Escherichia coli

Protein J, 2004 Jan, 23(1), 85 - 94
Activation parameters for the spontaneous and pressure-induced phases of the dissociation of single-ring GroEL (SR1) chaperonin; Panda M et al.; We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range 0.5-3.0 kbar . The kinetics were studied as a function of temperature in the range 15-35 degrees C . The dissociation processes at each pressure and temperature showed biphasic behavior . The slower rate (k1,obs) was confirmed to be the self-dissociation of SR1 at any specific temperature at atmospheric pressure . This dissociation was pressure independent and followed concentration-dependent first-order kinetics . The self-dissociation rates followed normal Eyring plots (In k1,obs/T vs . 1/T) from which the free energy of activation (deltaG++ = 22 +/- 0.3 kcal mol(-1)), enthalpy of activation (deltaH++ = 18 +/- 0.5 kcal mol(-1)), and entropy of activation (deltaS++ = -15 +/- 1 kcal mol(-1)) were evaluated . The effect of pressure on the dissociation rates resulted in nonlinear behavior (ln k2,obs vs . pressure) at all the temperatures studied indicating that the activation volumes were pressure dependent . Activation volumes at zero pressure (V++o) and compressibility factors (beta++) for the dissociation rates at the specific temperatures were calculated . This is the first systematic study where the self-dissociation of an oligomeric chaperonin as well as its activation parameters are reported.

Folia Microbiol (Praha), 2004, 49(1), 59 - 63
Genotyping of fimbrial adhesins in Escherichia coli strains isolated from Slovak piglets suffering from diarrhea; Vu-Khac H et al.; One-hundred sixty Escherichia coli isolates obtained from piglets with diarrhea from different parts of Slovakia were examined for the presence of genes coding for F4, F5, F6 and F41 fimbrial adhesins, and hemolytic activity . According to polymerase chain reaction tests 74 (46%) E . coli isolates were positive for primers that detected genes coding for fimbrial adhesins . Of these 74 isolates, 64 were positive for genes encoding for F4+, four for F5+, five for F6+, and one for both F41+ and F5+ adhesins.

Eur J Immunol, 2004 May, 34(5), 1451 - 60
Porphyromonas gingivalis lipopolysaccharides induce maturation of dendritic cells with CD14+CD16+ phenotype; Kanaya S et al.; Primary immune responses are initiated by dendritic cells (DC) that inform naive T helper cells about invading pathogens . DC undergo sequential events leading to irreversible maturation upon bacterial stimulation . To investigate the responses of DC during periodontal infection, we studied the effects of LPS from Porphyromonas gingivalis on DC . DC generated from human peripheral monocytes by culture with IL-4 and GM-CSF were incubated with P . gingivalis LPS (Pg LPS) or Escherichia coli LPS (Ec LPS) . Flow cytometry and real-time quantitative RT-PCR analysis revealed that Pg LPS, but not Ec LPS, preferentially up-regulated CD14 and CD16 expression at protein and mRNA levels . Furthermore, Pg LPS preferentially induced the secretion of soluble CD14 . CD1a, HLA-DR and CD54 were highly expressed on DC stimulated with both kinds of LPS; however, CD40, CD80, CD83 and CD86 expression on Pg LPS-stimulated DC was lower than on Ec LPS-stimulated DC . With regard to IL-6, IL-8, IL-10, IL-12 and RANTES production from DC and allogeneic T cell proliferation, Pg LPS was a weaker stimulator than Ec LPS . These results suggested that Pg LPS triggers maturation of DC with unique characteristics, which exhibited weak immunostimulatory activity and may contribute to induction of chronic inflammation at the site of periodontal infection.

Anal Biochem, 2004 May 15, 328(2), 196 - 202
General assay for sugar nucleotidyltransferases using electrospray ionization mass spectrometry; Zea CJ et al.; An electrospray ionization mass spectrometry-based assay has been developed to study the class of enzymes called sugar nucleotidyltransferases that couple sugar-1-phosphates and nucleotide triphosphates to form Leloir pathway glycosyl donors . The recombinant Escherichia coli and the commercially available yeast uridine-diphosphoglucose pyrophosphorylases were used as model systems . This technique allows the simultaneous and direct detection of the substrates and products without separation and, as described, is as sensitive as traditional coupled techniques . More importantly, the assay is capable of easily measuring kinetic values and inhibition constants for a range of natural and nonnatural substrates . This new assay was used to show for the first time that the reaction of the commercially available yeast uridine-diphosphoglucose pyrophosphorylase preparation is competitively inhibited by adenosine 5'-triphosphate (ATP), an observation that indicates a single active site that accepts both uridine 5'-triphosphate and ATP substrates.

Anal Biochem, 2004 May 15, 328(2), 147 - 54
Sesquiterpene lactones inhibit luciferase but not beta-galactosidase activity in vitro and ex vivo; Lindenmeyer MT et al.; Reporter enzymes such as firefly luciferase or beta-galactosidase of Escherichia coli are frequently used to study transcriptional activity of genes and to investigate the effects of novel compounds on gene or transcription factor activity . It is generally assumed that the activity of these enzymes is unaffected by the treatment conditions . Therefore, this factor is not considered when interpreting the data obtained . Biologically active compounds such as sesquiterpene lactones (SLs) have also been tested in reporter gene assays for their influence on gene expression . Here we show in in vitro and ex vivo experiments that SLs inhibit firefly luciferase activity probably by direct targeting of the enzyme while beta-galactosidase remains almost completely unaffected . The loss of luciferase activity after SL treatment could be an effect of their sulfhydryl-modifying potency and the subsequent alteration of the enzyme's tertiary structure . These results demonstrate that the effect of the test substance on the reporter enzyme used should be taken into consideration when the transcriptional effect of novel compounds is investigated.

Anal Biochem, 2004 May 15, 328(2), 139 - 46
Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography; Ohtomo R et al.; Polyphosphate is ubiquitous and has a variety of biochemical functions . Among polyphosphate quantification methods, an enzymatic assay using Escherichia coli polyphosphate kinase (PPK), in which polyphosphate is converted to adenosine 5'-triphosphate and quantified by luciferase assay, is the most specific and most sensitive . However, chain-length specificity of the assay has not been analyzed in detail so far . Ion chromatography equipped with an on-line hydroxide eluent generator enabled us to analyze polyphosphate up to 50 inorganic phosphate (P(i)) residues, and we employed this method to investigate the chain-length specificity of PPK in this study . Several fractions of short-chain polyphosphate were prepared by electrophoresis, and the chain-length distribution was analyzed before and after 1-6 h PPK reaction by ion chromatography . Polyphosphates longer than 23 P(i) residues were processed by PPK completely after 1 h incubation, but complete processing of those between 11 and 22 P(i) residues required 6h incubation . Limited processing of polyphosphates of 10 P(i) residues or shorter were observed even after 6h incubation . Metachromasy of Toluidine blue O, an alternative method for polyphosphate quantification, showed broader chain-length specificity although it was not as sensitive as the enzymatic assay . Combination of these two methods would be practically applicable to analysis of polyphosphate dynamics in living organisms.

Vet Immunol Immunopathol, 2004 May, 99(1-2), 99 - 111
Cloning and sequencing of a cDNA expressing a ribosomal P0 peptide from Culicoides nubeculosus (Diptera); Althaus H et al.; Insect bite dermal hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides spp . and sometimes Simulium spp . The aim of the investigation presented here was to identify allergens causing IBH . A cDNA library expressing recombinant Culicoides nubeculosus proteins was screened using affinity-purified serum from an IBH-affected horse . Screening of the library resulted in identification of one immunoreactive clone . The sequence of the cDNA insert was determined and revealed a 600 bp insert with an open reading frame coding for a 78 amino acid long protein, called rCul n 1 . Analysis of the deduced amino acid sequence revealed an identity of 67-78% to the C-terminal part of the 318 amino acid long ribosomal P0 protein from other Diptera . Furthermore, the 38 C-terminal amino acids displayed an identity of 57% with the C-terminal part of the acidic ribosomal protein P2 from Aspergillus fumigatus . The cDNA insert was subcloned and expressed as a {His}6-tagged protein in Escherichia coli and purified using Ni2(+)-chelate affinity chromatography . The 10kDa recombinant Cul n 1 protein bound the affinity-purified antibody fraction used for screening the expression library . Determination of IgE and IgG levels against rCul n 1 by ELISA in sera from 19 IBH-affected and 18 Swiss control horses and in sera from eight control horses living in Iceland showed no significant differences between the three groups of horses (median IgE levels = 60, 49 and 44 relative ELISA units, respectively) . rCul n 1 did not induce sulfidoleukotriene (sLT) release from peripheral blood leukocytes of IBH-affected horses (N = 5), although sLT release was induced with the Culicoides whole body extract .

Metab Eng, 2004 Apr, 6(2), 164 - 74
Effect of zwf gene knockout on the metabolism of Escherichia coli grown on glucose or acetate; Zhao J et al.; The mutant deficient in glucose-6-phosphate dehydrogenase (G6PDH) was constructed by disrupting zwf gene by one-step inactivation protocol using polymerase chain reaction primers . The knockout of zwf gene was shown to have different influence on the metabolism of Escherichia coli grown on glucose or acetate . The decreased rates of substrate uptake and CO(2) production were found for the mutant grown on acetate, whereas these two rates were increased during the growth on glucose . The metabolic flux analysis based on (13)C-labeling experiments indicates that the metabolism of the mutant grown on glucose is related to the higher flux via tricorboxylic acid (TCA) cycle to generate anabolic reducing equivalents normally provided by the oxidative pentose phosphate pathway . However, the metabolism of the mutant grown on acetate shows a lower flux towards the TCA cycle as compared with the parent strain . The decreased flux through TCA cycle is associated with an increased flux via the glyoxylate shunt, by which the carbon source can bypass the two decarboxylative steps of TCA cycle in which CO(2) is released, thus conserving more carbon for biosynthesis in response to the decreased uptake rate of the carbon source.

BMC Bioinformatics . 2004 Feb 18;5(1):15.
Network analysis of metabolic enzyme evolution in Escherichia coli; Light S et al.; BACKGROUND: The two most common models for the evolution of metabolism are the patchwork evolution model, where enzymes are thought to diverge from broad to narrow substrate specificity, and the retrograde evolution model, according to which enzymes evolve in response to substrate depletion . Analysis of the distribution of homologous enzyme pairs in the metabolic network can shed light on the respective importance of the two models . We here investigate the evolution of the metabolism in E . coli viewed as a single network using EcoCyc . RESULTS: Sequence comparison between all enzyme pairs was performed and the minimal path length (MPL) between all enzyme pairs was determined . We find a strong over-representation of homologous enzymes at MPL 1 . We show that the functionally similar and functionally undetermined enzyme pairs are responsible for most of the over-representation of homologous enzyme pairs at MPL 1 . CONCLUSIONS: The retrograde evolution model predicts that homologous enzymes pairs are at short metabolic distances from each other . In general agreement with previous studies we find that homologous enzymes occur close to each other in the network more often than expected by chance, which lends some support to the retrograde evolution model . However, we show that the homologous enzyme pairs which may have evolved through retrograde evolution, namely the pairs that are functionally dissimilar, show a weaker over-representation at MPL 1 than the functionally similar enzyme pairs . Our study indicates that, while the retrograde evolution model may have played a small part, the patchwork evolution model is the predominant process of metabolic enzyme evolution.

BMC Immunol . 2004 Mar 16;5(1):5.
Endothelial cells present antigens in vivo; Rothermel AL et al.; BACKGROUND: Immune recognition of vascular endothelial cells (EC) has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment . However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition . Here, we examined antigen presentation by EC in vivo by testing immune responses against E . coli beta-galactosidase (beta-gal) in two lines of transgenic mice that express beta-gal exclusively in their EC . TIE2-lacZ mice express beta-gal in all EC and VWF-lacZ mice express beta-gal in heart and brain microvascular EC . RESULTS: Transgenic and congenic wild type FVB mice immunized with beta-gal expression vector DNA or beta-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets . The immunized transgenic mice remained healthy, their EC continued to express beta-gal, and their blood vessels showed no histological abnormalities . In response to beta-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-gamma . Infection with recombinant vaccinia virus encoding beta-gal raised equivalent responses in transgenic and FVB mice . Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express beta-gal . These results suggested immunological ignorance of the transgene encoded EC protein . However, skin transplanted from TIE2-lacZ onto FVB mice lost beta-gal+ EC and the hosts developed beta-gal-specific antisera, demonstrating activation of host immune effector mechanisms . In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained beta-gal+ EC and no antisera developed, suggesting a tolerant host immune system . CONCLUSION: Resting, beta-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against beta-gal expressed by EC within transplanted skin . We conclude that EC effectively present intracellular "self" proteins to the immune system . However, antigen presentation by EC does not delete or anergize a large population of specific lymphocytes that respond to the same protein following conventional immunization with protein or expression vector DNA . These results clearly demonstrate striking context sensitivity in the immune recognition of EC, a subtlety that must be better understood in order to treat immune diseases and complications involving the vasculature.

J Agric Food Chem, 2004 May 5, 52(9), 2552 - 60
8S globulin of mungbean {Vigna radiata (L.) Wilczek}: cloning and characterization of its cDNA isoforms, expression in Escherichia coli, purification, and crystallization of the major recombinant 8S isoform; Bernardo AE et al.; Three isoforms of the cDNA of the major 8S globulin of mungbean, 8Salpha, 8Salpha', and 8Sbeta, were isolated, cloned, and characterized . The cDNA sequences of 8Salpha, 8Salpha', and 8Sbeta had open reading frames of 1362, 1359 or 1362, and 1359 bp, respectively, which code for 454, 453 or 454, and 453 amino acids corresponding to molecular weights of 51 973, 51 627 or 51 758, and 51 779, respectively . Homology in terms of cDNA and amino acid sequences was 91-92% between 8Salpha and 8Salpha', 87% between 8Salpha and 8Sbeta, and 86-88% between 8Salpha' and 8Sbeta . The signal peptide was found to be 1-25, 1-24 or 25, and 1-23 for 8Salpha, 8Salpha', and 8Sbeta, respectively, using the signalP website (Nielsen, H.; Engelbrecht, J.; Brunak, S.; von Heijne, G . Protein Eng . 1997, 10, 1-6) . The propeptide was determined to be IVHREN . A single site for glycosylation (N-X-S/T) was observed about 90 amino acids from the C terminus . Homology between mungbean 8S isoforms and other 7-8S proteins ranged from 45 to 68% within members of the legume family and 29 to 34% for crops of different species . The major isoform 8Salpha was expressed in Escherichia coli and purified by successive ammonium sulfate fractionation, hydrophobic interaction, and Mono Q column chromatography . The recombinant 8Salpha, but not the native form, was successfully crystallized producing rhombohedral crystals.

Chem Biol, 2004 Jan, 11(1), 10 - 2
Unraveling the pathway of lipoic acid biosynthesis; Booker SJ; Lipoic acid is almost universally required for aerobic metabolism . However, the mechanism for its synthesis and incorporation into proteins has remained elusive . A groundbreaking study published in the December issue of Chemistry & Biology uncovers critical features of the lipoic acid biosynthetic pathway.

Biophys J, 2004 May, 86(5), 3314 - 23
Cytomechanical properties of papaver pollen tubes are altered after self-incompatibility challenge; Geitmann A et al.; Self-incompatibility (SI) in Papaver rhoeas triggers a ligand-mediated signal transduction cascade, resulting in the inhibition of incompatible pollen tube growth . Using a cytomechanical approach we have demonstrated that dramatic changes to the mechanical properties of incompatible pollen tubes are stimulated by SI induction . Microindentation revealed that SI resulted in a reduction of cellular stiffness and an increase in cytoplasmic viscosity . Whereas the former cellular response is likely to be the result of a drop in cellular turgor, we hypothesize that the latter is caused by as yet unidentified cross-linking events . F-actin rearrangements, a characteristic phenomenon for SI challenge in Papaver, displayed a spatiotemporal gradient along the pollen tube; this suggests that signal propagation occurs in a basipetal direction . However, unexpectedly, local application of SI inducing S-protein did not reveal any evidence for localized signal perception in the apical or subapical regions of the pollen tube . To our knowledge this represents the first mechanospatial approach to study signal propagation and cellular responses in a well-characterized plant cell system . Our data provide the first evidence for mechanical changes induced in the cytoplasm of a plant cell stimulated by a defined ligand.

Biophys J, 2004 May, 86(5), 3230 - 40
Noninvasive auto-photoreduction used as a tool for studying structural changes in heme-copper oxidases by FTIR spectroscopy; Bettinger K et al.; We demonstrate an efficient Fourier transform infrared (FTIR) spectroscopic method, termed "auto-photoreduction," that uses anaerobic photo-induced internal electron transfer to monitor reaction-initiated changes of heme-copper oxidases . It can be applied without the use of either expensive electrochemical equipment, or caged compounds, which cause significant background signals . At high irradiation power, carbon monoxide is released from high-spin heme a of cytochrome c oxidase and heme o from cytochrome bo(3) . Photochemistry is initiated at wavelengths <355 nm, and the photochemical action spectrum has a maximum of 290 nm for cytochrome bo(3), which is consistent with the possible intermediate involvement of tyrosinate or an activated state of tyrosine . We propose that the final electron donors are proton channel water molecules . In the pH range of 4-9, the noninvasive auto-photoreduction method yields highly reproducible FTIR redox difference spectra within a broad range, resolving a number of vibrational changes outside the amide I region (1600-1640 cm(-1)) . Furthermore, it provides details of redox-induced changes in the spectral region between 1600 and 1100 cm(-1) . The auto-photoreduction method should be universally applicable to heme proteins.

Biophys J, 2004 May, 86(5), 3131 - 40
Conformation and dynamics of the {3-(13)C}Ala, {1-(13)C}Val-labeled truncated pharaonis transducer, pHtrII(1-159), as revealed by site-directed (13)C solid-state NMR: changes due to association with phoborhodopsin (sensory rhodopsin II); Yamaguchi S et al.; We have recorded (13)C NMR spectra of the {3-(13)C}Ala, {1-(13)C}Val-labeled pharaonis transducer pHtrII(1-159) in the presence and absence of phoborhodopsin (ppR or sensory rhodopsin II) in egg phosphatidylcholine or dimyristoylphosphatidylcholine bilayers by means of site-directed (amino acid specific) solid-state NMR . Two kinds of (13)C NMR signals of {3-(13)C}Ala-pHtrII complexed with ppR were clearly seen with dipolar decoupled magic angle spinning (DD-MAS) NMR . One of these resonances was at the peak position of the low-field alpha-helical peaks (alpha(II)-helix) and is identified with cytoplasmic alpha-helices protruding from the bilayers; the other was the high-field alpha-helical peak (alpha(I)-helix) and is identified with the transmembrane alpha-helices . The first peaks, however, were almost completely suppressed by cross-polarization magic angle spinning (CP-MAS) regardless of the presence or absence of ppR or by DD-MAS NMR in the absence of ppR . This is caused by an increased fluctuation frequency of the cytoplasmic alpha-helix from 10(5) Hz in the uncomplexed states to >10(6) Hz in the complexed states, leading to the appearance of peaks that were suppressed because of the interference of the fluctuation frequency with the frequency of proton decoupling (10(5) Hz), as viewed from the (13)C NMR spectra of {3-(13)C}Ala-labeled pHtrII . Consistent with this view, the (13)C DD-MAS NMR signals of the cytoplasmic alpha-helices of the complexed {3-(13)C}Ala-pHtrII in the dimyristoylphosphatidylcholine (DMPC) bilayer were partially suppressed at 0 degrees C due to a decreased fluctuation frequency at the low temperature . In contrast, examination of the (13)C CP-MAS spectra of {1-(13)C}Val-labeled complexed pHtrII showed that the (13)C NMR signals of the transmembrane alpha-helix were substantially suppressed . These spectral changes are again interpreted in terms of the increased fluctuation frequency of the transmembrane alpha-helices from 10(3) Hz of the uncomplexed states to 10(4) Hz of the complexed states . These findings substantiate the view that the transducers alone are in an aggregated or clustered state but the ppR-pHtrII complex is not aggregated . We show that (13)C NMR is a very useful tool for achieving a better understanding of membrane proteins which will serve to clarify the molecular mechanism of signal transduction in this system.

Biophys J, 2004 May, 86(5), 2883 - 95
Water dynamics and dewetting transitions in the small mechanosensitive channel MscS; Anishkin A et al.; The dynamics of confined water in capillaries and nanotubes suggests that gating of ion channels may involve not only changes of the pore geometry, but also transitions between water-filled and empty states in certain locations . The recently solved heptameric structure of the small mechanosensitive channel of Escherichia coli, MscS, has revealed a relatively wide (7-15 A) yet highly hydrophobic transmembrane pore . Continuum estimations based on the properties of pore surface suggest low conductance and a thermodynamic possibility of dewetting . To test the predictions we performed molecular dynamics simulations of MscS filled with flexible TIP3P water . Irrespective to the initial conditions, several independent 6-ns simulations converged to the same stable state with the pore water-filled in the wider part, but predominantly empty in the narrow hydrophobic part, displaying intermittent vapor-liquid transitions . The polar gain-of-function substitution L109S in the constriction resulted in a stable hydration of the entire pore . Steered passages of Cl(-) ions through the narrow part of the pore consistently produced partial ion dehydration and required a force of 200-400 pN to overcome an estimated barrier of 10-20 kcal/mole, implying negligibly low conductance . We conclude that the crystal structure of MscS does not represent an open state . We infer that MscS gate, which is similar to that of the nicotinic ACh receptor, involves a vapor-lock mechanism where limited changes of geometry or surface polarity can locally switch the regime between water-filled (conducting) and empty (nonconducting) states.

Biophys J, 2004 May, 86(5), 2862 - 70
Cysteine scanning of MscL transmembrane domains reveals residues critical for mechanosensitive channel gating; Levin G et al.; The mechanosensitive channel of large conductance (MscL), a bacterial channel, is perhaps the best characterized mechanosensitive protein . A structure of the Mycobacterium tuberculosis ortholog has been solved by x-ray crystallography, but details of how the channel gates remain obscure . Here, cysteine scanning was used to identify residues within the transmembrane domains of Escherichia coli MscL that are crucial for normal function . Utilizing genetic screens, we identified several mutations that induced gain-of-function or loss-of-function phenotypes in vivo . Mutants that exhibited the most severe phenotypes were further characterized using electrophysiological techniques and chemical modifications of the substituted cysteines . Our results verify the importance of residues in the putative primary gate in the first transmembrane domain, corroborate other residues previously noted as critical for normal function, and identify new ones . In addition, evaluation of disulfide bridging in native membranes suggests alterations of existing structural models for the "fully closed" state of the channel.

Biophys J, 2004 May, 86(5), 2846 - 61
Gating of the large mechanosensitive channel in situ: estimation of the spatial scale of the transition from channel population responses; Chiang CS et al.; Physical expansion associated with the opening of a tension-sensitive channel has the same meaning as gating charge for a voltage-gated channel . Despite increasing evidence for the open-state conformation of MscL, the energetic description of its complex gating remains incomplete . The previously estimated in-plane expansion of MscL is considerably smaller than predicted by molecular models . To resolve this discrepancy, we conducted a systematic study of currents and dose-response curves for wild-type MscL in Escherichia coli giant spheroplasts . Using the all-point histogram method and calibrating tension against the threshold for the small mechanosensitive channel (MscS) in each patch, we found that the distribution of channels among the subconducting states is significantly less dependent on tension than the distribution between the closed and conducting states . At -20 mV, all substates together occupy approximately 30% of the open time and reduce the mean integral current by approximately 6%, essentially independent of tension or P(o) . This is consistent with the gating scheme in which the major rate-limiting step is the transition between the closed state and a low-conducting substate, and validates both the use of the integral current as a measure of P(o), and treatment of dose-response curves in the two-state approximation . The apparent energy and area differences between the states deltaE and deltaA, extracted from 29 independent dose-response curves, varied in a linearly correlated manner whereas the midpoint tension stayed at approximately 10.4 mN/m . Statistical modeling suggests slight variability of gating parameters among channels in each patch, causing a strong reduction and correlated spread of apparent deltaE and deltaA . The slope of initial parts of activation curves, with a few channels being active, gave estimates of deltaE = 51 +/- 13 kT and deltaA = 20.4 +/- 4.8 nm(2), the latter being consistent with structural models of MscL, which predict deltaA = 23 nm(2).

Arch Biochem Biophys, 2004 May 15, 425(2), 133 - 46
Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity; Annalora A et al.; A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system . Purified enzyme was stable with retention of spectral and catalytic activity . The rate of 1,25-dihydroxyvitamin D(3) {1,25(OH)(2)D(3)} side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner . It was determined from substrate-induced spectral shifts that the 1 alpha- and 25-hydroxyl groups on vitamin D(3) metabolites and analogs were the major determinants for high-affinity binding to CYP24A1 . Lowest K(d) values were obtained for 1 alpha-vitamin D(3) (0.06 microM) and 1,25-dihydroxyvitamin D(3) (0.05 microM) whereas unmodified parental vitamin D(3) and the non-secosteroid 25-hydroxycholesterol had lower affinities with K(d) values of 1.3 and 1.9 microM, respectively . The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D(3) {24,25(OH)(2)D(3)} (0.43 microM) . Kinetic analyses of the two natural substrates 25-hydroxyvitamin D(3) {25(OH)D(3)} and 1,25-dihydroxyvitamin D(3) {1,25(OH)(2)D(3)} revealed similar K(m) values (0.35 and 0.38 microM, respectively), however, the turnover number was higher for 25(OH)D(3) compared to 1,25(OH)(2)D(3) (4.2 and 1 min(-1), respectively) . Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism . Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C(23) oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D(3) to calcitroic acid . Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C(23)-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C(24) oxidation of 1,24,25-trihydroxyvitamin D(3) . Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C(23) and C(24) oxidation reactions.

FEBS Lett, 2004 Apr 30, 564(3), 264 - 8
ABC transporter architecture and mechanism: implications from the crystal structures of BtuCD and BtuF; Locher KP et al.; ABC transporters are ubiquitous membrane proteins that facilitate unidirectional substrate translocation across the lipid bilayer . Over the past five years, new crystal structures have advanced our understanding of how ABC transporters couple adenosine triphosphate (ATP) hydrolysis to substrate transport . In the following, we will briefly review the results of these structural investigations and outline their mechanistic implications.

Int J Parasitol, 2004 May, 34(6), 683 - 92
EtCRK2, a cyclin-dependent kinase gene expressed during the sexual and asexual phases of the Eimeria tenella life cycle; Kinnaird JH et al.; EtCRK2, a cyclin-dependent kinase from the coccidian parasite, Eimeria tenella is closely related to eukaryotic cyclin-dependent kinases that regulate progression of the cell cycle and to several cyclin-dependent kinases identified in the Apicomplexa . Northern blot analyses revealed that EtCRK2 is transcribed during both asexual (first-generation schizogony) and sexual (oocyst sporulation) replicative phases of the parasite life cycle . In addition, it appears to be transcriptionally regulated during meiosis . Recombinant EtCRK2 produced in Escherichia coli has kinase activity which is significantly stimulated by the addition of vertebrate cyclin A . This cyclin-dependent kinase may play a significant role in regulating critical cell cycle events during both asexual proliferation and sexual development of the parasite.

J Mol Biol, 2004 May 14, 338(5), 1027 - 36
A combined transmembrane topology and signal peptide prediction method; Kall L et al.; An inherent problem in transmembrane protein topology prediction and signal peptide prediction is the high similarity between the hydrophobic regions of a transmembrane helix and that of a signal peptide, leading to cross-reaction between the two types of predictions . To improve predictions further, it is therefore important to make a predictor that aims to discriminate between the two classes . In addition, topology information can be gained when successfully predicting a signal peptide leading a transmembrane protein since it dictates that the N terminus of the mature protein must be on the non-cytoplasmic side of the membrane . Here, we present Phobius, a combined transmembrane protein topology and signal peptide predictor . The predictor is based on a hidden Markov model (HMM) that models the different sequence regions of a signal peptide and the different regions of a transmembrane protein in a series of interconnected states . Training was done on a newly assembled and curated dataset . Compared to TMHMM and SignalP, errors coming from cross-prediction between transmembrane segments and signal peptides were reduced substantially by Phobius . False classifications of signal peptides were reduced from 26.1% to 3.9% and false classifications of transmembrane helices were reduced from 19.0% to 7.7% . Phobius was applied to the proteomes of Homo sapiens and Escherichia coli . Here we also noted a drastic reduction of false classifications compared to TMHMM/SignalP, suggesting that Phobius is well suited for whole-genome annotation of signal peptides and transmembrane regions . The method is available at as well as at

Insect Biochem Mol Biol, 2004 May, 34(5), 493 - 500
Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana; Ampasala DR et al.; A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae . CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa . CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively . Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues . Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages . The highest level of CfTf expression was detected in the fat body . Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut . Expression of CfTf mRNA could be induced by bacteria but not fungi . Expression of CfTf mRNA was suppressed by iron load.

Bioorg Med Chem, 2004 May 15, 12(10), 2787 - 95
Acetyltransfer in natural product biosynthesis--functional cloning and molecular analysis of vinorine synthase; Bayer A et al.; Vinorine synthase (EC 2.3.1.160) catalyses the acetyl-CoA- or CoA-dependent reversible formation of the alkaloids vinorine (or 11-methoxy-vinorine) and 16-epi-vellosimine (or gardneral) . The forward reaction leads to vinorine, which is a direct biosynthetic precursor along the complex pathway to the monoterpenoid indole alkaloid ajmaline, an antiarrhythmic drug from the Indian medicinal plant Rauvolfia serpentina . Based on partial peptide sequences a cDNA clone was isolated and functionally expressed in Escherichia coli . The Km values of the native enzyme for gardneral and acetyl-CoA were determined to be 7.5 and 57 microM . The amino acid sequence of vinorine synthase has highest level of identity (28-31%) to that of Papaver salutaridinol acetyltransferase, Fragaria alcohol acyltransferase, and Catharanthus deacetylvindoline acetyltransferase involved in morphine, flavor, and vindoline biosynthesis, respectively . Vinorine synthase is a novel member of the BAHD superfamily of acyltransferases . Site-directed mutagenesis of 13 amino acid residues provided clear evidence that both, His160 and Asp164 of the consensus sequence HxxxD belong to the catalytic center . The mutations also showed that an amino acid triad is not characteristic of vinorine synthase . The experiments demonstrated the importance of the conserved motif SxL/I/VD near the N-terminus and the consensus sequence DFGWG near the C-terminal.

Biochem Biophys Res Commun, 2004 May 21, 318(1), 73 - 80
Cu+ distribution in metallothionein fragments; Salgado MT et al.; The differential distribution of Cu+ between separate alpha and beta domains of metallothionein (the isolated peptide fragments) and the rate of transfer of Cu+ between the two domains using copper-thiolate specific emission spectroscopy are reported . Kinetic data show the rate of transfer of Cu+ from the Cu6alpha to the Cd3beta domain is 2 x 10(-1) s(-1) while the transfer from Cu6beta to the Cd4alpha domain is much slower at 8 x 10(-3) s(-1), indicating the greater binding affinity of Cu+ for the MT beta domain . We report that the emission intensity of Cu6beta is 0.45 the emission intensity of Cu6alpha-MT . Lambda(max) is shown to be a probe of the environment of the Cu+ . A series of copper-containing domain intermediates to the formation of the filled Cu6S9-beta and Cu6S11-alpha-clusters are identified . A mechanism is proposed for the formation of Cu12(betaalpha)-MT that involves metal exchange reactions of Cu+ ions from the alpha to the beta domain with initial formation of a Cu4beta-cluster.

Biochem Biophys Res Commun, 2004 May 21, 318(1), 11 - 6
Groups on the side chain of T252 in Escherichia coli leucyl-tRNA synthetase are important for discrimination of amino acids and cell viability; Xu MG et al.; Leucyl-tRNA synthetase (LeuRS) catalyzes the leucylation of tRNA(Leu) . To maintain the fidelity of protein biosynthesis, LeuRS also catalyzes the editing reaction . In the present work, highly conserved T252 in the T-rich region within CP1 domain of Escherichia coli LeuRS was mutated to G, D, or E . Steady-state kinetic of aminoacylation, and combined editing assays indicated that not only the size of the amino acid but also the absence of hydrogen bonds between T252 and adjacent molecules may affect the editing . It is further confirmed by in vivo experiments using the temperature-sensitive strain KL231 (DeltaleuS), which revealed the arrested growth of bacterial cells bearing mutants with highly impaired editing activity in the presence of leucine analog.

Appl Radiat Isot, 2004 Jun, 60(6), 839 - 43
2-{(18)F}-fluoroisonicotinic acid hydrazide: biological evaluation in an acute infection model; Amartey JK et al.; We have synthesized 2-{(18)F}-fluoroisonicotinic acid hydrazide by nucleophilic displacement reaction on ethyl-2- (trimethylammonium)-isonicotinate precursor in acetonitrile . Kryptofix 222 was used as the phase transfer catalyst . The intermediate fluorinated ethyl ester reacted with hydrazine hydrate to produce the hydrazide . Excellent radiochemical yield was attained with total synthesis time of approximately 60 min . Biological evaluation was performed in bacterial cells and biodistribution in normal as well as E . coli infected CBA/J mice . It was found that the S . pneumoniae cells retained the radiotracer in an in vitro assay . The tracer showed positive localization at the infection/inflammation site in E . coli infected mice.

Fish Shellfish Immunol, 2004 May, 16(5), 571 - 9
Protection of Penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine; Witteveldt J et al.; Although invertebrates lack a true adaptive immune response, the potential to vaccinate Penaeus monodon shrimp against white spot syndrome virus (WSSV) using the WSSV envelope proteins VP19 and VP28 was evaluated . Both structural WSSV proteins were N-terminally fused to the maltose binding protein (MBP) and purified after expression in bacteria . Shrimp were vaccinated by intramuscular injection of the purified WSSV proteins and challenged 2 and 25 days after vaccination to assess the onset and duration of protection . As controls, purified MBP- and mock-vaccinated shrimp were included . VP19-vaccinated shrimp showed a significantly better survival (p<0.05) as compared to the MBP-vaccinated control shrimp with a relative percent survival (RPS) of 33% and 57% at 2 and 25 days after vaccination, respectively . Also, the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival when challenged two days after vaccination (RPS of 44% and 33%, respectively), but not after 25 days . These results show that protection can be generated in shrimp against WSSV using its structural proteins as a subunit vaccine . This suggests that the shrimp immune system is able to specifically recognize and react to proteins . This study further shows that vaccination of shrimp may be possible despite the absence of a true adaptive immune system, opening the way to new strategies to control viral diseases in shrimp and other crustaceans.

Bioelectrochemistry, 2004 Jun, 63(1-2), 79 - 85
Binding of single nucleotides to H+-ATP synthases observed by fluorescence resonance energy transfer; Steigmiller S et al.; F(0)F(1)-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate . The enzyme has three catalytic nucleotide binding sites, one on each beta-subunit; three non-catalytic binding sites are located mainly on each alpha-subunit . In order to observe substrate binding to the enzyme, the H(+)-ATP synthase from Escherichia coli was labelled selectively with the fluorescence donor tetramethylrhodamine (TMR) at position T106C of the gamma-subunit . The labelled enzymes were incorporated into liposomes and catalysed proton-driven ATP synthesis . The substrate ATP-Alexa Fluor 647 was used as the fluorescence acceptor to perform intermolecular fluorescence resonance energy transfer (FRET) . Single molecules are detected with a confocal set-up . When one ATP-Alexa Fluor 647 binds to the enzyme, FRET can be observed . Five stable states with different intermolecular FRET efficiencies were distinguished for enzyme-bound ATP-Alexa Fluor 647 indicating binding to different binding sites . Consecutive hydrolysis of excess ATP resulted in stepwise changes of the FRET efficiency . Thereby, gamma-subunit movement during catalysis was directly monitored with respect to the binding site with bound ATP-Alexa Fluor 647.

Bioelectrochemistry, 2004 Jun, 63(1-2), 43 - 7
Redox-triggered events in cytochrome c nitrite reductase; Gwyer JD et al.; Escherichia coli cytochrome c nitrite reductase is a homodimeric enzyme whose 10 heme centres range in reduction potential from ca . -30 to -320 mV . Protein film voltammetry (PFV) was performed to assess how the reactivity of the enzyme towards a number of small molecules was influenced by heme oxidation state . The experimental approach provided a high-resolution description of activity across the electrochemical potential domain by virtue of the fact that the enzyme sample was under the precise potential control of an electrode at all times . The current potential profiles displayed by nitrite reductase revealed that heme oxidation state has a profound, and often unanticipated, effect on the interactions with substrate molecules, nitrite and hydroxylamine, as well as the inhibitor, cyanide . Thus, PFV provides a powerful route to define redox-triggered events in this complex multi-centred redox enzyme.

Bioelectrochemistry, 2004 Jun, 63(1-2), 25 - 30
Electroanalytical determination of d(GCGAAGC) hairpin; Trnkova L et al.; Hairpins (or hairpin-like structures) may play a major role in expansion events of triplet repeat expansion diseases (X syndrome, Huntington's disease, Friedreich's ataxia) . The d(GCGAAGC) fragment has been found in the replication origins of phage phiX 174 and herpes simplex virus, in a promoter region of an Escherichia coli heat-shock gene, and in rRNA genes . The paper deals with the application of electrochemical methods to the determination of the DNA heptamer-d(GCGAAGC) which forms very stable hairpin structure in aqueous solutions . On mercury electrodes, this hairpin provides voltammetric reduction signals of adenine and cytosine, and oxidation signals of guanine . Both signals have been studied by cyclic voltammetry (CV), linear sweep voltammetry (LSV), and elimination voltammetry with linear scan (EVLS) in dependence on pH, accumulation time, scan rate, and loop sequences . The EVLS in combination with the adsorptive stripping was employed to the determination of the detection limit (LD) of this mini-hairpin (2 nM) . Multidimensional voltammetric data were worked up by Fourier Transform (FT) and for the first coefficient a confidence ellipse was calculated in order to drop out some outlier data . The same method was used also for detection limit determinations . The values of LD obtained by two approaches were compared.

Bioorg Chem, 2004 Jun, 32(3), 178 - 91
Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay; Nguyen KT et al.; A new spectrophotometric/fluorimetric assay for peptide deformylase (PDF) has been developed by coupling the PDF reaction with that of dipeptidyl peptidase I (DPPI) and using N-formyl-Met-Lys-AMC as substrate . Removal of the N-terminal formyl group by PDF renders the dipeptide an efficient substrate of DPPI, which subsequently removes the dipeptidyl units to release 7-amino-4-methylcoumarin as the chromophore/fluorophore . The PDF reaction is conveniently monitored on a UV-Vis spectrophotometer or a fluorimeter in a continuous fashion . The utility of the assay was demonstrated by determining the catalytic activity of PDF and the inhibition constants of PDF inhibitors . These studies revealed the slow-binding behavior of a previously reported macrocyclic PDF inhibitor . This method offers several advantages over the existing PDF assays and should be particularly useful for screening PDF inhibitors in the continuous fashion.

FEMS Microbiol Lett, 2004 May 1, 234(1), 133 - 7
A FhuA mutant of Escherichia coli is infected by phage T1-independent of TonB; Langenscheid J et al.; Infection of Escherichia coli K-12 by phages T1 and phi 80 requires the FhuA outer membrane protein and the TonB protein . Mutations in the N-terminal globular domain close to the predicted channel in the beta-barrel of FhuA were created . The FhuA Delta 107-111 N104K K110D L111P mutant and the FhuA(L(109)DPNGLK(110)) insertion mutant were sensitive to phage T1, but nearly resistant to phage phi 80 . FhuA Delta 107-111 N104K K110D L111P mediated phage T1 infection in a tonB mutant without formation of TonB-independent phage T1 host-range mutants . The FhuA mutants showed no altered sensitivity to phage T5 . Although the phages share overlapping binding sites in FhuA, the structural alterations elicited by the mutations resulted in very different phage sensitivities . In the FhuA deletion mutant, the TonB requirement for phage T1 infection was partially bypassed.

Int J Biochem Cell Biol, 2004 Jul, 36(7), 1348 - 64
Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties; Lakaye B et al.; Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation . We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28) . As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties . For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E . coli, produced the protein on a large scale and purified it to homogeneity . Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional . Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+ . With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP . The activity was maximum at pH 8.5 and very low at pH 6.0 . Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0 . Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+ . The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding . The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix.

Emerg Infect Dis, 2004 Mar, 10(3), 518 - 21
Enterotoxin-producing Escherichia coli O169:H41, United States; Beatty ME et al.; From 1996 to 2003, 16 outbreaks of enterotoxigenic Escherichia coli (ETEC) infections in the United States and on cruise ships were confirmed . E . coli serotype O169:H41 was identified in 10 outbreaks and was the only serotype in 6 . This serotype was identified in 1 of 21 confirmed ETEC outbreaks before 1996.

Biochemistry, 2004 May 4, 43(17), 5055 - 64
A monomeric human apolipoprotein E carboxyl-terminal domain; Fan D et al.; ApoE plays a critical role in lipoprotein metabolism and plasma lipid homeostasis through its high-affinity binding to the LDL-receptor family . In solution, apoE is an oligomeric protein and the C-terminal domain causes apoE's aggregation . The aggregation property presents a major difficulty for the structural determination of this protein . A high-level expression system of the apoE C-terminal domain is reported here . Using protein engineering techniques, we identified a monomeric, biologically active apoE C-terminal domain mutant . This mutant replaces five bulky hydrophobic residues in the region of residues 253-289 with either smaller hydrophobic or polar/charged residues (F257A, W264R, V269A, L279Q, and V287E) . The solubility of the mutant is significantly increased ( approximately 10-fold) . Cross-linking experiments indicate that this mutant is 100% monomeric even at 5 mg/mL . CD and guanidine hydrochloride denaturation results indicate that the mutant maintains an identical alpha-helical secondary structure and stability as compared with those of the wild-type protein . DMPC-binding assays demonstrate an identical vesicle clearance rate shared by both the mutant and the wild-type apoE C-terminal domain . In addition, electron microscopic results show identical recombinant HDL particles prepared with both the mutant and the wild-type proteins . These results indicate that residues F257, W264, V269, L279, and V287 are critical residues for aggregation but may not be important in maintaining the structure, stability, and lipid-binding activity of this apoE domain, suggesting that apoE may use different "epitopes" for its aggregation property, helical structure/stability, and lipid-binding activity . Finally, preliminary NMR data demonstrated that we have collected high-quality NMR spectra, allowing for an NMR structural determination of the apoE C-terminal domain.

Biochemistry, 2004 May 4, 43(17), 4906 - 12
Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro; Bacha U et al.; SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus . A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CL(pro) (also termed SARS-CoV 3CL(pro)) . We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CL(pro) as well as a truncated form containing only the catalytic domains . The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered . Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the alpha-helical domain . Differential scanning calorimetry indicates that the alpha-helical domain does not contribute to the structural stability of the catalytic domains . Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities . In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid . This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development . It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM . Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CL(pro) in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2003, 21(6), 353 - 6
{Study on the subcloning, expression and immunoprotection with Schistosoma japonicum CAI gene}; Luo XJ et al.; OBJECTIVE: To subclone and express the new gene of Schistosoma japonicum (Sj) CAI and evaluate the immunoprotective effect of the recombinant molecule . METHODS: The cDNA of SjCAI gene was subcloned into expression vector pGEX-5X-3 to form recombinants which were then used to transform to E . coli strain ER 2566 . Expression was induced by IPTG . The mice were vaccinated with the expressed protein and the immunoprotective effect was tested . RESULTS: Fusion protein of SjGST-CAI was highly expressed in E . coli as inclusion bodies . The worm reduction rate and the liver egg reduction rate in vaccination group of SjGST-CAI were 29.87% and 63.71%, respectively . CONCLUSION: SjCAI gene can be highly expressed in E . coli after subcloning into pGEX-5X-3 vector and the expressed fusion protein can induce immunoprotective effect against Sj in mice.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2003, 21(6), 345 - 8
{Preliminary identification of T cell epitopes on 22.6 kDa antigen of Schistosoma japonicum}; Wang XJ et al.; OBJECTIVE: To identify the T cell epitopes on 22.6 kDa antigen of Schistosoma japonicum (Sj22.6) . METHODS: The primary structure of Sj22.6 molecule was analysed using various predictive algorithms and a panel of 4 peptides were acquired . Their oligonucleotides were designed, synthesized and inserted into the multiple cloning site of plasmid pET-32c(+) . The recombinant plasmids were transformed into E . coli BL21 and identified by endonuclease digestion and sequencing . The positive clones containing the recombinant plasmids could express specific fusion proteins (trx-epitope, MW approximately 20 kDa) induced by IPTG . The fusion protein with 6 x His could be coupled with NTA resin specifically, and purified by elusion of the column with buffer containing imidazole . The purified fusion proteins were incubated with splenocytes of C3H mice and then, the proliferation of splenocytes was determined by 3H-TdR incorporation assay . RESULTS: The recombinant plasmids were constructed successfully and the positive clones containing the recombinant plasmids expressed specific fusion proteins . Three of the purified fusion proteins (P4, P5, P6) could stimulate the lymphocyte proliferation . CONCLUSION: Three T cell epitopes on Sj22.6 antigen were identified.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2003, 21(5), 279 - 81
{Cloning and expression of the signaling protein 14-3-3 of Toxoplasma gondii}; Du J et al.; OBJECTIVE: To clone and express the cell signaling protein 14-3-3 gene from Toxoplasma gondii RH strain . METHODS: Toxoplasma RH strain tachyzoites, which maintained by mouse passage, were harvested from ascites of mice and genomic DNA was prepared . A pair of primers were designed and synthesized based on the sequence of Toxo 14-3-3 cDNA . A specific fragment of Toxo 14-3-3 gene was obtained by RT-PCR amplification from Toxoplasma genomic DNA . The PCR products were ligated to pGEM-T . The EcoRI/Xho I restricted fragments, confirmed by PCR and EcoRI/XhoI digestion, were cloned into expression vector pET28a and the recombinants were transformed into E . coli BL21 . Fusion expression was induced by isopropyl-beta-D-thiogalactoside (IPTG) and confirmed by Western blotting with rabbit anti-Toxoplasma sera . RESULTS: The molecular size of Toxo 14-3-3 was 803 bp, which is highly homologous to the previous report cloned from the parasites of intestinal epithelial stage in cat . High expression was obtained in pET28a/Toxo 14-3-3/E . coli BL21 when confirmed by Western blotting . CONCLUSION: The recombinant construction of Toxo 14-3-3 was generated and expression was induced.

J Bone Miner Metab, 2004, 22(3), 176 - 84
Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone; Nampei A et al.; The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause oncogenic hypophosphatemic osteomalacia (OHO) . MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone mineralization . We developed a rabbit polyclonal antibody directed against recombinant human MEPE (rhMEPE) after cloning its cDNA from the cDNA library of a nasal tumor tissue causing OHO . Using this antibody, we analyzed the distribution of MEPE in human bones by immunohistochemistry . In bone specimens from normal subjects, MEPE was predominantly expressed by osteocytes and not by osteoblasts . In bone specimens from patients with osteomalacia, however, MEPE was focally expressed by deeply located osteocytes . We also compared the MEPE positivity of osteocytes in mineralized bone and non-mineralized osteoid obtained from patients with osteomalacia and osteoporosis . Among osteomalacia patients, MEPE positivity was seen in 87.5 +/- 8.6% of the osteocytes from mineralized bone compared with 7.8 +/- 6.4% of those from osteoid . Among osteoporosis patients, MEPE positivity was found in 95.3 +/- 0.5% of the osteocytes from mineralized bone compared with 4.9 +/- 5.7% of those from osteoid . MEPE was mainly expressed by osteocytes embedded in the matrix of mineralized bone from patients with osteomalacia or osteoporosis . Our data provide the first histological evidence that MEPE is predominantly expressed by osteocytes in human bone, with significant expression by osteocytes within mineralized bone.

Nat Genet, 2004 May, 36(5), 486 - 91 Epub 2004 Apr 25.
Just-in-time transcription program in metabolic pathways; Zaslaver A et al.; A primary goal of systems biology is to understand the design principles of the transcription networks that govern the timing of gene expression . Here we measured promoter activity for approximately 100 genes in parallel from living cells at a resolution of minutes and accuracy of 10%, based on GFP and Lux reporter libraries . Focusing on the amino-acid biosynthesis systems of Escherichia coli, we identified a previously unknown temporal expression program and expression hierarchy that matches the enzyme order in unbranched pathways . We identified two design principles: the closer the enzyme is to the beginning of the pathway, the shorter the response time of the activation of its promoter and the higher its maximal promoter activity . Mathematical analysis suggests that this 'just-in-time' (ref . 5) transcription program is optimal under constraints of rapidly reaching a production goal with minimal total enzyme production . Our findings suggest that metabolic regulation networks are designed to generate precision promoter timing and activity programs that can be understood using the engineering principles of production pipelines.

J Vet Med Sci, 2004 Mar, 66(3), 269 - 75
Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21; Muneta Y et al.; A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 microg/ml anti-porcine CD3 antibody for 48 hr . The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids . The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identity to the bovine, human, and murine IL-21, respectively . The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22-->q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby . The recombinant porcine mature IL-21 expressed by E . coli induced dose-dependent proliferation and IFN-gamma production from a human NK cell line, NK0 . The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs.

Plant Physiol, 2004 May, 135(1), 103 - 11 Epub 2004 Apr 23.
Folate biosynthesis in higher plants . cDNA cloning, heterologous expression, and characterization of dihydroneopterin aldolases; Goyer A et al.; Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants . This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli . The E . coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin . Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1-3, sharing 57%-73% identity), for which cDNAs were isolated . A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR . When overproduced in E . coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN . AtFolB3, however, could not be expressed in active form . Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme . Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance . Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families . The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 7046 - 51 Epub 2004 Apr 23.
Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination; Lundgren M et al.; Chromosome replication origins were mapped in vivo in the two hyperthermophilic archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus, by using microarray-based marker frequency analysis . Bidirectional replication was found to be initiated in near synchrony from three separate sites in both organisms . Two of the three replication origins in each species were located in the vicinity of a cdc6/orc1 replication initiation gene, whereas no known replication-associated gene could be identified near the third origin in either organism . In contrast to initiation, replication termination occurred asynchronously, such that certain replication forks continued to progress for >40 min after the others had terminated . In each species, all replication forks advanced at similar DNA polymerization rates; this was found to be an order of magnitude below that displayed by Escherichia coli and thus closer to eukaryotic elongation rates . In S . acidocaldarius, a region containing short regularly spaced repeats was found to hybridize aberrantly, as compared to the rest of the chromosome, raising the possibility of a centromere-like function.

Nucleic Acids Res . 2004 Apr 23;32(7):e66.
Control of siRNA expression using the Cre-loxP recombination system; Kasim V et al.; Gene silencing mediated by RNA interference (RNAi) was first discovered in Caenorhabditis elegans, and was subsequently recognized in various other organisms . In mammalian cells, RNAi can be induced by small interfering RNAs (siRNAs) . In earlier studies, our group developed a vector-based system for expression of siRNA under control of a polymerase III promoter, the U6 promoter, which can induce RNAi in living cells . We here describe a system for controlling the U6 promoter-driven expression of siRNA using the Cre-loxP recombination system . We constructed a 'Cre-On' siRNA expression vector which could be switched on upon excision catalyzed by Cre recombinase, which was delivered to cells directly from the medium as a fusion protein . An examination of the effectiveness of RNAi against a reporter gene revealed that addition of TAT-NLS-Cre (where NLS is a nuclear localization signal and TAT is a peptide of 11 amino acids derived from HIV) to the medium resulted in plasmid recombination, generation of siRNA and suppression of reporter activity . This system should allow us to induce RNAi in a spatially, temporally, cell type-specifically or tissue-specifically controlled manner and potentiate the improved application of RNAi in both an experimental and a therapeutic context.

J Biol Chem, 2004 Jun 25, 279(26), 26811 - 6 Epub 2004 Apr 23.
Elucidating the role of conserved glutamates in H+-pyrophosphatase of Rhodospirillum rubrum; Malinen AM et al.; H(+)-pyrophosphatase (H(+)-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes . All 50 of the known H(+)-PPase amino acid sequences contain four invariant glutamate residues . In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu(197), Glu(202), Glu(550), and Glu(649) in the H(+)-PPase of Rhodospirillum rubrum (R-PPase) . All residues were replaced with Asp and Ala . The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles . All substitutions, except E202A, generated enzymes capable of PP(i) hydrolysis and PP(i)-energized proton translocation, indicating that the negative charge of Glu(202) is essential for R-PPase function . The hydrolytic activities of all other PPase variants were impaired at low Mg(2+) concentrations but were only slightly affected at high Mg(2+) concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two- and three-metal pathways . Substitution of Glu(197), Glu(202), and Glu(649) resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions . Following the substitutions of Glu(550) and Glu(649), R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure . None of the substitutions affected the coupling of PP(i) hydrolysis to proton transport.

Oral Microbiol Immunol, 2004 Jun, 19(3), 182 - 7
Induction of calprotectin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils; Kido J et al.; Calprotectin, a major cytosolic protein of leukocytes, is detected in neutrophils, monocytes/macrophages, and epithelial cells . This protein is known to be a marker for several inflammatory diseases and is detected in inflammatory gingival tissue with periodontal disease . Recently, we found that the calprotectin level in gingival crevicular fluid from periodontitis patients was significantly higher than that of healthy subjects . However, the regulation of calprotectin in periodontal disease is unclear . In the present study, we investigated the effect of lipopolysaccharides of periodontopathic bacteria on calprotectin release from human neutrophils . Neutrophils from healthy donors were treated with lipopolysaccharides from Porphyromonas gingivalis (P-LPS), Actinobacillus actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, and Escherichia coli . Calprotectin of neutrophil was identified by immunoblotting and calprotectin amount was determined by ELISA . Two subunits (10 and 14 kDa) of calprotectin were observed in the cell and medium fractions from neutrophils . P-LPS increased calprotectin release from seven to 16 times the control level after 30 min and its effect appeared in a dose-dependent manner (10-1000 ng/ml) . Lipopolysaccharides from A . actinomycetemcomitans, P . intermedia, F . nucleatum, and E . coli also induced calprotectin release from neutrophils . These results suggest that lipopolysaccharides from periodontopathic bacteria induce calprotectin release from human neutrophils . Copyright Blackwell Munksgaard, 2004.

Trends Biochem Sci, 2004 Feb, 29(2), 55 - 7
Light traffic: photo-crosslinking a novel transport system; Palmer T et al.; The Tat protein transporter is found in the membranes of many bacteria and in plant chloroplasts . This highly unusual transport machine moves folded and often oligomeric substrate proteins across energy-conserving membranes . A recent paper reports the first use of a photo-crosslinking approach to dissect the early recognition events between the transporter and its substrate.

J Gen Virol, 2004 May, 85(Pt 5), 1181 - 9
Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein; Kukkonen SK et al.; The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts . The middle third (nt 2191-4344) could be expressed in E . coli and was used to immunize rabbits . The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells . The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells . Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 10(4) copies per cell at the peak level of expression . The antiserum was also used to detect the L protein in cell fractionation studies . In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction . The membrane association was confirmed with membrane flotation assays . To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L-EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system . L-EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

Antimicrob Agents Chemother, 2004 May, 48(5), 1856 - 64
Crystal structures of Escherichia coli topoisomerase IV ParE subunit (24 and 43 kilodaltons): a single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase; Bellon S et al.; Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome . The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication . Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-A resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-A resolution . The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures . We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM) . The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 micro M) . These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase . Additionally, the enzyme kinetic parameters were affected . In gyrase, the ATP K(m) increased approximately 5-fold and the V(max) decreased approximately 30% . In contrast, the topoisomerase IV ATP K(m) decreased by a factor of 6, and the V(max) increased approximately 2-fold from the wild-type values . These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.

Cell Microbiol, 2004 Jun, 6(6), 499 - 508
Developing RNase P ribozymes for gene-targeting and antiviral therapy; Trang P et al.; RNase P, a tRNA processing enzyme, contains both RNA and protein subunits . M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, recognizes its target RNA substrate mainly on the basis of its structure and cleaves a double stranded RNA helix at the 5' end that resembles the acceptor stem and T-stem structure of its natural tRNA substrate . Accordingly, a guide sequence (GS) can be covalently attached to the M1 RNA to generate a sequence specific ribozyme, M1GS RNA . M1GS ribozyme can target any mRNA sequence of choice that is complementary to its guide sequence . Recent studies have shown that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1 and human cytomegalovirus, and the BCR-ABL oncogenic mRNA in vitro and effectively reduce the expression of these mRNAs in cultured cells . Moreover, an in vitro selection scheme has been developed to select for M1 GS ribozyme variants with more efficient catalytic activity in cleaving mRNAs . When expressed in cultured cells, these selected ribozymes also show an enhance ability to inhibit viral gene expression and growth . These recent results demonstrate the feasibility of developing the M1GS ribozyme-based technology as a promising gene targeting approach for basic research and clinical therapeutic application.

Biochem J, 2004 Aug 1, 381(Pt 3), 823 - 9
Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli; Kim YH et al.; The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis . Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain . The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant . Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated . Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of aspartate, a key metabolic precursor of threonine . Synthesis of aspartase (aspartate ammonia-lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of aspartate to fumarate and ammonia . Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant . The far lower level of cystathionine beta-lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine . In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E . coli mutant.

J Nat Prod, 2004 Apr, 67(4), 678 - 81
New pigments from the terrestrial cyanobacterium Scytonema sp . collected on the Mitaraka inselberg, French Guyana; Bultel-Ponce V et al.; Inselbergs are hills rising abruptly from the surrounding plains where cyanobacteria are the only living organisms under conditions of intense solar radiation . A survival mechanism to prevent UV-damage has been associated with synthesis of the ultraviolet-screening, photostable sheath pigment scytonemin . The organic extract of Scytonema sp., collected on the Mitaraka inselberg, French Guyana, yielded three new pigments, tetramethoxyscytonemin (1), dimethoxyscytonemin (2), and scytonine (3), derived from the scytoneman skeleton of scytonemin . These structures were assigned mainly on the basis of (1)H and (13)C NMR and MS experiments.

J Nat Prod, 2004 Apr, 67(4), 672 - 4
New sesterterpenes from the Antarctic sponge Suberites sp; Lee HS et al.; Suberitenones C and D and suberiphenol, three new sesterterpenes of the suberitane class, were isolated from the sponge Suberites sp . collected from Antarctica . The structures of these compounds were determined on the basis of combined spectral and chemical analyses.

Cancer Genet Cytogenet, 2004 Feb, 149(1), 81 - 4
Detection of mutations in the cDNA of the proliferation marker Ki-67 protein in four tumor cell lines; Buban T et al.; The Ki-67 protein has an essential role in cell proliferation . It is present in all dividing cells of normal and tumor tissues, but absent in resting cells . At present, no data are available about any alterations in the gene of this protein that could contribute to its altered structure and function, resulting in tumor development . We therefore searched for mutations in the Ki-67 gene (MKI67) . cDNAs from four tumor cell lines derived from carcinoma of the cervix (HeLa), colon (CXF94, SW480), and lung (A549) were prepared . Defined parts of the cDNA were amplified by specific primers, cloned into pCRII-Blunt-TOPO vector, and replicated in Escherichia coli . The sequence of the amplified products were determined by automated fluorescence sequencing . Eight different mutations were characterized in the four cell lines tested . One is a deletion of a single base at position 1496 causing a truncated protein, the second is a A433T exchange is a silent mutation, and the remaining six mutations result in an amino acid change that might alter the conformation of the protein . Our results show that several mutations exist within the Ki-67 protein's cDNA in four tumor cell lines . These mutations might provide a genetic basis for tumor development.

Biotechnol Lett, 2004 Mar, 26(5), 385 - 91
Cloning and expression of rapeseed procruciferin in Escherichia coli and crystallization of the purified recombinant protein; Tandang MR et al.; Two rapeseed cruciferin cDNAs (cru2/3a and cru2/3b) were cloned and sequenced . A comparison of their DNA and protein sequences with other cruciferins, indicated cru2/3b to be a novel clone and, among them, an inherent and highly conserved sequence of twelve amino acids was identified . Procruciferin 2/3a and 2/3b were expressed in Eschericha coli, and procruciferin 2/3a was obtained in a soluble form . The expressed procruciferin 2/3a has a trimeric structure and formed crystals although the quality was not good, suggesting that this expression system is useful for protein engineering of procruciferin 2/3a.

Mol Immunol, 2004 Mar, 40(16), 1179 - 88
Expression and characterization of recombinant soluble monkey CD3 molecules: mapping the FN18 polymorphic epitope; Wang Z et al.; The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T cell depleting reagent when conjugated to binding site mutants of diphtheria toxin . This anti-CD3 antibody shares certain properties with its anti-human counterparts UCHT1, OKT3 and Leu-4 in that it precipitates two different CD3 chains from membrane detergent extracts . However, in contrast to human CD3, rhesus and cynomolgus monkeys display CD3 polymorphisms producing FN18 negative phenotypes . Using recently published sequence data, we have expressed the ectodomains of cynomolgus CD3-epsilon CD3-gamma and CD-delta chains in E . coli, and have refolded these chains separately and in pairs to produce CD3 homo- and heterodimeric proteins . These proteins were fractionated by anion exchange according to their differing isoelectric points and further identified by size differences on SDS gels . On the basis of ELISA, the FN18 epitope is restricted to the CD3-epsilongamma ectodomain heterodimer, CD-epsilondelta and CD-epsilonepsilon being non-reactive . Either of the two amino acid polymorphisms reported in the CD3-epsilon chain were sufficient to degrade the bioactivity of the CD3-epsilongamma towards FN18.

Acta Crystallogr D Biol Crystallogr, 2004 May, 60(Pt 5), 985 - 6 Epub 2004 Apr 21.
Detection of L-lactate in polyethylene glycol solutions confirms the identity of the active-site ligand in a proline dehydrogenase structure; Zhang M et al.; Polyethylene glycol (PEG) is often used in protein crystallography as a low-ionic-strength precipitant for crystallization and as a cryoprotectant for low-temperature data collection . Prompted by the discovery of an apparent L-lactate molecule bound in the active site of the Escherichia coli PutA proline dehydrogenase domain crystal structure, the L-lactate concentration of several PEG solutions was measured . 50%(w/v) solutions of PEGs with molecular weight 3000, 4000 and 8000 contain millimolar levels of L-lactate . In contrast, L-lactate was not detected in solutions of PEG monomethyl ethers or PEG 3350 . These results help to explain why L-lactate was present in the proline dehydrogenase domain crystal structure . This work also has implications for the crystallization of enzymes that bind L-lactate.

Acta Crystallogr D Biol Crystallogr, 2004 May, 60(Pt 5), 962 - 4 Epub 2004 Apr 21.
Expression, purification and preliminary crystallographic studies of a single-point mutant of Mos1 mariner transposase; Richardson JM et al.; A soluble single-point mutant of full-length Mos1 mariner transposase (MW = 40.7 kDa) has been overexpressed in Escherichia coli, purified to 95% homogeneity and crystallized . This provides the first example of the crystallization of a eukaryotic transposase . The native crystals diffract to 2.5 A resolution and show tetragonal symmetry, with unit-cell parameters a = b = 44.5, c = 205.6 A . Multiple-wavelength anomalous data from a selenomethionyl form of the protein and data from a heavy-atom derivative have been collected.

Acta Crystallogr D Biol Crystallogr, 2004 May, 60(Pt 5), 903 - 11 Epub 2004 Apr 21.
Structure of OsmC from Escherichia coli: a salt-shock-induced protein; Shin DH et al.; The crystal structure of an osmotically inducible protein (OsmC) from Escherichia coli has been determined at 2.4 A resolution . OsmC is a representative protein of the OsmC sequence family, which is composed of three sequence subfamilies . The structure of OsmC provides a view of a salt-shock-induced protein . Two identical monomers form a cylindrically shaped dimer in which six helices are located on the inside and two six-stranded beta-sheets wrap around these helices . Structural comparison suggests that the OsmC sequence family has a peroxiredoxin function and has a unique structure compared with other peroxiredoxin families . A detailed analysis of structures and sequence comparisons in the OsmC sequence family revealed that each subfamily has unique motifs . In addition, the molecular function of the OsmC sequence family is discussed based on structural comparisons among the subfamily members.

Methods Mol Biol, 2004, 265, 101 - 15
Geminivirus vectors for transient gene silencing in plants; Muangsan N et al.; Both RNA and DNA viruses have been engineered to serve as vectors for transient silencing in intact plants . Host gene sequences carried by the virus are seen by the plant as "foreign," and homologous gene-silencing machinery acts on both the viral vector RNA and the endogenous host gene mRNA . DNA viruses, such as geminiviruses, are advantageous for silencing because only their mRNAs are silenced and their DNA genomes continue to replicate and move . The conserved genome organization of geminiviruses and the fact that they can be cloned into Escherichia coli plasmids, propagated, and then inoculated into plants for infection simplifies the procedure for silencing specific chromosomal genes in intact plants . This chapter describes the development of a silencing vector from cabbage leaf curl virus for use in Arabidopsis and procedures for silencing two genes simultaneously.

Mol Pharmacol, 2004 May, 65(5), 1278 - 85
Activation of the anticancer prodrugs cyclophosphamide and ifosfamide: identification of cytochrome P450 2B enzymes and site-specific mutants with improved enzyme kinetics; Chen CS et al.; Cyclophosphamide (CPA) and ifosfamide (IFA) are oxazaphosphorine anticancer prodrugs metabolized by two alternative cytochrome P450 (P450) pathways, drug activation by 4-hydroxylation and drug inactivation by N-dechloroethylation, which generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde . CPA and IFA metabolism catalyzed by P450s 2B1, 2B4, 2B5, and seven site-specific 2B1 mutants was studied in a reconstituted Escherichia coli expression system to identify residues that contribute to the unique activities and substrate specificities of these enzymes . The catalytic efficiency of CPA 4-hydroxylation by rat P450 2B1 was 10- to 35-fold higher than that of rabbit P450 2B4 or 2B5 . With IFA, approximately 50% of metabolism proceeded via N-dechloroethylation for 2B1 and 2B4, whereas CPA N-dechloroethylation corresponded to only approximately 3% of total metabolism (2B1) or was absent (2B4, 2B5) . Improved catalytic efficiency of CPA and IFA 4-hydroxylation was obtained upon substitution of 2B1 Ile-114 by Val, and replacement of Val-363 by Leu or Ile selectively suppressed CPA N-dechloroethylation >or=90% . P450 2B1-V367A, containing the Ala replacement found in 2B5, exhibited only approximately 10% of wild-type 2B1 activity for both substrates . Canine P450 2B11, which has Val-114, Leu-363, and Val-367, was therefore predicted to be a regioselective CPA 4-hydroxylase with high catalytic efficiency . Indeed, P450 2B11 was 7- to 8-fold more active as a CPA and IFA 4-hydroxylase than 2B1, exhibited a highly desirable low K(m) (80-160 microM), and catalyzed no CPA N-dechloroethylation . These findings provide insight into the role of specific P450 2B residues in oxazaphosphorine metabolism and pave the way for gene therapeutic applications using P450 enzymes with improved catalytic activity toward these anticancer prodrug substrates.

Mol Pharmacol, 2004 May, 65(5), 1148 - 58
Cloning, expression, and characterization of three new mouse cytochrome p450 enzymes and partial characterization of their fatty acid oxidation activities; Wang H et al.; The mammalian CYP2C subfamily is one of the largest and most complicated in the cytochrome P450 superfamily . In this report, we describe the organization of the mouse Cyp2c locus, which contains 15 genes and four pseudogenes, all of which are located in a 5.5-megabase region on chromosome 19 . We cloned three novel mouse CYP2C cDNAs (designated CYP2C50, CYP2C54, and CYP2C55) from mouse heart, liver, and colon, respectively . All three cDNAs contain open reading frames that encode 490 amino acid polypeptides that are 57 to 95% identical to other CYP2Cs . The recombinant CYP2C proteins were expressed in Escherichia coli after N-terminal modification, partially purified, and shown to be active in the metabolism of both arachidonic acid (AA) and linoleic acid, albeit with different catalytic efficiencies and profiles . CYP2C50 and CYP2C54 metabolize AA to epoxyeicosatrienoic acids (EETs) primarily, and linoleic acid to epoxyoctadecenoic acids (EOAs) primarily, whereas CYP2C55 metabolizes AA to EETs and hydroxyeicosatetraenoic acids and linoleic acid to EOAs and hydroxyoctadecadienoic acids . Northern blotting and reverse transcription-polymerase chain reaction analysis reveal that CYP2C50 transcripts are abundant in liver and heart; CYP2C54 transcripts are present in liver, kidney, and stomach; and CYP2C55 transcripts are abundant in liver, colon, and kidney . Immunoblotting studies demonstrate that CYP2C50 protein is expressed in liver and heart, CYP2C54 protein is detected primarily in liver, and CYP2C55 protein is present primarily in colon . Immunohistochemistry reveals that CYP2C55 is most abundant in surface columnar epithelium in the cecum . We conclude that these new CYP2C enzymes are probably involved in AA and linoleic acid metabolism in mouse hepatic and extrahepatic tissues.

J Biol Chem, 2004 Jun 25, 279(26), 27410 - 21 Epub 2004 Apr 21.
Efficient intracellular delivery of a protein and a low molecular weight substance via recombinant polyomavirus-like particles; Abbing A et al.; Efficient encapsulation of foreign molecules like proteins and low molecular weight drugs into polyoma virus-like particles (capsoids) was achieved by the development of an anchoring technique based upon the specific interaction of the inner core protein VP2 with VP1 pentamers . A stretch of 49 amino acids of VP2 served as an anchor molecule, either expressed as a fusion protein with green fluorescent protein (GFP) or covalently linked to methotrexate (MTX) . The loaded capsoids showed regular morphology and stability for several months . GFP and MTX were internalized into cells in vitro, as was demonstrated by the detection of GFP and VP1 fluorescence in mouse fibroblasts and the cytostatic effect of intracellularly released MTX on leukemia T cells.

J Biol Chem, 2004 Jul 2, 279(27), 27957 - 64 Epub 2004 Apr 21.
Influence of GrpE on DnaK-substrate interactions; Brehmer D et al.; The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides . This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively . Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor . However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP . In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids . A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding . Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP . These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion.

Infect Immun, 2004 May, 72(5), 3022 - 30
Glycosylation of Anaplasma marginale major surface protein 1a and its putative role in adhesion to tick cells; Garcia-Garcia JC et al.; Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne rickettsial pathogen of cattle that multiplies in erythrocytes and tick cells . Major surface protein 1a (MSP1a) and MSP1b form the MSP1 complex of A . marginale, which is involved in adhesion of the pathogen to host cells . In this study we tested the hypothesis that MSP1a and MSP1b were glycosylated, because the observed molecular weights of both proteins were greater than the deduced molecular masses . We further hypothesized that the glycosylation of MSP1a plays a role in adhesion of A . marginale to tick cells . Native and Escherichia coli-derived recombinant MSP1a and MSP1b proteins were shown by gas chromatography to be glycosylated and to contain neutral sugars . Glycosylation of MSP1a appeared to be mainly O-linked to Ser/Thr residues in the N-terminal repeated peptides . Glycosylation may play a role in adhesion of A . marginale to tick cells because chemical deglycosylation of MSP1a significantly reduced its adhesive properties . Although the MSP1a polypeptide backbone alone was adherent to tick cell extract, the glycans in the N-terminal repeats appeared to enhance binding and may cooperatively interact with one or more surface molecules on host cells . These results demonstrated that MSP1a and MSP1b are glycosylated and suggest that the glycosylation of MSP1a plays a role in the adhesion of A . marginale to tick cells.

Infect Immun, 2004 May, 72(5), 2947 - 55
Erythrocyte invasion by Babesia bovis merozoites is inhibited by polyclonal antisera directed against peptides derived from a homologue of Plasmodium falciparum apical membrane antigen 1; Gaffar FR et al.; Apical membrane antigen 1 (AMA-1) is a micronemal protein secreted to the surface of merozoites of Plasmodium species and Toxoplasma gondii tachyzoites in order to fulfill an essential but noncharacterized function in host cell invasion . Here we describe cloning and characterization of a Babesia bovis AMA-1 homologue designated BbAMA-1 . The overall level of similarity of BbAMA-1 to P . falciparum AMA-1 was low (18%), but characteristic features like a transmembrane domain near the C terminus, a predicted short cytoplasmic C-terminal sequence with conserved sequence properties, and an extracellular domain containing 14 conserved cysteine residues putatively involved in disulfide bridge formation are typical of AMA-1 . Rabbit polyclonal antisera were raised against three synthetic peptides derived from the N-terminal region and domains II and III of the putative extracellular domain and were shown to recognize specifically recombinant BbAMA-1 expressed in Escherichia coli . Immunofluorescence microscopy showed that there was labeling of the apical half of merozoites with these antisera . Preincubation of free merozoites with all three antisera reduced the efficiency of invasion of erythrocytes by a maximum of 65% . Antisera raised against the N-terminal peptide detected a 82-kDa protein on Western blots and a 69-kDa protein in the supernatant that was harvested after in vitro invasion, suggesting that proteolytic processing and secretion take place during or shortly after invasion . A combination of two-dimensional Western blotting and metabolic labeling allowing direct identification of spots reacting with the BbAMA-1 peptide antisera together with the very low silver staining intensity of these spots indicated that very low levels of BbAMA-1 are present in Babesia merozoites.

Infect Immun, 2004 May, 72(5), 2648 - 58
Cloning and characterization of the gene encoding the major cell-associated phospholipase A of Legionella pneumophila, plaB, exhibiting hemolytic activity; Flieger A et al.; Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of amoebae, macrophages, and epithelial cells . The pathology of Legionella infections involves alveolar cell destruction, and several proteins of L . pneumophila are known to contribute to this ability . By screening a genomic library of L . pneumophila, we found an additional L . pneumophila gene, plaB, which coded for a hemolytic activity and contained a lipase consensus motif in its deduced protein sequence . Moreover, Escherichia coli harboring the L . pneumophila plaB gene showed increased activity in releasing fatty acids predominantly from diacylphospho- and lysophospholipids, demonstrating that it encodes a phospholipase A . It has been reported that culture supernatants and cell lysates of L . pneumophila possess phospholipase A activity; however, only the major secreted lysophospholipase A PlaA has been investigated on the molecular level . We therefore generated isogenic L . pneumophila plaB mutants and tested those for hemolysis, lipolytic activities, and intracellular survival in amoebae and macrophages . Compared to wild-type L . pneumophila, the plaB mutant showed reduced hemolysis of human red blood cells and almost completely lost its cell-associated lipolytic activity . We conclude that L . pneumophila plaB is the gene encoding the major cell-associated phospholipase A, possibly contributing to bacterial cytotoxicity due to its hemolytic activity . On the other hand, in view of the fact that the plaB mutant multiplied like the wild type both in U937 macrophages and in Acanthamoeba castellanii amoebae, plaB is not essential for intracellular survival of the pathogen.

Infect Immun, 2004 May, 72(5), 2618 - 27
Regulation of proinflammatory cytokine expression by Shiga toxin 1 and/or lipopolysaccharides in the human monocytic cell line THP-1; Harrison LM et al.; Infection with Shiga toxin (Stx)-producing bacteria and the subsequent release of Stxs and endotoxins into the bloodstream may damage blood vessels in the colon, kidneys, and central nervous system, leading to bloody diarrhea, acute renal failure, and neurological complications . The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) may contribute to the pathogenesis of Stx-induced vascular lesions by up-regulating toxin receptor expression on endothelial cells . We previously showed that macrophages treated with purified Shiga toxin 1 (Stx1) or lipopolysaccharides (LPS) secrete TNF-alpha and IL-1beta . Northern blot analysis revealed that treatment of the human monocytic cell line THP-1 with LPS induced a rapid and transient increase in steady-state TNF-alpha and IL-1beta transcripts . In contrast, Stx1 induced slower but prolonged elevations in cytokine transcripts . The presence of both stimulants resulted in optimal cytokine mRNA induction in terms of kinetics and prolonged expression . Compared to LPS, Stx1 was a poor inducer of IL-1beta protein expression, although levels of soluble IL-1beta induced by all treatments continually increased over 72 h . IL-1beta transcripts were not induced by Stx1 B-subunits . Using the transcriptional inhibitor actinomycin D, we determined that treatment with Stx1 or Stx1 plus LPS induced cytokine transcripts with increased stability compared to transcripts induced by LPS alone . For all treatments, IL-1beta mRNA decay was slower than TNF-alpha . Collectively, our data suggest that Stxs affect cytokine expression, in part, at the posttranscriptional level by stabilizing mRNAs . Optimal TNF-alpha expression occurs when both Stxs and LPS are present.

Infect Immun, 2004 May, 72(5), 2484 - 93
Involvement of lipoprotein NlpI in the virulence of adherent invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease; Barnich N et al.; Escherichia coli strain LF82 recovered from a chronic lesion of a patient with Crohn's disease (CD) is able to adhere to and invade cultured intestinal epithelial cells and to replicate within macrophages . One mutant selected for its impaired ability to invade epithelial cells had an insertion of a Tn phoA transposon within the nlpI gene encoding the lipoprotein NlpI . A NlpI-negative isogenic mutant showed a 35-fold decrease in its ability to adhere to and a 45-fold decrease in its ability to invade Intestine-407 cells, but its ability to survive and to replicate within macrophages was similar to that of wild-type strain LF82 . In addition, this mutant did not express flagella and synthesized very small amounts of type 1 pili . Downregulation of type 1 pili in the NlpI-negative mutant resulted from a preferential switch toward the OFF position of the invertible DNA element located upstream of the fim operon . The FimB and FimE recombinases act in concert to control the switch, and a large decrease in fimB and fimE mRNA levels was observed . The absence of flagellar structures correlated with a drastic 19-fold decrease in the fliC mRNA level, regardless of the FlhD(2)C(2) transcriptional regulator and of the sigma(28) transcription factor . The key role of NlpI in virulence is independent of type 1 pili and motility, since induced type 1 pilus expression and/or forced contact between bacteria and intestinal epithelial cells did not restore the ability of the NlpI mutant to adhere to and to invade intestinal epithelial cells.

Clin Cancer Res, 2004 Apr 15, 10(8), 2879 - 90
NY-ESO-1 protein formulated in ISCOMATRIX adjuvant is a potent anticancer vaccine inducing both humoral and CD8+ t-cell-mediated immunity and protection against NY-ESO-1+ tumors; Maraskovsky E et al.; NY-ESO-1 is a 180 amino-acid human tumor antigen expressed by many different tumor types and belongs to the family of "cancer-testis" antigens . In humans, NY-ESO-1 is one of the most immunogenic tumor antigens and NY-ESO-1 peptides have been shown to induce NY-ESO-1-specific CD8(+) CTLs capable of altering the natural course of NY-ESO-1-expressing tumors in cancer patients . Here we describe the preclinical immunogenicity and efficacy of NY-ESO-1 protein formulated with the ISCOMATRIX adjuvant (NY-ESO-1 vaccine) . In vitro, the NY-ESO-1 vaccine was readily taken up by human monocyte-derived dendritic cells, and on maturation, these human monocyte-derived dendritic cells efficiently cross-presented HLA-A2-restricted epitopes to NY-ESO-1-specific CD8(+) T cells . In addition, epitopes of NY-ESO-1 protein were also presented on MHC class II molecules to NY-ESO-1-specific CD4(+) T cells . The NY-ESO-1 vaccine induced strong NY-ESO-1-specific IFN-gamma and IgG2a responses in C57BL/6 mice . Furthermore, the NY-ESO-1 vaccine induced NY-ESO-1-specific CD8(+) CTLs in HLA-A2 transgenic mice that were capable of lysing human HLA-A2(+) NY-ESO-1(+) tumor cells . Finally, C57BL/6 mice, immunized with the NY-ESO-1 vaccine, were protected against challenge with a B16 melanoma cell line expressing NY-ESO-1 . These data illustrate that the NY-ESO-1 vaccine represents a potent therapeutic anticancer vaccine.

Exp Hematol, 2004 Feb, 32(2), 224 - 33
An embryonic-specific repressor element located 3' to the Agamma-globin gene influences transcription of the human beta-globin locus in transgenic mice; Katsantoni EZ et al.; OBJECTIVE: Persistent expression of the human fetal gamma-globin genes in the adult stage is often associated with naturally occurring deletions in the human beta-globin locus . The mapping of the 5' breakpoints of these deletions within the Agamma- to delta-globin intergenic region has suggested that regulatory elements involved in the silencing of the gamma-globin genes in the adult may be present . We previously identified two elements in this region, termed Enh and F, located 3' to the Agamma-globin gene acting as silencers in transient transfection assays . Here, we tested directly the in vivo significance of these elements in the developmental regulation of the human beta-like globin genes . MATERIALS AND METHODS . We selectively deleted both Enh and F elements in the context of a 185-kb human beta-globin locus PAC (P1 phage artificial chromosome) and tested the effects of this deletion on the expression of the human P-like globin genes in transgenic mice . RESULTS: The Enh/F deletion resulted in an increase in epsilon- and gamma-globin mRNA levels in the embryonic yolk sac stage of erythropoiesis, which appears to be due to an increase in the rate of transcription rather than to an increase in the number of cells transcribing the human globin locus . However, the human developmental switching from fetal gamma-globin to adult beta-globin gene expression in transgenic mice was not affected by this deletion . CONCLUSION: These results identify Enh and F as locus-wide regulatory elements capable of down-regulating transcription of the human beta-globin locus in an embryonic-specific manner.

Curr Opin Struct Biol, 2004 Feb, 14(1), 10 - 20
Catabolite activator protein: DNA binding and transcription activation; Lawson CL et al.; Recently determined structures of the Escherichia coli catabolite activator protein (CAP) in complex with DNA, and in complex with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) and DNA, have yielded insights into how CAP binds DNA and activates transcription . Comparison of multiple structures of CAP-DNA complexes has revealed the contributions of direct and indirect readout to DNA binding by CAP . The structure of the CAP-alphaCTD-DNA complex has provided the first structural description of interactions between a transcription activator and its functional target within the general transcription machinery . Using the structure of the CAP-alphaCTD-DNA complex, the structure of an RNA polymerase-DNA complex, and restraints from biophysical, biochemical and genetic experiments, it has been possible to construct detailed three-dimensional models of intact class I and class II transcription activation complexes.

Mol Microbiol, 2004 May, 52(3), 903 - 16
Tryptophan recycling is responsible for the interferon-gamma resistance of Chlamydia psittaci GPIC in indoleamine dioxygenase-expressing host cells; Wood H et al.; Comparative genomics indicates that vast differences in Chlamydia sp . host range and disease characteristics can be traced back to subtle variations in gene content within a region of the chromosome termed the plasticity zone . Genes required for tryptophan biosynthesis are located in the plasticity zone; however, the complement of genes encoded varies depending on the chlamydial species examined . Of the sequenced chlamydia genomes, Chlamydia psittaci GPIC contains the most complete tryptophan biosynthesis operon, encoding trpRDCFBA . Immediately downstream of the trp operon are genes encoding kynureninase and ribose phosphate pyrophosphokinase . Here, we show that, in GPIC, these genes are transcribed as a single transcript, the expression of which is regulated by tryptophan . Complementation analyses, using various mutant Escherichia coli isolates, indicate that the tryptophan biosynthesis, kynureninase and ribose phosphate pyrophosphokinase gene products are functional . Furthermore, growth of C . psittaci GPIC in HeLa cells, cultured in tryptophan-free medium, could be rescued by the addition of anthranilate, kynurenine or indole . In total, our results indicate that this complement of genes enables GPIC to recycle tryptophan and thus accounts for the interferon-gamma resistant phenotype displayed in indoleamine-2,3-dioxygenase-expressing host cells.

Mol Microbiol, 2004 May, 52(3), 861 - 72
A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli; Outten FW et al.; The suf and isc operons of Escherichia coli have been implicated in Fe-S cluster assembly . However, it has been unclear why E . coli has two systems for Fe-S cluster biosynthesis . We have examined the regulatory characteristics and mutant phenotypes of both operons to discern if the two operons have redundant functions or if their cellular roles are divergent . Both operons are similarly induced by hydrogen peroxide and the iron chelator 2,2'-dipyridyl, although by different mechanisms . Regulation of the isc operon is mediated by IscR, whereas the suf operon requires OxyR and IHF for the response to oxidative stress and Fur for induction by iron starvation . Simultaneous deletion of iscS and most suf genes is synthetically lethal . However, although the suf and isc operons have overlapping functions, they act as distinct complexes because the SufS desulphurase alone cannot substitute for the IscS enzyme . In addition, suf deletion mutants are more sensitive to iron starvation than isc mutants, and the activity of the Fe-S enzyme gluconate dehydratase is diminished in the suf mutant during iron starvation . These findings are consistent with the model that the isc operon encodes the housekeeping Fe-S cluster assembly system in E . coli, whereas the suf operon is specifically adapted to synthesize Fe-S clusters when iron or sulphur metabolism is disrupted by iron starvation or oxidative stress.

Mol Microbiol, 2004 May, 52(3), 661 - 75
Specific recognition of rpsO mRNA and 16S rRNA by Escherichia coli ribosomal protein S15 relies on both mimicry and site differentiation; Mathy N et al.; The ribosomal protein S15 binds to 16S rRNA, during ribosome assembly, and to its own mRNA (rpsO mRNA), affecting autocontrol of its expression . In both cases, the RNA binding site is bipartite with a common subsite consisting of a G*U/G-C motif . The second subsite is located in a three-way junction in 16S rRNA and in the distal part of a stem forming a pseudoknot in Escherichia coli rpsO mRNA . To determine the extent of mimicry between these two RNA targets, we determined which amino acids interact with rpsO mRNA . A plasmid carrying rpsO (the S15 gene) was mutagenized and introduced into a strain lacking S15 and harbouring an rpsO-lacZ translational fusion . Analysis of deregulated mutants shows that each subsite of rpsO mRNA is recognized by a set of amino acids known to interact with 16S rRNA . In addition to the G*U/G-C motif, which is recognized by the same amino acids in both targets, the other subsite interacts with amino acids also involved in contacts with helix H22 of 16S rRNA, in the region adjacent to the three-way junction . However, specific S15-rpsO mRNA interactions can also be found, probably with A(-46) in loop L1 of the pseudoknot, demonstrating that mimicry between the two targets is limited.

Mol Microbiol, 2004 May, 52(3), 613 - 9
Regulation of the Escherichia coli sigma-dependent envelope stress response; Alba BM et al.; The Escherichia colisigma(E)-dependent stress response pathway controls the expression of genes encoding periplasmic folding catalysts, proteases, biosynthesis enzymes for lipid A (a component of lipopolysaccharide or LPS) and other proteins known or predicted to function in or produce components of the envelope . When E . coli is subjected to heat or other stresses that generate unfolded envelope proteins, sigma(E) activity is induced . Four key players in this signal transduction pathway have been identified: RseA, an inner membrane sigma(E) antisigma factor; RseB, a periplasmic protein that binds to the periplasmic face of RseA; and the DegS and YaeL proteases . The major point of regulation, the interaction between sigma(E) and RseA, is primarily controlled by the stability of RseA . Envelope stress promotes RseA degradation, which occurs by a proteolytic cascade initiated by DegS . There is evidence that one sigma(E)-inducing stress (OmpC overexpression) directly activates DegS to cleave RseA . Secondarily, envelope stress may relieve RseB-mediated enhancement of RseA activity . Additional levels of control upon sigma(E) activity may become evident upon further study of this stress response pathway.

Biochem J, 2004 Jul 15, 381(Pt 2), 373 - 8
The water- and salt-stress-regulated Asr1 (abscisic acid stress ripening) gene encodes a zinc-dependent DNA-binding protein; Kalifa Y et al.; Tomato (Lycopersicon esculantum) ASR1 (abscisic acid stress ripening protein), a small plant-specific protein whose cellular mode of action defies deduction based on its sequence or homology analyses, is one of numerous plant gene products with unknown biological roles that become over-expressed under water- and salt-stress conditions . Steady-state cellular levels of tomato ASR1 mRNA and protein are transiently increased following exposure of plants to poly(ethylene glycol), NaCl or abscisic acid . Western blot and indirect immunofluorescence analysis with anti-ASR1 antibodies demonstrated that ASR1 is present both in the cytoplasmic and nuclear subcellular compartments; approx . one-third of the total ASR1 protein could be detected in the nucleus . Nuclear ASR1 is a chromatin-bound protein, and can be extracted with 1 M NaCl, but not with 0.5% Triton X-100 . ASR1, overexpressed in Escherichia coli and purified to homogeneity, possesses zinc-dependent DNA-binding activity . Competitive-binding experiments and SELEX (systematic evolution of ligands by exponential enrichment) analysis suggest that ASR1 binds at a preferred DNA sequence.

Lab Chip, 2001 Sep, 1(1), 50 - 5 Epub 2001 Aug 09.
On-chip culture system for observation of isolated individual cells; Inoue I et al.; To investigate the properties of isolated single cells with their environment, we developed the differential analysis method for single cells using an on-chip microculture system . The advantages of the system are, (i) . continuous cultivation of a series of isolated single cells or a group of cells under contamination free conditions, (ii) . continuous observation and comparison of those cells with 0.2 microm spatial resolution by a phase-contrast/fluorescent microscopy system with digital image processing . The core of the system is an n x n (n = 20-50) array of chambers, where each is 20-70 microm in diameter and 5-30 microm deep holes etched into a biotin-coated 0.17 mm thick glass slide . The biotin-coated glass slide is covered with the streptavidin coated cellulose semipermeable membrane, which is fixed on the surface of the glass slide by streptavidin-biotin attachment, separating those holes from the nutrient medium circulating through a 'cover chamber' above . A single cell or group of cells can thus be isolated from environment perfused with the same medium, and the medium in each chamber can be changed within the diffusion time (<1/30 s) . In addition, the microchamber volumes of specific cells or cell groups can be controlled by the sizes of the chambers . By using this system we found that the length of isolated Escherichia coli increased at 0.06 microm min(-1) between cell divisions regardless of the chamber volume, and that the cell concentration reached 10(12) cells ml(-1) under contamination free conditions . The system is thus particularly useful for one cell level analysis because the direct descendants of single cells can be cultured and compared in the isolated microchambers, and the physical properties of the cells in each microchamber can be continuously observed and compared.

Lab Chip, 2002 Nov, 2(4), 197 - 202 Epub 2002 Oct 25.
PDMS-glass hybrid microreactor array with embedded temperature control device . Application to cell-free protein synthesis; Yamamoto T et al.; A microreactor array was developed which enables high-throughput cell-free protein synthesis . The microreactor array is composed of a temperature control chip and a reaction chamber chip . The temperature control chip is a glass-made chip on which temperature control devices, heaters and temperature sensors, are fabricated with an ITO (indium tin oxide) resistive material . The reaction chamber chip is fabricated by micromolding of PDMS (polydimethylsiloxane), and is designed to have an array of reaction chambers and flow channels for liquid introduction . The microreactor array is assembled by placing the reaction chamber chip on the temperature control chip . The small thermal mass of the reaction chamber resulted in a short thermal time constant of 170 ms for heating and 3 s for cooling . The performance of the microreactor array was examined through the experiments of cell-free protein synthesis . By measuring the fluorescence emission from the products, it was confirmed that GFP (Green Fluorescent Protein) and BFP (Blue Fluorescent Protein) were successfully synthesized using Escherichia coli extract.

Lab Chip, 2002 Nov, 2(4), 179 - 87 Epub 2002 Nov 04.
High sensitivity PCR assay in plastic micro reactors; Yang J et al.; Small volume operation and rapid thermal cycling have been subjects of numerous reports in micro reactor chip development . Sensitivity aspects of the micro PCR reactor have not been studied in detail, however, despite the fact that detection of rare targets or trace genomic material from clinical and/or environmental samples has been a great challenge for microfluidic devices . In this study, a serpentine shaped thin (0.75 mm) polycarbonate plastic PCR micro reactor was designed, constructed, and tested for not only its rapid operation and efficiency, but also its detection sensitivity and specificity, in amplification of Escherichia coli (E . coli) K12-specific gene fragment . At a template concentration as low as 10 E . coli cells (equivalent to 50 fg genomic DNA), a K12-specific gene product (221 bp) was adequately amplified with a total of 30 cycles in 30 min . Sensitivity of the PCR micro reactor was demonstrated with its ability to amplify K12-specific gene from 10 cells in the presence of 2% blood . Specificity of the polycarbonate PCR micro reactor was also proven through multiplex PCR and/or amplification of different pathogen-specific genes . This is, to our knowledge, the first systematic study of assay sensitivity and specificity performed in plastic, disposable micro PCR devices.

Beijing Da Xue Xue Bao, 2004 Apr, 36(2), 185 - 9
{Expression of the partial protein encoded by mutated COL4A5 gene and analysis of the structure by circular dichroism}; Wang YF et al.; OBJECTIVE: To analyze the protein structure encoded by COL4A5 gene with a missense mutation and to discuss the effect of gene mutation on basic structure and predicted secondary structure of the encoded protein . METHODS: A fragment of cDNA with a g.3246G T mutation resulting in p.G1015V from an X-linked Alport patient, and that of corresponding cDNA from a control were expressed in E.coli . The recombinant and mutant polypeptide was a fragment of COL4A5, containing 158 amino acid residues with a glycine to valine substitution mutation in it . The secondary structure of the two recombinant proteins was analyzed using circular dichroism(CD) spectroscopy . RESULTS: CD spectra of the control exhibited a negative peak near 200 nm whereas that of the patient exhibited a negative peak near 220 nm . The magnitude of the negative peak of the patient decreased as compared with that of the control . Furthermore, secondary structure of the control polypeptide was mainly composed of beta-sheet and random coil without alpha-helix, whereas that of the patient presented 12.9% alpha-helix . CONCLUSION: Not only local structure of the substitution site but also folding kinetics of the entire alpha5 chain may be changed due to Gly-->Val substitution in Alport syndrome . We speculate that the abnormally folded polypeptide chain may not be assembled into the triple helix and the network of type IV collagen, or may be assembled into loosen triple-helix then degraded easily, resulting in the pathognomonic ultrastructural changes of the glomerular basement membrane.

Eur J Mass Spectrom (Chichester, Eng), 2004, 10(1), 89 - 99
Investigation of methods suitable for the matrix-assisted laser desorption/ionization mass spectrometric analysis of proteins from ribonucleoprotein complexes; Suh MJ et al.; A variety of protein isolation and purification techniques for ribonucleoprotein (RNP) complexes were investigated for their compatibility with downstream analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) . Ribosomal proteins from Escherichia coli 70S ribosomes were obtained using methods such as phenol extraction and precipitation by organic solvents or acids . Under optimal conditions, more than 90% of the expected ribosomal proteins were detected in a single MALDI-MS experiment . The most effective approach combined ribosome denaturation by buffer exchange with acid precipitation of the ribosomal ribonucleic acids . An improved acid precipitation approach, involving the sequential additions of acetic and trifluoroacetic acid, yielded more complete protein coverage while minimizing loss of ion signal from lower molecular weight proteins . With phenol extraction, substantial gains in ion abundance of higher molecular weight proteins are noted, although some of the lower molecular weight proteins were not efficiently extracted . These results illustrate several effective approaches for protein isolation from protein complexes such as RNPs that are MALDI-MS compatible, and these approaches should extend the use of MALDI-MS for proteomics-based analyses of other protein-nucleic acid complexes.

RNA, 2004 May, 10(5), 805 - 12
tRNA slippage at the tmRNA resume codon; Trimble MJ et al.; The bacterial ribosome does not initiate translation on the mRNA portion of tmRNA; instead translation that had begun on a separate mRNA molecule resumes at a particular triplet on tmRNA (the resume codon) . For at least two tRNAs that could pair with both the resume and -2 triplets on mutant tmRNAs, UAA (stop) as the second codon induced high-frequency -2 slippage on the resume codon in the P site . The frameshift product was not detected when the -2 base was altered . Deficiency for ribosomal L9 protein, which affects other cases of frameshifting, had no significant effect . A special feature of this frameshifting is its dependence on a particular context, that of the tmRNA resume codon; it failed on the same sequence in a regular mRNA, and, more strikingly, at the second tmRNA codon . This focuses attention on the peculiar features expected of the slippage-prone state, such as unusual E-site filling, that might make the P-site resume codon:anticodon interaction especially unstable . Keywords: tmRNA; ribosome; frameshift; E site; translation

RNA, 2004 May, 10(5), 795 - 804
Structure-function analysis of tRNA(Gln) in an Escherichia coli knockout strain; McClain WH et al.; The diverse and highly specific interaction between RNAs and proteins plays an essential role in many important biological processes . In the glutamine aminoacylation system, crystal structures of the free and ligated macromolecules have provided a description of the tRNA-protein interactions at the molecular level . This data lays the foundation for genetic, biochemical, and structural analyses to delineate the set of key interactions that governs the structure-function relationships of the two macromolecules . To this end the chromosomal tRNA(Gln) genes were disrupted in Escherichia coli to produce a tRNA(Gln) knockout strain that depends upon expression of a functional tRNA(Gln) from a plasmid for cell viability . Mutants of an inactive tester tRNA derived from tRNA(Ala) were generated by hydroxylamine mutagenesis, and the active derivatives were selected by their ability to support knockout cell growth . Two of the mutants contained substitutions in the first base pair of the acceptor stem that likely facilitate the formation of a hairpin loop that places A76 in the active site . The third mutation was located at position 13 in the D loop region of the tRNA, and suggests that an interaction with residue 13 contributes to a specific conformational change in unliganded GlnRS, which helps configure the enzyme active site in its catalytically proficient form . This work demonstrates the efficacy of an integrated approach that combines genetic selections and biochemical analyses with the physical data from crystal structures to reveal molecular steps that control the specificity of RNA-protein interactions.

RNA, 2004 May, 10(5), 772 - 8
Conserved but nonessential interaction of SRP RNA with translation factor EF-G; Sagar MB et al.; 4.5S RNA is essential for viability of Escherichia coli, and forms a key component of the signal recognition particle (SRP), a ubiquitous ribonucleoprotein complex responsible for cotranslational targeting of secretory proteins . 4.5S RNA also binds independently to elongation factor G (EF-G), a five-domain GTPase that catalyzes the translocation step during protein biosynthesis on the ribosome . Point mutations in EF-G suppress deleterious effects of 4.5S RNA depletion, as do mutations in the EF-G binding site within ribosomal RNA, suggesting that 4.5S RNA might play a critical role in ribosome function in addition to its role in SRP . Here we show that 4.5S RNA and EF-G form a phylogenetically conserved, low-affinity but highly specific complex involving sequence elements required for 4.5S binding to its cognate SRP protein, Ffh . Mutational analysis indicates that the same molecular structure of 4.5S RNA is recognized in each case . Surprisingly, however, the suppressor mutant forms of EF-G bind very weakly or undetectably to 4.5S RNA, implying that cells can survive 4.5S RNA depletion by decreasing the affinity between 4.5S RNA and the translational machinery . These data suggest that SRP function is the essential role of 4.5S RNA in bacteria.

J Biol Chem, 2004 Jul 2, 279(27), 28435 - 42 Epub 2004 Apr 20.
Preferential substrate binding orientation by the molecular chaperone HscA; Tapley TL et al.; HscA, a specialized bacterial hsp70-class chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif at positions 99-103 . We have used a site-directed fluorescence labeling and quenching strategy to determine whether HscA binds to IscU in a preferred orientation . HscA was selectively labeled on opposite sides of the substrate binding domain with the fluorescent probe bimane, and the ability of LPPVK-containing peptides having tryptophan at the N or C terminus to quench bimane fluorescence was measured . Quenching was highly dependent on the position of tryptophan in the peptide and the location of bimane on HscA implying a strong directional preference for peptide binding . Similar experiments showed that full-length IscU binds in the same orientation as IscU-derived peptides and that binding orientation is unaffected by the co-chaperone HscB . The preferred orientation of the HscA-IscU complex is the reverse of that previously described for peptide complexes of Escherichia coli DnaK and rat Hsc70 substrate binding domain fragments establishing that hsp70 isoforms can bind peptide/polypeptide substrates in different orientations.

J Biol Chem, 2004 Jun 25, 279(26), 27357 - 64 Epub 2004 Apr 20.
Identification of CRALBP ligand interactions by photoaffinity labeling, hydrogen/deuterium exchange, and structural modeling; Wu Z et al.; Cellular retinaldehyde-binding protein (CRALBP) functions in the retinal pigment epithelium (RPE) as an acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a substrate carrier for 11-cis-retinol dehydrogenase . Toward a better understanding of CRALBP function, the ligand binding cavity in human recombinant CRALBP (rCRALBP) was characterized by photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal and by high resolution mass spectrometric topological analyses . Eight photoaffinity-modified residues were identified in rCRALBP by liquid chromatography tandem mass spectrometry, including Tyr(179), Phe(197), Cys(198), Met(208), Lys(221), Met(222), Val(223), and Met(225) . Multiple different adduct masses were found on the photolabeled residues, and the molecular identity of each modification remains unknown . Supporting the specificity of photo-labeling, 50% of the modified residues have been associate with retinoid interactions by independent analyses . In addition, topological analysis of apo- and holo-rCRALBP by hydrogen/deuterium exchange and mass spectrometry demonstrated residues 198-255 incorporate significantly less deuterium when the retinoid binding pocket is occupied with 11-cis-retinal . This hydrophobic region encompasses all but one of the photo-labeled residues . A structural model of CRALBP ligand binding domain was constructed based on the crystal structures of three homologues in the CRAL-TRIO family of lipid-binding proteins . In the model, all of the photolabeled residues line the ligand binding cavity except Met(208), which appears to reside in a flexible loop at the entrance/exit of the ligand cavity . Overall, the results expand to 12 the number of residues proposed to interact with ligand and provide further insight into CRALBP ligand and protein interactions.

J Biol Chem, 2004 Jul 2, 279(27), 28358 - 66 Epub 2004 Apr 20.
Biochemical characterization of the Methanothermobacter thermautotrophicus minichromosome maintenance (MCM) helicase N-terminal domains; Kasiviswanathan R et al.; Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases . For the archaeal protein it was shown that the N-terminal portion of the protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity . Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described . Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for protein multimerization and that domain B is the main contact region with single-stranded DNA . It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.

Biochim Biophys Acta, 2004 Apr 12, 1655(1-3), 13 - 28
Proton-coupled electron transfer: a unifying mechanism for biological charge transport, amino acid radical initiation and propagation, and bond making/breaking reactions of water and oxygen; Chang CJ et al.; Redox-driven proton pumps, radical initiation and propagation in biology, and small-molecule activation processes all involve the coupling of electron transfer to proton transport . A mechanistic framework in which to interpret these processes is being developed by examining proton-coupled electron transfer (PCET) in model and natural systems . Specifically, PCET investigations are underway on the following three fronts: (1) the elucidation of the PCET reaction mechanism by time-resolved laser spectroscopy of electron donors and acceptors juxtaposed by a proton transfer interface; (2) the role of amino acid radicals in biological catalysis with the radical initiation and transport processes of E . coli ribonucleotide reductase (RNR) as a focal point; and (3) the application of PCET towards small-molecule activation with emphasis on biologically relevant bond-breaking and bond-making processes involving oxygen and water . A review of recent developments in each of these areas is discussed.

Mutat Res, 2004 May 9, 560(1), 49 - 55
Growth inhibition and mutagenesis induced in Escherichia coli by dihydropyrazines with DNA strand-cleaving activity; Takechi S et al.; Dihydropyrazine (DHP) causes DNA strand breaks in vitro . We evaluated the cytotoxic and genotoxic potential of DHP in Escherichia coli . DHP exposure dose-dependently caused inhibition of cell growth in the wild-type strain, death in recA and uvrB, and an increase in mutation frequency in uvrB . These findings indicate that DHP causes DNA strand breaks in vivo.

Mutat Res, 2004 May 9, 560(1), 27 - 32
ELF electromagnetic fields increase hydrogen peroxide (H2O2)-induced mutations in pTN89 plasmids; Koyama S et al.; We have examined the mutational effects of hydrogen peroxide (H(2)O(2)) in the presence and absence of an extremely low-frequency magnetic field (ELFMF), using pTN89 plasmids . Mutations were detected in the supF gene carried by these plasmids in Escherichia coli . The plasmids were either treated with H(2)O(2) (1microM) alone at 37 degrees C for 4h, or were exposed to an ELFMF (60Hz, 5millitesla (mT)) simultaneously with H(2)O(2) treatment . The mutation frequency was 2.28 x 10(-4) for H(2)O(2) treatment alone, and 5.81 x 10(-4) for ELFMF exposure with H(2)O(2) treatment . We did not observe any mutations using treatment with ELFMF exposure alone . This indicates that the ELFMF may potentiate H(2)O(2)-induced mutation . Sequence analysis of the supF mutant plasmids revealed that base substitutions, G: C-->A :T transitions and G:C-->T:A transversions were dominant in both treatment groups, and there was no difference in the mutation spectrum or the hotspots between the groups . Therefore, ELFMFs may interact and potentiate the damage induced by H(2)O(2), resulting in an increase in the number of mutations.

J Immunol Methods, 2004 Apr, 287(1-2), 203 - 15
Expression of the B subunit of the heat-labile enterotoxin of Escherichia coli in tobacco mosaic virus-infected Nicotiana benthamiana plants and its characterization as mucosal immunogen and adjuvant; Wagner B et al.; We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coli in tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants . We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal . The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA . Eighty-nine percent of N . benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments . The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots . rLTB from plant extracts of TMV-infected N . benthamiana leaves was purified to give 75 microg rLTB pentamers per gram fresh plant material and was capable of binding G(M)1 ganglioside . The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 microg per mouse) induced LTB-specific IgG1 antibodies . To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production . The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen . In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.

Mol Cell, 2004 Apr 23, 14(2), 195 - 205
The nuclear Hat1p/Hat2p complex: a molecular link between type B histone acetyltransferases and chromatin assembly; Ai X et al.; The yeast Hat1p/Hat2p type B histone acetyltransferase complex is localized to both the cytoplasm and nucleus . We isolate the nuclear form of the Hat1p/Hat2p complex and find that it copurifies with the product of the uncharacterized open reading frame YLL022C (named Hif1p) . The functional significance of the association of Hif1p with the Hat1p/Hat2p complex is confirmed by the observation that hif1Delta and hat1Delta strains display similar defects in telomeric silencing and DNA double-strand break repair . Hif1p is a histone chaperone that selectively interacts with histones H3 and H4 . Hif1p is also a chromatin assembly factor, promoting the deposition of histones in the presence of a yeast cytosolic extract . In vivo, the nuclear Hat1p/Hat2p/Hif1p complex is bound to acetylated histone H4, as well as histone H3 . The association of Hif1p with acetylated H4 requires Hat1p and Hat2p providing a link between type B histone acetyltransferases and chromatin assembly.

Mol Cell, 2004 Apr 23, 14(2), 175 - 82
Gene-specific modulation of TAF10 function by SET9-mediated methylation; Kouskouti A et al.; SET9 is a member of the SET domain-containing histone methyltransferase family that can specifically methylate histone 3 at lysine 4 position . Although nucleosomal histones are poor substrates for SET9, the active enzyme can stimulate activator-induced transcription . Here, we show that SET9 can monomethylate the TBP-associated factor TAF10 at a single lysine residue located at the loop 2 region within the putative histone-fold domain of the protein . Methylated TAF10 has an increased affinity for RNA polymerase II, pointing to a direct role of this modification in preinitiation complex formation . Reporter assays and studies on TAF10 null F9 cells expressing a methylation-deficient TAF10 mutant revealed that SET9-mediated methylation of TAF10 potentiates transcription of some but not all TAF10-dependent genes . This gene specificity correlated with SET9 recruitment . The promoter-specific effects of SET9-methylated TAF10 may have important implications regarding the biological function of SET domain-containing lysine methylases, whose primary targets have been presumed to be histones.

J Thromb Haemost, 2004 May, 2(5), 797 - 803
Construction and characterization of a recombinant plasminogen activator composed of an anti-fibrin single-chain antibody and low-molecular-weight urokinase; Hagemeyer CE et al.; BACKGROUND: Targeting of plasminogen activators to the fibrin component of a thrombus by antibodies directed against human fibrin can enhance their thrombolytic potency and clot specificity . OBJECTIVES: To overcome the disadvantages of chemical conjugation, we investigated whether the recombinant fusion of a single-chain antibody and a plasminogen activator results in an active bifunctional molecule that might be useful as a therapeutic agent . METHODS: The cDNA of low-molecular-weight single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scuPA(LMW)), was cloned from human endothelial cells and fused to a single-chain antibody specific for the 7 N-terminal amino acids (beta(15-22)) in the beta-chain of human fibrin (scFv(59D8)) . The fusion protein was purified using affinity chromatography with the beta(15-22)-peptide of human fibrin . RESULTS: Purified scFv(59D8)-scuPA(LMW) migrated as a 60-kDa band, which is consistent with a molecule composed of one scFv(59D8) and one scuPA(LMW) moiety . Both functions of the fusion molecule, fibrin-specific binding and plasminogen activation, were fully preserved . In human plasma clots, thrombolysis by scFv(59D8)-scuPA(LMW) is significantly faster and more potent compared with the clinically used urokinase . CONCLUSIONS: ScFv(59D8)-scuPA(LMW) constitutes a new recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency and relative fibrin selectivity, that can be produced with modern methods at low cost, large quantities and reproducible activity in Escherichia coli.

Adv Exp Med Biol, 2003, 538, 279 - 83; discussion 284
Orientation and motion of myosin light chain and troponin in reconstituted muscle fibers as detected by ESR with a new bifunctional spin label; Arata T et al.; Using electron spin resonance, we have studied dynamic structures of myosin neck domain and troponin C by site-directed spin labeling . We observed two broad but distinct orientations of a spin label attached specifically to a single cysteine (cys156) on the regulatoy light chain (RLC) of myosin in relaxed skeletal muscle fibers . The two probe orientations, separated by a 25 degrees axial rotation, did not change upon muscle activation, but orientational distributions became narrower substantially, indicating that a fraction of myosin heads undergoes a disorder-to-order transition of the myosin light chain domain upon force generation and muscle contraction . These results provide insight into the mechanism how myosin heads move their domains to translocate an actin filament . Site-directed spin-labeling was achieved by cysteine residues of human cardiac troponin C (TnC) . Spin dipole-dipole interaction showed that free TnC undergoes a global structural change (extended-to-compact) by Ca2+ or Mg2+ . The spectra from the spin labels at N-terminal half domain were broad and almost identical in parallel and perpendicular orientations of fiber, suggesting that the N-terminal of TnC molecule is flexible or disoriented with respect to the filament axis . We also succeeded, for the first time, in fixing the newly-synthesized bifunctional spin label rigidly on TnC molecule in solution (either in +/- Ca2+), giving a promise that we can determine the precise coordinate of the spin principal axis on protein surface.

Luminescence, 2004 Mar-Apr, 19(2), 78 - 84
Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans; Tian B et al.; Free radical scavenging effects of the cellular protein extracts from two strains of Deinococcus radiodurans and Escherichia coli against O2-, H2O2 and *OH were investigated by chemiluminescence (CL) methods . The cellular protein extracts of D . radiodurans R1 and KD8301 showed higher scavenging effects on O2- than that of E . coli . D . radiodurans R1 and KD8301 also strongly scavenged H2O2 with an EC50 (50% effective concentration) of 0.12 and 0.2 mg/mL, respectively, compared to that of E . coli (EC50 = 3.56 mg/mL) . The two strains of D . radiodurans were effective in scavenging *OH generated by the Fenton reaction, with EC50 of 0.059 and 0.1 mg/mL, respectively, compared to that of E . coli (EC50 > 1 mg/mL) . Results from the chemiluminescence assay of *OH-induced DNA damage and the plasmid pUC18 DNA double-strand break (DSB) model in vitro showed that D . radiodurans had remarkably inhibitory effect on the *OH-induced oxidative damage of DNA . The scavenging effects of D . radiodurans on reactive oxygen species (ROS) played an important role in the response to oxidation stress and preventing against DNA oxidative damage, and may be attributed to intracellular scavenging proteins, including superoxide dismutase (SOD) and catalase .

Protein Sci, 2004 May, 13(5), 1331 - 9
An efficient system for high-level expression and easy purification of authentic recombinant proteins; Catanzariti AM et al.; Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins . The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product . However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme . Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein . An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified . The expression system was tested using several proteins varying in size and complexity . These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.

Proc Natl Acad Sci U S A, 2004 Apr 27, 101(17), 6391 - 6 Epub 2004 Apr 19.
Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease; Bennett SE et al.; Escherichia coli nucleoside diphosphate kinase (Ndk) catalyzes ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the cognate diphosphate precursor . Recently, the Ndk polypeptide was reported to be a multifunctional base excision repair nuclease that processed uracil residues in DNA by acting sequentially as a uracil-DNA glycosylase inhibitor protein (Ugi)-sensitive uracil-DNA glycosylase, an apurinic/apyrimidiniclyase, and a 3'-phosphodiesterase {Postel, E . H . & Abramczyk, B . M . (2003) Proc . Natl . Acad . Sci . USA 100, 13247-13252} . Here we demonstrate that the E . coli Ndk polypeptide lacked detectable uracil-DNA glycosylase activity and, hence, was incapable of acting as a uracil-processing DNA repair nuclease . This finding was based on the following observations: (i) uracil-DNA glycosylase activity did not copurify with Ndk activity; (ii) Ndk purified from E . coli ung(-) cells showed no detectable uracil-DNA glycosylase activity; and (iii) Ndk failed to bind to a Ugi-Sepharose affinity column that tightly bound E . coli uracil-DNA glycosylase (Ung) . Collectively, these observations demonstrate that the E . coli Ndk polypeptide does not possess inherent uracil-DNA glycosylase activity.

Proc Natl Acad Sci U S A, 2004 May 18, 101(20), 7536 - 41 Epub 2004 Apr 19.
A truncated aminoacyl-tRNA synthetase modifies RNA; Salazar JC et al.; Aminoacyl-tRNA synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution . Analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl-tRNA synthetase paralogs . Here we report the characterization of a well conserved glutamyl-tRNA synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria . The E . coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain . Generation of a yadB disruption showed the gene to be dispensable for E . coli growth in rich and minimal media . Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNA(Asp) . Neither tRNA(Glu) nor tRNA(Gln) were substrates . In contrast to canonical aminoacyl-tRNA, glutamate was not esterified to the 3'-terminal adenosine of tRNA(Asp) . Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNA(Asp) . Glutamyl-queuosine, like canonical Glu-tRNA, was hydrolyzed by mild alkaline treatment . Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E . coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl-queuosine . Thus, this aminoacyl-tRNA synthetase fragment contributes to standard nucleotide modification of tRNA.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 6957 - 62 Epub 2004 Apr 19.
On the use of low-frequency normal modes to enforce collective movements in refining macromolecular structural models; Delarue M et al.; As more and more structures of macromolecular complexes get solved in different conditions, it has become apparent that flexibility is an inherent part of their biological function . Normal mode analysis using simplified models of proteins such as the elastic network model has proved very effective in showing that many of the structural transitions derived from a survey of the Protein Data Bank can be explained by just a few of the lowest-frequency normal modes . In this work, normal modes are used to carry out medium- or low-resolution structural refinement, enforcing collective and large-amplitude movements that are beyond the reach of existing methods . Refinement is carried out in reciprocal space with respect to the normal mode amplitudes, by using standard conjugate-gradient minimization . Several tests on synthetic diffraction data whose mode concentration follows the one of real movements observed in the Protein Data Bank have shown that the radius of convergence is larger than the one of rigid-body refinement . Tests with experimental diffraction data for the same protein in different environments also led to refined structural models showing drastic reduction of the rms deviation with the target model . Because the structural transition is described by very few parameters, over-fitting of real experimental data is easily detected by using a cross-validation test . The method has also been applied to the refinement of atomic models into molecular envelopes and could readily be used to fit large macromolecular complex rearrangements into cryo-electron microscopy-reconstructed images as well as small-angle x-ray scattering-derived envelopes.

Nucleic Acids Res, 2004 Apr 19, 32(7), 2158 - 70 Print 2004.
Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A; Cui S et al.; RecQ helicases are required for the maintenance of genome stability . Characterization of the substrate specificity and identification of the binding partners of the five human RecQ helicases are essential for understanding their function . In the present study, we have developed an efficient baculovirus expression system that allows us to obtain milligram quantities of recombinant RECQ1 . Our gel filtration and dynamic light scattering experiments show that RECQ1 has an apparent molecular mass of 158 kDa and a hydrodynamic radius of 5.4 +/- 0.6 nm, suggesting that RECQ1 forms dimers in solution . The oligomeric state of RECQ1 remains unchanged upon binding to a single-stranded (ss)DNA fragment of 50 nt . We show that RECQ1 alone is able to unwind short DNA duplexes (<110 bp), whereas considerably longer substrates (501 bp) can be unwound only in the presence of human replication protein A (hRPA) . The same experiments with Escherichia coli SSB show that RECQ1 is specifically stimulated by hRPA . However, hRPA does not affect the ssDNA-dependent ATPase activity of RECQ1 . In addition, our far western, ELISA and co-immunoprecipitation experiments demonstrate that RECQ1 physically interacts with the 70 kDa subunit of hRPA and that this interaction is not mediated by DNA.

J Cell Biol, 2004 Apr 26, 165(2), 213 - 22 Epub 2004 Apr 19.
F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis; van der Laan M et al.; The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity . Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase . We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC . Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers . Co-reconstituted SecYEG has no significant effect on the insertion efficiency . Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c . In conclusion, a novel membrane protein insertion pathway in E . coli is described in which YidC plays an exclusive role.

J Biol Chem, 2004 Jun 25, 279(26), 26846 - 57 Epub 2004 Apr 19.
The flavonoid baicalein inhibits fibrillation of alpha-synuclein and disaggregates existing fibrils; Zhu M et al.; The aggregation of alpha-synuclein has been implicated as a critical step in the development of Parkinson's disease . Parkinson's disease is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons from the substantia nigra; currently, no cure exists . Baicalein is a flavonoid with antioxidant properties; upon oxidation, it forms several products including quinones . We show here that low micromolar concentrations of baicalein, and especially its oxidized forms, inhibit the formation of alpha-synuclein fibrils . In addition, existing fibrils of alpha-synuclein are disaggregated by baicalein . The product of the inhibition reaction is predominantly a soluble oligomer of alpha-synuclein, in which the protein molecules have been covalently modified by baicalein quinone to form a Schiff base with a lysine side chain in alpha-synuclein . The binding of baicalein was abolished by conversion of the Tyr residues into Phe, demonstrating that Tyr is involved in the interaction of alpha-synuclein with baicalein . In disaggregation baicalein causes fragmentation throughout the length of the fibril . These observations suggest that baicalein and similar compounds may have potential as therapeutic leads in combating Parkinson's disease and that diets rich in flavonoids may be effective in preventing the disorder.

J Biol Chem, 2004 Jun 18, 279(25), 26307 - 13 Epub 2004 Apr 19.
Aggregates are the biologically active units of endotoxin; Mueller M et al.; For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past . Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E . coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates . We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins . We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios . Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406 . Similar results were obtained when using various phospholipids instead of compound 406 . These observations can only be understood by assuming that the active unit of endotoxins is the aggregate . We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.

J Biol Chem, 2004 Jun 18, 279(25), 26314 - 22 Epub 2004 Apr 19.
RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling; Mao H et al.; To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins . RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein . RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions . The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies . Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro . Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins . All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization . RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling . Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.

Biochem J, 2004 May 1, 379(Pt 3), 609 - 15
Complementation of the yeast deletion mutant DeltaNCE103 by members of the beta class of carbonic anhydrases is dependent on carbonic anhydrase activity rather than on antioxidant activity; Clark D et al.; In recent years, members of the beta class of CAs (carbonic anhydrases) have been shown to complement Delta NCE103, a yeast strain unable to grow under aerobic conditions . The activity required for complementation of Delta NCE103 by tobacco chloroplast CA was studied by site-directed mutagenesis . E196A (Glu196-->Ala), a mutated tobacco CA with low levels of CA activity, complemented Delta NCE103 . To determine whether restoration of Delta NCE103 was due to residual levels of CA activity or whether it was related to previously proposed antioxidant activity of CAs {Gotz, Gnann and Zimmermann (1999) Yeast 15, 855-864}, additional complementation analysis was performed using human CAII, an alpha CA structurally unrelated to the beta class of CAs to which the tobacco protein belongs . Human CAII complemented Delta NCE103, strongly arguing that CA activity is responsible for the complementation of Delta NCE103 . Consistent with this conclusion, recombinant NCE103 synthesized in Escherichia coli shows CA activity, and Delta NCE103 expressing the tobacco chloroplast CA exhibits the same sensitivity to H2O2 as the wild-type strain.

Biochemistry, 2004 Apr 27, 43(16), 4662 - 9
Role of residue 138 in the interdomain hinge region in transmitting allosteric signals for DNA binding in Escherichia coli cAMP receptor protein; Yu S et al.; The cAMP receptor protein (CRP) of Escherichia coli is a transcription factor . The affinity of CRP for a specific DNA sequence is significantly enhanced as a consequence of the binding of the allosteric effector, cAMP . The hinge region, particularly residues 136 and 138, connecting the cAMP and DNA binding domains of CRP has been proposed to play essential roles in transmitting the allosteric signals . To probe the specific role of residue 138, eight D138 mutants and wild-type CRP were tested for their ability to bind the lac26 and gallac26 promoter sequences in this study . A correlation was established between DNA binding affinity and side chain solvation free energy, namely, an increasing specific DNA affinity with an increasing hydrophilicity of the side chain of residue 138 . In addition, a linear correlation was found between DNA binding affinity and the energetics of subunit assembly . The ability of CRP to distinguish between cAMP and cGMP as an allosteric activator of DNA binding is weakened with higher energetics of subunit assembly . This correlation indicates that quaternary constraint leads to a constraint of the DNA binding domain . This observation is consistent with the concept that an optimum quaternary structural constraint is important in CRP exhibiting its allosteric properties . The stability of CRP was monitored by Trp fluorescence and circular dichroism in the presence of guanidine hydrochloride . These spectroscopic data revealed nonidentical denaturation profiles . Since the Trp residues are located exclusively in the beta-roll cyclic nucleotide binding domain, the denaturation profiles reveal the stability of the beta-roll structure . This study produces another linear correlation between DNA binding affinity in the presence of cAMP and cGMP and the stability of the beta-roll; namely, the stability of the beta-roll structure leads to a decrease in DNA binding affinity . All these correlations indicate the importance of structural stability and dynamics in the ability of CRP to manifest its intrinsic allosteric properties.

J Mol Biol, 2004 Feb 20, 336(3), 707 - 16
Crystal structure of the catalytic domain of human matrix metalloproteinase 10; Bertini I et al.; The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution . The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs . Furthermore, the binding mode of N-isobutyl-N-{4-methoxyphenylsulfonyl}glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.

J Mol Biol, 2004 Feb 20, 336(3), 673 - 93
Thermodynamic dissection of the polymerizing and editing modes of a DNA polymerase; Bailey MF et al.; DNA polymerases with intrinsic proofreading activity interact with DNA primer/templates in two distinct modes, corresponding to the complexes formed during the 5'-3' polymerization or 3'-5' editing of a nascent DNA chain . Thermodynamic measurements designed to quantify the energetic contributions of individual DNA-protein contacts in either the polymerizing or editing complexes are complicated by the fact that both species exist in solution and are not resolved in conventional DNA-protein binding assays . To overcome this problem, we have developed a new binding analysis that combines information from steady-state and time-resolved fluorescence experiments and uses the Klenow fragment of Escherichia coli DNA polymerase I (KF) and fluorescently labeled primer/template oligonucleotides as a model polymerase-DNA system . Steady-state fluorescence titrations are used to evaluate the overall affinity of KF for the primer/template, while time-resolved fluorescence anisotropy is used to quantify the equilibrium fractions of the primer/template bound in the polymerizing and editing modes . From a combined analysis of both data, the equilibrium constant and hence standard free energy change associated with each binding mode can be obtained unequivocally . This method is initially used to determine the equilibrium constants describing binding of a correctly base-paired primer/template to the 5'-3' polymerase and 3'-5' exonuclease sites of KF . It is then extended to quantify the extent to which these parameters are affected by the introduction of mismatches into the primer/template, and by rearrangement of specific side-chains in the exonuclease domain of the protein . While these perturbants were originally designed to demonstrate the utility of our new approach, they are also relevant in their own right since they have helped identify some hitherto unknown determinants of polymerase fidelity.

J Mol Biol, 2004 Feb 20, 336(3), 655 - 72
DNA double-strand breaks induce deletion of CTG.CAG repeats in an orientation-dependent manner in Escherichia coli; Hebert ML et al.; The influences of double-strand breaks (DSBs) within a triplet repeat sequence on its genetic instabilities (expansions and deletions) related to hereditary neurological diseases was investigated . Plasmids containing 43 or 70 CTG.CAG repeats or 43 CGG.CCG repeats were linearized in vitro near the center of the repeats and were transformed into parental, RecA-dependent homologous recombination-deficient, or RecBC exonuclease-deficient Escherichia coli . The resulting repair process considerably increased deletion of the repeating sequence compared to the circular DNA controls . Unexpectedly, the orientation of the insert relative to the unidirectional ColE1 origin of replication affected the amount of instability generated during the repair of the DSB . When the CTG strand was the template for lagging-strand synthesis, instability was increased, most markedly in the recA- strain . Results indicated that RecA and/or RecBC might play a role in DSB repair within the triplet repeat . Altering the length, orientation, and sequence composition of the triplet repeat suggested an important role of DNA secondary structures during repair intermediates . Hence, we hypothesize that ColE1 origin-dependent replication was involved during the repair of the DSB . A model is presented to explain the mechanisms of the observed genetic instabilities.

J Mol Biol, 2004 Feb 20, 336(3), 639 - 54
P53 inhibits strand exchange and replication fork regression promoted by human Rad51; Yoon D et al.; We explore the effects of p53 on strand exchange as well as regression of stalled replication forks promoted by human Rad51 . We have found that p53 specifically inhibits strand exchange mediated by human Rad51, but not by Escherichia coli RecA . In addition, we provide in vitro evidence that human Rad51 can promote regression of a stalled replication fork, and p53 also inhibits this fork regression . Furthermore, we show that two cancer-related p53 mutant proteins cannot inhibit strand exchange and fork regression catalyzed by human Rad51 . The results establish a direct functional link between p53 and human Rad51, and reveal that one of p53's functions in genome stabilization may be to prevent detrimental genome rearrangements promoted by human Rad51 . Thus, the results support the hypothesis that p53 contributes to genome stability by a transcription-independent modulation of homologous recombination.

Electrophoresis, 2004 Apr, 25(7-8), 949 - 58
Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations; Zhu Y et al.; Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E . coli) . Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential . A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly . Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers . PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis . The result is a pI versus MW map of bacterial protein content . IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E . coli strains.

Biochem Biophys Res Commun, 2004 May 14, 317(4), 1207 - 14
Cloning and expression of mitochondrial and protoflagellar creatine kinases from a marine sponge: implications for the origin of intracellular energy transport systems; Sona S et al.; Creatine kinase (CK) plays a central role in energy transactions in cells displaying high and variable rates of ATP turnover . Cytoplasmic and mitochondrial CK genes code for isoforms which are targeted to distinct intracellular compartments often in close physical proximity to sites of ATP hydrolysis or synthesis . In certain lower groups a third CK gene is present which codes for a flagellar CK isoform consisting of three complete, fused CK domains . Recent work has shown that cytoplasmic, mitochondrial, and flagellar CKs are present in protochordates and in deuterostome and protostome invertebrates . We report here that the marine sponge Tethya aurantia, a representative of the oldest of all multi-cellular animal groups, expresses three unique CK transcripts . One of these CK transcripts codes for protein that has a mitochondrial targeting sequence and in a phylogenetic analysis is positioned at the base of the cluster containing mitochondrial CK sequences from invertebrates, protochordates, and vertebrates; it is clearly a mitochondrial CK . When expressed in Escherichia coli the mitochondrial form from T . aurantia was found to be dimeric unlike all other mitochondrial CKs which are typically octameric . The other two T . aurantia transcripts code for proteins that appear to be more closely related to flagellar CKs . These protoflagellar CKs were found to be dimers when expressed in Escherichia coli . Sponges last shared a common ancestor with higher animals as long as one billion years ago . The antiquity of intracellular localization, as evidenced by the presence of a true mitochondrial CK and protoflagellar CKs in the sponge T . aurantia, indicates that physical constraints on cellular energy transport were key, early driving forces in the evolution of this key enzyme system.

Biochem Biophys Res Commun, 2004 May 14, 317(4), 1200 - 6
Disruption of the toxic conformation of the expanded polyglutamine stretch leads to suppression of aggregate formation and cytotoxicity; Popiel HA et al.; The polyglutamine (polyQ) diseases are a class of inherited neurodegenerative diseases including Huntington's disease, caused by the expansion of a polyQ stretch within each disease protein . This expansion is thought to cause a conformational change in the protein leading to aggregation of the protein, resulting in cytotoxicity . To analyze whether disrupting the toxic conformation of the polyQ protein can alter its aggregation propensity and cytotoxicity, we examined the effect of interruption of the expanded polyQ stretch by proline insertion, since prolines cause great alterations in protein conformation . Here, we show that insertion of prolines into the expanded polyQ stretch indeed disrupts its ordered secondary structure, leading to suppression of polyQ protein aggregation both in vitro and in cell culture, and reduction of cytotoxicity in correlation with the number of proline interruptions . Furthermore, we found that a short polyQ stretch with a proline interruption is able to inhibit aggregation of the expanded polyQ protein in trans . These results show that a gain in toxic conformation of the expanded polyQ protein is essential for aggregation and cytotoxicity, providing insight into establishing therapies against the polyQ diseases.

FEBS Lett, 2004 Apr 23, 564(1-2), 183 - 7
Enhancement of growth and cellulose accumulation by overexpression of xyloglucanase in poplar; Park YW et al.; Because the loosening of xyloglucan in the cell wall promotes plant growth (Takeda et al . (2002) Proc . Natl . Acad . Sci . USA 99, 9055-9060; Park et al . (2003) Plant J . 33, 1099-1106), we expressed Aspergillus xyloglucanase constitutively in Populus alba . The expression increased the length of stem even in the presence of sucrose . Increased stem growth was accompanied by a decrease in Young's elastic modulus in the growth zone but an increased elasticity in mature tissue . The increased internode length corresponded to an increase in cellulose content as well as specific gravity, showing that the removal of xyloglucan might cause an increase in cellulose density in the secondary xylem.

FEBS Lett, 2004 Apr 23, 564(1-2), 143 - 6
Functional complementation between the PDX1 vitamin B6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli; Wetzel DK et al.; The pathway for de novo vitamin B(6) biosynthesis has been characterized in Escherichia coli, however plants, fungi, archaebacteria, and most bacteria utilize an alternative pathway . Two unique genes of the alternative pathway, PDX1 and PDX2, have been described . PDX2 encodes a glutaminase, however the enzymatic function of the product encoded by PDX1 is not known . We conducted reciprocal transformation experiments to determine if there was functional homology between the E . coli pdxA and pdxJ genes and PDX1 of Cercospora nicotianae . Although expression of pdxJ and pdxA in C . nicotianae pdx1 mutants, either separately or together, failed to complement the pyridoxine mutation in this fungus, expression of PDX1 restored pyridoxine prototrophy to the E . coli pdxJ mutant . Expression of PDX1 in the E . coli pdxA mutant restored very limited ability to grow on medium lacking pyridoxine . We conclude that the PDX1 gene of the alternative B(6) pathway encodes a protein responsible for synthesis of the pyridoxine ring.

Brain Res Mol Brain Res, 2004 Apr 29, 124(1), 79 - 87
Increased expression of P/Q-type Ca2+ channel alpha1A subunit mRNA in cerebellum of N-type Ca2+ channel alpha1B subunit gene-deficient mice; Takahashi E et al.; The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity . Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice show normal behavior, presumably owing to compensation by the other Ca(2+) channels . In this study, we examined the mRNA expression of the P/Q-type Ca(2+) channel alpha(1A) subunit in cerebellum of alpha(1B)-deficient mice . The alpha(1A) subunit mRNA in homozygous alpha(1B)-deficient mice was expressed at a significantly higher level than in wild or heterozygous mice . To examine whether the increased expression is induced by a cis-regulatory element within the 5'-upstream region of the alpha(1A) subunit gene, we examined lacZ expression in alpha(1B)-deficient x alpha(1A)3.0-lacZ mice (carrying a 3.0-kb 5'-upstream fragment of the alpha(1A) subunit gene fused to Escherichia coli lacZ reporter gene), which express lacZ in granule but not in Purkinje cells, and in alpha(1B)-deficient x alpha(1A)6.3-lacZ mice (carrying a 6.3-kb 5'-upstream region fused to lacZ gene), which express lacZ in Purkinje but not in granule cells . The levels of lacZ expression in homozygous alpha(1B)-deficient x alpha(1A)3.0-lacZ mice were significantly higher than in wild or heterozygous mice, but no difference in lacZ expression level was found among wild, heterozygous and homozygous alpha(1B)-deficient x alpha(1A)6.3-lacZ mice . Therefore, a possible explanation of the normal behavior of alpha(1B)-deficient mice is that compensation by alpha(1A) subunit gene occurs and that the 3.0-kb 5'-upstream region of alpha(1A) subunit gene contains an enhancer cis-element(s) for compensation in cerebellar granule cells.

Biochim Biophys Acta, 2004 Apr 16, 1678(1), 47 - 56
Escherichia coli can tolerate insertions of up to 16 amino acids in the RNA polymerase alpha subunit inter-domain linker; Husnain SI et al.; The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alphaCTD) plays a key role in transcription initiation at many activator-dependent promoters and at UP element-dependent promoters . This domain is connected to the alpha N-terminal domain (alphaNTD) by an unstructured linker . To investigate the requirements of the alpha inter-domain linker to support growth of E . coli, we utilised a recently described technique for the substitution of the chromosomal rpoA gene, encoding alpha, by mutant rpoA alleles . We found that it was possible to replace wild-type rpoA by mutant alleles encoding alpha subunits containing inter-domain linkers that were longer by as many as 16 amino acids . However, using this method, it was not possible to transfer to the chromosome rpoA alleles encoding alpha subunits that contained an insertion of 32 amino acids or short deletions within the inter-domain linker . The effect of lengthening the alpha linker on activator-dependent and UP element-dependent transcription in the "haploid" rpoA system was shown to be qualitatively the same as observed previously in the diploid system . The ability of E . coli to tolerate insertions within the alpha inter-domain linker suggests that lengthening the alpha linker does not severely impair transcription of essential genes.

Crit Care Med, 2004 Mar, 32(3), 826 - 31
Role of peroxisome proliferator-activated receptor-gamma in the protection afforded by 15-deoxydelta12,14 prostaglandin J2 against the multiple organ failure caused by endotoxin; Collin M et al.; OBJECTIVE: The cyclopentenone prostaglandin 15-deoxydelta-prostaglandin J2 (15 d-PGJ2) exerts potent anti-inflammatory effects in vivo, which are in part due to the activation of peroxisome proliferator-activated receptor (PPAR)-gamma . Here we investigate the effects of 15 d-PGJ2 on the multiple organ injury/dysfunction associated with severe endotoxemia . DESIGN: Prospective, randomized study . SETTING: University-based research laboratory . SUBJECTS: Seventy anesthetized male Wistar rats . INTERVENTIONS: Rats received either Escherichia coli lipopolysaccharide (endotoxin, 6 mg/kg intravenously) or vehicle (saline, 1 mL/kg intravenously) . 15 d-PGJ2 (0.3 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered 30 mins before endotoxin . The selective PPAR-gamma antagonist GW9662 (0.3 mg/kg intravenously) or its vehicle (10% dimethyl sulfoxide) was given 45 mins before endotoxin . MEASUREMENTS AND MAIN RESULTS: Endotoxemia for 6 hrs increased serum concentrations of creatinine (indicator of renal dysfunction), aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, bilirubin (markers for hepatic injury and dysfunction), lipase (indicator of pancreatic injury), and creatine kinase (an indicator of neuromuscular skeletal muscle or cardiac injury) . The potent PPAR-gamma agonist 15 d-PGJ2 attenuated the increases in the serum concentrations of these variables, indicating a protective effect of 15 d-PGJ2 against the multiple organ injury/dysfunction caused by endotoxin . The specific PPAR-gamma antagonist GW9662 reduced the protective effects afforded by 15 d-PGJ2 . 15 d-PGJ2 did not affect the biphasic decrease in blood pressure or the increase in heart rate caused by endotoxemia . CONCLUSIONS: The potent PPAR-gamma agonist 15 d-PGJ2 reduces the multiple organ injury and dysfunction, but not the hypotension, caused by endotoxin in the rat . The mechanisms of the protective effect of this cyclopentenone prostaglandin are--at least in part--PPAR-gamma dependent, as the protection afforded by 15 d-PGJ2 was reduced by the PPAR-gamma antagonist GW9662 . We propose that 15 d-PGJ2 or other ligands for PPAR-gamma may be useful in treating organ injury associated with endotoxic shock.

Crit Care Med, 2004 Mar, 32(3), 801 - 5
Hemoadsorption removes tumor necrosis factor, interleukin-6, and interleukin-10, reduces nuclear factor-kappaB DNA binding, and improves short-term survival in lethal endotoxemia; Kellum JA et al.; OBJECTIVES: Previous studies have shown that inflammatory mediators can be removed from the circulation with hemofiltration and that adsorption plays an important role . Because adsorptive capacity of hollow-fiber dialyzers is limited, we sought to determine whether hemoadsorption using high surface area beads would result in greater mediator removal and improved survival in experimental sepsis . DESIGN: Randomized controlled laboratory experiment . SETTING: University laboratory . SUBJECTS: Sixty-six adult Sprague-Dawley rats . INTERVENTIONS: We conducted two ex vivo and two in vivo experiments . For in vivo experiments, we administered Escherichia coli endotoxin (20 mg/kg) by intravenous infusion and then randomized each animal to receive either hemoadsorption or a sham circuit for 4 hrs . Hemoadsorption was performed for 4 hrs using an arterial-venous circuit and a CytoSorb cartridge containing 10 g of polystyrene divinyl benzene copolymer beads with a biocompatible polyvinylpyrrolidone coating . Survival time was measured to a maximum of 12 hrs . In a separate set of experiments, we studied 12 animals using the same protocol except that we killed all animals at 4 hrs and removed standardized sections of liver for analysis of nuclear factor-kappaB DNA binding . MEASUREMENTS AND MAIN RESULTS: Mean survival time among hemoadsorption-treated animals was 629+/-114 vs . 518+/-120 mins for sham-treated animals (p <.01) . Overall survival (defined at 12 hrs) was also significantly better in the hemoadsorption group, seven of 20 vs . one of 20 (p <.05) . Plasma interleukin-6 and interleukin-10 concentrations and liver nuclear factor-kappaB DNA binding were significantly reduced by hemoadsorption . Ex vivo experiments showed no endotoxin adsorption but strengthened our in vivo observations by showing rapid adsorption of tumor necrosis factor, interleukin-6, and interleukin-10 . CONCLUSIONS: Hemoadsorption was associated with reduced inflammation and improved survival in this murine model of septic shock.

Crit Care Med, 2004 Mar, 32(3), 766 - 73
Novel endothelin receptor antagonist attenuates endotoxin-induced lung injury in sheep; Kuklin VN et al.; OBJECTIVE: To evaluate the cardiopulmonary effects of the novel endothelin receptor antagonist tezosentan in endotoxin-induced lung injury in sheep and to assess the dose response to tezosentan and endothelin-1 in healthy sheep . DESIGN: Prospective, randomized, controlled experimental study . SETTING: University animal laboratory . SUBJECTS: Twenty-one yearling sheep . INTERVENTIONS: Seventeen awake, chronically instrumented sheep were subjected to intravenous infusion of Ringer's lactate for 24 hrs . The animals were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide group (n = 7) receiving an intravenous infusion of Escherichia coli lipopolysaccharide 15 ng x kg x min, and a tezosentan group (n = 7) subjected to lipopolysaccharide and, from 4 hrs, an intravenous injection of tezosentan 3 mg/kg followed by infusion of 1 mg x kg x hr . In addition, four healthy sheep, exposed to an intravenous infusion of endothelin-1 at 20 ng x kg x min, after 1 hr received tezosentan in stepwise increasing doses of 0.5, 1, and 2 mg x kg x hr that were maintained for 1 hr each . After a 4-hr recovery, the sheep received infusions of tezosentan at the same dose rates as a pretreatment to endothelin-1 . MEASUREMENTS AND MAIN RESULTS: In the sham-operated sheep, all cardiopulmonary variables remained unchanged . Lipopolysaccharide caused pulmonary hypertension, increased extravascular lung water index, and induced arterial hypoxemia . Tezosentan decreased the increments in pulmonary vascular resistance and extravascular lung water index by as much as 60% and 70%, respectively . In parallel, tezosentan ameliorated arterial hypoxemia, increased cardiac index, attenuated the decrease in stroke volume index, and reduced systemic vascular resistance . Compared with the lipopolysaccharide group, tezosentan further increased plasma concentrations of endothelin-1 . In healthy animals, the administration of endothelin-1 induced systemic and pulmonary hypertension, increased extravascular lung water index, and evoked bradycardia and a decrease in cardiac index . These changes were attenuated by tezosentan infused at 1 and 2 mg x kg x hr . CONCLUSIONS: In an ovine model of endotoxin-induced lung injury, tezosentan ameliorates pulmonary hypertension, lung edema, cardiac dysfunction, and arterial hypoxemia . Tezosentan counteracts the hemodynamic effects of endothelin-1 in a dose-dependent manner.

J Biol Chem, 2004 Jul 2, 279(27), 28227 - 32 Epub 2004 Apr 16.
Three-dimensional structure of Wza, the protein required for translocation of group 1 capsular polysaccharide across the outer membrane of Escherichia coli; Beis K et al.; Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli . Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-A resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein . Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits . Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits . Wza is best described as a stack of two 4-A high rings with differing diameters providing a mushroom-like aspect from the side . The larger ring has a distinctive square shape with a diameter of 115 A, whereas the smaller is almost circular with a diameter of 90 A . In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of approximately 40 A in diameter . The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side . We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide.

J Bacteriol, 2004 May, 186(9), 2900 - 5
Polymerases leave fingerprints: analysis of the mutational spectrum in Escherichia coli rpoB to assess the role of polymerase IV in spontaneous mutation; Wolff E et al.; We compared the distribution of mutations in rpoB that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding dinB gene, strains with a chromosomal copy of dinB, strains with the F'128 plasmid, and strains with plasmid amplification of either the dinB operon (dinB-yafNOP) or the dinB gene alone . This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.

J Bacteriol, 2004 May, 186(9), 2891 - 6
Origin and diversity of alginate lyases of families PL-5 and -7 in Sphingomonas sp . strain A1; Miyake O et al.; Sphingomonas sp . strain A1 has three endotype alginate lyases (A1-I, A1-II {family PL-7}, and A1-III {family PL-5}), each of which is encoded by a single gene . In addition to those of these lyases, a gene (the A1-II' gene) showing significant identity with the A1-II gene was present in the bacterial genome and coded for an alginate lyase with broad substrate specificity . Since no expression of A1-II' was observed even in bacterial cells grown on alginate, the A1-II' gene was thought to be a silent gene derived from the A1-II gene, presumably through duplication, modification, and translocation.

J Bacteriol, 2004 May, 186(9), 2708 - 16
The protease Lon and the RNA-binding protein Hfq reduce silencing of the Escherichia coli bgl operon by H-NS; Dole S et al.; The histone-like nucleoid structuring protein H-NS represses the Escherichia coli bgl operon at two levels . H-NS binds upstream of the promoter, represses transcription initiation, and binds downstream within the coding region of the first gene, where it induces polarity of transcription elongation . In hns mutants, silencing of the bgl operon is completely relieved . Various screens for mutants in which silencing of bgl is reduced have yielded mutations in hns and in genes encoding the transcription factors LeuO and BglJ . In order to identify additional factors that regulate bgl, we performed a transposon mutagenesis screen for mutants in which silencing of the operon is strengthened . This screen yielded mutants with mutations in cyaA, hfq, lon, and pgi, encoding adenylate cyclase, RNA-binding protein Hfq, protease Lon, and phosphoglucose isomerase, respectively . In cyaA mutants, the cyclic AMP receptor protein-dependent promoter is presumably inactive . The specific effect of the pgi mutants on bgl is low . Interestingly, in the hfq and lon mutants, the downstream silencing of bgl by H-NS (i.e., the induction of polarity) is more efficient, while the silencing of the promoter by H-NS is unaffected . Furthermore, in an hns mutant, Hfq has no significant effect and the effect of Lon is reduced . These data provide evidence that the specific repression by H-NS can (directly or indirectly) be modulated and controlled by other pleiotropic regulators.

J Bacteriol, 2004 May, 186(9), 2664 - 72
Chemotactic signaling by an Escherichia coli CheA mutant that lacks the binding domain for phosphoacceptor partners; Jahreis K et al.; CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli . CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains . This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control . CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior . The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA . To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain . To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheADeltaP2 deletion mutants and examined their signaling properties in vitro and in vivo . We found that CheADeltaP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY . This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability . However, expression of additional P1 domains in the CheADeltaP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheADeltaP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability . These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.

J Bacteriol, 2004 May, 186(9), 2646 - 54
Evolution of the leucine gene cluster in Buchnera aphidicola: insights from chromosomal versions of the cluster; Sabater-Munoz B et al.; In Buchnera aphidicola strains associated with the aphid subfamilies Thelaxinae, Lachninae, Pterocommatinae, and Aphidinae, the four leucine genes (leuA, -B, -C, and -D) are located on a plasmid . However, these genes are located on the main chromosome in B . aphidicola strains associated with the subfamilies Pemphiginae and Chaitophorinae . The sequence of the chromosomal fragment containing the leucine cluster and flanking genes has different positions in the chromosome in B . aphidicola strains associated with three tribes of the subfamily Pemphiginae and one tribe of the subfamily Chaitophorinae . Due to the extreme gene order conservation of the B . aphidicola genomes, the variability in the position of the leucine cluster in the chromosome may be interpreted as resulting from independent insertions from an ancestral plasmid-borne leucine gene . These findings do not support a chromosomal origin for the leucine genes in the ancestral B . aphidicola and do support a back transfer evolutionary scenario from a plasmid to the main chromosome.

J Bacteriol, 2004 May, 186(9), 2603 - 11
HybF, a zinc-containing protein involved in NiFe hydrogenase maturation; Blokesch M et al.; HypA and HypB are maturation proteins required for incorporation of nickel into the hydrogenase large subunit . To examine the functions of these proteins in nickel insertion, the hybF gene, which is a homolog of hypA essential for maturation of hydrogenases 1 and 2 from Escherichia coli, was overexpressed, and the product was purified . This protein behaves like a monomer in gel filtration and contains stoichiometric amounts of zinc but insignificant or undetectable amounts of nickel and iron . In filter binding assays radioactively labeled nickel binds to HybF with a K(D) of 1.87 microM and in a stoichiometric ratio . To identify amino acid residues of HybF involved in nickel and/or zinc binding, variants in which conserved residues were replaced were studied . An H2Q replacement eliminated both in vivo activity and in vitro binding of nickel . The purified protein, however, contained zinc at the level characteristic of the wild-type protein . When E3 was replaced by Q, activity was retained, but an E3L exchange was detrimental . Replacement of each of the four conserved cysteine residues of a zinc finger motif reduced the cellular amount of HybF protein without a loss of in vivo activity, indicating that these residues play a purely structural role . A triple mutant deficient in the synthesis or activity of HypA, HybF, and HypB was constructed, and it exhibited the same responsiveness for phenotypic complementation by high nickel as mutants with a single lesion in one of the genes exhibited . The results are interpreted in terms of a concerted action of HypB and HybF in nickel insertion in which HybF (as well as its homolog, HypA) functions as a metallochaperone and HypB functions as a regulator that controls the interaction of HybF with the target protein.

J Bacteriol, 2004 May, 186(9), 2594 - 602
Restricted Mobility of Cell Surface Proteins in the Polar Regions of Escherichia coli; de Pedro MA et al.; The polar regions of the Escherichia coli murein sacculus are metabolically inert and stable in time . Because the sacculus and the outer membrane are tightly associated, we investigated whether polar inert murein could restrict the mobility of other cell envelope elements . Cells were covalently labeled with a fluorescent reagent, chased in dye-free medium, and observed by microscopy . Fluorescent material was more efficiently retained at the cell poles than at any other location . The boundary between high and low fluorescence intensity areas was rather sharp . Labeled material consisted mostly of cell envelope proteins, among them the free and murein-bound forms of Braun's lipoprotein . Our results indicate that the mobility of at least some cell envelope proteins is restrained at regions in correspondence with underlying areas of inert murein.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Apr, 24(4), 412 - 4
{Efficient soluble expression, purification and identification of the truncated SAG1 gene of Toxoplasma gondii in Escherichia coli}; Yan H et al.; OBJECTIVE: To express truncated SAG1 gene in Escherichia coli to obtain purified recombinant SAG1, and identify the immunoreactivity of the product . METHODS: The plasmid pET-30a(+)-trSAG1 was constructed, which was cut by Nco I and HindIII to obtain truncated SAG1 gene and inserted into pET-32a(+) cut by the same two restriction enzymes . After identification by restriction enzymes, the plasmid pET-32a(+)-trSAG1 was transformed into E.coli BL21, and the soluble product induced by 0.1 mmol/L IPTG for 4 hour before purification with Ni-NTA agarose, followed by identification of the purified recombinant protein with SDS-PAGE, Western-blotting and ELISA . RESULTS: Recombinant pET-32a(+)-trSAG1 was successfully constructed and high level of truncated SAG1 expression achieved in E.coli . SDS-PAGE showed that the expressed protein was approximately 40,000 in size and expressed in a soluble form that could be easily purified by Ni-NTA agarose . Western-blotting demonstrated that the purified product could be specifically recognized by sera from rabbits immunized with native antigen of T.gondii, and could also be recognized, as shown by ELISA, by sera from human with T.gondii infection . CONCLUSIONS: The truncated SAG1 gene was successfully expressed in E.coli in a soluble form, and the recombinant protein may be of value in the diagnosis of toxoplasmosis.

Eur J Haematol, 2004 Apr, 72(4), 273 - 9
A novel recombinant dual human SCF expressed in and purified from silkworm, Bombyx mori, possesses higher bioactivity than recombinant monomeric human SCF; Han J et al.; A novel recombinant dual human stem cell factor (rdhSCF) gene was constructed which consisted of a full-length hSCF cDNA plus a truncated hSCF cDNA (1-145 aa), linked by a peptide (GGGGSGGGGSGG) coding region . The rdhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the polyhedrin promoter control . Silkworm larvae infected with the recombinant virus expressed rdhSCF up to 15,800 units/mL in haemolymph . The specific activity of rdhSCF purified from the haemolymph was up to 3.0 x 10(6) units/mg, about 8.6 times as high as that of monomer rhSCF from Escherichia coli, and about 9.1 times as high as that of monomer rhSCF from insect cell . The binding affinity of rdhSCF to the cell surface receptor was higher than that of monomer rhSCF.

Biochem J, 2004 Jun 15, 380(Pt 3), 617 - 25
Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity; Lefebvre C et al.; At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids . In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays . The results show relevant modulations concerning several known and unknown genes . Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation . We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response . We have cloned the full-length cDNA and named the T . tessulatum gene as Tt-cysb . Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site . Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates . We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells . In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells . These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.

Chem Res Toxicol, 2004 Apr, 17(4), 529 - 36
Formation and reduction of aryl and heterocyclic nitroso compounds and significance in the flux of hydroxylamines; Kim D et al.; Cytochrome p450 (p450) 1A2 and NADPH-P450 reductase (NPR) catalyzed the oxidation of 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), with consumption of NADPH . The oxidation rate of NADPH by p450 1A2/NPR increased with time in the presence of IQ until depletion of NADPH . This unusual autocatalytic pattern of NADPH oxidation could be rationalized by formation of a nitroso derivative (IQ-N=O) and the subsequent reduction of the hydroxylamine (IQ-NHOH) and IQ-N=O, which would consume more NADPH . The formation of IQ-NHOH and IQ-N=O from IQ was confirmed using HPLC/MS . Reduction of IQ-NHOH and IQ-N=O was NPR-dependent but did not require p450 . Autocatalytic NADPH oxidation was also observed in the oxidation of other heterocyclic and arylamines . However, the N-hydroxyl and nitroso oxidation products of 2-aminofluorene and 4-aminobiphenyl were reduced nonenzymatically by NADPH, and NPR did not catalyze the reactions . We simulated the enzymatic kinetic model for possible pathways for IQ metabolism, which included the formation of IQ-N=O, using some kinetic parameters obtained from the experimental results . In the kinetic model, we could reproduce the similar curvature for NADPH oxidation and the formation of IQ-N=O, and the reduction of IQ-NHOH and IQ-N=O is required to explain the observed results for NADPH oxidation . Our results support a role for nitroso derivatives of HAAs in the unusual autocatalytic NADPH oxidation and may have relevance in terms of possible toxicities of the nitroso derivatives . Both IQ-NHOH and IQ-N=O were mutagenic in a bacterial tester system devoid of p450 and NPR; the mutagenicity of both was decreased by expression of NPR, consistent with the reduction of these compounds observed with purified NPR.

Arch Pharm Res, 2004 Mar, 27(3), 319 - 23
High-level expression of human cytochrome P450 3A4 by co-expression with human molecular chaperone HDJ-1 (Hsp40); Ahn T et al.; Cytochrome P450 (CYP) 3A4 is of great interest because of its important roles in the oxidation of numerous drugs and xenobiotics . HDJ-1, a molecular chaperone in human, is known to assist the correct folding of unfolded proteins . To achieve a high yield of recombinant human CYP3A4 in Escherichia coli, the CYP3A4 encoding gene was co-expressed with the chaperone HDJ-1, under the control of an inducible tac promoter in a bicistronic format . The levels of expression of the CYP3A4 in the bicistronic construct reached up to 715 nmol (liter culture)(-1) within 16 h at 37 degrees C, which was about a 3.3-fold increase compared to that of the CYP3A4 alone without the HDJ-1 . By co-expression with HDJ-1, the catalytic activity of CYP3A4 was also increased by approximately 15-fold . The amount of activity increase was similar to that of the CYP production at the whole cell level . The present over-expression system may be useful for the rapid production of large amounts of active CYP3A4 in E . coli.

Biometals, 2004 Apr, 17(2), 157 - 65
Effect of metal ions on the activity of the catalytic domain of calcineurin; Ping L et al.; Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB) . There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI) . It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions . Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme . In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E . coli . The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined . Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa . However, the activation degree of CNa by the metal ions was much higher than that of CNA . In term of different metal ions, the activating extents to CNA and CNa were different . To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa . No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments . Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified . These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa . The activating extents of metal ions via catalytic domain were higher than that via regulating fragment . The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.

J Pediatr Hematol Oncol, 2004 Apr, 26(4), 217 - 26
Asparaginase antibody and asparaginase activity in children with higher-risk acute lymphoblastic leukemia: Children's Cancer Group Study CCG-1961; Panosyan EH et al.; We investigated the anti-asparaginase antibody (Ab) and asparaginase enzymatic activity in the sera of 1,001 patients (CCG-1961) with high-risk acute lymphoblastic leukemia (HR-ALL) . Patients received nine doses of native Escherichia coli asparaginase during induction . Half of rapid early responders (RER) were randomly assigned to standard intensity arms and continued to receive native asparaginase . The other RER patients and all slow early responders received 6 or 10 doses of PEG-asparaginase . Serum samples (n = 3,193) were assayed for determination of asparaginase Ab titers and enzymatic activity . Three hundred ninety of 1,001 patients (39%) had no elevation of Ab among multiple evaluations-that is, were Ab-negative (<1.1 over negative control)-and 611 patients (61%) had an elevated Ab titer (>1.1) . Among these 611 patients, 447 had no measurable asparaginase activity during therapy . Patients who were Ab-positive but had no clinical allergies continued to receive E . coli asparaginase, the activity of which declined precipitately . No detectable asparaginase activity was found in 81 of 88 Ab-positive patients shortly after asparaginase injections (94% neutralizing Ab) . The Ab-positive patients with clinical allergies subsequently were given Erwinase and achieved substantial activity (0.1-0.4 IU/ml) . An interim analysis of 280 patients who were followed for 30 months from induction demonstrated that the Ab-positive titers during interim maintenance-1 and in delayed intensification-1 were associated with an increased rate of events . The CCG-1961 treatment schedule was very immunogenic, plausibly due to initially administrated native asparaginase . Anti-asparaginase Ab was associated with undetectable asparaginase activity and may be correlated with adverse outcomes in HR ALL.

Shock, 2004 May, 21(5), 484 - 91
Low-dose dexamethasone ameliorates circulatory failure and renal dysfunction in conscious rats with endotoxemia; Tsao CM et al.; Corticosteroids have long been proposed as a therapeutic adjuvant in septic renal dysfunction because of their anti-inflammatory properties and favorable results from animal experiments . However, some reports suggested the potential for harm associated with the administration of early high-dose corticosteroids in patients with severe sepsis and septic shock . Thus, we examined the effects of low-dose dexamethasone (0.01 and 0.1 mg/kg) on hemodynamics and renal function in conscious rats with endotoxemia . Intravenous injection of rats with endotoxin (E . coli lipopolysaccharide, LPS, 1 mg/kg) caused hypotension, vascular hyporeactivity, and tachycardia as well as renal dysfunction . Circulatory failure and renal dysfunction caused by LPS were significantly attenuated in the dexamethasone 0.1 mg/kg-treated group . The nitric oxide (NO) production in plasma and renal tissue and the iNOS protein expression in the kidney were suppressed by cotreatment of LPS rats with dexamethasone, 0.1 mg/kg . Light microscopy showed that 0.1 mg/kg dexamethasone reduced marked infiltration of neutrophils in renal tissues from LPS rats . Moreover, the survival rate at 18 h was significantly increased in the dexamethasone 0.1 mg/kg-treated group when compared with the LPS group . These results suggest that the beneficial effects of low-dose dexamethasone (0.1 mg/kg) in conscious rats with endotoxic shock are associated with amelioration of circulatory failure and renal dysfunction, and this is attributed to inhibition of NO production.

Shock, 2004 May, 21(5), 438 - 43
The hepatosplanchnic contribution to hyperlactatemia in endotoxic shock: effects of tissue ischemia; Creteur J et al.; We investigated the role of the hepatosplanchnic region in the hyperlactatemia observed during endotoxic shock . The study included 18 dogs anesthetized with pentobarbital and mechanically ventilated . After baseline measurements, including gut lactate production (GLP), liver lactate uptake (LLU), liver lactate extraction (LLE), and hepatosplanchnic lactate production (HSLP), each dog received 2 mg/kg of E . coli endotoxin . After a second set of measurements, cardiac tamponade was induced in 12 dogs (EDTX + Tamp) by repeated injections of normal saline into the pericardial sac to progressively reduce cardiac output and hepatic blood flow . The six remaining dogs served as septic controls (EDTX) . From a net lactate consumer before endotoxin infusion, the gut became a lactate producer after the endotoxin infusion, with GLP increasing from -11.4 +/- 27.0 to 32.9 +/- 38.2 x 10(-3) mEq/min (P < 0.05) . LLU increased from 48.1 +/- 26.2 to 86.6 +/- 45.2 x 10(-3) mEq/min (P < 0.05), so that LLE and HSLP did not change . In the EDTX + Tamp group, LLE became negative, and HSLP became positive only when hepatic oxygen delivery reached its critical value during cardiac tamponade . In the EDTX group, LLE remained positive and HSLP negative . In endotoxic shock, GLP is increased, but the liver can metabolize this additional load of lactate, so that the hepatosplanchnic area is not a major source of lactate unless the liver becomes profoundly hypoxic.

Nucleic Acids Res . 2004 Apr 15;32(7):e59.
Two-step total gene synthesis method; Young L et al.; In the post-genomic era, the ability to synthesize any arbitrary DNA sequence is increasingly in demand . A bottleneck in current gene synthesis technologies is the associated cost, due primarily to the high cost of oligonucleotides synthesis and post-synthesis sequencing . In the present paper, an improved method for low-cost gene synthesis that combines dual asymmetrical PCR and overlap extension PCR is presented, which enables any DNA sequence to be synthesized error free . Additionally, the method is easily amenable to automation.

J Biol Chem, 2004 Jun 18, 279(25), 26005 - 12 Epub 2004 Apr 14.
The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity; Levin MK et al.; Although helicases participate in virtually every cellular process involving nucleic acids, the details of their mechanism including the role of interaction between the subunits remains unclear . Here we study the unwinding kinetics of the helicase from hepatitis C virus using DNA substrates with a range of tail and duplex lengths . The binding of the helicase to the substrates was characterized by electron microscopy and fluorimetric titrations . Depending on the length of the ssDNA tail, one or more helicase molecules can be loaded on the DNA . Unwinding was measured under single-turnover conditions, and the results show that a monomer is active on short duplexes yet multiple molecules are needed to unwind long duplexes . Thus, increasing the ssDNA tail length increases the unwinding efficiency . The unwinding kinetics was modeled as a stepwise process performed by single or multiple helicase molecules . The model programmed in MATLAB was used for global fitting of the kinetics, yielding values for the rate of unwinding, processivity, cooperativity, step size, and occlusion site . The results indicate that a single hepatitis C virus helicase molecule unwinds DNA with a low processivity . The multiple helicase molecules present on the DNA substrate show functional cooperativity and unwind with greater efficiency, although they bind and release the substrate non-cooperatively, and the ATPase cycle of the helicase molecules is not coordinated . The functional interaction model explains the efficient unwinding by multiple helicases and is generally applicable.

J Biol Chem, 2004 Jun 18, 279(25), 26627 - 34 Epub 2004 Apr 15.
Identification and characterization of human and mouse ovastacin: a novel metalloproteinase similar to hatching enzymes from arthropods, birds, amphibians, and fish; Quesada V et al.; We have cloned and characterized human and mouse ovary cDNAs encoding a new protein of the astacin family of metalloproteinases, called ovastacin because of its predominant expression in ovarian tissues . Human and mouse ovastacins exhibit the same domain organization as other astacins, including signal sequence, propeptide, and metalloproteinase domain . However, ovastacins show an additional C-terminal domain of about 150 amino acids with no similarity to other ancillary domains present in the equivalent region of most astacins . Northern blot analysis of human tissues and cell lines revealed that ovastacin is only detected at significant levels in leukemia and lymphoma cells of different origin . In addition, RT-PCR analysis demonstrated that ovastacin is expressed in human and mouse ovary, in unfertilized mouse oocytes, and in 1.5-day-postcoitum preimplantation embryos . Further studies showed that superovulation caused a dramatic increase in the expression of mouse ovastacin, indicating that the production of this enzyme is under hormonal regulation . Human ovastacin was expressed in Escherichia coli and the purified recombinant protein hydrolyzed synthetic substrates used for assaying metalloproteinases . These activities were abolished by inhibitors of metalloproteinases, but not by inhibitors of other classes of proteases . On the basis of these results, we suggest that ovastacin could play in mammals a physiological function similar to that performed by hatching proteases in evolutionary distant species from arthropods to fish.

J Immunol Methods, 2004 Mar, 286(1-2), 187 - 201
Humanization of agonistic anti-human 4-1BB monoclonal antibody using a phage-displayed combinatorial library; Son JH et al.; Given the key role 4-1BB plays in the stimulation of T cells, humanization of agonistic anti-human 4-1BB monoclonal antibody (mAb) may have important clinical applications . In this paper, we present the humanization of agonistic anti-human 4-1BB mAb, BBK-4, using a phage display library . We first prepared the combinatorial library by incorporating murine and human alternative at positions representing buried residues that might affect the structural integrity of the antigen binding site . Six humanized single chain Fv (scFv) fragments were selected from the combinatorial library expressing phage-displayed humanized scFv . They were found to retain the epitope specificity of the original mAb but had affinities of lower than 1/10 of the original . In spite of the lower affinity, the humanized scFv coated on the surface expanded human peripheral blood mononuclear cells (PBMCs) in MLR similarly to the original mAb in the presence of anti-CD3 mAb . These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application.

BMC Plant Biol . 2004 Apr 15;4(1):5.
Synthesis of chlorophyll b: localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit; Eggink LL et al.; BACKGROUND: Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group . This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO) . The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein . The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly . RESULTS: Fluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy . However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane . The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center . Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center . A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042 . Fragmentation of the protein after incorporation of 125I- identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species . The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction . CONCLUSION: CAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C . reinhardtii cells . Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development . Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis.

Plant Cell, 2004 May, 16(5), 1178 - 90 Epub 2004 Apr 14.
SPIRAL1 encodes a plant-specific microtubule-localized protein required for directional control of rapidly expanding Arabidopsis cells; Nakajima K et al.; Highly organized interphase cortical microtubule (MT) arrays are essential for anisotropic growth of plant cells, yet little is known about the molecular mechanisms that establish and maintain the order of these arrays . The Arabidopsis thaliana spiral1 (spr1) mutant shows right-handed helical growth in roots and etiolated hypocotyls . Characterization of the mutant phenotypes suggested that SPR1 may control anisotropic cell expansion through MT-dependent processes . SPR1 was identified by map-based cloning and found to encode a small protein with unknown function . Proteins homologous to SPR1 occur specifically and ubiquitously in plants . Genetic complementation with green fluorescent protein fusion proteins indicated that the SPR1 protein colocalizes with cortical MTs and that both MT localization and cell expansion control are conferred by the conserved N- and C-terminal regions . Strong SPR1 expression was found in tissues undergoing rapid cell elongation . Plants overexpressing SPR1 showed enhanced resistance to an MT drug and increased hypocotyl elongation . These observations suggest that SPR1 is a plant-specific MT-localized protein required for the maintenance of growth anisotropy in rapidly elongating cells.

J Biol Chem, 2004 Jun 25, 279(26), 26932 - 8 Epub 2004 Apr 14.
Dimerization of CtIP, a BRCA1- and CtBP-interacting protein, is mediated by an N-terminal coiled-coil motif; Dubin MJ et al.; CtIP is a transcriptional co-regulator that binds a number of proteins involved in cell cycle control and cell development, such as CtBP (C terminus-binding protein), BRCA1 (breast cancer-associated protein-1), and LMO4 (LIM-only protein-4) . The only recognizable structural motifs within CtIP are two putative coiled-coil domains located near the N and C termini of the protein . We now show that the N-terminal coiled coil (residues 45-160), but not the C-terminal coiled coil, mediates homodimerization of CtIP in mammalian 293T cells . The N-terminal coiled coil did not facilitate binding to LMO4 and BRCA1 proteins in these cells . A protease-resistant domain (residues 27-168) that minimally encompasses the putative N-terminal coiled coil was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry . This region is predicted to contain two smaller coiled-coil regions . The CtIP-(45-160) dimerization domain is helical and dimeric, indicating that the domain does form a coiled coil . The two smaller domains, CtIP-(45-92) and CtIP-(93-160), also formed dimers of lower binding affinity, but with less helical content than the longer peptide . The hydrodynamic radius of CtIP-(45-160) is the same as those of CtIP-(45-92) and CtIP-(93-160), implying that CtIP-(45-160) does not form a single long coiled coil, but a more compact structure involving homodimerization of the two smaller coiled coils, which fold back as a four-helix bundle or other compact structure . These results suggest a specific model for CtIP homodimerization via its N terminus and contribute to an improved understanding of how this protein might assemble other factors required for its role as a transcriptional corepressor.

DNA Repair (Amst), 2004 May 4, 3(5), 515 - 25
A homolog of Escherichia coli RecA in mitochondria of the cellular slime mold Dictyostelium discoideum; Hasegawa Y et al.; The cellular slime mold Dictyostelium discoideum expresses a gene encoding a 452-amino-acid polypeptide that is 47% identical to Escherichia coli RecA . A recA-deficient E . coli, JE6651, was transformed by pYSN1, which was designed to express the truncated form of the D . discoideum gene, and used in suppression assays . The viability of the transformant, JE6651(pYSN1), increased following UV irradiation or mitomycin C treatment . Phage lambda (red(-) gam(-)), which required RecA activity for DNA packaging, formed plaques on a lawn of JE6651(pYSN1) . These results indicate that the gene product has a DNA recombination activity . Fluorescence of D . discoideum protein fused with GFP was detected in mitochondria . The gene disruption mutant was hypersensitive to UV-light (254nm), mitomycin C and H(2)O(2), indicating that D . discoideum recA is important for survival following exposure to DNA damaging agents.

Cell, 2004 Apr 16, 117(2), 199 - 209
Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed; Agashe VR et al.; Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E . coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood . Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation . Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro . This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes . While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E . coli remains inefficient . The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system . These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.

Biotechnol Bioeng, 2004 May 5, 86(3), 317 - 31
Using metabolic flux data to further constrain the metabolic solution space and predict internal flux patterns: the Escherichia coli spectrum; Wiback SJ et al.; Constraint-based metabolic modeling has been used to capture the genome-scale, systems properties of an organism's metabolism . The first generation of these models has been built on annotated gene sequence . To further this field, we now need to develop methods to incorporate additional "omic" data types including transcriptomics, metabolomics, and fluxomics to further facilitate the construction, validation, and predictive capabilities of these models . The work herein combines metabolic flux data with an in silico model of central metabolism of Escherichia coli for model centric integration of the flux data . The extreme pathways for this network, which define the allowable solution space for all possible flux distributions, are analyzed using the alpha-spectrum . The alpha-spectrum determines which extreme pathways can and cannot contribute to the metabolic flux distribution for a given condition and gives the allowable range of weightings on each extreme pathway that can contribute . Since many extreme pathways cannot be used under certain conditions, the result is a "condition-specific" solution space that is a subset of the original solution space . The alpha-spectrum results are used to create a "condition-specific" extreme pathway matrix that can be analyzed using singular value decomposition (SVD) . The first mode of the SVD analysis characterizes the solution space for a given condition . We show that SVD analysis of the alpha-spectrum extreme pathway matrix that incorporates measured uptake and byproduct secretion rates, can predict internal flux trends for different experimental conditions . These predicted internal flux trends are, in general, consistent with the flux trends measured using experimental metabolic flux analysis techniques .

Lasers Surg Med, 2004, 34(4), 352 - 9
Quantitative and qualitative changes of the seminiferous epithelium induced by Ga . Al . As . (830 nm) laser radiation; Taha MF et al.; BACKGROUND AND OBJECTIVES: Low level laser radiation stimulates both nucleic acid synthesis and cellular proliferation in E . coli, Hela tumor cells, fibroblasts, lymphocytes, and thyroid cells . It has been introduced as a therapeutic modality; nevertheless few studies have been carried out to determine the effects of laser radiation on the testes or spermatogenesis . The aim of this study was to determine the quantitative and qualitative changes of the seminiferous epithelium after Ga . Al . As . (830 nm) laser radiation . STUDY DESIGN/MATERIALS AND METHODS: The left testes of Sprague-Dawley rats were daily exposed to laser light for 15 days; so the cumulative doses used 28.05 and 46.80 J/cm(2) in two experimental groups . Sampling carried out 24 hours after the last treatment and samples were processed for LM and TEM study . RESULTS: The number of germ cells specially the pachytene spermatocytes and elongated spermatids increased after 28.05 J/cm(2) laser radiation . Ultrastructural features of germ and Sertoli cells in this group were similar to that of control; while laser irradiation at 46.80 J/cm(2) had a destructive effect on the seminiferous epithelium such as dissociation of immature spermatids and evident ultrastructural changes in them . CONCLUSIONS: The findings confirmed the existence of a biostimulatory threshold of applied laser energy and the importance of determining it for clinical applications . Moreover, it was revealed that low doses of laser light have a biostimulatory effect on the spermatogenesis and may provide benefits to the patients with oligospermia and azoospermia .

Protein Eng Des Sel, 2004 Mar, 17(3), 213 - 21 Epub 2004 Apr 13.
A carbohydrate binding module as a diversity-carrying scaffold; Cicortas Gunnarsson L et al.; The growing field of biotechnology is in constant need of binding proteins with novel properties . Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications . In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds . In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold . A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2 . Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method . Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C) . All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.

J Biol Chem, 2004 Jun 18, 279(25), 26411 - 6 Epub 2004 Apr 13.
High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine; Eneqvist T et al.; Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A . So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%) . Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)) . Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR . X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution . The apo structure is similar to human TTR with structural changes only at beta-strand D . This strand forms an extended loop conformation similar to the one in chicken TTR . The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4) . The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).

Genetics, 2004 Mar, 166(3), 1165 - 76
A dnaC mutation in Escherichia coli that affects copy number of ColE1-like plasmids and the PriA-PriB (but not Rep-PriC) pathway of chromosomal replication restart; Harinarayanan R et al.; Escherichia coli nusG and rho mutants, which are defective in transcription termination, are killed following transformation with several ColE1-like plasmids that lack the plasmid-encoded copy-number regulator gene rom because of uncontrolled plasmid replication within the cells . In this study, a mutation {dnaC1331(A84T)} in the dnaC gene encoding the replicative helicase-loading protein was characterized as a suppressor of this plasmid-mediated lethality phenotype . The mutation also reduced the copy number of the plasmids in otherwise wild-type strains . In comparison with the isogenic dnaC(+) strain, the dnaC mutant was largely unaffected for (i) growth on rich or minimal medium, (ii) tolerance to UV irradiation, or (iii) survival in the absence of the PriA, RecA, or RecB proteins . However, it was moderately SOS-induced and was absolutely dependent on both the Rep helicase and the PriC protein for its viability . A dnaC1331(A84T) dam mutant, but not its mutH derivative, exhibited sensitivity to growth on rich medium, suggestive of a reduced capacity in the dnaC1331(A84T) strains to survive chromosomal double-strand breaks . We propose that DnaC-A84T is proficient in the assembly of replication forks for both initiation of chromosome replication (at oriC) and replication restart via the Rep-PriC pathway, but that it is specifically defective for replication restart via the PriA-PriB pathway (and consequently also for replication of the Rom(-) ColE1-like plasmids).

Neurochem Int, 2004 Jul, 45(1), 171 - 8
Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats; Seki T et al.; The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions . Our previous studies have demonstrated that absence-like seizures of the tremor rat (tm/tm), one of the parent strains of SER, were inhibited by adenoviral transfer of the aspartoacylase (ASPA) gene, a deleted gene in the tremor rat . In the present study, we examined whether the adenoviral gene transfer of ASPA inhibited the tonic convulsions of SER . Replication-defective recombinant adenoviral vectors carrying the rat ASPA gene (AxASPA) or Escherichia coli beta-galactosidase gene (AxLacZ), as a control, were constructed . After it was confirmed that AxASPA-infected HeLa cells expressed ASPA in vitro, AxASPA or AxLacZ was administered into the left lateral ventricle of 11-week-old SER . The occurrence and duration of tonic convulsions in SER were evaluated before and after the administration of adenoviral vector . Intracerebroventricular administration of AxASPA (5 x 10(7) plaque forming units) transiently, but significantly, inhibited the occurrence of tonic convulsions in SER without affecting the duration per single convulsion 7 days after the administration . No inhibitory effects were observed 10 and 14 days after AxASPA administration . In contrast, the administration of AxLacZ did not have any effect on tonic convulsions in SER . Survival rates did not differ between AxASPA- and AxLacZ-treated SERs . Adenoviral gene transfer of ASPA, one of the deleted genes of SER, transiently rescued SERs from tonic convulsion, although it did not improve their survival time.

Neurochem Int, 2004 Jul, 45(1), 73 - 9
Expression of cystathionine beta-synthase, pyridoxal kinase, and ES1 protein homolog (mitochondrial precursor) in fetal Down syndrome brain; Shin JH et al.; Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized by somatic anomalies and mental retardation . The phenotype of DS is thought to result from overexpression of genes encoded on chromosome 21 . Although several studies reported mRNA levels of genes localized on chromosome 21, mRNA data cannot be simply extrapolated to protein levels . Furthermore, most protein data have been generated using immunochemical methods . In this study we investigated expression of three proteins (cystathionine beta-synthase (CBS), pyridoxal kinase (PDXK), ES1 protein homolog, mitochondrial precursor (ES1)) whose genes are encoded on chromosome 21 in fetal DS (n = 8; mean gestational age of 19.8 +/- 2.0 weeks) and controls (n = 7; mean gestational age of 18.8 +/- 2.2 weeks) brains (cortex) using proteomic technologies . Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied . Subsequent quantitative analysis of CBS and PDXK revealed levels comparable between DS and controls . By contrast, ES1 was two-fold elevated (P < 0.01) in fetal DS brain . This protein shows significant homology with the E . coli SCRP-27A/ELBB and zebrafish ES1 protein and contains a potential targeting sequence to mitochondria in its N-terminal region . Based on the assumption that structural similarities reflect functional relationship, it may be speculated that ES1 is serving a basic function in mitochondria . Although no function of the human ES1 protein is known yet, ES1 may be a candidate protein involved in the pathogenesis of the brain deficit in DS.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 May 25, 804(2), 413 - 20
Using capillary electrophoresis with laser-induced fluorescence to study the interaction of green fluorescent protein-labeled calmodulin with Ca2+- and calmodulin-binding protein; Zhang JF et al.; A separation using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was applied to the study of green fluorescent protein tagged calmoldulin (GFP-CaM) that was expressed from Escherichia coli and purified with Ni(2+)-nitrilotriacetate (Ni-NTA) resin column . It was found that GFP-CaM not only has good fluorescence properties under various conditions similar to GFP, but also retains its calcium-binding ability as the native CaM . GFP-CaM was separated and detected by CE-LIF within 10 min with a limit-of-detection (LOD) of 2 x 10(-10) M for an injection volume of 3 nl, higher than that of common chemical fluorescent-tagged protein method . The results indicated that, as a fluorescence probe, GFP could overcome the drawback of inefficient derivatization of chemical fluorescence probes . The interaction between the GFP-CaM and Ca(2+) was studied in detail using affinity capillary electrophoresis with laser-induced fluorescence and the dissociation constant (K(d)) between GFP-CaM and Ca(2+) was determined to be 1.2 x 10(-5), which is in good agreement with the literature values of untagged CaM (10(-6) to 10(-5)M) obtained by conventional method . As a preliminary application, the interaction between GFP-CaM and OsCBK was also investigated . The method makes it possible to screen the trace amounts of target proteins in crude extracts interacting with CaM under physiological conditions.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 May 25, 804(2), 327 - 35
Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process; Eon-Duval A et al.; Anion-exchange is the most popular chromatography technique in plasmid DNA purification . However, poor resolution of plasmid DNA from RNA often results in the addition of bovine-derived ribonuclease (RNase) A to degrade RNA impurities which raises regulatory concerns for the production of pharmaceutical-grade plasmid DNA . Low capacity for plasmid of most commercial media is another issue affecting the suitability of anion-exchange chromatography for large-scale processing . This study reports the use of anion-exchange chromatography to remove RNA in an RNase-free plasmid purification process . Resolution was achieved through careful selection of adsorbent and operating conditions as well as RNA reduction steps before chromatography . Dynamic capacity for plasmid was significantly increased (to 3.0mg/ml) so that it is now possible to envisage the large-scale manufacturing of therapeutic-grade plasmid DNA in the absence of added RNase using anion-exchange chromatography as a polishing step.

Anal Biochem, 2004 May 1, 328(1), 60 - 6
Construction of a whole-cell gene reporter for the fluorescent bioassay of nitrate; Taylor CJ et al.; The development of a whole-cell fluorescence-based biosensor for nitrate is reported . The sensor is Escherichia coli transformed with a plasmid (pPNARGFP) in which the promoter and regulatory regions of the membrane-bound nitrate reductase narGHJI operon (Pnar) are fused to a gfp gene encoding green fluorescent protein (GFP) . Pnar-gfp activity was measured at a range of nitrate concentrations using whole-cell GFP fluorescence . The bioassay conditions have been optimized so that the fluorescence intensity is proportional to the extracellular nitrate concentration . The developed bioassay has established that E . coli (pPNARGFP) can be used for the quantitative determination of nitrate in environmental waters without interference from other electron acceptors, e.g., nitrite, dimethyl sulfoxide, trimethylamine-N-oxide and fumerate, and azide, an inhibitor of redox-active proteins.

Anal Biochem, 2004 May 1, 328(1), 44 - 50
Microscale synthesis of isotopically labeled R-{6-xH}N5,N10-methylene-5,6,7,8-tetrahydrofolate as a cofactor for thymidylate synthase; Agrawal N et al.; A one-pot synthesis of isotopically labeled R-{6-xH}N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4F) is presented, where x=1, 2, or 3 represents hydrogen, deuterium, or tritium, respectively . The current procedure offers high-yield, high-purity, and microscale-quantity synthesis . In this procedure, two enzymes were used simultaneously in the reaction mixture . The first was Thermoanaerobium brockii alcohol dehydrogenase, which stereospecifically catalyzed a hydride transfer from C-2-labeled isopropanol to the re face of oxidized nicotinamide adenine dinucleotide phosphate to form R-{4-xH}-labeled reduced nicotinamide adenine dinucleotide phosphate . The second enzyme, Escherichia coli dihydrofolate reductase, used the xH to reduce 7,8-dihydrofolate (H2F) to form S-{6-xH}5,6,7,8-tetrahydrofolate (S-{6-xH}H4F) . The enzymatic reactions were followed by chemical trapping of S-{6-xH}H4F with formaldehyde to form the final product . Product purification was carried out in a single step by reverse phase high-pressure liquid chromatography separation followed by lyophilization . Two analytical methods were developed to follow the reaction progress . Finally, the utility of the labeled cofactor in mechanistic studies of thymidylate synthase is demonstrated by measuring the tritium kinetic isotope effect on the enzyme's second order rate constant.

Anal Biochem, 2004 May 1, 328(1), 14 - 21
Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061; Arias-Negrete S et al.; Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described . To obtain the crude bacterial lysate containing nitrate reductase activity, E . coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25% . Nitrate reductase activity was detected mainly in the crude preparation . To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay . Nitrate solutions were reduced to nitrite in a range of 60-70% . Importantly, no cofactors were necessary to perform nitrate reduction . The biological samples were first reduced with the nitrate reductase preparation . After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified . The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E . coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages . Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.

Arch Biochem Biophys, 2004 May 1, 425(1), 33 - 41
Access to the carbamate tunnel of carbamoyl phosphate synthetase; Kim J et al.; The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli revealed the existence of a molecular tunnel that has been proposed to facilitate the translocation of reaction intermediates between remotely located active sites . Five highly conserved glutamate residues, including Glu-25, Glu-383, Glu-577, Glu-604, and Glu-916, are close together in two clusters in the interior wall of the molecular tunnel that enables the intermediate carbamate to migrate from the site of synthesis to the site of utilization . Two arginines, Arg-306 and Arg-848, are located at either end of the carbamate tunnel and participate in the binding of ATP at each of the two active sites within the large subunit of CPS . The mutation of Glu-25 or Glu-577 results in a diminution in the overall rate of carbamoyl phosphate formation . Similar effects are observed upon mutation of Arg-306 and Arg-848 to alanine residues . The conserved glutamate and arginine residues may function in concert with one another to control entry of carbamate into the tunnel prior to phosphorylation to carbamoyl phosphate . The electrostatic environment of tunnel interior may help to stabilize the tunnel architecture and prevent decomposition of carbamate through protonation.

Immunol Lett, 2004 Apr 15, 92(3), 237 - 43
Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine; Zhu MS et al.; Induction of effective cytotoxic T lymphocyte (CTL) and/or a specific antibody against conserved viral proteins may be essential to the development of a safe and effective severe acute respiratory syndrome coronavirus (SARS-Cov) vaccine . DNA vaccination represents a new strategy for induction of humoral and cellular immune response . To determine the ability of SARS-Cov nucleoprotein (N protein) to induce antiviral immunity, in this report, we established a stable C2C12 line expressing SARS-Cov N protein, which was used as a target for specific CTL assay . We also expressed recombinant N proteins in Escherichia coli and prepared N protein-specific polyclonal antibodies . C3H/He mice were immunized with N protein-expressible pcDN-fn vector by intramuscular injections . We found that the DNA vaccination induced both N protein-specific antibody and specific CTL activity to the target . When C3H/He mice were immunized by three separate injections, high antibody titre (1:3200-1:6400, average titre is 1:4580) and high CTL activity (67.4+/-8.4% (E:T = 25:1), 69.6+/-6.7% (E:T = 50:1) and 71.8+/-6.2% (E:T = 100:1)) were observed . In the case of two vaccine injections, CTL activity was also high (56.6+/-12.7% (E:T = 25:1), 57.4+/-11.7% (E:T = 50:1) and 63.0+/-6.3% (E:T = 100:1)) However, antibody titres were much lower (1:200-1:3200, average titre is 1:980) . Our results suggest that SARS-Cov nucleocapsid gene might be a candidate gene for SARS DNA vaccination.

Biochem Biophys Res Commun, 2004 May 7, 317(3), 957 - 64
The 'effective number of codons' revisited; Fuglsang A; Frank Wright {Gene 87 (1990) 23} derived a formula for calculation of a quantity termed the 'effective number of codons' (Nc) based on codon homozygosities . This quantity is a number between 20 and 61 and tells to what degree the codon usage in a gene is biased, i.e., it approaches 20 codons for the extremely biased genes, and approaches 61 for the genes where all possible codons are used with no preference . Among the different measures of codon bias Nc is considered the most useful and has found widespread use in papers dealing with codon usage phenomena . In this paper, the mathematical behaviours of codon homozygosities and Nc are evaluated, using Escherichia coli as the model organism . The results indicate that the classical formula for calculation of Nc could appropriately be substituted under circumstances, where there is bias discrepancy, i.e., when one amino acid (or more) within a degeneracy group is associated with strong codon bias while at the same time others in the same degeneracy group have little bias . An alternative estimator, termed Nc, is proposed and tested against Nc, and performs better when there is such bias discrepancy.

Phytochemistry, 2004 Apr, 65(7), 837 - 46
O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase; Kota P et al.; Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates . 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli . OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT . However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa . Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives . Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.

Phytochemistry, 2004 Apr, 65(7), 801 - 11
The allene oxide cyclase of barley (Hordeum vulgare L.)--cloning and organ-specific expression; Maucher H et al.; The naturally occurring enantiomer of the various octadecanoids and jasmonates is established in a biosynthetic step catalyzed by the allene oxide cyclase (AOC) . The AOC converts an allene oxide formed by an allene oxide synthase (AOS) . Here, we show cloning and characterization of cDNAs encoding the AOC and a third AOS, respectively, in addition to the two AOSs previously published (Plant J . 21, 199-213, 2000) . The ORF of the AOC-cDNA of 717 bp codes for a protein of 238 amino acid residues carrying a putative chloroplast target sequence . Overexpression without chloroplast target sequence revealed AOC activity . The AOC was found to be a single copy gene which mapped on chromosome 6H . AOC mRNA accumulation appeared in leaf segments upon treatment with various jasmonates, octadecanoids and ABA or during stress such as treatment with sorbitol or glucose solutions . Infection with powdery mildew activated AOC expression in susceptible and resistant lines of barley which correlated with PR1b expression . Among different tissues of barley seedlings, the scutellar node and leaf base accumulated AOC mRNA preferentially which correlated with accumulation of mRNAs for other biosynthetic enzymes (lipoxygenases, AOSs) . AOC mRNA accumulation appeared also abundantly in parts of the root containing the tip and correlated with elevated levels of jasmonates . The data suggest a link of AOC expression and JA formation and support role of JA in stress responses and development of barley.

Allergy, 2004 May, 59(5), 526 - 32
Tomato profilin Lyc e 1: IgE cross-reactivity and allergenic potency; Westphal S et al.; BACKGROUND: To date, very little data are available about the nature of tomato allergens . Immunoglobulin E (IgE) cross-reactive profilins have been suggested to account for allergic symptoms in patients suffering from tomato allergy . METHODS: The cDNA of tomato profilin was amplified by reversely transcribed polymerase chain reaction (RT-PCR) from total RNA extracted from ripe tomato fruit . The gene was cloned into the pET101D expression plasmid and the protein was produced in Escherichia coli BL21 . Purification was performed via poly-l-proline (PLP) affinity chromatography . IgE reactivity of recombinant tomato profilin was investigated by immunoblot and enzyme-linked immunosorbent assay . IgE-inhibition studies were performed to analyse cross-reactivity with other profilins . To determine the allergenic activity of the recombinant protein, basophil histamine release assays using sera of patients with adverse reactions to tomato were performed . RESULTS: Profilin was identified as a new minor allergen in tomato fruits . The recombinant tomato profilin comprises 131 amino acids and high sequence identity to other allergenic food and pollen profilins . It was shown to be IgE-reactive with a prevalence of 22% (11/50) in tomato-allergic patients . In patients with tomato allergy and multiple sensitization to other foods and birch pollen, IgE directed against tomato profilin showed a strong cross-reactivity with profilins from plant food sources and birch pollen . The tomato profilin was able to induce mediator release from human basophils . CONCLUSION: The tomato profilin is a minor allergen in tomato fruit . Thus, it shows biological activity, as confirmed by in vitro histamine release assays with human basophils and thereby has the potential to account for clinical symptoms in tomato-allergic patients.

Res Commun Mol Pathol Pharmacol, 2002, 112(1-4), 145 - 57
Expression of the fragile histidine triad gene in normal rat tissues and human kidney cancer cell lines; Lee SH et al.; The fragile histidine triad (FHIT) gene located in human chromosome 3p14.2 is a candidate tumor suppressor gene that has a diadenosine triphosphate (Ap3A) hydrolase activity, but its role in carcinogenesis remains uncertain . To investigate the role of FHIT in normal cells, specific polyclonal antibodies to recombinant rat FHIT protein were generated . Immunoblot analysis revealed the 17-kd FHIT protein was most abundantly expressed in kidney and liver, whereas heart, skeletal muscle, and adrenal gland expressed trace amounts . In kidney, immunohistochemical staining was strongly observed in distal convoluted tubule and collecting duct during postnatal growth period . By a nested reverse transcription-PCR analysis of FHIT from 2 human kidney cancer cell lines, four abnormal-sized FHIT transcripts, with deletion and/or insertion, were detected . These were derived from the results of exon skipping, and/or insertion of FHIT intron 5 sequence, or selection of cryptic splice site within the FHIT cDNA sequence 179-180 . Taken together, our data indicate that FHIT expression is frequently altered in human kidney cancer cell lines by alternative splicing, and suggest that the FHIT protein may play a pivotal role in regulating intracellular metabolism of the distal convoluted tubule and collecting duct in maturity.

J Anim Sci, 2004 Apr, 82(4), 1100 - 7
High dietary phytase levels maximize phytate-phosphorus utilization but do not affect protein utilization in chicks fed phosphorus- or amino acid-deficient diets; Augspurger NR et al.; Four trials investigated the effect of high levels of three phytase enzymes on P and protein utilization in chicks . The three phytases were derived from Aspergillus (Fungal Phytase 1), Peniophora (Fungal Phytase 2), and E . coli . Within each assay, 8-d-old male chicks were given ad libitum access to their experimental diet for 10 to 14 d . For Trials 1, 2, and 3, the basal diet was a corn-soybean meal diet deficient in P that was analyzed to contain 23% CP and 0.38% total P (0.10% estimated available P, as-fed basis) . Phytase supplementation levels were based on the assessment of phytase premix activity (i.e., P release from Na phytate at pH 5.5 and 37 degrees C) . In Trial 1, supplementation of inorganic P from KH2PO4 (0 to 0.20%) resulted in a quadratic (P < 0.05) response in weight gain, gain:feed, and tibia ash concentration but a linear (P < 0.01) increase in tibia ash weight . Tibia ash was higher (P < 0.01) for chicks fed E . coli phytase than for those fed Fungal Phytase 1 at 500, 1,000, and 5,000 phytase units (FTU)/kg, but did not differ between these two phytases at 10,000 FTU/kg . In Trial 2, E . coli phytase supplementation at 1,000 FTU/kg maximized growth and bone responses, whereas addition of either of the two fungal phytases resulted in increasing responses up to 5,000 and 10,000 FTU/kg . Dietary addition of Fungal Phytase 2 resulted in the poorest (P < 0.01) responses among the three phytases . Escherichia coli phytase supplementation at 10,000 FTU/kg in Trial 3 resulted in tibia ash (millligrams) responses that were greater (P < 0.05) than those resulting from either 0.35% inorganic P supplementation or 10,000 FTU/kg of Fungal Phytase 1 or 2 . Trial 4 showed that E . coli phytase supplementation at either 500 or 10,000 FTU/ kg did not improve protein efficiency ratio (gain per unit of protein intake) of chicks fed low-protein soybean meal or corn gluten meal diets that were first-limiting in either methionine or lysine, respectively . These results demonstrate that high dietary levels of efficacious phytase enzymes can release most of the P from phytate, but they do not improve protein utilization.

J Virol, 2004 May, 78(9), 4508 - 16
A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase; Chen B et al.; The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes . We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B . Chen et al., J . Biol . Chem . 275:34946-34953, 2000) . We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase) . The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140 . The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains . Thus, the fusion protein is trimeric . When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase . Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC . The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.

J Biol Chem, 2004 Jun 25, 279(26), 27376 - 82 Epub 2004 Apr 12.
Leukotriene A4 hydrolase: identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates; Rudberg PC et al.; Leukotriene (LT) A(4) hydrolase is a bifunctional zinc metalloenzyme, which converts LTA(4) into the neutrophil chemoattractant LTB(4) and also exhibits an anion-dependent aminopeptidase activity . In the x-ray crystal structure of LTA(4) hydrolase, Arg(563) and Lys(565) are found at the entrance of the active center . Here we report that replacement of Arg(563), but not Lys(565), leads to complete abrogation of the epoxide hydrolase activity . However, mutations of Arg(563) do not seem to affect substrate binding strength, because values of K(i) for LTA(4) are almost identical for wild type and (R563K)LTA(4) hydrolase . These results are supported by the 2.3-A crystal structure of (R563A)LTA(4) hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function . For the aminopeptidase reaction, mutations of Arg(563) reduce the catalytic activity (V(max) = 0.3-20%), whereas mutations of Lys(565) have limited effect on catalysis (V(max) = 58-108%) . However, in (K565A)- and (K565M)LTA(4) hydrolase, i.e . mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (K(m) = 480-640%) . Together, our data indicate that Arg(563) plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site . In the aminopeptidase reaction, Arg(563) and Lys(565) seem to cooperate to provide sufficient binding strength and productive alignment of the substrate . In conclusion, Arg(563) and Lys(565) possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA(4) hydrolase.

Genome Res, 2004 May, 14(5), 917 - 24 Epub 2004 Apr 12.
Computational analysis of Plasmodium falciparum metabolism: organizing genomic information to facilitate drug discovery; Yeh I et al.; Identification of novel targets for the development of more effective antimalarial drugs and vaccines is a primary goal of the Plasmodium genome project . However, deciding which gene products are ideal drug/vaccine targets remains a difficult task . Currently, a systematic disruption of every single gene in Plasmodium is technically challenging . Hence, we have developed a computational approach to prioritize potential targets . A pathway/genome database (PGDB) integrates pathway information with information about the complete genome of an organism . We have constructed PlasmoCyc, a PGDB for Plasmodium falciparum 3D7, using its annotated genomic sequence . In addition to the annotations provided in the genome database, we add 956 additional annotations to proteins annotated as "hypothetical" using the GeneQuiz annotation system . We apply a novel computational algorithm to PlasmoCyc to identify 216 "chokepoint enzymes." All three clinically validated drug targets are chokepoint enzymes . A total of 87.5% of proposed drug targets with biological evidence in the literature are chokepoint reactions . Therefore, identifying chokepoint enzymes represents one systematic way to identify potential metabolic drug targets.

J Appl Microbiol, 2004, 96(5), 982 - 6
The expression of an R3 lipopolysaccharide-core by pathotypes of Escherichia coli; Chart H et al.; AIMS: To investigate the incidence of an R3 lipopolysaccharide (LPS)-core amplicon in a range of pathotypes of Escherichia coli, including Verocytotoxin-producing E . coli (VTEC), enteroaggregative E . coli (EAggEC) and enteropathogenic E . coli (EPEC) . METHODS AND RESULTS: A total of 100 strains of E . coli belonging to a range of pathotypes, including 41 strains of VTEC, were screened for the genes encoding the R3 LPS-core using PCR . Fifty-four per cent produced an amplicon with the R3 primer set . Of the 41 VTEC, 66% had an R3 LPS-core with a PCR product being observed with all strains belonging to serotypes O26:H11, O111ac:H- and O145:H25 . However, 46% of enteroaggregative E . coli and 50% of enteropathogenic E . coli were also shown to have an R3 LPS-core structure . CONCLUSIONS: Strains with an R3 LPS-core are widely distributed within the species E . coli . SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of E . coli with an R3 LPS-core structure appear not to be associated with a specific pathotype.

Plant J, 2004 Apr, 38(2), 276 - 84
The tandem complex of BEL and KNOX partners is required for transcriptional repression of ga20ox1; Chen H et al.; Summary Two interacting three amino acid loop extension (TALE) proteins of potato, StBEL5 (Solanum tuberosom BEL 5) and POTH1 (potato homeobox 1), mediate developmental processes by regulating phytohormone levels . Overexpression of either partner alone increased tuber yields by lowering gibberellin (GA) levels and increasing cytokinins . Gel shift assays demonstrated that StBEL5 and POTH1 bind to the regulatory region of ga20 oxidase1 (ga20ox1) from potato, a gene encoding a key enzyme in the GA biosynthetic pathway . In tandem, StBEL5 and POTH1 had a greater binding affinity for the ga20ox1 promoter than either protein alone . The StBEL5-POTH1 heterodimer bound specifically to a composite 10-bp sequence, containing two TGAC cores . Using a transcription assay, StBEL5 and POTH1 suppressed the activity of the ga20ox1 promoter by more than 50% . Dominant negative constructs containing the protein-binding domains of StBEL5 or POTH1 blocked the repression activity of StBEL5 or POTH1, respectively . The mutated ga20ox1 promoter that could be bound by the StBEL5 or POTH1 proteins individually but not by the StBEL5-POTH1 heterodimer also abolished the repression activity of StBEL5, POTH1, and the StBEL5-POTH1 heterodimer . These results indicate that the tandem interaction of StBEL5 and POTH1 is essential for regulation of the expression of their target gene.

Curr Protein Pept Sci, 2004 Apr, 5(2), 107 - 18
Complex II from a structural perspective; Horsefield R et al.; The super-macromolecular complex, succinate:quinone oxidoreductase (SQR, Complex II, succinate dehydrogenase) couples the oxidation of succinate in the matrix / cytoplasm to the reduction of quinone in the membrane . This function directly connects the Krebs cycle and the aerobic respiratory chain . Until the recent first report of the structure of SQR from Escherichia coli (E . coli) the structure-function relationships in SQR have been inferred from the structures of the homologous QFR, which catalyses the same reaction in the opposite direction . The structure of SQR from E . coli, analogous to the mitochondrial respiratory Complex II, has provided new insight into SQR's molecular design and mechanism, revealing the electron transport pathway through the enzyme . Comparison of the structures of SQR, QFR and other related flavoproteins shows how common amino acid residues at the interface of two domains facilitate the inter-conversion of succinate and fumarate . Additionally, the structure has provided a possible explanation as to why certain organisms utilise both SQR and QFR despite the fact that both can catalyse the inter-conversion of succinate and fumarate, in vitro and in vivo . Here we review how this structure has advanced our knowledge of this important enzyme and compare the structural information to other members of the Complex II superfamily and related flavoproteins.

Biochemistry, 2004 Apr 20, 43(15), 4583 - 91
Essential role of histidine 20 in the catalytic mechanism of Escherichia coli peptidyl-tRNA hydrolase; Goodall JJ et al.; The peptidyl-tRNA hydrolase (Pth) enzyme plays an essential role in recycling tRNA from peptidyl-tRNA that has prematurely dissociated from the ribosome . In this study of Escherichia coli Pth, the critical role of histidine 20 was investigated by site-directed mutagenesis, stopped-flow kinetic measurements, and chemical modification . The histidine residue at position 20 is known to play an important role in the hydrolysis reaction, but stopped-flow fluorescence measurements showed that, although the His20Asn Pth mutant enzyme was unable to hydrolyze the substrate, the enzyme retained the ability to bind peptidyl-tRNA . Chemical modification of Pth with diethyl pyrocarbonate (DEPC) showed that a residue, with a pK(a) value of 6.3, was essential for substrate hydrolysis and that the stoichiometry of inhibition was 0.70 +/- 0.06 mol of DEPC/mol of enzyme, indicating that modification of only a single residue by DEPC was responsible for the loss of activity . Parallel chemical modification studies with the His20Asn and Asp93Asn mutant enzymes showed that this essential residue was His20 . These studies indicate that histidine 20 acts as the catalytic base in the hydrolysis of peptidyl-tRNA by Pth.

Biochemistry, 2004 Apr 20, 43(15), 4559 - 67
FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli; Bramkamp M et al.; The KdpFABC complex of Escherichia coli, which belongs to the P-type ATPase family, has a unique structure, since catalytic activity (KdpB) and the capacity to transport potassium ions (KdpA) are located on different subunits . We found that fluorescein 5-isothiocyanate (FITC) inhibits ATPase activity, probably by covalently modifying lysine 395 in KdpB . In addition, we observed that the KdpFABC complex is able to hydrolyze p-nitrophenyl phosphate (pNPP) in a Mg(2+)-dependent reaction . The pNPPase activity is inhibited by FITC and o-vanadate . Low concentrations of ATP (1-30 microM) stimulate the pNPPase activity, while concentrations of >500 microM are inhibitory . This behavior can be explained either by a regulatory ATP binding site, where ATP hydrolysis is required, or by proposing an interactive dimer . The notion that FITC inhibits pNPPase and ATPase activity supports the idea that the catalytic domain of KdpB is much more compact than other P-type ATPases, like Na(+),K(+)-ATPase, H(+),K(+)-ATPase, and Ca(2+)-ATPase.

Biochemistry, 2004 Apr 20, 43(15), 4538 - 47
Kinetic mechanism of quinone oxidoreductase 2 and its inhibition by the antimalarial quinolines; Kwiek JJ et al.; Quinone oxidoreductase 2 (QR2) purified from human red blood cells was recently shown to be a potential target of the quinoline antimalarial compounds {Graves et al., (2002) Mol . Pharmacol . 62, 1364} . QR2 catalyzes the two-electron reduction of menadione via the oxidation of N-alkylated or N-ribosylated nicotinamides . To investigate the mechanism and consequences of inhibition of QR2 by the quinolines further, we have used steady-state and transient-state kinetics to define the mechanism of QR2 . Importantly, we have shown that QR2 when isolated from an overproducing strain of E . coli is kinetically equivalent to the enzyme from the native human red blood cell source . We observe ping-pong kinetics consistent with one substrate/inhibitor binding site that shows selectivity for the oxidation state of the FAD cofactor, suggesting that selective inhibition of the liver versus red blood cell forms of malaria may be possible . The reductant N-methyldihydronicotinamide and the inhibitor primaquine bind exclusively to the oxidized enzyme . In contrast, the inhibitors quinacrine and chloroquine bind exclusively to the reduced enzyme . The quinone substrate menadione, on the other hand, binds nonspecifically to both forms of the enzyme . Single-turnover kinetics of the reductive half-reaction are chemically and kinetically competent and confirm the inhibitor selectivity seen in the steady-state experiments . Our studies shed light on the possible in vivo potency of the quinolines and provide a foundation for future studies aimed at creating more potent QR2 inhibitors and at understanding the physiological significance of QR2.

Biochemistry, 2004 Apr 20, 43(15), 4530 - 7
Novel metabolism of 1 alpha,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1; Sawada N et al.; Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway {Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y . and Inouye, K . (2000) Eur . J . Biochem . 267, 6158-6165} . In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D(3) and 25,26,27-trinor-23-ene-1alpha(OH)D(3) as novel metabolites of 25(OH)D(3) and 1alpha,25(OH)(2)D(3), respectively . These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A1 produces much more of these metabolites than does rat CYP24A1 . We propose that the C(24)-C(25) bond cleavage occurs by a unique reaction mechanism including radical rearrangement . Namely, after hydrogen abstraction of the C-23 position of 1alpha,25(OH)(2)D(3), part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1alpha(OH)D(3), while a major part of them is converted into 1alpha,23,25(OH)(3)D(3) . Because the C(24)-C(25) bond cleavage abolishes the binding affinity of 1alpha,25(OH)D(3) for the vitamin D receptor, this reaction is quite effective for inactivation of 1alpha,25(OH)D(3).

Biochemistry, 2004 Apr 20, 43(15), 4454 - 63
Crystal structures of the catalytic domains of pseudouridine synthases RluC and RluD from Escherichia coli; Mizutani K et al.; The most frequent modification of RNA, the conversion of uridine bases to pseudouridines, is found in all living organisms and often in highly conserved locations in ribosomal and transfer RNA . RluC and RluD are homologous enzymes which each convert three specific uridine bases in Escherichia coli ribosomal 23S RNA to pseudouridine: bases 955, 2504, and 2580 in the case of RluC and 1911, 1915, and 1917 in the case of RluD . Both have an N-terminal S4 RNA binding domain . While the loss of RluC has little phenotypic effect, loss of RluD results in a much reduced growth rate . We have determined the crystal structures of the catalytic domain of RluC, and full-length RluD . The S4 domain of RluD appears to be highly flexible or unfolded and is completely invisible in the electron density map . Despite the conserved topology shared by the two proteins, the surface shape and charge distribution are very different . The models suggest significant differences in substrate binding by different pseudouridine synthases.

Avian Dis, 2004 Jan-Mar, 48(1), 189 - 95
Prevalence of pathogenic Escherichia coli in the broiler house environment; Jeffrey JS et al.; Matched sampling of Escherichia coli from broiler house litter and bird lesions of either cellulitis or colibacillosis was conducted to investigate the relationship of pathogenic E . coli to those found in the environment . Isolates were collected from six broiler flocks representing six geographically disparate ranches . Isolates were compared by flock for similarity in serotype and genotyped by pulsed-field gel electrophoresis . Serotyping revealed a considerable dissociation between the two groups of isolates . The prevalence of pathogenic E . coli that matched the environmental isolates from the same house was 0 to 3% . Statistical analysis of the serotype data showed a strong dependence of serotype on isolate source, indicating a high probability that a particular serotype would be found among lesions or litter but not in both groups . Genotyping of isolates on two farms supported the results of serotyping and provided differentiation of isolates that could not by typed by serology . These results suggested that the prevalence of pathogenic E . coli in the broiler house was independent of the prevalence of other commensal or environmental E . coli . Understanding the composition of E . coli populations in commercial poultry production may have bearing on the epidemiology and control of E . coli related diseases.

Anticancer Drugs, 2004 Feb, 15(2), 145 - 9
Special pharmacokinetics of dipetarudin suggests a potential antitumor activity of this thrombin inhibitor; Lopez ML et al.; Thrombin is a potent mitogen for many tumor cells, suggesting that this enzyme may be involved in tumor genesis and metastasis . Inhibition of thrombin expressed on the surface of tumor cells may improve outcomes in some tumor cases . For this reason, a thrombin inhibitor to be applied in antitumor therapy must have favorable pharmacokinetic attributes to exert its action as long as possible in the extravascular compartment of the extracellular space, with a short action intravascularly, avoiding bleeding and/or other undesirable side-effects . None of the thrombin inhibitors in clinical use has these properties . Here, we report for first time a direct thrombin inhibitor, named dipetarudin that could be very useful in antitumor therapy because of its pharmacokinetic behavior characterized by a rapid distribution in the extravascular space with a slow elimination from this compartment.

J Biol Chem, 2004 Jun 18, 279(25), 26159 - 66 Epub 2004 Apr 09.
Biochemical characterization of 2-Cys peroxiredoxins from Schistosoma mansoni; Sayed AA et al.; Peroxiredoxins are a large family of peroxidases that have important antioxidant and cell signaling functions . Genes encoding two novel 2-cysteine peroxiredoxin proteins were identified in the expressed sequence tag data base of the helminth parasite Schistosoma mansoni, a causative agent of schistosomiasis . The recombinant proteins showed peroxidase activity in vitro with a variety of hydroperoxides and used both the thioredoxin and the glutathione systems as electron donors . Steady-state kinetic analysis indicated that the new peroxiredoxins had saturable kinetics, whereas a previously identified schistosome peroxiredoxin was found to function with more typical unsaturable (ping-pong) kinetics . The catalytic efficiencies S . mansoni peroxiredoxins were similar to those for other peroxiredoxins studied (10(4)-10(5) m(-1) s(-1)) . Mutagenesis of S . mansoni peroxiredoxins indicated that glutathione dependence and kinetic differences were conferred by the C-terminal alpha-helix forming 22 amino acids . This is the first report of 2-cysteine peroxiredoxins efficiently utilizing reducing equivalents from both the thioredoxin and glutathione systems . Studies to determine the resistance to oxidative inactivation, important in regulating cell signaling pathways, showed that S . mansoni possess both bacterial-like resistant and mammalian-like sensitive peroxiredoxins . The susceptibility to oxidative inactivation was conferred by the C-terminal tail containing a tyrosine-phenylalanine motif . S . mansoni is the first organism shown to possess both robust and sensitive peroxiredoxins . The ability of schistosome peroxiredoxins to use alternative electron donors, and their variable resistance to overoxidation may reflect their presence in different cellular sites and emphasizes the significant differences in overall redox balance mechanisms between the parasite and its mammalian host.

J Biol Chem, 2004 Jun 25, 279(26), 27286 - 93 Epub 2004 Apr 09.
ICln, a novel integrin alphaIIbbeta3-associated protein, functionally regulates platelet activation; Larkin D et al.; A critical role for the conserved alpha-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin alpha(IIb)beta(3) . To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif . We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions . A number of potential integrin-binding proteins were identified . One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets . We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with alpha(IIb)beta(3) by surface plasmon resonance . The affinity of this interaction was 82.2 +/- 24.4 nm in a cell free assay . ICln co-immunoprecipitates with alpha(IIb)beta(3) in platelet lysates demonstrating that this interaction is physiologically relevant . Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates . Acyclovir (100 microm to 5 mm), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation . In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10-100 microm) also inhibits platelet function . Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.

J Biol Chem, 2004 Jul 2, 279(27), 28375 - 86 Epub 2004 Apr 09.
Human Kaposi's sarcoma herpesvirus processivity factor-8 functions as a dimer in DNA synthesis; Chen X et al.; In the absence of other proteins, the DNA polymerase (Pol-8) of Kaposi's sarcoma herpesvirus incorporates only several nucleotides from a primer template . However, association with the Kaposi's sarcoma herpesvirus processivity factor (PF-8) enables Pol-8 to incorporate thousands of nucleotides . Unlike the well described sliding clamp processivity factors, eukaryotic proliferating cell nuclear antigen and Escherichia coli beta-subunit, PF-8 and other herpesvirus processivity factors do not require a clamp loader or ATP to bind to template DNA . To begin to understand the mechanism used by PF-8 to achieve processivity, we have now purified PF-8 and demonstrated that it is a dimer both in solution and on the DNA . Mutational analysis of the PF-8 protein (396R) indicates that residues between 277 and 304 as well as the N-terminal 21 amino acids are required for dimerization . The results further correlate PF-8 dimerization with binding to Pol-8 and stabilizing Pol-8 on primer template . Notably, although removal of only 26 residues from the C terminus of PF-8 does not affect its ability to form dimers on DNA or to bind Pol-8, only short DNA chains (<100 nucleotides) are synthesized . This indicates that full-length PF-8 is necessary to enable Pol-8 to incorporate thousands of nucleotides . Interestingly, cross-linking of the processivity factor UL44 of cytomegalovirus reveals that it is a dimer in solution also.

Eukaryot Cell, 2004 Apr, 3(2), 483 - 94
Cryptosporidium parvum mitochondrial-type HSP70 targets homologous and heterologous mitochondria; Slapeta J et al.; A mitochondrial HSP70 gene (Cp-mtHSP70) is described for the apicomplexan Cryptosporidium parvum, an agent of diarrhea in humans and animals . Mitochondrial HSP70 is known to have been acquired from the proto-mitochondrial endosymbiont . The amino acid sequence of Cp-mtHSP70 shares common domains with mitochondrial and proteobacterial homologues, including 34 amino acids of an NH2-terminal mitochondrion-like targeting presequence . Phylogenetic reconstruction places Cp-mtHSP70 within the mitochondrial clade of HSP70 homologues . Using reverse transcription-PCR, Cp-mtHSP70 mRNA was observed in C . parvum intracellular stages cultured in HCT-8 cells . Polyclonal antibodies to Cp-mtHSP70 recognize a approximately 70-kDa protein in Western blot analysis of sporozoite extracts . Both fluorescein- and immunogold-labeled anti-Cp-mtHSP70 localize to a single mitochondrial compartment in close apposition to the nucleus . Furthermore, the NH2-terminal presequence of Cp-mtHSP70 can correctly target green fluorescent protein to the single mitochondrion of the apicomplexan Toxoplasma gondii and the mitochondrial network of the yeast Saccharomyces cerevisiae . When this presequence was truncated, the predicted amphiphilic alpha-helix was shown to be essential for import into the yeast mitochondrion . These data further support the presence of a secondarily reduced relict mitochondrion in C . parvum.

Vet Res Commun, 2004 Apr, 28(3), 209 - 24
Heterologous expression of a gene encoding a 35 kDa protein of Mycobacterium avium paratuberculosis in Escherichia coli; Basagoudanavar SH et al.; The full-length open reading frame coding for a potentially immunogenic 35 kDa protein of Mycobacterium avium paratuberculosis was generated using polymerase chain reaction technology . The gene was inserted in-frame into Escherichia coli expression plasmid pQE32 . The resulting recombinant plasmid pPMP35 was transformed into E . coli M15 . Analysis of the E . coli induced with isopropyl-beta-D-thiogalactopyranoside revealed that the protein accumulated into the cytoplasm as insoluble inclusion bodies . The level of expression of the recombinant 35 kDa protein (P35) was more than 30% of the total protein of E . coli cells . Expression of the recombinant protein was confirmed by immunoblotting . The P35 reacted with a rabbit antiserum raised against a sonicate of M . a . paratuberculosis . The protein was also recognized by serum from a goat with clinical paratuberculosis . Further, a polyclonal antiserum against P35 recognized a 35 kDa band in a membrane fraction of M . a . paratuberculosis . Also, the protein provoked a significant skin reaction in outbred guinea pigs sensitized with M . a . paratuberculosis, as well as in those sensitized with Mycobacterium avium . The results indicate that the 35 kDa protein of M . a . paratuberculosis is a membrane protein, having a role in the cellular immune response.

Verh K Acad Geneeskd Belg, 2004, 66(2), 97 - 150; discussion 150-3
Role of neutrophil polymorphonuclear leukocytes during bovine coliform mastitis: physiology or pathology?
Burvenich C, Monfardini E, Mehrzad J, Capuco AV, Paape MJ.
The review compiles some major findings concerning the inflammatory reaction in the mammary gland of dairy cows within the physiological context of the lactation cycle . The dual role of the PMN leukocyte in defense and tissue damage during experimentally induced coliform mastitis, especially around parturition and during early lactation, is highlighted . This disease affects many high producing cows in dairy herds and may cause several cases of death per year in the most severe cases . Most researchers now accept that the PMN is a key factor in the cows' defense against intramammary infection with E coli . During diapedesis of PMN into the mammary gland, several functionally important receptors are up-regulated, allowing for a more efficient phagocytosis and killing of invading pathogens . While PMN are phagocytosing and destroying the invading pathogens, they inadvertently release chemical mediators which induces swelling of secretory epithelium cytoplasm, sloughing of secretory cells, and decreased secretory activity . Permanent scarring will result in a loss of milk production . PMN's act as friends and as foes and are important components in the balance between mammary defense and damage . The mammary gland is a complex open self-regulatory system with a continuous flow of matter, energy and information . Metabolically, it has absolute priority over many other tissues except the brain . Self-regulation with change over time is characterized by a dynamic equilibrium between two mechanisms: homeostatic and homeorhetic . The defense against invaders by innate immunity and auto-repair of the damaged tissues are covered by homeostatic mechanisms while colostrogenesis and maintenance of milk secretion are controlled by mainly homeorhetic mechanisms . However, also innate immunity has to function and develop in time, depending on the lactation cycle, and its behavior and evolution in time in such a dynamical system is a challenge and a problem at the same time . In such a complex dynamic situation it is not surprising that physiology is not far away from pathology . E . coli mastitis can be a severe problem during the beginning of lactation whereas it is completely self-curing after peak lactation (8 weeks) . The approach to focus on the PMN doesn't mean that the defense of the mammary gland is more simple than in other tissues . The defense of mammary gland is characterized by its complexity and over the last years many data show that there are tight connections with the mononuclear cells in mammary gland tissue . Today it is known that T cells play a central role in orchestrating the immune response . However, because of the peculiar interest in the PMN of the authors during the last 10 years, the immunobiology of the mononuclear cells in the mammary gland is not covered.

Mikrobiologiia, 2004 Jan-Feb, 73(1), 51 - 6
{Inductive effect of hypoxanthine-xanthine oxidase system on lambda prophage}; Zhu SQ et al.; Hypoxanthine-xanthine oxidase (HX-XO) system is a classical system that can generate superoxide anions . The inductive effect of the HX-XO system for lambda prophage has been investigated in this study . The results showed that the system can induce lambda prophage from lysogenic state to lytic growth . The inductive effect was directly proportional to the concentration of HX and XO and inversely related to the time of preliminary incubation of HX with XO . The cell density of the lysogenic bacteria also greatly affected the inductive effect . The maximal PFU number of 2.9 x 10(4) PFU/ml was recorded at 0.86 mM HX, 1.6 x 10(-2) U/ml XO, and a cell density of 10(8) cells/ml . The inductive effect of the HX-XO system was inhibited in the suspensions by glutathione, superoxide dismutase, and catalase . The results provide evidence that free radicals are the primary factors in the induction of lambda prophage in lysogenic bacteria.

Chem Rec, 2004, 4(2), 72 - 82
Studies of Thermobifida fusca plant cell wall degrading enzymes; Wilson DB; SYNOPSIS: I have been studying the Thermobifida fusca cellulose degrading proteins for the past 25 years . In this period, we have purified and characterized the six extracellular cellulases and an intracellular beta- glucosidase used by T . fusca for cellulose degradation, cloned and sequenced the structural genes encoding these enzymes, and helped to determine the 3-dimensional structures of two of the cellulase catalytic domains . This research determined the mechanism of a novel class of cellulase, family 9 processive endoglucanases, and helped to show that there were two types of exocellulases, ones that attacked the non-reducing ends of cellulose and ones that attacked the reducing ends . It also led to the sequencing of the T . fusca genome by the DOE Joint Genome Institute . We have studied the mechanisms that regulate T . fusca cellulases and have shown that cellobiose is the inducer and that cellulase synthesis is repressed by any good carbon source . A regulatory protein (CelR) that functions in the induction control has been purified, characterized, and its structural gene cloned and expressed in E . coli . I have also carried out research on two rumen bacteria, Prevotella ruminicola and Fibrobacter succinogenes, in collaboration with Professor James Russell, helping to arrange for the genomes of these two organisms to be sequenced by TIGR, funded by a USDA grant to the North American Consortium for Genomics of Fibrolytic Ruminal Biology .

Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 5946 - 51 Epub 2004 Apr 08.
Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans; Matthies A et al.; Recent studies have identified the human genes involved in the biosynthesis of the molybdenum cofactor . The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese . The MOCS3 protein is believed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of the smaller subunit of molybdopterin (MPT) synthase . The MOCS3 rhodanese-like domain (MOCS3-RLD) was purified after heterologous expression in E . coli and was shown to catalyze the transfer of sulfur from thiosulfate to cyanide . In a defined in vitro system for the generation of MPT from precursor Z, the sulfurated form of MOCS3-RLD was able to provide the sulfur for the thiocarboxylation of MOCS2A, the small MPT synthase subunit in humans . Mutation of the putative persulfide-forming active-site cysteine residue C412 abolished the sulfurtransferase activity of MOCS3-RLD completely, showing the importance of this cysteine residue for catalysis . In contrast to other mammalian rhodaneses, which are mostly localized within mitochondria, MOCS3 in addition to the subunits of MPT synthase are localized in the cytosol.

Microbiology, 2004 Apr, 150(Pt 4), 1041 - 53
Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles; Trott DJ et al.; The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species . Little is known about the structure or protein constituents of the B . pilosicoli outer membrane (OM) . To identify surface-exposed proteins in this species, membrane vesicles were isolated from B . pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients . The membrane vesicles were separated into a high-density fraction (HDMV; rho=1.18 g cm(-3)) and a low-density fraction (LDMV; rho=1.12 g cm(-3)) . Both fractions were free of flagella and soluble protein contamination . LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B . pilosicoli OM protein) and was used as a source of antigens to produce mAbs . Five B . pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized . The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins . All three proteins were localized to the B . pilosicoli OM by immunogold labelling using specific mAbs . The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B . pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli . Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC . Recombinant BmpC partitioned predominantly in the OM fraction of E . coli strain SOLR . The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B . pilosicoli isolated from five different host species . Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B . pilosicoli.

Microbiology, 2004 Apr, 150(Pt 4), 877 - 83
Phosphorylation and DNA binding of the regulator DcuR of the fumarate-responsive two-component system DcuSR of Escherichia coli; Janausch IG et al.; The function of the response regulator DcuR of the DcuSR fumarate two-component sensory system of Escherichia coli was analysed in vitro . Isolated DcuR protein was phosphorylated by the sensory histidine kinase, DcuS, and ATP, or by acetyl phosphate . In gel retardation assays with target promoters (frdA, dcuB, dctA), phosphoryl DcuR (DcuR-P) formed a high-affinity complex, with an apparent K(D) (app . K(D)) of 0.2-0.3 microM DcuR-P, and a low-affinity (app . K(D) 0.8-2 microM) complex . The high-affinity complex was formed only with promoters transcriptionally-regulated by DcuSR, whereas low-affinity binding was seen also with some DcuSR-independent promoters . The binding site of DcuR-P at the dcuB promoter was determined by DNase I footprinting . One binding site of 42-52 nt (position -359 to -400/-410 nt upstream of the transcriptional start) was identified in the presence of low and high concentrations of DcuR-P . Non-phosphorylated DcuR, or DcuR-D56N mutated in the phosphoryl-accepting Asp56 residue, showed low-affinity binding to target promoters . DcuR-D56N was still able to interact with DcuS . DcuR-D56N increased the phosphorylation of DcuS and competitively inhibited phosphoryl transfer to wild-type DcuR.

J Biol Chem, 2004 Jun 18, 279(25), 26143 - 8 Epub 2004 Apr 08.
Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation; Yeh PY et al.; We previously reported that suppression of the MEK/ERK pathway increases drug resistance of SiHa cells . In this study, we further characterized the underlying mechanism of this phenomenon . Pretreatment of SiHa cells with MEK/ERK inhibitor enhanced cisplatin-induced NF-kappaB activation . However, results of immunoblotting analysis showed that neither cisplatin nor MEK/ERK inhibitors induced marked IkappaBalpha degradation, suggesting that suppression of the MEK/ERK signaling pathway may enhance cisplatin-induced NF-kappaB activation via mechanisms other than the conventional pathway . Previous findings that protein phosphatase 4 (PP4), a nuclear serine/threonine phosphatase, directly interacts with and activates NF-kappaB led us to examine the phosphorylation status of NF-kappaB p65 . Coincident with activation of NF-kappaB, cisplatin induced Ser phosphorylation but decreased Thr phosphorylation of NF-kappaB p65 . Suppression of the MEK/ERK pathway further enhanced cisplatin-induced Thr dephosphorylation but did not affect cisplatin-induced Ser phosphorylation of NF-kappaB p65 . Further, in parallel with Thr dephosphorylation, the protein level of nuclear PP4 was increased in cisplatin-treated cells and was further increased by suppression of the MEK/ERK pathway . SiHa cells were then transfected by a sense or an antisense PP4 gene . PP4-overexpressing cells showed a decrease in Thr phosphorylation of NF-kappaB p65 to nearly undetectable levels, and both basal and cisplatin-induced NF-kappaB activities were higher than those in parental cells . By contrast, cisplatin, either alone or with MEK/ERK inhibitors, induced little NF-kappaB activation in antisense PP4-transfected cells . Coprecipitated complex kinase assay revealed a fragment of NF-kappaB p65 (amino acids 279-444) to contain potential phosphorylation sites that directly interact with PP4 . Further studies by site-directed mutagenesis suggested that Thr(435) was the major phosphorylation site.

Eur J Med Chem, 2004 Apr, 39(4), 333 - 44
Design, synthesis and activity of bisubstrate, transition-state analogues and competitive inhibitors of aspartate transcarbamylase; Grison C et al.; Aspartate transcarbamylase initiates the de novo biosynthetic pathway for the production of the pyrimidine nucleotides, precursors of nucleic acids . This pathway is particularly active in rapidly growing cells and tissues . Thus, this enzyme has been tested as a potential target for antiproliferative drugs . In the present work, on the basis of its structural and mechanistic properties, a series of substrate analogues, including potential suicide-pseudosubstrates was synthesized and their putative inhibitory effects were tested using E . coli aspartate transcarbamylase as a model . Two of these compounds appear to be very efficient inhibitors of this enzyme.

Arch Inst Pasteur Tunis, 2002, 79(1-4), 11 - 7
Cloning of the rat CR3 alphaM (CD11b) subunit, expression and binding assay of recombinant isolated CD11b VA (A-domain) and ICAM-1 Ig modules; Zerria K et al.; The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex . It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration . We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library . The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart . We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E . coli as a soluble recombinant fusion protein with GST . Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E . coli as recombinant soluble (rs) fusion proteins with GST . Recombinant CD11b A-domain was released from the fusion protein by thrombin cut . Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain . These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.

Nat Genet, 2004 Apr, 36(4), 423 - 6
Pervasive joint influence of epistasis and plasticity on mutational effects in Escherichia coli; Remold SK et al.; The effects of mutations on phenotype and fitness may depend on the environment (phenotypic plasticity), other mutations (genetic epistasis) or both . Here we examine the fitness effects of 18 random insertion mutations in E . coli in two resource environments and five genetic backgrounds . We tested each mutation for plasticity and epistasis by comparing its fitness effects across these ecological and genetic contexts . Some mutations had no measurable effect in any of these contexts . None of the mutations had effects on phenotypic plasticity that were independent of genetic background . However, half the mutations had epistatic interactions such that their effects differed among genetic backgrounds, usually in an environment-dependent manner . Also, the pattern of mutational effects across backgrounds indicated that epistasis had been shaped primarily by unique events in the evolutionary history of a population rather than by repeatable events associated with shared environmental history.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2004 Mar, 35(2), 214 - 6
{Genotyping of EsBLs-producing Escherichia coli by pulsed-field gel electrophoresis in nosocomial infection}; Kang M et al.; OBJECTIVE: Using a genotying method which is accurate and rapid to study the molecular epidemiology of EsBLs-producing Escherichia coli in nosocomial infection . METHODS: After an analysis of clinical information to ascertain nosocomial infection, EsBLs were detected by phenotype confirmation test and pulsed-field gel electrophoresis (PFGE) . RESULTS: Three EsBLs-producing strains in ICU were verified as epidemiological-related because their dice coefficient index was higher than 95% . The others were verified as non-epidemiological-related since their different lanes were more than 6 . CONCLUSION: PFGE is a perfect molecular typing method to study epidemiolgy and analyze homology.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2004 Mar, 35(2), 149 - 53
{Construction of a reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state}; Fang DZ et al.; OBJECTIVE: To inquire into the mechanism of prothrombotic state (PTS) and the roles of liver therein by constructing a reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS . METHODS: The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization . The rat model of PTS was induced by a high-carbohydrate diet . Poly A+ mRNAs were isolated from PTS and control rats, and cDNAs were synthesized from the mRNAs . After digestion by means of Ras I, cDNAs 400-600 bp in size were obtained . For suppression subtractive hybridization, cDNAs from PTS rat were used as Driver and the cDNAs from control rat as Tester . The Tester was divided into two parts and ligated to adaptor 1 and adaptor 2R respectively . After two times of subtractive hybridization and two times of nested PCR, the products of the last PCR amplification were inserted into T/A plasmid vectors to transform the Escherichia coli JM109 cells . The transformed cells were incubated at 37 degrees C overnight on a LB agar plate containing ampicillin (50 micrograms/ml), IPTG and X-gal . The colonies were counted . RESULTS: 78% of the colonies were white and the reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state was successfully constructed . CONCLUSION: Prothrombotic state caused by malfunction of homeostasis and fibrinolysis is an important risk factor of cardiovascular disease . Liver plays important roles in the development of PTS, for the majority of the factors in the coagulation as well as fibrinolytic cascades are generated by the liver and secreted into the bloodstream . The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS, successfully constructed in the present study, provides an efficient way to further investigate the mechanism of PTS and the relevant liver functions.

Crit Care Med, 2004 Apr, 32(4), 1011 - 7
Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia; Hoffmann JN et al.; OBJECTIVE: To characterize microcirculatory actions of activated protein C in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction . DESIGN: Animal study using intravital microscopy . SETTING: Animal research facility . SUBJECTS: Male Syrian golden hamsters, 6-8 wks old with a body weight of 60-80 g . INTERVENTIONS: In skinfold preparations, endotoxemia was induced by intravenous administration of 2 mg/kg endotoxin (lipopolysaccharide, Escherichia coli) . Intravital microscopy allowed quantitative analysis of arteriolar and venular leukocyte adhesion and functional capillary density (cm) that served as a measure of microvascular perfusion failure . Activated protein C (APC group, n = 8, 24 microg/kg intravenously) was substituted continuously during 8 hrs after lipopolysaccharide, whereas endotoxemic buffer-treated animals (control, n = 7) served as controls . MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide increased leukocyte adhesion and decreased functional capillary density to 50% of baseline values (p <.01 vs . baseline) . Activated protein C treatment inhibited (p <.05) lipopolysaccharide-mediated leukocytic response and attenuated (p <.05) endotoxic perfusion failure in nutritive capillaries . CONCLUSIONS: Activated protein C-induced protection from lipopolysaccharide-mediated microcirculatory dysfunction was characterized in vivo for the first time . The impressive modification of leukocyte cross-talk indicates systemic anti-inflammatory activated protein C effects on leukocytes and the endothelium, subsequently improving capillary perfusion . These actions could represent the in vivo mechanism of activated protein C interactions observed in patients with severe sepsis.

Pediatr Infect Dis J, 2004 Apr, 23(4), 373 - 5
Reovirus type 2 isolated from cerebrospinal fluid; Hermann L et al.; An 8-week-old female infant presented with a history of active varicella complicated by Escherichia coli sepsis, oral thrush, hypoalbuminemia, intermittent fevers, diarrhea and feeding intolerance . Rhesus monkey kidney cells inoculated with cerebrospinal fluid revealed reovirus-like particles by electron microscopy . Virus neutralization and RNA-gel electrophoresis studies identified the isolated pathogen as reovirus serotype 2 . This report represents one of only a few to isolate reovirus from the central nervous system in humans.

J Clin Microbiol, 2004 Apr, 42(4), 1773 - 6
Detection of the Escherichia coli group 2 polysaccharide capsule synthesis Gene kpsM by a rapid and specific PCR-based assay; Johnson JR et al.; A rapid and simple PCR-based assay for detection of the group 2 capsule synthesis gene kpsM of Escherichia coli was designed and validated . When combined with the published group 2 primers (kpsIIf, 5'-GCGCATTTGCTGATACTGTTG-3'; kpsIIr, 5'-CATCCAGACGATAAGCATGAGCA-3'), the new primers (the kpsIIf primer and a new reverse primer K2r, 5'-AGGTAGTTCAGACTCACACCT-3') allowed specific identification by exclusion of the heretofore elusive K2 kpsM variant . The primers yielded the predicted amplicon when multiplexed with other primers and used under varied assay conditions, including a range of concentrations of individual reaction mixture ingredients and of annealing temperatures (from 54 to 64 degrees C).

J Clin Microbiol, 2004 Apr, 42(4), 1770 - 2
Characterization of an anonymous molecular marker strongly linked to Escherichia coli strains causing neonatal meningitis; Clermont O et al.; An anonymous 14.9-kb rrn-containing HindIII fragment is strongly linked to Escherichia coli strains causing neonatal meningitis . We show in this report that this fragment does not encode new virulence factors but lacks arpA, a gene common in avirulent E . coli strains, and we developed a PCR test to detect this fragment.

J Clin Microbiol, 2004 Apr, 42(4), 1570 - 6
Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus; Lu L et al.; Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by the SARS-associated coronavirus (SARS-CoV) . There are four major structural proteins in the SARS-CoV, including the nucleocapsid, spike, membrane, and small envelope proteins . In this study, two sets of truncated fragments of spike protein were generated, the first were approximately 210-bp nonoverlapping fragments and the second were overlapping segments of 750 to 900 bp . From these 23 fragments, we identified a fragment of 259 amino acids (amino acids 441 to 700) that is a major immunodominant epitope . This fragment was highly expressed, and the purified fragment C could detect all 33 SARS patient serum samples tested, collected from 7 to 60 days after the onset of fever, but had no reactivity with all 66 healthy human serum samples tested . Thus, fragment C of spike protein was identified as an immunodominant antigen and could be used for serological detection of SARS-CoV infection.

J Biol Chem, 2004 Jun 25, 279(26), 26823 - 9 Epub 2004 Apr 07.
De-epoxidation of violaxanthin in light-harvesting complex I proteins; Wehner A et al.; The conversion of violaxanthin (Vx) to zeaxanthin (Zx) in the de-epoxidation reaction of the xanthophyll cycle plays an important role in the protection of chloroplasts against photooxidative damage . Vx is bound to the antenna proteins of both photosystems . In photosystem II, the formation of Zx is essential for the pH-dependent dissipation of excess light energy as heat . The function of Zx in photosystem I is still unclear . In this work we investigated the de-epoxidation characteristics of light-harvesting complex proteins of photosystem I (LHCI) under in vivo and in vitro conditions . Recombinant LHCI (Lhcal-4) proteins were reconstituted with Vx and lutein, and the convertibility of Vx was studied in an in vitro assay using partially purified Vx de-epoxidase isolated from spinach thylakoids . All four LHCI proteins exhibited unique de-epoxidation characteristics . An almost complete Vx conversion to Zx was observed only in Lhca3, whereas Zx formation in the other LHCI proteins decreased in the order Lhca4 > Lhca1 > Lhca2 . Most likely, these differences in Vx de-epoxidation were related to the different accessibility of the respective carotenoid binding sites in the distinct antenna proteins . The results indicate that Vx bound to site V1 and N1 is easily accessible for de-epoxidation, whereas Vx bound to L2 is only partially and/or with the slower kinetics convertible to Zx . The de-epoxidation properties determined for the monomeric recombinant proteins were consistent with those obtained for isolated native LHCI-730 and LHCI-680 in the same in vitro assay and the de-epoxidation state found under in vivo conditions in native LHCIs.

Biochem J, 2004 Jul 1, 381(Pt 1), 185 - 93
Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase; Wu J et al.; An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector . The protein was overexpressed in Escherichia coli and purified to homogeneity . Recombinant A . thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa . Unlike previously studied bacterial KDOPSs, which are tetrameric, A . thaliana KDOPS appears to be a dimer in solution . The optimum temperature of the enzyme is 65 degrees C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity . The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme . Kinetic studies showed that the enzyme follows a sequential mechanism with K(m)=3.6 microM for phosphoenolpyruvate and 3.8 microM for D-arabinose 5-phosphate and kcat=5.9 s(-1) at 37 degrees C . On the basis of the characterization of A . thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

Plant Cell Rep, 2004 Aug, 23(1-2), 81 - 90 Epub 2004 Apr 07.
A geminivirus AYVV-derived shuttle vector for tobacco BY2 cells; Tamilselvi D et al.; We have developed a plant- Escherichia coli pASV shuttle vector from the essential elements of the Ageratum yellow vein virus (AYVV) . The geminivirus vector contains the AYVV genome with the coat-protein deletion, the E . coli vector backbone of pUC19, a unique cloning site and gene expression cassettes for plant selection and reporter gene activity . The replication of pASV vectors was compared in Nicotiana benthamiana and N . tabacum BY2 cells, and the latter were found to be suitable for long-term maintenance of the vectors in culture . The vector DNA was detected at regular intervals by PCR, beta-glucuronidase expression analysis and plasmid rescue during a 4-month culture period . A novel methylation-based PCR assay was carried out to show de novo replication for pASV-derived vectors in 2-month-old tobacco BY2 cell lines . This is the first report of the extrachromosomal replication of monopartite begomovirus with stability and foreign gene expression in long-term cell cultures.

J Biol Chem, 2004 Jun 11, 279(24), 25673 - 9 Epub 2004 Apr 06.
Analysis of sequence determinants of F1Fo-ATP synthase in the N-terminal region of alpha subunit for binding of delta subunit; Weber J et al.; The stator in F(1)F(o)-ATP synthase resists strain generated by rotor torque . In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(o) and to the alpha(3)beta(3) hexagon of F(1) . Previous work has shown that N-terminal residues of alpha subunit are involved in binding delta . A synthetic peptide consisting of the first 22 residues of alpha (alphaN1-22) binds specifically to isolated wild-type delta subunit with 1:1 stoichiometry and high affinity, accounting for a major portion of the binding energy between delta and F(1) . Residues alpha6-18 are predicted by secondary structure algorithms and helical wheels to be alpha-helical and amphipathic, and a potential helix capping box occurs at residues alpha3-8 . We introduced truncations, deletions, and mutations into alphaN1-22 peptide and examined their effects on binding to the delta subunit . The deletions and mutations were introduced also into the N-terminal region of the uncA (alpha subunit) gene to determine effects on cell growth in vivo and membrane ATP synthase activity in vitro . Effects seen in the peptides were well correlated with those seen in the uncA gene . The results show that, with the possible exception of residues close to the initial Met, all of the alphaN1-22 sequence is required for binding of delta to alpha . Within this sequence, an amphipathic helix seems important . Hydrophobic residues on the predicted nonpolar surface are important for delta binding, namely alphaIle-8, alphaLeu-11, alphaIle-12, alphaIle-16, and alphaPhe-19 . Several or all of these residues probably make direct interaction with helices 1 and 5 of delta . The potential capping box sequence per se appeared less important . Impairment of alpha/delta binding brings about functional impairment due to reduced level of assembly of ATP synthase in cells.

J Biol Chem, 2004 Jun 18, 279(25), 26462 - 8 Epub 2004 Apr 06.
Rapid evolution of beta-glucuronidase specificity by saturation mutagenesis of an active site loop; Geddie ML et al.; Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition . We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity . Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A . D . (2001) J . Mol . Biol . 305, 331-339) . Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E . coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-beta-d-xylopyranoside (pNP-xyl) . The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS . The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E . coli . Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening . One of the evolved proteins exhibited a approximately 200-fold improvement over the wild type in reactivity (k(cat)/K(m)) with pNP-xyl, with a 290,000-fold inversion of specificity . Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR . Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement . These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.

Mol Cell, 2004 Apr 9, 14(1), 57 - 66
Three-dimensional structures of translating ribosomes by Cryo-EM; Gilbert RJ et al.; Cryo-electron microscopy and image reconstruction techniques have been used to obtain three-dimensional maps for E . coli ribosomes stalled following translation of three representative proteins . Comparisons of these electron density maps, at resolutions of between 13 and 16 A, with that of a nontranslating ribosome pinpoint specific structural differences in stalled ribosomes and identify additional material, including tRNAs and mRNA . In addition, the tunnel through the large subunit, the anticipated exit route of newly synthesized proteins, is partially occluded in all the stalled ribosome structures . This observation suggests that significant segments of the nascent polypeptide chains examined here could be located within an expanded tunnel, perhaps in a rudimentary globular conformation . Such behavior could be an important aspect of the folding of at least some proteins in the cellular environment.

Cancer Biother Radiopharm, 2004 Feb, 19(1), 99 - 109
New strategies in polypeptide and antibody synthesis: an overview; Mertens N et al.; The synthesis of radioligands can benefit considerably from optimized recombinant protein production, both on the aspect of economy of production and on the level of improving the targeting and pharmacokinetics of the ligand . This paper first describes a general production optimization strategy, and then elaborates on a protein design strategy tailored to targeting applications . Production in Escherichia coli will benefit from economy of goods and time as compared to other organisms . In order to increase the chance of finding a successful production system in this host, we have assembled a large number of expression strategies in a single, uniform expression system (FastScreen) . The system allows rapid optimization of direct production of native proteins or via a fusion protein strategy with subsequent recovery of the desired protein . As an example of recombinant radioligand synthesis for improved targeting and clearing, a manifold of intermediate molecular size was synthesized by fusing one Fab and two single-chain variable fragments (scFv) antibody binding fragments into a trifunctional molecule (Tribody) . Due to the use of the specific heterodimerization of the Fab chains, trispecific, bispecific, or trivalent antibody derived targeting reagents can easily be obtained . Recombinant production techniques also allow for specific incorporation of amino acids favoring a site specific labeling (labeling tags).

Acta Virol, 2003, 47(4), 217 - 21
Enhancement of immune responses to the hepatitis B virus core protein through DNA vaccines with a DNA fragment encoding human IL-1beta 163-171 peptide; Shao HJ et al.; DNA vaccines have been widely used as effective means of eradicating a variety of viruses, parasites, bacteria as well as means of alleviating allergic and autoimmune diseases and tumors . As interleukin 1 (IL-1) plays an essential role in augmenting both cellular and humoral immune responses to foreign antigens, it may represent a good candidate for an adjuvant to DNA vaccines . Since the inflammatory activity of IL-1 may have a restricted application to DNA vaccines, we explored the possibility of augmenting immune response without unwanted inflammatory effect using IL-1beta 163-171 peptide, which is essential for IL-1 receptor 1 binding . A DNA fragment encoding the human IL-1beta 163-171 peptide of concern was fused to the Hepatitis B virus (HBV) core DNA vaccine, and injected into mice to analyze its immune responses . Compared with the control mice which received hepatitis B virus core antigen (HBcAg) alone, significant increase in not only the HBcAg-specific antibody response but also in T cell proliferation was observed in mice which received IL-1beta 163-171-HBcAg . These results suggest that the DNA fragment encoding the IL-1beta polypeptide of aa 163-171 might represent a good candidate for an adjuvant of DNA vaccines.

J Comput Aided Mol Des, 2003 Oct, 17(10), 699 - 710
Relative free energies of binding to thymidylate synthase of 2- and/or 4-thio and/or 5-fluoro analogues of dUMP; Jarmula A et al.; Free energy perturbation calculations have been applied to evaluate the relative free energies of binding of 2'-deoxyuridine-5'-monophosphate (dUMP) and its 2- and/or 4-thio and/or 5-fluoro analogues to the wild-type E . coli thymidylate synthase (ecTS) . The results accurately reproduce experimentally measured differences in the free energy of binding of dUMP versus 5-fluoro-dUMP to thymidylate synthase . They indicate that preferred binding of dUMP compared to 5-fluoro-dUMP in the binary complex is equally related to (i) more favorable electrostatic interactions of the dUMP molecule in the enzyme active site, and (ii) its less favorable solvation in the aqueous solution . The relative free energies of binding in the binary complex show moderate and qualitatively indistinguishable discrimination among the studied fluorinated and non-fluorinated 2- and/or 4-thio analogues of dUMP . The binding free energies of monothio analogues of dUMP and 5-fluoro-dUMP correspond quite well with experimentally measured activities of these nucleotides in the thymidylate synthase reaction . On the other hand, the binding free energies of both dithio analogues, 2,4-dithio-dUMP and 2,4-dithio-FdUMP, show lack of such correlation . The latter suggests that very low activities of the dithio analogues of dUMP and 5-fluoro-dUMP may relate more to the covalent reaction of these nucleotides within the ternary complex with TS and 5,10-methylenetetrahydrofolate, than to their pre-covalent binding . We speculate that a lack of substrate activity of 2,4-dithio-dUMP is related to the high aromaticity of its pyrimidine ring that prevents the Michael addition of the active site cysteine thiol to the pyrimidine C6 atom . A stronger affinity of the fluorinated analogues of dUMP to thymidylate synthase, compared to the non-fluorinated congeners, results from the fluorine substituent producing a local strain in the C6 region in the pyrimidine ring, thus sensitizing C6 to the Michael addition of the cysteine thiol.

Protein Eng Des Sel, 2004 Mar, 17(3), 277 - 84 Epub 2004 Apr 05.
An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases; Kim BS et al.; Bacterial type I polyketide synthases (PKSs) generate a structurally diverse group of natural products with a wide range of biological activities . Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade . Almost invariably only one or several specific hybrids are made at a time and tested for functionality . This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems . We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and the first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 Streptomyces venezuelae pikAI-deletion mutant . In the first step the plasmid sequence encoding the loading domain AT(0)-ACP(0) was replaced by a counter selectable marker, sacB . DNA family shuffling was then used to generate a diverse library of chimeric AT(0)-ACP(0) fragments, which were used to replace sacB by lambda-Red-mediated in vivo recombination in an Escherichia coli host . This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S.venezuelae BB138 . A bioassay of over 4000 of these transformants successfully revealed three different PikAI hybrids which were able to lead to pikromycin production . The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried . The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.

J Immunol, 2004 Apr 15, 172(8), 4733 - 43
A Toll-like receptor 2 ligand stimulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mitogen-activated protein kinase and c-Fos in dendritic cells; Dillon S et al.; The adaptive immune system can generate distinct classes of responses, but the mechanisms that determine this are poorly understood . In this study, we demonstrate that different Toll-like receptor (TLR) ligands induce distinct dendritic cell (DC) activation and immune responses in vivo . Thus, Escherichia coli LPS (TLR-4 stimulus), activates DCs to produce abundant IL-12(p70), but little IL-10, and stimulates Th1 and Tc1 responses . In contrast, Pam-3-cys (TLR-2 stimulus) elicits less IL-12(p70), but abundant IL-10, and favors Th2 and T cytotoxic 2 (Tc2) responses . These distinct responses likely occur via differences in extracellular signal-regulated kinase signaling in DCs . Thus, Pam-3-cys induces enhanced extracellular signal-regulated kinase signaling, compared with LPS, resulting in suppressed IL-12(p70) and enhanced IL-10 production, as well as enhanced induction of the transcription factor, c-Fos . Interestingly, DCs from c-fos(-/-) mice produce more IL-12(p70), but less IL-10, compared with control DCs . Therefore, different TLR ligands induce distinct cytokines and signaling in DCs, and differentially bias Th responses in vivo.

J Cell Biol, 2004 Apr, 165(1), 53 - 62 Epub 2004 Apr 05.
Role of YidC in folding of polytopic membrane proteins; Nagamori S et al.; YidC of Echerichia coli, a member of the conserved Alb3/Oxa1/YidC family, is postulated to be important for biogenesis of membrane proteins . Here, we use as a model the lactose permease (LacY), a membrane transport protein with a known three-dimensional structure, to determine whether YidC plays a role in polytopic membrane protein insertion and/or folding . Experiments in vivo and with an in vitro transcription/translation/insertion system demonstrate that YidC is not necessary for insertion per se, but plays an important role in folding of LacY . By using the in vitro system and two monoclonal antibodies directed against conformational epitopes, LacY is shown to bind the antibodies poorly in YidC-depleted membranes . Moreover, LacY also folds improperly in proteoliposomes prepared without YidC . However, when the proteoliposomes are supplemented with purified YidC, LacY folds correctly . The results indicate that YidC plays a primary role in folding of LacY into its final tertiary conformation via an interaction that likely occurs transiently during insertion into the lipid phase of the membrane.

J Biol Chem, 2004 Jun 18, 279(25), 26666 - 73 Epub 2004 Apr 02.
Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes; Li R et al.; Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin alpha and beta subunits may play an important role in integrin activation and clustering . As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type alpha(IIb) TM helix and single-site TM domain mutants . TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase . We found the amount of chloramphenicol acetyltransferase induced by the wild-type alpha(IIb) TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the alpha(IIb) TM domain oligomerizes in biological membranes . Mutating each of the alpha(IIb) TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization . Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG . Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of alpha(IIb) TM helix . These data were used to generate an atomic model of the alpha(IIb) homodimer, revealing a family of structures with right-handed crossing angles of 40 degrees to 60 degrees, consistent with a 4.0-residue periodicity, and with an interface rotated by 50 degrees relative to glycophorin A . Thus, although the alpha(IIb) TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.

J Biol Chem, 2004 Jun 18, 279(25), 26489 - 99 Epub 2004 Apr 02.
Heme distortion modulated by ligand-protein interactions in inducible nitric-oxide synthase; Li D et al.; The catalytic center of nitric-oxide synthase (NOS) consists of a thiolate-coordinated heme macrocycle, a tetrahydrobiopterin (H4B) cofactor, and an l-arginine (l-Arg)/N-hydroxyarginine substrate binding site . To determine how the interplay between the cofactor, the substrates, and the protein matrix housing the heme regulates the enzymatic activity of NOS, the CO-, NO-, and CN(-)-bound adducts of the oxygenase domain of the inducible isoform of NOS (iNOS(oxy)) were examined with resonance Raman spectroscopy . The Raman data of the CO-bound ferrous protein demonstrated that the presence of l-Arg causes the Fe-C-O moiety to adopt a bent structure because of an H-bonding interaction whereas H4B binding exerts no effect . Similar behavior was found in the CN(-)-bound ferric protein and in the nitric oxide (NO)-bound ferrous protein . In contrast, in the NO-bound ferric complexes, the addition of l-Arg alone does not affect the structural properties of the Fe-N-O moiety, but H4B binding forces it to adopt a bent structure, which is further enhanced by the subsequent addition of l-Arg . The differential interactions between the various heme ligands and the protein matrix in response to l-Arg and/or H4B binding is coupled to heme distortions, as reflected by the development of a variety of out-of-plane heme modes in the low frequency Raman spectra . The extent and symmetry of heme deformation modulated by ligand, substrate, and cofactor binding may provide important control over the catalytic and autoinhibitory properties of the enzyme.

Appl Environ Microbiol, 2004 Apr, 70(4), 2503 - 7
Frequency and distribution of tetracycline resistance genes in genetically diverse, nonselected, and nonclinical Escherichia coli strains isolated from diverse human and animal sources; Bryan A et al.; Nonselected and natural populations of Escherichia coli from 12 animal sources and humans were examined for the presence and types of 14 tetracycline resistance determinants . Of 1,263 unique E . coli isolates from humans, pigs, chickens, turkeys, sheep, cows, goats, cats, dogs, horses, geese, ducks, and deer, 31% were highly resistant to tetracycline . More than 78, 47, and 41% of the E . coli isolates from pigs, chickens, and turkeys were resistant or highly resistant to tetracycline, respectively . Tetracycline MICs for 61, 29, and 29% of E . coli isolates from pig, chickens, and turkeys, respectively, were >/=233 micro g/ml . Muliplex PCR analyses indicated that 97% of these strains contained at least 1 of 14 tetracycline resistance genes {tetA, tetB, tetC, tetD, tetE, tetG, tetK, tetL, tetM, tetO, tetS, tetA(P), tetQ, and tetX} examined . While the most common genes found in these isolates were tetB (63%) and tetA (35%), tetC, tetD, and tetM were also found . E . coli isolates from pigs and chickens were the only strains to have tetM . To our knowledge, this represents the first report of tetM in E . coli.

Appl Environ Microbiol, 2004 Apr, 70(4), 2354 - 66
Analysis of gene expression in Escherichia coli in response to changes of growth-limiting nutrient in chemostat cultures; Hua Q et al.; Studies of steady-state metabolic fluxes in Escherichia coli grown in nutrient-limited chemostat cultures suggest remarkable flux alterations in response to changes of growth-limiting nutrient in the medium (Hua et al., J . Bacteriol . 185:7053-7067, 2003) . To elucidate the physiological adaptation of cells to the nutrient condition through the flux change and understand the molecular mechanisms underlying the change in the flux, information on gene expression is of great importance . DNA microarray analysis was performed to investigate the global transcriptional responses of steady-state cells grown in chemostat cultures with limited glucose or ammonia while other environmental conditions and the growth rate were kept constant . In slow-growing cells (specific growth rate of 0.10 h(-1)), 9.8% of a total of 4,071 genes investigated, especially those involved in amino acid metabolism, central carbon and energy metabolism, transport system and cell envelope, were observed to be differentially expressed between the two nutrient-limited cultures . One important characteristic of E . coli grown under nutrient limitation was its capacity to scavenge carbon or nitrogen from the medium through elevating the expression of the corresponding transport and assimilation genes . The number of differentially expressed genes in faster-growing cells (specific growth rate of 0.55 h(-1)), however, decreased to below half of that in slow-growing cells, which could be explained by diverse transcriptional responses to the growth rate under different nutrient limitations . Independent of the growth rate, 92 genes were identified as being differentially expressed . Genes tightly related to the culture conditions were highlighted, some of which may be used to characterize nutrient-limited growth.

Vet Microbiol, 2004 Apr 19, 99(3-4), 287 - 94
Prevalence and deletion types of the pathogenicity island ETT2 among Escherichia coli strains from oedema disease and colibacillosis in pigs; Prager R et al.; Piglet pathogenic Escherichia coli encoding Shigatoxin 2e and F18 adhesins are the etiological agents of oedema disease as well as of non-oedema disease colibacillosis . In order to reveal virulence differences among this pathogen, the presence of the pathogenicity island (PAI) E . coli type three secretion system 2 (ETT2) was examined . Using PCR and Southern blot techniques for the identification of the right, the middle, and the left region of this 29.9kb large genetic element, the entire ETT2 was found among E . coli O138:H(-), O139:H1, and O147:H6 strains originated from cases of oedema disease in Germany between 1995 and 2001 and belonging to various clonal types . In contrast, non-oedema disease E . coli isolates (e.g . O8:H19, 101:H(-), O141:H4) contain deleted subtypes of ETT2 . These deletions cover the translocon part of the putative ETT2-encoded type III secretion apparatus . It is suggested that the entire ETT2 is associated with a particular virulence trait of piglet oedema disease E . coli (EDEC).

J Mol Biol, 2004 Apr 23, 338(2), 287 - 97
Solution structures of the inactive and BeF3-activated response regulator CheY2; Riepl H et al.; The chemotactic signalling chain to the flagellar motor of Sinorhizobium meliloti features a new type of response regulator, CheY2 . CheY2 activated by phosphorylation (CheY2-P) controls the rotary speed of the flagellar motor (instead of reversing the sense of rotation), and it is efficiently dephosphorylated by phospho-retrotransfer to the cognate kinase, CheA . Here, we report the NMR solution structures of the Mg(2+)-complex of inactive CheY2, and of activated CheY2-BeF(3), a stable analogue of CheY2-P, to an overall root mean square deviation of 0.042 nm and 0.027 nm, respectively . The 14 kDa CheY2 protein exhibits a characteristic open (alpha/beta)(5) conformation . Modification of CheY2 by BeF(3)(-) leads to large conformational changes of the protein, which are in the limits of error identical with those observed by phosphorylation of the active-centre residue Asp58 . In BeF(3)-activated CheY2, the position of Thr88-OH favours the formation of a hydrogen bond with the active site, Asp58-BeF(3), similar to BeF(3)-activated CheY from Escherichia coli . In contrast to E.coli, this reorientation is not involved in a Tyr-Thr-coupling mechanism, that propagates the signal from the incoming phosphoryl group to the C-terminally located FliM-binding surface . Rather, a rearrangement of the Phe59 side-chain to interact with Ile86-Leu95-Val96 along with a displacement of alpha4 towards beta5 is stabilised in S.meliloti . The resulting, activation-induced, compact alpha4-beta5-alpha5 surface forms a unique binding domain suited for specific interaction with and signalling to a rotary motor that requires a gradual speed control . We propose that these new features of response regulator activation, compared to other two-component systems, are the key for the observed unique phosphorylation, dephosphorylation and motor control mechanisms in S.meliloti.

Acta Pharmacol Sin, 2004 Apr, 25(4), 490 - 5
Effect of two human growth hormone receptor antagonists on glomerulosclerosis in streptozotocin-induced diabetic rats; Li W et al.; AIM: To explore the feasibility of human growth hormone (hGH) receptor antagonist in the treatment of end-stage diabetic renal complications . METHODS: Two hGH mutants, hGHA1 (Cys-hGH-del1-4, G120R, K168A, E174A, C182S, del186-191) and hGHA2 (hGH-H21A, G120R, E174A) were expressed in E coli . The IC50 (Mean+/-SD) values for the mutants for inhibiting 125I-hGH binding to rabbit growth hormone receptor were (65+/-10) ng for hGHA1, (27+/-5.6) ng for hGHA2, and (10+/-0.6) ng for wild type hGH, respectively . RESULTS: After treatment for 12 weeks, the renal histology analysis showed that treatment with hGHA2 at 4 mg/kg body weight daily markedly suppressed glomerulosclerosis in streptozotocin-induced diabetic Sprague-Dawley (SD) rats; hGHA1 at the same dosage slightly increased the renal damage compared with saline; while wild type hGH at 1 U/kg body weight daily severely worsened the glomerulo-sclerosis in diabetic SD rats . CONCLUSION: The data indicated that hGHA2 inhibited the end-stage glomerulosclerosis in diabetic rats, but hGHA1 mildly increased the glomerulosclerosis.

Eur J Biochem, 2004 Apr, 271(8), 1516 - 24
Crystal structures of HIV protease V82A and L90M mutants reveal changes in the indinavir-binding site; Mahalingam B et al.; The crystal structures of the wild-type HIV-1 protease (PR) and the two resistant variants, PR(V82A) and PR(L90M), have been determined in complex with the antiviral drug, indinavir, to gain insight into the molecular basis of drug resistance . V82A and L90M correspond to an active site mutation and nonactive site mutation, respectively . The inhibition (K(i)) of PR(V82A) and PR(L90M) was 3.3- and 0.16-fold, respectively, relative to the value for PR . They showed only a modest decrease, of 10-15%, in their k(cat)/K(m) values relative to PR . The crystal structures were refined to resolutions of 1.25-1.4 A to reveal critical features associated with inhibitor resistance . PR(V82A) showed local changes in residues 81-82 at the site of the mutation, while PR(L90M) showed local changes near Met90 and an additional interaction with indinavir . These structural differences concur with the kinetic data.

Eur J Biochem, 2004 Apr, 271(8), 1437 - 52
13C-, 15N- and 31P-NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins; Eisenreich W et al.; Thioredoxin reductase (TrxR) from Escherichia coli, the mutant proteins E159Y and C138S, and the mutant protein C138S treated with phenylmercuric acetate were reconstituted with {U-(13)C(17),U-(15)N(4)}FAD and analysed, in their oxidized and reduced states, by (13)C-, (15)N- and (31)P-NMR spectroscopy . The enzymes studied showed very similar (31)P-NMR spectra in the oxidized state, consisting of two peaks at -9.8 and -11.5 p.p.m . In the reduced state, the two peaks merge into one apparent peak (at -9.8 p.p.m.) . The data are compared with published (31)P-NMR data of enzymes closely related to TrxR . (13)C and (15)N-NMR chemical shifts of TrxR and the mutant proteins in the oxidized state provided information about the electronic structure of the protein-bound cofactor and its interactions with the apoproteins . Strong hydrogen bonds exist between protein-bound flavin and the apoproteins at C(2)O, C(4)O, N(1) and N(5) . The N(10) atoms in the enzymes are slightly out of the molecular plane of the flavin . Of the ribityl carbon atoms C(10alpha,gamma,delta) are the most affected upon binding to the apoprotein and the large downfield shift of the C(10gamma) atom indicates strong hydrogen bonding with the apoprotein . The hydrogen bonding pattern observed is in excellent agreement with X-ray data, except for the N(1) and the N(3) atoms where a reversed situation was observed . Some chemical shifts observed in C138S deviate considerably from those of the other enzymes . From this it is concluded that C138S is in the FO conformation and the others are in the FR conformation, supporting published data . In the reduced state, strong hydrogen bonding interactions are observed between C(2)O and C(4)O and the apoprotein . As revealed by the (15)N chemical shifts and the N(5)H coupling constant the N(5) and the N(10) atom are highly sp(3) hybridized . The calculation of the endocyclic angles for the N(5) and the N(10) atoms shows the angles to be approximately 109 degrees, in perfect agreement with X-ray data showing that the flavin assumes a bent conformation along the N(10)/N(5) axis of the flavin . In contrast, the N(1) is highly sp(2) hybridized and is protonated, i.e . in the neutral state . Upon reduction of the enzymes, the (13)C chemical shifts of some atoms of the ribityl side chain undergo considerable changes also indicating conformational rearrangements of the side-chain interactions with the apoproteins . The chemical shifts between native TrxR and C138S are now rather similar and differ from those of the two other mutant proteins . This strongly indicates that the former enzymes are in the FO conformation and the other two are in the FR conformation . The data are discussed briefly in the context of published NMR data obtained with a variety of flavoproteins.

Eur J Biochem, 2004 Apr, 271(8), 1426 - 36
Chaperone activity of cytosolic small heat shock proteins from wheat; Basha E et al.; Small Hsps (sHsps) and the structurally related eye lens alpha-crystallins are ubiquitous stress proteins that exhibit ATP-independent molecular chaperone activity . We studied the chaperone activity of dodecameric wheat TaHsp16.9C-I, a class I cytosolic sHsp from plants and the only eukaryotic sHsp for which a high resolution structure is available, along with the related wheat protein TaHsp17.8C-II, which represents the evolutionarily distinct class II plant cytosolic sHsps . Despite the available structural information on TaHsp16.9C-I, there is minimal data on its chaperone activity, and likewise, data on activity of the class II proteins is very limited . We prepared purified, recombinant TaHsp16.9C-I and TaHsp17.8C-II and find that the class II protein comprises a smaller oligomer than the dodecameric TaHsp16.9C-I, suggesting class II proteins have a distinct mode of oligomer assembly as compared to the class I proteins . Using malate dehydrogenase as a substrate, TaHsp16.9C-I was shown to be a more effective chaperone than TaHsp17.8C-II in preventing heat-induced malate dehydrogenase aggregation . As observed by EM, morphology of sHsp/substrate complexes depended on the sHsp used and on the ratio of sHsp to substrate . Surprisingly, heat-denaturing firefly luciferase did not interact significantly with TaHsp16.9C-I, although it was fully protected by TaHsp17.8C-II . In total the data indicate sHsps show substrate specificity and suggest that N-terminal residues contribute to substrate interactions.

Mol Microbiol, 2004 Apr, 52(2), 589 - 600
The histone-like nucleoid structuring protein H-NS represses the Escherichia coli bgl operon downstream of the promoter; Dole S et al.; Specificity of repression by the histone-like nucleoid structuring protein and pleiotropic regulator, H-NS, is exceptionally high in case of the Escherichia coli bgl (beta-glucoside) operon . Here we present evidence that H-NS represses the operon at two levels . The binding of H-NS to an upstream silencer results in an approximately threefold repression of the catabolite gene regulator protein (CRP) dependent bgl promoter . In addition, H-NS binds to a silencer region located approximately 600-700 base pairs downstream of the promoter, within the coding region of first gene, bglG, resulting in a approximately sevenfold further decrease of expression . Repression by H-NS at the downstream silencer requires termination factor Rho and is reduced by translation of the bglG mRNA, but is independent of the promoter . This suggests that H-NS induces polarity of transcription by acting as a roadblock to the elongating RNA polymerase . The control of the bgl operon by H-NS at two levels results in a highly specific repression.

Mol Microbiol, 2004 Apr, 52(2), 551 - 62
Requirement for RecFOR-mediated recombination in priA mutant; Grompone G et al.; Restart of arrested replication forks is an important process and PriA, the main Escherichia coli replication restart protein, is essential for viability under any condition that increases the frequency of fork arrest . In priA mutant, replication forks are arrested by spontaneously occurring roadblocks and blocked replication forks persist as a result of the defect in replication restart . In the present work, we analysed how recombination proteins contribute to the viability of the priA mutant . RecFOR-mediated homologous recombination occurs in a large fraction of priA mutant cells, indicating a frequent formation of DNA single strand gaps and their recombinational repair . This high level of homologous recombination renders the proteins that resolve Holliday junctions recombination intermediates essential for viability . When homologous recombination is blocked at early steps by recFOR or recA inactivation, exonuclease V-mediated DNA degradation is required for full viability of priA mutants, indicating that unrepaired gaps are broken and that DNA degradation of the broken DNA allows the formation of viable cells . Models for the formation of single strand DNA gaps consequently to a replication restart defect and for gap processing are proposed.

Mol Microbiol, 2004 Apr, 52(2), 437 - 49
Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP; Plumbridge J et al.; The chitobiose operon, chbBCARFG, encodes genes for the transport and degradation of the N-acetylglucosamine disaccharide, chitobiose . Chitobiose is transported by the phosphotransferase system (PTS) producing chitobiose-6P which is hydrolysed to GlcNAc-6P by the chbF gene product and then further degraded by the nagBA gene products . Expression of the chb operon is repressed by NagC, which regulates genes involved in amino sugar metabolism . The inducer for NagC is GlcNAc-6P . NagC binds to two sites separated by 115 bp and the transcription start point of the chb operon lies within the downstream NagC operator . In addition the chb operon encodes its own specific regulator, ChbR, an AraC-type dual repressor-activator, which binds to two direct repeats of 19 bp located between the two NagC sites . ChbR is necessary for transcription activation in the presence of chitobiose in vivo . Induction of the operon also requires CAP, which binds to a site upstream of the ChbR repeats . In the absence of chitobiose both NagC and ChbR act as repressors . Together these three factors cooperate in switching chb expression from the repressed to the activated state . The need for two specific inducing signals, one for ChbR to activate the expression of the operon and a second for NagC to relieve its repression, ensure that the chb operon is only induced when there is sufficient flux through the combined chb-nag metabolic pathway to activate expression of both the chb and nag operons.

Mol Microbiol, 2004 Apr, 52(2), 427 - 35
Dispensable PDZ domain of Escherichia coli YaeL essential protease; Bohn C et al.; In Escherichia coli, adaptation to extra-cytoplasmic stress relies on sigma(E) activation to induce a rescue pathway . Under non-stressed conditions, sigma(E) is sequestered by the inner membrane protein RseA . Extra-cytoplasmic stress activates DegS-dependent cleavage of RseA, rendering RseA sensitive to further degradation by the YaeL protease . YaeL contains two motifs characteristic of a family of metallo-proteases, as well as a periplasmic PDZ domain . We report results of mutational analyses of the YaeL domains . Surprisingly, expression in a strain depleted for wild-type YaeL or YaeL variants having a 40 amino acid deletion of the PDZ domain or amino acid substitutions of conserved amino acids of the YaeL PDZ domain did not affect cell viability . The proteolytic activity against RseA of these YaeL variants became independent of DegS . These observations suggest that the YaeL PDZ domain exerts a negative control on YaeL activity . Rather than being involved in substrate recognition, the PDZ domain of YaeL is likely to act as an inhibitor of proteolytic activity.

Mol Microbiol, 2004 Apr, 52(2), 413 - 26
Inversion within the haloalkaliphilic virus phi Ch1 DNA results in differential expression of structural proteins; Rossler N et al.; The sequence of phi Ch1 contains an open reading frame (int1) in the central part of its genome that belongs to the lambda integrase family of site-specific recombinases . Sequence similarities to known integrases include the highly conserved tetrad R-H-R-Y . The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR-L and IR-R), which are orientated in an inverted direction . Here, we show that a recombination active region exists in the genome of phi Ch1: the number of those repeats, non-homologous regions within the repeat clusters IR-L and IR-R and the orientation of the int1 gene vary in a given virus population . Within this study, we identified circular intermediates, composed of the int1 gene and the inwards orientated repeat regions IR-L and IR-R, which could be involved in the recombination process itself . IR-L and IR-R are embedded within ORF34 and ORF36 respectively . As a consequence of the inversion within this region of phi Ch1, the C-terminal parts of the proteins encoded by ORF34 and 36 are exchanged . Both proteins, expressed in Escherichia coli, interact with specific antisera against whole virus particles, indicating that they could be parts of phi Ch1 virions . Expression of the protein(s) in Natrialba magadii could be detected 98 h after inoculation, which is similar to other structural proteins of phi Ch1 . Taken together, the data show that the genome of phi Ch1 contains an invertible region that codes for a recombinase and structural proteins . Inversion of this segment results in a variation of these structural proteins.

Biochemistry, 2004 Apr 13, 43(14), 4375 - 84
Kinetic and chemical mechanism of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate isomeroreductase; Argyrou A et al.; 1-Deoxy-D-xylulose-5-phosphate (DXP) isomeroreductase catalyzes the isomerization and reduced nicotinamide adenine dinucleotide phosphate- (NADPH-) dependent reduction of DXP to generate 2-C-methylerythritol 4-phosphate (MEP) in the first committed step of the MEP pathway of isoprenoid biosynthesis . We have cloned the gene encoding the Mycobacterium tuberculosis DXP isomeroreductase, expressed the protein in Escherichia coli, and purified the enzyme to homogeneity using conventional column chromatography methods . DXP isomeroreductase is a metal ion-activated enzyme displaying superior specificity for Co(2+), good specificity for Mn(2+), and poor specificity for Mg(2+) . Although NADPH is preferred over reduced nicotinamide adenine dinucleotide (NADH) about 100-fold as evaluated by the relative k(cat)/K(m) values, the maximum turnover numbers are similar, suggesting that the 2'-phosphate of NADPH contributes predominantly to binding and not to catalysis . While k(cat) was independent of pH in the region 6.0 <or= pH <or= 8.75, k(cat)/K(act)(Mn)2+ decreased at low pH as two enzymatic groups with pK(a) values of 7.4 are protonated . These groups likely represent carboxylate groups that coordinate the divalent metal ion in the active site . The results also support an electrostatic role for the divalent metal ion in catalysis . The results of product inhibition studies and isotope effects suggest that the enzyme utilizes a steady-state random mechanism . Significant isotope effects were observed with {4S-(2)H}NAD(P)H, establishing that the enzyme promotes transfer of the C(4)-proS hydride of the reduced pyridine nucleotide . The magnitude of these primary deuterium kinetic isotope effects varied with metal ion and reduced pyridine nucleotide identities . The results are discussed in terms of significant differences in the commitment factors for the various metal ions and pyridine nucleotides.

Biochemistry, 2004 Apr 13, 43(14), 4356 - 65
Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains; Lansdon EB et al.; Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized . The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate . The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol . APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP) . The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ({APS}(opt)) of 15 microM . The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at {APS}(opt)) were 4.2 microM and 0.14 mM, respectively . At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1) . Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1) . The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM . The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling . Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i) . Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase . (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes . Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.

Biochemistry, 2004 Apr 13, 43(14), 4338 - 46
Activation of hemolysin toxin: relationship between two internal protein sites of acylation; Langston KG et al.; HlyC, hemolysin-activating lysine acyltransferase, catalyzes the acylation (from acyl-ACP) of Escherichia coli prohemolysin (proHlyA) on the epsilon-amino groups of specific lysine residues, Lys564 and Lys690 of the 1024-amino acid primary structure, to form hemolysin (HlyA) . The amino acid sequences flanking the two acylation sites are not homologous except that each has a glycine residue immediately preceding the lysine which is acylated; there are, however, numerous GK sequences throughout proHlyA that are not acylation sites . The substrate specificity of acylation was examined . ProHlyA-derived structures, altered by substantial deletions and separation of the acylation sites into two different peptides and site-directed mutation analyses of acylation sites, often served as internal protein acylation substrates, and the kinetics of the acylations were measured . The two sites of acylation of proHlyA functioned independently of one another with HlyC; there did not appear to be a common HlyC binding site or processivity of the enzyme between the sites . Acyl-HlyC was likely the enzyme form that interacted with the final acylation substrate . In a variety of constructs, the two acylation sites had similar K(m) values, but their V(max) values and catalytic efficiencies as substrates differed . Internal protein acylation was inhibited by specific small peptides mimicking the primary structure of each acylation site except that the crucial lysines were replaced with arginines; similar small peptides containing the crucial lysine, however, were not acylated.

Biochemistry, 2004 Apr 13, 43(14), 4304 - 12
In vitro processing of HIV-1 nucleocapsid protein by the viral proteinase: effects of amino acid substitutions at the scissile bond in the proximal zinc finger sequence; Tozser J et al.; The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein flanked by Gag sequences (r-preNC) was expressed in Escherichia coli and purified . HIV-1 proteinase cleaved r-preNC to the "mature" NCp7 form, which is comprised of 55 residues . Further incubation resulted in cleavages of NCp7 itself between Phe16 and Asn17 of the proximal zinc finger domain and between Cys49 and Thr50 in the C-terminal part . Kinetic parameters determined for the cleavage of oligopeptides corresponding to the cleavage sites in r-preNC correlated well with the sequential processing of r-preNC . Mutations of Asn17 were introduced to alter the susceptibility of NC protein to HIV-1 proteinase . While mutating Asn17 to Ala resulted in a protein which was processed in a manner similar to that of the wild type, mutating it to Phe or Leu resulted in proteins which were processed at a substantially higher rate at this site than the wild type . Mutation of Asn17 to Lys or Gly resulted in proteins which were very poorly cleaved at this site . Oligopeptides containing the same amino acid substitutions at the cleavage site of the proximal zinc finger domain were also tested as substrates of the proteinase, and the kinetic parameters agreed well with the semiquantitative results obtained with the protein substrates.

Biochemistry, 2004 Apr 13, 43(14), 4206 - 11
Thermodynamic characterization of the interaction of mutant UvrB protein with damaged DNA; Ma H et al.; During the DNA damage recognition of nucleotide excision repair in Escherichia coli the interaction of UvrB protein with damaged DNA ensures the recognition of differences in the intrinsic chemical structures of a variety of adduct molecules in DNA double helix . Our earlier study indicated that a single tyrosine-to-tryptophan mutation at residue 95 converted the UvrB to a protein {UvrB(Y95W)} that is able to bind to a structure-specific bubble DNA substrate, even in the absence of UvrA . Fluorescence spectroscopy therefore was adopted to investigate the biochemical properties and thermodynamics of DNA damage recognition by the mutant protein . We examined the binding of the UvrB(Y95W) mutant protein to a structure-specific 30 bp DNA substrate containing a single fluorescein which serves as both an adduct and a fluorophore . Binding of the protein to the substrate results in a significant reduction in fluorescence . By monitoring the fluorescence changes, binding isotherms were generated from a series of titration experiments at various physiological temperatures, and dissociation constants were determined . Analysis of our data indicate that interaction of UvrB(Y95W) protein with the adduct incurred a large negative change in heat capacity DeltaC(p)(o)(obs) (-1.1 kcal mol(-1) K(-1)), while the DeltaG(o)(obs) was relatively unchanged with temperature . Further study of the binding at various concentrations of KCl showed that on average only about 1.5 ion pairs were involved in formation of the UvrB-DNA complex . Together, these results suggested that hydrophobic interactions are the main driving forces for the recognition of DNA damage by UvrB protein.

Biochemistry, 2004 Apr 13, 43(14), 4196 - 205
DNA damage recognition of mutated forms of UvrB proteins in nucleotide excision repair; Zou Y et al.; The DNA repair protein UvrB plays an indispensable role in the stepwise and sequential damage recognition of nucleotide excision repair in Escherichia coli . Our previous studies suggested that UvrB is responsible for the chemical damage recognition only upon a strand opening mediated by UvrA . Difficulties were encountered in studying the direct interaction of UvrB with adducts due to the presence of UvrA . We report herein that a single point mutation of Y95W in which a tyrosine is replaced by a tryptophan results in an UvrB mutant that is capable of efficiently binding to structure-specific DNA adducts even in the absence of UvrA . This mutant is fully functional in the UvrABC incisions . The dissociation constant for the mutant-DNA adduct interaction was less than 100 nM at physiological temperatures as determined by fluorescence spectroscopy . In contrast, similar substitutions at other residues in the beta-hairpin with tryptophan or phenylalanine do not confer UvrB such binding ability . Homology modeling of the structure of E . coli UvrB shows that the aromatic ring of residue Y95 and only Y95 directly points into the DNA binding cleft . We have also examined UvrB recognition of both "normal" bulky BPDE-DNA and protein-cross-linked DNA (DPC) adducts and the roles of aromatic residues of the beta-hairpin in the recognition of these lesions . A mutation of Y92W resulted in an obvious decrease in the efficiency of UvrABC incisions of normal adducts, while the incision of the DPC adduct is dramatically increased . Our results suggest that Y92 may function differently with these two types of adducts, while the Y95 residue plays an unique role in stabilizing the interaction of UvrB with DNA damage, most likely by a hydrophobic stacking.

Biochemistry, 2004 Apr 13, 43(14), 4143 - 9
Molecular determinants of heritable vitamin E deficiency; Morley S et al.; Tocopherol transfer protein (TTP) is a key regulator of vitamin E homeostasis . TTP is presumed to function by transporting the hydrophobic vitamin between cellular compartments, thus facilitating its secretion to the extracellular space . Indeed, recombinant TTP demonstrates marked ability to facilitate tocopherol transfer between lipid bilayers . We report the biochemical characterization of six missense mutations TTP(1) that are found in human AVED patients . We expressed the H101Q, A120T, R192H, R59W, E141K, and R221W TTP mutants in Escherichia coli, and purified the proteins to homogeneity . We then characterized TTP and its mutant counterparts with respect to their affinity for RRR-alpha-tocopherol and to their ability to catalyze tocopherol transfer between membranes . We observe the R59W, E141K, and R221W mutations, associated with the severe, early-onset version of AVED, are impaired in tocopherol binding and transfer activity . Surprisingly, despite the profound clinical effect of the R59W, E141K, and R221W mutations in vivo, their impact on TTP activity in vitro is quite benign (2-3-fold reduction in transfer kinetics) . Furthermore, mutations associated with milder forms of the AVED disease, while causing pronounced perturbations in tocopherol homeostasis in vivo, are remarkably similar to the wild-type protein in the tocopherol transfer assays in vitro . Our data indicate that tocopherol transfer activity in vitro does not properly recapitulate the physiological functions of TTP . These findings suggest the possibility that the AVED syndrome may not arise from an inability of TTP to bind or to transfer alpha tocopherol, but rather from defects in other activities of the protein.

Biochemistry, 2004 Apr 13, 43(14), 4119 - 27
Pivotal role of Gly 121 in dihydrofolate reductase from Escherichia coli: the altered structure of a mutant enzyme may form the basis of its diminished catalytic performance; Swanwick RS et al.; The structure and folding of dihydrofolate reductase (DHFR) from Escherichia coli and the mutant G121V-DHFR, in which glycine 121 in the exterior FG loop was replaced with valine, were studied by molecular dynamics simulations and CD and fluorescence spectroscopy . The importance of residue 121 for the chemical step during DHFR catalysis had been demonstrated previously . High-temperature MD simulations indicated that while DHFR and G121V-DHFR followed similar unfolding pathways, the strong contacts between the M20 loop and the FG loop in DHFR were less stable in the mutant . These contacts have been proposed to be involved in a coupled network of interactions that influence the protein dynamics and promote catalysis {Benkovic, S . J., and Hammes-Schiffer, S . (2003) Science 301, 1196-1202} . CD spectroscopy of DHFR and G121V-DHFR indicated that the two proteins existed in different conformations at room temperature . While the thermally induced unfolding of DHFR was highly cooperative with a midpoint at 51.6 +/- 0.7 degrees C, G121V-DHFR exhibited a gradual decrease in its level of secondary structure without a clear melting temperature . Temperature-induced unfolding and renaturation from the urea-denatured state revealed that both proteins folded via highly fluorescent intermediates . The formation of these intermediates occurred with relaxation times of 149 +/- 4.5 and 256 +/- 13 ms for DHFR and G121V-DHFR, respectively . The fluorescence intensity for the intermediates formed during refolding of G121V-DHFR was approximately twice that of the wild-type . While the fluorescence intensity then slowly decayed for DHFR toward a state representing the native protein, G121V-DHFR appeared to be trapped in a highly fluorescent state . These results suggest that the reduced catalytic activity of G121V-DHFR is the consequence of nonlocal structural effects that may result in a perturbation of the network of promoting motions.

J Struct Biol, 2004 Jan-Feb, 145(1-2), 84 - 90
Fast automatic particle picking from cryo-electron micrographs using a locally normalized cross-correlation function: a case study; Rath BK et al.; Recent progress in single-particle reconstruction methods and cryo-EM techniques has led to the determination of macromolecular structures with unprecedented resolution . The number of particles that goes into the reconstruction is a key determinant in achieving high resolution . Interactive manual picking of particles from an electron micrograph is a very time-consuming, tedious, and inefficient process . We have implemented a fast automatic particle picking procedure in the SPIDER environment . The procedure makes use of template matching schemes and employs a recently developed locally normalized correlation algorithm based on Fourier techniques . As a test, we have used this procedure to pick 70S Escherichia coli ribosomes from a cryo-electron micrograph . Different search strategies including use of a circular mask and asymmetric masks for different orientations of the particle have been explored, and their relative efficiencies are discussed . The results indicate that the procedure can be optimally used to pick ribosomes in a fully automatic way within the limit of selecting less than 10% false positives while missing about 15% of true positives.

J Struct Biol, 2004 Jan-Feb, 145(1-2), 76 - 83
TYSON: robust searching, sorting, and selecting of single particles in electron micrographs; Plaisier JR et al.; We here present TYSON, a new program for automatic and semi-automatic particle selection from electron micrographs . TYSON employs a three-step strategy of searching, sorting and selecting single particles . In the first step, TYSON finds the positions of potential particles by one of three different methods: local averaging, template matching or local variance . The practical merits and drawbacks of these methods are discussed . In the second step, these potential particles are automatically sorted according to their probability of being true positives . Many criteria are provided for this sort . In the final -interactive- step, whole categories of poorly fitting false positives can be removed with a single mouse-click . We present results obtained using cryo-EM micrographs of both spherical virus particles and asymmetric particles . The procedures are fast and use of TYSON allowed, for example, some 20,000 particles to be selected in a single working day.

J Struct Biol, 2004 Jan-Feb, 145(1-2), 19 - 28
A two step approach for semi-automated particle selection from low contrast cryo-electron micrographs; Hall RJ et al.; Over recent years advances in cryo-electron microscopy for the study of macromolecular structure have resulted in resolutions in the range 10-15 A becoming routine . With this drive for increased resolution comes the need to collect larger datasets, commonly >10,000 particle images . Manual selection of particles from micrographs is often difficult and with such large numbers of particles now involved it is also laborious and a common bottleneck . Automated methods do exist but are normally restricted to specific samples or data, i.e., spherical particles, no aggregation, high contrast, and low noise . A two step approach has been developed that remains general and can be applied to low contrast, high noise micrographs of small molecules . Specifically, application of the approach is presented using micrographs of Escherichia coli RNA polymerase, which due to low contrast and the relatively small size of the molecule prove difficult to pick manually . To test the automated approach, independent reconstructions of RNA polymerase were carried out using manual and automatically picked data . The two reconstructions are shown to be comparable and the reconstruction from the automatically picked dataset is at a higher resolution, due to an increase in the number of particles picked.

Philos Trans R Soc Lond B Biol Sci, 2004 Jan 29, 359(1441), 61 - 9
Recombination and chromosome segregation; Sherratt DJ et al.; The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes . The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins . In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site-specific recombination system . Dimer resolution also requires FtsK, a division septum-located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction . FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes . The visualization of the replication/recombination-associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.

Philos Trans R Soc Lond B Biol Sci, 2004 Jan 29, 359(1441), 49 - 59
Interplay between DNA replication, recombination and repair based on the structure of RecG helicase; Briggs GS et al.; Recent studies in Escherichia coli indicate that the interconversion of DNA replication fork and Holliday junction structures underpins chromosome duplication and helps secure faithful transmission of the genome from one generation to the next . It facilitates interplay between DNA replication, recombination and repair, and provides means to rescue replication forks stalled by lesions in or on the template DNA . Insight into how this interconversion may be catalysed has emerged from genetic, biochemical and structural studies of RecG protein, a member of superfamily 2 of DNA and RNA helicases . We describe how a single molecule of RecG might target a branched DNA structure and translocate a single duplex arm to drive branch migration of a Holliday junction, interconvert replication fork and Holliday junction structures and displace the invading strand from a D loop formed during recombination at a DNA end . We present genetic evidence suggesting how the latter activity may provide an efficient pathway for the repair of DNA double-strand breaks that avoids crossing over, thus facilitating chromosome segregation at cell division.

Philos Trans R Soc Lond B Biol Sci, 2004 Jan 29, 359(1441), 25 - 30
Protein trafficking on sliding clamps; Lopez de Saro F et al.; The sliding clamps of chromosomal replicases are acted upon by both the clamp loader and DNA polymerase . Several other proteins and polymerases also interact with the clamp . These proteins bind the clamp at the same spot and use it in sequential fashion . First the clamp loader must bind the clamp in order to load it onto DNA, but directly thereafter the clamp loader must clear away from the clamp so it can be used by the replicative DNA polymerase . At the end of replication, the replicase is ejected from the clamp, which presumably allows the clamp to interact with yet other proteins after its use by the replicase . This paper describes how different proteins in the Escherichia coli replicase, DNA polymerase III holoenzyme, coordinate their traffic flow on the clamp . The mechanism by which traffic flow on the beta clamp is directed is based on competition of the proteins for the clamp, where DNA structure modulates the competition . It seems likely that the principles will generalize to a traffic flow of other factors on these circular clamp proteins.

Clin Exp Metastasis, 2004, 21(1), 65 - 74
Measurement of tumor load and distribution in a model of cancer-induced osteolysis: a necessary precaution when testing novel anti-resorptive therapies; Amhlaoibh RN et al.; The Arguello model of cancer metastasis to bone has been used extensively to study breast cancer-induced osteolytic disease . The effects of therapy on skeletal disease and on tumour burden in soft organs are traditionally measured using radiography and/or time-consuming histomorphometry, respectively . The purpose of this study was to develop a sensitive and efficient method for evaluating tumour burden in vivo using MDA-231 cells transduced with the E . coli lacZ gene (MDA-231BAG) . Osteolysis was measured by radiography and tumour burden was measured histomorphometrically or biochemically . In untreated mice, measurements of tumour burden in bone extracts using human cytokeratin-associated tissue polypeptide antigen (TPA) ELISA or E . coli beta-galactosidase (beta-gal) activity immunoassay reflected the extent of osteolytic disease as measured by radiography; however, tumour load could be detected before onset of osteolysis . When monitoring the effect of therapy (0.2 mg/kg ibandronate/day), radiography alone proved to be insufficient . Mice treated with the bisphosphonate ibandronate from time of inoculation with cancer cells had no radiologically visible signs of osteolysis but significant tumour load was measured in the bone extracts using these assays . Furthermore, beta-gal activity could be used as a measurement of tumour load in soft organs, and unlike other human breast cancer markers expressed by the MDA-231 cells in vitro, beta-gal activity was detected in the serum of mice with progressive disease . In conclusion, we describe an efficient model of breast cancer-induced osteolysis to quantify the effect of therapy on disease load and distribution, which could be beneficial in evaluating novel therapies for the treatment of the disease.

Nature, 2004 Apr 22, 428(6985), 868 - 71 Epub 2004 Apr 04.
Programmed population control by cell-cell communication and regulated killing; You L et al.; De novo engineering of gene circuits inside cells is extremely difficult, and efforts to realize predictable and robust performance must deal with noise in gene expression and variation in phenotypes between cells . Here we demonstrate that by coupling gene expression to cell survival and death using cell-cell communication, we can programme the dynamics of a population despite variability in the behaviour of individual cells . Specifically, we have built and characterized a 'population control' circuit that autonomously regulates the density of an Escherichia coli population . The cell density is broadcasted and detected by elements from a bacterial quorum-sensing system, which in turn regulate the death rate . As predicted by a simple mathematical model, the circuit can set a stable steady state in terms of cell density and gene expression that is easily tunable by varying the stability of the cell-cell communication signal . This circuit incorporates a mechanism for programmed death in response to changes in the environment, and allows us to probe the design principles of its more complex natural counterparts.

Methods Mol Biol, 2004, 261, 199 - 210
Mapping protein-ligand interactions by hydroxyl-radical protein footprinting; Loizos N; Hydroxyl-radical protein footprinting is a direct method to map protein sites involved in macromolecular interactions . The first step is to radioactively end-label the protein . Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand . Cleavage products are separated by high-resolution gel electrophoresis . The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl-radical cleavage . Molecular weight markers are electrophoresed on the same gel and hydroxyl-radical cleavage sites assigned by interpolation between the known cleavage sites of the markers . The results are presented in the form of a difference plot that show regions of the protein that change their susceptibility to cleavage while bound to a ligandPublication Types:

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