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 c