Biotechniques, 2003 Nov, 35(5), 976 - 9 Rapid development of gene-tagged microsatellite markers from bacterial artificial chromosome clones using anchored TAA repeat primers; Waldbieser GC et al.; We developed a technique to improve the efficiency of producing TAA repeat microsatellite markers linked to interspecific conserved genes . Template DNA was prepared from cultures derived from single bacterial artificial chromosome (BAC) colonies using a simple alkaline lysis miniprep . The presence of conserved genes in each BAC clone was verified by sequencing with gene-specific primers . The BAC templates were directly sequenced using short tandem repeat-anchored primers (STRAPs), consisting of TAA repeats with one or two unique 3' terminal bases . At least one STRAP provided sufficient 3' flanking sequence from each clone for the design of a BAC-specific primer . The BAC-specific primer was used to sequence back through the tandem repeat and obtain 5' flanking sequence, and a second BAC-specific primer was designed for microsatellite genotype analysis . This technique quickly provided microsatellite markers with an average of 15 tandem repeats for the BAC clones tested . The identification of polymorphic microsatellite loci in these clones permits the identification of alleles linked to candidate genes, placement of conserved genes on genetic linkage maps, and integration of linkage and physical maps. Infect Dis Obstet Gynecol, 2003, 11(2), 123 - 9 The effect of treating bacterial vaginosis on preterm labor; Tebes CC et al.; OBJECTIVE: Multiple studies suggest that bacterial vaginosis (BV) causes preterm labor; yet its routine treatment remains controversial . In order to help to elucidate this controversy, we performed a thorough review of studies with levels of evidence ranging from I to II-II . METHODS: We searched for all of the studies from the years 1994 to 2001 via Medline's database, including MD Consult and Ovid Mednet . RESULTS: Several trials discovered a decrease in the incidence of preterm labor when BV was treated, but most of those trials were performed on women with a history of preterm labor . However, the majority of trials reviewed advise against treatment of a general low-risk obstetric population, as there was no significant decrease in preterm labor . CONCLUSIONS: Therefore, based on the above studies and the current guidelines of the Centers for Disease Control and Prevention (CDC), treating pregnant women in high-risk populations who are diagnosed with BV provides the clinician with an opportunity to possibly prevent preterm labor in this population . In nulliparous women without a history of preterm birth, treatment is recommended if other risk factors are present (e.g . gonorrhea or chlamydia) . However, in the general low-risk populations, routine screening is not indicated. Infect Dis Obstet Gynecol, 2003, 11(2), 105 - 8 Bacterial sacroiliitis probably induced by lumbar epidural analgesia; Edelstein S et al.; BACKGROUND: Properly administered, lumbar epidural analgesia provides adequate pain relief during labor and delivery, and is considered to be a safe procedure with limited complications . The prevalence of infection after lumbar epidural analgesia is negligible . INTRODUCTION: Infection of the sacroiliac joint, although very close to the pucture area, has never been reported as a procedure complication . CASE: In this report, we describe a patient who experienced bacterial sacroiliitis a few days after lumbar epidural analgesia for labor . No portal of entry was identified, and we evoked a new potential risk factor that has never been proposed before, namely lumbar epidural analgesia . CONCLUSION: Sacroiliitis must be considered as a rare but serious complication of lumbar epidural analgesia. Nat Immunol, 2003 Dec, 4(12), 1247 - 53 Epub 2003 Nov 16. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility; Smith KD et al.; Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and activates host inflammatory responses . In this study, we examine the nature of the TLR5-flagellin interaction . With deletional, insertional and alanine-scanning mutagenesis, we precisely mapped the TLR5 recognition site on flagellin to a cluster of 13 amino acid residues that participate in intermolecular interactions within flagellar protofilaments and that are required for bacterial motility . The recognition site is buried in the flagellar filament, and monomeric flagellin, but not the filamentous molecule, stimulated TLR5 . Finally, flagellin coprecipitated with TLR5, indicating close physical interaction between the molecules . These studies demonstrate the exquisite ability of the innate immune system to precisely target a conserved site on flagellin that is essential for bacterial motility. Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13910 - 5 Epub 2003 Nov 17. Influence of topology on bacterial social interaction; Park S et al.; The environmental topology of complex structures is used by Escherichia coli to create traveling waves of high cell density, a prelude to quorum sensing . When cells are grown to a moderate density within a confining microenvironment, these traveling waves of cell density allow the cells to find and collapse into confining topologies, which are unstable to population fluctuations above a critical threshold . This was first observed in mazes designed to mimic complex environments, then more clearly in a simpler geometry consisting of a large open area surrounding a square (250 x 250 microm) with a narrow opening of 10-30 microm . Our results thus show that under nutrient-deprived conditions bacteria search out each other in a collective manner and that the bacteria can dynamically confine themselves to highly enclosed spaces. Oral Microbiol Immunol, 2003 Dec, 18(6), 401 - 4 The production of 6-deoxy-L-talan from Actinobacillus actinomycetemcomitans via bacterial coupling in vitro; Suzuki N et al.; A method of producing 6-deoxy-L-talan, the serotype c-specific polysaccharide antigen (SPA) from Actinobacillus actinomycetemcomitans,was established using a whole-cell reaction with two recombinant Escherichia coli strains . The production of serotype c-SPA was investigated using the dot blot assay with anti-A . actinomycetemcomitans NCTC 9710 serum after an 18-h reaction, starting with a solution containing the recombinant E . coli cells, alpha-d-glucose-1-phosphate, and dTTP . Moreover, examination of the time course for 6-deoxy-L-talan production proved that this system ran satisfactorily . This paper is the first report of a convenient method to readily produce the exopolysaccharide from A . actinomycetemcomitans in vitro. Oral Microbiol Immunol, 2003 Dec, 18(6), 350 - 8 Expression of MHC Class II, CD70, CD80, CD86 and pro-inflammatory cytokines is differentially regulated in oral epithelial cells following bacterial challenge; Han DC et al.; Oral epithelium may play a regulatory role in local immune responses when interacting with bacteria . The present study was undertaken to investigate the effects of selected bacterial pathogens found in periodontal and endodontic infections on oral epithelial cells . Expression of cell surface molecules (major histocompatibility complex (MHC) Class II, CD54, CD70, CD80 and CD86) and secretion of inflammatory cytokines (interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha) in response to selected bacterial challenge were examined on an immortalized oral epithelial cell line, HOK-18A and a skin epithelial cell line, HaCaT . Actinomyces viscosus, Actinomyces israelii, Fusobacterium nucleatum lipopolysaccharide (LPS) or primary human periradicular exudate from a granuloma were co-cultured with epithelial cells for 4 or 24 h . Subsequently, cell surface expression of MHC Class II, CD54, CD70, CD80 and CD86, along with pro-inflammatory cytokine levels were determined using flow cytometry, ELISA and RT-PCR . Results indicated that the selected oral bacteria have greater effects on oral versus skin epithelial cells . F . nucleatum increased MHC Class II and CD54 (ICAM-1) cell surface expression on HOK-18A and HaCaT cells . A . israelii also had enhancing effects on the expression of CD54 and MHC Class II . A . israelii and LPS induced a 2.8-fold (P < 0.001) and 4.4-fold (P < 0.005) TNF-alpha secretion, respectively, while F . nucleatum and LPS induced a 10-fold (P < 0.0004) and 6-fold (P < 0.01) IL-1beta secretion, respectively by HOK-18A . Interestingly, CD70, CD80, and CD86 were generally decreased upon bacteria and LPS challenge on HOK-18A . The effects of increased MHC Class II and decreased CD70 were also evident with challenge of human periradicular exudate on HOK-18A . The implications of the study are unique in that oral epithelial cells may play both activating and inhibitory roles in the host immune response towards infection by oral bacteria . We introduce a concept of 'dormancy' where the differential expression of key cell surface antigens on oral epithelial cells may keep the recruited immune effector cells in a state of unresponsiveness, thus contributing to the long term quiescent period observed in many periodontal and endodontic lesions. Nippon Rinsho, 2003 Nov, 61(11), 1892 - 6 {Potentiation of infectivity and pathogenesis of influenza virus by host and bacterial proteases}; Nagatake T; Infectivity and pathogenicity of influenza viruses are based on the interplay between the viral glycoprotein hemagglutinin (HA) and host proteases . HA receives its full biological activities by proteolytic cleavage of a precursor molecule at a definite cleavage site . Proteases selected by the Clara cells in the bronchial epithelia and many kind of bacteria, are virus activate proteases responsible for the cleavage activation and pathogenicity of influenza viruses . Although influenza in normal individuals is usually confined to the upper respiratory tract, the infection often develops into fatal pneumonia in aged patients, where bacterial infections often occur . Synergistic effects of bacterial infections on the pathogenesis of influenza viruses are described in regard to the cleavage activation of HA. Cell Mol Life Sci, 2003 Oct, 60(10), 2100 - 14 Bacterial oligopeptide-binding proteins; Monnet V; This review focuses on bacterial oligopeptide-binding proteins, which form part of the oligopeptide transport system belonging to the ATP-binding cassette family of transporters . Depending on the bacterial species, these binding proteins (OppA) capture peptides ranging in size from 2 to 18 amino acids from the environment and pass them on to the other components of the oligopeptide transport system for internalisation . Bacteria have developed several strategies to produce these binding proteins, which are periplasmic in Gram- bacteria and membrane-anchored in Gram+, with a higher stoichiometry (probably necessary for efficient transport) than the other components in the transport system . The expression of OppA-encoding genes is clearly modulated by external factors, especially nitrogen compounds, but the mechanisms of regulation are not always clear . The best-understood roles played by OppAs are internalisation of peptides for nutrition and recycling of muropeptides . It has, however, recently become clear that OppAs are also involved in sensing the external medium via specific or non-specific peptides. Cell Mol Life Sci, 2003 Oct, 60(10), 2034 - 52 The bacterial translocase: a dynamic protein channel complex; de Keyzer J et al.; The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway) . This route has been extensively studied in Escherichia coli and other bacteria . The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase . The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor . Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor . Translocation is driven by ATP hydrolysis and the proton motive force . In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation . Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process . Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane. J Appl Genet, 2003, 44(4), 561 - 84 High divergence rate of sequences located on different DNA strands in closely related bacterial genomes; Mackiewicz P et al.; One of the common features of bacterial genomes is a strong compositional asymmetry between differently replicating DNA strands (leading and lagging) . The main cause of the observed bias is the mutational pressure associated with replication . This suggests that genes translocated between differently replicating DNA strands are subjected to a higher mutational pressure, which may influence their composition and divergence rate . Analyses of groups of completely sequenced bacterial genomes have revealed that the highest divergence rate is observed for the DNA sequences that in closely related genomes are located on different DNA strands in respect to their role in replication . Paradoxically, for this group of sequences the absolute values of divergence rate are higher for closely related species than for more diverged ones . Since this effect concerns only the specific group of orthologs, there must be a specific mechanism introducing bias into the structure of chromosome by enriching the set of homologs in trans position in newly diverged species in relatively highly diverged sequences . These highly diverged sequences may be of varied nature: (1) paralogs or other fast-evolving genes under weak selection; or (2) pseudogenes that will probably be eliminated from the genome during further evolution; or (3) genes whose history after divergence is longer than the history of the genomes in which they are found . The use of these highly diverged sequences for phylogenetic analyses may influence the topology and branch length of phylogenetic trees . The changing mutational pressure may contribute to arising of genes with new functions as well. Mol Microbiol, 2003 Nov, 50(3), 763 - 70 Targeting of two signal transduction pathways to different regions of the bacterial cell; Wadhams GH et al.; Components of bacterial chemosensory pathways which sense via transmembrane receptors have been shown to localize to the cell poles . Many species, however, have operons encoding multiple putative chemosensory pathways, some including putative cytoplasmic receptors . In-genome fusions to single or multiple genes encoding components of two chemosensory pathways in Rhodobacter sphaeroides, cheOp2 and cheOp3, revealed that while sensory transducing proteins associated with transmembrane receptors and encoded on cheOp2 were targeted to the cell poles, the proteins associated with putative cytoplasmic receptors and encoded on cheOp3 were all targeted to a cytoplasmic cluster . No proteins were localized to both sites . These data show that bacteria target components of related pathways to different sites in the cell, presumably preventing direct cross-talk between the different pathways, but allowing a balanced response between extracellular and cytoplasmic signals . It also indicates that there is intracellular organization in bacterial cells, with specific proteins targeted and localized to cytoplasmic regions. Plant J, 2003 Nov, 36(3), 353 - 65 ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt; Godiard L et al.; Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species . We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana . One of these strains, 14.25, causes wilting symptoms in A . thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0) . Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity . Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R . solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5 . These loci explain about 90% of the resistance carried by the Col-0 accession . The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1 . Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R . solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways. Clin Microbiol Infect, 2003 Nov, 9(11), 1142 - 7 Spread of Stenotrophomonas maltophilia colonization in a pediatric intensive care unit detected by monitoring tracheal bacterial carriage and molecular typing; Lanotte P et al.; In our pediatric intensive care unit in Tours (France), intubated and ventilated inpatients are systematically monitored for tracheal bacterial colonization twice a week . This led us to detect five patients colonized with Stenotrophomonas maltophilia over a 4-month period . Molecular typing of the isolates using random amplified polymorphism DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) confirmed that four of the five isolates were genetically related . The strict isolation of carriers and improvements in hygiene measures stopped the spread . This systematic strategy prevented pulmonary nosocomial infections or allowed their early detection . Moreover, it has made it possible to assess the efficiency of care practices continuously. J Theor Biol, 2003 Dec 21, 225(4), 493 - 6 DNA-membrane interactions can localize bacterial cell center; Rabinovitch A et al.; In actively growing bacterial cells, the DNA exerts stress on the membrane, in addition to the turgor caused by osmotic pressure . This stress is applied through coupled transcription/translation and insertion of membrane proteins (so-called "transertion" process) . In bacillary bacteria, the strength of this interaction varies along cell length with a minimum at its midpoint, and hence can locate the cell's equator for the assembly of the FtsZ-ring. Biosens Bioelectron, 2003 Dec 15, 19(4), 325 - 30 Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles; Tanaka T et al.; We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes . Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers . The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA . The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions . The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate . BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate . This system permits the simultaneous heating and magnetic separation of 96 samples per assay . The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs. FEMS Microbiol Lett, 2003 Nov 7, 228(1), 45 - 9 Intragenomic heterogeneity between multiple 16S ribosomal RNA operons in sequenced bacterial genomes; Coenye T et al.; The availability of a large number of completely sequenced bacterial genomes allows the rapid and reliable determination of intragenomic sequence heterogeneity of 16S rRNA genes . In the present study we assessed the intragenomic sequence heterogeneity of 16S rRNA genes in 55 bacterial genomes, representing various phylogenetic groups . The total number of rRNA operons in genomes included ranged from 2 to 13 . The maximum number of nucleotides that were different between any pair of 16S rRNA genes within a genome ranged from 0 to 19 . The corresponding minimal similarity ranged from 100 to 98.74% . This indicates that the intragenomic heterogeneity between multiple 16S rRNA operons in these genomes is rather limited and is unlikely to have a profound effect on the classification of taxa . Among the multiple copies of the 16S rRNA genes present in the genomes included, 199 mutations were counted with transitions being the dominant type of mutations over the total length of the 16S rRNA gene . Most heterogeneity occurred in variable regions V1, V2, and V6. Biosens Bioelectron, 2003 Nov 30, 19(3), 249 - 59 Chemisorptions of bacterial receptors for hydrophobic amino acids and sugars on gold for biosensor applications: a surface plasmon resonance study of genetically engineered proteins; Luck LA et al.; This paper demonstrates potential applications of two periplasmic receptor proteins from E . coli as sensing elements for biosensors using the surface plasmon resonance (SPR) technique . These molecules, namely the aspartate to cysteine mutant of the leucine-specific receptor (LS-D1C) and the glutamine to cysteine mutant of the D-glucose/D-galactose receptor (GGR-Q26C) proteins, are chemisorbed on a thin (approximately 40 nm) Au film in neutral K2HPO4 buffers . Using angle and time resolved SPR measurements; we show that adsorption behaviors of both proteins are dominated by diffusion-free second order Langmuir kinetics . We also show that the protein-modified Au films exhibit measurable SPR shifts upon binding to their respective target ligands . According to these SPR data, the kinetics of ligand binding for both LS-D1C and GGR-Q26C are governed by irreversible first order diffusion limited Langmuir model . The utility of the SPR technique for studying reactions of biological molecules is further illustrated in this work. Genome, 2003 Oct, 46(5), 870 - 8 Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers; Nilmalgoda SD et al.; A BAC library of hexaploid wheat was constructed using the spring wheat cultivar Triticum aestivum L . 'Glenlea' . Fresh shoot tissue from 7- to 10-day-old seedlings was used to obtain HMW DNA . The library was constructed using the HindIII site of pIndigoBAC-5 and the BamHI site of pIndigoBAC-5 and pECBAC1 . A total of 12 ligations were used to construct the entire library, which contains over 650 000 clones . Ninety-six percent of the clones had inserts . The insert size ranged from 5 to 189 kb with an average of 79 kb . The entire library was gridded onto 24 high-density filters using a 5 x 5 array . A subset of these membranes was hybridized with two intergenic chloroplast probes and the percentage of clones containing chloroplast DNA (cpDNA) was calculated to be 2.2% . The genome coverage was estimated to be 3.1 x haploid genome equivalents, giving a 95.3% probability of identifying a clone corresponding to any wheat DNA sequence . BAC pools were constructed and screened using markers targeting the Glu-B1 locus (1BL), the hardness loci (5AS, 5BS, 5DS), the leaf rust resistance locus Lr1 (5DL), and the major fusarium head blight QTL locus located on 3BS . These markers were either locus-specific amplicons or microsatellites . A total of 49 BAC clones were identified for 14 markers giving an average of 3.5 clones/marker, thereby corroborating the estimated 3.1x genome coverage . An example using the gene encoding the HMW glutenin Bx7 is illustrated. Hepatobiliary Pancreat Dis Int, 2002 Feb, 1(1), 150 - 4 Early intrajejunal nutrition: bacterial translocation and gut barrier function of severe acute pancreatitis in dogs; Qin HL et al.; OBJECTIVE: To evaluate the effect of early intrajejunal nutrition in attenuating bacterial and/or endotoxin translocation and improving gut barrier function of severe acute pancreatitis (SAP) in dogs . METHODS: 15 dogs were divided into parenteral nutrition (PN) group (7 dogs) and early intrajejunal nutrition (EIN) group (8) . EIN was delivered nutrients via a needle jejunostomy catheter feeding at 48 h after operation . SAP model was induced by injecting 1 ml/kg of combined solution of 5% sodium taurocholate and 8000-10 000 BAEE units trypsin/ml into the pancreas via the pancreatic duct . Systemic blood samples were obtained before and 1, 3, 5, 7 d following SAP, and cultured by aerobic as well as anaerobic bacterial growth . Systemic plasma and portal vein endotoxin levels were quantified by the chromogenic limulus amebocyte lysate (LAL) technique . Portal vein blood and specimens of tissue from the mesenteriolum and mesocolon lymph nodes, lung, pulmonary portal lymph nodes, pancreatitistissue and periopancreas tissue were adopted before the experiment was finished . Aliquots of the homogenata were cultured as blood mentioned above to determine the magnitude of the bacteria . DNA, protein and the villi, the thickness of mucosa, and the whole bowel wall of the ileum and transverse colon were measured . RESULTS: The study showed that the levels of systemic plasma endotoxin and the magnitude of bacterial translocation to the portal and systemic blood and distant organ were reduced significantly in the EIN group as compared with the TPN group . The contents of Protein and DNA, the height of villi, the thickness of mucosa and whole bowel wall of the ileum and transverse colon in the EIN group were higher than those in the PN group . CONCLUSION: Our results suggested that EIN is safe and effective to be adopted by intrajejunal delivery of nutrients in SAP, decreases the occurrence of gut bacterial translocation, and improves the gut barrier function. Bioresour Technol, 2004 Feb, 91(3), 243 - 8 Stimulation of bacterial decolorization of an azo dye by extracellular metabolites from Escherichia coli strain NO3; Chang JS et al.; This study provides an attempt to use the extracellular metabolites of a dye-decolorizing strain, Escherichia coli strain NO3, as a biostimulator to entice E . coli strain NO3 into a beneficial mode of metabolism for an economically feasible decolorization . Addition of supernatants containing metabolites from growth and decolorization cultures triggered an enhancement of decolorization rates . Although extracellular metabolites play a crucial role for stimulating decolorization, they still cannot enable decolorization alone without involvement of biodecolorizers (E . coli strain NO3) . Substantial decreases in oxidation-reduction potential to negative levels were observed during anaerobic decolorization in the presence of the metabolites . Results of repeated batch cultures also showed that serial acclimation of E . coli strain NO3 significantly increased decolorization capability . Supplements of metabolites seem to derepress glucose inhibition on decolorization by E . coli strain NO3 . The metabolites produced from higher dye-concentration decolorization led to more enhancement of the bacterial decolorization. J Microbiol Methods, 2003 Dec, 55(3), 755 - 62 Comparison of fluorescence and resonance light scattering for highly sensitive microarray detection of bacterial pathogens; Francois P et al.; Microarrays have emerged as potential tools for bacterial detection and identification . Given their high parallelism, they might represent a breakthrough in current diagnostic methods, provided they can be coupled to simplified labeling protocols and detected with adequate sensitivities . We describe here a technique to directly label total bacterial RNA, thus avoiding the multiple steps and possible biases associated with enzymatic amplification (e.g . PCR) . We have then compared the performances of one white-light source and two laser-based fluorescence scanners for detection reliability and sensitivity . Our study reveals that nanoparticle-labeled bacterial RNA generates reproducible resonance light scattering signals that are at least 50 times more intense than state-of-the-art confocal-based fluorescence signals. Eur J Heart Fail, 2003 Oct, 5(5), 609 - 14 Invasive assessment of bacterial endotoxin and inflammatory cytokines in patients with acute heart failure; Peschel T et al.; AIMS: To test the hypothesis that during acute heart failure endotoxin might be increased in hepatic veins as a sign of bacterial or endotoxin translocation from the bowel into the blood stream . METHODS AND RESULTS: In patients with acute heart failure (NYHA IV; n=17) levels of endotoxin, soluble (s) CD14, tumor necrosis factor alpha (TNFalpha and interleukin 6 (IL6)) were measured in blood drawn from an antecubital vein on admission and compared with age-matched patients with stable chronic heart failure (n=21) and healthy volunteers (n=9) . All levels were systemically elevated during acute heart failure (all P<0.05); once patients were stable enough to undergo cardiac catheterization, endotoxin was found to be significantly higher in hepatic veins (0.62+/-0.05 EU/ml) than left ventricles (0.46+/-0.04 EU/ml; P<0.05), whereas sCD14, TNFalpha and IL6 were not different between these sites . At follow-up (29+/-6 days) endotoxin but not sCD14, TNFalpha or IL-6 was significantly lower as compared to baseline (P<0.05) . CONCLUSIONS: Higher levels of endotoxin in hepatic veins as compared to the left ventricle during acute heart failure are suggestive of bacterial or endotoxin translocation from the bowel into the blood stream . This may lead to new treatment strategies . The lack of difference in TNFalpha levels between the pulmonary artery and the left ventricle sheds doubt on the heart as a source of systemically elevated TNFalpha levels. Trends Microbiol, 2003 Nov, 11(11), 527 - 35 The outs and ins of bacterial type IV secretion substrates; Ding Z et al.; Bacteria use type IV secretion systems (T4SS) to translocate macromolecular substrates destined for bacterial, plant or human target cells . The T4SS are medically important, contributing to virulence-gene spread, genome plasticity and the alteration of host cellular processes during infection . The T4SS are ancestrally related to bacterial conjugation machines, but present-day functions include (i) conjugal transfer of DNA by cell-to-cell contact, (ii) translocation of effector molecules to eukaryotic target cells, and (iii) DNA uptake from or release to the extracellular milieu . Rapid progress has been made toward identification of type IV secretion substrates and the requirements for substrate recognition. Clin Tech Small Anim Pract, 2003 Aug, 18(3), 193 - 8 Bacterial corneal diseases in dogs and cats; Ollivier FJ; Corneal diseases are very common in small animals . Corneal disease associated with bacterial agents is frequent in the dog and maybe less frequent in the cat . The medical history, important steps of the ophthalmic examination, and the ophthalmic diagnostic tests that are relevant in such corneal conditions are outlined . Bacterial corneal diseases in dogs and cats are most commonly considered in one of two categories--bacterial ulcerative keratitis and corneal abscesses . The clinical aspects of these two entities as well as the therapeutic strategies available for general practitioners and ophthalmologists are discussed . Ulcerative keratitis is frequent; it represents the most common ocular diseases in dogs and cats . Because some of these corneal ulcers can be very severe, progress rapidly, and therefore are sight threatening, the crucial steps of their diagnosis and management are stressed . The use of a magnification system, fluorescein dye, and corneal cytology and culture, if indicated, is necessary for diagnosis at an early stage of the disease . The treatment of bacterial ulcerative keratitis should eradicate the infection, reduce or stop the corneal destruction and support the corneal structures, control the uveal reaction and the pain associated with it, and minimize the scarring . The prognosis depends on the stage and the severity of the corneal ulceration, the etiology of the condition, and the therapeutic choice . A close follow-up of animals with corneal ulceration is highly recommended because corneal ulcers can progress rapidly. Exp Physiol, 2003 Nov, 88(6), 747 - 54 Influence of pregnancy on plasma cytokines and the febrile response to intraperitoneal administration of bacterial endotoxin in rats; Fofie AE et al.; Rats have an attenuated febrile response to exogenous (e.g . bacterial endotoxin) and endogenous pyrogen (e.g . interleukin-1beta) near the term of pregnancy, the mechanism of which is unknown . The present experiments were carried out on 71 non-pregnant and 181 pregnant Sprague-Dawley rats to determine if basal levels of the endogenous antipyretic substance, interleukin-1 receptor antagonist (IL-1ra), change relative to interleukin-1beta (IL-1beta) throughout gestation . Furthermore, we have constructed complete Escherichia coli lipopolysaccharide (LPS) dose-core temperature response curves in non-pregnant and pregnant rats on days 10, 15 and 20 of gestation (term of gestation approximately 21 days) to determine if the attenuated febrile response near the term of pregnancy results from a simple shift of the dose-response relationship or results from a dampening of the overall dose-response relationship . Basal IL-1beta, as determined by ELISA on trunk blood from non-pregnant and pregnant rats on days 10, 15 and 20 of gestation (d10, d15, d20), did not change significantly during pregnancy . Basal IL-1ra, however, was increased significantly in d15 rats as compared to non-pregnant, and d10 and d20 rats . The attenuated febrile response near the term of pregnancy, as determined by biotelemetry, did not result from a simple shift of the E . coli LPS dose-core temperature response curve but rather a dampening of the overall dose-response relationship . The febrile responses to EC(50) and EC(100) doses of E . coli LPS were preceded by a period of hypothermia, and were delayed and attenuated near the term of pregnancy as compared to that observed early in pregnancy and in non-pregnant rats . Our data provide evidence that pregnant rats are more sensitive to the hypothermia-producing effects of E . coli LPS than are non-pregnant rats and allow us to speculate that elevated basal IL-1ra may play a role in mediating the attenuated febrile response to pyrogen on day 15 but not on day 20 of gestation in rats. Nucleic Acids Res, 2003 Nov 15, 31(22), 6409 - 18 A DNA translocation motif in the bacterial transcription--repair coupling factor, Mfd; Chambers AL et al.; The bacterial transcription-repair coupling factor, Mfd, is a superfamily II helicase that releases transcription elongation complexes stalled by DNA damage or other obstacles . Transcription complex displacement is an ATP-dependent reaction that is thought to involve DNA translocation without the strand separation associated with classical helicase activity . We have identified single amino acid substitutions within Mfd that disrupt the ability of Mfd to displace RNA polymerase but do not prevent ATP hydrolysis or binding to DNA . These substitutions, or deletion of the C-terminal 209 residues of Mfd, abrogate the ability of Mfd to increase the efficiency of roadblock repression in vivo . The substitutions fall in a region of Mfd that is homologous to the 'TRG' motif of RecG, a protein that catalyses ATP-dependent translocation of Holliday junctions . Our results define a translocation motif in Mfd and suggest that Mfd and RecG couple ATP hydrolysis to translocation of DNA in a similar manner. Appl Environ Microbiol, 2003 Nov, 69(11), 6399 - 404 Use of bacterial gamma-glutamyltranspeptidase for enzymatic synthesis of gamma-D-glutamyl compounds; Suzuki H et al.; An enzymatic method for synthesizing various gamma-D-glutamyl compounds efficiently and stereospecifically involving bacterial gamma-glutamyltranspeptidase (EC 2.3.2.2) with D-glutamine as a gamma-glutamyl donor was developed . With D-glutamine as a gamma-glutamyl donor instead of L-glutamine in gamma-glutamyltaurine synthesis, by-products such as gamma-glutamylglutamine and gamma-glutamyl-gamma-glutamyltaurine were not synthesized and the yield of gamma-glutamyltaurine dramatically increased from 25 to 71% . It was also shown that the purification could be simplified without these gamma-glutamyl by-products . The possibility of synthesizing various gamma-D-glutamyl compounds was also shown. Virus Res, 2003 Nov, 97(2), 57 - 63 Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome; Wang H et al.; Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides . Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC) . A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene . Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination . The DNA of budded viruses (BVs) was used to transform E . coli . One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced . For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6 . The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo . The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. Transgenic Res, 2003 Oct, 12(5), 631 - 4 Expression of green fluorescent protein from bacterial and plastid promoters in tobacco chloroplasts; Newell CA et al.; The expression of the green fluorescent protein reporter gene (gfp) from the bacterial trc and plastid rrn and psbA promoters has been compared in transplastomic tobacco plants produced by microprojectile bombardment . The homoplasmic nature of the regenerated plants was confirmed by Southern blot analysis . Northern blot analysis indicated that plants expressing gfp from the rrn promoter contained 3-fold more gfp RNA than plants containing the psbA promoter and 12-fold more than plants with the trc promoter . Immunoblot analysis and fluorescence spectroscopy indicated that plants expressing gfp from the rrn promoter contained approximately 90-fold more green fluorescent protein (GFP) than plants containing the psbA or trc promoters . This study demonstrates that the bacterial trc promoter is significantly weaker than the plastid rrn promoter for expression of gfp in tobacco chloroplasts. Transgenic Res, 2003 Oct, 12(5), 577 - 86 Induction of H2O2 in transgenic rice leads to cell death and enhanced resistance to both bacterial and fungal pathogens; Kachroo A et al.; Oxidative burst, mediated by hydrogen peroxide (H2O2), has been recognized as a key component of plant defense response during an incompatible interaction . To determine if elevated levels of H2O2 lead to cell death, activation of defense genes and enhanced resistance to diverse pathogens, transgenic rice plants expressing a fungal glucose oxidase gene (GOX) were generated using both constitutive and inducible expression systems . Constitutive or wound/pathogen-induced expression of GOX also allowed us to determine the effectiveness of these systems in conferring long lasting resistance to various pathogens . Both constitutive and wound/pathogen-induced expression of GOX lead to increases in the endogenous levels of H2O2, which in turn caused cell death . Elevated levels of H2O2 also activated the expression of several defense genes and these transgenic plants showed enhanced resistance to both bacterial and fungal pathogens . In comparison to inducible expression, constitutive expression of GOX resulted in 3-10-fold higher levels of the GOX transcript and the corresponding enzymatic activity . Such increased levels of GOX, which would result in elevated levels of H2O2, caused improper seed set and decreased seed viability in transgenic plants constitutively expressing GOX . Our results suggest that pathogen inducible expression of heterologous genes may be a practical and robust way of generating broad spectrum disease resistance. DNA Repair (Amst), 2003 Nov 21, 2(11), 1273 - 4 Excision repair--its bacterial beginnings; Cleaver JE; Nucleotide excision repair was first reported in 1964 by Setlow and Carrier and by Boyce and Howard-Flanders . These two reports clearly defined the existence in bacteria of a repair process that physically removed damaged sites, pyrimidine dimers, from the DNA in the form of acid-soluble fragments . These reports were the starting point for subsequent development of the whole field of DNA excision point. Gastroenterology, 2003 Nov, 125(5), 1388 - 97 Bacterial colonization leads to the colonic secretion of RELMbeta/FIZZ2, a novel goblet cell-specific protein; He W et al.; BACKGROUND & AIMS: Goblet cells are highly polarized exocrine cells found throughout the small and large intestine that have a characteristic morphology due to the accumulation of apical secretory granules . These granules contain proteins that play important physiologic roles in cellular protection, barrier function, and proliferation . A limited number of intestinal goblet cell-specific proteins have been identified . In this study, we investigate the expression and regulation of RELMbeta, a novel colon-specific gene . METHODS: The regulation of RELMbeta messenger RNA expression was determined in LS174T, Caco-2, and HT-29 cell lines in response to stimulation with interleukin 13 and lipopolysaccharide . Quantitative reverse-transcription polymerase chain reaction, immunoblots, and immunohistochemistry were used to examine the expression of RELMbeta in BALB/c and C.B17.SCID mice housed in conventional, germ-free, and gnotobiotic environments . RESULTS: Messenger RNA for RELMbeta is restricted to the undifferentiated, proliferating colonic epithelium . Immunohistochemistry shows that this protein is expressed in goblet cells located primarily in the distal half of the colon and cecum with lower levels detectable in the proximal colon . High levels of RELMbeta can be detected in the stool of mice and humans, where it exists as a homodimer under nonreducing conditions . Interestingly, the secretion of RELMbeta is dramatically reduced in germ-free mice . Furthermore, introduction of germ-free mice into a conventional environment results in enhanced expression and robust secretion of RELMbeta within 48 hours . CONCLUSIONS: These studies define a new goblet cell-specific protein and provide the first evidence that colon-specific gene expression can be regulated by colonization with normal enteric bacteria. Plant Cell Rep, 2004 Mar, 22(8), 569 - 75 Epub 2003 Nov 01. Use of asymmetric somatic hybridization for transfer of the bacterial blight resistance trait from Oryza meyeriana L . to O . sativa L . ssp . japonica; Yan CQ et al.; Bacterial blight is one of the major diseases affecting rice productivity . To improve the resistance of cultivated rice to bacterial blight, we introduced a bacterial blight resistance trait from Oryza meyeriana, a wild rice species, into an elite japonica rice cultivar (Dalixiang) using asymmetric somatic hybridization . One hundred and thirty-two independent lines were regenerated . The hybrid plants possessed several morphological features of the donor species, O . meyeriana . Random amplified polymorphic DNA analysis revealed that hybrid plants exhibited banding patterns derived from their parental genotypes . For the majority of the hybrids, resistance to bacterial blight pathogens was intermediate to that observed for O . meyeriana and O . sativa (cv . Dalixiang) . Four of the hybrid lines exhibited a high bacterial blight resistance, but it was less than that observed for O . meyeriana . These results demonstrate that O . meyeriana can be used as a good genetic source for improving bacterial blight resistance in commercial rice cultivars through asymmetric somatic hybridization. Appl Opt, 2003 Oct 20, 42(30), 6174 - 8 Laser-induced breakdown spectroscopy used to detect palladium and silver metal dispersed in bacterial cellulose membranes; Martin M et al.; The technique of laser-induced breakdown spectroscopy has been used for the first time to our knowledge for the identification of metals such as palladium and silver that were dispersed in bacterial cellulose membranes . These results for palladium-dispersed films have been correlated to a calibration curve obtained by use of atomic absorption spectroscopy and were found to be in good agreement . The experiments were conducted by use of wet and dry metal-doped membranes . The metal peaks obtained with a dry membrane are greater than five times higher in signal-to-background ratio than when metals are detected by a hydrated membrane . The advantage of this laser-based technique is that minimal sample handling and sample preparation are needed and measurements are completed in real time (a few seconds) . Hence this technique can be used for the detection of metals in dry membranes that would be used in the construction of electrode assemblies. Mol Biol (Mosk), 2003 Sep-Oct, 37(5), 916 - 23 {Biosynthesis of mini-antibodies to human ferritin in plant and bacterial producers}; Semeniuk EG et al.; A recombinant scFv antibody against human spleen ferritin was expressed as a barstar-fused protein in Escherichia coli and in Nicotiana tabacum plants and suspension cell cultures . As demonstrated by immunoblotting with antibarstar antibodies, direction of the recombinant protein to the endomembrane system of plant cells ensured its stability and solubility . Production of the recombinant protein did not differ between parental transgenic plants and their first-generation progeny . Fusion with barstar allowed not only immunochemical detection of the recombinant scFv antibody, but also their purification from the plant material by affinity chromatography with barnase-His6 immobilized on a metal-affinity carrier. Mol Biol (Mosk), 2003 Sep-Oct, 37(5), 834 - 42 {Search for alternative secondary structures of RNA, regulating expression of bacterial genes}; Liubetskaia EV et al.; Expression of many bacterial genes is regulated by formation of alternative secondary RNA structure within the leader mRNA sequence . Our algorithm designed to search for these structures (basing on analysis of one nucleotide sequence) was applied to analyze operons of amino acid biosynthesis in alpha- and gamma-proteobacteria . The attenuators of these operons are predicted for genomes of some poorly known gamma-proteobacteria including Shewanella putrefaciens, attenuators of the tryptophan operon in some alpha-proteobacteria are also predicted. Rev Neurol, 2003 Oct 16-31, 37(8), 722 - 5 {Spastic tetraparesis as a sequela of bacterial meningitis}; T-Barros SV et al.; AIMS: The aim of this study is to demonstrate the incidence of spastic tetraparesis (ST) in meningitis patients in the paediatric ICU, together with the associated variables, and establish comparisons with the existing literature . PATIENTS AND METHODS: We reviewed the medical records of patients who presented symptoms of meningitis and required hospital treatment in the Paediatric ICU at the Hospital de Clinicas de Porto Alegre, between January 1985 and June 2001 . In addition to the diagnosis of meningitis and the incidence of ST as a complication, we also examined the aetiological agent, sex, age at the moment of hospital admittance, length of time spent in hospital and treatment given in each case . RESULTS AND DISCUSSION . An incidence of 15.1% was found for cerebral palsy in the 112 cases of bacterial meningitis that were followed up clinically . In the patients with ST, the time spent in hospital was longer, and the frequency of seizures, intracranial hypertension and the protein concentration levels in CSF were higher (p<0.05). FEMS Microbiol Lett, 2003 Oct 24, 227(2), 243 - 7 Anaplasma phagocytophilum major surface protein-2 (Msp2) forms multimeric complexes in the bacterial membrane; Park J et al.; Anaplasma phagocytophilum 44-kDa major surface protein-2 (Msp2) mediates partial neutrophil adhesion and interactions . Since A . phagocytophilum 44-kDa monoclonal antibodies also react with 160- and 100-kDa bands, a putative adhesin complex was studied . After separate excision/immunoprecipitation of these three bands, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved each into three bands again with increased 44-kDa protein under reducing conditions suggesting oligomerization of Msp2 44-kDa monomers . With 9 M urea, each separately excised band was resolved only into 44-kDa monomers with three different pIs . With protein cross-linking, immunoblots showed four additional bands and increased high molecular mass band intensity, suggesting homo- and hetero-polymerization with other A . phagocytophilum proteins . Recognition of Msp2 complexes facilitates understanding of A . phagocytophilum-neutrophil adhesion. Surg Infect (Larchmt), 2003 Fall, 4(3), 281 - 7 Bacterial agents used for bioterrorism; Horn JK; BACKGROUND: Bacterial pathogens and their products are potential agents of biological terrorism and biological warfare . These agents can be deployed through simple aerosol delivery systems and thereby cause widespread disease and death . METHODS: This report is a review of bacterial species that have been employed for development of biological terrorism, relying on a system for classification of their threat developed by the Centers for Disease Control . RESULTS: Physicians must understand how to recognize early signs and symptoms caused by bacterial agents . Clinical findings often seen on presentation are emphasized along with a summary of therapeutic approaches . CONCLUSIONS: Initiation of immediate therapy and supportive care provides the best chance for survival from these potentially lethal and devastating infections . A high index of suspicion must be maintained, especially in the setting of a sudden influx of cases with what are often relatively nonspecific symptoms. Nature, 2003 Oct 30, 425(6961), 917 - 25 A gene expression atlas of the central nervous system based on bacterial artificial chromosomes; Gong S et al.; The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions . Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways . We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development . The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community. Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2294 - 6 The protein component of bacterial ribonuclease P flickers the metal ion response to the substrate shape preference of the ribozyme; Ando T et al.; The substrate shape specificity of the Escherichia coli ribonuclease P (RNase P) ribozyme depends on the concentration of magnesium ion . At 10 mM or more, it can cleave a hairpin substrate as well as a cloverleaf pre-transfer RNA (tRNA) . The results showed, however, that the holo enzyme cleaved the hairpin substrate at low concentrations of magnesium ion . Considering that the homologous E . coli tRNAs are resistant to internal cleavage by the RNase P, the phenomena suggest that this catalytic activity might take part in the removing the mis-folded RNAs in the cell. Int J Neurosci, 2003 Nov, 113(11), 1497 - 504 Evaluation of antioxidant status in children with acute bacterial meningitis and encephalitis; Caksen H et al.; Antioxidant status was investigated in children with acute bacterial meningitis and encephalitis to investigate the possible role of free radicals in children with meningitis and encephalitis . Our study included 16 children with acute bacterial meningitis, 13 with encephalitis, and 17 control subjects . Serum ceruloplasmin, uric acid, albumin, bilirubin superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) levels were studied in all subjects within 6 h of admission . There was a statistically significant difference between the groups for all parameters except for serum uric acid . All antioxidant activities except for albumin level were increased in the study groups . Albumin level was higher in the control group than those of meningitis and encephalitis groups . When the values of meningitis and encephalitis were compared, there was a statistically significant difference between the groups for serum SOD, GPx, ceruloplasmin, and albumin . In conclusion, our study showed that serum SOD, GPx, catalase, and ceruloplasmin were higher in children with acute bacterial meningitis and serum SOD, GPx, catalase, ceruloplasmin, and total bilirubin levels were increased in children with encephalitis . These findings suggest that antioxidant status was almost similar in both acute bacterial meningitis and encephalitis conditions in childhood. Mol Cell Biol, 1982 Dec, 2(12), 1628 - 32 Expression of bacterial beta-galactosidase in animal cells; An G et al.; A recombinant plasmid containing the gene for bacterial beta-galactosidase, situated close to the simian virus 40 early promoter, has been constructed . Transfection of CHO, L, and COS-1 cells with this plasmid led to the expression and appearance of the enzyme . Using this system, we have developed a series of promoter cloning vehicles capable of accepting promoter signals for animal genes. J Exp Med, 2003 Nov 3, 198(9), 1349 - 60 Epub 2003 Oct 27. Plasticity of repetitive DNA sequences within a bacterial (Type IV) secretion system component; Aras RA et al.; DNA rearrangement permits bacteria to regulate gene content and expression . In Helicobacter pylori, cagY, which contains an extraordinary number of direct DNA repeats, encodes a surface-exposed subunit of a (type IV) bacterial secretory system . Examining potential DNA rearrangements involving the cagY repeats indicated that recombination events invariably yield in-frame open reading frames, producing alternatively expressed genes . In individual hosts, H . pylori cell populations include strains that produce CagY proteins that differ in size, due to the predicted in-frame deletions or duplications, and elicit minimal or no host antibody recognition . Using repetitive DNA, H . pylori rearrangements in a host-exposed subunit of a conserved bacterial secretion system may permit a novel form of antigenic evasion. J Biol Chem, 2004 Jan 16, 279(3), 2231 - 41 Epub 2003 Oct 27. Evaluation of the RNA determinants for bacterial and yeast RNase III binding and cleavage; Lamontagne B et al.; Bacterial double-stranded RNA-specific RNase III recognizes the A-form of an RNA helix with little sequence specificity . In contrast, baker yeast RNase III (Rnt1p) selectively recognizes NGNN tetraloops even when they are attached to a B-form DNA helix . To comprehend the general mechanism of RNase III substrate recognition, we mapped the Rnt1p binding signal and directly compared its substrate specificity to that of both Escherichia coli RNase III and fission yeast RNase III (PacI) . Rnt1p bound but did not cleave long RNA duplexes without NGNN tetraloops, whereas RNase III indiscriminately cleaved all RNA duplexes . PacI cleaved RNA duplexes with some preferences for NGNN-capped RNA stems under physiological conditions . Hydroxyl radical footprints indicate that Rnt1p specifically interacts with the NGNN tetraloop and its surrounding nucleotides . In contrast, Rnt1p interaction with GAAA-capped hairpins was weak and largely unspecific . Certain duality of substrate recognition was exhibited by PacI but not by bacterial RNase III . E . coli RNase III recognized RNA duplexes longer than 11 bp with little specificity, and no specific features were required for cleavage . On the other hand, PacI cleaved long, but not short, RNA duplexes with little sequence specificity . PacI cleavage of RNA stems shorter than 27 bp was dependent on the presence of an UU-UC internal loop two nucleotides upstream of the cleavage site . These observations suggest that yeast RNase IIIs have two recognition mechanisms, one that uses specific structural features and another that recognizes general features of the A-form RNA helix. Curr Opin Chem Biol, 2003 Oct, 7(5), 586 - 91 Molecular mechanisms of bacterial quorum sensing as a new drug target; Suga H et al.; Bacterial quorum sensing evolved as a means for bacterial communities to rapidly and coordinately change genome expression patterns in response to environmental cues . Each cell in a community produces and responds to a signaling molecule called an autoinducer that serves as an indicator of the population density . A high concentration of autoinducer is associated with a large, often confined population, which requires altered gene expression for survival . Quorum sensing is one mechanism through which a bacterial population receives input from the environment and elicits an appropriate response . Because many pathogens require quorum sensing to produce virulence factors in response to association with a human host, the signaling pathway is a target for design of small-molecule inhibitors. Biotechniques, 2003 Oct, 35(4), 796 - 807 Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells; Montigny WJ et al.; Although bacterial artificial chromosomes (BACs) provide a well-characterized resource for the analysis of large chromosomal domains, low transfection rates have proven a significant limitation for their use in cell culture models . Using TP53 BAC clones that contain expression cassettes for enhanced green fluorescent protein or red fluorescent protein, we have examined conditions that promote BAC transfection in hamster, human, and mouse cell lines . Atomic force microscopy shows that BAC transfection efficiency correlates with the generation of small, highly condensed but dispersed lipid: BAC DNA transfection complexes . BAC DNA purity and concentration are critical for good transfection; debris from purification columns induces the formation of large aggregates that do not gain entry into the cell, and DNA concentrations must be optimized to promote intramolecular condensation rather than intermolecular linking, which also causes aggregation and diminished transfection efficiency . The expression of both markers and genes within BACs initially occurs at lower levels than observed with plasmids, requiring 3-5 days to evaluate the transfection results . We also show that BACs can be co-transfected with other BACs, which provides for increased experimental flexibility. Eur J Immunol, 2003 Nov, 33(11), 3164 - 74 Bacterial DNA activates human neutrophils by a CpG-independent pathway; Trevani AS et al.; Bacterial DNA stimulates macrophages, monocytes, B lymphocytes, NK cells, and dendritic cells in a CpG-dependent manner . In this work we demonstrate that bacterial DNA, but not mammalian DNA, induces human neutrophil activation as assessed by L-selectin shedding, CD11b upregulation, and stimulation of cellular shape change, IL-8 secretion, and cell migration . Induction of these responses is not dependent on the presence of unmethylated CpG motifs, as neutrophil stimulatory properties were neither modified by CpG-methylation of bacterial DNA nor reproduced by oligonucleotides bearing CpG motifs . We found that human neutrophils express Toll-like receptor (TLR) 9 mRNA . However, as expected for a CpG-independent mechanism, activation does not involve a TLR9-dependent signaling pathway; neutrophil stimulation was not prevented by immobilization of bacterial DNA or by wortmannin or chloroquine, two agents that inhibit TLR9 signaling . Of note, both single-stranded and double-stranded DNA were able to induce activation, suggesting that neutrophils might be activated by bacterial DNA at inflammatory foci even in the absence of conditions required to induce DNA denaturation . Our findings provide the first evidence that neutrophils might be alerted to the presence of invading bacteria through recognition of its DNA via a novel mechanism not involving CpG motifs. Hepatology, 2003 Nov, 38(5), 1210 - 8 Systemic, renal, and hepatic hemodynamic derangement in cirrhotic patients with spontaneous bacterial peritonitis; Ruiz-del-Arbol L et al.; Spontaneous bacterial peritonitis (SBP) is frequently associated with renal failure . This study assessed if systemic and hepatic hemodynamics are also affected by this condition . Standard laboratory tests, tumor necrosis factor alpha (TNF-alpha) in plasma and ascitic fluid, plasma renin activity (PRA) and norepinephrine (NE), and systemic and hepatic hemodynamics were determined in 23 patients with SBP at diagnosis and after resolution of infection . Eight patients developed renal failure during treatment . At diagnosis of infection, patients developing renal failure showed significantly higher values of TNF-alpha, blood urea nitrogen (BUN), PRA and NE, peripheral vascular resistance, and hepatic venous pressure gradient (HVPG) and lower cardiac output than patients not developing renal failure . During treatment, a significant reduction in cardiac output and arterial pressure and increase in PRA and NE, HVPG, and Child-Pugh score were observed in the first group but not in the second . Peripheral vascular resistance remained unmodified in both groups . Changes in PRA and NE correlated inversely with changes in arterial pressure and directly with changes in BUN, Child-Pugh score, and HVPG . Five patients in the renal failure group developed encephalopathy, and 6 died . In the group without renal failure, none of the patients developed encephalopathy or expired . In conclusion, patients with SBP frequently develop a rapidly progressive impairment in systemic hemodynamics, leading to severe renal and hepatic failure, aggravation of portal hypertension, encephalopathy, and death . This occurs despite rapid resolution of infection and is associated with an extremely poor prognosis. Am J Pathol, 2003 Nov, 163(5), 1961 - 8 Interleukin-12-independent down-modulation of cockroach antigen-induced asthma in mice by intranasal exposure to bacterial lipopolysaccharide; Lundy SK et al.; Several studies have shown that exposure to bacterial lipopolysaccharide (LPS) can either prevent or inhibit asthma in humans and laboratory rodents . Much emphasis has been placed on the role of cytokines and chemokines in the establishment and maintenance of allergic airway disease . Therefore, it is of interest to study the role of LPS in affecting airway pathology and lung cytokine and chemokine responses in the maintenance phase of asthma . Increasing doses of LPS were administered into the airways of mice presensitized with cockroach allergen (CRAg), then allergic airway disease parameters were assessed after CRAg challenge . Airway hyperresponsiveness after antigen challenge decreased at the highest dose of LPS tested, which was accompanied by a decrease in airway and lung eosinophils . However, a dramatic increase in lung inflammation because of neutrophil influx was observed . Measurement of cytokines in lungs of LPS-treated, CRAg-sensitized mice indicated that interleukin (IL)-12 levels were increased by LPS treatment in a dose-dependent manner, as were levels of several inflammatory chemokines . In contrast, levels of IL-4, IL-13, IL-5, and IL-10 were reduced in whole lung homogenates only of high-dose LPS-treated mice . Intranasal administration of neutralizing anti-IL-12 at the time of high-dose LPS challenge reduced lung IL-12, interferon-gamma, CXCL9, and CXCL10 but did not affect levels of the other chemokines or Th2-type cytokines, and did not restore AHR . These findings suggest that the amelioration of airway hyperresponsiveness observed in LPS-treated, CRAg-sensitized mice is coincident with an immune deviation of the lung inflammatory response, independent of IL-12. J Endotoxin Res, 2003, 9(5), 331 - 5 Receptor cluster formation during activation by bacterial products; Triantafilou M et al.; The recognition of bacterial products, such as lipopolysaccharide (LPS) by the innate immune system lead to a strong pro-inflammatory response that can eventually lead to fatal sepsis syndrome in humans . Although CD14 and TLR4 have been identified as the key molecules involved in LPS-induced signal transduction, accumulating evidence indicates that multiple receptors are also involved . Our group has recently identified a cluster of receptors, involving heat-shock proteins 70 and 90, chemokine receptor 4 as well as growth differentiation factor 5, that are formed following LPS stimulation . In addition, we present data demonstrating that these molecules associate with TLR4 and accumulate in membrane microdomains following LPS ligation . Our results suggest that the entire bacterial recognition is based around the recruitment of multiple signalling molecules, in addition to CD14 and TLRs, within the lipid rafts . We propose that different combinational associations of receptors within activation clusters determine the different responses to a variety of bacterial stimuli. Cornea, 2003 Nov, 22(8), 746 - 53 Culture plate temperature and delayed incubation effect on bacterial recovery; Rudometkin NJ et al.; PURPOSE: To discover if initial culturing conditions (plate temperature and time delay to incubation) adversely influence the recovery of organisms associated with bacterial keratitis . METHODS: The rate of temperature equilibration of culture plates taken from a refrigerator and placed in an incubator and left on the desk was evaluated with a digital thermometer . A standard inoculum for each of five organisms (S . aureus, S . pneumoniae, P . aeruginosa, E . aerogenes, K . oxytoca) isolated from human bacterial keratitis was spread evenly on blood agar plates at refrigerator (Tcold; 4 degrees C), room (Troom; 24 degrees C), and incubator (Twarm; 37 degrees C) temperatures . The plates were then kept at room temperature for 0, 1, 3, 5, and 8 hours before overnight incubation at 37 degrees C (S . pneumoniae under microaerophilic conditions), and the number of colony-forming units was counted . RESULTS: Cold plates took at least 15 minutes in an incubator to attain room temperature, and up to an hour when left on the desk . Increased organism recovery was found comparing both Twarm and Troom plates (6.2 to 24.8% and 7.0 to 14.7%, respectively, P<0.001) to Tcold plates for all organisms except S . pneumoniae (P=0.057) . Comparing Twarm plates to Troom plates demonstrated an increased recovery (P<0.001) for S . aureus . Delayed incubation resulted in decreased recovery for S . pneumoniae (P<0.001) . CONCLUSIONS: Culture plates should preferably be warmed at least to room temperature before inoculation, as well as promptly incubated to increase bacterial recovery from cases of septic keratitis. Science, 2003 Oct 24, 302(5645), 650 - 4 A new class of bacterial RNA polymerase inhibitor affects nucleotide addition; Artsimovitch I et al.; RNA polymerase (RNAP) is the central enzyme of gene expression . Despite availability of crystal structures, details of its nucleotide addition cycle remain obscure . We describe bacterial RNAP inhibitors (the CBR703 series) whose properties illuminate this mechanism . These compounds inhibit known catalytic activities of RNAP (nucleotide addition, pyrophosphorolysis, and Gre-stimulated transcript cleavage) but not translocation of RNA or DNA when translocation is uncoupled from catalysis . CBR703-resistance substitutions occur on an outside surface of RNAP opposite its internal active site . We propose that CBR703 compounds inhibit nucleotide addition allosterically by hindering movements of active site structures that are linked to the CBR703 binding site through a bridge helix. Vaccine, 2003 Nov 7, 21(31), 4539 - 44 Bacterial otitis media: a new non-invasive rat model; Tonnaer EL et al.; This study describes the development of a physiological rat model for otitis media . The model is based on the assumption that bacteria, intranasally introduced into the nasopharynx, will be transferred into the middle ear cavity during swallowing provided that the ambient air pressure is higher than the middle ear pressure . This model demonstrates that small pressure changes, generated in a pressure cabin under controlled conditions, can be used as driving force for the transfer of bacteria into the middle ear cavity resulting in bilateral otitis media . Because invasive techniques or biochemical agents are not applied, this model is suited to investigate immunological aspects of otitis media, including the effects of vaccination. Protein Sci, 2003 Nov, 12(11), 2588 - 96 The solution structure of the bacterial HSP70 chaperone protein domain DnaK(393-507) in complex with the peptide NRLLLTG; Stevens SY et al.; The Hsp70 family of molecular chaperones participates in a number of cellular processes, including binding to nascent polypeptide chains and assistance in protein (re)folding and degradation . We present the solution structure of the substrate binding domain (residues 393-507) of the Escherichia coli Hsp70, DnaK, that is bound to the peptide NRLLLTG and compare it to the crystal structure of DnaK(389-607) bound to the same peptide . The construct discussed here does not contain the alpha-helical domain that characterizes earlier published peptide-bound structures of the Hsp70s . It is established that removing the alpha-helical domain in its entirety does not affect the primary interactions or structure of the DnaK(393-507) in complex with the peptide NRLLLTG . In particular, the arch that protects the substrate-binding cleft is also formed in the absence of the helical lid . 15N-relaxation measurements show that the peptide-bound form of DnaK(393-507) is relatively rigid . As compared to the peptide-free state, the peptide-bound state of the domain shows distinct, widespread, and contiguous differences in structure extending toward areas previously defined as important to the allosteric regulation of the Hsp70 chaperones. Infect Immun, 2003 Nov, 71(11), 6420 - 5 Modulation of the bovine delayed-type hypersensitivity responses to defined mycobacterial antigens by a synthetic bacterial lipopeptide; Whelan AO et al.; The use of defined protein and peptide antigens can overcome specificity limitations of purified protein derivatives in the detection of bovine tuberculosis when the antigens are used in blood-based tests . Since the use of these specific antigens as skin test reagents could have practical advantages, we investigated the potential of Mycobacterium bovis-specific antigens to stimulate delayed-type hypersensitivity (DTH) responses in cattle experimentally infected with M . bovis . A cocktail of the recombinant antigens ESAT-6, MPB83, and MPB64 failed to stimulate in vivo DTH in cattle that had been experimentally infected with M . bovis despite the fact that the antigens were recognized in vitro by the same animals . However, it was possible to stimulate antigen-specific bovine DTH responses by using ESAT-6 in combination with a synthetic bacterial lipopeptide . This lipopeptide stimulated the release of the proinflammatory cytokine tumor necrosis factor alpha from monocyte-derived bovine dendritic cells in vitro, thereby providing a possible mechanism for its DTH-enhancing properties. Curr Opin Microbiol, 2003 Oct, 6(5), 512 - 8 Tracing the evolution of gene loss in obligate bacterial symbionts; Moran NA; The gamma-proteobacterial symbionts of insects are a model group for comparative studies of genome reduction . The phylogenetic proximity of these reduced genomes to the larger genomes of well-studied free-living bacteria has enabled reconstructions of the process by which genes and DNA are lost . Three genome sequences are now available for Buchnera aphidicola . Analyses of Buchnera genomes in comparison with those of related enteric bacteria suggest that extensive changes including large deletions, repetitive element proliferation and chromosomal rearrangements occurred initially, followed by extreme stasis in gene order and slow decay of additional genes . This pattern appears to be characteristic of symbiont evolution. Hepatogastroenterology, 2003 Sep-Oct, 50(53), 1700 - 3 Outcome of patients with inconsistent results from 13C-urea breath test and bacterial culture at the time of assessment of Helicobacter pylori eradication therapy in Japan; Nagahara Y et al.; BACKGROUND/AIMS: At the assessment of eradication therapy of Helicobacter pylori, the results of 13C-urea breath test and other methods such as bacterial culture are occasionally inconsistent . In this study, we examined the outcomes of inconsistent results . METHODOLOGY: Four hundred and four patients with peptic ulcer who were H . pylori-positive and who had completed eradication therapy were studied . Bacterial culture, rapid urease tests and 13C-urea breath test were performed between one and three months after the end of the therapy . The cut-off value for the 13C-urea breath test used originally in this study was 2.5 per mil . We investigated the outcome of inconsistent results by following up the patients every 6 to 12 months . RESULTS: At the initial assessment of eradication therapy, we observed inconsistent results with bacterial culture and 13C-urea breath test in 43 of 404 patients . Most of them (40 of 43) were culture-negative but urea breath test-positive, and the majority became negative for both tests . Based on the follow-up results, the optimum value for 13C-urea breath test at the assessment of eradication therapy was found to be 3.5 per mil . CONCLUSIONS: We found that outcomes of inconsistent results were variable, indicating the importance of the follow-up of patients after eradication therapy of H . pylori. Hepatogastroenterology, 2003 Sep-Oct, 50(53), 1542 - 6 Short and long-term effects of bacterial translocation due to obstructive jaundice on liver damage; Sakrak O et al.; BACKGROUND/AIMS: The present study was conducted to determine if obstructive jaundice promotes bacterial translocation and to evaluate the changes in hepatic histopathology in patients with benign biliary obstruction . METHODOLOGY: Between January 1996 and January 1998, 19 patients treated for benign biliary obstruction were studied . Fourteen patients with symptomatic cholelithiasis were taken as the control group . Patient characteristics, preoperative and post-operative laboratory tests with an interval of 7 days were recorded . In all patients, bile and mesenteric lymph nodes samples were taken for bacterial growth and histopathologic changes were studied on the liver excised during surgery . RESULTS: In the control group, bacterial growth was observed in the bile and mesenteric lymph nodes cultures in one (7.1%) and two patients (14.3%), respectively . In the study group, 8 patients (42%) had positive bile cultures and 12 patients (63.2%) had positive mesenteric lymph nodes cultures, respectively . Histopathologic examination of the liver revealed significant increase in the rate of periductal and portal fibrosis in the jaundiced patients, compared with control group (p < 0.001) . Postoperative complications in the study group were wound infection (3 cases), renal failure (2 cases), ARDS (1 cases) and intraabdominal abscess (1 cases) . In the control group, one patient had wound infection and one had atelectasis . Two patients with jaundice died of multiple organ failure and respiratory failure . In long-term follow-up (mean 17 months), when sclerosing cholangitis and secondary biliary cirrhosis developed in one patient each in the study group, no long-term complication occurred in the control group . CONCLUSIONS: Our clinical results demonstrate that extrahepatic biliary obstruction promotes bacterial translocation and this process is an important cause of morbidity and mortality in patients with jaundice . Also, obstructive jaundice subsequently leads to significant functional and morphological damage in the liver. Hepatogastroenterology, 2003 Sep-Oct, 50(53), 1415 - 8 Bone mineral density in patients with small intestinal bacterial overgrowth; Stotzer PO et al.; BACKGROUND/AIMS: Malabsorption has long been recognized as a cause of osteopenia, and mild forms of osteopenia are present in many gastrointestinal disorders . The aim of this study was to determine if osteopenia is common in patients with small intestinal bacterial overgrowth . METHODOLOGY: Bone mineral density was measured in fourteen patients with small intestinal bacterial overgrowth . Patients with obvious structural predisposing conditions such as previous gastric operations, small bowel strictures and small bowel diverticula, were excluded . Measurements were made in the distal right radius and ulna, in the hip and in the spine . The results were compared to those of a reference population . Radiographs of the spine were assessed for evidence of vertebral fractures . Blood samples were analyzed for serum concentrations of 25-hydroxyvitamin-D3 and 1,25-dihydroxyvitamin-D3, alkaline phosphatase activity, ionized calcium, intact parathyroid hormone and osteocalcin . All patients completed a questionnaire concerning, inter alia, previous fractures, past and current diseases, tobacco smoking and medication . RESULTS: Patients with small intestinal bacterial overgrowth had significantly low bone density in the femoral neck (p < 0.01) and in the lumbar spine (p < 0.05), compared to a reference population . Six of 14 (43%) patients had had fractures . CONCLUSIONS: Patients with small intestinal bacterial overgrowth have low bone mineral density . In patients with osteopenia of unknown origin, small intestinal bacterial overgrowth should be considered. Biol Proced Online, 2003, 5, 123 - 135 Epub 2003 May 1. Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins; Jacobsson K et al.; Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector . The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene . From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning . The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies . In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins. Can J Microbiol, 2003 Jul, 49(7), 418 - 24 Mechanisms of cadmium resistance in anaerobic bacterial enrichments degrading pentachlorophenol; Kamashwaran SR et al.; The mechanisms of heavy-metal resistance used by adapted sulfidogenic and methanogenic enrichments degrading pentachlorophenol in the presence of cadmium (Cd) were studied . The enrichment cultures adapted to and readily tolerated bioavailable Cd concentrations up to 50 ppm while degrading an equal concentration of pentachlorophenol . Both cultures removed >95% of the Cd from solution . Transmission electron micrographs revealed (i) . the presence of electron-dense particles surrounding the cells in the sulfidogenic enrichments and (ii) . the unusual clumping of cells and the presence of an exopolymer in the methanogenic enrichments . Energy dispersive X-ray analysis showed that the sulfidogenic enrichments removed Cd by extracellular precipitation of cadmium sulfide, while the methanogenic enrichment culture removed Cd by extracellular sequestration of Cd into the exopolymer. J Pharm Biomed Anal, 2003 Jul 14, 32(3), 495 - 503 Estimation of uncertainty in kinetic-colorimetric assay of bacterial endotoxins; Haishima Y et al.; The relative standard deviation (R.S.D.) of measurements is estimated in the kinetic-colorimetric assay of bacterial endotoxins without recourse to the usual repeated experiments . The measurements are the slopes of kinetic curves and two major factors are considered to cause the uncertainty of the measurements: (1) the pipetting of the sample and color development reagent; and (2) noise in the detection unit . The measurement R.S.D . is formulated as a function of endotoxin concentration . Two parameters (S.D . of the pipetted volumes and S.D . of the detector noise) are also required in the uncertainty equation, but no arbitrary coefficients are included . Since the S.D . values for pipettes and detector noise can be determined independently of the endotoxin assays, the measurement R.S.D . can be estimated by the above equation without repeating the assays . However, the calibration curve is necessary . The theoretical estimation is shown to be in good agreement with the experimental R.S.D . (n = 12) over a wide concentration range. Appl Microbiol Biotechnol, 2004 Apr, 64(2), 199 - 205 Epub 2003 Oct 15. Utilization of the buffering capacity of corn steep liquor in bacterial cellulose production by Acetobacter xylinum; Noro N et al.; Acetobacter xylinum BPR2001 produces water-insoluble bacterial cellulose (BC) . Using a pH sensor for the accurate control of pH, which is one of the most critical factors for efficient BC production, is difficult especially in a baffled shake-flask and an airlift reactor . The buffering capacity of corn steep liquor (CSL) was estimated by measuring beta (buffering capacity) values in advance and was used to maintain the pH within the optimal range during the production of BC . When CSL was added to either a shake-flask, a stirred-tank reactor or an airlift reactor, BC production was almost the same as that in cultivations where pH was controlled manually or by a pH sensor. Theor Appl Genet, 2004 Feb, 108(4), 663 - 9 Epub 2003 Oct 16. Construction of a HindIII Bacterial Artificial Chromosome library and its use in identification of clones associated with disease resistance in chickpea; Rajesh PN et al.; A chickpea ( Cicer arietinum L.) Bacterial Artificial Chromosome (BAC) library from germplasm line, FLIP 84-92C, was constructed to facilitate positional cloning of disease resistance genes and physical mapping of the genome . The BAC library has 23,780 colonies and was calculated to comprise approximately 3.8 haploid-genome equivalents . Studies on 120 randomly chosen clones revealed an average insert size of 100 kb and no empty clones . Colony hybridization using the RUBP carboxylase large subunit as a probe resulted in a very low percentage of chloroplast DNA contamination . Two clones with a combined insert size of 200 kb were isolated after the library was screened with a Sequence Tagged Microsatellite Site (STMS) marker, Ta96, which is tightly linked to a gene ( Foc3) for resistance to fusarium wilt caused by Fusarium oxysporum Schlechtend.: Fr . f . sp . ciceris (Padwick) race 3 at a genetic distance of 1 cM . Also, these two clones were analyzed with several resistance gene analog (RGA) markers . End sequencing of these clones did not identify repetitive sequences . The development of the BAC library will facilitate isolation of Foc3 and allow us to perform physical mapping of this genomic region where additional R genes against other races of the wilt causing pathogen are positioned. Cancer Res, 2003 Oct 1, 63(19), 6350 - 6 Coexpression of Helicobacter pylori's proteins CagA and HspB induces cell proliferation in AGS gastric epithelial cells, independently from the bacterial infection; De Luca A et al.; Adenocarcinoma of the stomach is the second most common cause of cancer mortality in the world . The purpose of this study was to evaluate the potential role in carcinogenesis of two secreted Helicobacter pylori's proteins, CagA and HspB, both shown to increase the risk of gastric carcinoma in patients infected with H . pylori-positive strain . The effects of these two proteins on cell kinetics and the ability to selectively affect the expression of cell cycle-related proteins by transfection of a human gastric epithelial cell line (AGS) were analyzed . Using a genomic library of H . pylori, we isolated and cloned CagA and HspB . The effects of the overexpression of these proteins on cell growth were analyzed in AGS cells by immunoblots, proliferation assay, and flow cytometry . Coexpression of CagA and HspB in AGS cells in the first 48 h caused an increase of the level of E2F transcription factor, cyclin D3, and phosphorylated retinoblastoma protein, all involved in the G(1)-S checkpoint of the cell cycle . Consistently, an increase of cell proliferation, corresponding to an augment of the fraction of the cells in the S-G(2)-M phase of the cell cycle, was also demonstrated . Moreover, an increase of c-jun protein levels, but not of c-fos, was also found after coexpression of CagA and HspB . All these data suggest that CagA and HspB, independently from the bacterial infection, have a direct effect on the cell growth of the gastric cells acting on the G(1)-S checkpoint of the cell cycle. Rapid Commun Mass Spectrom, 2003, 17(20), 2260 - 6 Electrospray ion-trap multistage mass spectrometry for characterisation of co-monomer compositional distribution of bacterial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) at the molecular level; Adamus G et al.; We report an electrospray ionisation multistage mass spectrometry (ESI-MSn) method that utilises molecular mass information for determination of sequence distribution and chemical structure of mass-selected macromolecules of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biopolyester, PHBH . On the basis of ESI-MSn studies of PHBH oligomers obtained by partial alkaline depolymerisation of natural PHBH containing 13-14 mol% of hydroxyhexanoate (HH) units, the microstructure of this bacterial copolyester was assessed up to the level of 28 repeat units . The subtle structural details of the PHBH were evaluated based on sequencing of individual macromolecular ions thus showing the utility of this technique for the analysis of biological copolyester macromolecules . It was confirmed that both HH and hydroxybutyrate (HB) units of the PHBH copolymer are randomly distributed . Eur J Obstet Gynecol Reprod Biol, 2003 Nov 10, 111(1), 83 - 7 New technology for diagnosis of bacterial vaginosis; Chaim W et al.; OBJECTIVE: To replace clinical diagnosis of bacterial vaginosis (BV) with a new and rapid analytical method based on ion mobility spectrometry (IMS) . IMS is an instrumental technique for identifying compounds and determining their concentrations, based on measurement of the velocity of ions drifting through air at atmospheric pressure under the influence of an electric field . The technique is particularly sensitive to amines taking less than 2 min . STUDY DESIGN: Clinical examination of 174 samples determined 22 BV-positive and 152 BV-negative samples . IMS analyzed and recorded biogenic amine emanation mobility spectra of the 174 samples of vaginal discharge from duplicate swabs . RESULTS: IMS confirmed 21 (true positive BV) samples with 1 false negative (21/22, sensitivity=95.5%) . Out of 152 samples, 150 were confirmed true BV-negative with 2 false positive samples (specificity=98.7%), PPV: 91.3%, NPV: 100% . CONCLUSIONS: The diagnostic procedure shows high accuracy and is technically simple and rapid . The trimethylamine level becomes an index of severity of the infection. Burns, 2003 Nov, 29(7), 733 - 8 Bacterial toxicosis/toxic shock syndrome as a contributor to morbidity in children with burn injuries; Brown AP et al.; When a child with a thermal injury suddenly becomes unwell, it is often difficult to obtain an accurate diagnosis in the early stages of the illness . Out of 71 children admitted to our burns unit over a 15-month period, 13 became acutely toxic . Most of the cases had relatively small burns and the exact reason for their sudden deterioration was not apparent in the immediate stages . Several children required admission to the intensive therapy unit and there was one death . The number of children experiencing this significant morbidity over a relatively short period of time prompted us to perform a detailed retrospective study to ascertain the exact cause of the deterioration and to identify any patterns in their presenting signs and symptoms.There was considerable variation in the clinical picture of the children who developed the toxicosis, however, several features were frequently observed which resembled those seen in the toxic shock syndrome (TSS) . In order to be more specific, we applied the criteria for TSS as defined by the Centres for Disease Control (CDC) to each of the 13 children . All of the criteria were noted in six children and the majority in the other seven . This was a much higher incidence than we would normally have expected . The clinical features of children with TSS are not always easy to distinguish from those observed in other illnesses . We therefore explored the possibility of alternative diagnoses that may have caused the toxicosis in these children.The mortality associated with TSS can be high, especially if there is a delay in recognition and subsequent management of the disease . Clinicians should be alerted to the fact that TSS is probably more prevalent than previously thought and should practise with a high index of suspicion in any child with a burn whose clinical condition suddenly deteriorates. Microbes Infect, 2003 Oct, 5(12), 1149 - 58 Fascination with bacteria-triggered cell death: the significance of Fas-mediated apoptosis during bacterial infection in vivo; Menaker RJ et al.; Increasing evidence indicates that bacterial pathogens have developed mechanisms to modulate the apoptotic signaling cascade of host cells and thereby cause disease . The Fas death receptor pathway is one of the most extensively investigated apoptotic signaling pathways . In this review we discuss the role of Fas signaling during the interplay between bacterial pathogens and the host in vivo. Zhonghua Gan Zang Bing Za Zhi, 2003 Sep, 11(9), 539 - 41 {Effects of cisapride on intestinal bacterial and endotoxin translocation in cirrhotic rats}; Zhang SC et al.; OBJECTIVES: To further investigate the effects of cisapride on intestinal bacterial overgrowth (IBO), bacterial and endotoxin translocation, intestinal transit and permeability in cirrhotic rats . METHODS: 25 normal control rats, 25 cirrhotic rats, 20 cirrhotic rats received saline, and 20 cirrhotic rats treated with cisapride were included in the study . All animals were assessed with many variables including bacterial and endotoxin translocation, IBO, intestinal transit and permeability . RESULTS: Bacterial translocation was found in 48%(12/25) cirrhotic rats and none of control rats . Among the 20 rats with IBO, there were 11 rats with bacterial translocation (BT) while only one rats occurred BT out of the 5 rats without IBO . Cirrhotic rats with IBO had a significantly higher rate of endotoxin translocation, higher intestinal permeability and longer intestinal transit than those without IBO . BT of a specific organism was always associated with IBO of that organism . Compared with the placebo group, cisapride-treated rats had lower rates of bacterial and endotoxin translocation and IBO, which had close relationship with shorter intestinal transit and lower permeability . CONCLUSION: Endotoxin and bacterial translocation in cirrhotic rats may be the result of IBO and higher permeability . IBO may be the result of longer transit . Cisapride which can accelerate intestinal transit and improve intestinal permeability is helpful in preventing and treating intestinal bacterial and endotoxin translocation. Pediatr Surg Int, 2003 Oct, 19(8), 573 - 7 Epub 2003 Oct 09. Pathogenesis of neonatal necrotizing enterocolitis: a study of the role of intraluminal pressure, age and bacterial concentration; Chan KL et al.; The pathogenesis of neonatal necrotizing enterocolitis (NEC) is unknown . Intestinal dilatation and preferred occurrence of NEC at sites of bacterial overgrowth (colon and ileum) are common findings . The study attempted to produce NEC with increasing intraluminal pressures and bacterial concentrations in two different aged groups of rats . First, 10-cm terminal ileum segments were isolated with intact vascular pedicles in 1-and 3-month-old rats, and a dose of 10(11) E . coli in 1 ml was injected into each segment . Intraluminal pressure was sustained for 1 h at 150, 100, 50 and 0 cmH(2)0, respectively, in four experimental groups ( n=6) . The isolated loop was then returned to the abdominal cavity and assessed grossly for NEC after 24 h . Histological examination was performed by a pathologist (KWC) who was blinded to the procedures . Second, the procedure was repeated with doses of 10(8), 10(5) and 0 bacteria/ml ( n=6) at intraluminal pressure of 100 cmH(2)0 in 1-month-old rats . Third, in another experimental group, oxygenation of the pedicled loop was assessed by oximetry as the intraluminal pressure increased and the findings were correlated with aortic blood pressure . The blood pressures (mean+/-SD) for 3- and 1-month-old rats were 110+/-6 and 72+/-4 mmHg, respectively . Hypoxia (<50% oxygen saturation) of the bowel was detected when the intraluminal pressure exceeded the mean blood pressure . The relative incidences of NEC in the bowel with intraluminal pressure above and below mean blood pressure were 100% (6/6) vs . 4% (1/24; P<0.05) in 3-month-old rats, and 100% (12/12) vs . 11% (2/18; P<0.05) in 1-month-old rats . There was no occurrence of NEC in bowel injected with 10(5) E . coli/ml and less at 100 cm intraluminal pressure . Increased intraluminal pressure results in bowel hypoxia and in the presence of adequate bacterial concentration predisposes to the development of NEC . Young age is associated with a lower threshold for increased intraluminal pressure leading to NEC. Pediatr Infect Dis J, 2003 Oct, 22(10), 895 - 903 Procalcitonin in pediatric emergency departments for the early diagnosis of invasive bacterial infections in febrile infants: results of a multicenter study and utility of a rapid qualitative test for this marker; Fernandez Lopez A et al.; BACKGROUND: Procalcitonin (PCT) is a potentially useful marker in pediatric Emergency Departments (ED) . The basic objectives of this study were to assess the diagnostic performance of PCT for distinguishing between viral and bacterial infections and for the early detection of invasive bacterial infections in febrile children between 1 and 36 months old comparing it with C-reactive protein (CRP) and to evaluate the utility of a qualitative rapid test for PCT in ED . METHODS: Prospective, observational and multicenter study that included 445 children who were treated for fever in pediatric ED . Quantitative and qualitative plasma values of PCT and CRP were correlated with the final diagnosis . To obtain the qualitative level of PCT the BRAHMS PCT-Q rapid test was used . RESULTS: Mean PCT and CRP values in viral infections were 0.26 ng/ml and 15.5 mg/l, respectively . The area under the curve obtained for PCT in distinguishing between viral and bacterial infections was 0.82 (sensitivity, 65.5%; specificity, 94.3%; optimum cutoff, 0.53 ng/ml), whereas for CRP it was 0.78 (sensitivity, 63.5%; specificity, 84.2%; optimum cutoff, 27.5 mg/l) . PCT and CRP values in invasive infections (PCT, 24.3 ng/ml; CRP 96.5 mg/l) were significantly higher than those for noninvasive infections (PCT, 0.32 ng/ml; CRP, 23.4 mg/l) . The area under the curve for PCT was 0.95 (sensitivity, 91.3%; specificity, 93.5%; optimum cutoff, 0.59 ng/ml), significantly higher (P < 0.001) than that obtained for CRP (0.81) . The optimum cutoff value for CRP was >27.5 mg/l with sensitivity and specificity of 78 and 75%, respectively . In infants in whom the evolution of fever was <12 h (n = 104), the diagnostic performance of PCT was also greater than that of CRP (area under the curve, 0.93 for PCT and 0.69 for CRP; P < 0.001) . A good correlation between the quantitative values for PCT and the PCT-Q test was obtained in 87% of cases (kappa index, 0.8) . The sensitivity of the PCT-Q test (cutoff >0.5 ng/ml) for detecting invasive infections and differentiating them from noninvasive infections was 90.6%, with a specificity of 83.6% . CONCLUSIONS: PCT offers better specificity than CRP for differentiating between the viral and bacterial etiology of the fever with similar sensitivity . PCT offers better sensibility and specificity than CRP to differentiate between invasive and noninvasive infection . PCT is confirmed as an excellent marker in detecting invasive infections in ED and can even make early detection possible of invasive infections if the evolution of the fever is <12 h . The PCT-Q test has a good correlation with the quantitative values of the marker. Protein Expr Purif, 2003 Oct, 31(2), 188 - 96 Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria; Simonet G et al.; The last decade, a new serine protease inhibitor family has been described in arthropods . Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5) and 11 additional locust peptides were identified by cDNA cloning . Furthermore, the light chain of the 155-kDa heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs) . These domains share a pattern of 6 conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa3-Cys) with the locust inhibitors . So far, for most of the PLD-related peptides the biological functions remain obscure . To obtain sufficient amounts of material to perform physiological experiments, we have optimised the production of SGPI-1-2 via a bacterial (Escherichia coli) expression system . The cDNA sequences encoding these peptides were inserted in the pMAL-2pX vector, downstream of the gene encoding the maltose-binding protein (including a signal peptide) . As a consequence, both peptides were expressed as fusion proteins (2-3 mg/l) and targeted to the periplasmic space . Following a one-step affinity purification, both fusion proteins were successfully cleaved by Factor Xa and after a methanol extraction, it took only one additional RP-HPLC run to purify both peptides to homogeneity . Finally, the formation of the disulphide bridges and the biological activity of the recombinant peptides were verified by mass spectrometry and a spectrophotometric protease inhibitor assay, respectively. Biotechnol Adv, 1990, 8(1), 185 - 205 Bacterial haloperoxidases and their role in secondary metabolism; Van Pee KH; Bacteria produce a large number of different halogenated secondary metabolites . Haloperoxidases are believed to be the enzymes responsible for the halogenation reaction . Two classes of haloperoxidases, heme and nonheme, were isolated from different bacteria and their role in the biosynthesis of halogenated secondary metabolites was investigated . Two genes of bacterial haloperoxidases were cloned and can now be used to produce large quantities of the enzymes. Biotechnol Adv, 1984, 2(2), 347 - 55 Bacterial degradation of ammonium lignosulfonate; Ammar E et al.; Bacterial strains were selected for their capacity to assimilate and to transform ammonium-lignosulfonate . Modification of the methyl content and of low molecular weight alkyl functions were demonstrated by gas-chromatography and HLPC analysis . Most of these strains completely degraded simple phenolic compounds related to lignosulfonate without inhibition by the carbohydrates present in the liquor . Further investigation suggested that the enzymes involved in the fission of the aromatic nuclei were constitutive in the strains tested. J Urol, 2003 Nov, 170(5), 2053 - 6 Prospective analysis of 16S rDNA as a highly sensitive marker for bacterial presence in Peyronie's disease plaques; Hauck EW et al.; PURPOSE: Previous studies have implicated an infectious agent induced pathogenesis in Peyronie's disease . To our knowledge an association with venereal diseases or other infectious diseases has not been demonstrated to date, although Peyronie's disease is an inflammatory disorder . In case of an infectious origin of the disorder bacteria or at least their fragments should occur in the plaque . We investigated prospectively the occurrence of 16S rDNA as a highly sensitive marker for the presence of bacteria in inflammatory processes . MATERIALS AND METHODS: In 19 patients with idiopathic Peyronie's disease biopsy of the tunica albuginea was sampled . A total of 16 men with no evidence of Peyronie's disease served as the control group . In these men tissue from the tunica albuginea was obtained during penile prosthesis implantation for erectile dysfunction or during a Nesbit procedure for congenital penile curvature . Screening for bacterial DNA was performed prospectively using a polymerase chain reaction targeting bacterial 16S rDNA . RESULTS: In the tunica albuginea specimen 16S rDNA was not detectable in patients with Peyronie's disease or in the control group . CONCLUSIONS: The results of this study do not indicate an association between Peyronie's disease and bacterial infection. Appl Environ Microbiol, 2003 Oct, 69(10), 6268 - 71 An alternative efficient procedure for purification of the obligate intracellular fish bacterial pathogen Piscirickettsia salmonis; Henriquez V et al.; Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome . Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile . Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P . salmonis DNA free from contaminating host cell DNA . In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis . The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P . salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell . This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter. Crit Rev Oral Biol Med, 2003, 14(5), 331 - 44 Molecular genetic analysis of the virulence of oral bacterial pathogens: an historical perspective; Kuramitsu HK; This review will focus on the impact of molecular genetic approaches on elucidating the bacterial etiology of oral diseases from an historical perspective . Relevant results from the pre- and post-recombinant DNA periods will be highlighted, including the roles of gene cloning, mutagenesis, and nucleotide sequencing in this area of research . Finally, the impact of whole-genome sequencing on deciphering the virulence mechanisms of oral pathogens, along with new approaches to control these organisms, will be discussed. J Biol Chem, 2003 Dec 26, 278(52), 52710 - 23 Epub 2003 Oct 06. A DNA pairing-enhanced conformation of bacterial RecA proteins; Haruta N et al.; The RecA proteins of Escherichia coli (Ec) and Deinococcus radiodurans (Dr) both promote a DNA strand exchange reaction involving two duplex DNAs . The four-strand exchange reaction promoted by the DrRecA protein is similar to that promoted by EcRecA, except that key parts of the reaction are inhibited by Ec single-stranded DNA-binding protein (SSB) . In the absence of SSB, the initiation of strand exchange is greatly enhanced by dsDNA-ssDNA junctions at the ends of DNA gaps . This same trend is seen with the EcRecA protein . The results lead to an expansion of published hypotheses for the pathway for RecA-mediated DNA pairing, in which the slow first order step (observed in several studies) involves a structural transition to a state we designate P . The P state is identical to the state found when RecA is bound to double-stranded (ds) DNA . The structural state present when the RecA protein is bound to single-stranded (ss) DNA is designated A . The DNA pairing model in turn facilitates an articulation of three additional conclusions arising from the present work . 1) When a segment of a RecA filament bound to ssDNA is forced into the P state (as RecA bound to the ssDNA immediately adjacent to dsDNA-ssDNA junction), the segment becomes "pairing enhanced." 2) The unusual DNA pairing properties of the D . radiodurans RecA protein can be explained by postulating this protein has a more stringent requirement to initiate DNA strand exchange from the P state . 3) RecA filaments bound to dsDNA (P state) have directly observable structural changes relative to RecA filaments bound to ssDNA (A state), involving the C-terminal domain. J Microbiol Methods, 2003 Nov, 55(2), 471 - 4 Consecutive analysis of bacterial PCR samples on a single electronic microarray; Zimmermann K et al.; For routine mass screening, the use of microarrays is hampered because one chip can only analyze one sample . 16S rRNA gene PCR products of several bacterial strains or mixtures thereof were consecutively loaded on a single electronic microarray and successfully analyzed using probes specific for the bacterial strains. J Microbiol Methods, 2003 Nov, 55(2), 411 - 8 Rapid screening method for quantitation of bacterial cell lipids from whole cells; Izard J et al.; Although specific lipids in bacteria can be quantitated, there is still a need to quantitate the total lipid content of a bacterial sample . The sulfo-phospho-vanillin reaction for quantitation of bacterial lipids has significant advantages over traditional methods for screening of engineered mutant strains . In this report we show that this methodology can be used directly on whole cell or homogenized biological material, without any extraction step . The cell components, and most of the reagents used for cell extraction, that were tested did not interfere with the reaction . The screening is based on the observation of physiologic variations using ratios of relative amounts: lipid/DNA and lipid/protein . Our results show that significant differences in those ratios can be detected when there is a modification of the phospholipid content of the cell . The sample manipulation required is minimal and could be autom