Biochemistry, 2004 Apr 13, 43(14), 4347 - 55 Ligand-induced changes in the conformational dynamics of a bacterial cytotoxic endonuclease; van den Bremer ET et al.; Knowledge about the conformational dynamics of a protein is key to understanding its biochemical and biophysical properties . In the present work we investigated the dynamic properties of the enzymatic domain of DNase colicins via time-resolved fluorescence and anisotropy decay analysis in combination with steady-state acrylamide quenching experiments . The dynamic properties of the apoenzyme were compared to those of the E9 DNase ligated to the transition metal ion Zn(2+) and the natural inhibitor Im9 . We further investigated the contributions of each of the two tryptophans within the E9 DNase (Trp22 and Trp58) using two single-tryptophan mutants (E9 W22F and E9 W58F) . Wild-type E9 DNase, E9 W22F, and E9 W58F, as well as Im9, showed multiple lifetime decays . The time-resolved and steady-state fluorescence results indicated that complexation of E9 DNase with Zn(2+) induces compaction of the E9 DNase structure, accompanied by immobilization of Trp22 along with a reduced solvent accessibility for both tryptophans . Im9 binding resulted in immobilization of Trp22 along with a decrease in the longest lifetime component . In contrast, Trp58 experienced less restriction on complexation of E9 DNase with Im9 and showed an increase in the longest lifetime component . Furthermore, the results point out that the Im9-induced changes in the conformational dynamics of E9 DNase are predominant and occur independently of the Zn(2+)-induced conformational effects. Medicina (Kaunas), 2004, 40(3), 260 - 4 Effect of bacterial stimulants on release of reactive oxygen metabolites from peripheral blood neutrophils in periodontitis; Zekonis G et al.; The aim of the present investigation was to explore the oxidative activity of peripheral blood polymorphonuclear neutrophils of periodontitis patients and of healthy subjects stimulated with non-opsonized E . coli and lipopolysaccharide of E . coli . MATERIAL AND METHODS: The leukocytes for this study were obtained from peripheral venous blood of 22 parodontitis patients and 16 healthy subjects . Oxidative activity of peripheral blood polymorphonuclear neutrophils was measured by method of the luminol-dependent chemiluminescence . RESULTS: The luminol-dependent chemiluminescence of stimulated neutrophils of periodontitis patients with non-opsonized E . coli increased less significantly (p<0.001) as compared to analogous chemiluminescence of control subjects (147126+/-8386 cpm and 189247+/-9134 cpm, respectively) . However, the luminol-dependent chemiluminescence of stimulated neutrophils of periodontitis patients with lipopolysaccharide was five times higher than that of the subjects with intact periodontal tissues and comprised 13261+/-1251 cpm and 2627+/-638 cpm, respectively . CONCLUSIONS: Our study results show a complex dependence of oxidative function of peripheral polymorphonuclear neutrophils of periodontitis patients upon the nature of stimulants . Therefore further attempts should be made to evaluate its significance in the etiopathogenesis of periodontal tissue diseases of inflammatory origin. Curr Opin Microbiol, 2004 Apr, 7(2), 198 - 202 Robust control in bacterial regulatory circuits; Goulian M; Robust control refers to regulatory systems that are insensitive to perturbations to the intra- or extra-cellular environment . It is generally believed that most cell regulatory circuits should possess some degree of robustness . Examples of robust control and the underlying mechanisms for achieving this robustness are only beginning to be uncovered . Various forms of robustness are associated with circuits based on negative and/or positive feedback, bi-functional enzymes, protein oligomerization and discrete or continuous control. Curr Opin Microbiol, 2004 Apr, 7(2), 163 - 7 Gating the bacterial mechanosensitive channels: MscS a new paradigm? Edwards MD, Booth IR, Miller S. Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock . In the millisecond timescale they must achieve the transition from tightly closed oligomers to large, relatively non-discriminating pores . The crystal structure for MscL, combined with genetic and biochemical analysis, provided the initial insights for the mechanism by which this structural transition might be made . Discovery of the gene for a second class of mechanosensitive channel, MscS, and its subsequent crystallisation, has provided a new paradigm for mechanosensation, enabling a deeper understanding of the mechanisms of sensing membrane tension. Curr Opin Microbiol, 2004 Apr, 7(2), 120 - 5 Mfd, the bacterial transcription repair coupling factor: translocation, repair and termination; Roberts J et al.; Mfd is a widely conserved bacterial protein that couples DNA repair with transcription . Mfd recognizes RNA polymerase stalled at a non-coding template site of DNA damage, disrupts the transcription complex to release the transcript and enzyme, and recruits the DNA excision repair machinery to the site . The mechanism of RNA release has been illuminated by the discovery that Mfd causes forward translocation of RNA polymerase, using an ATP-dependent motor that is highly homologous to that of the Holliday branch migration protein RecG. Curr Opin Microbiol, 2004 Apr, 7(2), 102 - 8 Regulation at complex bacterial promoters: how bacteria use different promoter organizations to produce different regulatory outcomes; Barnard A et al.; Most bacterial promoters are regulated by several signals . This is reflected in the complexity of their organization, with multiple binding sites for different transcription factors . Studies of a small number of complex promoters have revealed different distinct mechanisms that integrate the effects of multiple transcription factors. J Biotechnol, 2004 Apr 8, 109(1-2), 3 - 11 Bacterial expression and refolding of human trypsinogen; Hohenblum H et al.; The expression of recombinant trypsinogens from different mammalian origins in Escherichia coli typically leads to the formation of insoluble aggregates . This work describes the high level expression of human trypsinogen 1 in E . coli using the T7 expression system . Direct expression of trypsinogen was not possible, but the N-terminal fusion of the first 11 amino acids of the T7 protein 10 resulted in an expression level of 200 mg g(-1) bacterial dry mass . A refolding procedure was optimized, and a method using continuous feed of denatured product was developed . Thus the working concentration of trypsinogen could be raised four-fold, while the yield of active protein could be maintained at 20-35% . The refolded trypsinogen was converted to trypsin by autocatalytic activation, and the utility for the detachment of mammalian cells in culture was proven. Adv Drug Deliv Rev, 2004 Apr 19, 56(6), 779 - 94 Rafts and related glycosphingolipid-enriched microdomains in the intestinal epithelium: bacterial targets linked to nutrient absorption; Taieb N et al.; Plasma membrane microdomains such as lipid rafts or caveolae play a major role in host-pathogen interactions . Although this field of research has been extensively studied, two important points have been poorly addressed: (i) the molecular basis of raft-pathogen interactions, and (ii) the effect of such interactions on nutrient absorption . The aim of this review was to propose a biochemical analysis of bacterial adhesion to lipid raft components exposed on the mucosal surface of the intestinal epithelium . A special attention has been given to CH-pi interactions that allow the sugar rings of glycosphingolipids (GSL) to stack against aromatic side chains of bacterial adhesins and toxins . These interactions are controlled by cholesterol molecules intercalated between membrane GSL and/or by the presence of an alpha-OH group in the acyl chain of the ceramide backbone of GSL . In the second part of the review, we analysed the experimental data suggesting the involvement of lipid rafts in the intestinal absorption of nutrients, the mechanisms by which bacteria could impair intestinal functions, and possible therapeutic strategies based on the biochemistry of raft-pathogen interactions. Structure (Camb), 2004 Apr, 12(4), 703 - 15 X-Ray structure determination of three mutants of the bacterial photosynthetic reaction centers from Rb . sphaeroides; altered proton transfer pathways; Xu Q et al.; In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the reduction of a bound quinone molecule Q(B) is coupled with proton uptake . When Asp-L213 is replaced by Asn, proton transfer is inhibited . Proton transfer was restored by two second-site revertant mutations, Arg-M233-->Cys and Arg-H177-->His . Kinetic effects of Cd(2+) on proton transfer showed that the entry point in revertant RCs to be the same as in the native RC . The structures of the parental and two revertant RCs were determined at resolutions of 2.10, 1.80, and 2.75 A . From the structures, we were able to delineate alternate proton transfer pathways in the revertants . The main changes occur near Glu-H173, which allow it to substitute for the missing Asp-L213 . The electrostatic changes near Glu-H173 cause it to be a good proton donor and acceptor, and the structural changes create a cavity which accommodates water molecules that connect Glu-H173 to other proton transfer components. Infect Control Hosp Epidemiol, 2004 Mar, 25(3), 262 - 4 Ineffectiveness of handwashing with lotion soap to remove nosocomial bacterial pathogens persisting on fingertips: a major link in their intrahospital spread; Bottone EJ et al.; The effectiveness of five 30-second handwashes with a non-antiseptic lotion soap to remove nosocomial pathogens (10(8) CFU) applied to fingertips was studied . CFU for all species dropped rapidly after the first handwash; persistence (10 to 15 CFU) was maintained thereafter . Wiping hands with an antiseptic (70% isopropyl or 10% povidone-iodine) sponge removed persisters. Clin Chem Lab Med, 2004 Feb, 42(2), 192 - 7 Immunoglobulin G Fc(gamma) receptor expression on polymorphonuclear cells in bronchoalveolar lavage fluid of HIV-infected and HIV-seronegative patients with bacterial pneumonia; Armbruster C et al.; This study was designed to test the hypothesis that impaired neutrophil function might contribute to the development of bacterial pneumonia in patients with HIV-infection . Numbers of inflammatory cells and immunoglobulin G Fcgamma receptor (IgG FcgammaR) I, II, III levels were investigated in bronchoalveolar lavage (BAL) fluid of HIV-seronegative and HIV-infected patients with bacterial pneumonia . The 99 patients were classified into three groups: I: HIV-seronegative and pneumonia (n = 40); II: HIV-infected and pneumonia (n = 19); III: HIV-seronegative with other pulmonary diseases than pneumonia (n = 40) . The results of groups I and II, II and III, and I and III were compared . The percentage of alveolar macrophages was significantly lower (group II vs . III: p = 0.005, group I vs . III: p = 0.001), that of neutrophils increased significantly in patients with pneumonia (group II vs . III: p = 0.02, group I vs . III: p = 0.01) . Lymphocytes differed only between groups I and III (p = 0.04) . Although only the expression of FcgammaRI was significantly higher in HIV-seronegative pneumonia patients compared to those without pneumonia (p = 0.01), the mean expression of all three receptors was lower in the HIV-infected group, with that of FcgammaRI approaching statistical significance . This report provides first evidence that altered FcgammaR expression on BAL neutrophils might contribute to the increased susceptibility of HIV-infected patients to bacterial pneumonia. Genome, 2004 Apr, 47(2), 361 - 72 Soybean bacterial artificial chromosome contigs anchored with RFLPs: insights into genome duplication and gene clustering; Mudge J et al.; Surveying the soybean genome with 683 bacterial artificial chromosome (BAC) contiguous groups (contigs) anchored by restriction fragment length polymorphisms (RFLPs) enabled us to explore microsyntenic relationships among duplicated regions and also to examine the physical organization of hypomethylated (and presumably gene-rich) genomic regions . Numerous cases where nonhomologous RFLPs hybridized to common BAC clones indicated that RFLPs were physically clustered in soybean, apparently in less than 25% of the genome . By extension, we speculate that most of the genes are clustered in less than 275 M of the soybean genome . Approximately 40%-45% of this gene-rich portion is associated with the RFLP-anchored contigs described in this study . Similarities in genome organization among BAC contigs from duplicate genomic regions were also examined . Homoeologous BAC contigs often exhibited extensive microsynteny . Furthermore, paralogs recovered from duplicate contigs shared 86%-100% sequence identity. Genome, 2004 Apr, 47(2), 239 - 45 Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi); Qian Y et al.; The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species . We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey (Ateles geoffroyi) . A total of 193 152 BAC clones were generated in this library . The average insert size of the BAC clones was estimated to be 184.6 kb with the small inserts (50-100 kb) accounting for less than 3% and the non-recombinant clones only 1.2% . Assuming a similar genome size with humans, the spider monkey BAC library has about 11x genome coverage . In addition, by end sequencing of randomly selected BAC clones, we generated 367 sequence tags for the library . When blasted against human genome, they showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the library . This black-handed spider monkey BAC library would serve as a valuable resource in comparative genomic study and large-scale genome sequencing of nonhuman primates. J Org Chem, 2004 Mar 19, 69(6), 2137 - 46 Synthesis of a fragment of bacterial cell wall; Hesek D et al.; Cell wall is indispensable for survival of bacteria . This large molecular "mesh" encases the entire cytoplasm of bacteria, and it is comprised of repeating backbone units of N-acetyl-glucosamine (NAG)-N-acetyl-muramic acid (NAM) . A pentapeptide is attached to each of the lactyl units of the N-acetyl-muramic acid . The cell wall has both cross-linked and non-cross-linked components . In the present paper, we have devised a synthetic route for the preparation of a fragment of the cell wall comprised of a tetrasaccharide (NAG-NAM-NAG-NAM), along with the two appended peptides . We also report the syntheses of three glycosyl donors (compounds 5, 7, and 9) and three glycosyl acceptors (compounds 4, 6, and 8) based on the d-glucosamine structure as a building unit . The synthetic strategy that is disclosed is generally useful in construction of other natural products containing the d-glucosamine as a building block. Proc R Soc Lond B Biol Sci, 2004 Jan 22, 271(1535), 113 - 22 An ecological perspective on bacterial biodiversity; Horner-Devine MC et al.; Bacteria may be one of the most abundant and species-rich groups of organisms, and they mediate many critical ecosystem processes . Despite the ecological importance of bacteria, past practical and theoretical constraints have limited our ability to document patterns of bacterial diversity and to understand the processes that determine these patterns . However, recent advances in molecular techniques that allow more thorough detection of bacteria in nature have made it possible to examine such patterns and processes . Here, we review recent studies of the distribution of free-living bacterial diversity and compare our current understanding with what is known about patterns in plant and animal diversity . From these recent studies a preliminary picture is emerging: bacterial diversity may exhibit regular patterns, and in some cases these patterns may be qualitatively similar to those observed for plants and animals. Gastroenterology, 2004 Apr, 126(4), 1054 - 70 Mechanisms of cross hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells; Otte JM et al.; BACKGROUND & AIMS: Despite the ability to participate in immune responses and the continuous presence of bacteria and bacterial products, functional responses of intestinal epithelial cells (IEC) seem to be muted . Previously, tolerance to Toll-like receptors (TLRs) ligands has been described in monocytic cells . However, mechanisms in the intestine are unknown . METHODS: The effect of purified lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on expression and function of TLRs in intestinal epithelial cells (Colo205, SW480, T84) was assessed by Northern and Western blot and FACS analysis, kinase activity assays, immunohistochemistry, and ELISA . RESULTS: Expression of TLRs except 10 was detected in primary IEC and TLR1-10 in the cultured cells . Short-term stimulation with LPS or LTA activated proinflammatory signaling cascades in IEC, including phosphorylation of IRAK and MAP kinases and increased IL-8 secretion, whereas prolonged incubation resulted in a state of hyporesponsiveness with no reactivation of the cells by a second challenge with either substance detected . The cells remained responsive to tumor necrosis factor (TNF) . Hyporesponsive cells showed no alteration in expression of TLR or signaling molecules but revealed a decrease in TLR surface expression and IRAK activity . Toll-interacting protein (Tollip) mRNA and protein expression were increased in hyporesponsive cells, and overexpression of Tollip in IEC resulted in a significantly decreased proinflammatory response . CONCLUSIONS: Continuous presence of specific bacterial components results in a status of hyporesponsiveness in otherwise reactive IEC . Down-regulation of TLR surface expression and up-regulation of inhibitory Tollip with decreased phosphorylation of IRAK might all contribute to this hyporesponsiveness. Curr Microbiol, 2004 Apr, 48(4), 285 - 90 Factors affecting interpretation of restriction fragment length polymorphism (RFLP) patterns from PCR-amplified bacterial 16S rRNA genes: operon number and primer mismatching; Ramirez-Moreno S et al.; PCR methods have been shown to be biased by several factors . In the present study, we have developed a theoretic and practical approximation to elucidate how the presence of mismatches at the primers annealing regions and the different number of rDNA operons per cell can influence PCR and subsequent restriction fragment length polymorphism (RFLP) analyses from bacterial populations . We have performed RFLP analyses of 16S rRNA genes amplified by PCR from mixed bacterial cultures showing different primer identities and number of rDNA operons . Our results clearly corroborate that both factors, number of rDNA operons and primers identity, clearly influence the 16S rDNA-RFLP estimations . It has been demonstrated that a higher number of operons leads to a higher efficiency of detection, but a lower degree of primer complementarity implies a decrease in such efficiency. J Am Chem Soc, 2004 Apr 7, 126(13), 4132 - 44 A unified description of the electrochemical, charge distribution, and spectroscopic properties of the special-pair radical cation in bacterial photosynthesis; Reimers JR et al.; We apply our four-state 70-vibration vibronic-coupling model for the properties of the photosynthetic special-pair radical cation to: (1) interpret the observed correlations between the midpoint potential and the distribution of spin density between the two bacteriochlorophylls for 30 mutants of Rhodobacter sphaeroides, (2) interpret the observed average intervalence hole-transfer absorption energies as a function of spin density for six mutants, and (3) simulate the recently obtained intervalence electroabsorption Stark spectrum of the wild-type reaction center . While three new parameters describing the location of the sites of mutation with respect to the special pair are required to describe the midpoint-potential data, a priori predictions are made for the transition energies and the Stark spectrum . In general, excellent predictions are made of the observed quantities, with deviations being typically of the order of twice the experimental uncertainties . A unified description of many chemical and spectroscopic properties of the bacterial reaction center is thus provided . Central to the analysis is the assumption that the perturbations made to the reaction center, either via mutations of protein residues or by application of an external electric field, act only to independently modify the oxidation potentials of the two halves of the special pair and hence the redox asymmetry E0 . While this appears to be a good approximation, clear evidence is presented that effects of mutation can be more extensive than what is allowed for . A thorough set of analytical equations describing the observed properties is obtained using the Born-Oppenheimer adiabatic approximation . These equations are generally appropriate for intervalence charge-transfer problems and include, for the first time, full treatment of both symmetric and antisymmetric vibrational motions . The limits of validity of the adiabatic approach to the full nonadiabatic problem are obtained. Trends Genet, 2004 Mar, 20(3), 126 - 31 Global analysis of bacterial transcription factors to predict cellular target processes; Doerks T et al.; Whole-genome sequences are now available for >100 bacterial species, giving unprecedented power to comparative genomics approaches . We have applied genome-context methods to predict target processes that are regulated by transcription factors (TFs) . Of 128 orthologous groups of proteins annotated as TFs, to date, 36 are functionally uncharacterized; in our analysis we predict a probable cellular target process or biochemical pathway for half of these functionally uncharacterized TFs. Otolaryngol Pol, 2003, 57(6), 829 - 33 {Efficacy of clarithromycin (Fromilid) in treatment of uncomplicated acute bacterial rhinosinusitis in children}; Hassmann-Poznanska E et al.; The results of treatment of acute rhinosinusitis in children are presented . The study was based on retrospective analysis of data of 34 children treated with clarithromycin (Fromilid) . The clinical efficacy of this drug was confirmed by the study. Shanghai Kou Qiang Yi Xue, 1999 Mar, 8(1), 27 - 9 {Primary research of development of a mouse model for non-bacterial sialoadenitis}; Yu CQ et al.; OBJECTIVE: To develope a mouse model for non-bacterial sialoadenitis similar to SS.The model of organ-specific to the submandibular gland of mouse has developed for this study . METHODS: Several of the mouse strains were used to the development of non-bacterial sialoadenitis.The injection of syngeneic salivary gland tissue with CFA induced inflammation in submandibular gland in mice.Not all mice had a predictable response . RESULTS: C(57) mice (500microg/ml and 750microg/ml group )appear histologically to be more prove to develop sialoadenitis . CONCLUSION: An animal model of non-bacterial sialoadenitis had been set up,the non-bacterial sialoadentitis may be similar to the autoimmune process in human SS. Ann Biol Clin (Paris), 2004 Jan-Feb, 62(1), 7 - 14 {ATP-dependant proteolysis and bacterial pathogenesis}; Gaillot O; Proteolysis plays an central role in key metabolic pathways and cellular adaptation to environmental changes . It modulates the activity of regulatory peptides and eliminates misfolded or damaged proteins such as those generated by stress exposure . In eucaryotic cells ATP- dependent proteolysis is carried out by the 26S proteasome whose substrates are identified by ubiquitin tags . Conversely, bacteria possess several tagging systems and different ATP- dependent proteases . Bacterial ATP-dependent proteases carry distinct chaperone-ATPase and peptidase activities, either on the same molecule or on separate subunits . Although unrelated, all ATP-dependent proteases function according to a similar multistep scheme, from the docking of a substrate by the ATPase region to its proteolysis by the peptidase . Major bacterial ATP- dependent proteases include FtsH, Lon, HslUV and the Clp proteases . Clp proteases are multimeric complexes assembled into a structure centered on the proteolytic component ClpP . They are essential for quick adaptation to stress and regulate important developmental processes . Clp-mediated proteolysis is also required for disease progression and virulence of several bacterial pathogens, favoring survival in the host or modulating the activity of genuine virulence factors . Copyright John Libbey Eurotext 20003. Environ Sci Technol, 2004 Mar 1, 38(5), 1359 - 67 Stable isotope pulse-chasing and compound specific stable carbon isotope analysis of phospholipid fatty acids to assess methane oxidizing bacterial populations in landfill cover soils; Crossman ZM et al.; The oxidation of methane by bacteria residing in soils constitutes an important terrestrial methane sink . These bacteria are particularly abundant in the covering soils of landfill caps due to the supply of high concentrations of methane from the landfill below . Only about 0.1% of soil bacteria are amenable to available methods of culturing, resulting in the need for a method of in situ analysis . A combination of phospholipid fatty acid (PLFA) analysis and stable isotopic labeling has been employed in this investigation as a means of cultivation-independent bacterial analysis . Soil samples taken from the profiles of two landfill caps, one of clay and one of sand, were incubated with 13C-labeled methane . PLFAs were analyzed by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in order to determine their 13C content, from which the PLFA distribution of the methane-oxidizing bacteria was calculated . Neither landfill cap supported communities of bacteria capable of oxidizing ambient levels of methane but only those elevated levels that are usually attributable to landfills . The clay-capped landfill profile exhibited a change in the methane-oxidizing bacterial community with depth, whereas the sand-capped landfill site displayed a mixture of both type I and II methanotrophs throughout the profile . Two additional samples, taken from sites where methane production was evident, were particularly dominated by type II methanotrophic bacteria. Syst Appl Microbiol, 2004 Mar, 27(2), 175 - 85 Use of the genomic signature in bacterial classification and identification; Coenye T et al.; In this study we investigated the correlation between dinucleotide relative abundance values (the genomic signature) obtained from bacterial whole-genome sequences and two parameters widely used for bacterial classification, 16S rDNA sequence similarity and DNA-DNA hybridisation values . Twenty-eight completely sequenced bacterial genomes were included in the study . The correlation between the genomic signature and DNA-DNA hybridisation values was high and taxa that showed less than 30% DNA-DNA binding will in general not have dinucleotide relative abundance dissimilarity (delta*) values below 40 . On the other hand, taxa showing more than 50% DNA-DNA binding will not have delta* values higher than 17 . Our data indicate that the overall correlation between genomic signature and 16S rDNA sequence similarity is low, except for closely related organisms (16S rDNA similarity >94%) . Statistical analysis of delta* values between different subgroups of the Proteobacteria indicate that the beta- and gamma-Proteobacteria are more closely related to each other than to the other subgroups of the Proteobacteria and that the alpha- and epsilon-Proteobacteria form clearly separate subgroups . Using the genomic signature we have also predicted DNA-DNA binding values for fastidious or unculturable endosymbionts belonging to the genera Rickettsia, Wigglesworthia and Buchnera. Acta Paediatr, 2004 Feb, 93(2), 216 - 9 Procalcitonin serum levels in perinatal bacterial and fungal infection of preterm infants; Distefano G et al.; AIM: To determine reference values for procalcitonin (PCT) and C-reactive protein (CRP) for gestational age and to use these parameters as diagnostic markers of perinatal bacterial and fungal infection . METHODS: PCT and CRP serum levels were measured in a case-control study in a group of 35 low birthweight infants (< 34 wk of gestation) . 27 babies (77%) had clinical signs of infection confirmed by positive blood cultures and were compared to 8 (23%) uninfected matched patients . Seventeen (63%) of them had bacterial infection and 10 (37%) had fungal infection (Candida) . Serum PCT (Brahms Diagnostika) and CRP (Immunoassay Vitros 950) were measured serially at 3, 7 and 10d of life . RESULTS: At any time, PCT and CRP levels were significantly higher in neonates with perinatal infection (p < 0.05) (> 0.7 ng ml(-1) and > 1 mg dl(-1) respectively) . PCT showed a more rapid response to infection (9.3 +/- 1.5 ng ml(-1)) . especially to bacterial infection (10.8 +/- 1.4 ng ml(-1)), than CRP (1.5 +/- 0.5 mg dl(-1)) (sensitivity 99% vs 88%) . Lower sensitivity was noted for both parameters . PCT and CRP, to follow babies with fungal infection (6.7 +/- 0.8 ng ml(-1) and 0.9 +/- 0.7 mg dl(-1), respectively) (sensitivity 77% vs 58%) . CONCLUSION: This study gives PCT reference values in preterm babies with perinatal infection . In these babies, PCT seems to be more sensitive than CRP as a diagnostic marker of infection . Both parameters can be used alone or in combination for a better identification and follow-up of bacterial and fungal infection during the perinatal period. Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 4770 - 5 Epub 2004 Mar 24. Apoptosis or growth arrest: Modulation of tumor suppressor p53's specificity by bacterial redox protein azurin; Yamada T et al.; The tumor suppressor protein p53 is known to induce either apoptosis or growth arrest depending on cellular background . We have previously reported that a bacterial redox protein azurin induces apoptosis in J774 cell line-derived macrophages whereas a site-directed mutant M44KM64E azurin shows very little cytotoxicity and fails to induce apoptosis in J774 cells . We now report that purified M44KM64E mutant azurin protein can enter both J774 cells as well as the human breast cancer MCF-7 cells . Entry of M44KM64E mutant azurin in J774 cells causes strong inhibition of cell-cycle progression at the G1 to S phase and a higher level of transcription of the p21 gene . Corresponding to high p21 levels, the levels of cyclins and cyclin-dependent kinases were greatly lowered in M44KM64E mutant azurin-treated J774 cells . Interestingly, M44KM64E mutant azurin protein failed to elicit inhibition of cell-cycle progression in MCF-7 cells, presumably because of mutation at the retinoblastoma tumor suppressor protein that allows functional E2F formation in MCF-7 cells even in the presence of high intracellular p21 level . Thus, the WT azurin induces apoptosis but little inhibition of cell-cycle progression whereas the M44KM64E mutant azurin is deficient in the induction of apoptosis but mediates strong inhibition of cell-cycle progression, demonstrating the role of a single bacterial protein and its hydrophobic patch in modulating two important functions of p53. FEMS Microbiol Lett, 2004 Apr 1, 233(1), 115 - 23 Similarity of bacterial communities in sawdust- and straw-amended cow manure composts; Green SJ et al.; We analyzed bacterial communities in two cow manure composts derived from the same feed manure and composted in the same location, but composted with different carbon amendments, and in peat-based potting mixes amended with these composts . Bacterial communities were characterized by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of extracted DNAs, and population fingerprints generated for each sample were compared . Sequence analyses of dominant DGGE bands revealed that members of the phylum Bacteroidetes were the most dominant bacteria detected in this study (19 of 31 clones) . These analyses demonstrate that bacterial community profiles of individual composts were highly similar, as were profiles of compost-amended potting mixes . However, potting mix profiles differed substantially from the original compost profiles and from that of the peat base . These data indicate that highly similar bacterial populations were active in the two composts, and suggest that the effects of the initial carbon amendment on the mature compost bacterial communities were minor, while factors such as the feed manure and composting location may have been more influential. Curr Biol, 2004 Mar 23, 14(6), R242 - 4 Bacterial shape: concave coiled coils curve caulobacter; Margolin W; Bacterial cells exhibit a wide variety of shapes . Recent results indicate that the characteristic crescent shape of Caulobacter crescentus depends upon an inter-mediate filament-like protein that localizes to the concave side of the cell. Nature, 2004 Mar 25, 428(6981), 437 - 41 Functional interactions between receptors in bacterial chemotaxis; Sourjik V et al.; Bacterial chemotaxis is a model system for signal transduction, noted for its relative simplicity, high sensitivity, wide dynamic range and robustness . Changes in ligand concentrations are sensed by a protein assembly consisting of transmembrane receptors, a coupling protein (CheW) and a histidine kinase (CheA) . In Escherichia coli, these components are organized at the cell poles in tight clusters that contain several thousand copies of each protein . Here we studied the effects of variation in the composition of clusters on the activity of the kinase and its sensitivity to attractant stimuli, monitoring responses in vivo using fluorescence resonance energy transfer . Our results indicate that assemblies of bacterial chemoreceptors work in a highly cooperative manner, mimicking the behaviour of allosteric proteins . Conditions that favour steep responses to attractants in mutants with homogeneous receptor populations also enhance the sensitivity of the response in wild-type cells . This is consistent with a number of models that assume long-range cooperative interactions between receptors as a general mechanism for signal integration and amplification. J Virol Methods, 2004 May, 117(2), 169 - 77 Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus: bacterial expression and refolding; Das D et al.; Single chain antibodies (ScFvs) are heavy and light chain variable domains connected by an artificial linker . Because of their smaller size, ScFvs show improved tissue penetration in vivo and reduced immunogenicity, making them ideal for therapeutic applications . We have cloned a ScFv against western equine encephalitis (WEE) using rDNA technology . The ScFv was generated from a hybridoma cell line (11D2) specific to the WEE virus E1 glycoprotein and is arranged in the V(L)-V(H) orientation with a (gly(4)ser)(3) linker . This ScFv was engineered successfully with a biotin mimic tag (11 amino acid peptide) and cloned in the pET22b+ expression vector . The ScFv was expressed as a approximately 32kDa protein in Escherichia coli as inclusion bodies, with an estimated yield of 20-40 mg/l . Different refolding protocols were used to solubilise the inclusion bodies . Most of the functional ScFv was generated when the inclusion bodies were solubilized in a detergent, air oxidised in the presence of CuSO(4) and then denatured in urea buffer in comparison to other protocols . The product was renatured finally in Tris arginine buffer (pH 8.0) . Refolded protein was dialysed against phosphate buffer saline (PBS) (pH 7.3) to remove the Tris and arginine . Our refolding protocol generated up to a 50% yield of soluble protein, which retained antigen-binding activity with whole inactivated WEE virus as demonstrated by ELISA and Western blot analysis . This 11D2-biotin mimic ScFv complexed with streptavidin horseradish peroxidase (St-HRPO) will be useful as a detector reagent in the ultrasensitive ELISA detection of WEE virus antigen. J Autoimmun, 2004 May, 22(3), 217 - 25 A peptide that shares similarity with bacterial antigens reverses thrombogenic properties of antiphospholipid antibodies in vivo; Pierangeli SS et al.; OBJECTIVE: The factors causing production of antiphospholipid (aPL) antibodies remain unidentified . Recently, studies have shown that aPL and anti-beta2Glycoprotein I (anti-beta2GPI) antibodies with pathogenic properties can be generated with peptides from bacterial and viral origin, that mimic regions of beta2GPI . These data suggest a molecular mimicry between bacterial/viral antigens and self-proteins . In this study we examined the ability of a synthetic peptide (named peptide A, NTLKTPRVGGC) that shares similarity with common bacterial antigens, to reverse aPL-mediated thrombosis in mice in vivo . Peptide A is also found in region I/II of beta2GPI . A scrambled form of peptide A (named scA, GTKGCPNVRLT) was used as a control . METHODS AND RESULTS: Sera from 29 patients with APS bound to peptide A but not to peptide scA by ELISA in a dose-dependent fashion . Cardiolipin (CL) liposomes inhibited the binding of IgG-APS by ELISA to peptide A by 35% and to CL by 56% . The inhibition of binding to cardiolipin and to peptide A was enhanced by addition of beta2GPI to the liposomes . CL/peptide A liposomes but not peptide A alone inhibited the binding of IgG-APS to peptide A . beta2GPI alone did not inhibit binding of IgG-APS to peptide A, to beta2GPI or to CL . For the in vivo experiments, CD1 mice in groups of 20 were injected with affinity purified aPL antibodies or with control IgG-NHS twice intraperitoneally . Seventy hours after the first injection, and 30 min before the surgical procedure (induction of experimental thrombus) mice were infused i.v . in each group with either peptide A or with peptide scA . The femoral vein of the anesthetized mice were dissected to examine the dynamics of an induced thrombus in treated and control mice . The mean aCL titer of mice injected with aPL was 60 GPL units . Mice treated with aPL and infused with peptide scA produced significantly larger thrombi when compared to mice treated with IgG-NHS and peptide scA (2466+/-462 microm2 vs 772.5+/-626.4 microm2) . Treatment with peptide A significantly decreased thrombus size in mice injected with aPL antibodies (1063+/-890 microm2 compared to 2466+/-462 microm2) . CONCLUSION: The data indicates that a synthetic peptide that shares similarity with common bacterial antigens and with regions of beta2GPI is capable to inhibit thrombogenic properties of aPL in mice . This may have important implications in designing new modalities of prevention and/or treatment of thrombosis in APS. J Biol Chem, 2004 Jun 18, 279(25), 25959 - 65 Epub 2004 Mar 22. Ruthenium red-induced bundling of bacterial cell division protein, FtsZ; Santra MK et al.; The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process . Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro . In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly . Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 microm ruthenium red, respectively . In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers . Electron microscopic analysis showed that 4-10 microm of ruthenium red produced thick bundles of FtsZ polymers . The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ . Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles . Calcium inhibited the binding of ruthenium red to FtsZ . However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly . Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria. J Theor Biol, 2004 Apr 21, 227(4), 547 - 59 Bacterial shape maintenance: an evaluation of various models; Grover NB et al.; In this article, we examine a large number of combinations of growth models, with separate attention to cell volume, cylindrical surface-area, polar caps, nascent poles, onset of constriction, precision of cell division and interdivision-time dispersion, for Escherichia coli cells growing in steady state at various doubling times . Our main conclusion is striking, and quite general: exponential cylindrical surface-area growth is not possible, irrespective of the behaviour of cell volume, the polar regions, the nascent poles, or any other feature of cell growth-such cells never reach steady state . The same is true of linear cylindrical surface-area growth, regardless of when during the cell cycle the doubling in growth rate takes place . Only after the introduction of feedback into the surface-area growth law, do the cultures attain steady state, all of them . The other components of the models contribute only marginally to the properties of the steady state . Thus, whether the feedback applies just to the cylindrical portion of the cell or to its entire surface area affects only the coefficient of variation of cell radius and the radius-volume correlation . The dynamics of old-pole maintenance, constant area or constant shape, influences the radius-length and radius-volume correlations and, to a much lesser extent, the coefficients of variation of cell radius and length; how the nascent poles grow, whether linearly or exponentially, does not seem to matter at all . The absolute dimensions of the cells are set by the growth rate of the culture and have almost no effect when the feedback is taken to apply to the entire cell surface area; when it is limited to the cylindrical portion of the cell, however, both radius-length and radius-volume correlations increase with increasing doubling time . Comparison with published values was inconclusive . The nature of cell surface-area growth has therefore been settled, but whether the volume increases by simple-exponential or by pseudo-exponential growth, or whether the old poles maintain a constant shape or a constant area during the cell cycle, can be determined only with more precise experimental data . The form of nascent-pole growth is not resolvable by present techniques. J Biol Chem, 2004 May 28, 279(22), 23830 - 6 Epub 2004 Mar 22. Substrate-induced conformational change in bacterial complex I; Mamedova AA et al.; The mechanism coupling electron transfer and proton pumping in respiratory complex I (NADH-ubiquinone oxidoreductase) has not been established, but it has been suggested that it involves conformational changes . Here, the influence of substrates on the conformation of purified complex I from Escherichia coli was studied by cross-linking and electron microscopy . When a zero-length cross-linking reagent was used, the presence of NAD(P)H, in contrast to that of NAD+, prevented the formation of cross-links between the hydrophilic subunits of the complex, including NuoB, NuoI, and NuoCD . Comparisons using different cross-linkers suggested that NuoB, which is likely to coordinate the key iron-sulfur cluster N2, is the most mobile subunit . The presence of NAD(P)H led also to enhanced proteolysis of subunit NuoG . These data indicate that upon NAD(P)H binding, the peripheral arm of the complex adopts a more open conformation, with increased distances between subunits . Single particle analysis showed the nature of this conformational change . The enzyme retains its L-shape in the presence of NADH, but exhibits a significantly more open or expanded structure both in the peripheral arm and, unexpectedly, in the membrane domain also. Fertil Steril, 2004 Mar, 81(3), 662 - 9 Possible role of bacterial and viral infections in miscarriages; Matovina M et al.; OBJECTIVE: To determine the role of infections in miscarriages . Chorionic villi from aborted material were subjected to cytogenetic evaluation and analyzed for the presence of Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, human cytomegalovirus (HCMV), adeno-associated virus (AAV), and human papillomaviruses (HPV) . DESIGN: Retrospective study . SETTING: University hospital and academic research institution . MAIN OUTCOME MEASURE(S): Karyotyping and detection of bacterial and viral DNA by means of polymerase chain reaction (PCR) in placenta specimens . RESULT(S): In 54 (50%) of 108 samples the karyotype was normal, in 38 (35%) samples it was abnormal, and in 16 (15%) samples karyotype was undetermined . No U . urealyticum, M . hominis, HCMV, or AAV-2 DNA was detected, while C . trachomatis DNA was detected in one (1%) and HPV DNA in eight (7%) samples . No significant correlation of HPV-positive findings with karyotype status was established . CONCLUSION(S): Our findings do not support a role of C . trachomatis, U . urealyticum, M . hominis, HCMV, or AAV infections in miscarriages during the first trimester of pregnancy . However, further investigation should be made to determine a possible involvement of HPVs in the development of genetic abnormalities of the fetus and in miscarriages. Int Rev Cytol, 2004, 233, 93 - 134 The bacterial flagellar motor: structure and function of a complex molecular machine; Kojima S et al.; The bacterial flagellar motor harnesses ion flow to drive rotary motion, at speeds reaching 100000 rpm and with apparently tight coupling . The functional properties of the motor are quite well understood, but its molecular mechanism remains unknown . Studies of motor physiology, together with mutational and biochemical studies of the components, place significant constraints on the mechanism . Rotation is probably driven by conformational changes in membrane-protein complexes that form the stator . These conformational changes occur as protons move on and off a critical aspartate residue in the stator protein MotB, and the resulting forces are applied to the rotor protein FliG . The bacterial flagellum is a complex structure built from about two dozen proteins . Its construction requires an apparatus at the base that exports many flagellar components to their sites of installation by way of an axial channel through the structure . The sequence of events in assembly is understood in general terms, but not yet at the molecular level . A fuller understanding of motor rotation and flagellar assembly will require more data on the structures and organization of the constituent proteins. Toxicon, 2004 Jan, 43(1), 43 - 51 Bacterial expression, purification and functional characterization of a recombinant chimeric Fab derived from murine mAb BCF2 that neutralizes the venom of the scorpion Centruroides noxius hoffmann; Selisko B et al.; The murine monoclonal antibody BCF2 is able to neutralize the venom of the scorpion Centruroides noxius Hoffmann . A chimeric Fab of BCF2 (chFab-BCF2) comprising the variable regions of murine BCF2 and human constant regions was assembled . chFab-BCF2 was expressed as a soluble and functional protein in the periplasmic space of Escherichia coli . An expression yield of 1 mg/l was reached by combination of late-log-phase induction, rich culture medium, low expression temperature and addition of sucrose (0.3 M) to the culture medium . The addition of sucrose induced secretion of 60% of the protein into the medium . After expression for 23 h, a novel process was used to release the remaining periplasmic protein in situ consisting in the addition of lysozyme and sucrose up to 0.6 M (20%) directly to the culture medium . chFab-BCF2 was recovered by ammonium sulfate precipitation and purified in a single step by affinity chromatography using anti-human anti-F(ab')(2) IgG coupled to Sepharose-proteinG . Pure chFab-BCF2 maintained a similar nanomolar affinity as BCF2 to its cognate antigen, the Na(+)-channel-affecting toxin Cn2 . Recombinant chFab-BCF2 was able to neutralize Cn2 in vivo even at a molar ratio of 1:1, as well as the whole venom of C . noxius . Thus, it is a promising candidate to be used as a specific and efficient recombinant antidote against scorpion stings. BMC Microbiol . 2004 Mar 22;4(1):12. Library on a slide for bacterial comparative genomics; Zhang L et al.; BACKGROUND: We describe a novel application of microarray technology for comparative genomics of bacteria in which libraries of entire genomes rather than the sequence of a single genome or sets of genes are arrayed on the slide and then probed for the presence or absence of specific genes and/or gene alleles . RESULTS: We first adopted a 96-well high throughput working protocol to efficiently isolate high quality genomic DNA . We then optimized conditions to print genomic DNA onto a glass slide with high density (up to 15000 spots) and to sensitively detect gene targets in each genome spot using fluorescently labeled DNA probe . Finally, we created an E . coli reference collection array and probed it for the presence or absence of the hemolysin (hly) gene using a dual channel non-competing hybridization strategy . Results from the array hybridization matched perfectly with previous tests . CONCLUSIONS: This new form of microarray technology, Library on a Slide, is an efficient way for sharing and utilizing large strain collections in comparative genomic analyses. Biochemistry, 2004 Mar 30, 43(12), 3318 - 26 Absence of large-scale displacement of quinone QB in bacterial photosynthetic reaction centers; Breton J; Photosynthesis transforms light into chemical energy by coupling electron transfer to proton uptake at the quinone Q(B) . The possibility of initiating this process with a brief pulse of light and the known X-ray structure makes the photosynthetic bacterial reaction center a paradigm for studying coupled electron-proton transfer in biology . It has been established that electron transfer from the primary quinone Q(A) to Q(B) is gated by a protein conformational change . On the basis of a dramatic difference in the location of Q(B) in structures derived from crystals cooled to 90 K either under illumination or in the dark, a functional model for the gating mechanism was proposed whereby neutral Q(B) moves 4.5 A before receiving the electron from Q(A)(-) {Stowell, M . H . B., McPhillips, T . M., Rees, D . C., Soltis, S . M., Abresch, E., and Feher, G . (1997) Science 276, 812-816} . Isotope-edited FTIR difference spectroscopy of Q(B) photoreduction at 290 and 85 K is used to investigate whether Q(B) moves upon reduction . We show that the specific interactions of the carbonyl groups of Q(B) and Q(B)(-) with the protein at a single binding site remain identical at both temperatures . Therefore, the different locations of Q(B) reported in many X-ray crystal structures probably are unrelated to functional electron transfer from Q(A)(-) to Q(B). Nat Rev Microbiol, 2003 Nov, 1(2), 137 - 49 The versatile bacterial type IV secretion systems; Cascales E et al.; Bacteria use type IV secretion systems for two fundamental objectives related to pathogenesis--genetic exchange and the delivery of effector molecules to eukaryotic target cells . Whereas gene acquisition is an important adaptive mechanism that enables pathogens to cope with a changing environment during invasion of the host, interactions between effector and host molecules can suppress defence mechanisms, facilitate intracellular growth and even induce the synthesis of nutrients that are beneficial to bacterial colonization . Rapid progress has been made towards defining the structures and functions of type IV secretion machines, identifying the effector molecules, and elucidating the mechanisms by which the translocated effectors subvert eukaryotic cellular processes during infection. Nat Rev Microbiol, 2004 Jan, 2(1), 57 - 65 The regulation of bacterial transcription initiation; Browning DF et al.; Bacteria use their genetic material with great effectiveness to make the right products in the correct amounts at the appropriate time . Studying bacterial transcription initiation in Escherichia coli has served as a model for understanding transcriptional control throughout all kingdoms of life . Every step in the pathway between gene and function is exploited to exercise this control, but for reasons of economy, it is plain that the key step to regulate is the initiation of RNA-transcript formation. Genes Chromosomes Cancer, 2004 May, 40(1), 60 - 5 Delineation of a minimal region of deletion at 6q16.3 in follicular lymphoma and construction of a bacterial artificial chromosome contig spanning a 6-megabase region of 6q16-q21; Henderson LJ et al.; Regional deletions of 6q are frequent karyotypic alterations in malignant lymphoma and are associated with an adverse clinical outcome . One such region of recurrent deletion is 6q16-q21; however, the specific genes affected have not been identified . Our objective in this study was to identify cases with deletion of 6q16-q21 in follicular lymphoma and to define a minimal region of deletion . A physical map of 6q16.2-q21 was constructed using map information from both sequence-based and bacterial artificial chromosome (BAC) fingerprint-based maps . Forty-three BAC clones spanning a 6-Mb region of 6q16.2-q21 were identified and obtained from the RP-11 library . Selected BACs were fluorescence-labeled and hybridized to a series of 34 follicular lymphomas with a regional 6q deletion detected by G banding . Twenty-four cases with deletion of the 6q16.3 region were detected . A minimal deletion of 2.3 Mb was defined . Our study has identified a limited region of deletion of 6q16.3 that may implicate four known genes in follicular lymphoma and possibly in other cancers . A BAC contig spanning a 6-Mb region has been anchored to the 6q16.2-q21 region . This map represents a useful resource for gene identification in this region, not only in lymphoma but also in other neoplasms with 6q alterations . Infez Med, 1997, 5(3), 174 - 7 {Spontaneous bacterial peritonitis in patients with liver cirrhosis . Differential aspects with portal thrombosis}; Russo G et al.; In a series of 155 patients with decompensated liver cirrhosis spontaneous bacterial peritonitis (SBP) was diagnosed in 15 cases (9.7%) and portal thrombosis (PT) in 8 (5.1%) by mean of standard criteria . The main clinical and laboratory characteristics were similar in the two groups of patients; fever was more frequently recorded in the SBP patients . Cytological examination of the ascites showed an increase in total cell count and in neutrophils count higher in SBP group than in PT . The diagnosis of PT was confirmed by ultrasonography . The data suggest that a cytological examination of the ascites should be performed in all patients with decompensated cirrhosis and may contribute to the differential diagnosis of SBP or PT. Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 4805 - 9 Epub 2004 Mar 19. Beyond the diffusion limit: Water flow through the empty bacterial potassium channel; Saparov SM et al.; Water molecules are constrained to move with K+ ions through the narrow part of the Streptomyces lividans K+ channel because of the single-file nature of transport . In the presence of an osmotic gradient, a water molecule requires <10 ps to cross the purified protein reconstituted into planar bilayers . Rinsing K+ out of the channel, water may be 1,000 times faster than the fastest experimentally observed K+ ion and 20 times faster than the one-dimensional bulk diffusion of water . Both the anomalously high water mobility and its inhibition observed at high K+ concentrations are consistent with the view that liquid-vapor oscillations occur because of geometrical confinements of water in the selectivity filter . These oscillations, where the chain of molecules imbedded in the channel (the "liquid") cooperatively exits the channel, leaving behind a near vacuum (the "vapor"), thus far have only been discovered in hydrophobic nanopores by molecular dynamics simulations {Hummer, G., Rasaiah, J . C . & Noworyta, J . P . (2001) Nature 414, 188-190; and Beckstein, O . & Sansom, M . S . P . (2003) Proc . Natl . Acad . Sci . USA 100, 7063-7068}. Gene Ther, 2004 May, 11(10), 856 - 64 Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo; Chen ZY et al.; We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector . This suggested that bacterial DNA sequences played a role in episomal transgene silencing . To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms . In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups . In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups . All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact . In all groups, the relative amount of vector DNA in liver remained similar . Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements. Sex Transm Dis, 2004 Apr, 31(4), 236 - 8 A randomized, double-blind clinical trial of vaginal acidification versus placebo for the treatment of symptomatic bacterial vaginosis; Holley RL et al.; BACKGROUND AND OBJECTIVES: Vaginal acidification has been suggested as a means of normalizing the vaginal flora . GOAL: The purpose of this study was to determine the effectiveness of an acetic acid-based vaginal gel in the treatment of bacterial vaginosis (BV) . STUDY DESIGN: Forty-four patients with BV were enrolled in a randomized, double-blind clinic trial . Of these, 29 were evaluable . Patients were randomized to receive either 5 mL acetic acid gel (n = 14) or placebo gel (n = 15) intravaginally twice daily for 7 days . Clinical criteria and vaginal Gram stain scores were compared between the initial visit and at 2 weeks after initiating therapy . RESULTS: No significant differences were noted when comparing drug and placebo in terms of subjective or clinical improvement or improvement in Gram stain smears for bacterial vaginosis . CONCLUSION: Vaginal acidification with an acetic acid gel formulated to pH 3.9 to 4.1 was ineffective therapy for bacterial vaginosis. J Biol Chem, 2004 May 28, 279(22), 23782 - 9 Epub 2004 Mar 17. The Interactions of Allium sativum leaf agglutinin with a chaperonin group of unique receptor protein isolated from a bacterial endosymbiont of the mustard aphid; Banerjee S et al.; The homopteran sucking insect, Lipaphis erysimi (mustard aphid) causes severe damage to various crops . This pest not only affects plants by sucking on the phloem, but it also transmits single-stranded RNA luteoviruses while feeding, which cause disease and damage in the crop . The mannose-binding Allium sativum (garlic) leaf lectin has been found to be a potent control agent of L . erysimi . The lectin receptor protein isolated from brush border membrane vesicle of insect gut was purified to determine the mechanism of lectin binding to the gut . Purified receptor was identified as an endosymbiotic chaperonin, symbionin, using liquid chromatography-tandem mass spectrometry . Symbionin from endosymbionts of other aphid species have been reported to play a significant role in virus transmission by binding to the read-through domain of the viral coat protein . To understand the molecular interactions of the said lectin and this unique symbionin molecule, the model structures of both molecules were generated using the Modeller program . The interaction was confirmed through docking of the two molecules forming a complex . A surface accessibility test of these molecules demonstrated a significant reduction in the accessibility of the complex molecule compared with that of the free symbionin molecule . This reduction in surface accessibility may have an effect on other molecular interactive processes, including "symbionin virion recognition", which is essential for such symbionin-mediated virus transmission . Thus, garlic leaf lectin provides an important component of a crop management program by controlling, on one hand, aphid attack and on the other hand, symbionin-mediated luteovirus transmission. BMC Biol . 2004 Feb 10;2(1):2. Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site; Booth RE et al.; BACKGROUND: Glutaminyl cyclase (QC) forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins . We previously proposed the mammalian QC has some features in common with zinc aminopeptidases . We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP) and mutated the apparent active site residues to assess their role in QC catalysis . RESULTS: The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide . Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering) and the two glutamates (201 and 202), while the two aspartates (159 and 248) appear to play no catalytic role . ICP-MS analysis shows less than stoichiometric zinc (0.3:1) in the purified enzyme . CONCLUSIONS: We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues . In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity. Mol Phylogenet Evol, 2004 Jan, 30(1), 243 - 50 Decreasing the effects of horizontal gene transfer on bacterial phylogeny: the Escherichia coli case study; Escobar-Paramo P et al.; Phylogenetic reconstructions of bacterial species from DNA sequences are hampered by the existence of horizontal gene transfer . One possible way to overcome the confounding influence of such movement of genes is to identify and remove sequences which are responsible for significant character incongruence when compared to a reference dataset free of horizontal transfer (e.g., multilocus enzyme electrophoresis, restriction fragment length polymorphism, or random amplified polymorphic DNA) using the incongruence length difference (ILD) test of Farris et al . {Cladistics 10 (1995) 315} . As obtaining this "whole genome dataset" prior to the reconstruction of a phylogeny is clearly troublesome, we have tested alternative approaches allowing the release from such reference dataset, designed for a species with modest level of horizontal gene transfer, i.e., Escherichia coli . Eleven different genes available or sequenced in this work were studied in a set of 30 E . coli reference (ECOR) strains . Either using ILD to test incongruence between each gene against the all remaining (in this case 10) genes in order to remove sequences responsible for significant incongruence, or using just a simultaneous analysis without removals, gave robust phylogenies with slight topological differences . The use of the ILD test remains a suitable method for estimating the level of horizontal gene transfer in bacterial species . Supertrees also had suitable properties to extract the phylogeny of strains, because the way they summarize taxonomic congruence clearly limits the impact of individual gene transfers on the global topology . Furthermore, this work allowed a significant improvement of the accuracy of the phylogeny within E . coli. Plant Physiol, 2004 Apr, 134(4), 1317 - 26 Epub 2004 Mar 12. Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization; Gardiner J et al.; Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map . We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters . A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies . From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes . BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate . More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs . The majority of BACs identified (79%) were hybridized with one or two overgos . A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich . Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific . A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development . Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase . The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase . Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored . Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize. Genetics, 2004 Feb, 166(2), 823 - 33 A bacterial genetic screen identifies functional coding sequences of the insect mariner transposable element Famar1 amplified from the genome of the earwig, Forficula auricularia; Barry EG et al.; Transposons of the mariner family are widespread in animal genomes and have apparently infected them by horizontal transfer . Most species carry only old defective copies of particular mariner transposons that have diverged greatly from their active horizontally transferred ancestor, while a few contain young, very similar, and active copies . We report here the use of a whole-genome screen in bacteria to isolate somewhat diverged Famar1 copies from the European earwig, Forficula auricularia, that encode functional transposases . Functional and nonfunctional coding sequences of Famar1 and nonfunctional copies of Ammar1 from the European honey bee, Apis mellifera, were sequenced to examine their molecular evolution . No selection for sequence conservation was detected in any clade of a tree derived from these sequences, not even on branches leading to functional copies . This agrees with the current model for mariner transposon evolution that expects neutral evolution within particular hosts, with selection for function occurring only upon horizontal transfer to a new host . Our results further suggest that mariners are not finely tuned genetic entities and that a greater amount of sequence diversification than had previously been appreciated can occur in functional copies in a single host lineage . Finally, this method of isolating active copies can be used to isolate other novel active transposons without resorting to reconstruction of ancestral sequences. Biosens Bioelectron, 2004 Apr 15, 19(9), 977 - 85 Construction and characterization of novel dual stress-responsive bacterial biosensors; Mitchell RJ et al.; Using the genes for the green fluorescence protein and Xenorhabdus luminescens luciferase operon and the promoters for the recA and katG genes, two stress-responsive Escherichia coli biosensor strains have been constructed that can individually or concurrently respond to oxidative and genotoxic conditions . Strain DUO-1 carries the pRGDK1 plasmid, which has the recA::GFPuv4 and katG::luxCDABE fusion genes oriented divergently with each other, while in DUO-2, i.e., pRGDK2, they are in a tandem orientation, with the recA promoter showing run-though transcription of the katG::luxCDABE fusion . These two strains and their responses were characterized using several known hydroxyl radical-forming chemicals, e.g., hydrogen peroxide and cadmium chloride, along with some genotoxins, e.g., mitomycin C and methyl-N-nitro-N-nitrosoguanidine, and some general toxicants . Both strains showed an induction of green fluorescent protein (GFP) and bioluminescence when they experienced DNA and oxidative damage, respectively, while the tandem orientation of the two fusion genes within DUO-2 allowed it to also sensitively respond to genotoxins via the production of bioluminescence . However, the characteristics of DUO-2's bioluminescent response to each stress were easily distinguishable, making it useful for the detection of both stresses . Furthermore, tests with mixtures of chemicals showed that both DUO-1 and DUO-2 were responsive when chemicals causing oxidative or genotoxic stress were present as a single chemical or within complex chemical mixtures. Ther Apher Dial, 2003 Dec, 7(6), 504 - 9 Silver coating of dialysis catheters to reduce bacterial colonization and infection; Tobin EJ et al.; Complications resulting from infection remain a major problem for hemodialysis catheters, with significant numbers of catheters being removed due to catheter-related sepsis . Numerous strategies have been employed to reduce the occurrence of infection and improve long-term outcomes, with varying degrees of success . One promising approach is coating the external surface of catheters with silver using physical vapor deposition processes . This article reviews results of animal and clinical experiments conducted to assess efficacy and biocompatibility of silver-coated dialysis catheters . It is concluded that silver coatings can reduce bacterial colonization and occurrence of infection associated with these devices. J Biol Chem, 2004 Jul 2, 279(27), 28539 - 52 Epub 2004 Mar 11. A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt tolerance phenotype; Majee M et al.; l-myo-Inositol-1-phosphate synthase (EC 5.5.1.4, MIPS), an evolutionarily conserved enzyme protein, catalyzes the synthesis of inositol, which is implicated in a number of metabolic reactions in the biological kingdom . Here we report on the isolation of the gene (PINO1) for a novel salt-tolerant MIPS from the wild halophytic rice, Porteresia coarctata (Roxb.) Tateoka . Identity of the PINO1 gene was confirmed by functional complementation in a yeast inositol auxotrophic strain . Comparison of the nucleotide and deduced amino acid sequences of PINO1 with that of the homologous gene from Oryza sativa L . (RINO1) revealed distinct differences in a stretch of 37 amino acids, between amino acids 174 and 210 . Purified bacterially expressed PINO1 protein demonstrated a salt-tolerant character in vitro compared with the salt-sensitive RINO1 protein as with those purified from the native source or an expressed salt-sensitive mutant PINO1 protein wherein amino acids 174-210 have been deleted . Analysis of the salt effect on oligomerization and tryptophan fluorescence of the RINO1 and PINO1 proteins revealed that the structure of PINO1 protein is stable toward salt environment . Furthermore, introgression of PINO1 rendered transgenic tobacco plants capable of growth in 200-300 mm NaCl with retention of approximately 40-80% of the photosynthetic competence with concomitant increased inositol production compared with unstressed control . MIPS protein isolated from PINO1 transgenics showed salt-tolerant property in vitro confirming functional expression in planta of the PINO1 gene . To our knowledge, this is the first report of a salt-tolerant MIPS from any source. Gut, 2004 Apr, 53(4), 494 - 500 Increased antigen and bacterial uptake in follicle associated epithelium induced by chronic psychological stress in rats; Velin AK et al.; BACKGROUND: Chronic stress affects the course of inflammatory bowel disease and experimental colitis, and may also initiate intestinal inflammation in rats . AIM: To investigate the effects of stress on the M cell containing follicle associated epithelium, specialised in antigen uptake . SUBJECTS AND METHODS: Wistar rats were submitted to acute water avoidance stress for one hour or chronic water avoidance stress for 1 hour/day for 10 consecutive days . Permeability to (51)Cr-EDTA, horseradish peroxidase, and chemically killed Escherichia coli K-12 was studied in both villus and follicle associated epithelium in Ussing chambers . Segments were further examined by light, electron, and confocal microscopy . RESULTS: Acute stress increased horseradish peroxidase flux in villus as well as in follicle associated epithelium . Chronic stress further increased permeability to horseradish peroxidase in villus and follicle associated epithelium, in the latter by almost fourfold . Moreover, chronic stress induced over 30 times increased E coli passage in follicle associated epithelium whereas there was no significant increase in villus epithelium . Bacterial uptake was confirmed by confocal microscopy showing fluorescent bacteria penetrating and passing through the epithelial surface . CONCLUSIONS: These results show that the barrier function of follicle associated epithelium can be modulated, and that chronic stress enhances the uptake of luminal antigens and bacteria via the follicle associated epithelium . This can increase antigen exposure in Peyer's patches thereby having implications in the initiation of proinflammatory immune responses within the intestinal mucosa. Zhongguo Zhong Yao Za Zhi, 2003 Mar, 28(3), 199 - 201 {The current status of the determination of the bacterial endotoxin in traditional Chinese medicine}; Huo QL et al.; OBJECTIVE: To review the current status of the determination of the bacterial endotoxin in traditional chinese medicine injections with the limulus lysate test, and to evaluate the feasibility of this test for the determination of the bacterial endotoxin in these injections . METHOD: The data related to the topic was collected, analyzed and summarized . RESULT: The limulus lysate test was not available for most of traditional chinese medicine injections due to the interference . And the same injection may uield different test results because of different factories, batch numbers, different techniques and preparational conditions . CONCLUSION: There is still a long way to go to apply the limulus lysate test to the determination of the bacterial endotoxin in traditional chinese medicine injections. Chemosphere, 2004 May, 55(5), 775 - 80 Control of bacterial growth in water using synthesized inorganic disinfectant; Kim J et al.; Chlorine is a popular method for controlling bacterial growth in cooling towers . However, there are several drawbacks such as the difficulties in maintaining the disinfection efficacy particularly at high temperature and pH . In order to overcome these difficulties, an inorganic disinfectant based on silver and copper, which is called EEKO-BALL (commercial name), was recently developed . EEKO-BALL is made from specific ceramics and coating materials . This study was performed to evaluate the efficacy of EEKO-BALL and compare it with that of silver (Ag+) and copper (Cu2+) ions . In addition, a field study was undertaken to investigate the control of bacterial growth in several cooling tower systems . The results showed that the contact time required for inactivating 99% of E . coli was 15 min in the EEKO-BALL stock solution at 25 degrees C, pH 7.3, with 0.05 mgl(-1) Ag and 0.05 mgl(-1) Cu . EEKO-BALL was approximately four times more effective than silver and copper ions in inactivating E . coli at 25 degrees C, pH 7.3 . The control of bacterial growth in the cooling towers was found to be effective, lasting more than two months after a one-time installation of the EEKO-BALL . Overall, this study suggests that EEKO-BALL can effectively work as an inorganic disinfectant for bacterial growth control. Annu Rev Phytopathol, 1997, 35, 129 - 52 The role of hrp genes during plant-bacterial interactions; Lindgren PB; hrp genes control the ability of phytopathogenic bacteria to cause disease and to elicit hypersensitive reactions on resistant plants . Genetic and biochemical studies have demonstrated that Hrp proteins are components of Type III secretion systems, regulatory proteins, proteinaceous elicitors of the hypersensitive reaction, and enzymes needed for synthesis of periplasmic glucans . Significantly, Type III secretion systems are involved with the secretion of pathogenicity proteins in bacterial pathogens of animals . The transcriptional activation of a number of bacterial avirulence (avr) genes is controlled by Hrp regulatory proteins, and recent experimental evidence suggests that Avr proteins may be transported by Hrp secretion systems . It has also been hypothesized that pathogenicity and/or virulence gene products exit bacterial phytopathogens via Hrp pathways . Thus, hrp genes may be one of the most important groups of genes found in phytopathogenic bacteria in relationship to pathogenicity and host range. Hepatogastroenterology, 2004 Jan-Feb, 51(55), 171 - 5 Intestinal ischemia-reperfusion injury augments intestinal mucosal injury and bacterial translocation in jaundiced rats; Yuksek YN et al.; BACKGROUND/AIMS: The aim of this study was to evaluate local effects and degree of bacterial translocation related with intestinal ischemia-reperfusion injury in a rat obstructive jaundice model . METHODOLOGY: Thirty adult Sprague-Dawley rats (200-250 g) were divided into three groups; including Group 1 (jaundice group), Group 2 (jaundice-ischemia group) and Group 3 (ischemia group) . All rats had 2 laparotomies . After experimental interventions, tissue samples for translocation; liver and ileum samples for histopathological examination, 25 cm of small intestine for mucosal myeloperoxidase and malondialdehyde levels and blood samples for biochemical analysis were obtained . RESULTS: Jaundiced rats had increased liver enzyme levels and total and direct bilirubin levels (p<0.05) . Intestinal mucosal myeloperoxidase and malondialdehyde levels were found to be high in intestinal ischemia-reperfusion groups (p<0.05) . Intestinal mucosal damage was more severe in rats with intestinal ischemia-reperfusion after bile duct ligation (p<0.05) . Degree of bacterial translocation was also found to be significantly high in these rats (p<0.05) . CONCLUSIONS: Intestinal mucosa is disturbed more severely in obstructive jaundice with the development of ischemia and reperfusion . Development of intestinal ischemia-reperfusion in obstructive jaundice increases bacterial translocation. Proc Natl Acad Sci U S A, 2004 Mar 23, 101(12), 4320 - 4 Epub 2004 Mar 09. Identification of the bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in plants; Takahashi K et al.; Stringent control mediated by the bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) is a key regulatory process governing bacterial gene expression . By devising a system to measure ppGpp in plants, we have been able to identify ppGpp in the chloroplasts of plant cells . Levels of ppGpp increased markedly when plants were subjected to such biotic and abiotic stresses as wounding, heat shock, high salinity, acidity, heavy metal, drought, and UV irradiation . Abrupt changes from light to dark also caused a substantial elevation in ppGpp levels . In vitro, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp, demonstrating the existence of a bacteria-type stringent response in plants . Elevation of ppGpp levels was elicited also by treatment with plant hormones jasmonic acid, abscisic acid, and ethylene, but these effects were blocked completely by another plant hormone, indole-3-acetic acid . On the basis of these findings, we propose that ppGpp plays a critical role in systemic plant signaling in response to environmental stresses, contributing to the adaptation of plants to environmental changes. Mol Microbiol, 2004 Mar, 51(6), 1525 - 33 The bacterial Sm-like protein Hfq: a key player in RNA transactions; Valentin-Hansen P et al.; The conserved RNA-binding protein Hfq, originally discovered in Escherichia coli as a host factor for Qbeta replicase, has emerged as a pleiotropic regulator that modulates the stability or the translation of an increasing number of mRNAs . During the past 5 years, Hfq-mediated control has been an area of increasing focus because the protein has been linked to the action of many versatile RNA-based regulators that use basepairing interactions to regulate the expression of target mRNAs . The recent findings that Hfq assists in bimolecular RNA-RNA interactions and is similar structurally and functionally to eukaryotic Sm proteins have further fueled interest in this important post-transcriptional regulator . Here, we summarize the history of Hfq and highlight results that have led to an important gain in insight into the physiology, biochemistry and evolution of Hfq and its homologues. J Neurochem, 2004 Mar, 88(5), 1168 - 78 Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide; Rosenberger TA et al.; In a rat model of acute neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we measured brain activities and protein levels of three phospholipases A2 (PLA2) and of cyclo-oxygenase-1 and -2, and quantified other aspects of brain phospholipid and fatty acid metabolism . The 6-day intracerebral ventricular infusion increased lectin-reactive microglia in the cerebral ventricles, pia mater, and the glial membrane of the cortex and resulted in morphological changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles . LPS infusion increased brain cytosolic and secretory PLA2 activities by 71% and 47%, respectively, as well as the brain concentrations of non-esterified linoleic and arachidonic acids, and of prostaglandins E2 and D2 . LPS infusion also increased rates of incorporation and turnover of arachidonic acid in phosphatidylethanolamine, plasmenylethanolamine, phosphatidylcholine, and plasmenylcholine by 1.5- to 2.8-fold, without changing these rates in phosphatidylserine or phosphatidylinositol . These observations suggest that selective alterations in brain arachidonic acid metabolism involving cytosolic and secretory PLA2 contribute to early pathology in neuroinflammation. Eur J Biochem, 2004 Mar, 271(6), 1117 - 26 Chromophore selectivity in bacterial phytochromes: dissecting the process of chromophore attachment; Quest B et al.; Bacterial phytochromes (Bphs) are ancestors of the well characterized plant photoreceptors . Whereas plant phytochromes perform their photoisomerization exclusively via a covalently bound bilin chromophore, Bphs are variable in their chromophore selection . This is demonstrated in the cyanobacterium Calothrix PCC7601 that expresses two Bphs, CphA and CphB . CphA binds phycocyanobilin (PCB) covalently, whereas CphB, lacking the covalently binding cysteine of the plant phytochromes, carries biliverdin IXalpha (BV) as the chromophore . Our experiments elucidate the different modes of chromophore-protein interaction in CphA and CphB and offer a rationale for their chromophore selectivity . The tight binding of BV by CphB prevents PCB from competing for the binding cavity . Even when the chromophore-binding cysteine has been inserted (CphB-mutant L266C), PCB replaces BV very slowly, indicating the tight, but not irreversible binding of BV . The mutant CphB L266C showed a redox-sensitivity with respect to its PCB binding mode: under reducing conditions, the chromoprotein assembly leads to spectra indicative for a covalent binding, whereas absence of dithiothreitol or its removal prior to assembly causes spectra indicative for noncovalent binding . Regarding the CphB-type Bphs lacking the covalently binding cysteine, our results support the involvement of the succeeding histidine residue in chromophore fixation via a Schiff base-like bond between the bilin A-ring carbonyl and the histidine imidazole group . The assembly process and the stability of the holo-proteins were strongly influenced by the concentration of added imidazole (mimicking the histidine side-chain), making the attachment of the chromophore via the histidine more likely than via another cysteine of the protein. Eur J Biochem, 2004 Mar, 271(6), 1094 - 105 NF-kappaB- and c-Jun-dependent regulation of human cytomegalovirus immediate-early gene enhancer/promoter in response to lipopolysaccharide and bacterial CpG-oligodeoxynucleotides in macrophage cell line RAW 264.7; Lee Y et al.; The cytomegalovirus immediate-early (CMV IE) gene enhancer/promoter regulates the expression of immediate-early gene products and initiation of CMV replication . TNF-alpha and lipopolysaccharide (LPS) strongly activate the promoter, possibly involving NF-kappaB . CpG-oligodeoxynucleotides (CpG-ODNs), which contain unmethylated CpG dinucleotides in the context of particular base sequences, have gained attention because of their stimulating effects, via NF-kappaB, which have a strong innate immune response . To study the effects of LPS and CpG-ODNs, as well as the mechanisms of their actions regarding CMV IE enhancer/promoter activation, we used a macrophage cell line, RAW 264.7 . Stimulation of the cells with LPS or CpG-ODNs resulted in the activation of the CMV IE enhancer/promoter . We examined the involvement of NF-kappaB and c-Jun transcription factors by promoter deletion/site-specific mutation analysis and ectopic expression, and found them to have additive effects . Involvement of myeloid differentiation protein, an upstream regulator of NF-kappaB and c-Jun, was also investigated . Experimental results indicate that both LPS-induced and CpG-ODN-induced activations of CMV IE enhancer/promoter are mediated by Toll-like receptor signaling molecules . Several lines of evidence suggest the potential contribution of bacterial infection in CMV reactivation along with the potential application of CpG-ODNs in gene therapy as a stimulator for the optimal expression of target genes under the control of the CMV IE enhancer/promoter. Orthopade, 2004 Mar, 33(3), 297 - 304 {Bacterial osteitis . Special considerations in immunocompromised patients}; Niedhart C et al.; With increasing life expectancy and better medical competence, the number of older patients with multimorbidity is growing . Patients with a deficient immune system need more attention during diagnosis and treatment of osteomyelitis. Nucleic Acids Res, 2004 Mar 05, 32(4), 1548 - 54 Print 2004. Interaction of human and bacterial AlkB proteins with DNA as probed through chemical cross-linking studies; Mishina Y et al.; The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism . Three human homologs, ABH1, ABH2 and ABH3, have been identified, and two of them, ABH2 and ABH3, were shown to have similar repair activities to E.coli AlkB . However, ABH1 did not show any repair activity . It was suggested that ABH3 prefers single-stranded DNA and RNA substrates, whereas AlkB and ABH2 can repair damage in both single- and double-stranded DNA . We employed a chemical cross-linking approach to probe the structure and substrate preferences of AlkB and its three human homologs . The putative active site iron ligands in these proteins were mutated to cysteine residues . These mutant proteins were used to cross-link to different DNA probes bearing thiol-tethered bases . Disulfide-linked protein-DNA complexes can be trapped and analyzed by SDS-PAGE . Our results show that ABH2 and ABH3 have structural and functional similarities to E.coli AlkB . ABH3 shows preference for the single-stranded DNA probe . ABH1 failed to cross-link to the probes tested . This protein, unlike other AlkB proteins, does not seem to interact with DNA in its E.coli expressed form. J Microbiol Methods, 2004 Apr, 57(1), 17 - 22 Electrophoresis time impacts the denaturing gradient gel electrophoresis-based assessment of bacterial community structure; Sigler WV et al.; We investigated the impact of denaturing gradient gel electrophoresis (DGGE) run time on the assessment of bacterial community structure . Results indicated that increased electrophoresis run time (while maintaining 1000 volt-hours) resulted in dissimilar profiles, likely due to instability of the denaturing gradient . We recommend that DGGE run times be minimized to provide optimal band resolution, as extended electrophoresis times can greatly impact subsequent band-based analyses. J Mol Biol, 2004 Mar 19, 337(2), 485 - 96 Is 2-phosphoglycerate-dependent automodification of bacterial enolases implicated in their export? Boel G, Pichereau V, Mijakovic I, Maze A, Poncet S, Gillet S, Giard JC, Hartke A, Auffray Y, Deutscher J. We observed that in vivo and in vitro a small fraction of the glycolytic enzyme enolase became covalently modified by its substrate 2-phosphoglycerate (2-PG) . In modified Escherichia coli enolase, 2-PG was bound to Lys341, which is located in the active site . An identical reversible modification was observed with other bacterial enolases, but also with enolase from Saccharomyces cerevisiae and rabbit muscle . An equivalent of Lys341, which plays an important role in catalysis, is present in enolase of all organisms . Covalent binding of 2-PG to this amino acid rendered the enzyme inactive . Replacement of Lys341 of E.coli enolase with other amino acids prevented the automodification and in most cases strongly reduced the activity . As reported for other bacteria, a significant fraction of E.coli enolase was found to be exported into the medium . Interestingly, all Lys341 substitutions prevented not only the automodification, but also the export of enolase . The K341E mutant enolase was almost as active as the wild-type enzyme and therefore allowed us to establish that the loss of enolase export correlates with the loss of modification and not the loss of glycolytic activity. J Psychiatr Res, 2004 May-Jun, 38(3), 335 - 45 Maternal exposure to bacterial endotoxin during pregnancy enhances amphetamine-induced locomotion and startle responses in adult rat offspring; Fortier ME et al.; An increased incidence of schizophrenia has been associated with several perinatal insults, most notably maternal infection during pregnancy and perinatal hypoxia . This study used a rat model to directly test if maternal exposure to bacterial endotoxin (lipopolysaccharide, LPS) during pregnancy alters behaviors relevant to schizophrenia, in offspring at adulthood . The study also tested if postnatal anoxia interacted with gestational LPS exposure to affect behavior . At adulthood, offspring from dams administered LPS on days 18 and 19 of pregnancy showed significantly increased amphetamine-induced locomotion, compared to offspring from saline-treated dams . A period of anoxia on postnatal day 7 had no effect on amphetamine-induced locomotion and there was no interaction between effects of gestational LPS and postnatal anoxia on this behavior . Offspring from LPS-treated dams also showed enhanced acoustic startle responses as adults, compared to offspring from saline-treated dams . In offspring tested for pre-pulse inhibition (PPI) of acoustic startle and for apomorphine modulation of PPI, no effects of either gestational LPS or of postnatal anoxia and no interactions between LPS and anoxia were observed . It is concluded that maternal LPS exposure during pregnancy in the rat may be a useful model to study mechanisms responsible for effects of maternal infection on behaviors relevant to schizophrenia, in offspring. Biomacromolecules, 2004 Mar-Apr, 5(2), 445 - 52 Controlled sulfatation of natural anionic bacterial polysaccharides can yield agents with specific regenerating activity in vivo; Petit E et al.; The regenerating activities of chemically modified anionic bacterial polysaccharides by O-sulfonation were investigated using a in vivo model of rat injured muscle regeneration . Glucuronan (GA), a linear homopolysaccharide of -->4)-beta-D-GlcpA-(1--> residues partially acetylated at the C-3 and/or the C-2 position, and glucoglucuronan (GGA), a linear heteropolysaccharide of -->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1--> residues were sulfated . SO3-DMF sulfatation complex provided polysaccharides with different sulfur contents, however, a depolymerization occurred because we did not use large excess of pyridine to obtain pure modified polysaccharides . A regenerating activity on injured extensor digitorum longus (EDL) muscles on rats was obtained with these two sulfated anionic polymers . The position of sulfate groups on glucoglucuronan (primary or secondary alcohol) seems to have no influence on the biological activity by opposition to the degree of sulfatation both for the glucuronans and the glucoglucuronans . The yield of acetate groups in the glucuronan polymer modulated the specific activity. J Am Vet Med Assoc, 2004 Mar 1, 224(5), 739 - 42 Bacterial meningitis and brain abscesses secondary to infectious disease processes involving the head in horses: seven cases (1980-2001); Smith JJ et al.; OBJECTIVE: To determine clinical features of horses with bacterial meningitis or brain abscesses secondary to infectious disease processes involving the head . DESIGN: Retrospective study . ANIMALS: 7 adult horses . PROCEDURE: Medical records of Tufts University, the University of Pennsylvania, and the Livestock Disease Diagnostic Center (Lexington, Ky) were reviewed to identify adult (> 12 months old) horses in which a postmortem diagnosis of bacterial meningitis or brain abscess had been made . Horses were included in the study if an intracranial infection was confirmed, the horse had a primary infectious disease process involving the head, and there were no signs of systemic infection . RESULTS: 23 adult horses with bacterial meningitis or a brain abscess were examined during the study period, but only 7 met the criteria for inclusion in the study . Primary sites of infection included the paranasal sinuses, nasal cavity, periocular tissues, and submandibular lymph nodes . Three horses died suddenly prior to hospitalization, and 1 horse was hospitalized but died 7 days after the onset of neurologic abnormalities . The remaining 3 horses were euthanatized because of a rapid deterioration in clinical status . CONCLUSIONS AND CLINICAL RELEVANCE: Although rare, fatal intracranial complications can develop in horses with infectious diseases involving the head. J Mol Biol, 2004 Mar 12, 337(1), 15 - 30 Interactions of translational factor EF-G with the bacterial ribosome before and after mRNA translocation; Wilson KS et al.; A conserved translation factor, known as EF-G in bacteria, promotes the translocation of tRNA and mRNA in the ribosome during protein synthesis . Here, EF-G.ribosome complexes in two intermediate states, before and after mRNA translocation, have been probed with hydroxyl radicals generated from free Fe(II)-EDTA . Before mRNA translocation and GTP hydrolysis, EF-G protected a limited set of nucleotides in both subunits of the ribosome from cleavage by hydroxyl radicals . In this state, an extensive set of nucleotides, in the platform and head domains of the 30S subunit and in the L7/L12 stalk region of the 50S subunit, became more exposed to hydroxyl radical attack, suggestive of conformational changes in these domains . Following mRNA translocation, EF-G protected a larger set of nucleotides (23S rRNA helices H43, H44, H89, and H95; 16S rRNA helices h5 and h15) . No nucleotide with enhanced reactivity to hydroxyl radicals was detected in this latter state . Both before and after mRNA translocation, EF-G protected identical nucleotides in h5 and h15 of the 30S subunit . These results suggest that h5 and h15 may remain associated with EF-G during the dynamic course of the translocation mechanism . Nucleotides in H43 and H44 of the 50S subunit were protected only after translocation and GTP hydrolysis, suggesting that these helices interact dynamically with EF-G . The effects in H95 suggest that EF-G interacts weakly with H95 before mRNA translocation and strongly and more extensively with this helix following mRNA translocation. Womens Health Issues, 2004 Jan-Feb, 14(1), 14 - 8 Acceptability of a self-sampling technique to collect vaginal smears for gram stain diagnosis of bacterial vaginosis; Boskey ER et al.; To diagnose asymptomatic bacterial vaginosis (BV), self-sampled vaginal smears were collected during a study of risk factors for preterm birth in African American women . More than 90% of those women who were willing to participate in the interview portion of the study were also willing to provide a self-sampled vaginal smear . The smears are an acceptable and efficient way of detecting BV in an urban minority population. Int J Immunopathol Pharmacol, 2004 Jan-Apr, 17(1), 71 - 6 Involvement of reactive oxygen species in bacterial killing within epithelial cells; Battistoni A et al.; Several non-phagocytic cells can actively generate the superoxide anion by NAD(P)H oxidases resembling the enzymatic complex typical of phagocytes . Overexpression of periplasmic Cu,ZnSOD rescues invasive E . coli strains from killing within epithelial cells, suggesting that superoxide generation by such cells can oxidatively damage invading bacteria . Pre-treatment of HeLa cells with diphenyl iodonium or 4'-hydroxy-3'-methoxyacetophenone, two inhibitors of NAD(P)H oxidase, significantly enhances intracellular survival of wild type invasive E . coli cells . On the contrary, these inhibitors have no effect on the intracellular survival of an invasive E . coli strain engineered to overexpress Cu,ZnSOD . These results support the hypothesis that superoxide generation by a NAD(P)H oxidase-like complex can limit bacterial survival within epithelial cells and suggest that the role of periplasmic Cu,ZnSOD in bacterial infections is not simply that of conferring protection against the phagocytic oxidative burst. J Biol Chem, 2004 May 14, 279(20), 21327 - 33 Epub 2004 Mar 01. Flexibility and size heterogeneity of the LH1 light harvesting complex revealed by atomic force microscopy: functional significance for bacterial photosynthesis; Bahatyrova S et al.; Previous electron microscopic studies of bacterial RCLH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the Q(B) site of the RC to the quinone pool prior to reduction of the cytochrome bc(1) complex . We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size . This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape . This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature . Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions . The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides . The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (alpha(1)beta(1)Bchl(2)) comprising the LH1 complex . The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process. Anal Chem, 2004 Mar 1, 76(5), 1411 - 8 Real-time detection of bacterial contamination in dynamic aqueous environments using optical sensors; Ji J et al.; Here we describe on-line, real-time detection of waterborne bacteria using an optical sensor based on a starburst dendrimer film containing a lipophilic fluorophore . The sensor is constructed via covalent coupling between amine-terminated polyamidoamine dendrimer and silanized glass through an amide bond . The reporter molecule is embedded in the dendrimer layer through host-guest interaction . Real-time automated detection and quantitation of the bacteria are realized by using a charge-coupled detector camera and customized imaging and analysis software . The sensor responds to bacteria introduced to an aqueous flow system within 1 min . The limit of detection is approximately 10(4)cells/mL . The operational lifetime is more than 64 h, and the storage lifetime of the sensor is at least 7 months. Org Lett, 2004 Mar 4, 6(5), 803 - 6 1-methylidenesqualene and 25-methylidenesqualene as active-site probes for bacterial squalene:hopene cyclase; Tanaka H et al.; 1-methylidenesqualene and 25-methylidenesqualene were converted to 30-methylidenehop-22(29)-ene by squalene:hopene cyclase from Alicyclobacillus acidocaldarius . It was remarkable that both analogues generated the same product . The hopanyl intermediate cation, stabilized by the methylidene residue, enabled a rotation of the isobutenyl group at C-21 prior to the final proton elimination . In contrast, in the formation of hop-22(29)-ene, the final proton abstraction takes place regiospecifically from the Z-methyl group, which was verified by cyclization of (1,1,1,24,24,24-(2)H(6))squalene into (23,23,23,30,30,30-(2)H(6))hop-22(29)-ene . {reaction: see text} Biochem Biophys Res Commun, 2004 Mar 19, 315(4), 1097 - 103 Correlations between nucleotide frequencies and amino acid composition in 115 bacterial species; Bharanidharan D et al.; We studied the correlations between amino acid composition and mononucleotide and dinucleotide frequencies in 115 bacterial genomes of varying G+C content . Observed amino acid frequencies were compared with those expected from the actual mononucleotide and dinucleotide frequencies . Both mononucleotide and dinucleotide frequencies correlate well with the amino acid frequency, with dinucleotide frequencies doing so better . Despite the strong correlations, some of the observed amino acid frequencies, in particular for Arg, Val, Asp, Glu, Ser, and Cys, were consistently different from predicted values in all genomes . We suggest that this variation from predicted values is a consequence of selection pressure at the level of amino acids, while the close correspondence to the predictions in residues such as Thr, Phe, Lys, and Asn arises only from mutation and selection pressure at the level of the nucleic acid sequences. Arch Biochem Biophys, 2004 Jan 15, 421(2), 186 - 91 Proton NMR study of the heme environment in bacterial quinol oxidases; Zhang J et al.; The heme environment and ligand binding properties of two relatively large membrane proteins containing multiple paramagnetic metal centers, cytochrome bo3 and bd quinol oxidases, have been studied by high field proton nuclear magnetic resonance (NMR) spectroscopy . The oxidized bo3 enzyme displays well-resolved hyperfine-shifted 1H NMR resonance assignable to the low-spin heme b center . The observed spectral changes induced by addition of cyanide to the protein were attributed to the structural perturbations on the low-spin heme (heme b) center by cyanide ligation to the nearby high-spin heme (heme o) of the protein . The oxidized hd oxidase shows extremely broad signals in the spectral region where protons near high-spin heme centers resonate . Addition of cyanide to the oxidized bd enzyme induced no detectable perturbations on the observed hyperfine signals, indicating the insensitive nature of this heme center toward cyanide . The proton signals near the low-spin heme b558 center are only observed in the presence of 20% formamide, consistent with a critical role of viscosity in detecting NMR signals of large membrane proteins . The reduced bd protein also displays hyperfine-shifted 1H NMR signals, indicating that the high-spin heme centers (hemes b595 and d) remain high-spin upon chemical reduction . The results presented here demonstrate that structural changes of one metal center can significantly influence the structural properties of other nearby metal center(s) in large membrane paramagnetic metalloproteins. J Biol Chem, 2004 May 7, 279(19), 20186 - 93 Epub 2004 Feb 23. A redox-controlled molecular switch revealed by the crystal structure of a bacterial heme PAS sensor; Kurokawa H et al.; PAS domains, which have been identified in over 1100 proteins from all three kingdoms of life, convert various input stimuli into signals that propagate to downstream components by modifying protein-protein interactions . One such protein is the Escherichia coli redox sensor, Ec DOS, a phosphodiesterase that degrades cyclic adenosine monophosphate in a redox-dependent manner . Here we report the crystal structures of the heme PAS domain of Ec DOS in both inactive Fe(3+) and active Fe(2+) forms at 1.32 and 1.9 A resolution, respectively . The protein folds into a characteristic PAS domain structure and forms a homodimer . In the Fe(3+) form, the heme iron is ligated to a His-77 side chain and a water molecule . Heme iron reduction is accompanied by heme-ligand switching from the water molecule to a side chain of Met-95 from the FG loop . Concomitantly, the flexible FG loop is significantly rigidified, along with a change in the hydrogen bonding pattern and rotation of subunits relative to each other . The present data led us to propose a novel redox-regulated molecular switch in which local heme-ligand switching may trigger a global "scissor-type" subunit movement that facilitates catalytic control. Nucleic Acids Res, 2004 Feb 23, 32(4), 1335 - 44 Print 2004. The helix-turn-helix motif of bacterial insertion sequence IS911 transposase is required for DNA binding; Rousseau P et al.; The transposase of IS911, a member of the IS3 family of bacterial insertion sequences, is composed of a catalytic domain located at its C-terminal end and a DNA binding domain located at its N-terminal end . Analysis of the transposases of over 60 members of the IS3 family revealed the presence of a helix-turn-helix (HTH) motif within the N-terminal region . Alignment of these potential secondary structures further revealed a completely conserved tryptophan residue similar to that found in the HTH motifs of certain homeodomain proteins . The analysis also uncovered a similarity between the IS3 family HTH and that of members of the LysR family of bacterial transcription factors . This information was used to design site-directed mutations permitting an assessment of its role in transposase function . A series of in vivo and in vitro tests demonstrated that the HTH domain is important in directing the transposase to bind the terminal inverted repeats of IS911. Neuroscience, 2004, 124(3), 619 - 28 Combined toxicity of prenatal bacterial endotoxin exposure and postnatal 6-hydroxydopamine in the adult rat midbrain; Ling ZD et al.; We previously reported that injection of the Gram (-) bacteriotoxin, lipopolysaccharide (LPS), into gravid females at embryonic day 10.5 led to the birth of animals with fewer than normal dopamine (DA) neurons when assessed at postnatal days (P) 10 and 21 . To determine if these changes continued into adulthood, we have now assessed animals at P120 . As part of the previous studies, we also observed that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) was elevated in the striatum, suggesting that these animals would be more susceptible to subsequent DA neurotoxin exposure . In order to test this hypothesis, we injected (at P99) 6-hydroxydopamine (6OHDA) or saline into animals exposed to LPS or saline prenatally . The results showed that animals exposed to prenatal LPS or postnatal 6O