Microbial

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 6OHDA alone had 33% and 46%, respectively, fewer DA neurons than controls, while the two toxins combined produced a less than additive 62% loss . Alterations in striatal DA were similar to, and significantly correlated with (r(2)=0.833) the DA cell losses . Prenatal LPS produced a 31% increase in striatal TNFalpha, and combined exposure with 6OHDA led to an 82% increase . We conclude that prenatal exposure to LPS produces a long-lived THir cell loss that is accompanied by an inflammatory state that leads to further DA neuron loss following subsequent neurotoxin exposure . The results suggest that individuals exposed to LPS prenatally, as might occur had their mother had bacterial vaginosis, would be at increased risk for Parkinson's disease.

Bioorg Med Chem, 2004 Mar 1, 12(5), 935 - 47
A structure-based strategy to identify new molecular scaffolds targeting the bacterial ribosomal A-site; Foloppe N et al.; The need for novel antibiotics is widely recognized . A well validated target of antibiotics is the bacterial ribosome . Recent X-ray structures of the ribosome bound to antibiotics have shed new light on the binding sites of these antibiotics, providing fresh impetus for structure-based strategies aiming at identifying new ribosomal ligands . In that respect, the ribosomal decoding region of the aminoacyl-tRNA acceptor site (A-site) is of particular interest because oligonucleotide model systems of this site are available for crystallography, NMR and compound binding assays . This work presents how these different resources can be combined in a hierarchical screening strategy which has led to the identification of new A-site ligands . The approach exploits an X-ray structure of the A-site against which large and diverse libraries of compounds were computationally docked . The complementarity of the compounds to the A-site was assessed using a scoring function specifically calibrated for RNA targets . Starting from approximately 1 million compounds, the computational selection of candidate ligands allowed us to focus the experimental work on 129 compounds, 34 of which showed affinity for the A-site in a FRET-based binding assay . NMR experiments confirmed binding to the A-site for some compounds . For the most potent compound in the FRET assay, a tentative binding mode is suggested, which is compatible with the NMR data and the limited SAR in this series . Overall, the results validate the screening strategy.

Biochim Biophys Acta, 2004 Feb 24, 1670(3), 181 - 98
Tetrahydrobiopterin biosynthesis in white and brown adipose tissues is enhanced following intraperitoneal administration of bacterial lipopolysaccharide; Fujiwara K et al.; Tetrahydrobiopterin is an essential cofactor for nitric oxide synthase (NOS) . This study was undertaken to examine the effects of intraperitoneally injected lipopolysaccharide on tetrahydrobiopterin biosynthesis in murine white and brown adipose tissues . Tetrahydrobiopterin content, catalytic activity and mRNA expression level of GTP cyclohydrolase I (GCH), rate-controlling enzyme in de novo biosynthesis of tetrahydrobiopterin, in both adipose tissues were up-regulated by 500-microg lipopolysaccharide at 6 h after the injection . On the contrary, treatment of 3T3-L1 adipocytes with lipopolysaccharide alone did not affect GCH mRNA expression level, whereas the combination of lipopolysaccharide, tumor necrosis factor (TNF)-alpha, and interferon gamma induced the increase in expression levels of GCH mRNA and CD14 mRNA . Collectively, our results showed that tetrahydrobiopterin biosynthesis can be augmented by increased GCH activity caused by a synergistic effect of lipopolysaccharide and cytokines in white and brown adipose tissues . These observations support the view that tetrahydrobiopterin biosynthesis in the adipose tissues is a target of inflammatory events triggered by peripheral LPS injection.

Biochemistry, 2004 Mar 2, 43(8), 2178 - 87
Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor; Matsunami H et al.; The topa quinone (TPQ) cofactor of copper amine oxidase is produced by posttranslational modification of a specific tyrosine residue through the copper-dependent, self-catalytic process . We have site-specifically mutated three histidine residues (His431, His433, and His592) involved in binding of the copper ion in the recombinant phenylethylamine oxidase from Arthrobacter globiformis . The mutant enzymes, in which each histidine was replaced by alanine, were purified in the Cu/TPQ-free precursor form and analyzed for their Cu-binding and TPQ-generating activities by UV-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies . Among the three histidine-to-alanine mutants, only H592A was found to show a weak activity to form TPQ upon aerobic incubation with Cu(2+) ions . Also for H592A, exogenous imidazole rescued binding of copper and markedly promoted the TPQ formation . Accommodation of a free imidazole molecule within the cavity created in the active site of H592A was suggested by X-ray crystallography . Although the TPQ cofactor in H592A mutant was readily reduced with substrate, its catalytic activity was very low even in the presence of imidazole . Combined with the crystal structures of the mutant enzymes, these results demonstrate the importance of the three copper-binding histidine residues for both TPQ biogenesis and catalytic activity, fine-tuning the position of the essential metal.

Arzneimittelforschung, 2004, 54(1), 57 - 63
Prevention of recurrent upper respiratory tract infections in a community of cloistered nuns using a new immunostimulating bacterial lysate . A randomized, double-blind clinical trial; Tricarico D et al.; The aim of the present study was to evaluate the efficacy of the immunostimulating therapy with a new vaccine Ismigen in preventing recurrent infections of the upper respiratory tract in subjects belonging to a community of cloistered nuns . This product is a lysate obtained by mechanical lysis of bacteria (MLBL) usually responsible of respiratory tract infections . A randomized, double-blind, parallel groups, placebo controlled clinical trial was carried out in 47 nuns (age range 25-80 years) living in a cloistered religious community, suffering from recurrent infections of the upper respiratory tract . The 47 patients were allocated by randomization to two groups: Group A--The 24 patients (mean age SD: 48.12 +/- 14.25 years) of this group received one sublingual tablet of MLBL per day, for 10 consecutive days per month for 3 consecutive months (October, November and December 2001) . Group B--The 23 patients (mean age +/- SD: 49.04 +/- 14.73 years) of this group received daily one sublingual tablet of taste masked placebo, for 10 consecutive days per month for 3 consecutive months (October, November and December 2001) . At the end of the treatment period patients of both groups were followed up for further three months without any immunostimulating treatment . RESULTS: During and at the end of the treatment phase the number of respiratory infections (primary end-point) and their duration were statistically significantly lower in the MLBL group than in the placebo group . Moreover the administration of MLBL induced a marked reduction in the number of patients showing symptoms of infection in comparison to baseline and approximately 79 % of the patients showed an improvement of one or more of the evaluated symptoms . In the MLBL group a statistically significant increase of serum immunoglobulins (IgG +35%; IgM +86%; IgA +80 %) and salivary IgA (+110%) was found, in comparison to baseline; on the contrary no significant differences were observed in the placebo group . The beneficial effects of MLBL found in the treatment period were maintained also in the three-month follow-up . No adverse events associated with the treatment were found in both group . The results of this study demonstrate that MLBL is an efficacious and safe therapeutic option for the treatment and prevention of recurrent upper respiratory tract infections and that its use is recommended in subjects with a possible immune deficit.

Epidemiol Infect, 2004 Jan, 132(1), 61 - 5
A combined AFLP-multiplex PCR assay for molecular typing of Escherichia coli strains using variable bacterial interspersed mosaic elements; Horvath R et al.; The original method for molecular typing of E . coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay . The applicability of the method in the epidemiology of E . coli was tested on a group of 524 strains of human and veterinary origin . In the studied group 18 different genotypes were detected . Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E . coli.

Infez Med, 1995, 3(2), 61 - 70
{Bacterial eradication . Role of beta-lactamases}; Blandino G et al.; not available

Microbiol Immunol, 2004, 48(2), 91 - 6
Bacterial mRNA purification by magnetic capture-hybridization method; Pang X et al.; A magnetic capture-hybridization method was developed for purification of bacterial messenger RNA (mRNA) from total RNA by removing 5S, 16S and 23S ribosomal RNAs (rRNA) . The quality of mRNA was evaluated by A(260 nm) / A(280 nm) value, denatured gel electrophoresis and RT-PCR . The results showed that highly purified and intact mRNA was obtained by this method . The magnetic capture-hybridization is a rapid and simple method for bacterial mRNA purification and has promising potential for improving studies using bacterial mRNA.

Protein Sci, 2004 Mar, 13(3), 830 - 41
Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase; Yang ZW et al.; Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds . Its use in a screen to identify novel inhibitors of the enzyme NAD(+) synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme . Although no effects are observed on the enzyme's catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism . In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC) . In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, K(a), equal to 5.6 x 10(6)/M . DSC curve analysis is consistent with this finding . The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer <==> folded monomer <==> unfolded monomer . The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large K(a) down to approximately 10(6)/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried . A hypothetical structural mechanism to explain this effect is presented.

Front Biosci, 2004 May 01, 9, 1035 - 42
Mn2+ and bacterial pathogenesis; Zaharik ML et al.; Fe2+ has traditionally been considered the most important divalent cation involved in host-pathogen interactions . However, recent research indicates a previously unappreciated role for transition metal divalent cations other than Fe2+ during infection . Recent studies have identified an absolute requirement for Mn2+ in bacterial pathogens that are Fe2+-independent, indicating an important role for Mn2+ in pathogenesis . Potential roles for Mn2+ in pathogenesis include effects on the detoxification of reactive oxygen intermediates (ROIs), as a cofactor for enzymes involved in intermediary metabolism and signal transduction, and as a stimulus for virulence gene regulation . This review focuses on how these possible roles for Mn2+ may affect bacterial pathogenesis and the outcome of an infection.

J Biol Chem, 2004 Apr 30, 279(18), 18944 - 51 Epub 2004 Feb 19.
Bacterial penetration of bladder epithelium through lipid rafts; Duncan MJ et al.; Type 1 fimbriated Escherichia coli represents the most common human uropathogen, owing much of its virulence to invasion of the uroepithelium, which is highly impermeable due to the preponderance of uroplakins and highly ordered lipid components . We sought to elucidate the molecular basis for E . coli invasion of the bladder epithelium by employing human 5637 bladder epithelial cells, and we found the following: (i) intracellular E . coli associated with caveolae and lipid raft components; (ii) RNA(i) reduction of caveolin-1 expression inhibited bacterial invasion; (iii) a signaling molecule required for E . coli invasion was located in lipid rafts and physically associated with caveolin-1; (iv) bacterial invasion was inhibited by lipid raft disrupting/usurping agents . In the mouse bladder, the E . coli type 1 fimbrial receptor, uroplakin Ia, was located in lipid rafts, and lipid raft disruptors inhibited E . coli invasion . Cumulatively, E . coli uroepithelial invasion occurs through lipid rafts, which, paradoxically, contribute to bladder impermeability.

Water Res, 2004 Mar, 38(5), 1121 - 8
Mobilization of adsorbed copper and lead from naturally aged soil by bacterial extracellular polymers; Jensen-Spaulding A et al.; Sorption of pollutants is a dominant phase transfer process affecting the fate and transport of metals through the subsurface . The movement of contaminants is retarded by sorption to the stationary subsurface porous media and can seriously hinder remediation efforts . Research has shown that the binding of adsorbed metals becomes more pronounced the longer the contaminant is in the subsurface and the release rates of aged metal contaminants have not received the research attention given to freshly added metals in laboratory studies . Metal release rates are also influenced by the presence of dissolved ligands that compete with mineral soil surfaces by providing binding sites . Dissolved organic matter such as bacterial extracellular polymers are common in natural soil solutions and the metal binding properties of bacterial polymers are well established . Therefore, binding of metals to dissolved biopolymers may result in mobilization of an adsorbed metal . This is important for cases where the metals are assumed to be relatively immobile such as in the case of land applied biosolids . In addition, naturally occurring adherent bacteria commonly produce extracellular polymers and thus may modify the bioavailability of meal contaminants at the point of their attachment . In this study samples from three sites, one a land applied sludge test site, were used to investigate the ability of bacterial extracellular polymers to release metals from soils with long-term exposures . The presence of ?200mg/L bacterial extracellular polymer was found to increase the short-term (less than 350h) release of Cu and Pb by a factor of 2-4-fold.

Crit Care, 2004 Feb, 8(1), R12 - 20 Epub 2003 Nov 20.
Procalcitonin as a marker of bacterial infection in the emergency department: an observational study; Chan YL et al.; INTRODUCTION: Procalcitonin (PCT) has been proposed as a marker of infection in critically ill patients; its level is related to the severity of infection . We evaluated the value of PCT as a marker of bacterial infection for emergency department patients . METHODS: This prospective observational study consecutively enrolled 120 adult atraumatic patients admitted through the emergency department of a 3000-bed tertiary university hospital in May 2001 . Fifty-eight patients were infected and 49 patients were not infected . The white blood cell counts, the serum C-reactive protein (CRP) level (mg/l), and the PCT level (ng/ml) were compared between the infected and noninfected groups of patients . RESULTS: A white blood cell count >12,000/mm3 or <4000/mm3 was present in 36.2% of the infected patients and in 18.4% of the noninfected patients . The best cut-off serum levels for PCT and CRP, identified using the Youden's Index, were 0.6 ng/ml and 60 mg/l, respectively . Compared with CRP, PCT had a comparable sensitivity (69.5% versus 67.2%), a lower specificity (64.6% versus 93.9%), and a lower area under the receiver operating characteristic curve (0.689 versus 0.879) . PCT levels, but not CRP levels, were significantly higher in bacteremic and septic shock patients . Multivariate logistic regression identified that a PCT level >/= 2.6 ng/ml was independently associated with the development of septic shock (odds ratio, 38.3; 95% confidence interval, 5.6-263.5; P < 0.001) . CONCLUSIONS: PCT is not a better marker of bacterial infection than CRP for adult emergency department patients, but it is a useful marker of the severity of infection.

Virology, 2004 Jan 5, 318(1), 420 - 8
DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome; Meseda CA et al.; Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described . BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination . DNA from cells infected with the resulting recombinant virus was transformed into E . coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype . HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative . When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization . Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus . The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene . A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line . Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective . The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines . The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.

Diagn Microbiol Infect Dis, 2004 Feb, 48(2), 77 - 80
Reliability of interpretation of gram-stained vaginal smears by Nugent's scoring system for diagnosis of bacterial vaginosis; Zarakolu P et al.; This study was designed to assess reliability of interpretation of Gram-stained vaginal smears by using Nugent's scoring system for diagnosis of bacterial vaginosis (BV) across three different centers in Turkey: two in Ankara and one in Trabzon . The vaginal smears were collected from clients attending a family planning clinic in Trabzon, Turkey during October-December 1997 . One slide taken from each client was prepared according to the standard methods and enumerated . One evaluator from each center examined the slides independently for the presence of BV and none of them had access to the evaluation of the others . Out of 372 slides, 301 (81%) were found to be satisfactory for scoring by all three evaluators and included in the analysis . Nugent's scores from 1-10 reported from each evaluator were compared by Spearman correlation coefficients and Kappa statistics . The difference in the proportions of BV diagnosis in three centers was evaluated by chi2 test . There was good agreement for the interpretation of Gram-stained vaginal smears by Nugent's scoring system for diagnosis of BV . These results indicate that it is a reliable method in diagnosis of BV at different settings.

J Am Chem Soc, 2004 Feb 25, 126(7), 2042 - 9
Noncovalent interactions: defining cooperativity . Ligand binding aided by reduced dynamic behavior of receptors . Binding of bacterial cell wall analogues to ristocetin A; Williams DH et al.; Changes in the relative populations of the monomer and asymmetric dimer forms of ristocetin A, upon binding of two molecules of ligand, suggest that ligand binding is negatively cooperative with respect to dimerization . However, strong hydrogen bonds formed in the binding sites of the ligands are reinforced in the dimer relative to the monomer, and the barrier to dissociation of the dimer is increased upon binding of the ligands . It is concluded that the interactions which are common in the binding of both ligands are made with positive cooperativity with respect to those involved in dimerization . The conclusions are relevant to the binding of ligands to proteins, where ligand binding energy can be derived from stabilization of the protein in its ligand-bound form.

Boll Chim Farm, 2003 Dec, 142(10), 447 - 9
Treatment of patients with bacterial infections of the central nervous system--a pharmaco-economical analysis; Dimitrov D; The meningitis and the meningoencefalitis is 29% from all of the organic diseases of the Central Nerve System . The actuality of this problem is determined by the following factors: 1 . Social-only the childish group and the active group of workers among the adults are concerned; 2 . The diseases are taking their course seriously with a high percentage of lethality-30%; 3 . When there is untimely and inadequate therapy, there occurred additional manifestations of the disease.

Ann Periodontol, 2003 Dec, 8(1), 54 - 69
Associations between periodontal disease and risk for nosocomial bacterial pneumonia and chronic obstructive pulmonary disease . A systematic review; Scannapieco FA et al.; BACKGROUND: Several recent studies provide evidence that the oral cavity may influence the initiation and/or the progression of lung diseases such as pneumonia and chronic obstructive pulmonary disease (COPD) . RATIONALE: Studies have shown that poor oral hygiene and periodontal disease may foster colonization of the oropharyngeal region by respiratory pathogens, particularly in hospital or nursing home patients . If aspirated, these pathogens can cause pneumonia, one of the most common respiratory infections, especially in institutionalized subjects . Other cross-sectional epidemiologic studies point to an association between periodontal disease and COPD . This systematic review examines the literature to determine if interventions that improve oral hygiene reduce the rate of pneumonia in high-risk populations . FOCUSED QUESTION: Do periodontal diseases or other indicators of poor oral health influence the initiation/progression of pneumonia or other lung diseases? SEARCH PROTOCOL: MEDLINE, pre-MEDLINE, MEDLINE Daily Update, and the Cochrane Controlled Trials Register were searched to identify published studies that related variables associated with pneumonia and other lung disease to periodontal disease . Searches were performed for articles published in English from 1966 through March 2002 . INCLUSION CRITERIA: Randomized controlled clinical trials (RCTs), longitudinal, cohort, and case-control studies were included . Study populations included patients with any form of pneumonia or chronic obstructive pulmonary disease (COPD) and periodontal disease, as measured by assessments of gingival inflammation, probing depth, clinical attachment level, and/or radiographic bone loss, or oral hygiene indices . EXCLUSION CRITERIA: Limited to studies of humans . DATA COLLECTION AND ANALYSIS: The summary statistics used to analyze the RCTs included weighted mean differences in rates of disease between control and intervention groups . For cohort studies that measured differences in rates of disease between groups with and without oral disease, weighted mean differences, relative risks, or odds ratios were compared . A meta-analysis was performed on the 5 intervention studies to determine the relationship between oral hygiene intervention and rate of pneumonia in institutionalized patients . MAIN RESULTS: Of the initial 1,688 studies identified, 36 satisfied all inclusion criteria and were read . Of these, 21 (11 case-control and cohort studies {study population 1,413} and 9 RCTs {study population 1,759}) were included in the analysis . 1 . A variety of oral interventions improving oral hygiene through mechanical and/or topical chemical disinfection or antibiotics reduced the incidence of nosocomial pneumonia by an average of 40% . 2 . Several studies demonstrated a potential association between periodontal disease and COPD . REVIEWERS' CONCLUSIONS: 1 . Oral colonization by respiratory pathogens, fostered by poor oral hygiene and periodontal diseases, appears to be associated with nosocomial pneumonia . 2 . Additional large-scale RCTs are warranted to provide the medical community with further evidence to institute effective oral hygiene procedures in high-risk patients to prevent nosocomial pneumonia . 3 . The results associating periodontal disease and COPD are preliminary and large-scale longitudinal and epidemiologic and RCTs are needed.

Eur J Immunol, 2004 Jan, 34(1), 184 - 93
Antibody-mediated bacterial clearance from the lower respiratory tract of mice requires complement component C3; Pishko EJ et al.; To assess the contribution of complement to respiratory immunity in the context of a natural bacterial infection, we used mice genetically deficient in complement components and the murine pathogen Bordetella bronchiseptica . Complement component C3 was not required for the control of bacterial infection or for the generation of infection-induced protective immunity . However, C3-deficient (C3(-/-)) mice were severely defective, compared to wild type, in vaccine-induced protective immunity . Adoptively transferred immune serum from convalescent wild-type or C3(-/-) animals rapidly cleared B . bronchiseptica from the lungs of wild-type mice but did not affect its growth in C3(-/-) mice, indicating that the defect is not in the generation of protective immunity, but in its function . Immune serum was effective in C5-deficient mice but had little effect in the lungs of mice lacking either Fcgamma receptors (FcgammaR) or CR3, suggesting bacterial clearance is not via direct complement-mediated lysis . Together, these data indicate that complement is required for antibody-mediated clearance of Bordetella and suggest the mechanism involves C3 opsonization of bacteria for phagocytosis that is both CR3- and FcgammaR-dependent.

Br J Cancer, 2004 Feb 23, 90(4), 860 - 5
Identification and characterisation of constitutional chromosome abnormalities using arrays of bacterial artificial chromosomes; Cowell JK et al.; Constitutional chromosome deletions and duplications frequently predispose to the development of a wide variety of cancers . We have developed a microarray of 6000 bacterial artificial chromosomes for array-based comparative genomic hybridisation, which provides an average resolution of 750 kb across the human genome . Using these arrays, subtle gains and losses of chromosome regions can be detected in constitutional cells, following a single overnight hybridisation . In this report, we demonstrate the efficiency of this procedure in identifying constitutional deletions and duplications associated with predisposition to retinoblastoma, Wilms tumour and Beckwith-Wiedemann syndrome.

Cytogenet Genome Res, 2003, 102(1-4), 32 - 8
A comparative map of bovine chromosome 19 based on a combination of mapping on a bacterial artificial chromosome scaffold map, a whole genome radiation hybrid panel and the human draft sequence; Moore SS et al.; We have constructed a medium density physical map of bovine chromosome 19 using a combination of mapping loci on both a bovine bacterial artificial chromosome (BAC) scaffold map and a whole genome radiation hybrid (WGRH) panel . The resulting map contains 70 loci spanning the length of bovine chromosome 19 . Three contiguous groups of BACs were identified on the basis of multiple loci mapping to individual BAC clones . Bovine chromosome 19 was found in this study to be comprised almost entirely from regions of human chromosome 17, with a small region putatively assigned to human chromosome 10 . Fourteen breakpoints between the bovine and human chromosomes were detected, with a possibility of five more based on ordering of the WGRH map .

Fed Regist, 2004 Jan 5, 69(2), 255 - 67
Biological products; bacterial vaccines and toxoids; implementation of efficacy review . Final rule and final order; Food and Drug Administration et al.; The Food and Drug Administration (FDA) is amending the biologics regulations in response to the report and recommendations of the Panel on Review of Bacterial Vaccines and Toxoids with Standards of Potency (the Panel) . The Panel reviewed the safety, efficacy, and labeling of bacterial vaccines and toxoids that have standards of potency, bacterial antitoxins, and immune globulins . On the basis of the Panel's findings and recommendations, FDA is classifying these products as Category I (safe, effective, and not misbranded), Category II (unsafe, ineffective, or misbranded), or Category IIIB (off the market pending completion of studies permitting a determination of effectiveness).

Di Yi Jun Yi Da Xue Xue Bao, 2004 Feb, 24(2), 201 - 3
Bacterial growth characteristics in craniocerebral gunshot wound of cat in a hot and humid environment; Guo YW et al.; OBJECTIVE: To investigate the growth behavior of characteristics in craniocerebral gunshot wound of cats in a hot and humid environment . METHODS: Twenty-three cross-bred cats were randomly divided into 4 groups: group A, the gunshot wound control group at normal temperature, in which tissue sampling was performed immediately after the wounding; group B, another gunshot wound control group at normal temperature, in which the samples were taken 6 h after the wounding; group C, the gunshot wound group subjected to a hot and humid environment, in which the tissue samples were obtained 6 h after the wounding; group D, the control group without undergoing the wound . The tissues from the wound tract and the surrounding tissues were sampled for bacterial culture and counting . RESULTS: The bacterial counts of the tissues from the wound tract, the tissues within 5 mm and within 5-10 mm from wound tract varied insignificantly between groups A, B and C (P>0.05) . In each group, the bacterial counts declined in the tissues as the distance of the sampling sites from the wound tract increased (P<0.01) . The bacterial counts of the tissues from the wound tract and within 5 mm from the wound tract in group A, B and C were significantly different from those in group D (P<0.01) . CONCLUSION: Hot and humid environment does not significantly affect the bacterial growth in the craniocerebral gunshot wound within the first 6 h, which is a safe period against rapid bacterial growth and suitable for debridement.

Breast, 1999 Jun, 8(3), 149 - 51
Bacterial endocarditis associated with a skin-tunnelled catheter; Dawson LK et al.; Skin-tunnelled catheters have become an accepted method for establishing long-term central venous access in patients undergoing treatment for malignancies . They allow administration of continuous infusions of cytotoxic drugs, supplementation of fluids and blood products, total parenteral nutrition and access for the checking of blood tests . It is recognized that there are certain complications associated with their use including the risk of infection both of the exit site and tunnel, as a source of septicaemia, line-associated thrombosis (despite the use of prophylactic low dose warfarin and flushing of the line with heparin) and accidental dislodgement of the line . We report a case of bacterial endocarditis affecting the pulmonary valve shortly after removal of a skin-tunnelled catheter due to line-associated brachiocephalic venous thrombosis.

Curr Hematol Rep, 2004 Mar, 3(2), 121 - 7
Improving platelet safety: bacterial contamination of platelets; Brecher ME et al.; In developed countries, transfusion-transmitted bacterial contamination of platelets is the most common cause of fatalities related to transfusion-transmitted disease . Platelets, to maintain viability and function, must be stored at room temperature (20 degrees-24 degrees C), which creates an excellent growth environment for the proliferation of bacteria . Multiple studies have shown that 1:1000 to 1:2000 platelet units are bacterially contaminated . It is estimated that the risk of a bacterial-related death after a transfusion of a platelet unit ranges from 1:7500 to 1:100,000 . The use of bacterial detection to minimize the risk of transfusion-related bacterial sepsis is gaining favor around the world . Implementation of bacterial detection methods would improve safety for patients, result in an extension of the shelf life of platelets, and reduce the outdating of platelets, thus being a cost-saving safety intervention.

Anal Chem, 2004 Feb 15, 76(4), 1002 - 7
Relative quantitation of intact proteins of bacterial cell extracts using coextracted proteins as internal standards; Williams TL et al.; A method for quantitating protein expression using LC/MS of whole proteins is described . This method is based on the fact that some proteins present in cells are abundant universal proteins whose expression levels exhibit little variation . This method demonstrates that these coextracted proteins can be used as internal standards to which the other proteins in the sample can be compared . By comparing the intensities of a selected protein to marker proteins, or internal standards, a relative ratio is obtained . This ratio can then be used to determine the relative amount of protein expression between cellular extracts . The validity of this approach is described for a standard protein mixture, as well as, E . coli cells that were known to differentially express green fluorescent protein.

Mar Biotechnol (NY), 2000 Nov, 2(6), 571 - 6
Genomic bacterial artificial chromosome library of the Japanese flounder Paralichthys olivaceus; Katagiri T et al.; We have constructed a genomic bacterial artificial chromosome (BAC) library from homozygous cloned Japanese flounder Paralichthys olivaceus using the pBAC- lac vector . This BAC library consists of about 49,100 clones and is deposited in 128 microtiter plates with 384 wells . The average size of inserted DNA was calculated to be 165 kb . The BAC library was determined to cover 9 times the Japanese flounder haploid genome . The Japanese flounder genomic BAC library will be useful for gene isolation as well as quantitative trait loci (QTL) analysis.

Proc Natl Acad Sci U S A, 2004 Feb 17, 101(7), 2117 - 22 Epub 2004 Feb 09.
Crosslinking snapshots of bacterial chemoreceptor squads; Studdert CA et al.; The team signaling model for bacterial chemoreceptors proposes that receptor dimers of different detection specificities form mixed trimers of dimers . These receptor "squads" then recruit the cytoplasmic signaling proteins CheA and CheW to form ternary signaling teams, which typically cluster at the poles of the cell . We devised cysteine-directed in vivo crosslinking approaches to ask whether mixed receptor squads could form in the absence of CheA and CheW and, if so, whether the underlying structural interactions conformed to trimer-of-dimers geometry . One approach used cysteine reporters at positions in the serine (Tsr) and aspartate (Tar) receptors that should form disulfide-linked Tsr approximately Tar products when juxtaposed at the interface of a mixed trimer . Another approach used a cysteine reporter with trigonal geometry near the trimer contact region and a trifunctional maleimide reagent with a spacer length appropriate for capturing the three axial subunits in a trimer of dimers . Both approaches detected mixed receptor-crosslinking products in cells lacking CheA and CheW . Under these conditions, receptor methylation and ligand-binding state had no discernable effect on crosslinking efficiencies . Crosslinking with the trigonal reporter was rapid and did not increase with longer treatment times or higher reagent concentrations, suggesting that this method produces a short-exposure snapshot of the receptor population . The extent of crosslinking indicated that most of the cell's receptor molecules were organized in higher-order groups . Crosslinking in receptor trimer contact mutants correlated with their signaling behaviors, suggesting that trimers of dimers are both structural and functional precursors of chemoreceptor signaling teams in bacteria.

Biochemistry, 2004 Feb 17, 43(6), 1746 - 53
Compaction of a bacterial group I ribozyme coincides with the assembly of core helices; Perez-Salas UA et al.; Counterions are critical to the self-assembly of RNA tertiary structure because they neutralize the large electrostatic forces which oppose the folding process . Changes in the size and shape of the Azoarcus group I ribozyme as a function of Mg(2+) and Na(+) concentration were followed by small angle neutron scattering . In low salt buffer, the RNA was expanded, with an average radius of gyration (R(g)) of 53 +/- 1 A . A highly cooperative transition to a compact form (R(g) = 31.5 +/- 0.5 A) was observed between 1.6 and 1.7 mM MgCl(2) . The collapse transition, which is unusually sharp in Mg(2+), has the characteristics of a first-order phase transition . Partial digestion with ribonuclease T1 under identical conditions showed that this transition correlated with the assembly of double helices in the ribozyme core . Fivefold higher Mg(2+) concentrations were required for self-splicing, indicating that compaction occurs before native tertiary interactions are fully stabilized . No further decrease in R(g) was observed between 1.7 and 20 mM MgCl(2), indicating that the intermediates have the same dimensions as the native ribozyme, within the uncertainty of the data (+/-1 A) . A more gradual transition to a final R(g) of approximately 33.5 A was observed between 0.45 and 2 M NaCl . This confirms the expectation that monovalent ions not only are less efficient in charge neutralization but also contract the RNA less efficiently than multivalent ions.

Cent Eur J Public Health, 2003 Dec, 11(4), 238 - 9
Light microscopy observation of lytic enzymatic activity of the organisms associated with bacterial vaginosis; Demirezen S; In this study, cervico-vaginal smears taken from 500 patients were examined cytologically using the Papanicolaou technique . Seventeen of the 500 were classified as having bacterial vaginosis . Lytic enzymatic activity of the organisms on clue cells were determined at light microscopic level . The integrity of the cell and the smoothness of the cell membrane were disrupted . Small cavities on the cell membrane and hollows in the cytoplasm were observed . Due to the loss of cytoplasm, very narrow and thin tracks around the nucleus and in the cytoplasm resembling a cobweb were seen . It is suggested that these lytic cellular changes might be formed by the organisms on clue cells.

Mikrobiologiia, 2003 Nov-Dec, 72(6), 854 - 61
{Soil drying as a model for the action of stress factors on the natural bacterial population}; Rokitko PV et al.; The drying of soil samples reduced the abundance (especially of predominant species) and the diversity of bacteria isolated from these samples, making easier the isolation of rare bacterial species . Some bacterial species that were minor before soil drying became dominant in dried soil samples . In general, soil drying allowed the diversity of soil bacteria to be determined more adequately . The bacteria that were isolated from dried soil samples turned out to be resistant to gamma radiation (with LD90 = 2.8-4.6 kGy) and desiccation . It is concluded that soil drying may serve as a model for the action of stress factors on natural bacterial populations . The hypothesis that periodic desiccation was the primary cause of formation of bacterial radioresistance in nature is discussed.

Hepatology, 2004 Feb, 39(2), 484 - 91
A sequential study of serum bacterial DNA in patients with advanced cirrhosis and ascites; Frances R et al.; Bacterial translocation is currently considered the main pathogenic mechanism leading to spontaneous bacterial peritonitis in patients with advanced cirrhosis and ascites . However, to the authors' knowledge there is no information regarding the characteristics of this process in humans . The goals of the current study were to pursue partially identified bacterial DNA in blood (what the authors consider molecular evidence of bacterial translocation) through its relative quantification in a 72-hour study period by using real-time polymerase chain reaction (PCR) . A consecutive series of 17 patients with advanced cirrhosis and culture-negative, nonneutrocytic ascites were studied . Therapeutic paracentesis was performed at the time of admission, and blood samples were obtained at baseline and every 8 hours in a 3-day period . Bacterial DNA was detected by a PCR-based method, relatively quantified by real-time PCR, and identified by automated nucleotide sequencing . Seven of 17 patients demonstrated the simultaneous presence of bacterial DNA in blood and ascitic fluid at the time of admission . After therapeutic paracentesis was performed, bacterial DNA persisted in the blood for a minimum of 24 hours, and was reported to last as long as 72 hours in some patients . In addition, different patterns of bacterial DNA appearance and clearance from the blood were identified . The nucleotide sequencing process demonstrated that bacteria detected in the first sample were identical to those noted in subsequent detections over time . In conclusion, bacterial translocation is a single-species, dynamic process that appears to develop in a subgroup of patients with advanced cirrhosis.

Appl Environ Microbiol, 2004 Feb, 70(2), 845 - 9
New perspective on uncultured bacterial phylogenetic division OP11; Harris JK et al.; Organisms belonging to the OP11 candidate phylogenetic division of Bacteria have been detected only in rRNA-based sequence surveys of environmental samples . Preliminary studies indicated that such organisms represented by the sequences are abundant and widespread in nature and highly diverse phylogenetically . In order to document more thoroughly the phylogenetic breadth and environmental distribution of this diverse group of organisms, we conducted further molecular analyses on environmental DNAs . Using PCR techniques and primers directed toward each of the five described subdivisions of OP11, we surveyed 17 environmental DNAs and analyzed rRNA gene sequences in 27 clonal libraries from 14 environments . Ninety-nine new and unique sequences were determined completely, and approximately 200 additional clones were subjected to partial sequencing . Extensive phylogenetic comparisons of the new sequences to those representing other bacterial divisions further resolved the phylogeny of the bacterial candidate division OP11 and identified two new candidate bacterial divisions, OP11-derived 1 (OD1) and Sulphur River 1 (SR1) . The widespread environmental distribution of representatives of the bacterial divisions OD1, OP11, and SR1 suggests potentially conspicuous biogeochemical roles for these organisms in their respective environments . The information on environmental distribution offers clues for attempts to culture landmark representatives of these novel bacterial divisions, and the sequences are specific molecular signatures that provide for their identification in other contexts.

Nature, 2004 Feb 5, 427(6974), 561 - 5
Crystal structure and mechanism of a bacterial fluorinating enzyme; Dong C et al.; Fluorine is the thirteenth most abundant element in the earth's crust, but fluoride concentrations in surface water are low and fluorinated metabolites are extremely rare . The fluoride ion is a potent nucleophile in its desolvated state, but is tightly hydrated in water and effectively inert . Low availability and a lack of chemical reactivity have largely excluded fluoride from biochemistry: in particular, fluorine's high redox potential precludes the haloperoxidase-type mechanism used in the metabolic incorporation of chloride and bromide ions . But fluorinated chemicals are growing in industrial importance, with applications in pharmaceuticals, agrochemicals and materials products . Reactive fluorination reagents requiring specialist process technologies are needed in industry and, although biological catalysts for these processes are highly sought after, only one enzyme that can convert fluoride to organic fluorine has been described . Streptomyces cattleya can form carbon-fluorine bonds and must therefore have evolved an enzyme able to overcome the chemical challenges of using aqueous fluoride . Here we report the sequence and three-dimensional structure of the first native fluorination enzyme, 5'-fluoro-5'-deoxyadenosine synthase, from this organism . Both substrate and products have been observed bound to the enzyme, enabling us to propose a nucleophilic substitution mechanism for this biological fluorination reaction.

J Immunol, 2004 Feb 15, 172(4), 2382 - 8
The crystal structure of human CD1b with a bound bacterial glycolipid; Batuwangala T et al.; The human MHC class I-like molecule CD1b is distinctive among CD1 alleles in that it is capable of presenting a set of glycolipid species that show a very broad range of variation in the lengths of their acyl chains . A structure of CD1b complexed with relatively short acyl chain glycolipids plus detergent suggested how an interlinked network of channels within the Ag-binding groove could accommodate acyl chain lengths of up to 80 carbons . The structure of CD1b complexed with glucose monomycolate, reported in this study, confirms this hypothesis and illustrates how the distinctive substituents of intracellular bacterial glycolipids can be accommodated . The Ag-binding groove of CD1b is, uniquely among CD1 alleles, partitioned into channels suitable for the compact accommodation of lengthy acyl chains . The current crystal structure illustrates for the first time the binding of a natural bacterial lipid Ag to CD1b and shows how its novel structural features fit this molecule for its role in the immune response to intracellular bacteria.

Arq Gastroenterol, 2003 Apr-Jun, 40(2), 104 - 9 Epub 2004 Jan 16.
{The effect of bacterial lipopolysaccharide on the gastric emptying of rats: a pretreatment evaluation using dexamethasone and methylene blue}; Collares EF et al.; BACKGROUND: The nitric oxide might be a putative mediator of the decrease in gastric emptying induced by bacterial lipopolysaccharide in rats . AIM: For that, we evaluated the effect of the pretreatment intravenous with dexamethasone and methylene blue in the retardation process of gastric emptying induced by intravenous application of lipopolysaccharide in rats . Dexamethasone has been shown to inhibit the induction of NOS II (induced NO-synthase) while the methylene blue, that blocks the soluble guanylyl cyclase, inhibits nitric oxide synthases and, in addition, inactivates nitric oxide directly . MATERIAL AND METHODS: Male Wistar rats, specific patogenic free, were used after a 24 hour fast and 1 hour-water withdrawn . The pretreatment was performed using dexamethasone solutions (3 and 6 mg/kg), methylene blue (2 mg/kg) or sterile vehicle . The treatment consisted in the application of lipopolysaccharide (50 mug/kg) or vehicle . The time period between the pretreatment and treatment was 10 minutes, excluding the study with dexamethasone 6 mg/kg that was 1 hour . The gastric emptying was evaluated 1 hour after the lipopolysaccharide application, except for two studies with dexamethasone 3 mg/kg in which the time periods were 2 and 8 hours . A saline solution containing phenol red was used as the test meal . The gastric emptying was determined by measuring gastric retention 10 minutes after the orogastric infusion of the test meal . RESULTS: The pretreatment with dexamethasone or methylene blue and treatment with vehicle did not have effect in the gastric emptying comparing to the control group . We found that pretreatment with dexamethasone in the studies for 1 hour and 2 hours did not interfere in the retardation of the gastric emptying produced by endotoxin . Nevertheless, in the eighth period study with this drug there was a significant reduction of gastric retention in the endotoxin-treated animals in relation to the unpretreated ones . Meanwhile, the pretreatment with the methylene blue completely blocked the action of endotoxin on the gastric emptying in rats . CONCLUSION: These results suggest a possible involvement of nitric oxide on the effect of lipopolysaccharide in rat gastric emptying.

J Perinatol, 2004 Feb, 24(2), 100 - 4
Does umbilical cord care in preterm infants influence cord bacterial colonization or detachment?
Evens K, George J, Angst D, Schweig L.
OBJECTIVE: The purpose of the present study was to compare alcohol versus natural drying for umbilical cord care in preterm infants and to examine its effects on bacterial colonization and cord detachment . STUDY DESIGN AND METHODS: Eligible infants <34 weeks gestation admitted to the NICU were randomized to receive either umbilical cleansing with 70% isopropyl alcohol at each diaper change or natural drying . Umbilical stump cultures were performed at 12 to 24 hours, 72 hours, 7 days, and 14 days of age . RESULTS: A total of 109 infants were enrolled; 102 completed the study . Analyses revealed that the median cord detachment time was significantly shorter in the natural drying group compared to the alcohol group (13.0 versus 16.0 days; p=0.003) . There were no cases of local umbilical infection in either group . CONCLUSION: Based on the present study, it appears that natural drying is a safe and effective means of umbilical cord care in preterm infants.

Fungal Genet Biol, 2004 Mar, 41(3), 369 - 80
Construction of a bacterial artificial chromosome library, determination of genome size, and characterization of an Hsp70 gene family in Phytophthora nicotianae; Shan W et al.; The oomycete plant pathogen Phytophthora nicotianae causes diseases on a wide range of plant species . To facilitate isolation and functional characterization of pathogenicity genes, we have constructed a large-insert bacterial artificial chromosome (BAC) library using nuclear DNA from P . nicotianae H1111 . The library contains 10,752 clones with an average insert size of 90 kb and is free of mitochondrial DNA . The quality of the library was verified by hybridization with 37 genes, all of which resulted in the identification of multiple positive clones . The library is estimated to be 10.6 haploid genome equivalents based on hybridization of 23 single-copy genes and the genome size of P . nicotianae was estimated to be 95.5 Mb . Hybridization with a nuclear repetitive DNA probe revealed that 4.4% of clones in the library contained 28S rDNA . Hybridization of total genomic DNA to the library indicated that at least 39% of the BAC library contains repetitive DNA sequences . A BAC pooling strategy was developed for efficient library screening . The library was used to identify and characterize BAC clones containing an Hsp70 gene family whose four members were identified to be clustered within approximately 18 kb in the P . nicotianae genome based on the physical mapping of eight BACs spanning a genomic region of approximately 186 kb . The BAC library created provides an invaluable resource for the isolation of P . nicotianae genes and for comparative genomics studies.

Chemosphere, 2004 Apr, 55(2), 197 - 205
Total bacterial and fungal population after chlorpyrifos and quinalphos treatments in groundnut (Arachis hypogaea L.) soil; Pandey S et al.; Short-term inhibitory effect on the total bacterial population was observed after chlorpyrifos and quinalphos applications in the groundnut fields, which recovered within 60 days after seed treatment and by 45 days of soil treatment . The fungal population was significantly enhanced after chlorpyrifos treatment whereas quinalphos inhibited the fungal population during the initial days of treatment but no effect was observed after 60 days of treatment . The residues of chlorpyrifos and quinalphos in the treated soil were not persistent and their half-lives ranged from 7.0 to 9.2 days and 13.2 to 20.6 days, respectively.

J Biol Chem, 2004 Apr 23, 279(17), 17217 - 23 Epub 2004 Feb 02.
Activation of RAW264.7 macrophages by bacterial DNA and lipopolysaccharide increases cell surface DNA binding and internalization; McCoy SL et al.; Bacterial DNA containing unmethylated CpG motifs is a pathogen-associated molecular pattern (PAMP) that interacts with host immune cells via a toll-like receptor (TLR) to induce immune responses . DNA binding and internalization into cells is independent of TLR expression, receptor-mediated, and required for cell activation . The objective of this study was to determine whether exposure of immune cells to bacterial DNA affects DNA binding and internalization . Treatment of RAW264.7 cells with CpG oligodeoxynucleotide (ODN) for both 18 and 42 h resulted in a significant increase in DNA binding, whereas non-CpG ODN had no effect on DNA binding . Enhanced DNA binding was non-sequence-specific, inhibited by unlabeled DNA, showed saturation, was consistent with increased cell surface DNA receptors, and resulted in enhanced internalization of DNA . Treatment with Escherichia coli DNA or lipopolysaccharide (LPS) also resulted in a significant increase in DNA binding, but treatment with interleukin-1alpha, tumor necrosis factor-alpha, or phorbol 12-myristate 13-acetate had no effect on DNA binding . Soluble factors produced in response to treatment with CpG ODN or LPS did not affect DNA binding . These studies demonstrate that one consequence of activating the host innate immune response by bacterial infection is enhanced binding and internalization of DNA . During this period of increased DNA internalization, RAW264.7 cells were hypo-responsive to continued stimulation by CpG ODN, as assessed by tumor necrosis factor-alpha activity . We speculate the biological significance of increasing DNA binding and internalization following interaction with bacterial PAMPs may provide a mechanism to limit an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular environment.

J Mol Biol, 2004 Feb 13, 336(2), 331 - 42
Functional compatibility of elongation factors between mammalian mitochondrial and bacterial ribosomes: characterization of GTPase activity and translation elongation by hybrid ribosomes bearing heterologous L7/12 proteins; Terasaki M et al.; The mammalian mitochondrial (mt) ribosome (mitoribosome) is a bacterial-type ribosome but has a highly protein-rich composition . Almost half of the rRNA contained in the bacterial ribosome is replaced with proteins in the mitoribosome . Escherichia coli elongation factor G (EF-G Ec) has no translocase activity on the mitoribosome but EF-G mt is functional on the E.coli ribosome . To investigate the functional equivalency of the mt and E.coli ribosomes, we prepared hybrid mt and E.coli ribosomes . The hybrid mitoribosome containing E.coli L7/12 (L7/12 Ec) instead of L7/12 mt clearly activated the GTPase of EF-G Ec and efficiently promoted its translocase activity in an in vitro translation system . Thus, the mitoribosome is functionally equivalent to the E.coli ribosome despite their distinct compositions . The mt EF-Tu-dependent translation activity of the E.coli ribosome was also clearly enhanced by replacing the C-terminal domain (CTD) of L7/12 Ec with the mt counterpart (the hybrid E.coli ribosome) . This strongly indicates that the CTD of L7/12 is responsible for EF-Tu function . These results demonstrate that functional compatibility between elongation factors and the L7/12 protein in the ribosome governs its translational specificity.

Sex Transm Infect, 2004 Feb, 80(1), 63 - 7
Diagnosis of bacterial vaginosis: need for validation of microscopic image area used for scoring bacterial morphotypes; Larsson PG et al.; BACKGROUND: The diagnosis of bacterial vaginosis (BV) is often made according to Nugent's classification, a scoring system based on bacterial counting of Gram stained slides of vaginal secretion . However as the image area of the microscope field will influence the number of morphotypes seen there is a need to standardise the area . METHODS: A graph intended for recalculation of number of bacterial morphotypes seen by the observer using 1000 x magnification from various microscope set-ups was constructed and applied to data sets typical for scoring BV . The graph was used in recalculation of Nugent scores, which were also compared with the Ison/Hay scores to evaluate the consequences for the diagnosis of BV . RESULTS: The observed image area differed by 300% among the investigated microscope set-ups . In two different data sets, one treatment study and one screening study, a considerable change in the number of women classified as intermediate was seen when the graph was used to standardise the image area . The recalculated numbers were also compared to the Ison/Hay classification . Weighted kappa indexes between the different methods were 0.84, 0.88, and 0.90, indicating that the methods are comparable . CONCLUSION: Because of the considerable differences among image areas covered by different microscope set-ups used in Nugent and Ison/Hay scoring, there is a need to standardise the area in order to reach comparable scores reflecting the diagnosis of BV in different laboratories . The differences in the intermediate group will have a considerable effect on the results from both treatment and prevalence studies, even though the kappa indexes indicate very good agreement between the methods used.

Sex Transm Infect, 2004 Feb, 80(1), 58 - 62
Social and sexual risk factors for bacterial vaginosis; Smart S et al.; BACKGROUND: A number of sexual and social risk factors for bacterial vaginosis (BV) have been identified . However, many previous studies have used small numbers of patients, or highly selected or convenience samples, or poorly defined populations . This study aims to clarify potential sexual and non-sexual risk factors for BV . METHODS: Women attending the Sydney Sexual Health Centre with BV, between March 1991 and July 1999, were included . Controls were randomly selected women without BV . Information on the demographics, clinical findings, and sexual and non-sexual risk behaviours were extracted from the clinic database and analysed using SPSS and SAS . A logistic regression model was used to establish which associations with BV persisted . RESULTS: 890 women with BV and 890 controls were studied . Factors that were independently associated with BV were > or =3 male sexual partners in the past 12 months (OR = 1.60, 95% CI: 1.19 to 2.04), at least one female sexual partner in the past 12 months (OR = 2.1, p = 0.003), a past pregnancy (OR = 1.5, p<0.0006), and smoking . In contrast, women with BV were significantly less likely to have used hormonal contraception (OR = 0.60, 95% CI: 0.51 to 0.81) or to have used condoms consistently (OR = 0.5, 95% CI: 0.31 to 0.71) than controls . CONCLUSION: Our findings may be important for planning a preventive strategy for BV by discouraging smoking and increasing condom use and hormonal contraception among women.

Sex Transm Infect, 2004 Feb, 80(1), 8 - 11
Managing recurrent bacterial vaginosis; Wilson J; Bacterial vaginosis (BV) is the most frequently found condition of the female genital tract . It increases a woman's risk of acquiring HIV, is associated with increased complications in pregnancy, and may be involved in the pathogenesis of pelvic inflammatory disease . Yet there are many unanswered questions about its aetiology, making management of recurrent infection difficult and often idiosyncratic . This paper discusses the current knowledge and possible management of recurrent BV.

Bioinformatics, 2004 Mar 22, 20(5), 798 - 9 Epub 2004 Jan 29.
In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction; Bikandi J et al.; We have developed a website, which runs a software program that performs three basic tasks in completely sequenced bacterial genomes by in silico analysis: PCR amplification, amplified fragment length polymorphism (AFLP-PCR) and endonuclease restriction . For PCR, after selection of the genome and introduction of primers, fragment size, DNA sequence and corresponding open reading frame (ORF) identity of the resulting PCR product is computed . Plasmids of sequenced species may be included in the analysis . Theoretical AFLP-PCR analyzes similar parameters, and includes a suggestion tool providing a list of commercial restriction enzyme pairs yielding up to 50 amplicons in the selected genome . Endonuclease restriction analysis of complete genomes and plasmids calculates the number of restriction sites for endonucleases in a given genome . If the number of fragments is 50 or fewer, pulsed field gel electrophoresis image and restriction maps are illustrated . Other tools that have been included in this site are ORF search by name and DNA to protein translation as well as restriction digestion of user-defined DNA sequences . AVAILABILITY: This is a new molecular biology resource freely available over the Internet at http://www.in-silico.com

Anal Chem, 2004 Feb 1, 76(3), 599 - 603
Reagentless identification of single bacterial spores in aqueous solution by confocal laser tweezers Raman spectroscopy; Chan JW et al.; We demonstrate that optical trapping combined with confocal Raman spectroscopy using a single laser source is a powerful tool for the rapid identification of micrometer-sized particles in an aqueous environment . Optical trapping immobilizes the particle while maintaining it in the center of the laser beam path and within the laser focus, thus maximizing the collection of its Raman signals . The single particle is completely isolated from other particles and substrate surfaces, therefore eliminating any unwanted background signals and ensuring that information is collected only from the selected, individual particle . In this work, an inverted confocal Raman microscope is combined with optical trapping to probe and analyze bacterial spores in solution . Rapid, reagentless detection and identification of bacterial spores with no false positives from a complex mixed sample containing polystyrene and silica beads in aqueous suspension is demonstrated . In addition, the technique is used to analyze the relative concentration of each type of particle in the mixture . Our results show the feasibility for incorporating this technique in combination with a flow cytometric-type scheme in which the intrinsic Raman signatures of the particles are used instead of or in addition to fluorescent labels to identify cells, bacteria, and particles in a wide range of applications.

Biophys J, 2004 Feb, 86(2), 825 - 35
Anion pathway and potential energy profiles along curvilinear bacterial ClC Cl- pores: electrostatic effects of charged residues; Miloshevsky GV et al.; X-ray structures permit theoretical study of Cl(-) permeation along bacterial ClC Cl(-) pores . We determined the lowest energy curvilinear pathway, identified anion-coordinating amino acids, and calculated the electrostatic potential energy profiles . We find that all four bacterial ClC Cl(-) crystal structures correspond to closed states . E148 and S107 side chains form steric barriers on both sides of the crystal binding site in the StClC wild-type and EcClC wild-type crystals; both the EcClC(E148A) and EcClC(E148Q) mutants are blocked at the S107 site . We studied the effect that mutating the charge of some strongly conserved pore-lining amino acids has on the electrostatic potential energy profiles . When E148 is neutralized, it creates an electrostatic trap, binding the ion near midmembrane . This suggests a possible electrostatic mechanism for controlling anion flow: neutralize E148, displace the side chain of E148 from the pore pathway to relieve the steric barrier, then trap the anion at midmembrane, and finally either deprotonate E148 and block the pore (pore closure) or bring a second Cl(-) into the pore to promote anion flow (pore conductance) . Side-chain displacement may arise by competition for the binding site between the oxygens of E148 and the anion moving down the electrostatic energy gradient . We also find that the charge state of E111 and E113 may electrostatically control anion conductance and occupancy of the binding site within the cytoplasmic pore.

Ann Surg, 2004 Feb, 239(2), 257 - 64
Enteral administration of high-fat nutrition before and directly after hemorrhagic shock reduces endotoxemia and bacterial translocation; Luyer MD et al.; OBJECTIVE: To determine whether potential enhancement of endotoxin neutralization via high-fat enteral nutrition affects endotoxemia and bacterial translocation after hemorrhage . SUMMARY BACKGROUND DATA: Endotoxin and bacterial translocation due to gut barrier failure are important initiating events in the pathogenesis of sepsis after hemorrhage . Systemic inhibition of endotoxin activity attenuates bacterial translocation and distant organ damage . Triacylglycerol-rich lipoproteins constitute a physiological means of binding and neutralizing endotoxin effectively . We hypothesized that enhancement of triacylglycerol-rich lipoproteins via high-fat enteral nutrition would reduce endotoxemia and prevent bacterial translocation . METHODS: A rat model of nonlethal hemorrhagic shock was used . Hemorrhagic shock (HS) rats were divided into 3 groups: rats starved overnight (HS-S); rats fed with a low-fat enteral diet (HS-LF), and rats receiving a high-fat enteral diet (HS-HF) . RESULTS: Circulating triacylglycerol and apolipoprotein B, reflecting the amount of triacylglycerol-rich lipoproteins, were elevated in HS-HF rats compared with both HS-S rats (P <or= 0.005 and P <or= 0.05, respectively) and HS-LF rats (P <or= 0.005 and P <or= 0.05) . Circulating endotoxin was lower in HS-HF rats (7.2 +/- 10.2 pg/ml) compared with both HS-S rats (29.1 +/- 13.4 pg/ml, P <or= 0.005) and HS-LF rats (29.9 +/- 5.2 pg/ml, P <or= 0.005) . In line, bacterial translocation was lower in HS-HF rats (incidence 4/8 rats; median 3 {range 0-144} cfu/g) compared with both HS-S rats (8/8; 212 {60-483} cfu/g; P = 0.006), and HS-LF rats (8/8; 86 {30-209} cfu/g; P = 0.002) . CONCLUSION: This study is the first to show that high-fat enteral nutrition, leading to increased plasma triacylglycerol and apolipoprotein B levels, significantly decreases endotoxemia and bacterial translocation after hemorrhage.

Acta Microbiol Pol, 2003, 52(3), 245 - 60
Rearrangements between differently replicating DNA strands in asymmetric bacterial genomes; Mackiewicz D et al.; Many bacterial genomes are under asymmetric mutational pressure which introduces compositional asymmetry into DNA molecule resulting in many biases in coding structure of chromosomes . One of the processes affected by the asymmetry is translocation changing the position of the coding sequence on chromosome in respect to the orientation on the leading and lagging DNA strand . When analysing sets of paralogs in 50 genomes, we found that the number of observed genes which switched their positions on DNA strand is lowest for genomes with the highest DNA asymmetry . However, the number of orthologs which changed DNA strand increases with the phylogenetic distance between the compared genomes . Nevertheless, there is a fraction of coding sequences that stay on the leading strand in all analysed genomes, whereas there are no sequences that stay always on the lagging strand . Since sequences diverge very fast after switching the DNA strand, this bias in mobility of sequences is responsible, in part, for higher divergence rates among some of coding sequences located on the lagging DNA strand.

Acta Microbiol Pol, 2003, 52(3), 217 - 31
The properties and functions of bacterial aminopeptidases; Jankiewicz U et al.; Aminopeptidases are enzymes that release N-terminal amino residues from oligopeptides, polypeptides and proteins . The classification of aminopeptidases has often been based on mechanism of catalysis, structure of active site, substrate specificity kinetic and molecular properties . In terms of catalytic mechanism bacterial aminopeptidases can be divided into three main catalytic groups: metallo-, cysteine- and serine aminopeptidases . According to their substrate specificity the enzymes can be ordered into two sub-groups: having broad or narrow specificity . Almost half of the characterized aminopeptidases show a subunit structure . Enzymes having a quaternary structure are most often built of a combination of 2, 4, 6 subunits . Bacterial aminopeptidases may be localised in the cytoplasm, on membranes, associated with the envelope or secreted into the extracellular media . Regulation of the synthesis of aminopeptidases is assumed to take place mainly at the level of transcription . Most genes encoding the enzymes are monocistronic and contain a promotor characteristic for the genes transcribed by RNA polymerase associated with the factor sigma70 . Aminopeptidases play an important role in the initial and final steps of protein turnover and they are involved in several specific regulatory functions.

J Orthop Trauma, 2004 Feb, 18(2), 92 - 5
Head injury-associated bone fractures induce bacterial translocation: an experimental study; Oztuna V et al.; OBJECTIVES: To determine whether long bone fractures cause bacterial translocation and to investigate the effect of concomitant head trauma on this process . DESIGN: An in vivo animal model . SETTING: Animal Laboratory, University of Mersin School of Medicine, Mersin, Turkey . SUBJECTS: Male Sprague-Dawley rats (n = 60) . INTERVENTION: Sixty male Sprague-Dawley rats were divided into five groups: (1) . anesthesia only (control group, n = 12); (2) . anesthesia and tibia fracture (n = 12); (3) . anesthesia, tibia fracture, and femur fracture (n = 12); (4) . anesthesia, tibia fracture, femur fracture, and moderate head trauma (n = 12); and (5) . moderate head trauma only (n = 12) . After 24 hours, mesenteric lymph nodes, liver, spleen, ileum, and systemic blood samples were quantitatively cultured for aerobic organisms . MAIN OUTCOME MEASUREMENTS: Colony-forming unit per gram for bacteria count . RESULTS: The incidence of bacterial translocation was higher in groups that had fractures (4/12 in group 2; 5/12 in group 3) than in the control group (2/12); however, this did not reach statistical significance . There was a significant increase in the number of subjects with bacterial translocation in group 4 (9/12) compared with the control group and group 5 (3/12) (P = 0.0123, P = 0.0391) . CONCLUSIONS: Multiple fractures of long bones associated with head injury promote bacterial translocation.

Transplantation, 2004 Jan 27, 77(2), 177 - 83
Administration of nitric oxide with caspase inhibitors minimizes bacterial translocation in experimental intestinal transplantation; Azuara D et al.; BACKGROUND: Bacterial translocation (BT) has been suggested to be responsible for the high incidence of infections occurring after small-bowel transplantation (Trp) . Nitric oxide (NO) and apoptosis could affect cell demise . The aim of this study was to asses whether supplementation of University of Wisconsin (UW) solution with NO donors and apoptosis inhibitors can abolish BT in Trp . METHODS: The following experimental groups were studied: sham, Trp, intestinal transplantation, Trp+spermine NONOate (NONOs), and Trp+NONOs+caspase inhibitor Z-Val-Ala-Asp(Ome)-fluoromethylketone(Z-VAD-fmk) . Histologic analysis, caspase-3 activity, DNA fragmentation, and BT from graft to mesenteric lymph nodes, liver, and spleen were measured in tissue samples after transplantation . RESULTS: During intestinal transplantation, apoptosis and necrosis were increased, showing graft injury and high levels of BT . The rats treated with NONOs showed a histologic protection of transplanted graft and a decrease in BT despite caspase-3 and DNA fragmentation-inducing effects . Administration of caspase inhibitor Z-VAD to NONOs-treated rats reversed the NO apoptosis-inducing effects and showed the lowest levels of BT in all tissues . CONCLUSIONS: Exogenous administration of NO associated with the inhibition of apoptosis maintains the graft in optimal conditions in terms of BT and improves the histology of the graft after intestinal transplantation in rats.

Xenobiotica, 2004 Jan, 34(1), 49 - 60
Optimizing bacterial expression of catalytically active human cytochromes P450: comparison of CYP2C8 and CYP2C9; Boye SL et al.; 1 . Methods for the co-expression in Escherichia coli of human cytochrome P450 (CYP) 2C8 and CYP2C9 with NADPH-cytochrome P450 reductase (OxR) to produce a catalytically active system were compared . 2 . Approaches assessed were expression of a CYP:OxR fusion construct, bicistronic plasmids, simultaneous transformation with CYP and OxR plasmids, and separate expression of CYP and OxR with reconstitution of activity by mixing the bacterial membranes . Two N-terminal modifications (Delta3-20 and 17alpha-leader) of the individual P450s were additionally investigated . 3 . Each approach gave efficient expression of CYP2C8 and CYP2C9, but the bicistronic constructs under the expression conditions used gave low OxR expression and low catalytic activity . CYP expression was higher with the Delta3-20 construct for CYP2C9 and with the 17alpha-presequence construct for CYP2C8 . 4 . Using torsemide as substrate, all methods gave catalytically active systems with K(m) values similar to human liver microsomes . Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the Delta3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17alpha- presequence constructs . OxR co-expressed with CYP in the same membrane interacted with CYP to reconstitute activity more effectively than addition of exogenous OxR membranes . 5 . Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.

Biomaterials, 2004 May, 25(11), 2029 - 37
Role of chemical interactions in bacterial adhesion to polymer surfaces; Speranza G et al.; Development of biomaterial-related infections is attracting an increasing interest due to the significant percentage of implant failure in the hospital care . Recent literature puts in evidence the dependence of the infection risk on the different biomaterials used, because of the different interactions between material surface and micro-organisms . Despite this, the mechanisms underlying the adhesion of bacteria to the biomaterial surface are still unclear . Aim of this work is to study the initial events of the processes responsible for the bacterial adhesion on polymers in order to prevent the development of bacterial infections and the consequent failure and replacement of biomedical devices . Electrostatic and Lifshitz-van der Waals forces are usually considered responsible for the interactions at the biomaterial interface . A new term that involves Lewis acid-base interactions is here introduced to better describe the bacterial adhesion to the polymer surface . Two requirements are needed to test this hypothesis: the development of an ideal polymeric surface in terms of chemical and morphological properties and the choice of a specific bacterial strain to be utilized as "probe" . Experiments were worked out using an Escherichia coli (Gram-) strain that represent one of the principal isolates from infected biomaterial implants and its adhesion was investigated on polymers having different acid/basic character . The findings indicate that the bacterial adhesion is influenced by the chemical properties of the polymeric surface . These results may be interpreted taking into account a mechanism in which the acid/base (Lewis) interaction plays an important role.

Biochim Biophys Acta, 2004 Jan 30, 1608(1), 11 - 22
Disruption of a specific molecular interaction with a bound lipid affects the thermal stability of the purple bacterial reaction centre; Fyfe PK et al.; Relatively little is known about the functions of specific molecular interactions between membrane proteins and membrane lipids . The structural and functional consequences of disrupting a previously identified interaction between a molecule of the diacidic lipid cardiolipin and the purple bacterial reaction centre were examined . Mutagenesis of a highly conserved arginine (M267) that is responsible for binding the head-group of the cardiolipin (to leucine) did not affect the rate of photosynthetic growth, the functional properties of the reaction centre, or the X-ray crystal structure of the complex (determined to a resolution of 2.8 A) . However, the thermal stability of the protein was compromised by this mutation, part of the reaction centre population showing an approximately 5 degrees C decrease in melting temperature in response to the arginine to leucine mutation . The crystallised mutant reaction centre also no longer bound detectable amounts of cardiolipin at this site . Taken together, these observations suggest that this particular protein-lipid interaction contributes to the thermal stability of the complex, at least when in detergent micelles . These findings are discussed in the light of proposals concerning the unfolding processes that occur when membrane proteins are heated, and we propose that one function of the cardiolipin is to stabilise the interaction between adjacent membrane-spanning alpha-helices in a region where there are no direct protein-protein interactions.

Bioorg Med Chem Lett, 2004 Feb 9, 14(3), 695 - 9
Synthesis and evaluation of novel bacterial rRNA-binding benzimidazoles by mass spectrometry; He Y et al.; A series of novel benzimidazoles were efficiently synthesized using both solution- and solid-phase chemistry . These compounds were found to bind to the bacterial 16S ribosomal RNA A-site with micromolar affinities using unique mass spectrometry-based assays.

J Bioenerg Biomembr, 2003 Oct, 35(5), 409 - 18
Molecular properties of purified human uncoupling protein 2 refolded from bacterial inclusion bodies; Jekabsons MB et al.; One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies . Problems encountered with this approach include protein refolding, homogeneity, and stability . In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2) . N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (Kd) of 0.3-0.5 and 23-92 microM . Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a Kd of greater than 100 microM, suggesting that the low affinity site reflected binding of the tag . By direct titration, UCP2 bound {8-(14)C} ATP and {8-(14)C} ADP with Kds of 4-5 and 16-18 microM, respectively . Mg2+ (2 mM) reduced the apparent ATP affinity to 53 microM, an effect entirely explained by chelation of ATP; with Mg2+, Kd using calculated free ATP was 3 microM . A combination of gel filtration, Cu2+-phenanthroline cross-linking, and ultracentrifugation indicated that 75-80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated . We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg2+-free nucleotides: the Kd for ATP is about 3-5 microM and for Mant-ATP it is about 10 times lower; and (c) in C12E9 detergent, the monodisperse protein may be in dimeric form.

Theor Appl Genet . 2004 Jan 23; {Epub ahead of print}
Mapping, genetic effects, and epistatic interaction of two bacterial canker resistance QTLs from Lycopersicon hirsutum; Coaker GL et al.; Two quantitative trait loci (QTL) from Lycopersicon hirsutum, Rcm 2.0 and Rcm 5.1, control resistance to Clavibacter michiganensis subsp . michiganensis ( Cmm) . To precisely map both loci, we applied interval mapping techniques to 1,056 individuals in three populations exhibiting F(2) segregation . Based on a 1-LOD confidence interval, Rcm 2.0 mapped to a 14.9-cM interval on chromosome 2 and accounted for 25.7-34.0% of the phenotypic variation in disease severity . Rcm 5.1 mapped to a 4.3-cM interval on chromosome 5 and accounted for 25.8-27.9% of the phenotypic variation . Progeny testing of recombinant plants narrowed the QTL location for Rcm 2.0 to a 4.4-cM interval between TG537-TG091 and to a 2.2-cM interval between CT202-TG358 for Rcm 5.1 . A population of 750 individuals exhibiting F(2) segregation was used to detect epistasis between both loci using ANOVA and orthogonal contrasts ( P=0.027), suggesting that resistance was determined by additive gene action and an additive-by-additive epistatic interaction . A partial diallel mating design was used to confirm epistasis, advance superior genotypes, randomize genetic backgrounds, and create recombination opportunities . This crossing scheme created a more balanced population ( n=112) containing the nine F(2) genotypic classes . Parents in the diallel were selected from the previous population based on resistance, genotype at the Rcm 2.0 and Rcm 5.1 loci, and horticultural traits . A replicated trial using the diallel population confirmed additive-by-additive epistasis ( P<0.0001) . These results validate the gene action, intra -locus interaction, and map position of two loci controlling resistance to Cmm.

Plant Physiol, 2004 Feb, 134(2), 595 - 604 Epub 2004 Jan 22.
Plant and bacterial symbiotic mutants define three transcriptionally distinct stages in the development of the Medicago truncatula/Sinorhizobium meliloti symbiosis; Mitra RM et al.; In the Medicago truncatula/Sinorhizobium meliloti symbiosis, the plant undergoes a series of developmental changes simultaneously, creating a root nodule and allowing bacterial entry and differentiation . Our studies of plant genes reveal novel transcriptional regulation during the establishment of the symbiosis and identify molecular markers that distinguish classes of plant and bacterial symbiotic mutants . We have identified three symbiotically regulated plant genes encoding a beta,1-3 endoglucanase (MtBGLU1), a lectin (MtLEC4), and a cysteine-containing protein (MtN31) . MtBGLU1 is down-regulated in the plant 24 h after exposure to the bacterial signal, Nod factor . The non-nodulating plant mutant dmi1 is defective in the ability to down-regulate MtBGLU1 . MtLEC4 and MtN31 are induced 1 and 2 weeks after bacterial inoculation, respectively . We examined the regulation of these two genes and three previously identified genes (MtCAM1, ENOD2, and MtLB1) in plant symbiotic mutants and wild-type plants inoculated with bacterial symbiotic mutants . Plant (bit1, rit1, and Mtsym1) and bacterial (exoA and exoH) mutants with defects in the initial stages of invasion are unable to induce MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1 . Bacterial mutants (fixJ and nifD) and a subset of plant mutants (dnf2, dnf3, dnf4, dnf6, and dnf7) defective for nitrogen fixation induce the above genes . The bacA bacterial mutant, which senesces upon deposition into plant cells, and two plant mutants with defects in nitrogen fixation (dnf1 and dnf5) induce MtLEC4 and ENOD2 but not MtN31, MtCAM1, or MtLB1 . These data suggest the presence of at least three transcriptionally distinct developmental stages during invasion of M . truncatula by S . meliloti.

Clin Exp Immunol, 2004 Feb, 135(2), 219 - 25
Dietary soy phytoestrogens and ERalpha signalling modulate interferon gamma production in response to bacterial infection; Curran EM et al.; Diets rich in soy phytoestrogens have many potential health benefits but isoflavones such as genistein may suppress cell mediated immune function . The effect of dietary phytoestrogens on the host response to infection has not been extensively examined . Mice were fed a diet containing soy phytoestrogens and infected with Mycobacterium avium to establish a chronic infection and inflammatory response . As phytoestrogens may act through classical oestrogen receptors (ER), mice deficient in ERalpha signalling and wild type mice were evaluated for a panel of Type 1-associated cytokines (IFNgamma, IL-12 and IL-18) in the spleen . IFNgamma production in the spleen was increased approximately 4-fold in ERalpha-deficient mice fed a casein-based diet over wild type mice fed a casein-based diet (P < 0.05), suggesting a role for ERalpha in suppressing IFNgamma production . IL-18 levels in spleens of wild type mice were decreased compared to ERalpha-deficient mice on a casein diet . Splenic IL-12 and IL-18 levels were not affected in wild type and ERalpha-deficient mice on the phytoestrogen containing diets, with the exception that whole soy increased IL-12 levels in the tissues of ERalpha deficient mice . We conclude that ERalpha and dietary phytoestrogens can influence production of key regulatory cytokines in response to chronic bacterial infection.

J Environ Monit, 2004 Jan, 6(1), 65 - 70 Epub 2003 Dec 02.
Quantification of bacterial lipopolysaccharides (endotoxin) by GC-MS determination of 3-hydroxy fatty acids; Binding N et al.; A GC-MS method for the quantification of bacterial lipopolysaccharides (LPS, endotoxin) is presented . After hydrolytic cleavage of 3-hydroxy fatty acids (3-OH FAs) from the lipid A region of LPS, derivatisation of both the hydroxyl and the carboxyl group was performed in one step with a mixture of methyl-bis(trifluoracetamide)(MBTFA) and N-methyl-N-(tert-butyldimethylsilyl)trifluoracetamide (MTBSTFA) . Using GC-MS in the EI mode with selected ion monitoring (SIM) for analysis, baseline separation of 3-OH FAs (and of possibly interfering 2-OH FAs) was achieved . The sensitivity of the method (LOD 7-50 pg/injection for the different 3-OH FAs investigated) allows for the efficient quantification of LPS in occupational and environmental samples . Degradation of 3-OH FAs as well as of their derivatives during sample preparation and GC-MS separation as a possible source of errors in analytical methods based on 3-OH FA determination is reported for the first time . Thermal elimination of water from the underivatised 3-OH FAs and of trifluoroacetic acid from the derivatives was identified as the cause of degradation . The resulting alpha,beta-unsaturated compounds showing the same mass spectra as the 3-OH FA derivatives were detected as more or less prominent satellite peaks . By using alkaline instead of acidic hydrolysis and cool on-column instead of split/splitless injection, elimination was reduced to an acceptable level.

J Med Chem, 2004 Jan 29, 47(3), 509 - 18
Inhibition of the bacterial enoyl reductase FabI by triclosan: a structure-reactivity analysis of FabI inhibition by triclosan analogues; Sivaraman S et al.; To explore the molecular basis for the picomolar affinity of triclosan for FabI, the enoyl reductase enzyme from the type II fatty acid biosynthesis pathway in Escherichia coli, an SAR study has been conducted using a series of triclosan analogues . Triclosan (1) is a slow, tight-binding inhibitor of FabI, interacting specifically with the E.NAD(+) form of the enzyme with a K(1) value of 7 pM . In contrast, 2-phenoxyphenol (2) binds with equal affinity to the E.NAD(+) (K(1) = 0.5 microM) and E.NADH (K(2) = 0.4 microM) forms of the enzyme and lacks the slow-binding step observed for triclosan . Thus, removal of the three triclosan chlorine atoms reduces the affinity of the inhibitor for FabI by 70,000-fold and removes the preference for the E.NAD(+) FabI complex . 5-Chloro-2-phenoxyphenol (3) is a slow, tight-binding inhibitor of FabI and binds to the E.NAD(+) form of the enzyme (K(1) = 1.1 pM) 7-fold more tightly than triclosan . Thus, while the two ring B chlorine atoms are not required for FabI inhibition, replacement of the ring A chlorine increases binding affinity by 450,000-fold . Given this remarkable observation, the SAR study was extended to the 5-fluoro-2-phenoxyphenol (4) and 5-methyl-2-phenoxyphenol (5) analogues to further explore the role of the ring A substituent . While both 4 and 5 are slow, tight-binding inhibitors, they bind substantially less tightly to FabI than triclosan . Compound 4 binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3.2 and 240 nM, respectively, whereas compound 5 binds exclusively to the E.NADH enzyme complex with a K(2) value of 7.2 nM . Thus, the ring A substituent is absolutely required for slow, tight-binding inhibition . In addition, pK(a) measurements coupled with simple electrostatic calculations suggest that the interaction of the ring A substituent with F203 is a major factor in governing the affinity of analogues 3-5 for the FabI complex containing the oxidized form of the cofactor.

J Cardiovasc Surg (Torino), 2003 Dec, 44(6), 685 - 9
Ultrastructural changes of cardiac valves in bacterial endocarditis; Mirzaie M et al.; AIM: The principal objective of this study was to document morphological changes in valves with acute endocarditis in order to gain further knowledge of the pathogenesis of these diseases . METHODS: Scanning and transmission electron microscopic investigations were carried out on explanted human heart valves to reveal ultrastructural changes due to bacterial endocarditis . RESULTS: Bacterial inflammation endocarditis initially induced metaplasia of the endothelial cells which then lose contact with each other . In the 2nd phase of the disease, the collagen fibres are systematically removed whereby large cavities appear . In the 3rd phase, localised hyperplasia of collagen fibres was observed often resulting in the development of vegetation . The ultrastructural changes are uniform and independent of the bacterial species . CONCLUSION: Bacterial endocarditis is therefore a set of complex interactions between endothelial cells and bacteria which should be taken into consideration for the development of new therapeutic approaches.

J Immunol, 2004 Feb 1, 172(3), 1515 - 23
Expression of dual TCR on DO11.10 T cells allows for ovalbumin-induced oral tolerance to prevent T cell-mediated colitis directed against unrelated enteric bacterial antigens; Zhou P et al.; The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora . Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4(+) T cells (Treg) cells . We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice . SCID-bg mice given DO11.10 CD4(+)CD45RB(high) T cells developed colitis, and cotransferring DO11.10 CD45RB(low)CD4(+) T cells prevented CD4(+)CD45RB(high) T cell-induced colitis in the absence of OVA . The induction and prevention of disease by DO11.10 CD4(+) T cell subsets were associated with an increase in endogenous TCRalpha chain expression on Tg T cells . Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4(+)CD45RB(high) attenuated the colitis in association with increased TGF-beta and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag . OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB(high) T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells . The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag . Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells . Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.

Clin Chim Acta, 2004 Feb, 340(1-2), 149 - 52
Procalcitonin levels in plasma in oncohaematologic patients with and without bacterial infections; Ciaccio M et al.; BACKGROUND: The flogosis markers currently in use show both low sensitivity and specificity, particularly in neoplastic and degenerative diseases . Procalcitonin (PCT) is a pro-peptide of calcitonin produced mainly but not only in the C-cells of the thyroid glands and, as several studies show, PCT levels in plasma increase during infections . Bacterial infections are also the main cause of death in oncological patients . Furthermore, in patients with leukaemia in chemotherapy recovery, infections often induce relapses . The aim of the present study is to detect PCT levels in plasma in oncohaematologic patients with and without infections . METHODS: The study was carried out on 54 patients by a quantitative automated immunoassay . RESULTS: PCT plasma levels > or =0.5 ng were detected in 27 out of 30 patients (90,0%) with bacterial infections; 8 out of 9 patients (88,9%) with viral infections and in 12 out of 15 patients in the control group without statistically significant differences . CONCLUSIONS: The results, which differ from those in the literature, are discussed.

Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 396 - 402
Screening of Hsp105alpha-binding proteins using yeast and bacterial two-hybrid systems; Saito Y et al.; Hsp105alpha is a 105-kDa stress protein, which is expressed constitutively at especially high levels in the brain compared with other tissues in mammals, and is also induced by a variety of stressors . Recently, we have shown that Hsp105alpha binds to alpha-tubulin and prevents the heat-induced disaggregation of microtubules . To further elucidate the function of Hsp105alpha, we searched for Hsp105alpha-binding proteins by screening a mouse FM3A cell library and human and mouse brain cDNA libraries using the yeast and bacterial two-hybrid systems . We showed here that Hsp105alpha interacted with several cellular proteins, such as cofilin, dynein light chain 2A, alpha-adducin, ubiquitin activating enzyme E1, phosphoglycerate kinase 1, and platelet-activating factor acethylhydrolase alpha1-subunit . The interaction was validated by the results of a pull-down assay and indirect immunofluorescence analysis . The significance of Hsp105alpha and Hsp105alpha-binding proteins in cells was discussed.

Acta Chir Plast, 2003, 45(3), 89 - 94
The influence of moisture wound healing on the incidence of bacterial infection and histological changes in healthy human skin after treatment of interactive dressings; Koupil J et al.; In this article the authors discuss the problem faced by physicians when trying to use moisture-retentive dressing in pressure sores (decubitus ulcers) . First, they report the results of an in vitro study using a new model of experimental wound (radio-isotopic investigation) that assesses the release of Ringer's solution from interactive dressings continually during fourteen hours . Second, they perform an animal experiment that assesses the incidence of wound infection in defects treated conventionally or using interactive dressings . The defects treated with interactive pads had lower incidence of wound infection, and the process of wound healing was rapid . Finally, the authors discuss their experience in four paraplegic patients with decubitus ulcers where they used moisture-retentive dressing on ulcers and on the surrounding intact skin before surgical procedure to detect the possibility of maceration of healthy skin . Histological evaluation was performed in order to find microscopically changes after moisture healing . The changes of healthy skin were not significant after treatment of moisture-retentive dressings.

Trends Cell Biol, 1993 Jan, 3(1), 20 - 5
Participation of the bacterial membrane in DNA replication and chromosome partition; Funnell BE; The concept that the bacterial membrane plays an active role in the regulation of DNA replication and in segregation, or 'partition', of the bacterial chromosome at cell division was proposed in 1963 . Membrane participation offered a relatively simple way to coordinate replication and partition . Some of the details of this model have been confirmed, while others have been changed . In fact, it appears that the membrane may play several distinct roles in these processes, and recent experiments have begun to identify the complexity of membrane involvement.

Mol Microbiol, 2004 Feb, 51(3), 711 - 20
Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A resolution; Hilsenbeck JL et al.; Colicin B (55 kDa) is a cytotoxic protein that recognizes the outer membrane transporter, FepA, as a receptor and, after gaining access to the cytoplasmic membranes of sensitive Escherichia coli cells, forms a pore that depletes the electrochemical potential of the membrane and ultimately results in cell death . To begin to understand the series of dynamic conformational changes that must occur as colicin B translocates from outer membrane to cytoplasmic membrane, we report here the crystal structure of colicin B at 2.5 A resolution . The crystal belongs to the space group C2221 with unit cell dimensions a = 132.162 A, b = 138.167 A, c = 106.16 A . The overall structure of colicin B is dumbbell shaped . Unlike colicin Ia, the only other TonB-dependent colicin crystallized to date, colicin B does not have clearly structurally delineated receptor-binding and translocation domains . Instead, the unique N-terminal lobe of the dumbbell contains both domains and consists of a large (290 residues), mostly beta-stranded structure with two short alpha-helices . This is followed by a single long ( approximately 74 A) helix that connects the N-terminal domain to the C-terminal pore-forming domain, which is composed of 10 alpha-helices arranged in a bundle-type structure, similar to the pore-forming domains of other colicins . The TonB box sequence at the N-terminus folds back to interact with the N-terminal lobe of the dumbbell and leaves the flanking sequences highly disordered . Comparison of sequences among many colicins has allowed the identification of a putative receptor-binding domain.

Mol Genet Genomics, 2004 Feb, 271(1), 111 - 20 Epub 2004 Jan 17.
Genome-wide analysis of defense-responsive genes in bacterial blight resistance of rice mediated by the recessive R gene xa13; Chu Z et al.; Defense responses triggered by dominant and recessive disease resistance (R) genes are presumed to be regulated by different molecular mechanisms . In order to characterize the genes activated in defense responses against bacterial blight mediated by the recessive R gene xa13, two pathogen-induced subtraction cDNA libraries were constructed using the resistant rice line IRBB13--which carries xa13--and its susceptible, near-isogenic, parental line IR24 . Clustering analysis of expressed sequence tags (ESTs) identified 702 unique expressed sequences as being involved in the defense responses triggered by xa13; 16% of these are new rice ESTs . These sequences define 702 genes, putatively encoding a wide range of products, including defense-responsive genes commonly involved in different host-pathogen interactions, genes that have not previously been reported to be associated with pathogen-induced defense responses, and genes (38%) with no homology to previously described functional genes . In addition, R-like genes putatively encoding nucleotide-binding site/leucine rich repeat (NBS-LRR) and LRR receptor kinase proteins were observed to be induced in the disease resistance activated by xa13 . A total of 568 defense-responsive ESTs were mapped to 588 loci on the rice molecular linkage map through bioinformatic analysis . About 48% of the mapped ESTs co-localized with quantitative trait loci (QTLs) for resistance to various rice diseases, including bacterial blight, rice blast, sheath blight and yellow mottle virus . Furthermore, some defense-responsive sequences were conserved at similar locations on different chromosomes . These results reveal the complexity of xa13-mediated resistance . The information obtained in this study provides a large source of candidate genes for understanding the molecular bases of defense responses activated by recessive R genes and of quantitative disease resistance.

Eur Surg Res, 2004 Jan-Feb, 36(1), 45 - 52
Effect of different enteral nutrients on bacterial translocation in experimental obstructive jaundice; Kuru B et al.; Obstructive jaundice leads to bacterial translocation (BT) by disruption of the gut barrier, intestinal microecology, and impaired host immune defence . The objective of the present study is to investigate the effects of different enteral nutrients on BT that is induced by obstructive jaundice in rats . Eighty male Wistar-Albino rats were randomly assigned into 4 groups . Group 1: 20 rats underwent laparotomy, common bile duct (CBD) was not actually ligated and transected, but sham ligation of CBD was performed . Groups 2-4: 60 rats underwent laparotomy, CBD ligation and transection . Group 1 and 2 rats were given rat chow, group 3 rats were fed a glutamine and arginine supplemented enteral diet, and group 4 rats were fed an arginine, m-RNA and omega-3 supplemented enteral diet, an immunonutrient . Rats in groups 3 and 4 had significantly less BT to mesenteric lymph nodes compared to rats in group 2 (p = 0.001) . These findings suggest that oral administration of an arginine and glutamine supplemented diet and immunonutrition reduce BT in rats with obstructive jaundice .

Biosci Biotechnol Biochem, 2003 Dec, 67(12), 2664 - 7
Stereochemistry of 2-phenylethylamine oxidation catalyzed by bacterial copper amine oxidase; Uchida M et al.; The stereochemical course of the reaction catalyzed by a copper amine oxidase from Arthrobacter globiformis has been investigated using 2-phenylethylamine stereospecifically deuterium-labeled at the C1 position . Measurements of deuterium content in the product, phenylacetaldehyde, by gas chromatography-mass spectrometry revealed stereospecific abstraction of the pro-S hydrogen during the enzymatic oxidation, as predicted from the structure modeling for the enzyme-bound substrate.

Biosci Biotechnol Biochem, 2003 Dec, 67(12), 2652 - 4
Cellulose as extracellular polysaccharide of hot spring sulfur-turf bacterial mat; Ogawa K et al.; The carbohydrate fraction of a hot spring sulfur-turf bacterial mat was shown to contain cellulose by the examination of neutral sugar composition, methylation analysis, and the identification of free oligosacchrides obtained from an acetolyzate of the desulfurized sulfur-turf mat . This suggested that the sulfur-oxidizing bacteria composing the sulfur-turf were producers of cellulose.

J Bacteriol, 2004 Feb, 186(3), 866 - 9
Transcriptional profiling of colicin-induced cell death of Escherichia coli MG1655 identifies potential mechanisms by which bacteriocins promote bacterial diversity; Walker D et al.; We report the transcriptional response of Escherichia coli MG1655 to damage induced by colicins E3 and E9, bacteriocins that kill cells through inactivation of the ribosome and degradation of chromosomal DNA, respectively . Colicin E9 strongly induced the LexA-regulated SOS response, while colicin E3 elicited a broad response that included the induction of cold shock genes, symptomatic of translational arrest . Colicin E3 also increased the transcription of cryptic prophage genes and other laterally acquired mobile elements . The transcriptional responses to both these toxins suggest mechanisms that may promote genetic diversity in E . coli populations, pointing to a more general role for colicins in adaptive bacterial physiology than has hitherto been realized.

J Bacteriol, 2004 Feb, 186(3), 811 - 7
The trans-acting protein interacting with the DNA motif proximal to the transcriptional start site of plant L-asparaginase is bacterial sarcosine oxidase; Jones WT et al.; A trans-acting protein interacting with a specific sequence motif proximal to the transcriptional start site of the L-asparaginase promoter has been observed previously (E . Vincze, J . M . Reeves, E . Lamping, K . J . F . Farnden, and P . H . S . Reynolds, Plant Mol . Biol . 26:303-311, 1994) . Gel retardation experiments in which protein extracts of Mesorhizobium loti and developing nodules were used suggested a bacterial origin for the repressor binding protein (rep2037) . Nodulation tests were performed by using different Fix(-) Tn5 mutants of M . loti . Analyses of these mutants revealed a correlation between the presence of Mesorhizobium in the nodule-like structures and the ability of nodule protein extracts to bind the repressor binding domain (RBD) . Through the use of mutated RBD sequences, the RBD sequence was identified as CTAAAAT . The repressor protein was isolated from M . loti NZP2037 by multiple chromatographic procedures and affinity separation by using concatemers of RBD attached to magnetic beads . Sequencing of the recovered protein resulted in identification of the repressor protein as the sarcosine oxidase alpha subunit . This was confirmed by expression of the gene encoding the M . loti alpha subunit of sarcosine oxidase in Escherichia coli . When the expressed peptide was bound to RBD, the gel retardation result was identical to the result obtained with rep2037 from M . loti strain NZP2037.

Injury, 2004 Jan, 35(1), 35 - 43
Bacterial translocation, endotoxaemia and apoptosis following Pringle manoeuvre in rats; Filos KS et al.; BACKGROUND: Intraoperative occlusion of the hepatoduodenal ligament (Pringle manoeuvre (Pm)) is often employed for the reduction of blood loss during liver surgery . No data exist to date on the effects of Pm on mucosal barrier dysfunction, systemic bacterial translocation (BT), endotoxaemia and apoptosis . MATERIALS AND METHODS: Sixty-five male Wistar rats in three groups: I (n=25) controls, II (n=20) sham operation, III (n=20) occlusion of the hepatoduodenal ligament (Pm) . Tissue samples from mesenteric lymph nodes (MLNs), liver, lungs and spleen were analysed after 30 min and at 24 h . Endotoxin was measured in portal and aortic blood and routine haematological and biochemical parameters were measured before and after Pm . RESULTS: No differences were found in the blood parameters before and after Pm, but a significant increase in contaminated MLNs and liver was noted . All cultured bacteria were enteric in origin . Portal and aortic endotoxin were significantly increased . Overall the ileal architecture remained intact in all specimens studied and no significant pathology was observed . The ABC increased after Pm significantly (P<0.01) . CONCLUSION: Normothermic Pm of 30 min duration results in immediate and delayed gut barrier failure by significantly increasing BT and endotoxaemia which might be attributed to portal stasis leading to intestinal congestion as well as temporary liver ischaemia . Apoptosis increased significantly 30 min after performing the Pm.

Insect Mol Biol, 2004 Feb, 13(1), 37 - 44
Characterization of an Aedes aegypti bacterial artificial chromosome (BAC) library and chromosomal assignment of BAC clones for physical mapping quantitative trait loci that influence Plasmodium susceptibility; Jimenez LV et al.; Previous studies have confirmed a genetic basis for susceptibility of mosquitoes to Plasmodium parasites . Here we describe our efforts to characterize a bacterial artificial chromosome genomic library for the yellow fever mosquito, Aedes aegypti, and to identify BAC clones containing genetic markers that define quantitative trait loci (QTL) for Plasmodium gallinaceum susceptibility . This library (NDL) was prepared from the Ae . aegypti Liverpool strain and consists of 50 304 clones arrayed in 384-well microplates . We used PCR analysis with oligonucleotide primer pairs specific to 106 genetic markers (as sequence-tagged sites or STS) to screen the NDL library . Each STS identified between one and thirteen independent clones with an average of 3.3 clones . The average insert size was 122 kb and therefore the NDL library provides approximately 7.87-fold genome coverage . The availability of the NDL library should greatly facilitate physical mapping efforts, including positional cloning of QTL for traits of interest such as Plasmodium susceptibility and for whole genome sequence determination and assembly.

Clin Infect Dis, 2004 Feb 1, 38(3), 384 - 90 Epub 2004 Jan 14.
Reducing intracranial pressure may increase survival among patients with bacterial meningitis; Lindvall P et al.; We reported findings concerning continuous intracranial pressure (ICP) and cerebral perfusion pressure (CPP) measurements and mortality in patients with severe bacterial meningitis treated on the basis of an ICP-targeted approach . Eighteen patients with severe bacterial meningitis were admitted for neurointensive care at Umea University Hospital (Umea, Sweden) . In 15 patients, ICP was measured continuously through an ICP measuring device . During care, all patients but one developed intracranial hypertension with an ICP of >or=15 mm Hg (14 {93%} of 15 patients) . Ten (67%) of 15 patients survived and were discharged, and 5 patients (33%) died . Mean ICP was significantly higher and CPP was markedly decreased in nonsurvivors, compared with survivors . Among the survivors, ICP was gradually reduced . Treatment of patients with severe bacterial meningitis should include neurointensive care and continuous ICP measurement . Increased ICP may be reduced by using the ICP-targeted therapy that closely resembles the "Lund concept."

Dev Cell, 2004 Jan, 6(1), 3 - 4
DNA segregation by bacterial actin homologs; Pogliano J; The bacterial actin homolog ParM catalyzes segregation of plasmid DNA in E . coli . Recent studies now suggest a model in which ParM forms actin-like filaments between two plasmid molecules, thereby providing the driving force for plasmid DNA separation.

Spine, 2004 Jan 15, 29(2), 164 - 9
Study of bacterial translocation from gut after paraplegia caused by spinal cord injury in rats; Liu J et al.; STUDY DESIGN: Normal rats and paraplegic rats with and injured spinal cord were used to study bacterial translocation from gut . OBJECTIVE: To investigate whether bacterial translocation from gut occurs after spinal cord injury . SUMMARY OF BACKGROUND DATA: It has been demonstrated that trauma and operation can lead to gastrointestinal paralysis; disturbance of gastrointestinal motility following trauma may cause bacterial overgrowth in gastrointestinal tract and increase the incidence of bacterial translocation . However, bacterial translocation from gut after spinal cord injury has not been studied . METHODS: Under aseptic manipulation, samples of blood were collected for bacterial cultures and endotoxin determination . In the meantime, samples of mesenteric lymph node, spleen, and liver were collected for bacterial culture . The jejunum and ileum were observed by light and electron microscope . RESULTS: Endotoxemia and bacterial translocation appeared 24 hours and 48 hours correspondingly after spinal cord injury complicated with paraplegia . CONCLUSION: Bacterial translocation from gut would occur after spinal cord injury in rats, which indicated that antibiotics should be administered to paraplegic patients with spinal cord injury as soon as possible to prevent potential bacterial translocation.

Exp Cell Res, 2004 Jan 1, 292(1), 231 - 40
Bacterial GroEL-like heat shock protein 60 protects epithelial cells from stress-induced death through activation of ERK and inhibition of caspase 3; Zhang L et al.; Bacterial heat shock proteins (hsps) can have various effects on human cells . We investigated whether bacterial hsp60s can protect epithelial cells from cell death by affecting the mitogen-activated protein kinase (MAPK) signal pathways . Cell protection was studied by adding bacterial hsp60s to skin keratinocyte cultures (HaCaT cell line) before UV radiation . The results show that hsp60 significantly protected against UV radiation-induced cell death . Effects of UV radiation and exogenous hsp60 on phosphorylation of MAPKs and on activation of caspase 3 were examined by Western blot analysis . UV radiation strongly induced phosphorylation of p38 MAPK and formation of active caspase 3 . A p38 inhibitor, SB 203580, totally blocked UV radiation-mediated activation of caspase 3 . Preincubation with hsp60 strongly induced phosphorylation of ERK1/2 and inhibited UV radiation-mediated activation of caspase 3 . PD 98059, a specific inhibitor of the ERK1/2 pathway, blocked this inhibitory effect of exogenous hsp60 . Studies on the association between activity of MAPKs or caspase 3 and cell death showed that the ERK1/2 pathway inhibitor reversed protective effect of hsp60 while specific inhibition of p38 and caspase 3 reduced cell death . These results indicate that in HaCaT cells UV radiation mediates cell death through activation of p38 followed by caspase 3 activation . Exogenous hsp60 partially protects against UV radiation-mediated epithelial cell death through activation of ERK1/2, which inhibits caspase 3 activation.

Obstet Gynecol Clin North Am, 2003 Dec, 30(4), 685 - 94
Gynecologic consequences of bacterial vaginosis; Schwebke JR; BV is a prevalent sexually associated infection linked to several gynecologic complications and acquisition of STDs and acquisition and transmission of HIV . It seems that normalization of the vaginal flora may be effective for preventing short-term complications . The implications of screening and treating BV to prevent long-term complications are less clear and may depend on the availability of more effective treatment regimens.

Mar Biotechnol (NY), 2003 Jul-Aug, 5(4), 380 - 7
Purification of sulfated fucoglucuronomannan lyase from bacterial strain of Fucobacter marina and study of appropriate conditions for its enzyme digestion; Sakai T et al.; A marine bacterial strain, Fucobacter marina, produced extracellular sulfated fucoglucuronomannan (SFGM) lyase when cultivated in the presence of crude SFGM obtained from fucoidan of Kjellmaniella crassifolia (brown algae) by cetyl pyridinium chloride fractionation . For the SFGM lyase assay, SFGM fraction separated from K . crassifolia fucoidan by anion exchange column chromatography was used as the substrate . The extracellular SFGM lyase was purified to homogeneity on an electrophoresis gel with 4240-fold purity at 13.8% yield . The enzyme proved to be a monomer, since gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis gave the same relative molecular mass of 67,000 . The enzyme specifically digested SFGM but did not digest any other uronic-acid-containing polysaccharides tested . The optimum conditions for the enzyme reaction were around pH 7.5, 43 degrees C, and 0.4 M NaCl concentration . The enzyme was strongly inhibited by CuCl(2) and ZnCl(2), and also by some sulfhydryl reagents.

Kidney Int, 2004 Feb, 65(2), 654 - 60
Icodextrin-induced peritonitis: study of five cases and comparison with bacterial peritonitis; Toure F et al.; BACKGROUND: An epidemic of aseptic peritonitis related to the presence of peptidoglycan contaminant in some batches of icodextrin solution (Extraneal, Baxter Healthcare Corporation) occurred in Europe in the first six months of 2002 . METHODS: By case-control study we examined the clinical and biologic features of 5 patients with icodextrin-induced peritonitis (group AP) and compared them with 7 patients with bacterial peritonitis (group BP) recruited in our clinical center between January and June 2002 . RESULTS: Diagnosis of icodextrin-induced peritonitis was confirmed in all cases by a positive reintroduction test with contaminated batches of icodextrin . No recurrence was observed on re-exposure to icodextrin free of peptidoglycan . Skin tests were positive with contaminated icodextrin in 2 of 5 patients, while they were negative with icodextrin solution free of peptidoglycan (<0.6 ng/mL) . During peritonitis, serum level of C-reactive protein (CRP) was lower in group AP (42.4 +/- 34 mg/L) than in group BP (135 +/- 59 mg/L) (P= 0.01) . Leukocyte number in peritoneal dialysis effluent was lower in group AP (284 +/- 101/mm3), with a lower neutrophil/monocyte ratio (N/M = 0.67) than in group BP (1410 +/- 973/mm3; N/M = 4) (P < 0.05) . A low number of peritoneal fluid eosinophilia (11 +/- 8%) was detected in group AP . CONCLUSION: Icodextrin-induced peritonitis was associated with a burst of intraperitoneal cytokines . The phenotype of peritoneal neutrophils was different between aseptic and bacterial peritonitis, indicating that inflammatory stimuli that activate neutrophils in both types of peritonitis are clearly distinct . Finally, peritoneal injury measured by weight gain, peritoneal permeability, and CA125 concentration seemed to be less severe during icodextrin-induced peritonitis than during bacterial peritonitis.

Clin Diagn Lab Immunol, 2004 Jan, 11(1), 216 - 8
Bacterial expression of a human monoclonal antibody-alkaline phosphatase conjugate specific for Entamoeba histolytica; Tachibana H et al.; We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli . In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E . coli was designed and constructed . The E . coli PhoA gene was fused to the 3' terminus of the gene encoding the heavy-chain Fd region . The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of E . histolytica, were used as human antibody genes . When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of E . histolytica and developed with a substrate, the trophozoites appeared to be stained . These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E . histolytica and that the antibody can be used to detect E . histolytica antigen without the use of chemically conjugated secondary antibodies.

Biomacromolecules, 2004 Jan-Feb, 5(1), 144 - 52
Comblike complexes of bacterial poly(gamma,d-glutamic acid) and cationic surfactants; Perez-Camero G et al.; The ability of microbally produced poly(gamma,d-glutamic acid) to form stable polyelectrolyte-opposite charged surfactant complexes was investigated . A sonicated sample of polyacid with a molecular weight about 10(5) Da and a content of d enantiomer higher than 90% was used in this study . Nearly stoichiometric complexes of poly(gamma,d-glutamate) anions and alkyltrimethylammonium cations bearing linear alkyl chains with even numbers of carbon atoms from 12 up to 22 were "synthesized" by precipitation from equimolar mixtures of aqueous solutions of the two components . All complexes were found to adopt stratified supramolecular structures made of alternating layers of poly(gamma,d-glutamate) and surfactant with a periodicity increasing from 3.2 up to 4.3 nm according to the length of the alkyl side chain . No definite evidence indicative of the conformation adopted by the main chain in these complexes could be afforded . In all cases, the alkyl chains are in an extended conformation and oriented normal or nearly normal to the layer planes . Polymethylene chains with more than 16 carbon atoms were partially crystallized in the complexes in a separated paraffinic phase, whereas no crystallinity was detected for shorter lengths . The crystallized paraffinic phases were found to melt reversibly at temperatures between 40 and 70 degrees C . This process was found to happen with a concomitant expansion-contraction that amounts between 2 and 8% of the long period of the structure but without significant alteration of the layered arrangement.

J Hepatobiliary Pancreat Surg, 2003, 10(6), 415 - 8
Bacterial translocation and its prevention in acute pancreatitis; Dervenis C et al.; In recent years, bacterial translocation from the gut onto pancreatic necrosis has been proposed as the main cause of pancreatic infection and the consequent sepsis . Failure of the intestinal barrier, together with bacterial overgrowth due to motility changes and immunosuppression, constitute the pathways of the continuous pancreatic contamination from bacterial translocation in patients with severe acute pancreatitis . Selective decontamination, by using a combination of oral and intravenous antibiotics, has been reported to decrease the incidence of sepsis and the related mortality . Immunostimulation is another action to be taken to enhance the ability of the immune system to prevent bacterial translocation, by the entrapment and killing, by enterocytes, of the bacteria trying to translocate through the bowel wall . To keep the mucosal barrier function intact is one of the main issues in the prevention of bacterial translocation . This could be achieved by the adequate delivery of oxygen and nutrient supplementation . Enteral nutrition is a key factor, as it has been proven to maintain mucosal integrity, along with preventing deterioration of the immune function of the intestine.

Z Naturforsch {C}, 2003 Nov-Dec, 58(11-12), 843 - 9
Antioxidative responses of tobacco expressing a bacterial glutathione reductase; Lederer B et al.; Reports on stress response of tobacco expressing a bacterial glutathione reductase (GR) do not agree . To clarify this situation we investigated several parameters using the tobacco BelW3 line and its transformant BelW3gor expressing an E . coli GR . This alteration in the activity of GR led to an ambiguous modification of the antioxidative system . In contrast to the wild type, the transgenic tobacco suffered lipid peroxidation under moderate light intensities, while it was found to be more resistant towards oxidative stress induced by paraquat or hydrogen peroxide . Transcript levels for violaxanthin deepoxidase and cytosolic Cu-Zn-superoxide dismutase were strongly reduced in BelW3gor plants as compared to BelW3.

Appl Environ Microbiol, 2004 Jan, 70(1), 610 - 2
Simple and reliable method to precipitate proteins from bacterial culture supernatant; Caldwell RB et al.; A simple and reliable method for precipitating protein from bacterial culture supernatants based on a pyrogallol red-molybdate-methanol (PRMM) protocol has been developed and applied for the analysis of proteins secreted by a bacterial type III secretion system . PRMM-based precipitation has been shown to be more efficient and robust than are conventional protocols.

Adv Cancer Res, 2003, 90, 91 - 125
High-resolution analysis of genetic events in cancer cells using bacterial artificial chromosome arrays and comparative genome hybridization; Cowel JK et al.; Chromosome analysis of cancer cells has been one of the primary means of identifying key genetic events in the development of cancer . The relatively low resolution of metaphase chromosomes, however, only allows characterization of major genetic events that are defined at the megabase level . The development of the human genome-wide bacterial artificial chromosome (BAC) libraries that were used as templates for the human genome project made it possible to design microarrays containing these BACs that can theoretically span the genome uninterrupted . Competitive hybridization to these arrays using tumor and normal DNA samples reveals numerical chromosome abnormalities (deletions and amplifications) that can be accurately defined depending on the density of the arrays . At present, we are using arrays with 6,000 BACs, which provide an average resolution of less than 700 kb . Analysis of tumor DNA samples using these arrays reveals small deletions and amplifications that were not detectable by chromosome analysis and provides a global view of these genetic changes in a single hybridization experiment in 24 hours . The extent of the genetic changes can then be determined precisely and the gene content of the affected regions established . These arrays have widespread application to the analysis of cancer patients and their tumors and can detect constitutional abnormalities as well . The availability of these high-density arrays now provides the opportunity to classify tumors based on their genetic fingerprints, which will assist in staging, diagnosis, and even prediction of response to therapy . Importantly, subtle genetic changes that occur consistently in tumor cell types may eventually be used to stratify patients for clinical trials and to predict their response to custom therapies.

Biosens Bioelectron, 2004 Feb 15, 19(7), 653 - 60
Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein; Hsieh HV et al.; The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes . Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase . Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor . By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants . The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation . The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP . Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM) . One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5mM . These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices.

Biotechnol Appl Biochem, 2004 Oct, 40(Pt 2), 157 - 65
Herpes simplex virus VP22-human papillomavirus E2 fusion proteins produced in mammalian or bacterial cells enter mammalian cells and induce apoptotic cell death; Roeder GE et al.; Infection by high-risk HPV (human papillomavirus) is supposed to be the primary cause of cervical cancer . The HPV E2 protein (E2) is a DNA-binding protein that regulates viral gene expression and is required for efficient viral replication . Overexpression of the E2 protein in cervical cancer cells can induce growth arrest and/or apoptotic cell death, suggesting that E2 might be useful in the treatment of this disease . In the present study, we show that VP22 (herpes simplex virus VP22 protein) can be used to deliver E2 to target cells . VP22-E2 fusion proteins induce apoptosis in transiently transfected HPV-transformed cervical carcinoma cell lines . However, VP22-E2 fusion proteins do not kill COS-7 cells, probably because these cells constitutively express the simian-virus-40 T antigen and this protein sequesters the tumour suppressor protein p53 . When COS-7 cells producing VP22-E2 are seeded into cultures of HPV-transformed cells, VP22-E2 enters the non-producing cells and induces apoptosis . VP22-E2 proteins produced in bacterial cells can also enter cervical cancer cells and induce apoptosis in a dose-dependent manner . Our results suggest that local delivery of VP22-E2 fusion proteins could be used to treat cervical cancer and other HPV-associated diseases.

Microb Ecol, 2003 Aug, 46(2), 177 - 86
The rate of change of a soil bacterial community after liming as a function of temperature; Pettersson M et al.; The response of a bacterial community to liming of a forest humus soil (pH 4.9 increased to pH 7.5) was studied in the laboratory at three temperatures (5, 20, and 30 degrees C) . As a comparison an unlimed soil (pH 4.9) and a soil limed in the field 15 years ago (pH around 6) were also included . The bacterial community tolerance of pH was measured using TdR incorporation . The pH of the bacterial suspensions (bacteria directly extracted from soil) was altered to 3.6 and 8.3 using different buffers before measuring TdR incorporation . The logarithmic ratio between TdR incorporation at 8.3 and 3.6 was then used as an indicator of the community pH tolerance . The rate of changes in the community tolerance to pH after liming was fastest for the soil incubated at 30 degrees C, but only minor differences in rate of change could be seen between samples incubated at 5 and 20 degrees C . Changes in phospholipid fatty acid (PLFA) pattern after increasing the pH were most rapid for the bacterial community in the soil incubated at 30 degrees C followed by the soil incubated at 20 degrees C, whereas no changes could be seen in the PLFA pattern of the soil incubated at 5 degrees C, even after 82 days' incubation . Thus, the changes in the PLFA pattern were considerably slower than the changes in bacterial community tolerance to pH measured using TdR incorporation.

Int J Urol, 2004 Feb, 11(2), 97 - 102
Therapeutic effect of transurethral needle ablation in non-bacterial prostatitis: chronic pelvic pain syndrome type IIIa; Chiang PH et al.; AIM: Non-bacterial prostatitis is difficult to manage with conventional treatment . This study was undertaken to evaluate the therapeutic effect of transurethral needle ablation (TUNA) on men with chronic inflammatory non-bacterial prostatitis . METHODS: Thirty-two patients with non-bacterial prostatitis (type IIIa) were treated with TUNA . The TUNA procedure, which uses radiofrequency energy, heats the prostate tissue to approximately 90-110 degrees C over a 5-min period . Evaluation consisted of a prostatitis symptom severity score chart, the monitoring of the leukocyte count in the expressed prostatic secretion (EPS) and a subjective global assessment . RESULTS: The decrease in the prostate symptom severity score chart at 3 and 6 months compared with the baseline assessment was statistically significant . Analysis of the leukocyte levels in the EPS in 14 patients was available . All 14 patients had a decrease in the EPS leukocyte count 3 months after treatment . However, six of these men (43%) still had EPS leukocyte levels above the normal indices (>10 white blood cells per high-power field) . A second session of TUNA on these partial responders resulted in three of the six men obtaining a normal EPS leukocyte count . At 6 months following treatment, complete, partial and poor improvement in terms of subjective global assessment were noted in 60, 35 and 5% of patients, respectively . No major complications, including those of sexual dysfunction or retrograde ejaculation, were noted in this cohort . CONCLUSIONS: Transurethral needle ablation appears to be an easy, safe and effective treatment for men with chronic inflammatory non-bacterial prostatitis.

Biotechnol Bioeng, 2004 Jan 5, 85(1), 29 - 33
Construction of high-density bacterial colony arrays and patterns by the ink-jet method; Xu T et al.; We have developed a method for fabricating bacterial colony arrays and complex patterns using commercially available ink-jet printers . Bacterial colony arrays with a density of 100 colonies/cm(2) were obtained by directly ejecting Escherichia coli (E . coli) onto agar-coated substrates at a rapid arraying speed of 880 spots per second . Adjusting the concentration of bacterial suspensions allowed single colonies of viable bacteria to be obtained . In addition, complex patterns of viable bacteria as well as bacteria density gradients were constructed using desktop printers controlled by a simple software program .

Anim Biotechnol, 2003 Nov, 14(2), 145 - 53
Construction and characterization of a novel 13.34-fold chicken bacterial artificial chromosome library; Liu W et al.; A chicken bacterial artificial chromosome (BAC) library consisting of 138,240 clones was constructed in vector pBeloBAC11 with genomic DNA isolated from female white-silk chicken . An average insert size of 118 kb was estimated from 452 randomly isolated clones, which indicate the library to be approximate 13.34-fold genome coverage . For the demonstration of the probability to pick out any unique genes or DNA markers from the library, 8 single-copy genes were screened out and the positive clones were yielded between 2 and 15 with an average of 11.125, in agreement with the estimated high genomic coverage of this library . Positive superpools were obtained for 40 microsatellite markers selected from different regions of chicken genome . The number of positive superpools for each marker varies from 1 to 15 with an average of 9.475.

Plant Cell Physiol, 2003 Dec, 44(12), 1359 - 67
Expression of a bacterial xylose isomerase in potato tubers results in an altered hexose composition and a consequent induction of metabolism; Urbanczyk-Wochniak E et al.; Here we investigate the role of hexoses in the metabolism of the developing potato (Solanum tuberosum) tuber by the expression of a bacterial xylose isomerase which catalyzes the interconversion of glucose and fructose . Previously, we found that glycolysis was induced in transgenic tubers expressing a yeast invertase in the cytosol and postulated that this was due either to the decreased levels of sucrose or to effects downstream of the sucrose cleavage . In the present study xylose isomerase was expressed under the control of the tuber-specific patatin promoter . Selected transformants exhibited minor changes in the levels of tuber glucose and fructose but not in sucrose . Analysis of the enzyme activities of the glycolytic pathway revealed minor yet significant increases in the maximal catalytic activities of aldolase and glyceraldehyde 3-phosphate dehydrogenase but no increase in the activities of other enzymes of glycolysis . These lines were also characterized by an elevated tuber number, glycolytic and sucrose synthetic fluxes and in some metabolite levels downstream of glycolysis . When considered together these data suggest that the perturbation of hexose levels can result in increased glycolytic and sucrose (re)synthetic fluxes in the potato tuber even in the absence of changes in the level of sucrose . The consequences of altering hexose levels in the tuber are, however, not as severe as those observed following perturbation of the level of tuber sucrose.

Ital Heart J, 2003 Nov, 4(11), 816 - 8
Vasculitis mimicking bacterial endocarditis; Calachanis M et al.; Fever of unknown origin is one of the most intriguing issues in clinical practice . One of the most feared diagnoses, especially in patients with known valvular disease, is endocarditis . The differential diagnosis of fever is often complicated by the clinical-pathological overlap between the systemic inflammatory response in different types of pathologies such as infectious, autoimmune or neoplastic disorders . We report a case of a patient presenting with fever, cutaneous nodules and malaise, with a known mitral valve prolapse and moderate regurgitation, in which the diagnosis of Wegener's granulomatosis was finally made.

J Biol Chem, 2004 Mar 19, 279(12), 10955 - 61 Epub 2003 Dec 29.
Role of the transmembrane domain in the stability of TrwB, an integral protein involved in bacterial conjugation; Hormaeche I et al.; TrwB is an integral membrane protein encoded by the conjugative plasmid R388 . TrwB binds ATP and is essential for R388-directed bacterial conjugation . The protein consists of a cytosolic domain, which contains an ATP-binding site, and a transmembrane domain . The complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, TrwBDeltaN70 . The availability of intact and truncated forms of the protein provides a convenient system to study the role of the transmembrane domain in the stability of TrwB . Protein denaturation was achieved by heat, in the presence of guanidinium HCl, or under low salt conditions . In all three cases TrwB was significantly more stable than TrwBDeltaN70 with other conditions being the same . IR spectroscopy of the native and truncated forms revealed significant differences between them . In addition, it was found that TrwBDeltaN70 was stabilized in dispersions of non-ionic detergent, suggesting the presence of hydrophobic patches on the surface of the truncated protein . IR spectroscopy also confirmed the conformational stability provided by the detergent . These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane . In addition, this study indicates that the truncated soluble parts of two-domain membrane proteins may not reflect the physiological conformation of their native counterparts.

Acta Paediatr, 2003 Nov, 92(11), 1272 - 6
Beta-glucuronidase in the diagnosis of bacterial meningitis and response to treatment; Beratis NG et al.; AIM: Beta-glucuronidase activity is increased in the cerebrospinal fluid (CSF) of patients with bacterial meningitis . The aim of this study was to investigate the beta-glucuronidase activity in the cell-free CSF of bacterial meningitis and its course during treatment, and compare it with other CSF parameters . METHODS: The beta-glucuronidase activity, cell number, protein concentration and CSF/blood glucose ratio were measured in 43 consecutive infants and children with bacterial meningitis, and 97 control subjects . Patients had one or two follow-up lumbar punctures . RESULTS: The beta-glucuronidase activity was increased early in bacterial meningitis, even when the other CSF parameters were undisturbed . Before treatment, the median activity in affected children was 136 micromoles 4-methylumbelliferone l(-1) h(-1) (range 44-826) and in controls 14 (7-23) . In all patients who improved, the activity was lower in the follow-up CSF samples . Six to 12 h after starting treatment, the median activity was already reduced by 59% . The other CSF parameters showed a variability during the first 24 h of treatment independently of the course of the disease . Multiple comparisons of the CSF parameters in 17 patients who had two follow-up punctures showed that the beta-glucuronidase activity was the best prognostic index . CONCLUSION: Beta-glucuronidase activity in the CSF is a reliable indicator of bacterial meningitis, which can identify the response to treatment early in the course of illness . The enzyme activity is increased early in the disease, even when the other laboratory parameters from the CSF remain normal.

Hepatogastroenterology, 2003 Nov-Dec, 50(54), 2133 - 6
Prognosis of patients with liver cirrhosis and spontaneous bacterial peritonitis; Jepsen P et al.; BACKGROUND/AIMS: Patients with liver cirrhosis and ascites have a high risk of spontaneous bacterial peritonitis, but the prognostic impact of spontaneous bacterial peritonitis has not been well examined . METHODOLOGY: Patients with liver cirrhosis and ascites were included at the time of their first paracentesis during hospitalization in the Department of Hepatology, Aarhus University Hospital, Denmark, between September 1992 and September 2000 . Cox regression was used to estimate the mortality of patients with spontaneous bacterial peritonitis (ascites leukocyte count > or = 250 per mm3) relative to controls without spontaneous bacterial peritonitis . Furthermore, we used Cox regression to estimate the change in mortality when controls developed spontaneous bacterial peritonitis during follow-up . RESULTS: Of 286 patients, 76 (27%) had spontaneous bacterial peritonitis at the first paracentesis . The mortality ratio of patients with spontaneous bacterial peritonitis relative to controls was 1.0 (95% confidence interval 0.7-1.5) after adjustment for age, gender, comorbidity, and alcohol abuse . Of the 210 controls, 42 (20%) were found to have spontaneous bacterial peritonitis at a later paracentesis . Their mortality rate more than doubled with the onset of spontaneous bacterial peritonitis . CONCLUSIONS: Spontaneous bacterial peritonitis at the first paracentesis did not affect the prognosis of patients with liver cirrhosis, whereas development of spontaneous bacterial peritonitis during follow-up doubled the mortality risk . This may be due to a longer diagnostic delay in those who developed spontaneous bacterial peritonitis during follow-up.

Proc Natl Acad Sci U S A, 2004 Jan 13, 101(2), 482 - 7 Epub 2003 Dec 26.
Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization; Aharoni A et al.; Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis . However, their structure and mechanism of action are poorly understood . PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance . Family shuffling and screening lead to the first PON variants that express in a soluble and active form in Escherichia coli . We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities . In particular, we have evolved PON1 variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis . Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members.

Luminescence, 2003 Nov-Dec, 18(6), 330 - 3
Ecabet sodium attenuates reactive oxygen species produced by neutrophils after priming with bacterial lipopolysaccharides; Munakata W et al.; The pathogenic roles of reactive oxygen species (ROS) have been implicated in ulcerative colitis (UC) . The aim of this study was to examine the effects of ecabet sodium on ROS produced by human neutrophils, particularly after being primed by bacterial lipopolysaccharides (LPS) . Neutrophils were isolated from six healthy volunteers . Each well of a 96-well microplate received neutrophil suspension (1.0 x 10(5) cells) and the plates were incubated at 37 degrees C for 30 min with or without E . coli LPS (f.c . 0.001 ng/ micro L) . Ecabet sodium (f.c . 0-5.0 mg/mL) was added before starting or after finishing the incubation . Neutrophils were stimulated by opsonized zymosan (OZ; 1.0 mg/mL) or calcium ionophore (A21837; 0.3 micro mol/L) and luminol-dependent chemiluminescence response was measured using a Lumi Box H-1000 . Ecabet sodium attenuated ROS production at a concentration of 5.0 mg/mL (p < 0.05) in LPS-primed neutrophils . However, attenuating effects were not significantly different when ecabet sodium was added before or after the incubation with E . coli LPS . Ecabet sodium may have some attenuating effects on ROS produced by human neutrophils even after neutrophils are primed by bacterial LPS . These results may explain, in part, the therapeutic effects of ecabet sodium for UC .

J Pediatr Surg, 2004 Jan, 39(1), 10 - 5
Glucocorticoid treatment down-regulates chemokine expression of bacterial cholangitis in cholestatic rats; Hsieh CS et al.; BACKGROUND: Postoperative cholangitis is common after operation for biliary atresia . Empirical pulse therapy with glucocorticoid is effective in reversing some detrimental clinical manifestations, but the rationale for such a therapy still is not substantiated . METHODS: Adult male rats were divided into groups according to the treatment: sterile normal saline (NS) or Escherichia coli (EC, 1 mL containing 10(8) cells of ATCC 25922 strain), 1 mL, were infused into the proximal choledochostomy (PC) tube 2 weeks after ligation of the PC tube (bile duct ligation, BDL), then immediate tube-tube choledocho-choledochostomy (biliary drainage, BD) was constructed . A high dose of dexamethasone (DEX, intraperitoneal injection; 2 mg/kg of body weight) was given after BD in treatment groups . Histopathology of the liver, as well as liver chemokine mRNA expression and serum chemokine levels, were studied 24 hours after treatment . RESULTS: Inflammatory cell infiltration to the liver was retarded with DEX treatment, which was correlated with a significantly lower expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) mRNA in the liver (P =.006) . Serum IL-8 and MCP-1 levels were also significantly down-regulated with DEX treatment (P = 0.008) . CONCLUSIONS: Glucocorticoid treatment is effective in modulating IL-8 and MCP-1 expression and ameliorating inflammatory cell infiltration in rat liver with bacterial cholangitis and cholestasis.

Bioinformatics, 2004 Jan 1, 20(1), 67 - 74
Distribution of words with a predefined range of mismatches to a DNA probe in bacterial genomes; Melko OM et al.; MOTIVATION: Hybridization of oligonucleotides with longer nucleotide sequences is an essential step in nucleic acid biosynthesis in vitro and in vivo, in oligonucleotide-based diagnostics, and in therapeutic applications of oligonucleotides . A major factor determining sensitivity and selectivity of hybridization is the number of base pair mismatches that occur in an ungapped alignment of the oligonucleotide (probe) and a longer sequence (target) . RESULTS: The k-distance match count between the probe and the target is defined as the number of ungapped alignments between the two sequences that have exactly k mismatches, and the k-neighbor match count is defined as the sum of the j-distance match counts for j between 0 and k . We derive a novel formula for the probability of a k-distance match . This formula is based on the assumption that the target is strand-symmetric Bernoulli text (i.e . nucleotides are independently, identically distributed in the target and satisfy Chargaff's second parity rule) . Our model predicts that the GC-content in both the probe and the target significantly affects the match count expectation . The ratio of k-neighbor match counts in two distinct genomes for a given probe is a measure of its specificity . We calculated such ratios for pairs of bacterial genomes with different combinations of length, GC-content and phylogenetic distance . Examination of the extreme values of these ratios indicates that probes with a high discriminative power exist for each tested pair.

Bioresour Technol, 2004 Apr, 92(2), 111 - 9
Use of poly(lactic acid) amendments to promote the bacterial fixation of metals in zinc smelter tailings; Edenborn HM; The ability of poly(lactic acid) (PLA) to serve as a long-term source of lactic acid for bacterial sulfate reduction activity in zinc smelter tailings was investigated . Solid PLA polymers mixed in water hydrolyzed abiotically to release lactic acid into solution over an extended period of time . The addition of both PLA and gypsum was required for indigenous bacteria to lower redox potential, raise pH, and stimulate sulfate reduction activity in highly oxidized smelter tailings after one year of treatment . Bioavailable cadmium, copper, lead and zinc were all lowered significantly in PLA/gypsum treated soil, but PLA amendments alone increased the bioavailability of lead, nickel and zinc . Similar PLA amendments may be useful in constructed wetlands and reactive barrier walls for the passive treatment of mine drainage, where enhanced rates of bacterial sulfate reduction are desirable.

Protein Sci, 2004 Jan, 13(1), 230 - 9
The N-terminal domain (IF2N) of bacterial translation initiation factor IF2 is connected to the conserved C-terminal domains by a flexible linker; Laursen BS et al.; Bacterial translation initiation factor IF2 is a multidomain protein that is an essential component of a system for ensuring that protein synthesis begins at the correct codon within a messenger RNA . Full-length IF2 from Escherichia coli and seven fragments of the protein were expressed, purified, and characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) methods . Interestingly, resonances of the 6 kD IF2N domain located at the extreme N terminus of IF2 can be clearly identified within the NMR spectra of the full-length 97-kD protein . (15)N NMR relaxation rate data indicate that (1) the IF2N domain is internally well ordered and tumbles in solution in a manner that is independent of the other domains of the IF2 protein, and (2) the IF2N domain is connected to the C-terminal regions of IF2 by a flexible linker . Chemical shifts of resonances within the isolated IF2N domain do not significantly differ from those of the corresponding residues within the context of the full-length 97-kD protein, indicating that IF2N is a structurally independent unit that does not strongly interact with other regions of IF2 . CD and NMR data together provide evidence that Domains I-III of IF2 have unstructured and flexible regions as well as substantial helical content; CD data indicate that the helical content of these regions decreases significantly at temperatures above 35 degrees C . The features of structurally well-ordered N- and C-terminal domains connected by a flexible linker with significant helical content are reminiscent of another translation initiation factor, IF3.

Mol Cell, 2003 Dec, 12(6), 1477 - 87
Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism; Moller-Jensen J et al.; Bacterial DNA segregation takes place in an active and ordered fashion . In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles . Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation . ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM . Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments . Thus, the process of R1 plasmid segregation in E . coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells . In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

J Immunol, 2004 Jan 1, 172(1), 302 - 9
Dermal enhancement: bacterial products on intact skin induce and augment organ-specific autoimmune disease; Riminton DS et al.; The skin is both an essential barrier for host defense and an important organ of immunity . In this study, we show that the application of cholera toxin to intact mouse skin induces and enhances autoimmune diseases affecting organs at distant anatomic sites, whereas its administration by the mucosal route has been reported to have the opposite effect . First, the CNS autoantigen myelin oligodendrocyte glycoprotein 35-55, when applied repeatedly with cholera toxin to the intact skin of healthy C57BL/6 mice, induced relapsing paralysis with demyelinating immunopathologic features similar to multiple sclerosis . Second, the application of cholera toxin in the absence of autoantigen exacerbated the severity of conventional experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein in CFA . Third, the application of cholera toxin to the intact skin of NOD/Lt mice, with or without insulin B peptide 9-23, exacerbated insulitis and T lymphocyte-derived IFN-gamma and IL-4 production in the islets of Langerhans, resulting in an increased incidence and rate of onset of autoimmune diabetes . The data presented in this study highlight the different outcomes of adjuvant administration by different routes . Because dermal application of cholera toxin, and other bacterial products with similar adjuvant activities, is being developed as a clinical vaccination strategy, these data raise the possibility that it could precipitate autoimmune disease in genetically susceptible humans.

J Mol Biol, 2004 Jan 16, 335(3), 761 - 73
The first structure of a bacterial class B Acid phosphatase reveals further structural heterogeneity among phosphatases of the haloacid dehalogenase fold; Calderone V et al.; AphA is a periplasmic acid phosphatase of Escherichia coli belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases . The crystal structure of AphA has been determined at 2.2A and its resolution extended to 1.7A on an AuCl(3) derivative . This represents the first crystal structure of a class B bacterial phosphatase . Despite the lack of sequence homology, the AphA structure reveals a haloacid dehalogenase-like fold . This finding suggests that this fold could be conserved among members of the DDDD superfamily of phosphohydrolases . The active enzyme is a homotetramer built by using an extended N-terminal arm intertwining the four monomers . The active site of the native enzyme, as prepared, hosts a magnesium ion, which can be replaced by other metal ions . The structure explains the non-specific behaviour of AphA towards substrates, while a structure-based alignment with other phosphatases provides clues about the catalytic mechanism.

Eur J Biochem, 2004 Jan, 271(1), 163 - 72
Active-site residues and amino acid specificity of the bacterial 4'-phosphopantothenoylcysteine synthetase CoaB; Kupke T; In bacteria, coenzyme A is synthesized in five steps from d-pantothenate . The Dfp flavoprotein catalyzes the synthesis of the coenzyme A precursor 4'-phosphopantetheine from 4'-phosphopantothenate and cysteine using the cofactors CTP and flavine mononucleotide via the phosphopeptide-like compound 4'-phosphopantothenoylcysteine . The synthesis of 4'-phosphopantothenoylcysteine is catalyzed by the C-terminal CoaB domain of Dfp and occurs via the acyl-cytidylate intermediate 4'-phosphopantothenoyl-CMP in two half reactions . In this new study, the molecular characterization of the CoaB domain is continued . In addition to the recently described residue Asn210, two more active-site residues, Arg206 and Ala276, were identified and shown to be involved in the second half reaction of the (R)-4'-phospho-N-pantothenoylcysteine synthetase . The proposed intermediate of the (R)-4'-phospho-N-pantothenoylcysteine synthetase reaction, 4'-phosphopantothenoyl-CMP, was characterized by MALDI-TOF MS and it was shown that the intermediate is copurified with the mutant His-CoaB N210H/K proteins . Therefore, His-CoaB N210H and His-CoaB N210K will be of interest to elucidate the crystal structure of CoaB complexed with the reaction intermediate . Wild-type His-CoaB is not absolutely specific for cysteine and can couple derivatives of cysteine to 4'-phosphopantothenate . However, no phosphopeptide-like structure is formed with serine . Molecular characterization of the temperature-sensitive Escherichia coli dfp-1 mutant revealed that the residue adjacent to Ala276, Ala275 of the strictly conserved AAVAD(275-279) motif, is exchanged for Thr.

Clin Pediatr (Phila), 2003 Nov-Dec, 42(9), 797 - 805
A comparison of time to positive culture and time to clinical identification of serious bacterial infection in infants; Brown JC et al.; To compare the time to positive culture with the time to clinical detection of serious bacterial infection (SBI) in young infants, a retrospective case series of 949 infants age 0-60 days, who had a body fluid cultured in the emergency department or within 24 hours of admission was analyzed . Times to first report of positive culture and first clinical diagnosis of SBI were compared . Of 44 infants with positive cultures, 48% were clinically diagnosed with SBI at first evaluation . Of 21 infants with cultures reported positive after 24 hours, 14 were already diagnosed with SBI . Infections that altered therapy were identified after 24 and 36 hours in 4 infants and 1 infant, respectively . In infants with SBIs, the time to positive culture is longer than the time to identification of infection.

J Biol Chem, 2004 Mar 19, 279(12), 11582 - 92 Epub 2003 Dec 16.
Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer: expression and mutational analysis of two cyanophycin synthetase and cyanophycinase gene clusters in heterocyst-forming cyanobacterium Anabaena sp . PCC 7120; Picossi S et al.; Two gene clusters each encoding the cyanophycin-metabolism enzymes cyanophycin synthetase and cyanophycinase are found in the heterocyst-forming cyanobacterium Anabaena sp . PCC 7120 . In cluster cph1, the genes cphB1 and cphA1 were expressed in media containing ammonium, nitrate, or N(2) as nitrogen sources, but expression was higher in the absence of combined nitrogen taking place both in vegetative cells and heterocysts . Both genes were cotranscribed from three putative promoters located upstream of cphB1, and, additionally, the cphA1 gene was expressed monocistronically from at least two promoters located in the intergenic cphB1-cphA1 region . Both constitutive promoters and promoters dependent on the global nitrogen control transcriptional regulator NtcA were identified . In cluster cph2, the cphB2 and cphA2 genes, which are found in opposite orientations, were expressed as monocistronic messages in media containing ammonium, nitrate, or N(2), but expression was higher in the absence of ammonium . Expression of the cph2 genes was lower than that of cph1 genes . Analysis of cph gene insertional mutants indicated that cluster cph1 genes contributed more than cluster cph2 genes to cyanophycin accumulation in the whole filament as well as in heterocysts . Diazotrophic growth was more severely impaired in cyanophycinase than in cyanophycin synthetase mutants, indicating that cyanophycin, although normally synthesized in the heterocysts, is not required for heterocyst function and that the inability to degrade this polymer is detrimental for the diazotrophic growth of the cyanobacterium.

Arch Biochem Biophys, 2004 Jan 1, 421(1), 149 - 58
Cloning, sequence analysis, and purification of choline oxidase from Arthrobacter globiformis: a bacterial enzyme involved in osmotic stress tolerance; Fan F et al.; Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, one of a limited number of compounds that accumulate to high levels in the cytoplasm of cells to prevent dehydration and plasmolysis in adverse hyperosmotic environments . In the present study, the highly GC rich codA gene encoding for choline oxidase was cloned from genomic DNA of Arthrobacter globiformis strain ATCC 8010 and expressed to high yields in Escherichia coli strain Rosetta(DE3)pLysS . The resulting enzyme was purified to high levels in a single chromatographic step using DEAE-Sepharose, as shown by SDS-PAGE analysis . Denaturation and mass spectroscopic analyses showed that the covalent linkage between the FAD cofactor and the protein is preserved in recombinant choline oxidase, consistent with protein flavinylation being a self-catalytic process . The enzyme was shown to be a homodimer of 120,000 Da by size-exclusion chromatography and to be active with both choline and betaine aldehyde as substrate . Sequencing analysis indicated that the nucleotide sequence of codA originally reported in GenBank contains seven flaws, resulting in a translated protein with a significantly altered amino acid sequence between position 298 and 410.

Acta Obstet Gynecol Scand, 2004 Jan, 83(1), 27 - 36
Cost-effectiveness of screening and treatment for bacterial vaginosis in early pregnancy among women at low risk for preterm birth; Kekki M et al.; BACKGROUND: Bacterial vaginosis (BV) is an important risk factor for preterm birth . BV is detected in 10-30% of pregnant women and is often asymptomatic . Treatment of BV during pregnancy seems to reduce the risk of preterm delivery among high-risk women . We performed a cost-effectiveness analysis of screening and treatment for BV in early pregnancy among asymptomatic women at low risk for preterm delivery . METHODS: A decision tree was built with two arms . For the screening (and treatment) arm the probabilities were derived from our earlier randomized trial on screening and treatment for BV, consisting of BV-positive women treated with intravaginal clindamycin cream or placebo and also of BV-negative pregnant women . The probabilities of outcomes among these women were collected from antenatal clinic records and hospital records, and for the no-screening arm mainly from the Finnish Perinatal Statistics . The outcomes considered were preterm delivery, mode of delivery, peripartum infections and postpartum complications . The unit costs associated with these outcomes were mainly based on disease-related groups (DRGs) . No-screening was compared with two screening programs (one with clindamycin, the other with metronidazole treatment) and subjected to sensitivity analyses . RESULTS: There was no significant difference between screening and no-screening strategies in the costs and in the rate of preterm deliveries but the screening strategy produced significantly fewer peripartum infections and postpartum complications . Sensitivity analyses suggested that the screening strategy may become cost-saving if the rate of preterm deliveries exceeds 3% . CONCLUSION: Screening and treatment for BV in early pregnancy may not reduce costs compared to no-screening in a population at low risk for preterm birth but would produce, at the same cost, more health benefits in terms of fewer peripartum infections and postpartum complications . However, it may be cost-saving if the rate of preterm deliveries is higher than 3%.

Curr Opin Struct Biol, 2003 Dec, 13(6), 722 - 30
Structure and mechanism of bacterial dehalogenases: different ways to cleave a carbon-halogen bond; de Jong RM et al.; The dehalogenases make use of fundamentally different strategies to cleave carbon-halogen bonds . The structurally characterized haloalkane dehalogenases, haloacid dehalogenases and 4-chlorobenzoate-coenzyme A dehalogenases use substitution mechanisms that proceed via a covalent aspartyl intermediate . Recent X-ray crystallographic analysis of a haloalcohol dehalogenase and a trans-3-chloroacrylic acid dehalogenase has provided detailed insight into a different intramolecular substitution mechanism and a hydratase-like mechanism, respectively . The available information on the various dehalogenases supports different views on the possible evolutionary origins of their activities.

J Nanobiotechnology . 2003 Dec 15;1(1):6.
Emerging Applications of Bacterial Spores in Nanobiotechnology; Ricca E et al.; Bacterial spores are robust and dormant life forms with formidable resistance properties, in part, attributable to the multiple layers of protein that encase the spore in a protective and flexible shield . The coat has a number of features pertinent to the emerging field of nanobiotechnology including self-assembling protomers and the capacity for engineering and delivery of foreign molecules . This review gives an account of recent progress describing the use of the spore, and specifically, the spore coat as a vehicle for heterologous antigen presentation and protective immunization (vaccination) . As interest in the spore coat increases it seems likely that they will be exploited further for drug and enzyme delivery as well as a source of novel self-assembling proteins.

Biochem J, 2004 Apr 1, 379(Pt 1), 183 - 90
Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution; Agrimi G et al.; The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria . They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix . SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins . The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity . By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed . This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC) . Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple . SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria . The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine . This is the first report describing the identification and characterization of the human SAMC and its gene.

Biochemistry, 2003 Dec 23, 42(50), 14762 - 73
Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins; Siebert HC et al.; Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates . These complex carbohydrates often present more than one potential lectin-binding site in a single chain . Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue . The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans . It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc . Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy . Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively) . To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons . Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues . Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results . The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design . In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15830 - 5 Epub 2003 Dec 12.
Induction of colitis by a CD4+ T cell clone specific for a bacterial epitope; Kullberg MC et al.; It is now well established that the intestinal flora plays an important role in the pathogenesis of inflammatory bowel disease (IBD) . However, whether bacteria serve as the sole target of the immune response in this process or whether they act indirectly by triggering an anti-self response is still unclear . We have previously shown that specific pathogen-free IL-10-deficient (IL-10 KO) mice develop a T helper (Th1)-cytokine associated colitis after experimental infection with Helicobacter hepaticus . We here show that H . hepaticus Ag (SHelAg)-specific CD4+ Th1 clones transfer disease to H . hepaticus-infected T cell-deficient RAG KO hosts . Importantly, uninfected recipients of the SHelAg-specific clones did not develop intestinal inflammation, and a control Schistosoma mansoni-specific Th1 clone did not induce colitis upon transfer to infected RAG KO mice . The disease-inducing T cell clones recognized antigen(s) (Ag) specifically expressed by certain Helicobacter species as they responded when stimulated in vitro with H . hepaticus and Helicobacter typhlonius Ag, but not when cultured with Ag preparations from Helicobacter pylori, various non-helicobacter bacteria, or with cecal bacterial lysate from uninfected mice . Characterization of the Ag specificity of one of the clones showed that it reacts uniquely with a 15-mer peptide epitope on the flagellar hook protein (FlgE) of H . hepaticus presented by I-Ab . Together, our results demonstrate that colitis can be induced by clonal T cell populations that are highly specific for target Ag on intestinal bacteria, suggesting that an aberrant T cell response directed against gut flora is sufficient to trigger IBD.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15528 - 33 Epub 2003 Dec 12.
Domain movements of HAP2 in the cap-filament complex formation and growth process of the bacterial flagellum; Maki-Yonekura S et al.; The cap at the growing end of the bacterial flagellum is essential for its growth, remaining stably attached while permitting the insertion of flagellin transported from the cytoplasm through the narrow central channel . We analyzed the structure of the isolated cap in its frozen hydrated state by electron cryomicroscopy . The 3D density map now shows detailed features of domains and their connections, giving reliable volumes and masses, making assignment of the domains to the amino acid sequence possible . A model of the cap-filament complex built with an atomic model of the filament allows a quantitative analysis of the cap domain movements on cap binding and rotation that promotes the efficient self assembly of flagellin during the filament growth process.

J Biol Chem, 2004 Mar 5, 279(10), 9379 - 88 Epub 2003 Dec 12.
Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA . Silencing of the p110alpha isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1alpha,25-dihydroxycholecalciferol and bacterial lipopolysaccharide; Lee JS et al.; Studying mononuclear phagocyte cell biology through genetic manipulation by non-viral transfection methods has been challenging due to the dual problems of low transfection efficiency and the difficulty in obtaining stable transfection . To overcome this problem, we developed a system for mediating RNA interference in monocytic cells . The p110alpha isoform of phosphoinositide 3-kinases (PI3Ks) was silenced using a lentiviral vector expressing short hairpin RNA (shRNA) . This resulted in the generation of stable THP-1 and U-937 monocytic cell lines deficient in p110alpha . Notably, p110alpha was silenced without affecting levels of either the other class I(A) PI3K catalytic subunits p110beta and p110delta, or the p85alpha regulatory subunit . The role of p110alpha in mediating cell adherence was examined . Monocyte adherence induced in response to either lipopolysaccharide (LPS) or 1alpha,25-dihydroxycholecalciferol (D(3)) was blocked by the PI3K inhibitor LY294002 . However, although adherence induced in response to D(3) was sensitive to silencing of p110alpha, LPS-induced adherence was not . Expression of the monocyte differentiation marker CD11b was also induced by D(3) in a PI3K-dependent manner and gene silencing using shRNA showed that p110alpha was also required for this effect . Taken together, these findings demonstrate that LPS and D(3) use distinct isoforms of class I(A) PI3K to induce functional responses and that lentiviral-mediated delivery of shRNA is a powerful approach to study monocyte biology.

Obstet Gynecol, 2003 Nov, 102(5 Pt 1), 927 - 33
Bacterial vaginosis in sexually experienced and non-sexually experienced young women entering the military; Yen S et al.; OBJECTIVE: To estimate the prevalence of bacterial vaginosis by Nugent Gram stain criteria in a nonclinic national sample of young women entering recruit training; to examine clinical associations with bacterial vaginosis; and to evaluate the performance of a pH test card and Papanicolaou smear against Gram stain as screening tools for bacterial vaginosis . METHODS: A cross-sectional study of 1938 women was conducted . Self-collected vaginal swabs were applied to a colorimetric pH test card and a glass slide for Gram stain evaluation according to the Nugent criteria . Papanicolaou smears and samples for sexually transmitted diseases screening were collected during routine entry pelvic examinations . RESULTS: Bacterial vaginosis prevalence was 27%, with 28% in sexually experienced and 18% in non-sexually experienced women (P = .001) . Bacterial vaginosis prevalence was 11% in Asian/Pacific Islanders, which was lower than in other nonwhite ethnic groups (P = .004) . Clinically, bacterial vaginosis was directly related to multiple sexual partners (P = .026), self-report of vaginal discharge (P = .001), self-report of vaginal odor (P < .001), and concurrent Chlamydia trachomatis infection (P = .002), and inversely related to hormonal contraceptive use (P = .013) . Vaginal discharge did not achieve statistical significance in multivariate analysis . Compared with the Nugent criteria, the sensitivities and specificities for bacterial vaginosis diagnosis were as follows: colorimetric pH test: 72% and 67%; Papanicolaou smear: 72% and 79%, respectively . CONCLUSION: Among these diverse young women, bacterial vaginosis occurs commonly in both sexually experienced and inexperienced young women and differs by race and ethnicity . The pH colorimetric test and Papanicolaou smear performed moderately well as screening tools for bacterial vaginosis . The inverse relationship of bacterial vaginosis with hormonal contraceptive use and its direct relationship with C . trachomatis need further study.

Ann Chim, 2003 Sep-Oct, 93(9-10), 771 - 5
Preliminary studies on bacterial decolorization of H-acid based azo dye-reactive black 5; Mohanty S et al.; A co-culture acclimatized to H-acid was used to degrade Reactive Black 5 (RB 5), a bis azo dye having central H-acid function . The effect of substrate concentration, pH and medium composition on the decolorization has been investigated . Decolorization was found independent of pH . Luria-Bertani broth favored decolorization over Yeast Extract; however further decolorization experiments have been conducted using Yeast Extract . The Michaelis-Menten Kinetic model is found to describe the dependence of specific decolorization rate on the RB 5 dye concentration.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15481 - 5 Epub 2003 Dec 11.
A macroscopic scale model of bacterial flagellar bundling; Kim M et al.; Escherichia coli and other bacteria use rotating helical filaments to swim . Each cell typically has about four filaments, which bundle or disperse depending on the sense of motor rotation . To study the bundling process, we built a macroscopic scale model consisting of stepper motor-driven polymer helices in a tank filled with a high-viscosity silicone oil . The Reynolds number, the ratio of viscous to elastic stresses, and the helix geometry of our experimental model approximately match the corresponding quantities of the full-scale E . coli cells . We analyze digital video images of the rotating helices to show that the initial rate of bundling is proportional to the motor frequency and is independent of the characteristic relaxation time of the filament . We also determine which combinations of helix handedness and sense of motor rotation lead to bundling.

Carbohydr Res, 2003 Nov 14, 338(23), 2679 - 86
Substitution pattern of 3-deoxy-D-manno-oct-2-ulosonic acid in bacterial lipopolysaccharides investigated by methylation analysis of whole LPS; Rybka J et al.; 3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) is a constituent of the inner core part of bacterial lipopolysaccharides (LPS) . This sugar may contribute to biological activities of the LPS, the type of substitution of Kdo is thus of importance and this work is aimed at the evaluation of a method for monitoring the substitution of Kdo in LPS . The procedure consists of three steps, namely permethylation of the lipopolysaccharide, with iodomethane and sodium methylsulfinylmethanide or NaOH in Me(2)SO, or with methyl triflate, then the product is methanolysed with HCl in MeOH and acetylated with acetic anhydride in pyridine . The resulting partially methylated acetates of Kdo methyl glycosides were analyzed by gas-liquid chromatography-electron impact ionization mass spectrometry (GLC-MS) . For several derivatives of Kdo, specific GLC retention times and MS fragmentation patterns were determined . Lipopolysaccharides from several bacterial strains were isolated and analyzed with three different methods of methylation . The complete solubilization of the LPS in the acid form allows diminishing possible undermethylation . Sodium methylsulfinylmethanide is the most efficient agent in the permethylation of the whole LPS, of all the tested procedures . Methylation with methyl triflate allows the detection of base labile substituents on Kdo residues.

Carbohydr Res, 2003 Nov 14, 338(23), 2539 - 47
Immunology of bacterial polysaccharide antigens; Weintraub A; Carbohydrates in the form of capsular polysaccharides and/or lipopolysaccharides are the major components on the surface of bacteria . These molecules are important virulence factors in many bacteria isolated from infected persons . Immunity against these components confers protection against the disease . However, developing vaccines based on polysaccharides is difficult and several problems have to be solved . First of all, most of the bacterial polysaccharides are T-lymphocyte independent antigens . Anti-polysaccharide immune response is characterised by lack of T-lymphocyte memory, isotype restriction and delayed ontogeny . Children below 2 years of age and elderly respond poorly to polysaccharide antigens . Secondly, the wide structural heterogeneity among the polysaccharides within and between species is also a problem . Thirdly, some bacterial polysaccharides are poor immunogens in humans due to their structural similarities with glycolipids and glycoproteins present in man . The T-lymphocyte independent nature of a polysaccharide may be overcome by conjugating the native or depolymerised polysaccharide to a protein carrier . Such neoglycoconjugates have been proven to be efficient in inducing T-lymphocyte dependent immunity and to protect both infants as well as elderly from disease . Another approach to circumvent the T-lymphocyte independent property of polysaccharides is to select peptides mimicking the immunodominant structures . Several examples of such peptides have been described.

Carbohydr Res, 2003 Nov 14, 338(23), 2477 - 89
Physicochemical properties of bacterial glycopolymers in relation to bioactivity; Brandenburg K et al.; An overview is given on the physicochemical properties of bacterial glycopolymers, i.e., pure oligo- and polysaccharides as well as glycolipids . Data from analysis of the chemical and physicochemical properties of various sugar polymers are summarized . Furthermore, data are presented on the thorough characterization of the most important class of bacterial glycopolymers, the lipopolysaccharides (LPS) . These data comprise the chemical characterization, the gel to liquid crystalline phase transition behaviour of their acyl chains, the ultrastructural studies of their morphology, and the investigation of the types of aggregate structures present above the critical micellar concentration (CMC) . Furthermore, the relevance of these data with respect to an understanding of the various biological effects elicited by LPS is discussed.

Carbohydr Res, 2003 Nov 14, 338(23), 2459 - 75
Characterisation of bacterial polysaccharides: steps towards single-molecular studies; Sletmoen M et al.; Techniques used in studies of polysaccharides, including chemical composition, linkage pattern, and higher order structures are in constant development . They provide information necessary for understanding of the polysaccharide properties and functions . Here, recent advancements in studies of the polysaccharides at the single-molecule level are highlighted . Over the last few years, single-molecule techniques such as force spectroscopy have improved in sensitivity and can today be used to detect forces in the pN range . In addition, these techniques can be used to investigate properties of single molecules close to physiological conditions . The challenges in the interpretation of the observations are aided by control experiments using well-characterised polysaccharides and by data provided by complementary methods . This field is expected to have increasing impact on the further advancement of the molecular understanding of the role of polysaccharides in various biological processes such as recognition and cell adhesion.

J Health Psychol, 2003 Nov, 8(6), 693 - 704
Illness perceptions in people with acute bacterial gastro-enteritis; Parry SD et al.; Functional gastro-intestinal disorders (FGID) like irritable bowel syndrome (IBS) are common and can develop after gastro-enteritis . Illness representations may be important influences on the development of post-infectious FGIDs . Here, we studied both the relationship between prior chronic symptoms (FGIDs) and illness perception during an acute illness (bacterial gastro-enteritis) as well as the relationship between illness perception during an acute illness (bacterial gastro-enteritis) and the subsequent development of chronic abdominal symptoms . Two hundred and seventeen people with recent gastro-enteritis completed a questionnaire asking about gut symptoms consistent with a diagnosis of IBS, functional dyspepsia or functional diarrhoea and the Illness Perception Questionnaire . Those without a prior FGID were followed up and completed a similar gut questionnaire at six months . People with a prior FGID had significantly more symptoms and scored significantly higher on the timeline and consequence scores than those without . People who developed a FGID had a non-significantly higher number of symptoms and higher consequence and timeline scores than those who did not . Neither comparative group differed in the control/cure scores or causation scores . The implications of the findings are discussed.

Genomics, 2004 Jan, 83(1), 66 - 75
Functional analysis of bacterial artificial chromosomes in mammalian cells: mouse Cdc6 is associated with the mitotic spindle apparatus; Illenye S et al.; Bacterial artificial chromosomes (BACs) provide a well-characterized resource for studying the functional organization of genes and other large chromosomal domains . To facilitate functional studies in cell cultures, we have developed a simple approach for generating stable cell lines with variable copy numbers of any BAC . Here we describe hamster cell lines with BAC transgenes that express mouse Cdc6 at levels that correlate with BAC copy number; show that mouse Cdc6 is regulated normally during the cell cycle, binds chromatin, and is degraded during apoptosis; and report a novel fraction of Cdc6 that associates with the spindle apparatus during mitosis . With RNA interference to assess genetic complementation by BAC alleles, this system will facilitate functional studies on large chromosomal domains at variable copy number in mammalian cell models.

Eur J Contracept Reprod Health Care, 2003 Sep, 8(3), 135 - 8
Frequency of bacterial vaginosis among women attending for intrauterine device insertion at an inner-city family planning clinic; Tosun I et al.; The aim of this study was to investigate the rate of bacterial vaginosis in women attending an inner-city family planning clinic for intrauterine device (IUD) insertion . In a population of 86 women, eight (9.3%) and 20 (23.2%) were found to have bacterial vaginosis according to the criteria of Amsel and Nugent, respectively . Sensitivity, specificity, positive and negative predictive values were calculated in relation to bacterial vaginosis for Amsel's criteria . The detection of clue cells demonstrated excellent sensitivity (85%) . Positive amine test and vaginal discharge demonstrated poor sensitivity (50% and 55%, respectively) . Our results suggest that Gram staining of vaginal specimens may be of use to identify the presence of bacterial vaginosis prior to IUD insertion.

Calcif Tissue Int, 2003 Sep, 73(3), 232 - 6
Systemic effects on bone healing of a new hyaluronic acid-like bacterial exopolysaccharide; Zanchetta P et al.; Critical Size Defect (CSD) technique was used to evaluate the systemic activities on bone regeneration capacity of a newly discovered hyaluronic acid-like exopolysaccharide synthesized by a bacteria originating from a deep sea hydrothermal vent . Some systemic effects were previously detected on earlier experiments . A 5 mm diameter hole was made on each parietal bone of male rats . The right hole was filled with 0.5 mg of a new bacterial exopolysaccharide referenced HE 800, while the left hole remained free of any treatment . After 21 days, the holes and surrounding tissues were examined by direct examination, X-rays, and histological staining . Using HE 800, bone healing was almost complete after only 21 days in the treated hole and always complete in the control side by some systemic effect . Neovascularization was also observed along with an organized trabecular bone on both sides . No abnormal bone growth or conjunctival abnormalities were noticed . At the end of the experiment, 90.1% ( +/- 5.2) bone healing (n = 20) was observed on the treated side; conversely, the control side animals demonstrate an amazing healing 100% (+/- 0.5) by a systemic effect.

Epidemiol Mikrobiol Imunol, 2003 Nov, 52(4), 147 - 51
{Bacterial cellular communication in relation to pathogenicity}; Hostacka A et al.; The authors briefly summarize data on cell-cell communication (quorum sensing) as well as principles of simple methods for detection of N-acyl homoserine lactones.

J Biol Chem, 2004 Feb 27, 279(9), 7495 - 504 Epub 2003 Dec 02.
Inducible expression, enzymatic activity, and origin of higher plant homologues of bacterial RelA/SpoT stress proteins in Nicotiana tabacum; Givens RM et al.; All living cells possess adaptive responses to environmental stress that are essential to ensuring cell survival . For motile organisms, this can culminate in avoidance or attractile behavior, but for sessile organisms such as plants, stress adaptation is a process of success or failure within the confines of a given environment . Nearly all bacterial species possess a highly evolved system for stress adaptation, known as the "stringent response." This ancient and ubiquitous regulatory response is mediated by production of a second messenger of general stress, the nucleotide guanosine-3',5'-(bis)pyrophosphate (ppGpp), which mediates reprogramming of the global transcriptional output of the cell . Accumulation of ppGpp is stress-induced through the enzymatic activation of the well known bacterial ppGpp synthetases, RelA and SpoT . We have recently discovered homologues of bacterial relA/spoT genes in the model plant Nicotiana tabacum . We hypothesize that these homologues (designated RSH genes for RelA/SpoT homologues) serve a stress-adaptive function in plants analogous with their function in bacteria . In support of this hypothesis, we find 1) inducibility of tobacco RSH gene expression following treatment with jasmonic acid; 2) bona fide ppGpp synthesis activity of purified recombinant Nt-RSH2 protein, and 3) a wide spread distribution of RSH gene expression in the plant kingdom . Phylogenetic analyses identifies a distinct phylogenetic branch for the plant RSH proteins with two subgroups and supports ancient symbiosis and nuclear gene transfer as a possible origin for these stress response genes in plants . In addition, we find that Nt-RSH2 protein co-purifies with chloroplasts in subcellular fractionation experiments . Taken together, our findings implicate a direct mode of action of these ppGpp synthetases with regard to plant physiology, namely regulation of chloroplast gene expression in response to plant defense signals.

Appl Environ Microbiol, 2003 Dec, 69(12), 7073 - 82
High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure; Paloheimo M et al.; A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides . By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures . The carriers tested were the T . reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T . reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof . The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide . The recombinant Xyn11A produced had properties similar to those of the native xylanase . It constituted 6 to 10% of the total proteins secreted by the transformants . About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed . Even in the best Xyn11A producers, the levels of the fusion mRNAs were only approximately 10% of the level of cel7A (cbh1) mRNA in the untransformed host strain.

Trends Biochem Sci, 2003 Dec, 28(12), 628 - 31
Bacterial mimics of eukaryotic GTPase-activating proteins (GAPs); Litvak Y et al.; Bacterial GTPase-activating proteins (GAPs) subvert their host's eukaryotic Rho GTPases to their own advantage . Studies of bacterial GAPs extend our understanding of the action of eukaryotic GAPs, provide new tools for studies of cytoskeletal dynamics and offer new targets for anti-bacterial drugs.

W V Med J, 2003 Jul-Aug, 99(4), 154 - 5
Case report: bacterial tracheitis in an adult female; Stuchell B et al.; Bacterial tracheitis is an extremely rare entity, long considered to be a disease of pediatrics . However, cases continue to be reported among adult patients . We present the case of a 19-year-old female patient who presented to our Emergency Department (ED) with bacterial tracheitis . Other adult cases of bacterial tracheitis as reported in the literature from 1981 to the present are discussed.

J Comput Chem, 2004 Jan 30, 25(2), 160 - 8
Conformation-dependent intermolecular interaction energies of the triphosphate anion with divalent metal cations . Application to the ATP-binding site of a binuclear bacterial enzyme . A parallel quantum chemical and polarizable molecular mechanics investigation; Gresh N et al.; We have explored the conformation-dependent interaction energy of the triphosphate moiety, a key constituent of ATP and GTP, with a closed-shell divalent cation, Zn2+, used as a probe . This was done using the SIBFA polarizable molecular mechanics procedure . We have resorted to a previously developed approach in which triphosphate is built out from its elementary constitutive fragments, and the intramolecular, interfragment, interaction energies are computed simultaneously with their intermolecular interactions with the divalent cation . This approach has enabled reproduction of the values of the intermolecular interaction energies from ab initio quantum-chemistry with relative errors <3% . It was extended to the complex of a nonhydrolyzable analog of ATP with the active site of a bacterial enzyme having two Mg2+ cations as cofactors . We obtained following energy-minimization a very close overlap of the ATP analog over its position from X-ray crystallography . For models of the ATP analog-enzyme complex encompassing up to 169 atoms, the values of the SIBFA interaction energies were found to match their DFT counterparts with relative errors of <2% .

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2279 - 82 Epub 2003 Nov 27.
Bacterial expression, purification and preliminary X-ray crystallographic characterization of the invertase inhibitor Nt-CIF from tobacco; Hothorn M et al.; Acid invertases catalyzing the breakdown of sucrose are regulated at the post-translational level by extracellular inhibitory proteins of 16-20 kDa molecular weight in a pH-dependent manner . Little is known about the characteristics of the underlying protein-protein interaction . Here, the expression, purification, characterization, crystallization and initial X-ray analysis of a biologically active invertase inhibitor Nt-CIF from tobacco is reported . Four crystal forms covering a wide pH range have been obtained and data sets at resolutions higher than 2.5 A have been collected.

J Gen Virol, 2003 Dec, 84(Pt 12), 3393 - 403
Generation and precise modification of a herpesvirus saimiri bacterial artificial chromosome demonstrates that the terminal repeats are required for both virus production and episomal persistence; White RE et al.; Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus, and shares considerable homology with the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus . The generation of herpesvirus mutants is a key facet in the study of virus biology . The use of F-factor-based bacterial artificial chromosomes (BACs) to clone and modify the genomes of herpesviruses has enhanced the variety, precision and simplicity of mutant production . Here we describe the cloning of the genome of HVS non-transforming strain A11-S4 into a BAC . The cloning of the BAC elements disrupts open reading frame (ORF) 15 but the HVS-BAC can still replicate at levels similar to wild-type virus, and can persistently infect fibroblasts . The HVS-BAC was modified by RecA-mediated recombination initially to substitute reporter genes and also to delete the terminal repeats (TR) . After deletion of the TR, the HVS-BAC fails to enter a productive virus lytic cycle, and cannot establish a persistent episomal infection when transfected into fibroblast cell lines . This shows that while ORF 15 is dispensable for virus function in vitro, the TR is required for both virus latency and lytic virus production . In addition, the HVS-BAC promises to be a valuable tool that can be used for the routine and precise production and analysis of viral mutants to further explore gammaherpesvirus biology.

J Clin Pathol, 2003 Dec, 56(12), 972 - 5
Water transport becomes uncoupled from K+ siphoning in brain contusion, bacterial meningitis, and brain tumours: immunohistochemical case review; Saadoun S et al.; Specimens of normal human brain, contused brain, brain with bacterial meningitis, and brain tumours were immunolabelled for aquaporin 4 (AQP4) and Kir4.1 . In normal brain tissue, AQP4 and Kir4.1 were detected around the microvessels . In pathological brain tissue, AQP4 was upregulated in astrocytes in oedematous regions and Kir4.1 was upregulated in astrocytes in damaged brain . Changes in alpha syntrophin expression paralleled those of AQP4 and Kir4.1 . The following hypothesis is proposed: in astrocytes, under normal conditions, AQP4 couples water transport with Kir4.1 mediated K+ siphoning, but in pathological states, AQP4 facilitates the flow of brain oedema fluid, and Kir4.1 buffers increased extracellular K+.

J Neuroimmunol, 2003 Dec, 145(1-2), 148 - 53
Chemotactic activity of CXCL5 in cerebrospinal fluid of children with bacterial meningitis; Zwijnenburg PJ et al.; CXCL5 (epithelial-cell-derived neutrophil-activating protein (ENA-)78) is a CXC-chemokine that specifically acts on neutrophils . To obtain insight into the extent of local presence and action of CXCL5 during bacterial meningitis, we measured its concentrations in cerebrospinal fluid (CSF) of patients with culture-proven bacterial meningitis (n=14), aseptic meningitis (n=6), and controls (n=32) and compared these results with levels of other CXC-chemokines, CXCL8- (interleukin-8) and CXCL1-related oncogene (growth-related oncogene (GRO)-alpha) . Patients with bacterial meningitis had profoundly elevated CSF concentrations of all three chemokines . CXCL5 was not detectable in patients with aseptic meningitis or control subjects . CSF from patients with bacterial meningitis exerted chemotactic activity towards neutrophils, which was partially inhibited by neutralizing antibodies against CXCL5 and CXCL8, but not CXCL1 . CSF from controls exerted minor chemotactic activity, which could be strongly enhanced by the addition of recombinant CXCL5, CXCL8 or CXCL1 . During bacterial meningitis, CXCL5 is elevated in CSF, where it is involved in the recruitment of neutrophils to the central nervous system.

Biochim Biophys Acta, 2003 Dec 3, 1618(1), 25 - 32
Proteorhodopsin in living color: diversity of spectral properties within living bacterial cells; Kelemen BR et al.; Proteorhodopsin is a family of over 50 proteins that provide phototrophic capability to marine bacteria by acting as light-powered proton pumps . The potential importance of proteorhodopsin to global ocean ecosystems and the possible applications of proteorhodopsin in optical data storage and optical signal processing have spurred diverse research in this new family of proteins . We show that proteorhodopsin expressed in Escherichia coli is functional and properly inserted in the membrane . At high expression levels, it appears to self-associate . We present a method for determining spectral properties of proteorhodopsin in intact E . coli cells that matches results obtained with detergent-solubilized, purified proteins . Using this method, we observe distinctly different spectra for protonated and deprotonated forms of 21 natural proteorhodopsin proteins in intact E . coli cells . Upon protonation, the wavelength maxima red shifts between 13 and 53 nm . We find that pKa values between 7.1 and 8.5 describe the pH-dependent spectral shift for all of the 21 natural variants of proteorhodopsin . The wavelength maxima of the deprotonated forms of the 21 natural proteorhodopsins cluster in two sequence-related groups: blue proteorhodopsins (B-PR) and green proteorhodopsins (G-PR) . The site-directed substitution Leu105Gln in Bac31A8 proteorhodopsin shifts this G-PR's wavelength maximum to a wavelength maximum the same as that of the B-PR Hot75m1 proteorhodopsin . The site-directed substitution Gln107Leu in Hot75m1 proteorhodopsin shifts this B-PR's wavelength maximum to a wavelength maximum as that of Bac31A8 proteorhodopsin.

Neuroscience, 2003, 122(4), 1073 - 80
The bacterial endotoxin lipopolysaccharide enhances seizure susceptibility in mice: involvement of proinflammatory factors: nitric oxide and prostaglandins; Sayyah M et al.; Central nervous system (CNS) inflammation in cases such as head trauma, infection and stroke has been associated with the occurrence of epileptic seizures . Microglia, the principal immune cells in the brain, readily become activated in response to injury, infection or inflammation . The bacterial endotoxin lipopolysaccharide (LPS) induces the activation of microglia and the production of proinflammatory factors including nitric oxide (NO) and prostaglandins (PGs) . We examined the effect of LPS on seizure susceptibility of mice, by using the sensitive test, threshold of clonic seizures induced by i.v . infusion of pentylenetetrazole . LPS decreased the seizure threshold in a dose- and time-dependent manner . Pretreatment of mice with the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester or cyclooxygenase inhibitor, piroxicam or the opioid receptor antagonist, (-)-naloxone completely reversed the proconvulsant effect of LPS.These results indicate that NO, PGs and endogenous opioid peptides seem to be involved in LPS-induced decrease in seizure threshold.

J Mol Biol, 2003 Dec 12, 334(5), 993 - 1008
Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system; Gimble FS et al.; The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering . To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates . In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized . There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme . A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs . Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases . Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.

J Mol Biol, 2003 Dec 12, 334(5), 919 - 31
Eclipse-synchrony relationship in Escherichia coli strains with mutations affecting sequestration, initiation of replication and superhelicity of the bacterial chromosome; Olsson JA et al.; Initiation of replication from oriC on the Escherichia coli chromosomes occurs once and only once per generation at the same cell mass per origin . During rapid growth there are overlapping replication cycles, and initiation occurs synchronously at two or more copies of oriC . Since the bacterial growth can vary over a wide range (from three divisions per hour to 2.5 hours or more per division) the frequency of initiation should change in coordination with bacterial growth . Prevention of reinitiation from a newly replicated origin by temporary sequestration of the hemi-methylated GATC-sites in the origin region provides the molecular/genetic basis for the maintenance of the eclipse period between two successive rounds of replication . Sequestration is also believed to be responsible for initiation synchrony, since inactivation of either the seqA or the dam gene abolishes synchrony while drastically reducing the eclipse . In this work, we attempted to examine the functional relationship(s) between the eclipse period and the synchrony of initiation in E.coli strains by direct measurements of these parameters by density-shift centrifugation and flow-cytometric analyses, respectively . The eclipse period, measured as a fraction of DNA-duplication times, varied continuously from 0.6 for the wild-type E.coli K12 to 0.1 for strains with mutations in seqA, dam, dnaA, topA and gyr genes (all of which have been shown to cause asynchrony) and their various combinations . The asynchrony index, a quantitative indicator for the loss of synchrony of initiation, changed from low (synchronous) to high (asynchronous) values in a step-function-like relationship with the eclipse . An eclipse period of approximately 0.5 generation time appeared to be the critical value for the switch from synchronous to asynchronous initiation.

Fungal Genet Biol, 2004 Jan, 41(1), 23 - 32
Contig assembly and microsynteny analysis using a bacterial artificial chromosome library for Epichloë festucae, a mutualistic fungal endophyte of grasses; Kutil BL et al.; We constructed and characterized a bacterial artificial chromosome (BAC) library for Epichloe festucae, a genetically tractable fungal plant mutualist . The 6144 clone library with an average insert size of 87kb represents at least 18-fold coverage of the 29 Mb genome . We used the library to assemble a 110kb contig spanning the putative ornithine decarboxylase (odc) ortholog and subsequently expanded it to 228kb with a single walking step in each direction . Furthermore, we evaluated conservation of microsynteny between E . festucae and some model filamentous fungi by comparing sequence available from a 43kb region at the end of one BAC to publicly available fungal genome sequences . Orthologs to the 13 contiguous open reading frames (ORFs) identified in E . festucae are syntenic in Neurospora crassa and Magnaporthe grisea occurring in small sets of two, three or four colinear ORFs . This library is a valuable resource for research into traits important for the development and maintenance of a plant-fungus mutualistic symbiosis.

Bioelectrochemistry, 2003 Oct, 61(1-2), 1 - 8
Electrochemistry of nano-scale bacterial surface protein layers on gold; Handrea M et al.; The mechanism of the recrystallization of nano-scale bacterial surface protein layers (S-layers) on solid substrates is of fundamental interest in the understanding and engineering of biomembranes and e.g . biosensors . In this context, the influence of the charging state of the substrate had to be clarified . Therefore, the electrochemical behaviour of the S-layers on gold electrodes has been investigated by in-situ electrochemical quartz microbalance (EQMB) measurements, scanning force microscopy (SFM) and small-spot X-ray photoelectron spectroscopy (SS-XPS) of potentiostatically emersed substrates . It was shown that the negatively charged bonding sites of the S-layer units (e.g . carboxylates) can bond with positively charged Au surface atoms in the positively charged electrochemical double layer region positive of the point of zero charge ( approximately -0.8 V vs . saturated mercury-mercurous sulphate electrode) . Surface conditions in other potential regions decelerated the recrystallization and fixation of S-layers . Time-resolved in-situ and ex-situ measurements demonstrated that two-dimensional S-layer crystal formation on gold electrodes can occur within few minutes in contrast to hours common in self-assembled monolayer (SAM) generation . These results proved that the recrystallization and fixation of 2D-crystalline S-layers on an electronic conductor can be influenced and controlled by direct electrochemical manipulation.

Metab Eng, 2003 Oct, 5(4), 255 - 63
Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production; Ravanello MP et al.; Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed . Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1 . This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB . Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B . napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed . Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI . The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct . However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together . This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling . This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with enhanced agronomic, animal feed and human nutritional values.

Int Endod J, 2003 Nov, 36(11), 733 - 9
Effect of different irrigation solutions and calcium hydroxide on bacterial LPS; Tanomaru JM et al.; AIM: To evaluate the effect of biomechanical preparation with different irrigating solutions and calcium hydroxide dressing in dog root canals containing bacterial endotoxin (lipopolysaccharides; LPS) . METHODOLOGY: One hundred and forty premolar roots from seven dogs were filled with Escherichia coli LPS for 10 days (three roots were lost during histological processing) . The following irrigating solutions were used for biomechanical preparation: 1% (group I, n = 20), 2.5% (group II, n = 19) and 5% sodium hypochlorite (group III, n = 19), 2% chlorhexidine digluconate (group IV, n = 20) and physiological saline solution (group V, n = 19) . In group VI (n = 20), the LPS solution was maintained in the root canal during the entire experiment and in group VII (n = 20), after biomechanical preparation with saline solution, the root canals were filled with a calcium hydroxide dressing (Calen; control) . After 60 days, the animals were sacrificed and the following parameters of periapical disease were evaluated: (a) inflammatory infiltrate, (b) periodontal ligament thickness, (c) cementum resorption and (d) bone resorption . Scores were given and data were analysed statistically with the Kruskal-Wallis and Dunn tests (P < 0.05) . RESULTS: Histopathological evaluation showed that groups I-VI had more inflammatory infiltrate, greater periodontal ligament thickening and greater cementum and bone resorption (P < 0.05) compared to group VII, which received the calcium hydroxide intracanal dressing . CONCLUSIONS: Biomechanical preparation with the irrigating solutions did not inactivate the effects of the endotoxin but the calcium hydroxide intracanal dressing did appear to inactivate the effects induced by the endotoxin in vivo.

Cell Microbiol, 2003 Dec, 5(12), 887 - 99
Helicobacter pylori-induced homotypic phagosome fusion in human monocytes is independent of the bacterial vacA and cag status; Rittig MG et al.; Following reports that a VacA+cag+ toxigenic but not a VacA-cag- non-toxigenic Helicobacter pylori strain induced homotypic phagosome fusion in murine macrophages, we addressed that phenomenon in human cells . Mononuclear phagocytes and epitheloid cells were challenged with H . pylori strains of different vacA and cag genotypes and with VacA- and Cag- isogenic mutants, and chased in the absence or presence of signal transduction modulators . Electron microscopy revealed that, in monocytes: (i) homotypic phagosome fusion was frequently induced by all live H . pylori strains investigated but not by exogenous VacA; (ii) phagosomes containing bacteria fused, but not those containing latex beads; (iii) fusion resulted in communal compartments resembling giant multivesicular bodies; and (iv) formation of these compartments was blocked by inhibiting the host cell regulators PI 3-kinase, phospholipase C and p42 MAP kinase . Whereas some internalized bacteria remained viable 1 h after uptake, none survived a 24 h period . In contrast to monocytes, infected epitheloid cells rarely developed communal compartments . In combination, these results demonstrate that, in human monocytes, the H . pylori-induced homotypic phagosome fusion depends on neither the vacuolating cytotoxin VacA nor the cag pathogenicity island of H . pylori and does not result in prolonged intracellular survival.

Indian J Pediatr, 2003 Oct, 70(10), 799 - 801
Bacterial antigen detection test in meningitis; Das BK et al.; OBJECTIVE: To evaluate the role of bacterial antigen detection test in cerebrospinal fluid (CSF) for a rapid etiological diagnosis of bacterial meningitis . METHODS: The study included 36 cases of bacterial meningitis and 14 controls . Latex particle agglutination test (LPA test) for detection of bacterial antigen was done in the CSF using slidex meningitis kit (Biomeriux, France) . RESULTS: Using LPA test, an etiological diagnosis could be made in 83% cases of bacterial meningitis . In contrast, CSF Gram stain and culture showed 36% and 6% positivity, respectively . The sensitivity and specificity of LPA test were 83% and 100%, respectively . The common etiological organisms were S . pneumoniae, H . influenzae type b and N . meningitidis A . S . pneumoniae was encountered in all age groups while H . influenzae type b was found only below one year of age . CONCLUSIONS: LPA test is a rapid and superior diagnostic tool as compared to CSF Gram stain and culture . The study recommends LPA test as an adjunct laboratory test for rapid etiological diagnosis of bacterial meningitis for prompt institution of proper antibiotics.

Trends Immunol, 2003 Dec, 24(12), 652 - 8
Lessons from Nod2 studies: towards a link between Crohn's disease and bacterial sensing; Girardin SE et al.; Nod2 (Card15) belongs to the family of the recently described Nod molecules, which also includes the closely related protein Nod1 (Card4) . Nod proteins have been initially described as intracellular activators of the caspase and NF-kappaB signaling pathways . Recent progress has enabled research to demonstrate genetically that NOD2 (CARD15) is involved in the predisposition to Crohn's disease and Blau syndrome . In addition, biochemical evidence has unraveled the role of Nod1 (Card4) and Nod2 (Card15) as intracellular sensors of bacterial peptidoglycan . Together, studies on Nod2 (Card15) provide a conceptual link between inflammatory disorders, such as Crohn's disease and Blau syndrome, and bacterial sensing.

Transfusion, 2003 Dec, 43(12), 1677 - 82
Immunochemical detection of prion protein on dipsticks prepared with crystalline bacterial cell-surface layers; Volkel D et al.; BACKGROUND: Transmissible spongiform encephalopathy (TSE) represents a spectrum of diseases affecting humans and animals . A definitive diagnosis of TSEs is only possible by postmortem identification of pathologic prion protein in brain tissue that has been treated with protease . The pathologic protein is detected by Western blot analysis or ELISA methods . The bovine spongiform encephalopathy crisis and occurrence of a new variant of CJD has increased demand for rapid and simple assays . STUDY DESIGN AND METHODS: A dipstick assay has been developed for prion diagnosis based on a sandwich ELISA specific for prion protein, and crystalline bacterial cell-surface layers (S-layers) were used as an immobilization matrix . The usefulness of the dipstick assay was evaluated by determining the detection limit, comparison with other methods, and analysis of CJD samples . RESULTS: The sensitivity of the prion dipsticks was similar to that published for time-resolved fluorescence ELISA methods . After protease treatment, pathologic prion protein could be detected specifically . CONCLUSION: The dipstick assay is a sensitive and specific test useful for the detection of prion protein . The simplicity of the S-layer dipstick lends itself to a variety of potential applications including field diagnostics.

Infect Immun, 2003 Dec, 71(12), 7170 - 2
Deletion of Mycobacterium tuberculosis sigma factor E results in delayed time to death with bacterial persistence in the lungs of aerosol-infected mice; Ando M et al.; The stress-induced extracytoplasmic sigma factor E (SigE) of Mycobacterium tuberculosis shows increased expression after heat shock, sodium dodecyl sulfate treatment, and oxidative stress, as well as after phagocytosis in macrophages . We report that deletion of sigE results in delayed lethality in mice without a significant reduction of bacterial numbers in lungs.

Infect Immun, 2003 Dec, 71(12), 7014 - 22
Orally administered CpG oligodeoxynucleotide induces production of CXC and CC chemokines in the gastric mucosa and suppresses bacterial colonization in a mouse model of Helicobacter pylori infection; Raghavan S et al.; Bacterial DNA and unmethylated CpG oligodeoxynucleotides (CpG ODN) are known to be potent stimulators of the innate immune system in vitro and in vivo . We therefore investigated if oral administration of CpG ODN could enhance innate immunity in the gastric mucosa and control the extent of Helicobacter pylori infection in mice . Intragastric administration of a single dose of CpG ODN significantly increased local production of the CC chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES and the CXC chemokine gamma interferon-inducible protein 10 in the stomach and/or the small intestine . Importantly, intragastric administration of CpG ODN to mice with an already established H . pylori infection, in the absence of any coadministered antigen, was found to reduce the bacterial load in the stomach compared to the load in H . pylori-infected control mice, while similar administration of non-CpG ODN had no effect on the bacterial load . The reduction in the bacterial numbers in the stomachs of mice treated with CpG ODN was associated with enhanced infiltration of immune cells and increased RANTES production in the gastric mucosa compared to the infiltration of immune cells and RANTES production in H . pylori-infected control animals . These findings suggest that intragastric administration of CpG ODN without antigen codelivery may represent a valuable strategy for induction of innate immunity against H . pylori infection.

FEMS Microbiol Rev, 2003 Dec, 27(5), 559 - 92
Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs; Korner H et al.; The Crp-Fnr regulators, named after the first two identified members, are DNA-binding proteins which predominantly function as positive transcription factors, though roles of repressors are also important . Among over 1200 proteins with an N-terminally located nucleotide-binding domain similar to the cyclic adenosine monophosphate (cAMP) receptor protein, the distinctive additional trait of the Crp-Fnr superfamily is a C-terminally located helix-turn-helix motif for DNA binding . From a curated database of 369 family members exhibiting both features, we provide a protein tree of Crp-Fnr proteins according to their phylogenetic relationships . This results in the assembly of the regulators ArcR, CooA, CprK, Crp, Dnr, FixK, Flp, Fnr, FnrN, MalR, NnrR, NtcA, PrfA, and YeiL and their homologs in distinct clusters . Lead members and representatives of these groups are described, placing emphasis on the less well-known regulators and target processes . Several more groups consist of sequence-derived proteins of unknown physiological roles; some of them are tight clusters of highly similar members . The Crp-Fnr regulators stand out in responding to a broad spectrum of intracellular and exogenous signals such as cAMP, anoxia, the redox state, oxidative and nitrosative stress, nitric oxide, carbon monoxide, 2-oxoglutarate, or temperature . To accomplish their roles, Crp-Fnr members have intrinsic sensory modules allowing the binding of allosteric effector molecules, or have prosthetic groups for the interaction with the signal . The regulatory adaptability and structural flexibility represented in the Crp-Fnr scaffold has led to the evolution of an important group of physiologically versatile transcription factors.

Biochim Biophys Acta, 2003 Oct 31, 1617(1-2), 89 - 95
Aggregation properties of mycolic acid molecules in monolayer films: a comparative study of compounds from various acid-fast bacterial species; Hasegawa T et al.; Three kinds of mycolic acids (MAs) (alpha-, keto and methoxy-MAs) extracted from several species of mycobacteria were used to prepare monolayer films on water, and the surface pressure-area (pi-A) isotherms of the monolayers have been compared, so that the monolayer characteristics of the MAs as in cell walls would be revealed, since the monolayer molecular aggregation is related to drug permeability via the molecular packing . It was expected that the limiting molecular areas of the isotherms would be changed only a little, which reflects the minor difference in chemical structure and conformation of the mycobacteria . Nevertheless, the results are largely different from the expectation, and two greatly different patterns of the limiting molecular area have been observed . In a new model for elucidation of the results, two parts in an MA molecule are separately considered, and both contributions to the molecular unfolding by the monolayer compression have been suggested . This model is found to be useful to totally understand the isotherm behaviors of MAs . The relationship between monolayer properties and chemical structures for MAs has been summarized for the first time.

Gene, 2003 Dec 4, 321, 47 - 56
A structural and functional study of plastid RNAs homologous to catalytic bacterial RNase P RNA; de la Cruz J et al.; Ribonuclease P (RNase P), the ubiquitous enzyme required for 5' maturation of transfer RNA, is a ribonucleoprotein containing an essential RNA subunit . This RNA (P RNA) is the catalytic component of RNase P in Bacteria and some Archaea . A putative P RNA is encoded in the chloroplast genome of three algae: Cyanophora paradoxa, Porphyra purpurea and Nephroselmis olivacea . In no case, the P RNAs from the plastids were active in vitro in conditions where bacterial and some archaeal P RNAs are functional . By using lead-ion-induced hydrolysis, we conclude that the catalytic deficiency is most likely due to the perturbation of the global structure of the plastid P RNAs compared to the bacterial counterpart . As a consequence, the plastid P RNAs are unable to bind to the precursor tRNA substrates . We discuss these results in the context of plastid and RNase P evolution.

Bioessays, 2003 Dec, 25(12), 1150 - 3
Investigating protein-protein interfaces in bacterial transcription complexes: a fragmentation approach; Burrows PC; Transcription initiation by sigma(54)-RNA polymerase (RNAP) relies explicitly on a transient interaction with a complex molecular machine belonging to the AAA+ (ATPases associated with various cellular activities) superfamily . Members of the AAA+ superfamily convert chemical energy derived from NTP hydrolysis to a mechanical force used to remodel their target substrate . Recently Bordes and colleagues,1 using a protein fragmentation approach, identified a unique sequence within sigma(54)-dependent transcriptional activators that constitutes a sigma(54)-binding interface . This interface is not static, but subject to nucleotide-dependent movement which may represent a common mechanism for controlling output that has been adopted by other AAA+ proteins .

J Vet Med B Infect Dis Vet Public Health, 2003 Nov, 50(9), 447 - 50
Removal of hair surrounding the teat and associated bacterial counts on teat skin surface, in milk, and intramammary infections; Silk AS et al.; The effectiveness of monthly removal of hair surrounding teats on the reduction of teat skin surface bacteria, and the incidence of intramammary infection (IMI), was studied for 10 months in a dairy farm . A split udder design was used where hair was removed on one side, left or right, with the other side serving as a control . Controls and treatment sides were randomly applied in a systematic fashion to 218 cows . Standard milking time pre- and post-milking hygiene practices were applied to all udders during the trial . Collection of teat skin swab solutions preceded aseptic collection of milk samples, performed at monthly intervals, immediately prior to milking . Teat skin bacterial counts did not differ between control and treated teats . Incidences of IMI were similar for treatment when compared with control mammary quarters, as measured by total or by pathogen type . In a second study, the effect of hair removal on the bacterial content of milk was determined using 40 cows . Treatments and allocations were as described . Udder half milk, milk from both mammary quarters of each udder half, was combined and diverted into separate buckets . Buckets were thoroughly cleaned and sanitized between milkings . A portion of bucket milk was collected 24 h after removal of udder hair . The total milk bacterial counts, and counts of psychrotrophs and thermoduric organisms were not reduced by udder hair removal . Results do not suggest that removal of udder hair leads to an improvement in milk quality as determined by milk bacterial content in the herd studied.

Lett Appl Microbiol, 2003, 37(6), 458 - 62
Generation of an Escherichia coli lysA targeted deletion mutant by double cross-over recombination for potential use in a bacterial growth-based lysine assay; Li X et al.; AIMS: To generate a stable Escherichia coli lysine auxotroph for the lysine bioavailability assay . METHODS AND RESULTS: An E . coli lysine auxotrophic strain was constructed by deleting the entire lysA gene and replacing it with a gene that confers resistance to ampicillin (bla) . The linear DNA contained 50 bp homologous sequence of upstream of lysA in one end and 50 bp of downstream of lysA in the other end . CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The deltalysA::bla strain exhibited a linear response to lysine supplementation and can be used for quantifying lysine.

Bull Exp Biol Med, 2003 Aug, 136(2), 129 - 31
Effect of bacterial endotoxin on secretion and synthesis of vasopressin during saline load in rats; Liskina EB et al.; Secretion and synthesis of vasopressin was studied in adult male Wistar rats receiving lipopolysaccharide in a dose of 250 microg/100 g body weight and subjected to moderate osmotic stimulation (2% NaCl perorally for 6 days) . Lipopolysaccharide stimulated secretion of vasopressin into the blood . It should be emphasized that the content of vasopressin mRNA in gigantocellular nuclei of the hypothalamus decreased, which probably reflected intensification of its translation . The observed changes and slight increase in transcription of the vasopressin gene (determined by the content of heterogeneous nuclear RNA) provide intensive secretion of this neurohormone into the blood.

Biol Cell, 2003 Nov, 95(8), 527 - 33
Bacterial lipopolysaccharide modulates protein kinase C signalling in Lymnaea stagnalis haemocytes; Walker AJ et al.; Our knowledge of cell signalling pathways in the molluscan immune system and their response to immunological challenge is currently poor . The present study focused on the Protein Kinase C (PKC) pathway in the immune cells (haemocytes) of Lymnaea stagnalis and its response following exposure to bacterial lipopolysaccharide (LPS) . Western blotting of haemocyte proteins with either anti-PKC (pan) or anti-phospho-PKC (Ser 660) antibodies revealed the presence of two PKC-like immuno-reactive proteins of approximately 76 and 85 kDa . Challenge of haemocytes with LPS transiently increased the phosphorylation of the 85 kDa isoform, with a 2.2-fold increase in phosphorylation levels at 5 min and a return to basal levels after 20 min . This LPS-mediated response was blocked following treatment of haemocytes with GF109203X . PKC activities measured in anti-phospho-PKC immunocomplexes following haemocyte treatment with LPS and GF109203X correlated well with the observed PKC phosphorylation levels . These data show for the first time that the activity of the PKC pathway in molluscan immune cells is modulated by LPS, as it is in mammals, and suggest that cell signalling in the innate immune response may have been conserved through evolution.

FEBS Lett, 2003 Nov 27, 555(1), 170 - 5
Collection and characterisation of bacterial membrane proteins; Saidijam M et al.; A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described . As an illustration we report the cloning of the putative alpha-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities . The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity . This strategy for overexpression and purification is extended to additional membrane proteins from H . pylori and from other bacteria.

FEBS Lett, 2003 Nov 27, 555(1), 45 - 50
Proton transfer pathways and mechanism in bacterial reaction centers; Paddock ML et al.; The focus of this minireview is to discuss the state of knowledge of the pathways and rates of proton transfer in the bacterial reaction center (RC) from Rhodobacter sphaeroides . Protons involved in the light driven catalytic reduction of a quinone molecule QB to quinol QBH2 travel from the aqueous solution through well defined proton transfer pathways to the oxygen atoms of the quinone . Three main topics are discussed: (1) the pathways for proton transfer involving the residues: His-H126, His-H128, Asp-L210, Asp-M17, Asp-L213, Ser-L223 and Glu-L212, which were determined by a variety of methods including the use of proton uptake inhibiting metal ions (e.g . Zn2+ and Cd2+); (2) the rate constants for proton transfer, obtained from a 'chemical rescue' study was determined to be 2 x 10(5) s(-1) and 2 x 10(4) s(-1) for the proton uptake to Glu-L212 and QB-*, respectively; (3) structural studies of altered proton transfer pathways in revertant RCs that lack the key amino acid Asp-L213 show a series of structural changes that propagate toward L213 potentially allowing Glu-H173 to participate in the proton transfer processes.

Biochem Biophys Res Commun, 2003 Dec 5, 312(1), 223 - 8
Bacterial (CYP101) and mitochondrial P450 systems-how comparable are they?
Schiffler B, Bernhardt R.
The bacterial CYP101 system and mitochondrial P450 systems show high similarity . Both systems contain the same protein components, a FAD containing reductase, a ferredoxin of the {2Fe2S} type, and a cytochrome P450 . At a first glance they seem to be comparable but there are considerable differences among both proteins . Thus, the ferredoxin components of the two systems display significant structural homology but cannot substitute for each other in functional assays . Going into more detail, pronounced differences between the two systems that affect their biological functions are found.

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