfer the 2'-phosphate from ligated tRNA to NAD, producing mature tRNA and ADP ribose-1' '-2' '-cyclic phosphate . To address how Tpt1 protein recognizes substrate RNAs, we measured the steady-state kinetic parameters of Tpt1 protein with 2'-phosphorylated ligated tRNA and a variety of related substrates . Tpt1 protein has a high apparent affinity for ligated tRNA (K(m,RNA), 0.35 nM) and a low turnover rate (k(cat), 0.3 min(-1)) . Tpt1 protein recognizes both tRNA and the internal 2'-phosphate of RNAs . Steady-state kinetic analysis reveals that as RNAs lose structure and length, K(m,RNA) and k(cat) both increase commensurately . For a 2'-phosphorylated octadecamer derived from the anticodon stem-loop of ligated tRNA, K(m,RNA) and k(cat) are 5- and 8-fold higher, respectively, than for ligated tRNA, whereas for a simple substrate like pApA(p)pA, K(m,RNA) and k(cat) are 430- and 150-fold higher, respectively . Tpt1 is not detectably active on a trimer with a terminal 5'- or 3'-phosphate and is very inefficient at removal of a terminal 2'-phosphate unless there is an adjacent 3'-phosphate or phosphodiester . The K(m,NAD) for Tpt1 is substrate dependent: K(m,NAD) is 10 microM with ligated tRNA, 200 microM with pApA(p)pA, and 600 microM with pApApA(p) . Preliminary analysis of KptA, a functional Tpt1 protein homologue from Escherichia coli, reveals that KptA protein is strikingly similar to yeast Tpt1 in its kinetic parameters, although E . coli is not known to have a 2'-phosphorylated RNA substrate.

Biochemistry, 2001 Nov 20, 40(46), 14069 - 80
IscA, an alternate scaffold for Fe-S cluster biosynthesis; Krebs C et al.; An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity . Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form . Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mossbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies . Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins . Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile {4Fe-4S}(2+) cluster per (Nif)IscA homodimer, via a transient {2Fe-2S}(2+) cluster intermediate . The cluster assembly process was monitored temporally using UV-vis absorption and Mossbauer spectroscopy, and the intermediate {2Fe-2S}(2+)-containing species was additionally characterized by resonance Raman spectroscopy . The Mossbauer and resonance Raman properties of the {2Fe-2S}(2+) center are consistent with complete cysteinyl ligation . The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one {2Fe-2S}(2+) or one {4Fe-4S}(2+) per homodimer suggest that both cluster types are subunit bridging . In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur . The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions . On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed . It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.

Biochemistry, 2001 Nov 20, 40(46), 13964 - 71
Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter; Coggshall KA et al.; The bacterial outer membrane transporter for vitamin B(12), BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane protein TonB . This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box . In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P . In the wild-type protein, the Ton box is folded into the barrel of the transporter . The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm . In the wild-type protein, vitamin B(12) induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box . Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique . The failure of these mutants to execute the B(12) transport cycle may be a result of the altered conformation of the Ton box.

Biochemistry, 2001 Nov 20, 40(46), 13925 - 32
Differential regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase isoforms; Tokumitsu H et al.; We have previously demonstrated that the alpha isoform of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KKalpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca(2+)/CaM {Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R . (2000) J . Biol . Chem . 275, 20090-20095} . In this study, we find that rat brain extract contains Ca(2+)/CaM-independent CaM-KK activity . This result is consistent with an enhanced Ca(2+)/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KKbeta isoform . By using various truncation mutants of CaM-KKbeta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity . A CaM-KKbeta deletion mutant of this domain shows a significant increase of Ca(2+)/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity . The activities of CaM-KKalpha and CaM-KKbeta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca(2+)/CaM, suggesting that the autoinhibitory regions of CaM-KKalpha and CaM-KKbeta are functional . These results establish for the first time that residues 129-151 of CaM-KKbeta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity.

Biochemistry, 2001 Nov 20, 40(46), 13876 - 87
Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli; Hirschman A et al.; We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP . We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases . Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)) . Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+) . The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+) . The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA . Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA . Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP) . Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution . We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction.

Biochemistry, 2001 Nov 20, 40(46), 13833 - 9
A GCN4 variant with a C-terminal basic region binds to DNA with wild-type affinity; Hollenbeck JJ et al.; Basic-region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a leucine zipper dimerization domain and a basic region that directly contacts DNA . In all naturally occurring bZip proteins, the basic region is positioned N-terminal to the leucine zipper . We have designed a series of model bZip peptides in which the basic region of the yeast transcriptional activator GCN4 is placed C-terminal to its leucine zipper . DNA-binding studies demonstrate that the optimal reverse GCN4 (rGCN4) peptide is able to bind specifically and with wild-type affinity to DNA despite this unnatural arrangement of the two subdomains . These results suggest that a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the dimerization domain is unlikely.

Oncogene, 2001 Oct 25, 20(48), 7096 - 7
Induction of endogenous beta-galactosidase by ionizing radiation complicates the analysis of p53-LacZ transgenic mice; Coates PJ et al.; Studies of the response of p53-lacZ transgenic mice have uncovered an unexpected induction of endogenous acid-beta-galactosidase activity following whole body irradiation . Strong induction of endogenous enzyme activity is seen in a variety of mouse strains commonly used in the production of transgenes . The induction of endogenous enzyme activity therefore complicates the analysis of p53-lacZ transgenes and may also influence the analysis of radiation responses in other lacZ-reporter mice.

J Biol Chem, 2002 Jan 18, 277(3), 1749 - 54 Epub 2001 Nov 09.
Miscoordination of the iron-sulfur clusters of the anaerobic transcription factor, FNR, allows simple repression but not activation; Scott C et al.; The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis . Environmental oxygen is sensed by the acquisition of oxygen-labile {4Fe-4S} clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase . Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters . An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire {4Fe-4S} clusters in vitro that enhance site-specific DNA binding . Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively . Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V . Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.

Vox Sang, 2001 Oct, 81(3), 194 - 8
Isolation of human scFv antibody fragments against ABO blood group antigens from a phage display library; Santos-Esteban E et al.; BACKGROUND AND OBJECTIVES: Phage display has proven very useful for the isolation of antibodies against a number of antigens . We used this technology to isolate scFv antibody fragments against A and B red blood cell antigens . MATERIALS AND METHODS: Phages from a phage display library were selected using unmodified red blood cells as a source of antigen . Bound phages were absorbed onto cells lacking the antigen of interest and used to infect Escherichia coli cells . Phages were rescued and assayed for specificity by enzyme-linked immunosorbent assay (ELISA) . RESULTS: After several rounds of panning and subtraction on red blood cells, one anti-A and one anti-B human scFv antibody fragments were isolated . CONCLUSION: Isolation of anti-A and anti-B scFv antibody fragments on whole cells is an alternative method of obtaining antibodies against native cell-surface antigens.

Mol Microbiol, 2001 Oct, 42(2), 519 - 26
The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response; Ingmer H et al.; Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid . Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication . Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid-borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess . We show that delayed cell division occurs by RepA-mediated induction of the SOS response and can be reversed by over-expression of the host DNA primase, DnaG . The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites . The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls--potentially pre-empting plasmid loss in cultures of dividing cells.

Mol Microbiol, 2001 Oct, 42(2), 483 - 94
Orientational control of fimE expression in Escherichia coli; Sohanpal BK et al.; Phase-variable expression of type 1 fimbriae is, in part, controlled by site-specific DNA inversion of the fim switch in Escherichia coli . Of the two fim recombinases (FimB and FimE) that catalyse the inversion reaction, FimE exhibits a strong bias for phase switching from the ON to the OFF orientation . The specificity associated with fimE is the result of two different mechanisms: (i) FimE exhibits a preference for the invertible element in the ON orientation as substrate for recombination; (ii) the invertible element in the OFF orientation acts in cis to inhibit recombinase activity (orientational control) . We show here that the invertible element negatively regulates fimE, even though expression of a fimE-lacZYA transcriptional fusion is unaffected by orientational control . The fimE transcript extends into the invertible region and hence switch ON-specific and switch OFF-specific mRNA contain different sequences . Furthermore, we show that orientational control is suppressed by the insertion of a structured RNA (tRNA(Gly)) between fimE and the fim switch, indicating that the switch OFF-specific mRNA is inactivated by 3' to 5' degradation . Analysis of the fim switch reveals that it contains two inhibitory elements that exert orientational control independently.

Mol Microbiol, 2001 Oct, 42(2), 331 - 44
Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm; Journet L et al.; Several proteins of the Tol/Pal system are required for group A colicin import into Escherichia coli . Colicin A interacts with TolA and TolB via distinct regions of its N-terminal domain . Both interactions are required for colicin translocation . Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR . This interaction is specific to colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the colicin translocation step . TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm . The interaction involves part of the periplasmic domain of TolR and a small region of the colicin A N-terminal domain . This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the colicin A N-terminal domain and appear to be arranged linearly in the colicin sequence . Multiple contacts with periplasmic-exposed Tol proteins are therefore a general principle required for group A colicin translocation.

Biochemistry (Mosc), 2001 Sep, 66(9), 984 - 8
Activation of the Escherichia coli SoxRS-regulon by nitric oxide and its physiological donors; Vasil'eva SV et al.; Activation of the Escherichia coli SoxRS-regulon by nitric oxide (NO) and its physiological donors (S-nitrosothiol (GS-NO) and dinitrosyl iron complexes with glutathione (DNIC(glu)) and cysteine (DNIC(cys)) ligands) has been studied . To elucidate the molecular mechanisms of signal transduction via nitrosylation of Fe-S-centers in SoxR, the ability of pure NO and NO-producing agents to activate the SoxRS-regulon in E . coli cells bearing a soxS::lacZ operon (promoter) fusion has been compared . EPR spectroscopy of whole cells has been used to monitor the formation of inducible protein-DNIC complexes . DNIC(cys), GS-NO, and pure NO appeared to be potent inducers of soxS expression, whereas DNIC(glu) was considerably less efficient . Thus, lower in vitro stability of DNIC(cys) was in contrast with its higher biological activity . Pretreatment of the cells with o-phenanthroline, a chelating agent for iron, prevented soxS expression by GS-NO . Treatment of intact E . coli cells with DNIC, GS-NO, and NO at equimolar concentration 150 microM resulted in formation of a single EPR-detectable DNIC-type signal with g = 2.03 . The initial stage in the SoxR transcription activity is supposed to include two steps: first, DNIC primers are formed from exogenous NO and free iron, and then these DNIC disintegrate SoxR {2Fe-2S} clusters and thus activate SoxRS-regulon transcription.

Biochemistry (Mosc), 2001 Sep, 66(9), 973 - 8
Role of glutathione in the response of Escherichia coli to osmotic stress; Smirnova GV et al.; The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity . A mutant in gamma-glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain . The unfavorable effect of the gsh mutation on osmoadaptation of growing E . coli cells was more pronounced at low concentrations of K+ in the medium . An increase in osmolarity caused an increase in the intracellular content of glutathione . Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage . The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation . Changes in the level of intracellular K+ were similarly biphasic: a rapid increase in the K+ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage . In mutant gshA cells adapted to osmotic shock, the intracellular K+ level was markedly higher than in the parental strain cells . The possible role of glutathione in the response of E . coli to osmotic shock is discussed.

Dev Cell, 2001 Aug, 1(2), 187 - 99
Two zinc finger proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in C . elegans; Detwiler MR et al.; Oocytes are released from meiotic prophase I arrest through a process termed oocyte maturation . We present here a genetic characterization of oocyte maturation, using C . elegans as a model system . We show that two TIS11 zinc finger-containing proteins, OMA-1 and OMA-2, express specifically in maturing oocytes and function redundantly in oocyte maturation . Oocytes in oma-1;oma-2 mutants initiate but do not complete maturation and arrest at a defined point in prophase I . Two maturation signal-induced molecular events, including the maintenance of activated MAP kinase, do not occur in Oma oocytes . The Oma prophase arrest is released by inactivation of a MYT-1-like kinase, suggesting that OMA-1 and OMA-2 function upstream of MYT-1 as positive regulators of prophase progression during meiotic maturation.

DNA Seq, 2001 Jul, 12(1), 39 - 51
Molecular cloning, sequencing analysis and expression of the catalase-peroxidase gene from Halobacterium salinarum; Long S et al.; The gene encoding catalase-peroxidase was cloned from chromosomal DNA from the Archaea, Halobacterium salinarum . The nucleotide sequence of a 3.5 kb fragment, containing the catalase-peroxidase gene and its flanking regions was determined . A 2.16 kb open reading frame was obtained, encoding the enzyme which was comprised of 720 amino acid residues with a calculated molecular weight of 80 kDa . The deduced amino acid sequence of the H . salinarum catalase-peroxidase showed a high degree of identity to other bifunctional catalase-peroxidases . A transcriptional start site was identified 183 bp upstream of the translational start codon . Southern blot analysis indicated that catalase-peroxidase was a single copy gene . The Archaeal catalase-peroxidase gene was expressed in Escherichia coli, and the expressed fusion protein exhibited both catalase and peroxidase activities.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 406 - 9
{Constructing and expression of three zinc-fingers peptide with specific DNA recognition property in Escherichia coli}; Zhang SX et al.; For investigating the DNA binding property of classical zinc finger protein Zif268, an in vivo transcription interference experiment was once utilized to develop a genetic selection assay . By screening a library in which the key amino acids of the third zinc finger from Zif268 were randomized, some single fingers with new binding specificity were obtained . In this study, by combining the single fingers, two three-finger peptides cDNA ZF123 and 2ZF123 were constructed by an over-lap PCR technique using the DNA binding domain of Zif268 as the template . After three times PCR, the products were inserted into pUC18 for cloning . The ZF123 and 2ZF123 cDNA were also inserted into pGEX-2T for expression in Escherichia coli after sequencing confirmation . The result showed that the three-finger peptides were expressed at a high level in E . coli JM109 . The fusion protein GST-ZF123/2ZF123 have the relative molecular weight of 34.0 kD and consisted about 20% of the total soluble cell protein as detected by SDS-PAGE . After supersonic treatment, the soluble part of the bacterial extract was purified . After two additional thrombin cleavage and Sepharose 4B affinity purification steps, the free three-fingers peptide proteins were also obtained . The construction and obtaining of the three-fingers peptide cDNA and its products will facilitate the in vivo and in vitro DNA binding specificity study and the design of the hybrid transcription factors.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 396 - 9
{Cloning and expression of MGMT cDNA and analysis of the DNA repair activity of the recombinant protein}; Zhu YW et al.; A cDNA encoding human O6-methylguanine-DNA methyltranferase (MGMT) was cloned from Hela S3 cells and the sequence is identical with the published data . The MGMT cDNA was inserted into the expression vector pET-21a and transformed into E . coli BL21 (DE3) . A 24 kD protein was expressed after IPTG induction . Essays using lethal dose of alkylating agents indicate that expression of MGMT protein can repair the DNA mutations of the recombinant bacteria produced by alkylating agents.

Arch Microbiol, 2001 Nov, 176(5), 370 - 6
Expression of the pho regulon interferes with induction of the uhpT gene in Escherichia coli K-12; Hoffer SM et al.; The uptake of hexose 6-phosphates in Escherichia coli is mediated by the transporter UhpT, the synthesis of which is induced by the presence of glucose 6-phosphate (glucose 6-P) in the medium . Since this protein functions as an anion exchanger, it is generally assumed to be geared for the use of sugar phosphates as a carbon source . However, the question was unresolved whether this transporter can also provide the cells with glucose 6-P as a phosphate source . It is demonstrated in this work that UhpT-mediated glucose 6-P uptake does not allow the cells to grow on glucose 6-P as phosphate source . Hence, the expression of UhpT under phosphate limitation would not be particularly advantageous and some form of interaction between the uhp system and the Pi-limitation-inducible pho regulon, the products of which are involved in phosphate acquisition, may be anticipated . Indeed, the use of an uhpT-lacZ fusion revealed that much higher concentrations of the inducer glucose 6-P were required to elevate uhpT transcription when the pho regulon was expressed . This interference was the result of degradation of glucose 6-P by one of the products of the pho regulon, the periplasmic enzyme alkaline phosphatase . The specific form of interaction between the Pho and Uhp systems is designated inducer degradation.

Circ Res, 2001 Nov 9, 89(10), 874 - 81
Interaction between PEVK-titin and actin filaments: origin of a viscous force component in cardiac myofibrils; Kulke M et al.; The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes . Here, human N2-B-cardiac PEVK was expressed in Escherichia coli and tested-along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains-for a possible interaction with actin filaments . In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin . The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments . High {Ca(2+)} reversed the PEVK effect . PEVK concentrations >/=10 microgram/mL caused actin bundling . Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength . In cosedimentation assays, PEVK-titin interacted weakly with actin at 0 degrees C, but more strongly at 30 degrees C, suggesting involvement of hydrophobic interactions . To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period . The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude . The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca(2+)-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap . Steady-state passive force dropped only after longer exposure to gelsolin . We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils . The interaction could also oppose shortening during contraction.

Trends Plant Sci, 2001 Nov, 6(11), 510 - 3
An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana; Leyman B et al.; Trehalose accumulation has been documented in many organisms, such as bacteria and fungi, where it serves a storage and stress-protection role . Although conspicuously absent in most plants, trehalose biosynthesis genes were discovered recently in higher plants . We have uncovered a family of 11 TPS genes in Arabidopsis thaliana, one of which encodes a trehalose-6-phosphate (Tre6P) synthase, and a subfamily of which might encode the still elusive Tre6P phosphatases . A regulatory role in carbon metabolism is likely but might not be restricted to the TPS control of hexokinase activity as documented for yeast . Incompatibility between high trehalose levels and chaperone-assisted protein folding might be a reason why plants have evolved to accumulate some alternative stress-protection compounds to trehalose.

J Org Chem, 2001 Nov 16, 66(23), 7770 - 5
Biosynthesis of terpenoids: efficient multistep biotransformation procedures affording isotope-labeled 2C-methyl-D-erythritol 4-phosphate using recombinant 2C-methyl-D-erythritol 4-phosphate synthase; Hecht S et al.; This paper describes the recombinant expression of the ispC gene of Escherichia coli specifying 2C-methyl-D-erythritol 4-phosphate synthase in a modified form that can be purified efficiently by metal-chelating chromatography . The enzyme was used for the preparation of isotope-labeled 2C-methyl-D-erythritol 4-phosphate employing isotope-labeled glucose and pyruvate as starting materials . The simple one-pot methods described afford numerous isotopomers of 2C-methyl-D-erythritol 4-phosphate carrying (3)H, (13)C, or (14)C from commercially available precursors . The overall yield based on the respective isotope-labeled starting material is approximately 50%.

J Org Chem, 2001 Nov 16, 66(23), 7561 - 7
Synthesis and biological activity of N-sulfonylphosphoramidates: probing the electrostatic preferences of alkaline phosphatase; Burlingham BT et al.; N-Sulfonylphosphoramidates have been synthesized to investigate the electrostatic requirements for binding to alkaline phosphatase . Alkyl- and aryl N-benzylated sulfonamides were phosphorylated with bromophosphates or synthesized via phosphoramidite chemistry in moderate yields (44-77%.) The resulting tribenzylated N-sulfonylphosphoramidates may be deprotected in one step to give the free acids in quantitative yields . Physical data of N-sulfonylphosphoramidates, including pK(a)'s and stability toward hydrolysis, were determined . Inhibition data suggests that AP does not bind trianionic N-sulfonylphosphoramidates better than dianionic N-sulfonylphosphoramidates, although N-sulfonylphosphoramidates are bound tighter than N-phenylphosphoramidate . k(cat) for the hydrolysis of N-sulfonylphosphoramidates by bovine and E . coli alkaline phosphatases is 10-60% that of p-nitrophenyl phosphate.

Anal Biochem, 2001 Nov 15, 298(2), 314 - 21
Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay; Fu TF et al.; An enzymatic method for determination of B6 vitamers is presented . In this method pyridoxal 5'-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme . The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH . The rate of the cycle is directly proportional to the amount of active holoserine hydroxymethyltransferase, which is a measure of the amount of pyridoxal 5'-phosphate in the original sample . The cycle operates about 50 times per minute giving a 100-fold enhancement of NADPH production with respect to original pyridoxal 5'-phosphate content . Other B6 vitamers are converted to pyridoxal 5'-phosphate by a preincubation with a combination of pyridoxal kinase and pyridoxine 5'-phosphate oxidase . A complete analysis of B6 vitamers can be completed in less than 1 h and the assay is linear in the 2- to 50-pmol range of pyridoxal 5'-phosphate . The method is applied to the determination of the B6 vitamer pools in extracts of Escherichia coli . The results show that the pool of pyridoxal 5'-phosphate that is not bound to proteins is large enough to account for product inhibition of both pyridoxal kinase and pyridoxine 5'-phosphate oxidase .

Genome Inform Ser Workshop Genome Inform, 2000, 11, 185 - 95
Two-phase partition method for simulating a biological system at an extremely high speed; Kurata H et al.; To accelerate the calculation speed for simulating a biological system, we proposed a novel simulation method, the two-phase partition method, which calculated molecular processes at a higher speed than any other proposed method . This method divides a biological system, which can be described by chemical reaction equations, into two-phases: the binding and reaction phases . We demonstrated the capability of the two-phase partition method to simulate a complex biological system at an extremely high speed and clarified the accuracy of the simulation . The two-phase partition method is very useful for simulating complex interactions among proteins and DNAs.

Genome Inform Ser Workshop Genome Inform, 2000, 11, 141 - 50
Protein sequence-structure alignment based on site-alignment probabilities; Miyazawa S; A protein sequence-structure alignment method for database searches is examined on how effectively this method together with a simple scoring function previously developed can identify compatibilities between sequences and structures of proteins . The scoring function consists of pairwise contact energies, repulsive packing potentials of residues for overly dense arrangement and short-range potentials for secondary structures . Pairwise contact interactions in a sequence-structure alignment are evaluated in a mean field approximation on the basis of probabilities of site pairs to be aligned . Gap penalties are assumed to be proportional to the number of contacts at each residue position, and as a result gaps will be more frequently placed on protein surfaces than in cores . In addition to minimum energy alignments, we use probability alignments made by successively aligning site pairs in order by pairwise alignment probabilities . Results show that the present energy function and alignment method can detect well both folds compatible with a given sequence and, inversely, sequences compatible with a given fold . Probability alignments consisting of most reliable site pairs only can yield small root mean square deviations, and including less reliable pairs increases the deviations . Remarkably, by this method some individual sequence-structure pairs are detected having only 5-20% sequence identity.

Crit Care Med, 2001 Nov, 29(11), 2185 - 93
Effects of levosimendan, a novel inotropic calcium-sensitizing drug, in experimental septic shock; Oldner A et al.; OBJECTIVE: Levosimendan is a novel inodilator that improves cardiac contractility by sensitizing troponin C to calcium . This drug has proved to be effective in treating advanced congestive heart failure but has not been evaluated in septic settings . The purpose of the present study was to study the effects of this drug in a porcine model of endotoxemia . DESIGN: Prospective experimental study . SUBJECTS: Fourteen landrace pigs . INTERVENTIONS: All animals were anesthetized and catheterized for measurement of central and pulmonary hemodynamics . Ultrasonic flow probes were placed around the renal artery and portal vein to measure blood flow . A tonometer was placed in the ileum to measure mucosal pH . Levosimendan was given to six animals as a bolus (200 microg x kg(-1)) followed by a continuous infusion (200 microg x kg(-1) x hr(-1)) . Thirty minutes after onset of levosimendan treatment, all animals received endotoxin (20 microg x kg(-1) x hr(-1) for 3 hrs) . MEASUREMENTS AND MAIN RESULTS: At baseline, levosimendan induced a systemic vasodilation with a reduction in blood pressure and an increase in heart rate . A tendency to an increase in cardiac index did not reach statistical significance (p =.055) . Cardiac index and systemic oxygen delivery were markedly improved in the levosimendan group during endotoxemia . Systemic vascular resistance and blood pressure were reduced in the levosimendan group . The latter parameter, however, was only different from the control group during the initial phase of endotoxin shock but not at the late, most pronounced phase of shock . Levosimendan also efficiently attenuated endotoxin-induced pulmonary hypertension . Portal venous blood flow and gut oxygen delivery were improved, but no concomitant reduction in endotoxin-induced intestinal mucosal acidosis was observed . Renal blood flow was unaffected, as was the endotoxin-induced increase in plasma endothelin-1-like immunoreactivity . These findings support previous reports of calcium desensitization as a potential component in septic myocardial depression . Furthermore, the vasodilatory properties of this drug were well tolerated in the current model of hypodynamic endotoxin shock, and they may have contributed to improved regional blood flow as seen in the gut as well as improved systemic perfusion by means of reduced biventricular afterload . CONCLUSION: Pretreatment with levosimendan in pigs subjected to endotoxin shock improved cardiac output and systemic and gut oxygen delivery . In addition, pulmonary hypertension largely was attenuated without any adverse effects on gas exchange . These results are promising in several aspects, but the role of levosimendan in the treating circulatory failure in sepsis remains to be established.

Microbiology, 2001 Nov, 147(Pt 11), 3149 - 58
The elements of the locus of enterocyte effacement in human and wild mammal isolates of Escherichia coli: evolution by assemblage or disruption?
Sandner L, Eguiarte LE, Navarro A, Cravioto A, Souza V.
Escherichia coli is an excellent model for studying the evolution of pathogenicity since within one species various genes can be found in pathogenic islands and plasmids causing a wide spectrum of virulence . A collection of 122 strains from different human and wild mammal hosts were analysed by PCR and Southern hybridization for the presence of a subset of the genes included in the LEE (locus of enterocyte effacement) . In the PCR analysis, two markers (cesT/eae and espB genes) were found together in more strains (25.4%) than either were found alone . The cesT/eae gene was less frequently found alone (8.2%) than was the espB gene (15.6%) . Four regions of the LEE were analysed in a subsample of 25 strains using Southern hybridization . The four regions were all present (44%), all absent (12%) or present in different combinations (44%) in a given strain . The flanking regions of the LEE showed the highest rate of hybridization (in 72% of the strains) . The results indicate that the LEE is a dynamic genetic entity, both the complete gene cluster and the individual genes . The genes that comprise this locus seem to be horizontally acquired (or lost) in an independent way and may control other functions in non-pathogenic E . coli lineages . In this way, horizontal transfer may allow the gradual stepwise construction of gene cassettes facilitating coordinate regulation and expression of novel functions.

Microbiology, 2001 Nov, 147(Pt 11), 3141 - 8
Binding to sulfatide and enterotoxicity of various Escherichia coli STb mutants; Labrie V et al.; Binding of the 48 amino acid polypeptide of the mature heat-stable Escherichia coli enterotoxin b (STb) to the functional receptor sulfatide (SFT) constitutes the first step in inducing secretory diarrhoea in the intestinal lumen of animals . The NMR structure of this toxin dictated the choice of amino acids for site-directed mutagenesis to delineate the binding site of STb to SFT . Amino acids facing the solvent either in the loop or the hydrophobic alpha-helix were selected . Seventeen site-specific mutants of STb toxin were produced and purified by high-pressure liquid chromatography . Enterotoxicity of the 17 mutants was determined using a rat loop assay and binding was evaluated using a microtitre plate binding assay . Both hydrophobic and electrostatic interactions are important for STb attachment . When mutations (F37K, I41S and M42S) were introduced into the hydrophobic alpha-helix to lessen hydrophobicity, binding activity and enterotoxicity decreased by more than sixfold . The loop defined by C21 and C36 also made specific contributions . Mutants generated at basic residues (K22, K23 and R29) within this region exhibited both reduced binding activities and reduced toxic activities . For all STb mutants constructed and analysed, when binding to SFT was reduced, a reduction in toxicity equivalent or greater was noted, indicating that binding to SFT is a step that precedes the toxic effect observed for STb toxin . Significantly, when the negatively charged D30 was substituted for either alanine or valine, the binding to SFT was about twice that of native STb, whereas the enterotoxicity was reduced by half.

Microbiology, 2001 Nov, 147(Pt 11), 3093 - 104
N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli; Self WT et al.; The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers . The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator . Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon . Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family . A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro . The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level . Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence . Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein . Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression . The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro . Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate . Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex . The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

Microbiology, 2001 Nov, 147(Pt 11), 3071 - 81
Monomer-dimer control of the ColE1 P(cer) promoter; Chatwin HM et al.; XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss . In addition to XerCD, recombination requires the accessory factors ArgR and PepA . The promoter P(cer), located centrally within cer, is also required for stable plasmid maintenance . P(cer) is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division . It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides . This study has shown that ArgR does not act as a transcriptional repressor of P(cer) in plasmid monomers . P(cer) is unusual in that the -35 and -10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity . An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp . These observations are consistent with the hypothesis that P(cer) activation involves realignment of the -35 and -10 sequences within a recombinational synaptic complex . This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

Microbiology, 2001 Nov, 147(Pt 11), 3005 - 13
Deletion of one of two Escherichia coli genes encoding putative Na+/H+ exchangers (ycgO) perturbs cytoplasmic alkali cation balance at low osmolarity; Verkhovskaya ML et al.; Two genes in the Escherichia coli genome, b4065 (yjcE) and b1191 (ycgO), are similar to genes encoding eukaryotic Na+/H+ exchangers . Mutants were constructed in which yjcE (GRN11), ycgO (GRF55) or both (GRD22) were inactivated . There was no change in respiration-driven Na+ efflux in any of the mutants when grown in media containing 50-500 mM Na+ . The only striking finding was that growth of GRF55 was impaired at low osmolarity . In complex low-salt medium, GRF55 grew at a wild-type rate for three to four generations but then stopped; the growth was partially recovered after a pause, the length of which was dependent on salt concentration . Measurement of cytoplasmic alkali cations showed that an abrupt loss of about one-half of the intracellular K+ preceded the pause . When grown in low-salt medium with only 20 mM added Na+, GRF55 also lost the ability to maintain a sodium concentration gradient . However, this phenomenon appears to be a secondary effect of the ycgO deletion . The double mutant GRD22 has the same properties as GRF55; no additional effect was found . The data indicate that neither ycgO nor yjeE participates in respiration-driven Na+ extrusion . Instead, ycgO is required for growth at low osmolarity . Hence it is concluded that ycgO participates in cell volume regulation, and accordingly it is suggested that ycgO be renamed cvrA.

Microbiology, 2001 Nov, 147(Pt 11), 2981 - 9
An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation; Liu X et al.; Expression of the major outer-membrane porins in Escherichia coli is transcriptionally controlled during nutrient limitation . Expression of ompF was more than 40-fold higher under glucose limitation than under nitrogen (ammonia) limitation in chemostat cultures at the same growth rate . In contrast, ompC expression was higher under N limitation . The basis of regulation by nutrient limitation was investigated using mutations affecting expression of porin genes . The influence of cyaA, rpoS, ackA and pta, as well as the two-component envZ-ompR system, was studied under glucose and N limitation in chemostat cultures . A major contributor to low ompF expression under N limitation was negative control by the RpoS sigma factor . RpoS levels were high under N limitation and loss of RpoS resulted in a 19-fold increase in ompF transcription, but little change was observed with ompC . Lack of RpoS under glucose limitation had a lesser stimulatory effect on ompF expression . Porin production was minimally dependent on EnvZ under N limitation due to OmpR phosphorylation by acetyl phosphate . Evidence obtained with pta and ackA mutants suggested that the acetyl phosphate level also regulates porins independently and indirectly via RpoS and other pathways . pta-envZ double mutants had a residual level of porin transcription, implicating alternative means of OmpR phosphorylation under nutrient limitation . Another critical factor in regulation was the level of cAMP, as a cyaA mutant hardly expressed ompF under glucose limitation but boosted ompC . In addition, the role of DNA-binding proteins encoded by hns and himA was tested under glucose limitation: the hns mutation reduced the glucose-limitation peak, but the himA mutation suppressed the hns effect, suggesting a complex web of interrelationships between the DNA-binding proteins . Indeed, multiple inputs and no single regulator were responsible for the high peak of ompF expression under glucose limitation.

J Biol Chem, 2002 Feb 1, 277(5), 3447 - 55 Epub 2001 Nov 07.
U2 small nuclear RNA is a substrate for the CCA-adding enzyme (tRNA nucleotidyltransferase); Cho HD et al.; The CCA-adding enzyme builds and repairs the 3' terminus of tRNA . Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3'-terminal CCA, as do all mature tRNAs; the other 35% ends in 3' CC or possibly 3' C . The 3'-terminal A of U2 snRNA cannot be encoded because the 3' end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide . The first detectable U2 snRNA precursor contains 10-16 extra 3' nucleotides that are removed by one or more 3' exonucleases . Thus, if 3' exonuclease activity removes the encoded 3' CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3'-terminal A, CA, or CCA of U2 snRNA . We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3'-terminal A, CA, or CCA to human U2 snRNA lacking 3'-terminal A, CA, or CCA . The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs . We suggest that the 3' stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.

J Biol Chem, 2002 Jan 11, 277(2), 1203 - 9 Epub 2001 Nov 07.
On the molecular basis of the thermal sensitivity of an Escherichia coli topA mutant; Wang Y et al.; Studies of two temperature-sensitive Escherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity . We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17 . We find that the supD allele in this strain had lost its temperature sensitivity . The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive . Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 degrees C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 degrees C, a nonpermissive temperature . Our results thus implicate an indispensable role of DNA topoisomerase I in E . coli cells grown in media of any osmolarity.

J Biol Chem, 2002 Jan 11, 277(2), 1614 - 8 Epub 2001 Nov 07.
Influence of DNA sequence on the positioning of RecA monomers in RecA-DNA cofilaments; Volodin AA et al.; We show that certain DNA sequences have the ability to influence the positioning of RecA monomers in RecA-DNA complexes . A tendency for RecA monomers to be phased was observed in RecA protein complexes with several oligonucleotides containing a recombinational hotspot sequence, the chi-site from Escherichia coli . This influence was observed in both the 5' to 3' and 3' to 5' directions with respect to chi . A 5'-end phosphate group and probably some other features in DNA also influence the phasing of RecA monomers . We conclude that natural DNAs contain a number of features that influence the positioning of RecA monomers . The ability of specific DNA sequences to influence the positioning of RecA monomers demonstrates some specificity in the binding of individual bases at different sites within a RecA monomer and, most likely, reflects the stereochemical non-equivalence of these sites . The possible biological implications of the phasing of RecA monomers in presynaptic DNA-protein cofilaments are discussed.

J Biol Chem, 2002 Jan 25, 277(4), 2498 - 504 Epub 2001 Nov 07.
In vitro repression of the gal promoters by GalR and HU depends on the proper helical phasing of the two operators; Lewis DE et al.; Repression of transcription initiation from the two gal promoters, P1 and P2, requires binding of GalR protein to two flanking operators, O(E) and O(I), binding of HU to a site, hbs, located between the two operators, and supercoiled DNA template . Previous experiments suggested that repression involves the interaction of two DNA-bound GalR proteins, which generates a 113-bp DNA loop encompassing the promoter region . Interaction between two DNA-bound proteins would be allowed if the binding sites on DNA are properly aligned . To test the idea that the observed repression of gal transcription in vitro is mediated by DNA looping, we investigated the effect of changing the relative angular orientation of O(E) and O(I) in the DNA helix . We found that repression is a periodic function of the distance between the two operator sites . Since repression recurred commensurate with DNA helical repeat, we conclude that the observed in vitro repression is mediated by DNA looping and the in vitro conditions reflect the in vivo situation.

J Biol Chem, 2002 Feb 1, 277(5), 3186 - 94 Epub 2001 Nov 07.
The helix-turn-helix motif of the coliphage 186 immunity repressor binds to two distinct recognition sequences; Shearwin KE et al.; The CI protein of coliphage 186 is responsible for maintaining the stable lysogenic state . To do this CI must recognize two distinct DNA sequences, termed A type sites and B type sites . Here we investigate whether CI contains two separate DNA binding motifs or whether CI has one motif that recognizes two different operator sequences . Sequence alignment with 186-like repressors predicts an N-terminal helix-turn-helix (HTH) motif, albeit with poor homology to a large master set of such motifs . The domain structure of CI was investigated by linker insertion mutagenesis and limited proteolysis . CI consists of an N-terminal domain, which weakly dimerizes and binds both A and B type sequences, and a C-terminal domain, which associates to octamers but is unable to bind DNA . A fusion protein consisting of the 186 N-terminal domain and the phage lambda oligomerization domain binds A and B type sequences more efficiently than the isolated 186 CI N-terminal domain, hence the 186 C-terminal domain likely mediates oligomerization and cooperativity . Site-directed mutation of the putative 186 HTH motif eliminates binding to both A and B type sites, supporting the idea that binding to the two distinct DNA sequences is mediated by a variant HTH motif.

Annu Rev Genet, 2001, 35, 243 - 74
Homologous recombination near and far from DNA breaks: alternative roles and contrasting views; Smith GR; Double-strand breaks and other lesions in DNA can stimulate homologous genetic recombination in two quite different ways: by promoting recombination near the break (roughly within a kb) or far from the break . Recent emphasis on the repair aspect of recombination has focused attention on DNA interactions and recombination near breaks . Here I review evidence for recombination far from DNA breaks in bacteria and fungi and discuss mechanisms by which this can occur . These mechanisms include entry of a traveling entity ("recombination machine") at a break, formation of long heteroduplex DNA, priming of DNA replication by a broken end, and induction of recombination potential in trans . Special emphasis is placed on contrasting views of how the RecBCD enzyme of Escherichia coli promotes recombination far (tens of kb) from a double-strand break . The occurrence of recombination far from DNA breaks and of correlated recombination events far apart suggests that "action at a distance" during recombination is a widespread feature among diverse organisms.

Annu Rev Genet, 2001, 35, 53 - 82
Recombinational DNA repair of damaged replication forks in Escherichia coli: questions; Cox MM; It has recently become clear that the recombinational repair of stalled replication forks is the primary function of homologous recombination systems in bacteria . In spite of the rapid progress in many related lines of inquiry that have converged to support this view, much remains to be done . This review focuses on several key gaps in understanding . Insufficient data currently exists on: (a) the levels and types of DNA damage present as a function of growth conditions, (b) which types of damage and other barriers actually halt replication, (c) the structures of the stalled/collapsed replication forks, (d) the number of recombinational repair paths available and their mechanistic details, (e) the enzymology of some of the key reactions required for repair, (f) the role of certain recombination proteins that have not yet been studied, and (g) the molecular origin of certain in vivo observations associated with recombinational DNA repair during the SOS response . The current status of each of these topics is reviewed.

J Mol Biol, 2001 Nov 9, 313(5), 1171 - 9
Formation of helical hairpins during membrane protein integration into the endoplasmic reticulum membrane . Role of the N and C-terminal flanking regions; Hermansson M et al.; The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a common structural element in integral membrane proteins . Previous studies on the sequence determinants of helical hairpin formation have focussed on the role of polar and charged residues placed centrally in a long stretch of hydrophobic residues, and have yielded a "propensity scale" for the relative efficiency with which different residues promote the formation of helical hairpins . In this study, we shift our attention to the role of charged residues flanking the hydrophobic stretch . Clusters of charged residues are known to hinder membrane translocation, and thus flanking charged residues may conceivably force a long hydrophobic segment to form a helical hairpin even if there are no or only weakly turn-promoting residues in the hydrophobic stretch . We indeed find that Lys and, more surprisingly, Asp residues strongly affect helical hairpin formation when placed next to a poly-Leu-based transmembrane segment . We also find that a cluster of four consecutive Lys residues can affect the efficiency of helical hairpin formation even when placed approximately 30 residues downstream of the hydrophobic stretch . These observations have interesting implications for the way we picture membrane protein topogenesis within the context of the endoplasmic reticulum (ER) translocon .

J Mol Biol, 2001 Nov 9, 313(5), 1093 - 102
Channeling of ammonia in glucosamine-6-phosphate synthase; Teplyakov A et al.; Glucosamine-6-phosphate synthase catalyses the first and rate-limiting step in hexosamine metabolism, converting fructose 6-phosphate into glucosamine 6-phosphate in the presence of glutamine . The crystal structure of the Escherichia coli enzyme reveals the domain organisation of the homodimeric molecule . The 18 A hydrophobic channel sequestered from the solvent connects the glutaminase and isomerase active sites, and provides a means of ammonia transfer from glutamine to sugar phosphate . The C-terminal decapeptide sandwiched between the two domains plays a central role in the transfer . Based on the structure, a mechanism of enzyme action and self-regulation is proposed . It involves large domain movements triggered by substrate binding that lead to the formation of the channel .

J Mol Biol, 2001 Nov 9, 313(5), 1059 - 72
Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 A resolution; Trapani S et al.; ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) (ATP: oxaloacetate carboxylyase (transphosphorylating), EC 4.1.1.49) is a key enzyme involved in the catabolism of glucose and amino acids in the parasite Trypanosoma cruzi, the causative agent of Chagas' disease . Due to the significant differences in the amino acid sequence and substrate specificity of the human enzyme (PEPCK (GTP-dependent), EC 4.1.1.32), the parasite enzyme has been considered a good target for the development of new anti-chagasic drugs . We have solved the crystal structure of the recombinant PEPCK of T . cruzi up to 2.0 A resolution, characterised the dimeric organisation of the enzyme by solution small angle X-ray scattering (SAXS) and compared the enzyme structure with the known crystal structure of the monomeric PEPCK from Escherichia coli . The dimeric structure possesses 2-fold symmetry, with each monomer sharing a high degree of structural similarity with the monomeric structure of the E . coli PEPCK . Each monomer folds into two complex mixed alpha/beta domains, with the active site located in a deep cleft between the domains . The two active sites in the dimer are far apart from each other, in an arrangement that seems to permit an independent access of the substrates to the two active sites . All residues of the E . coli PEPCK structure that had been found to interact with substrates and metal cofactors have been found conserved and in a substantially equivalent spatial disposition in the T . cruzi PEPCK structure . No substrate or metal ion was present in the crystal structure . A sulphate ion from the crystallisation medium has been found bound to the active site . Solution SAXS data suggest that, in solutions with lower sulphate concentration than that used for the crystallisation experiments, the actual enzyme conformation may be slightly different from its conformation in the crystal structure . This could be due to a conformational transition upon sulphate binding, similar to the ATP-induced transition observed in the E . coli PEPCK, or to crystal packing effects . The present structure of the T . cruzi PEPCK will provide a good basis for the modelling of new anti-chagasic drug leads .

Biochem Biophys Res Commun, 2001 Nov 16, 288(5), 1141 - 8
DnaA protein Lys-415 is close to the ATP-binding site: ATP-pyridoxal affinity labeling; Kubota T et al.; Binding of ATP, but not of ADP, activates Escherichia coli DnaA protein for replicational initiation of the chromosome . To elucidate this switching mechanism, we used the affinity-labeling agent ATP-pyridoxal, which forms a covalent bond with the Lys residue located at or near the gamma-phosphate of ATP . ATP-pyridoxal inhibited the ATP binding for DnaA protein, with a competitive mode . Binding stoichiometry was 0.28 ATP-pyridoxal/DnaA molecule, a value consistent with that of ATP . Thus, ATP-pyridoxal was a potent antagonist for the DnaA ATP-binding site . The labeled DnaA protein was inactive for minichromosome replication in vitro, suggesting that conformation of the region is important for DnaA activity . Isolation of the labeled, tryptic fragment and the Edman degradation revealed that ATP-pyridoxal modified Lys-415 . Thus, this residue is likely close to the bound ATP . Since Lys-415 is located in the DNA-binding domain, these findings imply internal interaction between the domains for ATP binding and DNA binding .

Arch Virol, 2001, 146(9), 1753 - 63
Study of the polymerization step of the rolling circle replication of peach latent mosaic viroid; Pelchat M et al.; We have developed an in vitro transcriptional assay using Escherichia coli RNA polymerase to initiate the replication of peach latent mosaic viroid (PLMVd) . Regardless of the polarity of the PLMVd strand used as template, initiation in vitro occurred at the same hairpin structure . These initiation sites correspond to the 5'-ends of two small (280 nt) PLMVd-related RNAs found in infected peach leaves . Using a series of truncated PLMVd-derived transcripts, we have demonstrated that the viroid domain composed solely of the self-complementary hammerhead sequences is sufficient to trigger polymerase-driven replication in vitro . These data suggest that the bacterial-like RNA polymerase from peach chloroplasts catalyzes PLMVd replication.

Arch Virol, 2001, 146(9), 1693 - 704
Transcriptional and translational expression kinetics of the UL25 homologue of bovine herpesvirus 1.1; Desloges N et al.; We investigated whether the bovine herpesvirus 1.1 (BHV1) ORF, a homologue of the herpes simplex virus 1 (HSV1) UL25 gene, represented a functional gene . The BHV1 UL25 ORF, which is located at positions 60602<--62398 of the viral genome, generated a 4.5 kb transcript accumulating at low abundance as soon as 3 hours p.i . after which the levels increased up to 12h p.i . and remained constant up to 24 hours p.i . In addition, UL25 transcription initiated at 303 bases upstream from the translation initiation codon, corresponding to 26 and 354 b downstream from putative TATA and CAAT boxes, respectively, thus providing evidence that these elements function as the UL25 promoter . Western blotting of BHV1-infected cell lysates, using a BHV1-UL25 specific antiserum generated against a T7-Tag/UL25 fusion recombinant protein expressed in E . coli, detected a 63 kDa protein of the expected size (63,083 Da) whose expression profile followed that of its transcript . The synthesis of the 63 kDa protein was abrogated by a DNA synthesis inhibitor, unambiguously demonstrating that the viral specific protein expressed from the BHV1 UL25 ORF belongs to the gamma2 class.

J Periodontol, 2001 Oct, 72(10), 1413 - 9
Mechanisms of Actinobacillus actinomycetemcomitans-induced expression of interleukin-8 in gingival epithelial cells; Sfakianakis A et al.; BACKGROUND: Gingival epithelial cells (GEC) are the first cells of the periodontium to encounter known periodontal pathogens, such as Actinobacillus actinomycetemcomitans (A.a.) and, therefore, the role of this pathogen in the initiation of the inflammatory response is critical . However, little is known about the interactions of A.a . with GEC . In the present study, the mechanisms by which extracts from A.a . induced expression of the chemotactic cytokine interleukin-8 (IL-8) in GEC, in vitro, were examined . METHODS: An established GEC line, PP, was co-cultured with sonicated extracts of A.a . under various in vitro experimental conditions, and the IL-8 secretion was determined with enzyme-linked immunosorbent assay . RESULTS: A.a . extracts induced a time- and dose-dependent expression of IL-8 from the cells . Dose-response studies indicated that the highest IL-8 secretion (7-fold, P < 0.01) was at the level of 50 micrograms/ml of A.a . extract . Time-course studies revealed a dramatic increase of IL-8 expression after 12 hours of continuous stimulation . Pretreatment with polymyxin B (lipopolysaccharide {LPS} inhibitor) did not reduce the IL-8 expression induced by A.a . extracts (P > 0.10) . The introduction of p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 markedly inhibited (> 75%, P < 0.01) A.a.-induced expression of IL-8 . It is concluded that A.a . extracts upregulated the basal IL-8 expression in GEC . CONCLUSIONS: The effect was LPS-independent and involved a p38 MAPK signal transducing pathway . Understanding mechanisms of proinflammatory cytokine induction is important in periodontal pathology as it may lead to novel therapeutic approaches for periodontitis, thus controlling host inflammatory responses.

Shock, 2001 Nov, 16(5), 398 - 402
Glutamine reduces cytokine release, organ damage, and mortality in a rat model of endotoxemia; Wischmeyer PE et al.; Clinical trials have demonstrated that glutamine (GLN) supplementation can decrease infectious morbidity and improve survival in a number of settings of critical illness . The mechanism of this protection remains unclear . The objective of this study was to evaluate the effect of GLN on cytokine release, organ injury, and survival from endotoxin-induced septic shock . Endotoxemia was induced in Male Sprague-Dawley rats by intravenous administration of 5 mg/kg Escherichia coli lipopolysaccharide (LPS) . Concomitantly, animals were fluid resuscitated with a lactated ringers (LR) solution and given GLN (0.75 g/kg i.v.) or LR alone . Blood samples were obtained at multiple time points post-LPS injury for cytokine analysis . Survival rates were monitored for 72 h . Organ injury was evaluated in a separate set of animals via pathologic exam of tissues harvested 6 h post-LPS injury . A single dose of GLN significantly attenuated the release of TNF-alpha at 2 h (P < 0.005) and IL-1 beta at 4 h (P < 0.0001) . This attenuation of cytokine release was associated with a significant decrease in mortality (P < 0.003) . Pathologic exam demonstrated significant protection of both lung and small bowel tissue by GLN . Blood gas values 6-h post-LPS injury showed increased PaO2 and bicarbonate concentration in GLN treated animals . These data indicate that GLN can significantly attenuate pro-inflammatory cytokine release, protect against end-organ damage, and decrease mortality from endotoxemia . GLN confers protection even when administered at the onset of endotoxemia, rather then as pre-treatment . Thus, one explanation for the clinical benefits observed from GLN-supplementation may be related to the attenuation of pro-inflammatory cytokines.

Shock, 2001 Nov, 16(5), 368 - 72
Beat-to-beat evaluation of cardiac function in low-dose endotoxemia using a conscious sheep model; Abel FL et al.; The effects of very low doses of endotoxin (20 ng/kg/h for 8 h) were evaluated in a conscious sheep model in which introducers for catheters for monitoring pressure and ventricular dimensions had been previously inserted so that the studies could be performed without anesthesia and without a previous thoracotomy . Analog data was obtained at hourly intervals for 10 h and again at 24 h and was used to construct a beat-to-beat analysis of left ventricular performance . Only minimal effects were observed on heart rate, end diastolic pressure, arterial pressure, cardiac output, or cardiac work, although there was a significant rise in pulmonary artery pressure at 1 h of infusion . Despite the absence of changes in heart rate, preload, or afterload, maximal dp/dt decreased significantly by 4 h and remained decreased for the 10-h observation period; it returned to normal at 24 h . End systolic elastance decreased at 6 h and Pmax/EDV, a new indicator of performance, also decreased at 6 and 9 h . Thus, systolic performance decreased . Negative dp/dt did not change, but time for relaxation from 80% to 20% of peak ventricular pressure increased significantly at 5, 6, and 8 h . Plasma TNF-alpha was also measured and showed a significant rise at 2 h, but rapidly decreased thereafter . These results indicate an early depression of myocardial contractility and distensibility at doses of endotoxin insufficient to produce measurable effects on arterial pressure or cardiac output.

J Biol Chem, 2002 Jan 11, 277(2), 1158 - 65 Epub 2001 Nov 06.
Drosophila melanogaster glutamate-cysteine ligase activity is regulated by a modifier subunit with a mechanism of action similar to that of the mammalian form; Fraser JA et al.; Glutamate-cysteine ligase (GCL) plays an important role in regulating glutathione homeostasis . In mammals, it comprises a catalytic (GCLC) and modifier (GCLM) subunit . The existence of a modifier subunit in invertebrates has not been described to date . We now demonstrate that GCL from Drosophila melanogaster has a functional modifier subunit (DmGCLM) . A putative DmGCLM was obtained as an expressed sequence tag with 27% identity to human GCLM at the amino acid level . D . melanogaster GCLC (DmGCLC) and the candidate DmGCLM were expressed separately in Escherichia coli, purified, mixed, and then subjected to gel filtration, where they eluted as an approximately 140-kDa complex . DmGCLC co-immunoprecipitated with DmGCLM from S2 cell extracts, suggesting that they also associate in vivo . Enzyme kinetic analyses showed that DmGCLC has a K(m) for glutamate of 2.88 mm, but when complexed with DmGCLM, the K(m) for glutamate is 0.45 mm . Inhibition of DmGCLC activity by glutathione was found to be competitive with respect to glutamate (K(i) = 0.03 mm), whereas inhibition of the GCL complex was mixed (K(i) = 0.67 mm), suggesting allosteric effects . In accordance with this, DmGCLC and DmGCLM have the ability to form reversible intermolecular disulfide bridges . A further mechanism for control of D . melanogaster GCL was found to be induction of DmGCLC by tert-butylhydroquinone in S2 cells . DmGCLM levels were, however, unaffected by tert-butylhydroquinone.

J Biol Chem, 2002 Feb 1, 277(5), 3158 - 67 Epub 2001 Nov 06.
Cloning expression and characterization of methionine adenosyltransferase in Leishmania infantum promastigotes; Reguera RM et al.; Methionine adenosyltransferase (MAT) catalyzes the synthesis of s-adenosylmethionine (AdoMet), a metabolite that plays an important role in a variety of cellular functions, such as methylation, sulfuration, and polyamine synthesis . In this study, genomic DNA from the protozoan parasite Leishmania infantum was cloned and characterized . L . infantum MAT, unlike mammalian MAT, is codified by two identical genes in a tandem arrangement and is only weakly regulated by AdoMet . L . infantum MAT mRNA is expressed as a single transcript, with the enzyme forming a homodimer with tripolyphosphatase in addition to MAT activity . Expression of L . infantum MAT in Escherichia coli proves that the MAT and tripolyphosphatase activities are functional in vivo . MAT shows sigmoidal behavior and is weakly inhibited by AdoMet, whereas tripolyphosphatase activity has sigmoidal behavior and is strongly activated by AdoMet . Plasmids containing the regions flanking MAT2 were fused immediately upstream and downstream of the luciferase-coding region and transfected into L . infantum . Subsequent examination of luciferase activity showed that homologous expression in L . infantum promastigotes was dramatically dependent on the presence of polypyrimidine tracts and a spliced leader junction site upstream of the luciferase gene, whereas downstream sequences appeared to have no bearing on expression.

J Bacteriol, 2001 Dec, 183(23), 6961 - 4
Carboxy-terminal region involved in activity of Escherichia coli TolC; Yamanaka H et al.; The Escherichia coli TolC acts as a channel tunnel in the transport of various molecules across the outer membrane . Partial-deletion studies of tolC revealed that the region extending from the 50th to the 60th amino acid residue from the carboxy terminus plays an important role in this transport activity of TolC.

J Bacteriol, 2001 Dec, 183(23), 6957 - 60
The rlmB gene is essential for formation of Gm2251 in 23S rRNA but not for ribosome maturation in Escherichia coli; Lovgren JM et al.; In Saccharomyces cerevisiae, the rRNA Gm2270 methyltransferase, Pet56p, has an essential role in the maturation of the mitochondrial large ribosomal subunit that is independent of its methyltransferase activity . Here we show that the proposed Escherichia coli ortholog, RlmB (formerly YjfH), indeed is essential for the formation of Gm in position 2251 of 23S rRNA . However, a DeltarlmB mutant did not show any ribosome assembly defects and was not outgrown by a wild-type strain even after 120 cell mass doublings . Thus, RlmB has no important role in ribosome assembly or function in E . coli.

J Bacteriol, 2001 Dec, 183(23), 6908 - 16
Isolation and characterization of Escherichia coli tolC mutants defective in secreting enzymatically active alpha-hemolysin; Vakharia H et al.; This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin . Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event . Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules . Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain . Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release . Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed alpha-helical domain, while the remaining two mapped within the outer membrane-embedded beta-barrel domain of TolC . A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.

J Bacteriol, 2001 Dec, 183(23), 6841 - 51
HcwA, an autolysin, is required for heterocyst maturation in Anabaena sp . strain PCC 7120; Zhu J et al.; In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N(2) . Synthesis of the heterocyst envelope polysaccharide is dependent on the gene hepA in Anabaena sp . strain PCC 7120 . In search of genes that are involved in the regulation of hepA, we transposon mutagenized strain DR1069, which bears a chromosomal hepA::luxAB fusion . One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of hepA in response to nitrogen deprivation . In HNL3, transposon Tn5-1058 is inserted within gene hcwA, a constitutively expressed open reading frame whose predicted product resembles N-acetylmuramoyl-L-alanine amidases . Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon . The induction of hepA in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type hcwA . Moreover, HcwA expressed in Escherichia coli exhibits wall-lytic activity . These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.

Neurosci Lett, 2001 Nov 13, 314(1-2), 53 - 6
Central cardiovascular responses induced by interleukin 1 beta and tumor necrosis factor alpha infused into nucleus tractus solitarii, nucleus parabrachialis medialis and third cerebral ventricle of normotensive rats; Mollace V et al.; The effect of IL-1 beta and TNF alpha infused into nucleus tractus solitari (NTS), nucleus parabrachialis medialis (NPBmed) and third cerebral ventricle of normotensive rats on blood pressure (BP) and heart rate (HR) was investigated.Microinfusion of IL-1 beta and TNF alpha into the third cerebral ventricle and NPBmed of normotensive rats produced a dose-dependent hypotensive and bradycardic response . A similar cardiovascular response was produced by infusion of IL1 beta into NTS but not by TNF alpha.When rats were pre-treated with Escherichia coli lipopolisaccharide (LPS), an enhancement of cardiovascular response elicited by IL-1 beta and TNF alpha was found . Thus, IL-1 beta and TNF alpha produce cardiovascular responses when infused into specific areas of the CNS . This effect is potentiated by LPS and this may explain the alteration in cardiovascular regulation which can be observed in diseases in which an excess of circulating endotoxins and cytokines may occur.

Biochimie, 2001 Sep, 83(9), 873 - 82
The looped domain organization of the nucleoid in histone-like protein defective Escherichia coli strains; Brunetti R et al.; We have investigated the major Escherichia coli histone-like proteins (H-NS, HU, FIS, and IHF) as putative factors involved in the maintenance of the overall DNA looped arrangement of the bacterial nucleoid . The long-range architecture of the chromosome has been studied by means of an assay based on in vivo genomic fragmentation mediated by endogenous DNA gyrase in the presence of oxolinic acid . The fragmentation products were analysed by CHEF electrophoresis . The results indicate that in vivo a large fraction of the bacterial chromatin constitutes an adequate substrate for the enzyme . DNA fragments released upon oxo-treatment span a size range from about 1000 kb to a limit-size of about 50 kb . The latter value is in excellent agreement with the average size reported for bacterial chromosomal domains . The DNA gyrase-mediated fragmentation does not appear to be significantly altered in strains depleted in histone-like proteins as compared to an E . coli wild type strain . This suggests that these proteins may not represent critical determinants for the maintenance of the supercoiled loop organisation of the E . coli chromosome.

J Mol Biol, 2001 Nov 2, 313(4), 813 - 29
Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases; Sayre PH et al.; Crystal structures of four pyrrolo(2,3-d)pyrimidine-based antifolate compounds, developed as inhibitors of thymidylate synthase (TS) in a strategy to circumvent drug-resistance, have been determined in complexes with their in vivo target, human thymidylate synthase, and with the structurally best-characterized Escherichia coli enzyme, to resolutions of 2.2-3.0 A . The 2.9 A crystal structure of a complex of human TS with one of the inhibitors, the multi-targeted antifolate LY231514, demonstrates that this compound induces a "closed" enzyme conformation and leads to formation of a covalent bond between enzyme and substrate . This structure is one of the first liganded human TS structures, and its solution was aided by mutation to facilitate crystallization . Structures of three other pyrrolo(2,3-d)pyrimidine-based antifolates in complex with Escherichia coli TS confirm the orientation of this class of inhibitors in the active site . Specific interactions between the polyglutamyl moiety and a positively charged groove on the enzyme surface explain the marked increase in affinity of the pyrrolo(2,3-d)pyrimidine inhibitors once they are polyglutamylated, as mediated in vivo by the cellular enzyme folyl polyglutamate synthetase .

J Mol Biol, 2001 Nov 2, 313(4), 751 - 64
Distortion of DNA junctions imposed by the binding of resolving enzymes: a fluorescence study; Fogg JM et al.; Junction-resolving enzymes are nucleases that are specific for the structure of the four-way DNA junction . The binding of RuvC of Escherichia coli and Hjc of Sulfolobus solfataricus can be followed by an increase in the fluorescence anisotropy of Cy3 terminally attached to one of the helical arms of a four-way junction . By contrast, there was no change in fluorescein anisotropy with the binding of single dimers of these proteins . Fluorescence resonance energy transfer has therefore been used between fluorescein and Cy3 fluorophores attached to the ends of helical arms to analyse the global structure of the junction on protein binding . The results indicate that both enzymes induce a marked change in the global DNA conformation on the binding of a single dimer . The structure of the protein-junction complexes is independent of the presence or absence of divalent metal ions, unlike that of the protein-free junction . The structures of the RuvC and Hjc complexes are different, but both represent a significant opening of the structure compared to the stacked X-structure of the protein-free junction in the presence of magnesium ions . This protein-induced opening is likely to be important in the function of these enzymes .

J Biomol Struct Dyn, 2001 Oct, 19(2), 219 - 36
Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain; Alexandrovich A et al.; The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E . coli have been determined using a combination of high resolution NMR, ultracentrifugation, 15N NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry . The subunit molecular weight is 7,438 kDa., compared with 14.5+/-1.0 kDa . determined by equilibrium sedimentation, indicating a dimeric structure . The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface . The Stokes radius of the protein decreases from a high plateau value (ca . 22 A) at protein concentrations greater than 4 microM to about 18 A at concentrations less than 0.1 microM . The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca . 71.5 degrees C at 36 microM) . The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively . The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with deltaH1=23 kJ mol(-1) . K1(298)=0.4 microM and deltaH2= 184 kJ mol(-1) . This is in good agreement with direct calorimetric analysis of the thermal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol(-1) and a van't Hoff enthalpy change of 354 kJ mol(-1), confirming the dimer to monomer unfolding . The thermodynamic data can be reconciled with the observed mode of dimerisation . 15N NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein . The role of dimerisation of this domain in the excision repair mechanism is discussed.

J Biomol Struct Dyn, 2001 Oct, 19(2), 215 - 8
Convergent evolution of MutS and topoisomerase II for clamping DNA crossovers and stacked Holliday junctions; Timsit Y; This study shows that topoisomerase II and MutS proteins share a structural motif that has, in its dimeric form, a suitable geometry for clamping the two arms of either right-handed DNA crossovers or their isostructural stacked Holliday junctions . This defines a new protein family selected by convergent evolution for sensing DNA topology and binding recombination intermediates . This study also proposes that MutS binding on 2-fold right-handed crossover provides a mechanism for strand discrimination during DNA translocation.

Antibiot Khimioter, 2001, 46(7), 19 - 22
{Immunotropic activity of panaxans--bioglycans isolated from ginseng}; Smolina TP et al.; Immunomodulating activity of panaxanes--polysaccharides isolated from the roots and culture of Panax ginseng was studied . Effects of both preparations were analogous . Profilaxy use of panaxanes provided increased resistance to coli-sepsis in mice, increased neutrophiles and macrophages phagocytosis, stimulated humoral and cell immune factors and induced important regulating cytokins--interferone gamma and tumor necrosis factor.

Antibiot Khimioter, 2001, 46(7), 14 - 8
{1,3;1,6-beta-D-glucan translam: results of studying and prospects for application}; Ivanushko LA et al.; The results of translam chemical structure and biological activity investigation are presented . Translam is a new original semisynthetic polysaccharide of marine origin . The preparation demonstrated potent treatment effect in experimental radiation disease . It had preventing effect at experimental bacterial infections, stimulated hematopoiesis, had effect on humoral and cell immunity and on factors of nonspecific organism resistance.

Receptors Channels, 2001, 7(4), 319 - 28
Molecular model of the Escherichia coli Na1/H1 antiporter NhaA; Ravna AW et al.; A three-dimensional electron density projection map of the ion-coupled membrane protein Escherichia coli Na+/H+ antiporter (NhaA) was recently published . Based on this projection map, and previous biophysical studies determining the assignment of the 12 transmembrane alpha-helices (TMHs), a three-dimensional molecular model of the NhaA was constructed, using interactive molecular graphics and energy calculations . The diuretic drug, amiloride, was docked into the model and putative interacting amino acids were identified . The model suggests that the pH dependent activity of NhaA may be explained by charge changes in the intracellular loop between TMH8 and TMH9 which alter the positions of TMHs 4, 5 and 11 relative to each other, such that a pore area of the transporter protein is opened.

Free Radic Res, 2001 Aug, 35(2), 159 - 66
Selective interaction of tirapazamine with DNA bases and DNA . A comparison of cyclic voltammetry and electrolysis techniques; Tocher JH; An electrochemical model has been used to study the reductive activation of the hypoxic cell cytotoxin tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine-1,4-dioxide) . Cyclic voltammetry and controlled potential electrolysis have been used to generate and study the 1-electron reduction product, the assumed biologically active species . Cyclic voltammetry of tirapazamine in dimethylformamide shows a quasi-reversible 1-electron reduction with the product showing a tendency to participate in a following chemical reaction . Controlled potential electrolysis to generate the 1-electron reduction product was unsuccessful due to the formation of a new redox-active species at less negative reduction potentials . However, the cyclic voltammetry of tirapazamine in the presence of E . coli DNA shows a decrease in the lifetime of the radical anion, signifying direct interaction with the DNA . The radical lifetime also decreased in the presence of adenine, thymine and guanine, but increased upon addition of cytosine and ribose . The study shows that cyclic voltammetry is an extremely useful tool for investigating the interaction between bio-reductive drugs and biological target molecules.

Free Radic Res, 2001 Oct, 35(4), 427 - 34
Proposed reductive metabolism of artemisinin by glutathione transferases in vitro; Mukanganyama S et al.; Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge . It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum . It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins . Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases . Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides . Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs . Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver . Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1 . Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB . Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase . We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase . The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.

Free Radic Res, 2001 Jul, 35(1), 73 - 84
Ascorbic acid and N-acetylcysteine improve in vitro the function of lymphocytes from mice with endotoxin-induced oxidative stress; De la Fuente M et al.; Oxidative stress associated with reactive oxygen species (ROS) and cytokines produced by immune cells, which is involved in septic shock caused by endotoxin, can be controlled to a certain degree by antioxidants with free radical scavenging action . N-acetylcysteine (NAC) and ascorbic acid (AA) are ROS scavengers that improve the immune response, and modulate macrophage function in mice with endotoxin-caused oxidative stress . Therefore, we have investigated the in vitro effects of these antioxidants on the functions of lymphocytes from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of E . coli lipopolysaccharide (LPS) (100 mg/kg) . Adherence to tissues and chemotaxis (the earliest two functions of lymphocytes in the immune response), as well as ROS levels and TNF alpha production were determined in the presence or absence of NAC or AA (0.001, 0.01, 0.1, 1 and 2.5 mM) in lymphocytes from peritoneum, axillary nodes, spleen and thymus obtained at several times (2, 4, 12 and 24 hours) after LPS injection . Endotoxic shock decreases the chemotaxis of lymphocytes from all the above localizations and increases their adherence, TNF alpha and ROS production . These changes in lymphocyte function were counteracted by NAC and AA, bringing these functions to values near those of control animals . Our data suggest that lymphocytes are important targets of endotoxins contributing to oxidative stress by septic shock, and that antioxidants can preserve the function of lymphocytes, preventing the homeostatic disturbances caused by endotoxin.

J Drug Target, 2001 Apr, 9(2), 123 - 39
Folate-PEG-folate-graft-polyethylenimine-based gene delivery; Benns JM et al.; Folate-polyethylene glycol-folate-grafted-polyethylenimine (FPF-g-PEI) was synthesized by linking folic acid to both ends of a mono-functional PEG and then grafting to PEI . The graft ratio was determined using Beer's law by measuring the UV absorbance at 363 nm . The pH profile, diameter and shape of the carriers were determined . Transfection efficiencies were optimized in normal smooth muscle cells (SMC) and CT-26 colon adenocarcinoma cells using plasmid DNA encoding luciferase reporter gene . Free folic acid was shown to inhibit transfection with FPF-2.3 g-PEI at neutral charge ratio . Relative toxicity between PEI and the modified carrier was measured using MTT colorimetric assay . Therapeutic potential of pmIFN-gamma complexed with these polymeric carriers in terms of gene expression was determined at protein and mRNA levels using ELISA and RT-PCR . FPF-g-PEI was determined to have 2.3 folate-PEG-folate (FPF) linear polymers grafted to each PEI molecule . The average molecular weight was measured to be approximately 33,500 Mw and the pH profile was characteristic of endosomal disruption capacity . Atomic Force Microscopy (AFM) and Dynamic Laser Light Scattering (DLLS) indicated FPF-2.3 g-PEI and PEI (at 2 w/w ratio) efficiently condensed plasmid DNA resulting in oblique spheroid polyplexes with a mean diameter of approximately 150 nm . FPF-2.3 g-PEI was superior to PEI in terms of cytotoxicity and transfection efficiency in cancer cells . Smooth muscle cells showed no specificity for folate tethered complexes, where PEI/pLuc complexes yielded higher efficiencies.

Receptors Channels, 2001, 7(5), 401 - 11
In vitro binding characteristics and affinity for sulfatide of Escherichia coli STb enterotoxin; Beausoleil HE et al.; It has previously been demonstrated that sulfatide (3'-sulfogalactosyl-ceramide), present at the surface of epithelial cells of the small intestine of pigs, interacts with the thermostable enterotoxin b (STb) produced by ETEC, and that this molecule is implicated in the mechanism of action of the toxin . However, few things are known about the affinity and physical characteristics of the interaction between these two macromolecules . In this study, using a microtiter plate binding assay (MPBA), we showed that STb toxin has a strong specificity for sulfatide and that this binding is dose-dependent and saturable . A very weak binding occurred with galactosyl-ceramide whereas attachment to 3'-sulfolactosyl-ceramide corresponded to 76% of the binding to sulfatide . STb toxin was shown to possess a lectin-like property; a significative binding was observed when a terminal beta-galactose was present in the glycosphingolipids tested and an increased binding was observed in presence of a sulfate group in position 3 on the galactose . These findings suggest that a sulfated galactosyl residue seems to represent the epitope recognized by the toxin . The reaction between sulfatide and STb toxin is also time and temperature dependent and is not affected by pH . The interaction was not inhibited by free sugars, sulfated polymers, glycolipids or free ions, but was partly inhibited by high concentrations of charged sugars . STb-sulfatide binding process is a low affinity interaction, as demonstrated by the determined Kd of 2-6 +/- 1.5 microM.

Food Chem Toxicol, 2001 Dec, 39(12), 1191 - 7
Effects of Brussels sprouts extracts on hydrogen peroxide-induced DNA strand breaks in human lymphocytes; Zhu CY et al.; Aqueous Brussels sprouts extracts inhibit oxidation of isolated DNA in vitro, possibly through scavenging oxygen radicals . We have studied the effect of preincubating human lymphocytes with aqueous extracts of raw, cooked and autolysed Brussels sprouts and the glucosinolate, sinigrin, on hydrogen peroxide-induced DNA damage, strand breaks and base oxidation, in vitro by means of the Comet assay . DNA repair enzymes endonuclease III (EndoIII) and formamidopyrimidine-DNA glycosylase (FPG) were used to examine the levels of oxidised pyrimidines and purines in DNA, respectively . Aqueous extracts of cooked and autolysed Brussels sprouts and sinigrin decreased DNA strand breaks in human lymphocytes exposed to 100 microM H2O2 for 5 min on ice, although the level of EndoIII and FPG sensitive sites was not reduced . The maximum inhibition was by 38 and 39% at concentrations of cooked and autolysed extracts of 10 microg/ml and 5 microg/ml, respectively, whereas the inhibitory effect decreased with increasing concentrations up to 100 microg/ml . The maximum inhibition by sinigrin was by 54% at 2 microg/ml . Extracts of raw Brussels sprouts or green beans had no DNA-protective effect . The results indicate that compounds, including sinigrin, in cooked and autolysed Brussels sprouts can enhance lymphocyte resistance towards H2O2-induced DNA strand breaks in vitro.

Plant J, 2001 Oct, 28(1), 83 - 94
The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase-like protein sinapoylglucose:choline sinapoyltransferase; Shirley AM et al.; Serine carboxypeptidase-like (SCPL) proteins have traditionally been assigned roles in the hydrolytic processing of proteins; however, several SCPL proteins have recently been identified as catalysts in transacylation reactions of plant secondary metabolism . The novel functions of these enzymes suggest a catalytic diversity for plant SCPL proteins that extends beyond simple hydrolysis reactions . Characterization of the Arabidopsis sng2 (sinapoylglucose accumulator 2) mutant has identified another SCPL protein involved in plant secondary metabolism . The sng2 mutant was isolated by screening seed extracts for altered levels of sinapate esters, a group of phenylpropanoid compounds found in Arabidopsis and some other members of the Brassicaceae . Homozygous sng2 seeds accumulate sinapoylglucose instead of sinapoylcholine, and have increased levels of choline and decreased activity of the enzyme sinapoylglucose:choline sinapoyltransferase (SCT) . Cloning of the SNG2 gene by a combination of map-based and candidate gene approaches demonstrates that SCT is another member of the growing class of SCPL acyltransferases involved in plant secondary metabolism.

Plant J, 2001 Oct, 28(1), 73 - 82
Light-dependent gene expression for proteins in the respiratory chain of potato leaves; Svensson AS et al.; Expression of genes for respiratory chain dehydrogenases was investigated in potato (Solanum tuberosum L . cv . Desiree) leaves . The recently characterized 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) . As leaves develop from young to mature, the nda1 transcript level increases, accompanied by an elevation in immunodetected NDA protein and internal rotenone-insensitive NADH oxidation . The other investigated transcripts, proteins and NAD(P)H oxidation activities were essentially unchanged . A variation in transcript level, specific for nda1, is seen at different times of the day with highest expression in the morning . This variation also influences the apparent developmental induction . Further, the nda1 mRNA in leaves specifically and completely disappears during dark treatment, with a rapid re-induction when plants are returned to light . Corresponding immunodetected NDA protein is specifically decreased in mitochondria isolated from dark-treated plants, accompanied by a lower capacity for internal rotenone-insensitive NADH oxidation . Complete light dependence and diurnal changes in expression have previously not been reported for genes encoding respiratory chain proteins . Qualitatively similar to NDA, the alternative oxidase showed developmental induction and light dependence . In addition to the specific change in nda1, a general, slower down-regulation in darkness was seen for the other NAD(P)H dehydrogenase genes . The nda1 expression during development, and in response to light, indicates a specific role of the encoded enzyme in the photosynthetically associated mitochondrial metabolism.

Lett Appl Microbiol, 2001 Nov, 33(5), 371 - 6
Decimal reduction times of Pyrodinium bahamense var . compressum and Escherichia coli in chlorine- and ultraviolet-treated seawater; Azanza MP et al.; AIMS: Decimal reduction times (D-values) of the vegetative cells of Pyrodinium bahamense var . compressum and Escherichia coli in ultraviolet- and chlorine-treated seawater were established . METHODS AND RESULTS: The cells of the test organisms were exposed to ultraviolet- and chlorine-treated seawater and maintained at 20-35 ppt salinity and 20 to 35 degrees C . The dinoflagellate cells which cause Paralytic Shellfish Poisoning (PSP) were found to be more resilient than the bacterial cells . Ultraviolet treatment was found to be more effective than chlorine to both test organisms . Irreversible morphological changes in the treated dinoflagellate cells were noted, including protoplast discoloration, cellular membrane leakage and damage to the thecal armour . CONCLUSIONS: The vegetative cells of both test organisms in seawater were more sensitive to ultraviolet treatment than to chlorine exposure . Generally, the dinoflagellate cells were less susceptible than bacterial cells to both disinfection treatments . SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study may have significant implications in depuration procedures for molluscs and cleaning protocols for ballast waters of ships.

Biochem J, 2001 Nov 15, 360(Pt 1), 173 - 7
Identification of a developmentally regulated iron superoxide dismutase of Trypanosoma brucei; Kabiri M et al.; An iron superoxide dismutase (FeSOD) gene of the protozoan parasite Trypanosoma brucei has been cloned and its gene product functionally characterized . The gene encodes a protein of 198 residues which shows 80% identity with FeSODs from other trypanosomatids . Inhibitor studies with purified recombinant FeSOD expressed in Escherichia coli confirmed that the enzyme is an iron-containing SOD . The FeSOD is developmentally regulated in the parasite, expression being lowest in the cell-cycle-arrested, short stumpy bloodstream forms . Differential expression of the FeSOD protein contrasts with only minor quantitative changes in the FeSOD mRNA, indicating post-transcriptional regulation of the enzyme . As the level of FeSOD increases during differentiation of cell-cycle-arrested short stumpy into dividing procyclic forms, it is suggested that the enzyme is only required in proliferating stages of the parasite for the elimination of superoxide radicals which are released during the generation of the iron-tyrosyl free-radical centre in the small subunit of ribonucleotide reductase.

Biochemistry, 2001 Nov 13, 40(45), 13744 - 52
Evidence for intraprotein charge transfer during the transport activity of the melibiose permease from Escherichia coli; Ganea C et al.; Electrogenic activity associated with the activity of the melibiose permease (MelB) of Escherichia coli was investigated by using proteoliposomes containing purified MelB adsorbed onto a solid-supported membrane . Transient currents were selectively recorded by applying concentration jumps of Na+ ions (or Li+) and/or of different sugar substrates of MelB (melibiose, thio-methyl galactoside, raffinose) using a fast-flow solution exchange system . Characteristically, the transient current response was fast, including a single decay exponential component (tau approximately 15 ms) on applying a Na+ (or Li+) concentration jump in the absence of sugar . On imposing a Na+ (or Li+) jump on proteoliposomes preincubated with the sugar, a sugar jump in a preparation preincubated with the cation, or a simultaneous jump of the cation and sugar substrates, the electrical transients were biphasic and comprised both the fast and an additional slow (tau approximately 350 ms) decay components . Finally, selective inactivation of the cosubstrate translocation step by acylation of MelB cysteins with N-ethyl maleimide suppressed the slow response components and had no effect on the fast transient one . We suggest that the fast transient response reflects charge transfer within MelB during cosubstrate binding while the slow component is associated with charge transfer across the proteoliposome membrane . From the time course of the transient currents, we estimate a rate constant for Na+ binding in the absence and presence of melibiose of k > 50 s(-1) and one for melibiose binding in the absence of Na+ of k approximately 10 s(-1).

Biochemistry, 2001 Nov 13, 40(45), 13617 - 22
Mutation at active site lysine 212 to arginine uncouples the glycosylase activity from the lyase activity of human endonuclease III; Liu X et al.; The human endonuclease III (hNTH1) is an important DNA glycosylase with associated abasic lyase activity . We previously demonstrated that the K212Q mutant was totally inactive, while the K212R mutant had reduced DNA glycosylase/lyase activity and could form a covalent complex with the substrate DNA upon reduction . We further characterized the biochemical properties of this K212R mutant protein . NH2- (N-) terminal sequencing in combination with mass spectrometry of the peptide-DNA adduct suggested that "opportunistic" lysine(s) in the lysine-rich N-terminal tail formed a Schiff base which might result in beta-elimination . However, simultaneous substitution of Lys-75 with Gln and deletion of first 72 residues in the N-terminal tail could not cause further alteration in the glycosylase reaction or beta-elimination event . Nonetheless, the time kinetics of K212R and its subsequent mutants showed glycosylase activity without any detectable AP-lyase activity during the first 10 min of the reaction . These results suggest that a single point mutation at the active site (K212R) uncoupled the glycosylase activity from the lyase activity . We propose that the uncoupled reaction carried out by K212R is a result of direct attack either by the nonionized form of the guanidino group of arginine which forms an unstable Schiff base that hydrolyzes prior to the beta-elimination event or by hydroxide ion to cleave the glycosylic bond . In either case this reaction is followed by a secondary beta-elimination event performed by random lysine residues primarily from the N-terminal tail region.

Biochemistry, 2001 Nov 13, 40(45), 13607 - 16
Insights into the catalytic mechanism of HlyC, the internal protein acyltransferase that activates Escherichia coli hemolysin toxin; Worsham LM et al.; Hemolysin, a toxic protein secreted by pathogenic Escherichia coli, is converted from nontoxic prohemolysin, proHlyA, to toxic hemolysin, HlyA, by an internal protein acyltransferase, HlyC . Acyl-acyl carrier protein (ACP) is the essential acyl donor . The acyltransferase reaction proceeds through two partial reactions and entails formation of a reactive acyl-HlyC intermediate {Trent, M . S., Worsham, L . M., and Ernst-Fonberg, M . L . (1999) Biochemistry 38, 9541-9548} . The ping pong kinetic mechanism implied by these findings was validated using two different acyl-ACP substrates, thus verifying the independence of the previously demonstrated two partial reactions . Assessments of the stability of the acyl-HlyC intermediate revealed an increased stability at pH 8.6 compared to more acidic pHs . Mutations of a single conserved histidine residue essential for catalysis gave minimal activity when substituted with a tyrosine residue and no activity with a lysine residue . Unlike numerous other His23 mutants, however, the H23K enzyme showed significant acyl-HlyC formation although it was unable to transfer the acyl group from the proposed amide bond intermediate to proHlyA . These findings are compatible with transient formation of acyl-His23 during the course of HlyC catalysis . The effects of several other single site-directed mutations of conserved residues of HlyC on different portions of the reaction progress were examined using a 39 500 kDa fragment of proHlyA which was a more effective substrate than intact proHlyA.

Biochemistry, 2001 Nov 13, 40(45), 13538 - 47
Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase; Greasley SE et al.; Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide transformylase (GAR Tfase), which incorporate key features of the folate cofactor and the beta-GAR substrate, typically exhibit K(i)'s in the picomolar range . However, these compounds have reduced bioavailability due to the incorporation of a negatively charged phosphate moiety that prevents effective cellular uptake . Thus, a folate analogue that is capable of adduct formation with the substrate on the enzyme active site could lead to a potent GAR Tfase inhibitor that takes advantage of the cellular folate transport systems . We synthesized a dibromide folate analogue, 10-bromo-10-bromomethyl-5,8,10-trideazafolic acid, that was an intermediate designed to assemble with the substrate beta-GAR on the enzyme active site . We have now determined the crystal structure of the Escherichia coli GAR Tfase/MAI complex at 1.6 A resolution to ascertain the nature and mechanism of its time-dependent inhibition . The high-resolution crystal structure clearly revealed the existence of a covalent adduct between the substrate beta-GAR and the folate analogue (K(i) = 20 microM) . However, the electron density map surprisingly indicated a C10 hydroxyl in the adduct rather than a bromide and suggested that the multisubstrate adduct is not formed directly from the dibromide but proceeds via an epoxide . Subsequently, we demonstrated the in situ conversion of the dibromide to the epoxide . Moreover, synthesis of the authentic epoxide confirmed that its inhibitory, time-dependent, and cytotoxic properties are comparable to those of the dibromide . Further, inhibition was strongest when the dibromide or epoxide is preincubated with both enzyme and substrate, indicating that inhibition occurs via the enzyme-dependent formation of the multisubstrate adduct . Thus, the crystal structure revealed the successful formation of an enzyme-assembled multisubstrate adduct and highlighted a potential application for epoxides, and perhaps aziridines, in the design of efficacious GAR Tfase inhibitors.

Biochemistry, 2001 Nov 13, 40(45), 13439 - 47
Role of the conserved phenylalanine 181 of NADPH-cytochrome P450 oxidoreductase in FMN binding and catalytic activity; Paine MJ et al.; NADPH-cytochrome P450 oxidoreductase (P450 reductase, EC 1.6.2.4) is an essential component of the P450 monooxygenase complex and binds FMN, FAD, and NADPH cofactors . Residues Tyr140 and Tyr178 are known to be involved in FMN binding . A third aromatic side chain, Phe181, is also located in the proximity of the FMN ring and is highly conserved in FMN-binding proteins, suggesting an important functional role . This role has been investigated by site-directed mutagenesis . Substitution of Phe181 with leucine or glutamine decreased the cytochrome c reductase activity of the enzyme by approximately 50% . Ferricyanide reductase activity was unaffected, indicating that the FAD domain was unperturbed . The mutant FMN domains were expressed in Escherichia coli, and the redox potentials and binding energies of their complexes with FMN were determined . The affinity for FMN was decreased approximately 50-fold in the Leu181 and Gln181 mutants . Comparison of the binding energies of the wild-type and mutant enzymes in the three redox states of FMN suggests that Phe181 stabilizes the FMN-apoprotein complex . The amide 1H and 15N resonances of the Phe181Leu FMN domain were assigned; comparison of their chemical shifts with those of the wild-type domain indicated that the effect of the substitution on FMN affinity results from perturbation of two loops which form part of the FMN binding site . The results indicate that Phe181 cooperates with Tyr140 and Tyr178 to play a major role in the binding and stability of FMN.

Biochemistry, 2001 Nov 13, 40(45), 13430 - 8
Structural and spectroscopic analysis of the F393H mutant of flavocytochrome P450 BM3; Ost TW et al.; In the preceding paper in this issue {Ost, T . W . B., Miles, C . S., Munro, A . W., Murdoch, J., Reid, G . A., and Chapman, S . K . (2001) Biochemistry 40, 13421-13429}, we have established that the primary role of the phylogenetically conserved phenylalanine in flavocytochrome P450 BM3 (F393) is to control the thermodynamic properties of the heme iron, so as to optimize electron-transfer both to the iron (from the flavin redox partner) and onto molecular oxygen . In this paper, we report a detailed study of the F393H mutant enzyme, designed to probe the structural, spectroscopic, and metabolic profile of the enzyme in an attempt to identify the factors responsible for causing the changes . The heme domain structure of the F393H mutant has been solved to 2.0 A resolution and demonstrates that the histidine replaces the phenylalanine in almost exactly the same conformation . A solvent water molecule is hydrogen bonded to the histidine, but there appears to be little other gross alteration in the environment of the heme . The F393H mutant displays an identical ferric EPR spectrum to wild-type, implying that the degree of splitting of the iron d orbitals is unaffected by the substitution, however, the overall energy of the d-orbitals have changed relative to each other . Magnetic CD studies show that the near-IR transition, diagnostic of heme ligation state, is red-shifted by 40 nm in F393H relative to wild-type P450 BM3, probably reflecting alteration in the strength of the iron-cysteinate bond . Studies of the catalytic turnover of fatty acid (myristate) confirms NADPH oxidation is tightly coupled to fatty acid oxidation in F393H, with a product profile very similar to wild-type . The results indicate that gross conformational changes do not account for the perturbations in the electronic features of the P450 BM3 heme system and that the structural environment on the proximal side of the P450 heme must be conformationally conserved in order to optimize catalytic function.

J Bioenerg Biomembr, 2001 Jun, 33(3), 169 - 77
Complex I: a chimaera of a redox and conformation-driven proton pump?
Friedrich T.
From phylogenetic sequence analysis, it can be concluded that the proton-pumping NADH:ubiquinone oxidoreductase (complex I) has evolved from preexisting modules for electron transfer and proton translocation . It is built up by a peripheral NADH dehydrogenase module, an amphipatic hydrogenase module, and a membrane-bound transporter module . These modules, or at least part of them, are also present in various other bacterial enzymes . It is assumed that they fulfill a similar function in complex I and related enzymes . Based on the function of the individual modules, it is possible to speculate about the mechanism of complex I . The hydrogenase module might work as a redox-driven proton pump, while the transporter module might act as a conformation-driven proton pump . This implies that complex I contains two energy-coupling sites . The NADH dehydrogenase module seems to be involved in electron transfer and not in proton translocation.

Adv Space Res, 2001, 27(9), 1593 - 8
Mathematical modeling of response of ecosystems with different structure to external impact; Shirobokova IM et al.; A mathematical model was used to study the response of ecosystems of different structures to external impact . The response was measured as a sensitivity coefficient: the magnitude of the system's response vs . the change of the factor in the inflow . The formula has been obtained to calculate the sensitivity coefficient for ecosystems containing different numbers of trophic links . The derived sensitivity coefficients demonstrate that the degree of compensation for the external impact can differ depending on the type of system regulation and the length of the trophic chain . E . g . the sensitivity coefficient decreases with complexity of trophic links in an ecosystem for top-down controlled systems and impact of degree of openness on sensitivity e.g . closed ecosystems show higher sensitivity then fully open ecosystem to impacts also bottom-up control system show less sensitivity then top-down . Grant numbers: N99-04-96017, N25 . c 2001 . COSPAR . Published by Elsevier Science Ltd . All rights reserved.

J Parasitol, 2001 Oct, 87(5), 1218 - 21
Schistosoma mansoni proteases Sm31 (cathepsin B) and Sm32 (legumain) are expressed in the cecum and protonephridia of cercariae; Skelly PJ et al.; Adult Schistosoma mansoni parasites live in the bloodstream of their vertebrate hosts where they consume red blood cells . Hemoglobin, released from the ingested red blood cells, is degraded by a variety of parasite proteases, including Sm31 (cathepsin B) and Sm32 (schistosome legumain) . In this study the localization pattern of the Sm31 and Sm32 enzymes in cercariae (the infectious life cycle stage) was examined . Antibodies generated against recombinant Sm31 and Sm32 recognize their respective proteins in Western blots of soluble parasite extracts . Highest levels are seen in adult female extracts, whereas the level of both proteins is below detection in cercarial extracts . However, in fixed, whole cercariae, both proteins are seen in the cecum and protonephridia . In the cecum, the staining pattern has a granular appearance, suggesting that the proteins are packaged in vesicles . In the protonephridial system, Sm31 and Sm32 are detected in all 8 flame cells in the cercarial body and in both flame cells in the cercarial tail . The distribution of the 2 proteins differs in the flame cells . Examination of immunostained cercariae using laser scanning confocal microscopy shows that whereas Sm31 is located in the tubule cell, Sm32 is found in both the tubule cell and its adjoining cap cell . These findings suggest that the proteins are involved in the proposed excretory and osmoregulatory roles of flame cells.

Yi Chuan Xue Bao, 2001, 28(10), 971 - 9
Construction of a novel display vector deriving from CS3 fimbriae of human enterotoxigenic Escherichia coli; Gao RK et al.; Based on the prediction of the hydrophilicity, epitopes, secondary structure and flexibility of the CS3 subunit, a novel vector pCSX72 which permits the insertion of foreign epitopes into CS3 at the position of 72nd aa was constructed . Two epitopes, the VP1 of FMDV and a ten-peptides epitope of C-myc, were displayed with it respectively . Compared with the two previously-constructed vectors, the vector pCSX72 expressed the hybrid fimbriae in higher level . Mice produced dual immune response against the CS3 and the inserted epitopes when they were immunized by injecting the live recombinant bacteria intraperitoneally.

J Nutr, 2001 Nov, 131(11 Suppl), 3082S - 6S
Protection by dietary compounds against mutation in a transgenic rodent; de Boer JG; One of the most relevant biomarkers of genotoxicity and, potentially, carcinogenesis is the occurrence of mutations . Data indicate that carcinogens are highly specific with regard to their target tissue in inducing both tumors and mutations . This specificity may reflect the dependence on tissue-specific metabolic activation, the organ-specific environment or both . Ideally, therefore, mutation should be determined in a real animal rather than in a cell culture system . The lacI transgenic rodent model provides such a system . We have used this model to investigate tissue, species and sex specificity of mutation induced by selected dietary carcinogens and to examine how some compounds may alter the induction of mutation . We have studied mutation using several chemicals, including the dietary heterocyclic amine 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP), the environmentally important aromatic hydrocarbon benzo{a}pyrene and the food contaminant aflatoxin B1 . We have shown that the mutagenic potency of these chemicals can be modulated by other dietary compounds, including green tea and conjugated linoleic acid, and the dioxin 2,3,7,8-tetrachlorodibenzo{b,e}{1,4}dioxin (TCDD) . These results demonstrate that the lacI transgenic rodent is a useful model for the study of chemoprevention in vivo.

J Nutr, 2001 Nov, 131(11), 2994S - 3004S
Genetic approaches to studying protein synthesis: effects of mutations at Psi516 and A535 in Escherichia coli 16S rRNA; Lee K et al.; A genetic system for the study of ribosomal RNA function and structure was developed . First, the ribosome binding sequence of the chloramphenicol acetyltransferase gene and the message binding sequence of 16S ribosomal RNA were randomly mutated and alternative highly functional sequences were selected and characterized . From this set of mutants, a single clone was chosen and subjected to a second round of mutagenesis to optimize the specificity of the system . In the resulting system, plasmid-encoded ribosomes efficiently and exclusively translate specific mRNA containing the appropriate ribosome binding sequences . This system allows facile isolation and analysis of mutations that would normally be lethal and allows direct selection of rRNA mutants with predetermined levels of ribosome function . The system was used to examine the effects of mutations at the sole pseudouridine (Psi) in Escherichia coli 16S rRNA which is located at position 516 of the conserved 530 loop . The nucleotide opposite Psi516 in the hairpin, A535, was also mutated . The data show that a pyrimidine (Psi or C) is required at position 516, while substitutions at position 535 reduce ribosome function by < 50% . A requirement for base pair formation between Psi516 and A535 was not indicated.

J Nutr, 2001 Nov, 131(11), 2978S - 82S
Quality control of the elongation step of protein synthesis by tmRNP; Wower J et al.; Trans-translation is a quality-control process, activated upon premature termination of protein elongation, which recycles stalled ribosomes and degrades incomplete polypeptides . These functions are facilitated by transfer-messenger RNA (tmRNA, also called 10Sa RNA or SsrA RNA), a small stable RNA molecule encoded by the SsrA gene found in bacteria, chloroplasts and mitochondria . Most tmRNAs consist of a tRNA- and an mRNA-like domain connected by up to four pseudoknots . Comparative sequence analysis provided the first insight into tmRNA secondary and three-dimensional structure . Studies of the E . coli tmRNA in vitro and in vivo demonstrated that tmRNA functions as a ribonucleoprotein (RNP) complex with elongation factor Tu (EF-Tu), protein SmpB and ribosomal protein S1 . The tRNA-like and mRNA-like activities of tmRNA mark prematurely terminated proteins for degradation by attaching to their C-termini peptide tags, which are recognized by numerous proteases . Studies aimed at understanding the details of the molecular mechanisms of trans-translation are ongoing.

J Biol Chem, 2002 Feb 1, 277(5), 3424 - 32 Epub 2001 Nov 02.
The peroxisome proliferator-induced cytosolic type I acyl-CoA thioesterase (CTE-I) is a serine-histidine-aspartic acid alpha /beta hydrolase; Huhtinen K et al.; Long-chain acyl-CoA thioesterases hydrolyze long-chain acyl-CoAs to the corresponding free fatty acid and CoASH and may therefore play important roles in regulation of lipid metabolism . We have recently cloned four members of a highly conserved acyl-CoA thioesterase multigene family expressed in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which are regulated via the peroxisome proliferator-activated receptor alpha (Hunt, M . C., Nousiainen, S . E . B., Huttunen, M . K., Orii, K . E., Svensson, L . T., and Alexson, S . E . H . (1999) J . Biol . Chem . 274, 34317-34326) . Sequence comparison revealed the presence of putative active-site serine motifs (GXSXG) in all four acyl-CoA thioesterases . In the present study we have expressed CTE-I in Escherichia coli and characterized the recombinant protein with respect to sensitivity to various amino acid reactive compounds . The recombinant CTE-I was inhibited by phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, suggesting the involvement of serine and histidine residues for the activity . Extensive sequence analysis pinpointed Ser(232), Asp(324), and His(358) as the likely components of a catalytic triad, and site-directed mutagenesis verified the importance of these residues for the catalytic activity . A S232C mutant retained about 2% of the wild type activity and incubation with (14)C-palmitoyl-CoA strongly labeled this mutant protein, in contrast to wild-type enzyme, indicating that deacylation of the acyl-enzyme intermediate becomes rate-limiting in this mutant protein . These data are discussed in relation to the structure/function of acyl-CoA thioesterases versus acyltransferases . Furthermore, kinetic characterization of recombinant CTE-I showed that this enzyme appears to be a true acyl-CoA thioesterase being highly specific for C(12)-C(20) acyl-CoAs.

Bioelectrochemistry, 2001 Nov, 54(2), 101 - 5
Twelve hours exposure to inhomogeneous high magnetic field after logarithmic growth phase is sufficient for drastic suppression of Escherichia coli death; Ishizaki Y et al.; When Escherichia coli B was aerobically grown at 43 degrees C in a medium whose concentration was one-fourth that of the Luria-Bertani (LB) medium supplemented with 1.5 g/l of glutamic acid, drastic cell death was observed after the end of the logarithmic growth phase . However, when the same experiment was conducted under inhomogeneous 5.2-6.1 T magnetic field, cell death was extremely suppressed and the ratio of viable cell number under high magnetic field to that under geomagnetic field reached as much as 100,000 . When the magnetic field exposure was restricted to 12 h after the logarithmic growth phase, a similar high degree of suppressive effect on the death was observed . The findings that the amount of sigma S protein encoded by the rpoS gene under the high magnetic field was larger than that under the geomagnetic field, and that the magnetic field effect disappeared when the rpoS gene-deficient strain was cultivated under the high magnetic field, suggest the interaction of magnetic field with a stationary phase specific gene.

J Biochem Biophys Methods, 2001 Oct 30, 49(1-3), 533 - 52
Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption; Reichert U et al.; Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli . In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate . EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution . A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml) . Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application . In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run . On this scale, 19 l of a benzonase-treated E . coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed . bed height, 5 x 10(-4) m/s fluid velocity) . After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH . This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.

Clin Chim Acta, 2001 Nov, 313(1-2), 125 - 31
Full-length cloning and 3'-terminal portion expression of human perforin cDNA; Li F et al.; BACKGROUND: Perforin (also known as pore-forming protein, PFP) is one of the main effector molecules which natural killer cells (NK) and cytotoxic T lymphocytes (CTL) utilize to kill their targets both in vivo and in vitro . We report the full length of human perforin cDNA, which was cloned from liver tissue . RESULTS: Sequencing analysis showed that there were discrepancies of four nucleotides and three amino acids compared with previously published sequence of human PFP . The cDNA fragment was then inserted into fusion protein expressive vector pGEX-2T to construct a recombinant expressive plasmid . The C-terminal truncated 125 amino acids polypeptide (410-534aa) of human perforin (hPFP-C) was selectively expressed in a form of fusion protein . Under the induction of IPTG, GST/hPFP-C fusion protein was expressed in E . coli BL21 (DE3) . The fusion protein GST/hPFP-C was purified by affinity chromatography with glutathione agarose . The recombinant hPFP-C obtained by thrombin cleavage showed a significant hemolytic activity when tested with rabbit erythrocytes . CONCLUSION: These results suggest that the domain responsible for lytic function lies not only in the N-terminal portion but also in the C-terminal portion of perforin molecule . The recombinant hPFP-C protein will be useful as a highly purified biological factor for immunological, pathological and structural studies.

Appl Biochem Biotechnol, 2001 Aug, 95(2), 93 - 101
Cloning and expression of L-asparaginase gene in Escherichia coli; Wang Y et al.; The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences . Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E . coli host strains . The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain . The recombinant plasmid in E . coli AS1.357 remained stable after 72 h of cultivation and 5 h of heat induction without selective pressure . The ASN gene of E . coli AS1.357 was sequenced and had high homology compared to the reported data.

J Dairy Res, 2001 Aug, 68(3), 357 - 67
Colostral whey concentrate supplement increases complement activity in the sera of neonatal calves; Rokka S et al.; We evaluated the effect of a commercial bovine colostral whey on the complement-mediated immune responses of calves . Two groups of neonatal calves were fed, in addition to whole milk (WM) and pooled colostrum (PC), different amounts of a commercial immunoglobulin concentrate made from pooled colostral whey (Ig-C) for the first two feedings post natum . The control group was fed WM and PC only . Serum samples were obtained at the ages of 2, 7, 14 and 30 d . Bacteriolytic activity against complement-sensitive Escherichia coli JM103 and opsonic activity against complement-lysis-resistant E . coli IH3080 strains were studied, as well as the levels of C3 complement component and E . coli JM103 specific antibodies in the sera . Groups fed Ig-C had 2-3 times higher bacteriolytic activity than the control group of both the classic (P < 0.005) and alternative pathways (P < 0.0001) at days 2 and 7 post natum . This effect is obviously not caused solely by the antibodies ingested but also involves other unknown colostral factors, possibly lectins . The opsonisation capacities of the sera correlated well with the amounts of immunoglobulins ingested (P < 0.05) at days 2-14 . The levels of C3 component in sera did not differ between the groups . In the group fed the largest amount of immunoglobulins levels of E . coli JM103-specific antibodies were highest (P < 0.0001) . It can thus be concluded that the antibody independent complement activities of serum can be increased substantially by feeding colostral whey concentrate to calves during their first days of life.

Nucleic Acids Res, 2001 Nov 1, 29(21), E105 - 5
A versatile in vivo footprinting technique using 1,10-phenanthroline-copper complex to study important cellular processes; Basak S et al.; A number of reagents have been used to define the sequence-specific protein-DNA contacts by footprinting analysis . We report a new in vivo technique using the complex of 1,10-phenanthroline and copper {(OP(2))Cu} as a probe to study various intracellular DNA-protein interactions in whole cells . The versatility of the protocol is demonstrated by applying the technique to address various processes . The protocol is applied to (i) detect structural alterations in DNA as a result of single base substitution, (ii) footprint site-specific DNA-binding proteins, (iii) analyze promoter occupancy by RNA polymerase and (iv) analyze molecular interactions during transcription initiation . The results demonstrate that in vivo (OP)(2)Cu probing is a useful tool in studying important cellular processes involving DNA-protein interactions and has potential applications in post-genomic research.

Comp Biochem Physiol A Mol Integr Physiol, 2001 Nov, 130(4), 653 - 63
Interleukin-12 prevents diaphragm muscle deterioration in a septic animal model; Nakahata E et al.; The effects of an intravenous injection of Interleukin-12 (IL-12) after endotoxin administration and without endotoxin administration on diaphragm muscle were studied using Wistar rats . Three treatment groups, namely a control (Saline+endotoxin) group, an IL-12+endotoxin group and an IL-12 only group were studied . E . coli endotoxin (30 mg/kg) was injected intraperitoneally 5 min after Saline or IL-12 (0.25 microg) injection . In the control group, the force-frequency curves, twitch tension (TT) and slope during contraction time (TT/CT) were significantly lower at 4 h than those at 0 h due to endotoxin (P<0.001, P<0.01 and P<0.01, respectively), and NO production was increased at 4 h as shown by NADPH diaphorase staining . In the IL-12+endotoxin group, the decrement of the force-frequency curves, TT and TT/CT induced by endotoxin at 4 h were significantly prevented compared with those of the control group (P<0.001, P<0.05 and P<0.05, respectively), and NO production was blocked at 4 h . In the IL-12 only group, the force-frequency curves were decreased in the range of high frequency and IL-12 resulted in NO production . Furthermore, the positive muscle fibers detected by NADPH diaphorase staining were classified as type I and IIa muscle fibers by ATPase staining in the control and IL-12 only groups . It is concluded that IL-12 prevents the deterioration of diaphragm muscle contraction induced by endotoxin by reducing NO production in type I and IIa muscle fibers . These results suggest that IL-12 and endotoxin may interfere with each other.

J Biotechnol, 2002 Jan 31, 93(1), 15 - 26
Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems; Li M et al.; The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated . In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C . The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated . The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed . Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated . All of the studied enzymes partitioned unevenly in these polymer/salt systems . The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis . Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis . Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis . A 22% gain in maltose yield was obtained as a result of the increased productivity . This work is the first reported application of thermoseparating polymer ATPS in the processing of starches . These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.

J Biotechnol, 2002 Jan 31, 93(1), 1 - 14
Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins; Bandmann N et al.; Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants . One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags . However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency . In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated . Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology . A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system . After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones . The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein . The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.

Biochim Biophys Acta, 2001 Oct 18, 1549(2), 174 - 8
The distal heme pocket of Escherichia coli flavohemoglobin probed by infrared spectroscopy; Bonamore A et al.; The infrared absorption spectra of ferric cyanide and ferrous carbonmonoxy Escherichia coli flavohemoglobin have been measured in order to probe the fine structural properties of the distal heme pocket, characterized by the presence of a tyrosine in position B10 and a glutamine in position E7 . The stretching frequency of iron bound cyanide occurs at 2136 cm(-1), an unusually high value if compared to other heme proteins . The infrared spectrum of the CO bound derivative displays two peaks centered at 1960 cm(-1) and at 1909 cm(-1) respectively . H(2)O effects have been studied in both the ferric cyanide and ferrous CO derivatives in order to establish the presence of a distal hydrogen bonding to the iron bound ligand . The observed isotope shifts indicate that in the ferric cyanide derivative a hydrogen bond is donated from a residue in the distal pocket to the biatomic ligand whereas in the ferrous carbon monoxy derivative only the 1909 cm(-1) component is most likely hydrogen bonded to the phenolic group of TyrB10.

Biochim Biophys Acta, 2001 Oct 31, 1521(1-3), 73 - 80
Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region; Morigen et al.; Initiation of chromosomal replication in Escherichia coli is dependent on availability of the initiator protein DnaA . We have introduced into E . coli cells plasmids carrying the chromosomal locus datA, which has a high affinity for DnaA . To be able to monitor oriC initiation as a function of datA copy number, we introduced a minichromosome which only replicates from oriC, using a host cell which replicates its chromosome independently of oriC . Our data show that a moderate increase in datA copy number is accompanied by increased DnaA protein synthesis that allows oriC initiation to occur normally, as measured by minichromosome copy number . As datA gene dosage is increased dnaA expression cannot be further derepressed, and the minichromosome copy number is dramatically reduced . Under these conditions the minichromosome was maintained by integration into the chromosome . These findings suggest that the datA locus plays a significant role in regulating oriC initiation, by its capacity to bind DnaA . They also suggest that auto regulation of the dnaA gene is of minor importance in regulation of chromosome initiation.

Biochim Biophys Acta, 2001 Oct 31, 1521(1-3), 59 - 65
The macrophage-induced gene (mig) of Mycobacterium avium encodes a medium-chain acyl-coenzyme A synthetase; Morsczeck C et al.; The macrophage-induced gene (mig) of Mycobacterium avium has been associated with virulence, but the functions of the gene product were still unknown . Here we have characterized the Mig protein by biochemical methods . A plasmid with a histidine-tagged fusion protein was constructed for expression in Escherichia coli . Mig was detected as a 60 kDa protein after expression and purification of the recombinant gene product . The sequence of the fusion gene and of the parent gene in M . avium were reexamined . This confirmed that the mig gene encodes a 550 amino acid protein (58 kDa) instead of a 295 amino acid protein (30 kDa) as predicted before . The 550 amino acid Mig exhibits a high degree of homology to bacterial acyl-CoA synthetases . Two artificial 30 kDa derivatives of Mig were expressed and purified as histidine-tagged fusion proteins in E . coli . These proteins and the 58.6 kDa histidine-tagged Mig protein were analysed for activity with an acyl-CoA synthetase assay . Among the three investigated proteins, only the 58.6 kDa Mig exhibited detectable activity as an acyl-CoA synthetase (EC 6.2.1.3) with saturated medium-chain fatty acids, unsaturated long-chain fatty acid and some aromatic carbon acids as substrates . Enzymatic activity could be inhibited by 2-hydroxydodecanoic acid, a typical inhibitor of medium-chain acyl-CoA synthetases . We postulate a novel medium-chain acyl-CoA synthetase motif . We have investigated the biochemical properties of Mig and suggest that this enzyme is involved in the metabolism of fatty acid during mycobacterial survival in macrophages.

BMC Microbiol . 2001;1(1):25 . Epub 2001 Oct 09.
Lactoferrin and free secretory component of human milk inhibit the adhesion of enteropathogenic Escherichia coli to HeLa cells; de Araujo AN et al.; BACKGROUND: Diarrhoea caused by Escherichia coli is an important cause of infant morbidity and mortality in developing countries . Enteropathogenic Escherichia coli (EPEC) is considered one of the major causes of diarrhoea in children living in developing countries . The ability of diarrhoeagenic strains of E . coli to adhere to and colonize the intestine is the first step towards developing the disease . EPEC strains adhere to enterocytes and HeLa cells in a characteristic pattern known as localized adherence.Many epidemiological studies of diarrhoea have shown that breast-feeding protects infants from intestinal infections . Both immunoglobulin and non-immunoglobulin elements of human milk are thought to contribute to the protection from diarrhoeal agents . RESULTS: The effects of human milk and its protein components on the localized adherence of EPEC were investigated . Non-immunoglobulin components of human milk responsible for the inhibition of EPEC adhesion to HeLa cells were isolated by chromatographic fractionation of human whey proteins . Besides secretory immunoglobulin A, which has been previously reported to affect the adhesion of EPEC, free secretory component (fSC) and lactoferrin (Lf) were isolated . Even in concentrations lower than those usually found in whole milk, fSC and Lf were able to inhibit the adhesion of EPEC . alpha-lactalbumin was also isolated, but showed no activity on EPEC adhesion . CONCLUSIONS: This study demonstrated that the immunoglobulin fraction, the free secretory component and lactoferrin of human milk inhibit EPEC adhesion to HeLa cells . These results indicate that fSC and Lf may be important non-specific defence factors against EPEC infections.

J Virol, 2001 Dec, 75(23), 11373 - 83
Autonomous role of 3'-terminal CCCA in directing transcription of RNAs by Qbeta replicase; Tretheway DM et al.; We have studied transcription in vitro by Qbeta replicase to deduce the minimal features needed for efficient end-to-end copying of an RNA template . Our studies have used templates ca . 30 nucleotides long that are expected to be free of secondary structure, permitting unambiguous analysis of the role of template sequence in directing transcription . A 3'-terminal CCCA (3'-CCCA) directs transcriptional initiation to opposite the underlined C; the amount of transcription is comparable between RNAs possessing upstream (CCA)(n) tracts, A-rich sequences, or a highly folded domain and is also comparable in single-round transcription assays to transcription of two amplifiable RNAs . Predominant initiation occurs within the 3'-CCCA initiation box when a wide variety of sequences is present immediately upstream, but CCA or a closely similar sequence in that position results in significant internal initiation . Removal of the 3'-A from the 3'-CCCA results in 5- to 10-fold-lower transcription, emphasizing the importance of the nontemplated addition of 3'-A by Qbeta replicase during termination . In considering whether 3'-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that tRNA(His) is the only stable Escherichia coli RNA with 3'-CCCA . In vitro-generated transcripts corresponding to tRNA(His) served as poor templates for Qbeta replicase; this was shown to be due to the inaccessibility of the partially base-paired CCCA . These studies demonstrate that 3'-CCCA plays a major role in the control of transcription by Qbeta replicase and that the abundant RNAs present in the host cell should not be efficient templates.

EMBO J, 2001 Nov 1, 20(21), 6140 - 9
Clue to damage recognition by UvrB: residues in the beta-hairpin structure prevent binding to non-damaged DNA; Moolenaar GF et al.; UvrB, the ultimate damage-recognizing component of bacterial nucleotide excision repair, contains a flexible beta-hairpin rich in hydrophobic residues . We describe the properties of UvrB mutants in which these residues have been mutated . The results show that Y101 and F108 in the tip of the hairpin are important for the strand-separating activity of UvrB, supporting the model that the beta-hairpin inserts between the two DNA strands during the search for DNA damage . Residues Y95 and Y96 at the base of the hairpin have a direct role in damage recognition and are positioned close to the damage in the UvrB-DNA complex . Strikingly, substituting Y92 and Y93 results in a protein that is lethal to the cell . The mutant protein forms pre- incision complexes on non-damaged DNA, indicating that Y92 and Y93 function in damage recognition by preventing UvrB binding to non-damaged sites . We propose a model for damage recognition by UvrB in which, stabilized by the four tyrosines at the base of the hairpin, the damaged nucleotide is flipped out of the DNA helix.

EMBO J, 2001 Nov 1, 20(21), 5908 - 18
EcfE, a new essential inner membrane protease: its role in the regulation of heat shock response in Escherichia coli; Dartigalongue C et al.; We have identified a new protease in Escherichia coli, which is required for its viability under normal growth conditions . This protease is anchored in the inner membrane and the gene encoding it has been named ecfE, since it is transcribed by Esigma(E) polymerase . Multicopy expression of the ecfE gene was found to turn down expression of both Esigma(E)- and Esigma(32)-transcribed promoters . Purified EcfE degrades both heat shock sigma factors RpoE and RpoH in vitro . EcfE has a zinc binding domain at the N-terminus, a PDZ-like domain in the middle and a highly conserved tripeptide, LDG, at the C-terminus . These features are characteristic of members of a new class of proteases whose activity occurs close to the inner membrane or within the inner membrane . Temperature-sensitive mutants of this gene were isolated mapping to the catalytic site and other domains that exhibited constitutively elevated levels of both heat shock regulons.

EMBO J, 2001 Nov 1, 20(21), 5853 - 62
Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein; Ostergaard H et al.; To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein . Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence . Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements . The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment . By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence . The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines . In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

J Biotechnol, 2002 Jan 18, 92(3), 251 - 8
Preparation and characterization of PEGylated adducts of recombinant human tumor necrosis factor-alpha from Escherichia coli; Li W et al.; This study was conducted to get an insight into monomethoxypolyethylene glycol (MPEG) modified recombinant human TNF-alpha (rhTNF-alpha) derived from gene cloning and expression . The purification and modification processes were integrated together to make the scale-up production of PEG-modified rhTNF-alpha practical . Capillary electrophoresis was demonstrated to be a highly efficient tool in the biochemical characterization of the PEGylated products compared to slab electrophoresis . The cytotoxicity of MPEG-modified rhTNF-alpha was studied in vitro towards L929 cell line and found to decrease gradually along with the increase in the reaction ratio between the activated MPEG and the native rhTNF-alpha . The MPEG-modified rhTNF-alpha was more resistant to proteinase degradation in vitro than the native . When MPEG chains were released partially from the MPEG-modified rhTNF-alpha by alkaline pretreatment, the cytotoxicity of the MPEG-modified rhTNF-alpha was enhanced, which was in contrast to the native . These results would be helpful to explain the disagreement between the high bioavailability of MPEG-modified TNF-alpha in vivo and its decreased cytotoxicity in vitro.

J Biotechnol, 2002 Jan 18, 92(3), 237 - 49
Effect of glycine on the cell yield and growth rate of Escherichia coli: evidence for cell-density-dependent glycine degradation as determined by (13)C NMR spectroscopy; Han L et al.; Addition of selected amino acids could be a means to improve production of recombinant proteins in industrial processes . We found that glycine increased the maximum specific growth rate of Escherichia coli from 0.67 to 0.78 h(-1), and the cell yield from 0.57 to 0.98 g dry weight per g substrate, when supplemented to batch cultures in a glucose-mineral medium . Maximum effect occurred at pH 6.8, at a glycine concentration of 6-12 mmol l(-1), and at cell densities below 1.15 g dry weight l(-1) (0D(610).3) . When glycine was added to a culture at a cell density of 1.15 g l(-1) or above, no growth promoting effect of glycine was seen . The 'glycine effect' was not due to CO(2) produced by the glycine cleavage system (GCV), and the lack of effect at higher cell densities was not masked by acetate accumulation, but coincided with increased acetate production . The metabolism of glycine was further investigated in cultures supplied with {2-(13)C} labelled glycine, and the redistribution of label in the {1-(13)C}, {2-(13)C}, and {1,2-(13)C} isotopomeres of excreted acetate was analysed by 13C NMR . The NMR data revealed that very little degradation of glycine occurred at cell densities below 1.15 g l(-1) . Simultaneously the biosynthesis of serine and glycine was repressed as judged by the absence of {2-(13)C} acetate, implying that added glycine was used as a source of glycine, serine, one-carbon units, and threonine . At cell densities above 1.15 g l(-1), 53% of the consumed glycine carbon was excreted as acetate . Degradation of glycine was associated with an increased uptake rate, cleavage by GCV, and degradation of both glycine-derived serine, and glucose-derived serine to pyruvate . This switch in metabolism appears to be regulated by quorum sensing.

J Biotechnol, 2002 Jan 18, 92(3), 205 - 15
A comparison of the process issues in expressing the same recombinant enzyme periplasmically in Escherichia coli and extracellularly in Streptomyces lividans; Pierce JJ et al.; The choice of a host for the production of a biological molecule will have a significant effect on isolation and purification procedures employed . This paper makes a comparison between the production of a single enzyme, a recombinant alpha-amylase, in Escherichia coli and Streptomyces lividans, on a small scale . It defines the differences in the cultivation and in the isolation stages and also describes the impact of the expression system on later downstream processing steps . At the cultivation stage, the specific productivity of the E . coli in units per gram per hour is four times that of the S . lividans while the total biomass yields are of the same order . The initial volume for downstream processing of S . lividans is six-fold larger and the total protein released into the extracellular medium is three times greater than E . coli, however, the recoverable yield from the E . coli is a fifth of that obtained from the S . lividans and requires three additional stages prior to chromatography . Even with these stages the final specific activity is 64% of the S . lividans . The results indicate the need to consider the whole process when making such comparisons.

Toxicon, 2002 Feb, 40(2), 185 - 91
Trypan blue uptake by chinese hamster ovary cultured epithelial cells: a cellular model to study Escherichia coli STb enterotoxin; Beausoleil HE et al.; The thermostable enterotoxin b (STb) produced by enterotoxigenic Escherichia coli strains is responsible for diarrheal diseases mainly in weaning piglets . For now, the only available assay for biological activity of STb toxin was in the animal host (i.e . piglet) or in an animal model (i.e . rat, mouse) . In this study, we developed a cellular model for the study of the biological activity of STb enterotoxin . Using a trypan blue vital stain method, we showed that STb-treated cells of three out of the five cell lines tested absorbed more vital stain than their controls . Of all the cell lines tested, the chinese hamster's ovary derivated cells (CHO) were the most sensitive, absorbing 50% more trypan blue than their control . Maximal stain uptake was observed after 2h . We then evaluated the trypan blue uptake for 16 STb mutants, produced in a previous work, on the CHO cell lines in order to compare it with the in vivo rat loop assay data . Interestingly, we observed a good correlation between the two bioassays . In fact, the biological activity observed in the rat could be correlated with the trypan blue uptake by the CHO cells (R(2)=0.78) for STb toxin and the 16 mutants . Using the variance analysis statistical test, we determined that the correlation between the two bioassays is significant (F(c)> or =F(0.005)) . These results suggest that the trypan blue uptake bioassay could represent a new method to evaluate the biological activity and facilitate the elucidation of the mechanism of action of E . coli STb enterotoxin.

Atherosclerosis, 2001 Nov, 159(1), 85 - 91
Recombinant proapoA-I(Lys107del) shows impaired lipid binding associated with reduced binding to plasma high density lipoprotein; Huang W et al.; In the present study apoA-I (Lys 107del), a naturally occurring human apoA-I variant with a deletion of Lys 107, was expressed in E . coli to examine the effect of this mutation on lipid binding, cholesterol efflux and lecithin:cholesterol acyltranferase (LCAT) activation . Dimyristoyl phosphatidylcholine (DMPC) binding studies revealed slow interaction of proapoA-I(Lys107del) with DMPC relative to normal proapoA-I . After preincubation with human plasma lipoprotein (d<1.225 g/ml) for 1 h at 37 degrees C, 125I-labeled normal proapoA-I chromatographed as a single peak with the high density lipoprotein (HDL) fraction, whereas 125I-labeled proapoA-I(Lys107del) chromatographed with both HDL and free proapoA-I (26% of the radioactivity) . Circular dichroism measurements showed that the alpha-helical content of lipid-bound proapoA-I (Lys107del) was reduced to 64 versus 73% of normal proapoA-I . Non-denaturing gradient gel electrophoresis of reconstituted HDL assembled with either proapoA-I(Lys107del) or normal proapoA-I showed that the mutation led to the formation of a second population of smaller rHDL particles . DMPC/proapoA-I(Lys107del) and normal DMPC/proapoA-I complexes exhibited a similar capacity to promote cholesterol efflux from fibroblasts . ProapoA-I (Lys107del) also activated LCAT similar to wild type proapoA-I and human plasma apoA-I . We conclude that deletion of Lys 107 substantially alters the lipid binding properties of the protein, which correlated with reduced binding to plasma HDL in vitro, but did not affect the capacity of the mutant/lipid complex to promote cholesterol efflux or activate LCAT.

Mol Urol, 2001 Spring, 5(1), 37 - 43
Nitric oxide synthase gene therapy for erectile dysfunction: comparison of plasmid, adenovirus, and adenovirus-transduced myoblast vectors; Tirney S et al.; BACKGROUND AND PURPOSE: Nitric oxide (NO) has been recognized as an important transmitter for genitourinary tract function . This transmitter mediates smooth muscle relaxation and is essential for erection . The objective of our research was to determine whether overexpression of nitric oxide synthase (NOS) in the corpus cavernosum of the penis would correct erectile dysfunction . MATERIALS AND METHODS: We introduced the inducible form of the enzyme NOS (iNOS) into the corpus cavernosum of adult (250-300 g) male Sprague-Dawley rats by injecting a solution of plasmid, adenovirus, or adenovirus-transduced myoblast cells (adeno-myoblast) (N = 3-5 each group) . We also injected plasmid, adenovirus, and adeno-myoblast encoding the expression of the beta-gatactosidase reporter gene . RESULTS: We noted expression of beta-galactosidase throughout the corpora cavernosum after injection of each of the three solutions . Staining was greatest for adeno-myoblast followed by adenovirus and then plasmid . The basal intracavernous pressure (ICP) of iNOS-treated animals (adenovirus and adenovirus-transduced myoblast) increased to 55 +/- 23 cm H(2)O v 5 +/- 6 H(2)O in naive animals (P = 0.001) . Stimulation of the cavernous nerve (15 Hz, 1.5 msec, 10-40 V, 1 min) resulted in a twofold increase in ICP (adenovirus and adeno-myoblast) from the basal level of the iNOS-treated animals . Direct in situ measurement of NO demonstrated release of 1 to 1.3 microM NO in the adeno-myoblast-treated penis . CONCLUSION: Myoblast-mediated gene therapy was more successful in delivering iNOS into the corpus cavernosum than were the direct adenovirus or plasmid transfection methods . Gene therapy of NOS may open new avenues of treatment for erectile dysfunction . Control of NOS expression would be necessary to prevent priapism.

Biochem Biophys Res Commun, 2001 Nov 9, 288(4), 1052 - 8
The Escherichia coli SOS gene sbmC is regulated by H-NS and RpoS during the SOS induction and stationary growth phase; Oh TJ et al.; sbmC, an Escherichia coli gene, belongs to the SOS regulon, whose product is involved in cell susceptibility to microcin B17 and its expression is induced at the onset of the stationary growth phase . In the present work, we have investigated the regulation of sbmC expression during SOS induction and the stationary growth phase using a single-copy sbmC'-'lacZ fusion . The SOS induction of sbmC is profoundly diminished in the hns mutant and less diminished in the rpoS mutant . The strain with hns, rpoS double mutation, showed a similar level of sbmC induction to that of a strain with hns single mutation . Mutation in rpoS or hns causes the repression of the sbmC gene during the stationary growth phase . The sbmC expression in the rpoS mutant strain was approximately twofold lower than that in the hns mutant and the rpoS hns double mutant showed a similar level of sbmC expression to mutants deficient in rpoS alone . Interestingly, the sbmC'-'lacZ expression in the exponential growth phase was not derepressed in the hns mutant background . Transformation of hns and rpoS mutants with plasmids carrying histone-like nucleoid protein (H-NS) and RpoS effectively restored the sbmC expression to the wild-type level, respectively . The gel mobility shift assay showed that purified H-NS protein directly bound with a high affinity to a DNA fragment carrying the sbmC promoter region . These findings demonstrate that H-NS regulates the sbmC expression via H-NS's direct binding to the promoter region . In conclusion, our data suggest that H-NS and RpoS regulate a stationary phase-inducible sbmC gene in E . coli .

Biochem Biophys Res Commun, 2001 Nov 9, 288(4), 975 - 80
Inhibition of vitronectin-mediated haptotaxis and haptoinvasion of MG-63 cells by domain 5 (D5(H)) of human high-molecular-weight kininogen and identification of a minimal amino acid sequence; Kamiyama F et al.; We found that human kinin-free high-molecular-weight kininogen (kf-HK) significantly inhibited vitronectin-mediated migration (haptotaxis) and invasive potentiation (haptoinvasion) of osteosarcoma (MG-63) cells but that HK, LK, the common heavy chain of HK and LK, and the light chain (D6(H)) of HK had no inhibitory effect . Recombinant GST-D5(H) (histidine-rich region of HK) obtained from Escherichia coli . (BL21) also inhibited both haptotaxis and haptoinvasion to about 30% of the control level in a dose-dependent manner . These findings suggest that a specific region of D5(H) is responsible for the inhibition of cell haptotaxis and haptoinvasion . Among the seven synthetic peptides covering D5(H), peptide H(479)KHGHGHGKHKNKGK(493) (P-5) inhibited both haptotaxis and haptoinvasion in a dose-dependent manner, suggesting that P-5 could possibly be utilized to prevent primary and secondary metastases of tumor cells .

Biochem Biophys Res Commun, 2001 Nov 9, 288(4), 809 - 18
The specificity of the myelin basic protein gene promoter studied in transgenic mice; Asipu A et al.; The myelin basic proteins (MBPs) are a family of polypeptides that are predominantly expressed in the nervous system where they play a major role in myelination . We have generated four lines of transgenic mice carrying a transgene in which 1.34 kb of the 5'-flanking sequence of the mouse MBP gene was fused upstream of the coding region of the Escherichia coli lac Z gene in order to investigate developmental and tissue-specific expression of the MBP gene . Expression of both the lacZ transgene and the endogenous MBP gene followed a common developmental pattern in mouse brain . Transgene expression was detected in primary oligodendrocytes, but not in type 2 astrocytes . In addition, the lacZ gene product was expressed in epithelial cells of certain nonneural tissues, namely kidney, epididymis, ureter, and seminal vesicles . The ectopic expression of the transgene was associated with the development of DNase I hypersensitive sites at the site of insertion which was found to be within the intron 1 region of the endogenous MBP gene . The results reported here strongly suggest that the 1.34-kb 5'-flanking region of the MBP gene contains cis-regulatory elements that confer developmental regulation of the MBP gene, although this region appears to lack elements that restrict its expression to the nervous system .

Biol Chem, 2001 Sep, 382(9), 1335 - 42
Duocalins: engineered ligand-binding proteins with dual specificity derived from the lipocalin fold; Schlehuber S et al.; Anticalins comprise a novel class of receptor proteins with predetermined ligand specificities which were engineered using the lipocalin fold . Attractive features of these artificial ligand-binding proteins include their small size and monomeric nature, being composed of a single polypeptide chain . Here we report the construction of a functional fusion protein from two independent anticalins, a so-called duocalin . The gene for the fusion protein was assembled from nucleotide sequences encoding an anticalin with fluorescein specificity on the one hand and an anticalin with digoxigenin specificity on the other . Both engineered lipocalins were previously selected from a random library prepared on the basis of the bilin-binding protein, a natural lipocalin abundant in insects . The corresponding fusion protein was expressed in a secretable form in E . coli cells and isolated from the periplasmic fraction using the Strep-tag method . The major fraction of the purified protein appeared to possess the proper pattern of altogether four disulphide bonds . The ligand-binding behaviour of the fusion protein was investigated both by solid phase ELISA and in fluorescence titration experiments . Our results demonstrate that the novel fusion protein has retained both ligand specificities . Up to now, dimerized ligand-binding proteins were mostly derived from recombinant antibody fragments . Compared with those constructs the duocalins, either with bispecific or with bivalent target recognition properties, should provide useful reagents for various purposes in biotechnology.

J Cardiothorac Vasc Anesth, 2001 Oct, 15(5), 593 - 602
A double-blind study to evaluate the safety of recombinant human hemoglobin in surgical patients during general anesthesia; Hayes JK et al.; OBJECTIVE: To evaluate recombinant human hemoglobin (rHb1.1) in patients undergoing surgery involving general anesthesia; examine rHb1.1 for toxicity, including renal dysfunction and hypertension; and measure plasma concentrations of rHb1.1 over time . DESIGN: Prospective, double-blinded, randomized, placebo-controlled study . SETTING: University medical center hospital . PARTICIPANTS: Eighteen patients having surgery under general anesthesia . INTERVENTIONS: One of 4 escalating doses of rHb1.1 or normal saline (control) was administered by continuous infusion to patients receiving general anesthesia for elective surgical procedures . Total rHb1.1 doses ranged from 4.7 to 25.6 g . MEASUREMENTS AND MAIN RESULTS: Clinical and laboratory data, including vital signs monitoring, hematology (white blood cell and reticulocyte count, hemoglobin, hematocrit, erythrocyte sedimentation rates, and coagulation values), renal function (serum creatinine and blood urea nitrogen), hepatic function (mean and indirect bilirubin), pancreatic function (serum amylase and lipase), and antibodies (IgG and IgM) to Escherichia coli protein, were collected at specified intervals for 7 days after infusion of rHb1.1 . No serious adverse events occurred . The most frequently observed clinical event occurred during the first 24 hours after infusion and was primarily associated with surgery and anesthetic administration . A slightly higher incidence of hypertension, symptoms suggestive of pyrogenicity, mildly elevated total and indirect bilirubin, and elevated pancreatic enzymes was observed in rHb1.1 treatment groups when compared with control . Hypertension resolved within 7 hours, and laboratory values returned to normal levels by day 7 . CONCLUSION: Although the elevations in pancreatic enzymes seen in some rHb1.1-treated patients remain unexplained, the safety profile of rHb1.1 appears to be acceptable . These results support the continued clinical evaluation and development of rHb1.1 .

J Ind Microbiol Biotechnol, 2001 Oct, 27(4), 246 - 51
Branched-chain fatty acid biosynthesis in Escherichia coli; Smirnova N et al.; Beta-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) catalyzes the first elongation step in straight-chain fatty acid (SCFA) biosynthesis in Escherichia coli . Overproduction of the corresponding KASIII gene, or the Brassica napus KASIII gene has previously been observed to lead to an increase in the amount of shorter-chain fatty acids produced by E . coli . In this study it is shown that overexpression of the KASIII gene, which initiates branched-chain fatty acid (BCFA) in Streptomyces glaucescens, does not lead to a change in the fatty acid profiles of E . coli . E . coli produces trace levels of BCFAs when grown in the presence of isobutyric acid, but the amounts of these are not significantly altered by expression of the S . glaucescens KASIII gene . In contrast, the amounts of BCFAs produced from isobutyryl CoA in vitro by E . coli cell-free extracts can be increased at least four-fold by the presence of the S . glaucescens KASIII . These observations suggest that in vivo production of isopalmitate by E . coli expressing the S . glaucescens KASIII is limited by availability of the appropriate BCFA biosynthetic primers.

Cancer Gene Ther, 2001 Oct, 8(10), 713 - 8
Efficient and cancer-selective gene transfer to hepatocellular carcinoma in a rat using adenovirus vector with iodized oil esters; Shiba H et al.; Gene therapy for cancer requires efficient, selective gene transfer to cancer cells . In gene therapy for hepatocellular carcinoma (HCC), gene transfer is efficient for small tumors, but not for large tumors . The delivery of anticancer agents and of iodized oil esters as embolic agents through tumor-feeding arteries is known as transarterial embolization . We speculate that genes may be efficiently and selectively transferred for HCC using iodized oil esters because these esters may remain together with a genetic vector within HCC selectively . Hence, we have studied the effect of iodized oil esters on adenovirus vector-mediated gene transfer for HCC in vivo . A rat model of HCC induced with diethylnitrosamine and phenobarbital was injected with either AxCALacZ, which expresses the beta-galactosidase of Escherichia coli, or AxCALacZ and iodized oil esters into the hepatic artery . Histological comparisons revealed that the beta-galactosidase expression in the rats with HCC injected with AxCALacZ and iodized oil esters was greater (P<.0001) in small tumors (P=.0046) and large tumors (P=.0023), and more selective (P=.0229) than in only AxCALacZ-injected rats . These results suggest that iodized oil esters are injected into hepatic artery together with adenovirus vector, and that genes may be efficiently and cancer-selectively transferred to HCC.

Cancer Gene Ther, 2001 Oct, 8(10), 695 - 704
Enhanced anti-tumor effects of herpes simplex virus thymidine kinase/ganciclovir system by codelivering monocyte chemoattractant protein-1 in hepatocellular carcinoma; Sakai Y et al.; The therapeutic efficacy of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system in many types of tumors is unsatisfactory due to the insufficient spread of gene transfer and insufficient cell killing . In the current study, we investigated whether adenovirally delivered monocyte chemoattractant protein (MCP)-1 potentiates the antitumor effects of the HSV-tk/GCV system in hepatocellular carcinoma (HCC) cells . Subcutaneous tumor foci of the human HCC cell line, HuH7, established in athymic mice were directly transduced with a recombinant adenovirus (rAd) harboring an HSV-tk gene driven by a human alpha-fetoprotein promoter, followed by GCV administration . Subsequently, another rAd expressing MCP-1 under the universal CAG promoter was injected . The growth of tumors was markedly suppressed by codelivering HSV-tk and MCP-1 genes compared to that by either HSV-tk/GCV or MCP-1 delivery . In the tumor tissues, monocyte/macrophage infiltration was detected immunohistochemically . The antitumor effects of the rAd expressing MCP-1 were markedly reduced by the administration of carrageenan, a compound known to inactivate macrophage . These results indicate that adenovirally delivered MCP-1 enhanced the antitumor effects of the HSV-tk/GCV system synergistically by recruitment/activation of macrophages in tumor tissues, suggesting an effective immunotherapy for HCC and other lineages of tumors when used adjuvantly with a suicide gene.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13025 - 30 Epub 2001 Oct 30.
The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand binding studies reveal the location of the active site; Kelly MJ et al.; Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes . By applying a combination of residue-specific isotope labeling and protein deuteration strategies tailored to yield specific information, we were able to determine the solution structure and study structure-activity relationships of 3,4-dihydroxy-2-butanone-4-phosphate synthase, a 47-kDa enzyme from the Escherichia coli riboflavin biosynthesis pathway and an attractive target for novel antibiotics . Our investigations of the enzyme's ligand binding by NMR and site-directed mutagenesis yields a conclusive picture of the location and identity of residues directly involved in substrate binding and catalysis . Our studies illustrate the power of state-of-the-art NMR techniques for the structural characterization and investigation of ligand binding in protein complexes approaching the 50-kDa range in solution.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13172 - 6 Epub 2001 Oct 30.
Simulating pseudogene evolution in vitro: determining the true number of mutations in a lineage; Vartanian JP et al.; Hypermutagenic PCR has been used to simulate pseudogene evolution of the Escherichia coli R67 dihydrofolate reductase gene . Each time the most divergent clone was used as template for another round of hypermutagenesis . After six rounds, with an average mutation rate of 0.05 per base per round, up to a 46% nucleic acid sequence variation was achieved . For a few clones the protein information content could be annihilated . As the intermediates were cloned and sequenced, it was possible to establish the real lineage and compute the true number of mutations . Not surprisingly the true number of forward and back mutations as well as variable sites exceeded those based on comparing any single intermediate to the initial sequence . However, the true number of forward and backward mutations, as well as the number of variable sites, increased linearly with sequence divergence from the original sequence, suggesting an empirical means to correct for branch lengths.

J Biol Chem, 2002 Jan 25, 277(4), 2763 - 72 Epub 2001 Oct 30.
Molecular characterization of the starfish inositol 1,4,5-trisphosphate receptor and its role during oocyte maturation and fertilization; Iwasaki H et al.; The release of calcium ions (Ca(2+)) from their intracellular stores is essential for the fertilization of oocytes of various species . The calcium pools can be induced to release Ca(2+) via two main types of calcium channel receptor: the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor . Starfish oocytes have often been used to study intracellular calcium mobilization during oocyte maturation and fertilization, but how the intracellular calcium channels contribute to intracellular calcium mobilization has never been understood fully, because these molecules have not been identified and no specific inhibitors of these channels have ever been found . In this study, we utilized a novel IP(3)R antagonist, the "IP(3) sponge," to investigate the role of IP(3) during fertilization of the starfish oocyte . The IP(3) sponge strongly and specifically competed with endogenous IP(3)R for binding to IP(3) . By injecting IP(3) sponge into starfish oocyte, the increase in intracellular calcium and formation of the fertilization envelope were both dramatically blocked, although oocyte maturation was not blocked . To investigate the role of IP(3)R in the starfish oocyte more precisely, we cloned IP(3)R from the ovary of starfish, and the predicted amino acid sequence indicated that the starfish IP(3)R has 58-68% identity to mammalian IP(3)R types 1, 2, and 3 . We then raised antibodies that recognize starfish IP(3)R, and use of the antibodies to perform immunoblot analysis revealed that the level of expression of IP(3)R remained unchanged throughout oocyte maturation . An immunocytochemical study, however, revealed that the distribution of starfish IP(3)R changes during oocyte maturation.

J Biol Chem, 2002 Jan 4, 277(1), 259 - 66 Epub 2001 Oct 30.
Domain structure of the HSC70 cochaperone, HIP; Velten M et al.; The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches . HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli . NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains . After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively . Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape . Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation . Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity . Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding.

Clin Diagn Lab Immunol, 2001 Nov, 8(6), 1213 - 9
Prevalence of hepatitis E virus antibodies in Canadian swine herds and identification of a novel variant of swine hepatitis E virus; Yoo D et al.; Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation . In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds . A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in Escherichia coli . These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada . From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody . The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%) . By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs . The nucleotide sequence identity between the U.S . swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America . Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein . Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.

Clin Diagn Lab Immunol, 2001 Nov, 8(6), 1126 - 30
Mapping of Escherichia coli H27-specific epitope from H-specific polypeptides; Seah JN et al.; A murine monoclonal antibody (MAb) reactive to H27 flagellin antigen was produced and characterized . Forty-nine partially purified native H-type flagellins were used to evaluate the specificity of the MAb . The fliC gene of H27 is 1,464 bp in length (487 amino acids {aa}; 50.88 kDa) . The central variable region (CVR) of the H27 flagellin gene was defined by comparison with flagellin sequences derived from H8, H34, and H49 . To study the distribution of antigenic epitopes, the CVR covering amino acid residues 70 to 457 (388 aa) was dissected into seven overlapping fragments . Fragments carrying the H-type-specific antigenic determinants were identified by H27-specific antiserum . Polyclonal antibodies raised against different H-type flagellin proteins were used to determine the cross-reactive determinants . Three fragments, spanning amino acid residues 240 to 380, which carried the potential H-specific determinants were used for MAb production . A MAb specific to H27 was produced, and the specific epitope was mapped to amino acid residues 330 to 340 . In this study, we produced MAbs from predetermined H27-specific polypeptides and used whole flagellin in enzyme-linked immunosorbent assays to circumvent the interference of anti-glutathione S-transferase antibodies . These factors when combined could help to improve the identification of the desired MAb.

Diagn Microbiol Infect Dis, 2001 Sep-Oct, 41(1-2), 43 - 9
Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase; Carnevale S et al.; Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1) . In this study we tested its potential as antigen for the serologic diagnosis of F . hepatica infections by enzyme-linked immunosorbent assay (ELISA) . The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases . The sensitivity and specificity of the rproCL1-ELISA were 100% . We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals . Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis.

Biochim Biophys Acta, 2001 Oct 3, 1528(2-3), 101 - 6
Improved heterologous expression of human glutathione transferase A4-4 by random silent mutagenesis of codons in the 5' region; Nilsson LO et al.; Glutathione transferase A4-4 (GST A4-4) is involved in the detoxication of lipid peroxidation products such as alkenals . The human enzyme has been heterologously expressed in Escherichia coli, but for more extensive characterization of the enzyme the expression level had to be elevated . A clone providing up to 8-fold higher yields was created, by screening an expression library with random silent mutations in the 5' region of the cDNA encoding GST A4-4.

J Immunol Methods, 2001 Nov 1, 257(1-2), 175 - 84
Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression; Morino K et al.; We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens . An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system . The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation . In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP) . In addition, a His-tag was inserted between CL and GFP (or RFP) . We describe the utility of this system using Caenorhabditis elegans embryo as a model.

J Immunol Methods, 2001 Nov 1, 257(1-2), 163 - 73
Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries; Christmann A et al.; We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells . A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector . A carboxyterminally truncated intimin, an adhesin from enteropathogenic E . coli, serves as a carrier protein to present foreign peptides on the surface of E . coli K12 cells . Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS) . Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence . A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum . Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies . Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns) . This demonstrates that gene-fragment libraries displayed on E . coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E . coli surface exposed antigenic peptide.

J Immunol Methods, 2001 Nov 1, 257(1-2), 117 - 22
T7 displayed peptides as targets for selecting peptide specific scFvs from M13 scFv display libraries; Castillo J et al.; M13 scFv display libraries are frontline research tools due to the rapidity in which target binding scFv can be obtained . Simple modifications of the standard affinity selection and amplification protocol allow for multiple targets to be panned simultaneously . Target peptide or protein production can become the rate-limiting step in a high throughput approach to antibody selection . To overcome this obstacle and to save both time and money, we have displayed the target peptides on T7 phage particles . In this paper we demonstrate that these particles can be used directly to select peptide specific scFv . We have discovered that the selection is most efficient when a linker separates the display from the T7 coat protein and when a continuous subtraction is employed . We have used antibodies selected in this fashion to characterize target proteins in cell lysates, immuno-precipitations and immuno-histochemistry.

J Biochem (Tokyo), 2001 Nov, 130(5), 627 - 35
Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach; Farh L et al.; In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin . Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster . The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion . The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme . In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis . The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content . From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity . Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding . The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively . The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site . Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.

Surg Today, 2001, 31(9), 785 - 90
Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene increases the sensitivity of esophageal cancer cells to 5-fluorouracil; Nakamura H et al.; Esophageal cancer is associated with the poorest prognosis among the digestive tract cancers, and chemotherapy is the treatment of choice for many patients . In this study, we experimentally introduced an Escherichia coli-derived uracil phosphoribosyltransferase (UPRT) gene to cultured esophageal cancer cell lines to potentiate the antitumor effects of a representative anticancer drug, 5-fluorouacil (5-FU) . UPRT is a pyrimidine salvage enzyme that catalyzes the synthesis of uridine monophosphate from uracil and PRPP . The UPRT gene was transduced into five cultured esophageal cancer cell lines, TE1, TE2, TE3, NUEC1, and T.T, using an adenovirus vector . It was confirmed that the sensitivities of all cultured cell lines to 5-FU were increased in vitro . Subsequently, the T.T line was subcutaneously inoculated into nude mice to induce tumors, after which 5-FU was administered intraperitoneally . When a UPRT gene-recombinant adenovirus vector was directly injected into the tumors, tumor proliferation was markedly inhibited compared with that in the group treated with 5-FU alone, suggesting potentiation of 5-FU sensitivity by UPRT gene transduction in vivo . Therefore, we potentiated the effects of commercially available anticancer drugs by gene transduction . Our method may prove useful as a new form of cancer gene therapy in the future.

Environ Sci Technol, 2001 Oct 15, 35(20), 4139 - 44
Escherichia coli disinfection by electrohydraulic discharges; Ching WK et al.; We study the survival of single-strain Escherichia coli colonies in aqueous media exposed to 5.5 kV, 90 kA electrohydraulic discharges (EHD) . The probability of survival (Pn) of a 4 x 10(7) cfu mL(-1) E . coli population after n consecutive EHDs follows a logit distribution: In(Pn/ 100 - Pn) = 1.329 - 1.579 ln n with r2 = 0.993 that corresponds to lethal doses of LD50 = 2.2 and LD90 = 10.5 EHDs . Considering that the reactor is thoroughly mixed during each discharge and that LD50 = 0.9 values are nearly independent of E . coli concentrations in the range of 2 x 10(3) < or = E coli/cfu mL(-1) < or = 3 x 10(6), we ascribe the nonexponential Pn decay of single-strain E . coli colonies to a shielding phenomenon where inactive cells protect the successively smaller numbers of viable cells in the EHD . The qualitatively similar concentration dependence observed for survival under 254 nm of radiation, in contrast with the lower resistance of denser colonies to 20 kHz power ultrasound and the delayed onset of extracellular beta-D-galactosidase activity in bacterial populations already decimated by EHDs, support the view that UV radiation is the dominant disinfection agent generated by electrohydraulic discharges.

Biotechnol Annu Rev, 2001, 7, 1 - 30
Purification of plasmids for gene therapy and DNA vaccination; Prazeres DM et al.; This chapter covers the different aspects of the production and purification of plasmids for gene therapy and DNA vaccination . Process issues are extensively covered and complemented with information related to plasmid DNA structure, vector construction, product specifications and quality assurance and control.

J Pediatr Surg, 2001 Nov, 36(11), 1623 - 8
Ascending cholangitis provokes IL-8 and MCP-1 expression and promotes inflammatory cell infiltration in the cholestatic rat liver; Hsieh CS et al.; BACKGROUND/PURPOSE: Postoperative cholangitis is one of the most common complications after bile duct reconstruction . The pathogenesis and early consequences of ascending cholangitis still are unidentified . METHODS: Male Sprague-Dawley rats were divided into 5 treatment groups: control (n = 4), blood sampling and liver biopsy only; group I, {BDL/Eschericha coli; n = 6}, ligation of common bile duct (BDL) for a week, followed by Roux-en-Y choledochojejunostomy (RYCJ) and injection of E coli (ATCC 25922) into Roux limb after 24 hours; group II, {BDL/NS; n = 5}, same procedures as in group I, with injection of normal saline (NS) into Roux limb; group III, {SBDL/E coli; n = 6}, primary RYCJ was constructed 1 week after sham ligation of common bile duct (SBDL) followed by the same treatment as group I; Group IV, {SBDL/N.S; n = 6}, same procedures as in group III, but injecting NS into Roux limb . All animals were killed after 24 hours of treatment . Blood was sampled for culture and serum cytokine levels . The liver was harvested for quantitative bacterial culture, as well as for MCP-1, interleukin (IL)-8 (CINC in the rat) and transforming growth factor beta1 mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR) and for immunohistochemistry . The choledochojejunostomy was resected for culture . Serum cytokine levels were detected by ELISA kits . RESULTS: A significant increase of E coli ATCC 25922, occurred in the livers of group I rats, compared with group IV (P =.037) . MCP-1 expression increased in all groups, compared with control (P =.000) . The IL-8 mRNA expression was significantly higher in group I than in control (P =.021) . The expression of TGF-beta1 mRNA was similar among the groups (P =.361), consistent with the immunohistochemistry results . The serum MCP-1 and IL-8 levels were higher in the 4 groups than in the control (P =.000) and were significantly higher in group I than in group IV (P =.001) . CONCLUSIONS: This study found that a significant colonization of E coli of the same strain was present in the cholestatic rat liver injected into the Roux limb, which was associated with a higher expression of liver MCP-1 and IL-8 mRNA, a significant increase of serum MCP-1 and IL-8, and a more evident inflammatory cell infiltration into the porta hepatis .

Curr Microbiol, 2001 Dec, 43(6), 452 - 6
PCR genome walking identifies a genetic locus comprising two heat shock genes (hslV and hslU) from Leptospira borgpetersenii serovar hardjobovis; Lin M et al.; PCR walking on genomic DNA of Leptospira borgpetersenii serovar hardjobovis has identified a genetic locus comprising two heat shock genes (hslV and hslU) . This is the first molecular study on hslV and hslU in a Leptospira species . The hslV gene has an open reading frame (ORF) of 543 bp coding for a 180-amino acid polypeptide (19.476 kD) with 49-62% identity to other bacterial HslV homologs . Forty-three nucleotides downstream from the hslV stop codon was found a second heat shock gene hslU with an ORF of 1401 bp encoding a 53.139-kD polypeptide of 467 amino acids with 49-56% identity to the HslU homologs from other bacteria . An Escherichia coli-like ribosome binding site (RBS) and a final sigma32-like heat shock promoter sequence were present upstream of both heat shock genes . Identification of the hslV and hslU genes in a Leptospira species and the high degree of similarity of the encoded proteins to the E . coli homologs suggest that the encoded HslV and HslU proteins function as forming an ATP-dependent protease complex as in E . coli.

Arch Microbiol, 2001 Oct, 176(4), 255 - 63
Cloning and characterization of a second acid phosphatase from Sinorhizobium meliloti strain 104A14; Deng S et al.; Sinorhizobium meliloti has two nonspecific periplasmic acid phosphatases . The NapD enzyme has been previously described, and a second acid phosphatase, NapE, is described in this report . NapE was partially purified from an S . meliloti napD mutant and characterized with respect to molecular mass and substrate range . As predicted from SDS-PAGE analysis, the subunit molecular mass of NapE is approximately 35.8 kDa and gel filtration experiments estimated the native molecular mass to be approximately 70 kDa, indicating that the active enzyme is a homodimer . NapE demonstrated significant activity with p-nitrophenyl phosphate, phenyl phosphate, and alpha-naphthyl-phosphate . The pH optimum was between 4.5 and 5.0 . The gene encoding NapE was also sequenced and the inferred amino acid sequence from the predicted ORF was found to be 60% identical and 75% similar to that encoded by napD . An S . meliloti napE mutant was constructed and assessed for symbiotic competence . This mutant did not differ from the wild-type parent strain in nodulation and symbiotic efficiency.

Nat Struct Biol, 2001 Nov, 8(11), 984 - 9
Crystal structure of a DinB family error-prone DNA polymerase from Sulfolobus solfataricus; Silvian LF et al.; A new group of error-prone DNA polymerases overcomes the blockage posed to normal DNA replication by damaged template bases, suggesting an active site with a loose, flexible pocket that accommodates aberrant DNA structures . We have determined a 2.8 A resolution crystal structure of the Sulfolobus solfataricus Dbh protein, a DNA translesion polymerase closely related to Escherichia coli DNA polymerase IV and human polymerase kappa . A high error rate is observed for the Dbh polymerase in a range of 10(-2)-10(-3) for all 12 base substitution mispairs . The crystal structure of Dbh reveals an overall architecture resembling other DNA polymerases but has unique features that are likely to contribute to error-prone synthesis, including -1 frameshifting mutations.

J Immunol Methods, 2001 Dec 1, 258(1-2), 169 - 81
Single-chain antibody against the common epitope of mutant p53: isolation and intracytosolic expression in mammalian cells; Govorko D et al.; The peptide epitope FRHSVV is cryptic in wild-type p53 and is exposed in many types of mutant p53 molecules isolated from various tumors . Mutant p53 marked by this epitope abrogates a tumor-suppressor function of wild-type p53 and possibly contributes to the transforming potential of other oncogenic processes . We report here the construction of a single-chain scFv antibody gene library derived from the mRNA of a mouse immunized with the epitope peptide FRHSVV which mimics the common epitope in p53 mutant protein molecules . The scFv was presented by phage display . The selected antibody gene, named ME1, was found to bind to the mutant p53 protein but not to the wild-type p53 protein . Preliminary studies show that the ME1 gene is expressed in the cytosol of mammalian cells . These findings suggest that the ME1 single-chain antibody may be useful as a tool for clarifying the role of mutant p53 in tumor transformation, especially in cells heterozygous in p53, and possibly for gene therapy of tumors.

J Immunol Methods, 2001 Dec 1, 258(1-2), 97 - 109
Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function; Henson SE et al.; CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement . We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21 . A sequence coding for a biotinylation signal peptide was added to the 3' end of the human C3dg cDNA . The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme . Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly . Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor . Two assays were used to assess the functional capacity of the recombinant C3dg . First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells . Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody . These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.

FEBS Lett, 2001 Oct 26, 507(2), 225 - 30
Dynamic flexibility in the Escherichia coli genome; Tsai L et al.; Empirical rules based on tetranucleotide parameters were presented to predict the structural parameters twist (Omega), roll (rho), tilt (tau) and slide (D(y)) . A statistical mechanical model was used to analyze the flexibility of the Escherichia coli genome . The replication terminus region displayed a low level of flexibility . A strong correlation can be seen between G+C content and flexibility . Average flexibilities in the coding regions were found to be significantly larger than those in non-coding regions . The flexible characteristics in the 5'-neighborhood of the coding regions and in three class sigma promoter sequences in the E . coli genome were also analyzed.

FEBS Lett, 2001 Oct 26, 507(2), 220 - 4
Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli; Drew DE et al.; Escherichia coli is one of the most widely used vehicles to overexpress membrane proteins (MPs) . Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies . Overexpression of MPs in the cytoplasmic membrane is strongly favoured to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies . Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion versus inclusion body formation of overexpressed MPs in E . coli . Furthermore, we show that an overexpressed MP can be recovered from a MP-GFP fusion using a site specific protease . This makes GFP an excellent tool for large-scale MP target selection in structural genomics projects.

FEBS Lett, 2001 Oct 26, 507(2), 128 - 32
Nucleotide requirements for CDX2 binding to the cis promoter element mediating intestine-specific expression of guanylyl cyclase C; Di Guglielmo MD et al.; Guanylyl cyclase C (GC-C), specifically expressed by intestinal epithelial cells, is the receptor for the Escherichia coli heat-stable enterotoxin that causes diarrhea . Tissue-specific expression of GC-C is mediated by the intestinal transcriptional regulator CDX2 . This trans-activating protein regulates intestine-specific expression by binding to a critical sequence in the proximal promoter of GC-C . The precise nucleotide elements mediating CDX2 binding to promoter elements remain undefined . Several nuclear proteins form complexes with a DNA probe containing the promoter element of GC-C mediating CDX2 binding . The present study examined the nucleotide requirements in the consensus binding site and flanking regions in the cis element that mediates specific CDX2 binding to the promoter of GC-C . These studies identified seven core base pairs in the critical promoter element mediating tissue-specific expression of GC-C that are required for CDX2 binding . In addition, base pairs flanking this core sequence contribute to and are required for CDX2 recognition . These studies describe the precise nucleotide sequence within the GC-C promoter that comprises the CDX2 binding site required for intestine-specific expression.

Mol Cell, 2001 Oct, 8(4), 795 - 806
BCR/ABL regulates mammalian RecA homologs, resulting in drug resistance; Slupianek A et al.; RAD51 is one of six mitotic human homologs of the E . coli RecA protein (RAD51-Paralogs) that play a central role in homologous recombination and repair of DNA double-strand breaks (DSBs) . Here we demonstrate that RAD51 is important for resistance to cisplatin and mitomycin C in cells expressing the BCR/ABL oncogenic tyrosine kinase . BCR/ABL significantly enhances the expression of RAD51 and several RAD51-Paralogs . RAD51 overexpression is mediated by a STAT5-dependent transcription as well as by inhibition of caspase-3-dependent cleavage . Phosphorylation of the RAD51 Tyr-315 residue by BCR/ABL appears essential for enhanced DSB repair and drug resistance . Induction of the mammalian RecA homologs establishes a unique mechanism for DNA damage resistance in mammalian cells transformed by an oncogenic tyrosine kinase.

Mol Cell, 2001 Oct, 8(4), 734 - 6
A step backward in advancing DNA replication: rescue of stalled replication forks by RecG; Dillingham MS et al.; In the October 5 issue of Cell, Singleton et al . report the crystal structure of RecG protein bound to an analog of a stalled DNA replication fork . This structure shows how RecG can recognize branched DNA structures and suggests a mechanism for fork reversal, an early event in recombination-dependent reinitiation of DNA replication.

Mol Cell, 2001 Oct, 8(4), 730 - 2
The ins and outs of GroEL-mediated protein folding; Weissman JS; Two papers recently published in Cell investigate the role of protein encapsulation by GroEL in assisting folding . The first shows how encapsulation can "smooth" the folding landscape, accelerating folding of some proteins . The second defines a confinement-independent pathway, which allows GroEL to assist folding of substrates too large to be encapsulated.

Int J Radiat Biol, 2001 Nov, 77(11), 1095 - 108
Reaction of guanyl radicals in plasmid DNA with biological reductants: chemical repair of DNA damage produced by the direct effect of ionizing radiation; Milligan JR et al.; PURPOSE: It has been previously argued that the use of the one-electron oxidants (SCN)2(*-) and Br2(*-) with plasmid DNA leads to the formation of DNA guanyl radicals . These guanyl radical species are intermediates in the DNA damage produced by processes such as photo-ionization and ionizing irradiation . The present paper evaluates the use of thallium(II) ions (Tl(II)OH(+)) as the one-electron oxidant, and also determines rate constants for the reduction (repair) of guanyl radicals in plasmid DNA by a variety of reducing agents including the biologically important compounds ascorbate and glutathione . MATERIALS AND METHODS: Aqueous solutions of plasmid DNA containing 10(-3) mol dm(-3) thiocyanate or thallous ions and a reducing agent (azide, nitrite, ferrocyanide, hexachloroiridate(III), iodide, ascorbate, glutathione, glutathione disulphide, methionine, tyrosine, 5-hydroxyindole-3-acetic acid, 10(-7)-10(-4) mol dm(-3)) were irradiated with 137Cs gamma-rays (662 keV) . After irradiation, the plasmid was incubated with the E . coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG) . Strand break yields after incubation were quantified by means of agarose gel electrophoresis . RESULTS: High yields of FPG-sensitive sites produced by the oxidants (SCN)2(*-) and Tl(II)OH(+) were strongly attenuated by the presence of the reducing agents . CONCLUSIONS: From the results, it is possible to arrive at estimates of the rate constants for the reduction of the DNA guanyl radical by the reducing agents . Values lie in the range 10(4)-10(7) dm(3) mol(-1) s(-1) . Using the values for ascorbate and glutathione, it is possible to estimate an upper limit on the order of milliseconds for the lifetime of DNA guanyl radicals under cellular conditions . The implication is that there may well be a significant chemical repair of DNA base damage by the direct effect of ionizing radiation.

Eur J Biochem, 2001 Nov, 268(21), 5639 - 46
Role of a highly conserved YPITP motif in 2-oxoacid:ferredoxin oxidoreductase: heterologous expression of the gene from Sulfolobus sp.strain 7, and characterization of the recombinant and variant enzymes; Fukuda E et al.; 2-Oxoacid:ferredoxin oxidoreductase from Sulfolobus sp . strain 7, an aerobic and thermoacidophilic crenoarchaeon, catalyses the coenzyme A-dependent oxidative decarboxylation of pyruvate and 2-oxoglutarate, a cognate Zn-7Fe-ferredoxin serving as an electron acceptor . It comprises two subunits, a (632 amino acids) and b (305 amino acids) . To further elucidate its structure and function, we constructed a gene expression system . The wild-type recombinant enzyme was indistinguishable from the natural one in every criterion investigated . A series of variants was constructed to elucidate the role of the YPITP-motif (residues 253-257) in subunit a, which is conserved universally in the 2-oxoacid:ferredoxin oxidoreductase (OFOR) family . Single amino-acid replacements at Y253 and P257 by other amino acids caused a drastic loss of enzyme activity . T256, the hydroxyl group of which has been proposed to be essential for binding of the 2-oxo group of the substrate in the Desulfovibrio africanus enzyme, was unexpectedly replaceable with Ala, the kcat and Km for 2-oxoglutarate being approximately 33% and approximately 51%, respectively, as compared with that of the wild-type enzyme . Replacement at other positions resulted in a significant decrease in the kcat of the reaction while the Km for 2-oxoacid was only slightly affected . Thus, the YPITP-motif is essential for the turnover of the reaction rather than the affinity toward 2-oxoacid.

Eur J Biochem, 2001 Nov, 268(21), 5633 - 8
Isolation and functional analysis of a cDNA encoding a myrcene synthase from holm oak (Quercus ilex L.); Fischbach RJ et al.; An 859-bp cDNA segment of a terpene synthase gene was amplified by PCR from the evergreen sclerophyllous holm oak (Quercus ilex L.) using heterologous primers for conserved regions of terpene synthase genes (TPS) in dicotyledonous plants . Based on the sequence of this segment, homologous primers were designed for amplification by RACE-PCR of a cDNA segment carrying the monoterpene synthase gene myrS . The gene encodes a protein of 597 amino acids including an N-terminal putative plastid transit peptide . The gene without the segment encoding the transit peptide was cloned by PCR into a bacterial expression vector . Expression in Escherichia coli yielded an active monoterpene synthase, which converted geranyl diphosphate (GDP) predominantly into the acyclic monoterpene myrcene and to a very small extent into cyclic monoterpenes . Sequence comparison with previously cloned monoterpene synthases revealed that the myrcene synthase from Q . ilex belongs to the TPSb subfamily.

Environ Microbiol, 2001 Sep, 3(9), 588 - 99
Short- and long-term changes in proteome composition and kinetic properties in a culture of Escherichia coli during transition from glucose-excess to glucose-limited growth conditions in continuous culture and vice versa; Wick LM et al.; To investigate the ability of Escherichia coli K12 MG1655 to cope with excess and limitation of a carbon and energy source, we studied the changes in kinetic properties and two-dimensional (2D) gel protein patterns of an E . coli culture . The population was transferred from glucose-excess batch to glucose-limited continuous culture (D = 0.3 h(-1)), in which it was cultivated for 500 h (217 generations) and then transferred back to glucose-excess batch culture . Two different stages to glucose-limitation were recognized: a short-term physiological adaptation characterized by a general effort in enhancing the cell's substrate scavenging ability and mutations resulting in a population exhibiting increased glucose affinity . Physiological short-term adaptation to glucose-limitation was achieved by upregulation of 12 proteins, namely MglB, MalE, ArgT, DppA, RbsB, YdcS, LivJ (precursor), UgpB (precursor), AceA, AldA, AtpA and GatY . Eight of these proteins are periplasmic binding proteins of ABC transporters . Most of them are not involved in glucose transport regulons, but rather in chemotaxis and transport of other substrates, whereas MalE and MglB have previously been shown to belong to transport systems important in glucose transport under glucose-limited conditions . Evolution under low glucose concentration led to an up to 10-fold increase in glucose affinity (from a K(s) of 366 +/- 36 microg l(-1) at the beginning to 44 +/- 7 microg l(-1)) . The protein pattern of a "500-h-old" continuous culture showed a highly increased expression of MglB and MalE as well as of the regulator protein MalI . When adapted cells taken from the "500-h-old" continuous culture were transferred to batch culture, an increased expression of MalE was observed, compared with cells from un-adapted batch-grown cells . Otherwise, no significant changes were observed in the protein pattern of batch-grown populations before and after 500 h of evolution in the glucose-limited continuous culture.

Biochemistry, 2001 Nov 6, 40(44), 13302 - 9
Structural evidence that the methionyl aminopeptidase from Escherichia coli is a mononuclear metalloprotease; Cosper NJ et al.; The Co and Fe K-edge extended X-ray absorption fine structure (EXAFS) spectra of the methionyl aminopeptidase from Escherichia coli (EcMetAP) have been recorded in the presence of 1 and 2 equiv of either Co(II) or Fe(II) (i.e., {Co(II)_(EcMetAP)}, {Co(II)Co(II)(EcMetAP)}, {Fe(II)_(EcMetAP)}, and {Fe(II)Fe(II)(EcMetAP)}) . The Fourier transformed data of both {Co(II)_(EcMetAP)} and {Co(II)Co(II)(EcMetAP)} are dominated by a peak at ca . 2.05 A, which can be fit assuming 5 light atom (N,O) scatterers at 2.04 A . Attempts to include a Co-Co interaction (in the 2.4-4.0 A range) in the curve-fitting parameters were unsuccessful . Inclusion of multiple-scattering contributions from the outer-shell atoms of a histidine-imidazole ring resulted in reasonable Debye-Waller factors for these contributions and a slight reduction in the goodness-of-fit value (f ') . These data suggest that a dinuclear Co(II) center does not exist in EcMetAP and that the first Co atom is located in the histidine-ligated side of the active site . The EXAFS data obtained for {Fe(II)_(EcMetAP)} and {Fe(II)Fe(II)(EcMetAP)} indicate that Fe(II) binds to EcMetAP in a similar site to Co(II) . Since no X-ray crystallographic data are available for any Fe(II)-substituted EcMetAP enzyme, these data provide the first glimpse at the Fe(II) active site of MetAP enzymes . In addition, the EXAFS data for {Co(II)Co(II)(EcMetAP)} incubated with the antiangiogenesis drug fumagillin are also presented.

Proteomics, 2001 Aug, 1(8), 987 - 1000
Investigation of the applicability of a sequential digestion protocol using trypsin and leucine aminopeptidase M for protein identification by matrix-assisted laser desorption/ionization--time of flight mass spectrometry; Doucette A et al.; An investigation of the applicability of a sequential digestion procedure involving endo- and exoprotease digestion of proteins is reported . The procedure involves the digestion of a protein sample with trypsin, yielding peptide fragments characteristic of the protein . The resulting mixture of peptide fragments is then subjected to N-terminal sequencing with leucine aminopeptidase M (LAP), with matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis of the various digestion products . Several proteins in solution, as well as gel extracted proteins were subjected to this sequential enzyme digestion procedure . The results of these experiments reveal that LAP will preferentially cleave specific peptides of the trypsin digested sample with high efficiency, while leaving other peptides undigested . Also, the length of the amino acid sequence tags that can be generated with this method is limited; the longest sequence tag generated from a single tryptic peptide was four amino acids, even though the digestion was allowed to proceed for long times . In the experiments, N-terminal digestion products were detected as early as two minutes, or as late as 90 minutes, following the addition of LAP to the sample . The method was shown to be effective for subpicomole starting quantities of protein, although with some loss in digestion efficiency at lower concentrations of starting material . This method is useful in providing additional sequence information to increase the level of confidence in protein identification, as illustrated in the identification of bacterial proteins fractionated by high-performance liquid chromatography . In some instances, this method can provide additional sequence information where post source decay and nanospray mass spectrometry failed to generate fragment ion spectra . This is illustrated in an example where the procedure was applied to a membrane protein, CD9, that had been isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis . Although the sequential digestion procedure requires more human intervention, it is a straightforward method and can be readily implemented.

Proteomics, 2001 Aug, 1(8), 946 - 54
Analysis of protein sequences and protein complexes by matrix-assisted laser desorption/ionization mass spectrometry; Belghazi M et al.; In the context of proteome analysis, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) can fulfil the two tasks of primary structure verification and protein identification . As an illustration of the first of these tasks, the sequence of Eschericha coli isoleucyl-tRNA synthetase, a protein with 15 reported sequence conflicts, has been established by means of MALDI mass mapping . The identification of mitochondrial proteins participating in a yeast supramolecular complex exhibiting NADH dehydrogenase activity highlights the performances of MALDI-MS for the second task . The spectral suppression phenomenon occurring for complex peptide mixtures analysed by MALDI is discussed, as well as the role of post-source decay analysis for confident protein identification.

Curr Microbiol, 2001 Oct, 43(4), 278 - 83
Biochemical characterization of glucosylglycerol-phosphate synthase of Synechocystis sp . strain PCC 6803: comparison of crude, purified, and recombinant enzymes; Hagemann M et al.; Glucosylglycerol-phosphate synthase (GGPS), the key enzyme of the glucosylglycerol biosynthesis in salt-stressed cells of Synechocystis, was biochemically analyzed in crude extracts, after partial purification by FPLC and after overexpression of the gene ggpS in Escherichia coli and purification to homogenity of the recombinant protein, respectively . These GGPS preparations behaved similarly with regard to temperature stability, pH optimum, Mg2+ dependence, inhibition by phosphates, and Km values, but differed in their dependence on NaCl concentration: crude enzyme needed activation by addition of NaCl, whereas both partially-purified and recombinant GGPS showed high activities independent of the NaCl concentration.

Curr Microbiol, 2001 Oct, 43(4), 260 - 4
Cloning, characterization, and functional expression in Escherichia coli of argH encoding argininosuccinate lyase in the cyanobacterium Nostoc sp . strain PCC 73102; Troshina O et al.; A gene argH, encoding argininosuccinate lyase (ASL), has been cloned from a cosmid library of the filamentous cyanobacterium Nostoc sp . strain PCC 73102 . The argH open reading frame encodes a protein comprised of 461 amino acids with a calculated molecular mass of 51,349 Da . Protein sequence comparisons reveal significant similarities of the Nostoc PCC 73102 ASL to related proteins from other organisms . In an Escherichia coli delta argH strain, the Nostoc PCC 73102 ASL expressed from a recombinant plasmid could restore the ability to grow on medium without arginine . Moreover, cell extracts show a specific ASL activity of 16.2 nmoles of urea x min(-1) x (mg protein)(-1) . Partially purified, His-tagged ASL runs as a 53-kDa protein band in SDS-PAGE and about 215-kDa protein in native-PAGE, suggesting that the native protein is a tetramer.

Mol Genet Genomics, 2001 Oct, 266(2), 313 - 7
BipA is required for growth of Escherichia coi K12 at low temperature; Pfennig PL et al.; The bipA gene encodes a ribosome-associated GTPase postulated to be involved in regulatory functions in enteropathogenic Escherichia coli . Previous studies demonstrated that BipA is tyrosine phosphorylated in EPEC strains, but not in E . coli strain K12 . Results presented here indicate that BipA function is required at low temperatures in E . coli K12, suggesting a regulatory role independent of phosphorylation and of pathogenicity.

Mol Genet Genomics, 2001 Oct, 266(2), 260 - 70
Complex control of the developmental regulatory locus brlA in Aspergillus nidulans; Han S et al.; brlA is a primary regulator of asexual development in Aspergillus nidulans . Activation of brlA is necessary and sufficient for conidiophore development . It is known that brlA produces two overlapping transcripts, designated brlAalpha and brlAbeta . We found that expression of brlA is subject to complex regulation, in that activation of the two brlA transcripts is regulated at different levels . While brlAalpha is regulated at the transcriptional level, brlAbeta is regulated at both the transcriptional and translational levels . brlAalpha expression requires both abaA and brlA, but overexpression of brlAbeta can induce brlAalpha in an abaA mutant . brlAbetamuORF, a short ORF located upstream of the brlA initiator codon, regulates expression of brlA by damping translation of the brlAbeta ORF, and translational repression of brlA expression prevents premature development in A . nidulans . Transcriptional control of brlAbeta is apparently independent of BrlA . In order to understand better the transcriptional control of brlAalpha and brlAbeta, we have made 5' deletions in the essential approximately 2-kb upstream control sequences that regulate brlAbeta transcription and fused them to the E . coli lacZ reporter gene . Various deletions in this region resulted in only minor changes in the regulation of beta-galactosidase expression . The results of the deletion experiments indicate that there are probably several cis-acting control sequences involved in the regulation of brlAbeta . As a complementary approach, we fused various fragments of the 2034-bp brlAbeta and 754-bp brlAalpha control sequences to an otherwise inactive amdS::lacZ fusion, in order to search for regions that are sufficient to place the reporter under developmental control . We identified two approximately 600-bp brlAbeta fragments extending from -2901 to -2293 and -967 to -414, respectively, and a approximately 150-bp brlAalpha segment from -271 to -127, that confer activity on the inactive amdS promoter . brlA is overexpressed in an abaA null mutant and one site for abaA-dependent repression is apparently located in the -742 to -414 brlAbeta fragment . This indicates that abaA-mediated repression of brlA expression occurs through control of brlAbeta, but apparently involves a mechanism that does not require AbaA binding to brlA(p) sequences, because there are no AbaA binding sites in this region.

Mol Genet Genomics, 2001 Oct, 266(2), 207 - 15
Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli; Kim SR et al.; DNA polymerase IV (pol IV) in Escherichia coli is a member of a novel family of DNA polymerases (the DinB/UmuC/Rad30/Rev1 super-family or the DNA polymerase Y family) . Although expression of the dinB gene encoding DNA pol IV is known to result in an enhancement of untargeted mutagenesis, it remains uncertain whether DNA pol IV is involved in a variety of lesion-induced mutagenesis (targeted mutagenesis), and the relationship between expression levels of dinB and the mutagenesis that DNA pol IV promotes has not been investigated thoroughly . Here, we report that DNA pol IV is involved in -1 frameshift mutagenesis induced by 4-nitroquinoline N-oxide (4-NQO) and that the expression level of the chromosomal pol IV gene is 6-12 times higher than those for other SOS-inducible DNA polymerases in E . coli, i.e., DNA pol II (PolB) or DNA pol V (UmuDC), respectively . Interestingly, the dinB gene is present not only on the chromosome but also on the F' plasmid in the E . coli CC108 strain . In this strain, 750 molecules of DNA pol IV are expressed from the F' dinB gene in the uninduced state and 250 molecules are expressed from the chromosomal gene . These cellular expression levels strongly affect -1 frameshifts induced by 4-NQO in runs of six guanine bases: mutagenicity was highest in the strain CC108, followed by strains YG2242 (chromosome deltadinB/F' dinB+), YG2247 (chromosome dinB+/F' deltadinB) and FC1243 (chromosome deltadinB/F' deltadinB) . The incidence of untargeted -1 frameshifts was reduced by two-thirds on deletion of dinB from the F' episome . The chromosomal dinB gene appeared to have little or no effect on the untargeted mutagenesis . These results suggest that DNA pol IV efficiently mediates targeted mutagenesis by 4-NQO, and that the cellular levels of expression substantially affect targeted and untargeted mutagenesis.

Mol Genet Genomics, 2001 Oct, 266(2), 167 - 79
Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA; Kawakami H et al.; In Escherichia coli, an interaction between the replication initiator DnaA and the sliding clamp protein, the beta subunit (DnaN) of DNA polymerase III, is required to regulate the chromosomal replication cycle . We report here that colony formation by, and cell division of, the temperature (42 degrees C)-sensitive dnaN59 mutant are inhibited at 34-35 degrees C when DnaA is moderately (4-to 8-fold ) overexpressed, although chromosomal replication and the beta subunit-dependent regulation of DnaA activity are not significantly inhibited . Immunoblotting analysis revealed that the beta subunit is abundant (present at a level of about 5000 dimers per cell) at 34 degrees C, and its concentration per unit cell volume was practically unaffected in the dnaN59 mutant by the overexpression of DnaA . The dnaN mutant cells that overexpress DnaA become filamentous at 34 degrees C via an sfiA-independent pathway, different from that activated by the SOS response . This filamentation is accompanied by inhibition of nucleoid partition and FtsZ ring formation . In the dnaN59 mutant, oversupply of DnaA may disturb the coordinated action of cell cycle-regulating molecules, thus leading to the inhibition of these events.

Mutagenesis, 2001 Nov, 16(6), 461 - 5
Mutagenicity of a single 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene in Escherichia coli located in a GGC sequence; Bacolod MD et al.; 1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-amino- pyrene (dG(AP)) . Mutational spectra of randomly introduced dG(AP) in Escherichia coli included different types of mutations, which depended on the base sequence surrounding the adduct . In earlier works we investigated the DNA sequence context effects of the adduct in repetitive CpG and non-repetitive CpGpC sequences . In the current work this adduct was incorporated into a non-repetitive GpGpC sequence in single-stranded M13mp7L2 DNA with the adduct located at either the 5' or 3' G . Potent genotoxicity of dG(AP) was evident from a significant reduction in the population of progeny phage following replication of these constructs in repair-competent E.coli cells . However, progeny derived from the 3'-G(AP) construct were much larger than those from the 5'-G(AP) construct . We suspect that a more facile translesion synthesis past the adduct at the 3' G relative to that at the 5' G, presumably due to a difference in conformation of dG(AP) in these two sites, might be responsible for this effect . With both adducted constructs, >95% of the progeny did not show any mutations at or near the adduct site, indicating highly efficient error-free translesion synthesis . However, a small population of mutants with one base deletions and base substitutions were detected . While the adduct induced -1 frameshifts (<1%) in each G site, base substitutions (1-2%), exhibiting predominantly G-->C transversions, were detected only when the adduct was located at the 5' G . A comparison of the data from this study with a prior study in the CpGpC sequence suggests that dG(AP) mutagenesis is highly sensitive to the local DNA sequence and that a 5'-pyrimidine base might be important for targeted base substitutions by this adduct in E.coli.

J Biol Chem, 2002 Jan 4, 277(1), 830 - 5 Epub 2001 Oct 26.
Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica; Choi KB et al.; S-adenosyl-L-methionine:coclaurine N-methyltransferase (CNMT) converts coclaurine to N-methylcoclaurine in isoquinoline alkaloid biosynthesis . The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5'- and 3'-rapid amplification of cDNA ends (RACE) . The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-L-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase . A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed . Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that Coptis CNMT has quite broad substrate specificity, i.e . not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline . The evolution of N-methyltransferases in secondary metabolism is discussed based on sequence similarity.

FEMS Microbiol Lett, 2001 Oct 16, 204(1), 155 - 61
Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction; Precigou S et al.; A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding nitrile hydratase (NHase) . Primer design was based on the highly conserved sequences found in the coding region of the alpha-subunit gene corresponding to the metal-binding site . Purified genomic DNA from bacterial strains or directly from soil can serve as the target for the PCR, thus affording a simple and rapid method for screening NHase genes . The primer pairs, NHCo1/NHCo2 and NHFe1/NHFe2 yield PCR products corresponding to a partial coding sequence of cobalt and iron NHase genes, respectively . Using the PCR method, both types of iron- and cobalt-NHase-encoding genes were detected in DNA from pure cultures and soil samples . Furthermore consensus primers allowed rapid cloning and expression of novel NHases in Escherichia coli.

FEMS Microbiol Lett, 2001 Oct 16, 204(1), 105 - 10
Metabolism of phenylpropionic acid in enteropathogenic Escherichia coli belonging to serogroup O111 and its application for diagnosis; Irino K et al.; We evaluated a biochemical assay based on the ability to metabolise beta-phenylpropionic acid (PPA) as a diagnostic aid in the identification of typical enteropathogenic Escherichia coli (EPEC) strains . A total of 1061 E . coli strains of serogroups O55, O111, and O119 were initially characterised regarding their H types (serotypes) and the presence of EPEC DNA sequences, eae, EAF, and bfpA . In case of the serogroup O111 strains, 84.6% carried the typical EPEC markers, and the great majority of those (98.1%) were PPA-positive . In contrast, only 0.9% of the serogroups O55 and O119 strains carrying the typical EPEC markers (53.6% and 75.4%, respectively) were PPA-positive . We conclude that the PPA test is a useful method to detect typical EPEC strains only among strains of the O111 serogroup.

FEMS Microbiol Lett, 2001 Oct 16, 204(1), 75 - 9
A novel locus of enterocyte effacement (LEE) pathogenicity island inserted at pheV in bovine Shiga toxin-producing Escherichia coli strain O103:H2; Jores J et al.; We describe a locus of enterocyte effacement (LEE) which is part of a new pathogenicity island (PAI) detected in the bovine Shiga toxin-producing Escherichia coli strain RW1374 (O103:H2) . This PAI is at least 80 kb in size and inserted in the vicinity of the pheV tRNA gene at 67 min of the E . coli chromosome . Furthermore, the PAI differs from the previously described LEEs by unique flanking regions at both sides, which harbor one copy each of an insertion element in an inverted orientation that is 96% identical to insertion site (IS)629 . In addition, a 5-kb PAI-specific sequence downstream of the LEE core region and adjacent to the E . coli K12 region is duplicated upstream of the LEE core region as well . The duplicated sequences are more than 80% identical to each other and consist partially of prophage sequences.

FEMS Microbiol Lett, 2001 Oct 16, 204(1), 65 - 9
STa-induced translocation of protein kinase C from cytosol to membrane in rat enterocytes; Ganguly U et al.; Escherichia coli heat stable enterotoxin (STa) binds to isolated rat intestinal epithelial cells and triggers a cascade reaction including increase of intracellular calcium levels ({Ca(2+)}(i)) and membrane bound protein kinase C (PKC) activity . In response to STa, the cytosolic PKC activity falls from 110 to 35 nmol with increase of membrane bound PKC activity from 15 to 78 nmol . Furthermore, the increase of PKC activity induced by STa treatment was always preceded by an increase in {Ca(2+)}(i) . Cytosolic {Ca(2+)}(i) was significantly higher (161 nM) in STa treated cells as compared to untreated cells (51.3 nM) . In addition, immunoblot performed on extracts of STa treated rat enterocytes with a monoclonal antibody against PKC alpha showed a prominent band of PKC alpha . Translocation of PKC alpha could be blocked by dantrolene, a drug which inhibits the mobilisation of {Ca(2+)}(i) from the intracellular store . Our results, therefore, provide evidence for the role of {Ca(2+)}(i) in STa treated cells for the translocation of PKC alpha from cytosol to membrane.

Virus Res, 2001 Dec 4, 81(1-2), 113 - 23
A 'minimal' approach in design of flavivirus infectious DNA; Mishin VP et al.; The 'infectious DNA' approach, which is based on in vivo transcription of (+)RNA virus genome cDNA cassettes from eukaryotic promoters in transfected cells, became a popular alternative to the classical scheme in the infectious clone methodology . Its use, however, is often limited by the instability of plasmids due to a transcriptional activity of eukaryotic promoters in Escherichia coli resulting in synthesis of products toxic for the bacterial host . Using a highly unstable representative infectious clone of Japanese encephalitis (JE) flavivirus, we tested a new approach in design of such problematic 'infectious DNA' constructs, which is based on minimizing unwanted transcription in the bacterial host . A plasmid containing full genome size JE cDNA under control of the minimal cytomegalovirus (CMV) promoter can be propagated in E . coli with growth and stability characteristics similar to that of constructs controlled by the T7 promoter . Transfection of this plasmid into susceptible cells leads to the establishment of a productive infectious cycle . Reinsertion of the CMV enhancer at the 3'-end of the JE cassette substantially increased the specific infectivity without affecting the stability and growth characteristics of the construct . This approach can be useful when stabilization of infectious clones by modification of a viral cDNA cassette is not the feasible or suitable alternative.

FEBS Lett, 2001 Oct 19, 507(1), 35 - 8
A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe; Taki M et al.; A small and highly fluorescent non-natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin . The positions were directed by a CGGG/CCCG four-base codon/anticodon pair . The non-natural mutants were obtained in excellent yields and some of them retained strong biotin-binding activity . The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding . These unique properties indicate that the atnDap is the most suitable non-natural amino acid for a position-specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.

FEBS Lett, 2001 Oct 19, 507(1), 16 - 20
GFP-like chromoproteins as a source of far-red fluorescent proteins; Gurskaya NG et al.; We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins . An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission lambda(max)=615-640 nm) . Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H . crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm . A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling . Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications . In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.

EMBO J, 1986 Jun, 5(6), 1143 - 8
Interaction of turnip yellow mosaic virus Val-RNA with eukaryotic elongation factor EF-1 {alpha} . Search for a function; Joshi RL et al.; The 3'-terminal tRNA-like structure in turnip yellow mosaic virus (TYMV) RNA can be adenylated by tRNA nucleotidyltransferase and subsequently aminoacylated by valyl-tRNA synthetase.Here we present evidence that TYMV Val-RNA can form a stable complex with eukaryotic wheat germ elongation factor EF-1alpha and GTP: the Val-RNA is protected by EF-1alpha. . GTP against digestion by RNase A . By affinity chromatography of TYMV Val-RNA fragments on immobilized EF-1alpha . GTP, it has been established that the valylated aminoacyl RNA domain, which in TYMV RNA is formed by the 3' half of the tRNA-like region, is sufficient for complex formation with EF-1alpha . GTP . The aminoacyl RNA domain is equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T stem and loop . In line with these results, the aminoacyl RNA domain in TYMV Val-RNA complexed to EF-1 alpha . GTP is resistant to digestion by RNase A . It is also shown that the TYMV RNA replicase (RNA-dependent RNA polymerase) isolated from TYMV-infected Chinese cabbage leaves does not contain tRNA nucleotidyltransferase, valyl-tRNA synthetase or EF-1alpha . This suggests that interaction of TYMV RNA with EF-1alpha is not mandatory for replicase activity.

Proteomics, 2001 Jan, 1(1), 54 - 65
Comparison of three different fluorescent visualization strategies for detecting Escherichia coli ATP synthase subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Berggren KN et al.; The correlation between protein molecular weight and the number of lysine or basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F1F0-ATP synthase and the translated open reading frame of E . coli . A relatively poor correlation between protein molecular weight and the number of cysteine residues was observed in all cases . The ability of amine-reactive, thiol-reactive and basic amino acid-binding fluorophores to detect the eight subunits of F1F0-ATP synthase complex was assessed using 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively . Though experimentally none of the fluorophores provided accurate estimates of the subunit stoichiometry of this complex, MDPF and SYPRO Ruby protein gel stain were capable of semiquantitative detection of every subunit . MBB, however, failed to detect subunits a, b and c of the hydrophobic F0 complex, as well as subunit epsilon of the F1 complex . All three fluorescent detection procedures permitted subsequent identification of representative subunits by peptide mass profiling using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) . The use of thiol-reactive fluorophores for the global analysis of protein expression profiles does not appear to be advisable as a significant number of proteins have few or no cysteine residues, thus escaping detection.

Proteomics, 2001 Jan, 1(1), 42 - 53
Chromatographic separations as a prelude to two-dimensional electrophoresis in proteomics analysis; Butt A et al.; Current methods of proteome analysis rely almost solely on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by the excision of individual spots and protein identification using mass spectrometry (MS) and database searching . 2-D PAGE is denaturing in both dimensions and, thus, cannot indicate functional associations between individual proteins . Moreover, less abundant proteins are difficult to identify . To simplify the proteome, and explore functional associations, nondenaturing anion exchange column chromatography was used to separate a soluble protein extract from Escherichia coli . Successive fractions were then analysed using 2-D PAGE and selected spots from both the gels for the start material and the fractionated material were quantified and identified by peptide mass fingerprinting using a MALDI-TOF mass spectrometer . Enrichments of up to 13-fold were attained for individual protein spots and peptide mass fingerprints were of significantly higher quality after chromatographic separation . The marked anomalies between predicted p/and column elution position contrasted with the almost perfect correlation with migration distance on isoelectric focusing (IEF) and were explored further for basic proteins.

RNA, 2001 Oct, 7(10), 1464 - 75
Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome; Baginsky S et al.; In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome . This complex has an important role in processing and degradation of RNA . Chloroplasts contain an exoribonuclease homologous to E . coli PNPase . Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E . coli degradosome . Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex . Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography . These results suggest that chloroplast PNPase exists as a homo-multimer complex . No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography . Database analysis of proteins homologous to E . coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E . coli protein that is involved in assembly of the degradosome . Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast . Thus, RNA processing and degradation in this organelle differ in several respects from those in E . coli.

RNA, 2001 Oct, 7(10), 1403 - 15
pH-dependent conformational flexibility within the ribosomal peptidyl transferase center; Muth GW et al.; A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation . Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes . A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH . Here we present three independent lines of evidence in support of the alternative interpretation . First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection . Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner . Third, within E . coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base . The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement . It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis . Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known.

Int J Med Microbiol, 2001 Sep, 291(4), 277 - 85
Modulation of host cell signalling by enteropathogenic and Shiga toxin-producing Escherichia coli; Kresse AU et al.; The majority of Escherichia coli strains are harmless symbionts in the intestinal tract . However, there are several pathogenic forms, which are responsible for various diseases in humans and live stock . In this review we discuss the interactions between Shiga toxin-producing E . coli and enteropathogenic E . coli and their target host cells, describing their strategies to activate specific cellular signalling pathways which lead to subversion of critical physiological functions . We mainly concentrate on those pathogenic mechanisms that are dependent on a functional type III secretion system, but we also briefly discuss additional factors that contribute to the specific pathogenic profiles of Shiga toxin-producing E . coli and enreropathogenic E . coli.

Biotechniques, 2001 Oct, 31(4), 948 - 50, 952-3
High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/ translation system; Martin GA et al.; The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E . coli lysate for an in vitro transcription/translation reaction . In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h . We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems . We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells . The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria . Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction . This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.

Pflugers Arch, 2001 Sep, 442(6), 920 - 7
Endotoxin and cytokines alter contractile protein expression in cardiac myocytes in vivo; Patten M et al.; Release of bacterial endotoxin and cytokines induce cardiac failure during sepsis . We investigated the direct effects of E . coli endotoxin (lipopolysaccharide, LPS) and cytokines induced by LPS on the cardiac myocyte gene program . For in vivo-experiments adult Wistar rats were given 600 microg/day LPS i.v . for 24 h or 7 days . In addition, cultured adult rat cardiac myocytes were treated with LPS, interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNFalpha), interferon-gamma (IFNgamma) or IL-6 for 24 h . mRNA expression was evaluated for cardiac-alpha-actin (cAct), skeletal-alpha-actin (skAct), beta- and alpha-myosin heavy chain (MHC) . LPS induced betaMHC-mRNA 3.6-fold and repressed alphaMHC 2.7-fold and cAct 2.5-fold after 24 h in vivo . Up-regulation of betaMHC (3-fold) and repression of cAct (2.5-fold) were still observed after 7 days LPS infusion, whereas alphaMHC-mRNA levels had returned to normal . At the protein level, increased expression of betaMHC by LPS treatment occurred already after 24 h and was maintained thereafter . LPS had no influence on skAct-mRNA . Similar changes in contractile protein mRNA expression were observed in LPS-treated cardiomyocytes in culture, whereas the tested cytokines either activated (IL-1beta, IFNgamma) or repressed (TNFalpha, IL-6) both MHC-isoforms and cAct . In conclusion, LPS and proinflammatory cytokines induce changes in contractile protein expression that may contribute to the acute heart failure observed during endotoxaemia.

Hepatology, 2001 Nov, 34(5), 918 - 25
Transforming growth factor beta and activin tonically inhibit DNA synthesis in the rat liver; Ichikawa T et al.; The present study was conducted to assess the role of transforming growth factor beta (TGF-beta) and activin(s) in the regulation of the mass of the liver . To this end, we eliminated TGF-beta or activin signaling in intact rat liver by adenovirus-mediated transfer of the gene encoding truncated type II TGF-beta receptor (AdextTR) or truncated type II activin receptor (AdextAR) . In intact rat liver that received a single application of either AdextTR or AdextAR via the portal vein, DNA synthesis as assessed by bromodeoxy uridine (BrdU) labeling was induced . In AdextTR- or AdextAR-treated rats, nuclear labeling was significantly higher than that in AdexLacZ, adenovirus vector encoding Escherichia coli beta-galactosidase gene, or saline-treated rats at 3, 5, 7, and 9 days of infusion . The peak of the BrdU labeling was observed after 7 days of infusion and the labeling decreased thereafter . Apoptosis of hepatocytes, assessed by the terminal deoxynucleotidyl transferase (TdT)-mediated, dUTP-biotin nick-end labeling method was detected after 9 days of infusion . Immunoreactivity of TGF-beta and activin A increased in the liver after the blockade of the activin or TGF-beta signaling . TGF-beta and activin A may have been up-regulated when the action of these ligands was blocked . These results indicate that blockade of the action of either TGF-beta or activin leads to the initiation of DNA synthesis in intact liver . TGF-beta and activin tonically inhibit hepatocyte growth even in intact liver and may play a critical role in the maintenance of constant liver mass.

J Infect Dis, 2001 Nov 15, 184(10), 1336 - 40 Epub 2001 Oct 15.
Inhibition of purified recombinant reverse transcriptase from wild-type and zidovudine-resistant clinical isolates of human immunodeficiency virus type 1 by zidovudine, stavudine, and lamivudine triphosphates; Duan C et al.; Cross-resistance between zidovudine, stavudine, and lamivudine was studied, using purified recombinant reverse transcriptase from a zidovudine-susceptible and -resistant pair of clinical isolates of human immunodeficiency virus type 1 . The zidovudine-resistant isolate exhibited low-level cross-resistance to both stavudine and lamivudine in drug susceptibility assays . Enzyme from the resistant isolate demonstrated reduced inhibition by zidovudine triphosphate and stavudine triphosphate and, to a lesser extent, lamivudine triphosphate . These findings provide additional evidence at the viral and enzyme level for cross-resistance between zidovudine and stavudine, and they suggest a possible effect of zidovudine resistance on susceptibility to lamivudine.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1726 - 8 Epub 2001 Oct 25.
Crystallization and preliminary X-ray diffraction analysis of glutathione-dependent dehydroascorbate reductase from spinach chloroplasts; Mizohata E et al.; Glutathione-dependent dehydroascorbate reductase (GSH-DHAR) catalyzes the reduction of dehydroascorbate to ascorbate using reduced glutathione as the electron donor . GSH-DHAR from spinach chloroplasts produced in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method . The crystals were monoclinic, space group C2, with unit-cell parameters a = 98.25, b = 39.96, c = 106.86 A, beta = 110.46 degrees . The asymmetric unit contained two molecules, giving a crystal volume per enzyme mass (V(M)) of 2.06 A(3) Da(-1) and a solvent content of 40.3% . A full set of X-ray diffraction data were collected to 2.2 A Bragg spacing from three native crystals with an overall R(merge) of 6.5% and a completeness of 93.4%.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1722 - 5 Epub 2001 Oct 25.
Expression, purification and crystallization of Aspergillus nidulans NmrA, a negative regulatory protein involved in nitrogen-metabolite repression; Nichols CE et al.; The NmrA repressor protein of Aspergillus nidulans was overproduced in Escherichia coli and purified to homogeneity . Gel-exclusion chromatography showed that NmrA was monomeric in solution under the buffer conditions used . The protein was crystallized in three forms, belonging to trigonal, monoclinic and hexagonal space groups . Two of these crystal forms (A and B) diffract to high resolution and thus appear suitable for structure determination . Crystal form A belongs to space group P3((1))21, with unit-cell parameters a = b = 76.8, c = 104.9 A . Crystal form B belongs to space group C2, with unit-cell parameters a = 148.8, b = 64.3, c = 110.2 A, beta = 121.8 degrees.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1686 - 8 Epub 2001 Oct 25.
Crystallization and preliminary X-ray studies of Trp138Phe/Val185Thr xylose isomerases from Thermotoga neapolitana and Thermoanaerobacterium thermosulfurigenes; Kim YS et al.; Xylose isomerases from Thermotoga neapolitana (TNXI) and Thermoanaerobacterium thermosulfurigenes (TTXI) share 70.4% sequence identity and are thermostable . The double mutants Trp138Phe/Val185Thr of TNXI and TTXI have higher catalytic efficiencies than TNXI and TTXI, respectively . The Trp138Phe/Val185Thr TNXI and TTXI mutants were overexpressed in Escherichia coli strain BL21(DE3) and purified . Crystals of the two proteins were grown with polyethylene glycol 8000 as the major precipitant by the hanging-drop vapour-diffusion method . Crystals of the TNXI mutant were obtained in the absence of substrate, in complex with glucose and in complex with fructose . Crystals of the TTXI mutant were obtained complexed with glucose . Diffraction data were collected at 1.9, 2.1 and 2.1 A resolution for the fructose-TNXI mutant, glucose-TNXI mutant and substrate-unbound TNXI mutant, respectively . The diffraction data for the glucose-TTXI mutant were collected at 2.0 A resolution . The crystals belong to the orthorhombic space groups C222(1) (TNXI mutant) and P2(1)2(1)2(1) (TTXI mutant) . The TNXI and TTXI mutant crystals contain two and four monomers in the asymmetric unit, respectively.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1671 - 3 Epub 2001 Oct 25.
Expression, purification, crystallization and preliminary crystallographic analysis of recombinant pteridine reductase of Trypanosoma cruzi; Schormann N et al.; The recombinant version of Trypanosoma cruzi pteridine reductase was expressed in Escherichia coli, purified to homogeneity from the soluble fraction of bacterial extract by metal-chelate affinity chromatography and crystallized in the presence of the cofactor (NADPH) and an inhibitor (methotrexate) at 295 K using sodium acetate as precipitant . The crystals are trigonal, belonging to space group P3(1) (or P3(2)), with unit-cell parameters a = 74.35, c = 179.96 A under cryogenic conditions . The asymmetric unit contains a tetramer, with a corresponding V(M) of 2.3 A(3) Da(-1)and a solvent content of 46% . Native data have been collected to 2.1 A resolution using Cu Kalpha X-rays.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1650 - 1 Epub 2001 Oct 25.
Overexpression, purification, crystallization and preliminary crystallographic studies on a thermostable beta-glycosidase from Thermus nonproteolyticus HG102; He XY et al.; A thermostable beta-glycosidase (Tn-gly) from Thermus nonproteolyticus HG102 has been cloned and overexpressed in Escherichia coli . The recombinant enzyme, with a molecular mass of 48.9 kDa, was purified to homogeneity . It can hydrolyze a wide range of oligosaccharides and perform transglycosylation . Crystals of the recombinant enzyme were grown by the hanging-drop vapour-diffusion technique with MPD and NaCl as precipitants . They belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 66.7, b = 94.6, c = 176.5 A.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1646 - 9 Epub 2001 Oct 25.
Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of Cu,Zn superoxide dismutase from Peking duck; Liu W et al.; The cDNA encoding Peking duck Cu,Zn superoxide dismutase (dSOD) was cloned and sequenced . The recombinant enzyme was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion technique . Trigonal crystals of dSOD were obtained at 278 K at low ionic strength and around neutral pH . These crystals belong to space group P3(2)21, with unit-cell parameters a = 124.4, c = 163.5 A, gamma = 120 degrees . The asymmetric unit contains four dimers (eight monomers of Cu,Zn dSOD) and has a 56% solvent content, with a V(M) of 2.8 A(3) Da(-1) . On a Rigaku R-AXIS IIc image-plate area-detector system, the crystal diffracted to 2.9 A . Unusual supermolecular double-helix packing with 9(2)2 non-crystallographic symmetry in crystals has been observed in the initial structural analysis.

J Biol Chem, 2001 Dec 28, 276(52), 48956 - 60 Epub 2001 Oct 25.
Vibrational structure of dihydrofolate bound to R67 dihydrofolate reductase; Deng H et al.; R67 is a Type II dihydrofolate reductase (DHFR) that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate by facilitating the addition of a proton to N5 of DHF and the transfer of a hydride ion from NADPH to C6 . Because this enzyme is a plasmid-encoded DHFR from trimethoprim-resistant bacteria, extensive studies on R67 with various methods have been performed to elucidate its reaction mechanism . Here, Raman difference measurements, conducted on the ternary complex of R67.NADP(+).DHF believed to be an accurate mimic of the productive DHFR.NADPH.DHF complex, show that the pK(a) of N5 in the complex is less than 4 . This is in clear contrast to the behavior observed in Escherichia coli DHFR, a substantially more efficient enzyme, where the pK(a) of bound DHF at N5 is increased to 6.5 compared with its solution value of 2.6 . A comparison of the ternary complexes in R67 and E . coli DHFRs suggests that enzymic raising of the pK(a) at N5 can significantly increase the catalytic efficiency of the hydride transfer step . However, R67 shows that even without such a strategy an effective DHFR can still be designed.

Appl Environ Microbiol, 2001 Nov, 67(11), 5335 - 8
Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury; Bae W et al.; Synthetic phytochelatins (ECs) are a new class of metal-binding peptides with a repetitive metal-binding motif, (Glu-Cys)(n)Gly, which were shown to bind heavy metals more effectively than metallothioneins . However, the limited uptake across the cell membrane is often the rate-limiting factor for the intracellular bioaccumulation of heavy metals by genetically engineered organisms expressing these metal-binding peptides . In this paper, two potential solutions were investigated to overcome this uptake limitation either by coexpressing an Hg(2+) transport system with (Glu-Cys)(20)Gly (EC20) or by directly expressing EC20 on the cell surface . Both approaches were equally effective in increasing the bioaccumulation of Hg(2+) . Since the available transport systems are presently limited to only a few heavy metals, our results suggest that bioaccumulation by bacterial sorbents with surface-expressed metal-binding peptides may be useful as a universal strategy for the cleanup of heavy metal contamination.

Appl Environ Microbiol, 2001 Nov, 67(11), 5325 - 7
Concentrations of copper thought to be toxic to Escherichia coli can induce the viable but nonculturable condition; Grey B et al.; We have determined that concentrations of copper considered to be toxic can induce a fraction of a population of Escherichia coli to enter the viable but nonculturable (VBNC) condition . Copper-induced VBNC cells could be resuscitated for up to 2 weeks after entering the VBNC state.

J Biomol Screen, 2001 Feb, 6(1), 39 - 46
FlashPlate scintillation proximity assays for characterization and screening of DNA polymerase, primase, and helicase activities; Earnshaw DL et al.; DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies . Replication proteins are conventionally assayed using precipitation/filtration or gel-based techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats . We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents . This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities . The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors . In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells . Various adaptations were employed for each enzyme activity . For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of {(3)H}-dNTPs onto the growing primer 3' end . For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of {(3)H}-rNTPs or by subsequent polymerase extension with {(3)H}-dNTPs from unlabeled primers . For DNA helicase, unwinding of a {(33)P}-labeled oligonucleotide complementary to the tethered oligonucleotide was measured . This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.

Microvasc Res, 2001 Nov, 62(3), 315 - 26
Molecular properties of fibrin-based matrices for promotion of angiogenesis in vitro; Hall H et al.; The molecular properties of fibrin-based matrices, such as fibrillar structure and covalent modifications with adhesion domains, influence the angiogenic behavior of human umbilical vein endothelial cells (HUVECs) in vitro . The fibrillar structure of fibrin-based matrices was influenced by pH but not by covalent incorporation of exogenous adhesion domains . Native fibrin-based matrices polymerized at pH 10 formed organized and longitudinally oriented fibrin fibrils, which provided a good angiogenic substrate for endothelial cells . Furthermore, upon covalent incorporation of the model ligand L1Ig6, which binds to the integrin most prominently expressed on the surface of angiogenic endothelial cells, alpha(v)beta3, these matrices became angiogenesis-promoting when polymerized at physiological pH . The amount of incorporation of L1Ig6 into the matrices depended on the fibrinogen concentration on all three fibrin chains . Soluble forms of L1Ig6 diffused rapidly out of the matrix . Most important, L1Ig6-modified matrices were very specific in inducing the angiogenic phenotype of HUVECs, whereas control cells did not differentiate on these matrices . Our results indicate that artificial extracellular matrices can influence cell behavior in two ways . One way is based on the three-dimensional fibril structure of the matrix molecules themselves, and the other is due to providing specific binding sites for direct cell-matrix interactions that lead to the activation of second-messenger cascades and thus promoting angiogenic differentiation .

Am J Med Sci, 2001 Oct, 322(4), 222 - 8
A shock toxin that produces disseminated intravascular coagulation and multiple organ failure; Hardaway RM et al.; OBJECTIVE: To introduce a new concept in the etiology and treatment of traumatic and septic shock . It describes 3 types of shock: (1) hypovolemic shock, (2) traumatic shock, and (3) septic shock . BACKGROUND: The mortality of septic shock in both total number and mortality rate has been increasing over the past 40 years despite major advances in diagnosis and treatment, including a number of "magic bullets." Trauma is the No . 1 cause of death in persons under the age of 44 and the No . 3 cause of all deaths . Traumatic shock has been assumed to be caused by hypovolemia; however, many traumatic shock patients die with a normal blood volume, usually after several days . Septic shock in pigs using an injection of killed Escherichia coli organisms produced disseminated intravascular coagulation (DIC) . Control pigs treated with plasminogen activator survived . Septic shock in humans also treated with plasminogen activator showed excellent results . Traumatic shock studied in pigs showed excellent results with plasminogen activator . A normal blood volume was maintained with the use of intravenous fluids . Traumatic shock in humans also treated by plasminogen activator showed excellent results . The improvement in PaO2 and other parameters demonstrated in these studies provides a new possibility in the treatment of trauma and/or sepsis induced acute respiratory distress syndrome (ARDS) . DIC is almost always present in traumatic and septic shock and probably in the course of ARDS and multiple organ failure . The DIC is probably initiated by tissue cell or bacterial cell destruction, which liberates a thrombogenic aminophospholipid that forms the inner layer of all cell walls.

Fresenius J Anal Chem, 2001 Sep, 371(2), 276 - 81
Analysis of single-cell differences by use of an on-chip microculture system and optical trapping; Wakamoto Y et al.; A method is described for continuous observation of isolated single cells that enables genetically identical cells to be compared; it uses an on-chip microculture system and optical tweezers . Photolithography is used to construct microchambers with 5-microm-high walls made of thick photoresist (SU-8) on the surface of a glass slide . These microchambers are connected by a channel through which cells are transported, by means of optical tweezers, from a cultivation microchamber to an analysis microchamber, or from the analysis microchamber to a waste microchamber . The microchambers are covered with a semi-permeable membrane to separate them from nutrient medium circulating through a "cover chamber" above . Differential analysis of isolated direct descendants of single cells showed that this system could be used to compare genetically identical cells under contamination-free conditions . It should thus help in the clarification of heterogeneous phenomena, for example unequal cell division and cell differentiation.

J Biol Chem, 2002 Jan 18, 277(3), 1653 - 61 Epub 2001 Oct 24.
DNA polymerase I of Mycobacterium tuberculosis: functional role of a conserved aspartate in the hinge joining the M and N helices; Arrigo CJ et al.; The highly conserved GXD sequence present in the Mycobacterium tuberculosis DNA polymerase I corresponds to a hinge region in the finger subdomain connecting M and N helices of Escherichia coli pol I . An examination of the crystal structures of pol I family polymerases reveals that the invariant aspartate of the hinge forms a salt bridge with the conserved arginine of the O-helix and an H-bond with Gln-708 . To clarify the role of this region, we generated and characterized conserved and nonconserved mutant derivatives of this aspartate, the preceding glutamate and the Gln in TB pol I . For comparison, D732A mutein of pol I was also included . The muteins representing conserved aspartate (Asp-707 of TB pol I or Asp-732 of pol I) showed a strong K(m)((dNTP)) effect and minor alteration in K(d)((DNA)), with about 10-20-fold decrease in overall catalytic efficiency . The TB muteins, E706A and Q683A, have less pronounced deviations from the wild-type enzyme . Further examination of D707A of TB pol I showed no alteration in the processivity or the dideoxynucleotide sensitivity patterns . However, both TB pol D707A and homologous E . coli D732A failed to form a stable E.DNA.dNTP ternary complex . These results suggest that the aspartate in the hinge region is catalytically important and is required for dNTP binding and in the formation of a prepolymerase ternary complex.

Gastroenterology, 2001 Nov, 121(5), 1191 - 202
Increases in guanylin and uroguanylin in a mouse model of osmotic diarrhea are guanylate cyclase C-independent; Steinbrecher KA et al.; BACKGROUND & AIMS: Guanylin and uroguanylin are peptide hormones that are homologous to the diarrhea-causing Escherichia coli enterotoxins . These secretagogues are released from the intestinal epithelia into the intestinal lumen and systemic circulation and bind to the receptor guanylate cyclase C (GC-C) . We hypothesized that a hypertonic diet would result in osmotic diarrhea and cause a compensatory down-regulation of guanylin/uroguanylin . METHODS: Gut-to-carcass weights were used to measure fluid accumulation in the intestine . Northern and/or Western analysis was used to determine the levels of guanylin, uroguanylin, and GC-C in mice with osmotic diarrhea . RESULTS: Wild-type mice fed a polyethylene glycol or lactose-based diet developed weight loss, diarrhea, and an increased gut-to-carcass ratio . Unexpectedly, 2 days on either diet resulted in increased guanylin/uroguanylin RNA and prohormone throughout the intestine, elevated uroguanylin RNA, and prohormone levels in the kidney and increased levels of circulating prouroguanylin . GC-C-deficient mice given the lactose diet reacted with higher gut-to-carcass ratios . Although they did not develop diarrhea, GC-C-sufficient and -deficient mice on the lactose diet responded with elevated levels of guanylin and uroguanylin RNA and protein . A polyethylene glycol drinking water solution resulted in diarrhea, higher gut-to-carcass ratios, and induction of guanylin and uroguanylin in both GC-C heterozygous and null animals . CONCLUSIONS: We conclude that this model of osmotic diarrhea results in a GC-C-independent increase in intestinal fluid accumulation, in levels of these peptide ligands in the epithelia of the intestine, and in prouroguanylin in the kidney and blood.

Free Radic Biol Med, 2001 Nov 1, 31(9), 1090 - 100
The peroxiredoxin gene family in Drosophila melanogaster; Radyuk SN et al.; Five peroxiredoxin genes have been identified in Drosophila melanogaster on the basis of a genome-wide search . Three of the genes (DPx-4156, DPx-4783, and DPx-5037) fall into the 2-Cys subgroup, while the other two (DPx-2540 and DPx-6005) belong to the 1-Cys subgroup . Using cDNAs, all five were expressed in E . coli and the purified recombinant proteins were shown to reduce H(2)O(2) in the presence of dithiothreitol . The three 2-Cys Prx were also shown to be active in the thioredoxin system and were, consequently, classified as thioredoxin peroxidases . Antisera raised against the DPx-4783 recombinant protein crossreacted with all family members and recognized protein species of the predicted sizes (22-27 kD) . All five family members, when individually overexpressed in Drosophila S2 cells, conferred some resistance to H(2)O(2) treatment, as measured by cell viability . Functional diversification of the Drosophila peroxiredoxin family members was suggested by two lines of evidence: (i) the patterns of mRNA accumulation varied for the different genes during development and (ii) recombinant proteins fused to an epitope tag and overexpressed in Drosophila cells, differed in subcellular localizations--three proteins occurred in the cytosol, one was localized to the mitochondria, and one was found to be secreted.

Zhonghua Gan Zang Bing Za Zhi, 2001 Oct, 9(5), 291 - 3
{Adenovirus induced acute hepatitis in non-human primates after liver- directed gene therapy}; Lu H et al.; OBJECTIVE: To define the role of lymphocyte subsets and investigate the efficiency of immunosuppression regimen in acute hepatitis in non-human primates after adenovirus mediated gene therapy . METHODS: Six rhesus monkeys were infused with E-1 deleted adenovirus (Ad) expressing E coli lacZ gene or luciferase by various routes . Four of 6 animals were immunosuppressed by cyclophosphamide and predenisone . Two monkeys were transfected with a lacZ containing plasmid with lipofectamine as the control . Lymphocyte subsets CD3, CD4, CD8, CD20 and MHC molecule beta2-microglobulin(beta2-MG) and HLA-DR in liver tissues were studied using immunohistochemical staining . RESULTS: Staining of beta2-MG and HLA-DR on the membranes of hepatocyte and increased numbers of CD3+, CD4+ and CD8+ T-lymphocytes were detected in the livers after gene transfer, while B-lymphocytes were absent . The monkeys developed a mild to moderate transient hepatitis . This was accompanied by adenovirus-mediated T-cell proliferation and neutralizing antibodies to adenovirus . Drug-induced immune suppression enhanced the ability of adenovirus- mediated gene transfer . The development of acute hepatitis and the accompanying immune abnormalities were delayed in immunosuppressed monkeys until after discontinuation of immunosuppressive therapy . Lipofectamine-mediated gene transfer was inefficient and no immune response and liver damage were observed in the livers . CONCLUSIONS: Increased numbers of beta2-MG, HLA-DR, CD3, CD4 and CD8 antigen positive cells are presented in the monkey livers after adenovirus-mediated gene therapy and induce mild to moderate transient hepatic inflammation . Immunosuppression regimen may prolong transgene expression and delay the development of acute adenoviral hepatitis.

Res Vet Sci, 2001 Jun, 70(3), 247 - 53
Oedema disease is associated with metabolic acidosis and small intestinal acidosis; Nabuurs MJ et al.; Limited information is available about the pathogenesis and pathophysiology of oedema disease (OD) . Oedema disease is caused by specific enterotoxemic Escherichia coli (SLTIIv-toxin producing) strains; however, the same strains are also found in non-afflicted pigs . Furthermore, it is unclear how the 80 kDa SLTIIv-toxin can pass the intestinal barrier . In the present paper, piglets showing signs of acute OD were anaesthetised, instrumented and cardiovascular and intestinal parameters were determined at 0, 1, 2 and 3 hours . Healthy piglets from the same herd were used as a control . Cardiac output, blood pH and bicarbonate, small intestinal intramucosal pH, and (pulmonary) blood pressure were significantly lower in OD-pigs than in control pigs . It is concluded that OD is associated with metabolic and intestinal acidosis . Intestinal acidosis is known to increase macromolecular permeability . This suggests that once OD has developed, influx of SLTIIv-toxin into the blood stream is facilitated, thus perpetuating the disease . Since intestinal permeability appears to be central in OD, it is argued that post-weaning events increase intestinal permeability and predispose individuals to OD.

Protein Expr Purif, 2001 Nov, 23(2), 348 - 58
Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase; Barber MJ et al.; A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus . The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography . The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups . The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains . Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins . Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence . The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities indicating efficient electron transfer from FAD to heme and retention of physiological function . This work represents the first successful bacterial expression of a soluble, catalytically competent, rat hepatic cytochrome b(5)-cytochrome b(5) reductase fusion protein that retains the functional properties characteristic of the individual heme and flavin domain .

Protein Expr Purif, 2001 Nov, 23(2), 338 - 47
Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones; Levy R et al.; Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm . Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, the redox potential of the cytoplasm becomes comparable to that of the mammalian endoplasmic reticulum, thus allowing the formation of disulfide bonds in certain complex proteins (P . H . Bessette et al., 1999, Proc . Natl . Acad . Sci . USA 96, 13703-13708} . Here, we investigate the expression of a Fab antibody fragment in the bacterial cytoplasm . The effect of coexpressing cytoplasmic chaperones (GroEL/ES, trigger factor, DnaK/J), as well as signal sequenceless versions of periplasmic chaperones (DsbC and Skp), was examined . Skp coexpression was shown to have the most significant effect (five- to sixfold increase) on the yield of correctly folded Fab . A maximum yield of 0.8 mg Fab/L/OD(600) Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments .

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