J Immunol, 2000 Oct 1, 165(7), 3647 - 55 Bacterial lipopolysaccharide, TNF-alpha, and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in CD14+ monocytes through distinct pathways that activate NK-kappa B; Lyakh LA et al.; To facilitate the study of signaling pathways involved in myeloid dendritic cell (DC) differentiation, we have developed a serum-free culture system in which human CD14+ peripheral blood monocytes differentiate rapidly in response to bacterial LPS, TNF-alpha, or calcium ionophore (CI) . Within 48-96 h, depending on the inducing agent, the cells acquire many immunophenotypical, morphological, functional, and molecular properties of DC . However, there are significant differences in the signaling pathways used by these agents, because 1) LPS-induced, but not CI-induced, DC differentiation required TNF-alpha production; and 2) cyclosporin A inhibited differentiation induced by CI, but not that induced by LPS . Nevertheless, all three inducing agents activated members of the NF-kappaB family of transcription factors, including RelB, suggesting that despite differences in upstream elements, the signaling pathways all involve NF-kappaB . In this report we also demonstrate and offer an explanation for two observed forms of the RelB protein and show that RelB can be induced in myeloid cells, either directly or indirectly, through a calcium-dependent and cyclosporin A-sensitive pathway. Vet Clin North Am Small Anim Pract, 2000 Sep, 30(5), 1103 - 17 Canine systemic bacterial infections; Dziezyc J; Although systemic bacterial diseases are an uncommon cause of uveitis in dogs, they should be included in the differential diagnoses for uveitis . A work-up for uveitis should include tests for B . canis and B . burgdorferi . If an aqueous centesis is performed, Leptospira titers or PCR can be performed on the aqueous humor and the serum . Better documentation of the role of Leptospira and B . burgdorferi in uveitis in the dog is needed . Any suspected cases should be worked-up thoroughly . If the dog does prove to be positive for the organism, the case should be submitted to a peer-reviewed journal. Intensive Care Med, 2000 Aug, 26(8), 1082 - 8 Serum and ascitic procalcitonin levels in cirrhotic patients with spontaneous bacterial peritonitis: diagnostic value and relationship to pro-inflammatory cytokines; Viallon A et al.; OBJECTIVE: To assess the potential role of procalcitonin and pro-inflammatory cytokines, TNF-alpha, and IL-6, in the diagnosis of spontaneous bacterial peritonitis (SBP) . DESIGN: Prospective study . SETTING: The emergency unit of a teaching hospital . PATIENTS: We included 21 patients with SBP and 40 patients with sterile ascitic fluid . INTERVENTIONS: None . MEASUREMENTS AND MAIN RESULTS: For the diagnosis of SBP, the best markers were serum levels of procalcitonin with a cut-off value of 0.75 ng/ml, a sensitivity of 95%, a specificity of 98%, and ascitic fluid levels of IL-6 with a cut-off value of 5,000 ng/ml, a sensitivity of 100%, and a specificity of 88% . C-reactive protein and serum polymorphonuclear count have low sensitivity/specificity at 62/92% and 57/90%, respectively . From 21 patients with SBP, ascitic fluid to serum ratio of TNF-alpha and IL-6 was greater than to 2 in all cases with a mean at 6.2 +/- 6.5 and 34 +/- 31, respectively . By contrast, this ratio for procalcitonin was less than 1 in all cases with a mean at 0.31 +/- 0.25 . We found no correlation between procalcitonin levels and cytokine levels in either ascitic fluid or serum . CONCLUSIONS: Serum procalcitonin level may become a useful marker for the diagnosis of SBP in cirrhotic patients . The low ratio of ascitic fluid to serum procalcitonin supports the hypothesis that procalcitonin is not produced intraperitoneally. J Bacteriol, 2000 Nov, 182(21), 6091 - 8 The mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial growth in the presence of transferrin or lactoferrin; Freestone PP et al.; Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum . Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of Escherichia coli . Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth . Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels . Norepinephrine also caused the loss of iron from lactoferrin . The pharmacologically inactive metabolite norepinephrine 3-O-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium . Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin . Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine . Norepinephrine-stimulated growth in medium containing (55)Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells . Moreover, norepinephrine-stimulated growth in medium containing {(3)H}norepinephrine indicated concomitant uptake of norepinephrine . In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity ((55)Fe or (3)H) associated with bacterial cells . A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed. J Biol Chem, 2001 Jan 5, 276(1), 406 - 12 Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase; Emanuelli M et al.; A 1329-base pair clone isolated from a human placenta cDNA library contains a full-length 837-base pair coding region for a 31.9-kDa protein whose deduced primary structure exhibits high homology to consensus sequences found in other NMN adenylyltransferases . Northern blotting detected a major 3.1-kilobase mRNA transcript as well as a minor 4.1-kilobase transcript in all human tissues examined . In several cancer cell lines, lower levels of mRNA expression were clearly evident . The gene encoding the human enzyme was mapped to chromosome band 1p32-35 . High efficiency bacterial expression yielded 1.5 mg of recombinant enzyme/liter of culture medium . The molecular and kinetic properties of recombinant human NMN adenylyltransferase provide new directions for investigating metabolic pathways involving this enzyme. Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 1261 - 4 Bacterial DNA methylation and gene transfer efficiency; Allamane S et al.; The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer . These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences . These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm . In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown . Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria . Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer . On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart . Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency . However, the persistence of the use of (dam(+), dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in eukaryotic cells or organisms . J Surg Res, 2000 Oct, 93(2), 247 - 56 Role of nitric oxide in hemorrhagic shock-induced bacterial translocation; Hua TC et al.; BACKGROUND: Hemorrhagic shock-induced bacterial translocation is an etiologic factor in the pathogenesis of multiple system organ damage . Excessive production of nitric oxide (NO) during hemorrhagic shock may lead to cellular injury and gut barrier failure that promotes bacterial translocation . We investigated the effect of aminoguanidine (AG) and N(G)-nitro-l-arginine methyl ester (l-NAME), both inhibitors of NO synthase, on hemorrhagic shock- induced bacterial translocation in the rat . MATERIALS AND METHODS: Anesthetized male Sprague-Dawley rats were subjected to a hemorrhagic shock protocol for 30 min followed by intravenous injection (1 mL/kg body wt) with normal saline, AG (100 mg/kg), or l-NAME (10 mg/kg) . Tissues/organs were examined histologically for damage and bacterial translocation . Plasma nitrate/nitrite was measured using a procedure based on the Griess reaction, and nitric oxide synthase (NOS) expression was determined immunohistochemically . RESULTS: The shocked animals treated with saline died within 90 min, and deaths were associated with 100% bacterial translocation, increased tissue/organ damage, and elevated nitrate/nitrite production . In contrast, both AG and l-NAME increased the survival time of shocked rats to >72 h, abrogated bacterial translocation, reduced tissue/organ damage, and prevented excessive nitrate/nitrite production and upregulation of expression of endothelial NOS and inducible NOS . CONCLUSIONS: Prevention of bacterial translocation by pharmacologic agents such as aminoguanidine and l-NAME could be an important therapeutic approach to lessen mortality rates following hemorrhagic shock . Acta Med Port, 2000 May-Jun, 13(3), 77 - 80 {Evaluation of microscopy methods for the diagnosis of bacterial vaginosis}; Mota A et al.; The diagnosis of Bacterial Vaginosis has always been controversial . During many years, the laboratory diagnosis of this syndrome was based on the criteria of Amsel et al (1983) . This includes many factors, such us aqueous vaginal discharge, positive KOH test and the presence of clue-cells in a wet mount or Gram stain . Lately, a new diagnostic method only based on laboratory findings was performed by Nugent et al (1991), which has the advantage of being more objective and rapid . It is also easy to be used in any laboratory or even in a doctor's room . In this study, we evaluated 74 Gram stained vaginal smears and compared both Amsel et al (1983) and Nugent et al (1991) methods . Bacterial Vaginosis was diagnosed in 28% by Amsel et al (1983) criteria and in 31% by the Nugent et al (1991) criteria . The latter method seems to have a higher efficacy in diagnosing Bacterial Vaginosis, although both techniques together diagnose a higher number of cases. FEBS Lett, 2000 Oct 6, 482(3), 185 - 8 Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes; Ramachandiran V et al.; Five different coding sequences of bacterial or eukaryotic origin in plasmids under the T7 promoter were expressed in a cell-free system derived from Escherichia coli . Translation on E . coli ribosomes resulted in a full-length product only in four of the five coding sequences tested . A unique pattern of less than full-length polypeptides was generated in each case . Many of these polypeptides on E . coli ribosomes reacted with a puromycin derivative, cytidylic acid-puromycin, which was radioactively labeled . Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site . Certain nascent peptides could be shifted into full-length protein indicating that they resulted from translational pausing . In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell-free system resulted in a 80-90% full-length product with no evidence for nascent polypeptides and translational pausing. FEMS Microbiol Lett, 2000 Oct 15, 191(2), 187 - 90 In situ assay for identifying inhibitors of bacterial transglycosylase; Branstrom AA et al.; An in situ transglycosylase assay has been developed using endogenously synthesized lipid II . The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc . In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited . Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II . This reaction was inhibited by moenomycin, a known transglycosylase inhibitor . In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected . Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established. Biophys J, 2000 Oct, 79(4), 2066 - 74 Selectivity in lipid binding to the bacterial outer membrane protein OmpF; O'Keeffe AH et al.; The outer membrane porin OmpF from Escherichia coli has been reconstituted into lipid bilayers of defined composition, and fluorescence spectroscopy is used to characterize its interaction with the surrounding lipid . OmpF is a trimer within the membrane . It contains two Trp residues per monomer, Trp(214) at the lipid-protein interface and Trp(61) at the trimer interface . The fluorescence of Trp-214 in the mutant W61F is quenched by dibromostearoylphosphatidylcholine (di(Br(2)C18:0)PC), whereas the fluorescence of Trp(61) in the mutant W214F is not quenched by di(Br(2)C18:0)PC when fluorescence is excited directly through the Trp rather than through the Tyr residues . Measurements of relative fluorescence quenching for OmpF reconstituted into mixtures of lipid X and di(Br(2)C18:0)PC have been analyzed to give the binding constant of lipid X for OmpF, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC) . The phosphatidylcholine showing the strongest binding to OmpF is dimyristoyloleoylphosphatidylcholine (di(C14:1)PC) with binding constants decreasing with either increasing or decreasing fatty acyl chain length . Comparison with various theories for hydrophobic matching between lipids and proteins suggests that in the chain length range from C14 to C20, hydrophobic matching is achieved largely by distortion of the lipid bilayer around the OmpF, whereas for chains longer than C20, distortion of both the lipid bilayer and of the protein is required to achieve hydrophobic matching . Phosphatidylcholine and phosphatidylethanolamine bind with equal affinity to OmpF, but the affinity for phosphatidylglycerol is about half that for phosphatidylcholine. Microb Ecol, 2000 Aug, 40(2), 114 - 124 Bacterial Growth Rate and Marine Virus-Host Dynamics; Middelboe M; The dynamics of a marine virus-host system were investigated at different steady state growth rates in chemostat cultures and the data were analyzed using a simple model . The virus-host interactions showed strong dependence on host cell growth rate . The duration of the infection cycle and the virus burst size were found to depend on bacterial growth rate, and the rate of cell lysis and virus production were positively correlated with steady state growth rate in the cultures (r(2) > 0.96, p < 0.05) . At bacterial growth rates of 0.02 to 0.10 h(-1) in the chemostats the virus burst size increased from 12 +/- 4 to 56 +/- 4, and the latent period decreased from 2.0 to 1.7 h . Resistant clones of the host strain were present in the cultures from the beginning of the experiment and replaced the sensitive host cells following viral lysis in the cultures . Regrowth of resistant cells correlated significantly (r(2) = 1.000, p < 0.02) with the lysis rate of sensitive cells, indicating that release of viral lysates stimulated growth of the non-infected, resistant cells . The constructed model was suitable for simulating the observed dynamics of the sensitive host cells, viruses and resistant clones in the cultures . The model was therefore used in an attempt to predict the dynamics of this virus-host interaction in a natural marine environment during a certain set of growth conditions . The simulation indicated that a steady state relationship between the specific viruses and sensitive and resistant bacterial clones may occur at densities that are reasonable to assume for natural environments . The study demonstrates that basic characterization and modeling of specific virus-host interactions may improve our understanding of the behavior of bacteria and viruses in natural systems. Am J Perinatol, 2000, 17(2), 83 - 8 Bacterial vaginosis and cervical dilation and effacement at 24-29 weeks' gestation; Pastore LM et al.; The purpose of this study was to investigate the association between bacterial vaginosis (BV) and cervical dilation and effacement, as measures of impending preterm delivery . The Pregnancy, Infection, and Nutrition Study collected genital tract specimens and documented cervical change from 807 eligible women between 24 and 29 weeks' gestation . BV was assessed with Nugent-scored vaginal smears, and analyzed in relation to cervical measurements . At 24-29 weeks' gestation, <7% of women had a dilated cervix, 31% had a cervix < or =2 cm, and 17.3% had BV . Unadjusted analyses found no associations between BV and cervical measurements . Adjusted logistic regression suggested an association between BV and cervical effacement among women with a sexually transmitted disease (STD) earlier in pregnancy (odds ratio = 1.9, 95% CI 0.8-4.3) . Stratified analyses for BV/dilation also suggested interaction with STDs . Overall, BV was not association with cervical dilation or effacement at 24-29 weeks' gestation. Bull Exp Biol Med, 2000 Jun, 129(6), 553 - 5 Effects of vagotomy and bacterial lipopolysaccharide on food intake and expression of cyclooxygenase-2 mRNA in rat brain vessels; Sergeev VG et al.; Effects of bilateral subdiaphragmatic vagotomy on food intake and expression of cyclooxygenase-2 mRNA in cerebral vessels in rats intraperitoneally injected with bacterial lipopolysaccharide were studied using in situ hybridization technique . Low doses of lipopolysaccharide decreased food intake in sham-operated animals, but did not affect this parameter in vagotomized rats . Comparison of hybridization signals in brain slices showed that low doses of endotoxin did not affect expression of cyclooxygenase-2 mRNA in vessels of control and experimental animals . High doses of lipopolysaccharide reduced food intake in vagotomized and sham-operated rats and elevated cyclooxygenase-2 mRNA expression in vascular endothelial cells of the brain parenchyma and meninges . The data suggest that the vagus nerve activates central structures responsible for manifestation of anorexia after intraperitoneal injection of low doses of lipopolysaccharide . High doses of endotoxin activate the vagus-independent mechanism of cyclooxygenase-2 synthesis in the endothelium of cerebral vessels . It is assumed that prostaglandins synthesized by cyclooxygenase-2 diffuse into the brain parenchyma and cause anorexia by activating target nerve structures. Otolaryngol Head Neck Surg, 2000 Oct, 123(4), 363 - 7 Analysis of aerobic bacterial strains found in chronic rhinosinusitis using the polymerase chain reaction; Keech DR et al.; INTRODUCTION: Rhinosinusitis is a common disease affecting an estimated 14% of the population . Although there is general agreement in the literature regarding acute rhinosinusitis, chronic rhinosinusitis is not as well studied, and no consensus has been reached regarding the bacterial etiology . The goal of this study was to test chronic rhinosinusitis mucosal specimens using the polymerase chain reaction (PCR) for aerobic bacterial content and to compare the results with standard culture data . RESULTS: Routine culture samples grew 50% aerobic bacteria, whereas PCR detected 62% aerobic bacteria contamination . CONCLUSION: PCR detected more bacteria in mucosal samples than in standard culture, but standard culture of this mucosa reflects the general aerobic bacteria found in chronic rhinosinusitis, with no predominant species of aerobic bacteria . SIGNIFICANCE: The analysis of chronic rhinosinusitis mucosa with the PCR method should give a more accurate picture of the bacteria found in chronic rhinosinusitis so that proper therapy can be instituted. J Hepatol, 2000 Sep, 33(3), 423 - 9 Prevalence and prognostic value of hepatocellular carcinoma in cirrhotic patients presenting with spontaneous bacterial peritonitis; Llovet JM et al.; BACKGROUND/AIMS: This study examined the prognostic power of hepatocellular carcinoma in patients presenting an episode of spontaneous bacterial peritonitis treated with 3rd generation cephalosporins or quinolones, and subsequent prophylaxis with norfloxacin until death or transplantation . METHODS: The study comprises the prospective evaluation of 168 consecutive cirrhosis patients presenting an episode of spontaneous bacterial peritonitis . RESULTS: Hepatocellular carcinoma was diagnosed in 35 out of the 168 (20%) patients included in the study (10 single; 25 advanced tumors) . Renal impairment developed in 82 patients . Resolution of infection was achieved in 90% of the cases, the hospital survival being 70% . Renal impairment, advanced tumor stage, albumin, and GGT showed independent prognostic value for hospital mortality . At the end of follow-up 101 patients had died, the 1- and 2-year survival being 36% and 31%, respectively . Four variables independently predicted survival: advanced tumor (OR: 3.9; p=0.00001), renal impairment (OR: 2.1; p=0.00001), bilirubin (OR: 1.6; p=0.02) and creatinine (OR: 1.3; p=0.03) . Advanced tumor retained independent predictability in patients surviving hospitalization (OR: 7.5; p=0.0001), the 6-month survival being significantly lower in patients with advanced tumor (12% vs 57%, p<0.00001) . CONCLUSION: The prevalence of hepatocellular carcinoma in cirrhotic patients with spontaneous bacterial peritonitis is high, and its presence should be actively sought . Advanced tumor impairs both hospital and long-term survival, and should be considered in the design of future trials. Phys Rev Lett, 2000 Mar 27, 84(13), 3017 - 20 Particle diffusion in a quasi-two-dimensional bacterial bath; Wu XL et al.; We study the effect of bacterial motion on micron-scale beads in a freely suspended soap film . Given the sizes of bacteria and beads, the geometry of the experiment is quasi-two-dimensional . Large positional fluctuations are observed for beads as large as 10 microm in diameter, and the measured mean-square displacements indicate superdiffusion in short times and normal diffusion in long times . Though the phenomenon is similar to Brownian motions of small particles, its physical origin is different and can be attributed to the collective dynamics of bacteria. Phys Rev Lett, 2000 Feb 14, 84(7), 1627 - 30 Chiral self-propulsion of growing bacterial macrofibers on a solid surface; Mendelson NH et al.; Supercoiling motions that accompany the growth of bacterial macrofibers (multicellular filamentous structures formed in B . subtilis by cell division without separation) are responsible for rolling, pivoting, and walking of fibers on a surface . Fibers possess a fulcrum about which they pivot and step in a chiral manner; forces and torques associated with cell growth, when blocked by friction, result in self-propulsion . The elastic engine that drives macrofiber motions generates torques estimated as microdyn cm and femtowatts of power; optical trapping studies yield a first direct measurement of the Young's modulus of the bacterial cell wall, the engine's "working fluid," of ca . 0.05 GPa. Genetics, 2000 Oct, 156(2), 833 - 8 Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial artificial chromosome contig reveals the role of chromosomal duplications in the expansion of the Brassica rapa genome; Jackson SA et al.; Comparative genome studies are important contributors to our understanding of genome evolution . Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes . Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques . We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative physical mapping between Arabidopsis thaliana and Brassica rapa . A set of six bacterial artificial chromosomes (BACs) representing a 431-kb contiguous region of chromosome 2 of A . thaliana was mapped on both chromosomes and DNA fibers of B . rapa . This DNA fragment has a single location in the A . thaliana genome, but hybridized to four to six B . rapa chromosomes, indicating multiple duplications in the B . rapa genome . The sizes of the fiber-FISH signals from the same BACs were not longer in B . rapa than those in A . thaliana, suggesting that this genomic region is duplicated but not expanded in the B . rapa genome . The comparative fiber-FISH mapping results support that chromosomal duplications, rather than regional expansion due to accumulation of repetitive sequences in the intergenic regions, played the major role in the evolution of the B . rapa genome. Immunology, 2000 Sep, 101(1), 46 - 52 Poly-guanosine motifs costimulate antigen-reactive CD8 T cells while bacterial CpG-DNA affect T-cell activation via antigen-presenting cell-derived cytokines; Lipford GB et al.; Pathogen-derived pattern recognition ligands like lipopolysaccharide (LPS) and bacterial cytidine-guanosine (CpG)-DNA not only activate dendritic cells and macrophages but are also mitogenic for B cells . Less clear are the claimed effects of CpG-DNA on T cells, which range from direct activation, costimulation, or indirect transient activation via antigen-presenting cell (APC)-derived interferon type I (IFN type I) . Here we demonstrate that CpG-DNA sequence specifically triggers macrophages to produce IFN type I, interleukin (IL)-12, IL-6 and tumour necrosis factor (TNF), but lacks the ability to directly costimulate T cells . Strikingly, poly-guanosine (poly-G) extensions to CpG-containing oligonucleotides (ODN) abolished the macrophage stimulatory potential yet generated T-cell costimulatory activities . In fact, independently of CpG-motifs, poly-G-ODN displayed the ability to costimulate T cells . Costimulation was operative on CD8 T cells but not CD4 T cells . Poly-G-mediated costimulation resulted in IL-2-driven T-cell proliferation and induced cytolytic T cells . Overall the data imply that poly-G motifs costimulate antigen reactive CD8 T cells, while CpG-DNA motifs fail to do so but may affect T-cell activation via APC derived cytokines such as IFN type I. J Vet Intern Med, 2000 Sep-Oct, 14(5), 534 - 41 Quantitative bacterial cultures and cytological examination of bronchoalveolar lavage specimens in dogs; Peeters DE et al.; Cytology and quantitative bacterial cultures of lower respiratory tract secretions are widely used in human medicine to differentiate airway infection from simple bacterial colonization . A retrospective study was conducted to determine the usefulness of quantitative aerobic cultures and Gram stain intracellular bacteria counts from bronchoalveolar lavage (BAL) specimens in dogs in diagnosing lower respiratory tract infection (LRTI) and to determine whether chronic bronchitis is associated with marked bacterial growth in dogs . The threshold determined to define clinically relevant bacterial growth was 1.7 x 10(3) colony-forming units per milliliter of BAL fluid . We used this threshold and found that diagnostic sensitivity and specificity were 86% and 100%, respectively . With a threshold for infection of >2 intracellular bacteria observed in any of 50 fields, microscopic examination of Gram stain BAL preparations had a sensitivity of 71% and a specificity of 97% in establishing LRTI . There was a high correlation between bacterial morphology on BAL Gram stain and bacterial cultures . Combining the results of intracellular bacteria counts from the BAL Gram stain with those from the quantitative cultures, the sensitivity in diagnosing LRTI was 87% and the specificity was 97% . BAL quantitative cultures as well as quantitating intracellular bacteria on Gram stain BAL cytology were revealed to be useful in identifying LRTI in dogs . Chronic bronchitis does not appear to be associated with marked bacterial growth in dogs. Appl Environ Microbiol, 2000 Oct, 66(10), 4372 - 7 Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions; Normander B et al.; An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents . The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA . Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days . Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil . No change in bacterial community composition was observed in relation to plant age . Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane . The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA . The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere. Appl Environ Microbiol, 2000 Oct, 66(10), 4361 - 5 Bacterial functional redundancy along a soil reclamation gradient; Yin B et al.; A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates . Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest . Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU) . BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site . The number and diversity of bacterial groups responding to four carbon substrates (L-serine, L-threonine, sodium citrate, and alpha-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate . These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils . They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning. Appl Environ Microbiol, 2000 Oct, 66(10), 4237 - 46 Bacterial community structure associated with a dimethylsulfoniopropionate-producing North Atlantic algal bloom; Gonzalez JM et al.; The bacteria associated with oceanic algal blooms are acknowledged to play important roles in carbon, nitrogen, and sulfur cycling, yet little information is available on their identities or phylogenetic affiliations . Three culture-independent methods were used to characterize bacteria from a dimethylsulfoniopropionate (DMSP)-producing algal bloom in the North Atlantic . Group-specific 16S rRNA-targeted oligonucleotides, 16S ribosomal DNA (rDNA) clone libraries, and terminal restriction fragment length polymorphism analysis all indicated that the marine Roseobacter lineage was numerically important in the heterotrophic bacterial community, averaging >20% of the 16S rDNA sampled . Two other groups of heterotrophic bacteria, the SAR86 and SAR11 clades, were also shown by the three 16S rRNA-based methods to be abundant in the bloom community . In surface waters, the Roseobacter, SAR86, and SAR11 lineages together accounted for over 50% of the bacterial rDNA and showed little spatial variability in abundance despite variations in the dominant algal species . Depth profiles indicated that Roseobacter phylotype abundance decreased with depth and was positively correlated with chlorophyll a, DMSP, and total organic sulfur (dimethyl sulfide plus DMSP plus dimethyl sulfoxide) concentrations . Based on these data and previous physiological studies of cultured Roseobacter strains, we hypothesize that this lineage plays a role in cycling organic sulfur compounds produced within the bloom . Three other abundant bacterial phylotypes (representing a cyanobacterium and two members of the alpha Proteobacteria) were primarily associated with chlorophyll-rich surface waters of the bloom (0 to 50 m), while two others (representing Cytophagales and delta Proteobacteria) were primarily found in deeper waters (200 to 500 m). Microbes Infect, 2000 Aug, 2(10), 1171 - 80 Mechanisms of disposal of bacterial lipopolysaccharides by animal hosts; Elsbach P; Much of the very extensive literature describing the (bio)chemistry and biology of bacterial lipopolysaccharides (LPS, endotoxin) has dealt with the properties of these molecules as potent triggers of host responses . This brief review will focus on what has been learned recently about mechanisms by which the host can dispose of LPS and counter its often excessive stimulatory effects. Plant Cell, 2000 Sep, 12(9), 1783 - 94 The bacterial elicitor flagellin activates its receptor in tomato cells according to the address-message concept; Meindl T et al.; flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, acts as a potent elicitor in plants . Here, we have used an iodinated derivative of flg22 ((125)I-labeled Tyr-flg22) as a molecular probe for the flagellin receptor in tomato cells . This radioligand showed rapid binding to a single class of specific, saturable, high-affinity receptor sites in intact cells and membrane preparations . Binding, although essentially nonreversible under physiological conditions, was not covalent, and chemical cross-linking was required to specifically label a single polypeptide of 115 kD . Intact flagellin and elicitor-active flagellin peptides but not biologically inactive analogs efficiently competed for binding of radioligand . Peptides lacking the C terminus of the conserved domain, previously found to act as competitive antagonists of elicitor action in tomato cells, also competed for binding of radioligand . Thus, this novel, high-affinity binding site exhibited all the characteristics expected of a functional receptor of bacterial flagellin . For a model of receptor activation, we propose a two-step mechanism according to the address-message concept, in which binding of the N terminus (address) is the first step and activation of responses with the C terminus (message) is the second step. J Biol Chem, 2000 Dec 29, 275(52), 41150 - 5 Structure-function relationships of a novel bacterial toxin, hemolysin E . The role of alpha G; Atkins A et al.; The novel pore-forming toxin hemolysin E (HlyE, ClyA, or SheA) consists of a long four-helix bundle with a subdomain (beta tongue) that interacts with target membranes at one pole and an additional helix (alpha(G)) that, with the four long helices, forms a five-helix bundle (tail domain) at the other pole . Random amino acid substitutions that impair hemolytic activity were clustered mostly, but not exclusively, within the tail domain, specifically amino acids within, adjacent to, or interacting with alpha(G) . Deletion of amino acids downstream of alpha(G) did not affect activity, but deletions encompassing alpha(G) yielded insoluble and inactive proteins . In the periplasm Cys-285 (alpha(G)) is linked to Cys-87 (alpha(B)) of the four-helix bundle via an intramolecular disulfide . Oxidized HlyE did not form spontaneously in vitro but could be generated by addition of Cu(II) or mimicked by treatment with Hg(II) salts to yield inactive proteins . Such treatments did not affect binding to target membranes nor assembly into non-covalently linked octameric complexes once associated with a membrane . However, gel filtration analyses suggested that immobilizing alpha(G) inhibits oligomerization in solution . Thus once associated with a membrane, immobilizing alpha(G) inhibits HlyE activity at a late stage of pore formation, whereas in solution it prevents aggregation and consequent inactivation. Pflugers Arch, 2000, 440(5 Suppl), R72 - 4 Interleukin-8 and procalcitonin in early diagnosis of early severe bacterial infection in critically ill neonates; Bonac B et al.; We studied the value of serum interleukin-8 (IL-8) and procalcitonin (PCT) in the early diagnosis of early severe bacterial infection in 58 critically ill ventilated neonates . ELISA was used for determining IL-8 and immunoluminometric assay for PCT . IL-8 and PCT were compared with routinely used serum C-reactive protein (CRP) . Neonates were divided into four groups: Ia--proven severe bacterial infection (n = 9), Ib--clinical sepsis (n = 16), II--respiratory distress without bacterial infection (n = 12), and III--various types of neonatal distress (n = 21) . Sera were collected on admission, at 24 h and 48 h after admission . There was no significant difference between groups Ia and Ib for either parameter at any time interval . Significant difference was found between group Ia+b (septic neonates) and group II for PCT and CRP at 24 and 48 h, but not for IL-8 . There was no difference between group Ia+b and group III except for CRP at 24 h . Diagnostic accuracy was best for PCT on admission and for CRP at 24 h . Serum PCT and IL-8 are not specific markers for early severe bacterial infection in critically ill neonates and are not better than CRP. Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 413 - 21 Re-emerging structures: continuing crystallography of the bacterial reaction centre; Fyfe PK et al.; The reaction centre is nature's solar battery, and is found in a number of variations on a common theme in plants, algae and photosynthetic bacteria . During the last 20 years, a combination of X-ray crystallography, spectroscopy and mutagenesis has provided increasingly detailed insights into the mechanism of light energy transduction in the bacterial reaction centre . This mini-review looks at the application of X-ray crystallography to the bacterial reaction centre, focussing in particular on recent information on the structural consequences of site-directed mutagenesis, the roles played by water molecules in the reaction centre, the mechanism of ubiquinone reduction, and studies of the phospholipid environment of the protein. Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 325 - 30 A novel protein transport system involved in the biogenesis of bacterial electron transfer chains; Berks BC et al.; The Tat system is a recently discovered bacterial protein transport pathway that functions primarily in the biosynthesis of proteins containing redox active cofactors . Analogous transport systems are found in plant organelles . Remarkably and uniquely the Tat system functions to transported a diverse range of folded proteins across a biological membrane, a feat that must be achieved without rendering the membrane freely permeable to protons and other ions . Here we review the operation of the bacterial Tat system and propose a model for the structural organisation of the Tat preprotein translocase. Mamm Genome, 2000 Oct, 11(10), 899 - 905 Human and mouse mitochondrial orthologs of bacterial ClpX; Corydon TJ et al.; We have determined the cDNA sequence and exon/intron structure of the human CLPX gene encoding a human ortholog of the E . coli ClpX chaperone and protease subunit . The CLPX gene comprises 14 exons and encodes a 633-amino acid-long precursor polypeptide . The polypeptide contains an N-terminal putative mitochondrial transit peptide, and expression of a full-length ClpX cDNA tagged at its C-terminus (Myc-His) shows that the polypeptide is transported into mitochondria . FISH analysis localized the CLPX gene to human Chromosome (Chr) 15q22.1-22.32 . This localization was refined by radiation hybrid mapping placing the CLPX gene 4.6 cR distal to D15S159 . Murine ClpX cDNA was sequenced, and the mouse Clpx locus was mapped to a position between 31 and 42 cM offset from the centromere on mouse Chr 9 . Experimental observations indicate the presence of a pseudogene in the mouse genome and sequence variability between mouse ClpX cDNAs from different strains . Alignment of the human and mouse ClpX amino acid sequences with ClpX sequences from other organisms shows that they display the typical modular organization of domains with one AAA(+) domain common to a large group of ATPases and several other domains conserved in ClpX orthologs linked by non-conserved sequences . Notably, a C-4 zinc finger type motif is recognized in human and mouse ClpX . This motif of so far unknown function is present only in a subset of the known ClpX sequences. Arch Virol, 2000, 145(8), 1639 - 57 Bacterial lipoprotein based expression vectors as tools for the characterisation of African swine fever virus (ASFV) antigens; Leitao A et al.; African swine fever virus (ASFV) is the causative agent of an important pig disease for which protective mechanisms are still poorly understood . The present work was aimed at the characterisation of ASFV antigens using previously reported vectors that allow their expression as fusion proteins with the bacterial lipoprotein OprI . Several recombinant clones induced SLA-restricted, ASFV-specific lymphoproliferation and one (A2) was demonstrated to stimulate ASFV-specific CTL activity in vitro, in opposition to the effect of UV inactivated virus . The nucleotide sequence of the fragment cloned in A2 showed 99% identity with a portion of the G1340L ORF of the BA71V isolate, and the expressed fusion lipoprotein induced specific antibodies in vivo . Blood mononuclear leukocytes from a pig immunised with outer membrane preparations from A2 showed to reduce strongly (99.6%) the ASFV yield in cultures of autologous macrophages . However, after inoculation with virulent virus the pig developed acute fatal ASF . Overall our results show that OprI based expression vectors are valuable tools to screen viral antigens in terms of their capacity to trigger immune competent cells. Life Sci, 2000 Jun 16, 67(4), 365 - 72 Mercaptoethylguanidine attenuates inflammation in bacterial meningitis in rabbits; Irazuzta JE et al.; Reactive oxygen and nitrogen species participate in the inflammatory process during meningitis . Among them, superoxide, nitric oxide (NO), and their reaction product peroxynitrite exert cytotoxic effects . Mercaptoethylguanidine (MEG) exerts beneficial effects in in vivo inflammatory conditions by scavenging peroxynitrite and inhibiting the inducible NO synthase . This study was designed to investigate whether MEG may attenuate inflammation and brain injury in experimental meningitis . Meningitis increased nitrite/nitrate, and protein content in the cerebrospinal fluid (CSF) . In the brain tissue high levels of malondialdehyde and formation of nitrotyrosine indicated lipid peroxidation and nitrosative stress, respectively . Myeloperoxidase activity was increased indicating accumulation of neutrophils into the brain parenchyma . Treatment with MEG decreased nitrite/nitrate levels whereas it did not affect the bacterial clearance from the CSF . Furthermore, treatment with MEG markedly reduced brain tissue levels of myeloperoxidase and malondialdehyde . These data demonstrate that MEG could have a therapeutic role in meningitis. Respir Med, 2000 Sep, 94(9), 881 - 7 Lower airway bacterial colonization in asymptomatic smokers and smokers with chronic bronchitis and recurrent exacerbations; Qvarfordt I et al.; Bacterial colonization of the lower airways in patients with chronic bronchitis (CB) has been described mainly in patients with co-existing chronic obstructive pulmonary disease (COPD) . Although smoking has been identified as a risk factor for bacterial colonization it is not known whether asymptomatic smokers (AS) can be colonized . The aim of this study was to study lower airway bacterial colonization in smokers with stable CB and recurrent exacerbations and compare with AS and healthy never-smokers (NS) . Thirty-nine smokers with CB and recurrent exacerbations (median FEV1 85% of predicted normal), 10 AS and 10 NS, underwent bronchoscopy and a two-step bronchoalveolar lavage (BAL) procedure where the first portion (20 ml, 'pre-BAL') was recovered separately from the rest (140 ml, 'BAL') . The degree of oropharyngeal contamination of pre-BAL and BAL samples was evaluated by cytology . Semiquantitative bacterial cultures were performed on all samples . Higher bacterial numbers than 10(3) colony-forming units (cfu) x ml(-1) in BAL were found only in the two smoking groups . Using 10(3) cfu x ml(-1) as cut-off, 6/10 (60%) in the AS-, and 7/35 (20%) in the CB-group were colonized in the lower airways . In all, 29% of all smokers had bacterial colonization . Only bacteria belonging to the normal oropharyngeal flora were found . The proportion of samples with oropharyngeal contamination was significantly lower in BAL than in pre-BAL (5% vs . 21%, P=0.039) . The proportion of sterile samples was significantly higher in BAL than in pre-BAL (49% vs . 26%, P=0.002) . Lower airway bacterial colonization was found both in asymptomatic smokers and in patients with CB . Colonization with potential respiratory pathogens is uncommon in patients with CB and recurrent exacerbations without severe airflow obstruction . The two-step BAL procedure seems to decrease oropharyngeal contamination. Nature, 2000 Sep 14, 407(6801), 177 - 9 Bacterial photosynthesis in surface waters of the open ocean; Kolber ZS et al.; The oxidation of the global ocean by cyanobacterial oxygenic photosynthesis, about 2,100 Myr ago, is presumed to have limited anoxygenic bacterial photosynthesis to oceanic regions that are both anoxic and illuminated . The discovery of oxygen-requiring photosynthetic bacteria about 20 years ago changed this notion, indicating that anoxygenic bacterial photosynthesis could persist under oxidizing conditions . However, the distribution of aerobic photosynthetic bacteria in the world oceans, their photosynthetic competence and their relationship to oxygenic photoautotrophs on global scales are unknown . Here we report the first biophysical evidence demonstrating that aerobic bacterial photosynthesis is widespread in tropical surface waters of the eastern Pacific Ocean and in temperate coastal waters of the northwestern Atlantic . Our results indicate that these organisms account for 2-5% of the photosynthetic electron transport in the upper ocean. J Chromatogr A, 2000 Sep 1, 891(1), 85 - 92 Fast quantitation of recombinant plasminogen activator inhibitor type 1 in bacterial lysates by micropellicular reversed-phase liquid chromatography; O'Keefe DO et al.; A rapid reversed-phase HPLC assay is described for quantitating recombinant plasminogen activator inhibitor type 1 (PAI-1) in cultures of Escherichia coli . The assay utilized a short nonporous micropellicular C18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid . The selective resolution of recombinant PAI-1 from major host proteins occurred within 1.3 min . Regeneration of initial chromatography conditions was fast and allowed successive injections every 3 min . The assay's limit of detection for recombinant PAI-1 was 0.5 microg in 5-microl injections of bacterial lysates and the assay was linear to 20 microg . The assay's apparent recovery was between 94 and 108% throughout the quantitative range . In a direct comparison to gel electrophoresis the reversed-phase assay proved superior in monitoring recombinant PAI-1 titers in cultures of E . coli. RNA, 2000 Sep, 6(9), 1257 - 66 Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification; Trottier M et al.; All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA . The genome is subsequently inserted into the procapsid . The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs . Recently, we found that pRNA dimers are building blocks of pRNA hexamers . Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+ . The chemical-modification pattern of the monomer is compared to that of the dimer . The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges . However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas . It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification . Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape . The dimer was twice as long as the monomer . Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation . Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization . This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging. RNA, 2000 Sep, 6(9), 1212 - 25 Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA; Schmitz M et al.; We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy . The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme . Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii . The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6) . We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations . In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt . Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins . The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant . The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+. Int J STD AIDS, 2000 Sep, 11(9), 603 - 6 Bacterial vaginosis and smoking; Hellberg D et al.; This study aimed to investigate if there is an association between bacterial vaginosis (BV) and smoking . This cohort study included 956 randomly chosen, apparently healthy women at 2 family planning and one youth clinic . Of the 956 women, 131 women fulfilled the criteria for BV and the remaining 825 served as a control group . BV, BV-associated bacteria and gynaecological infections were diagnosed . Structured personal interviews concerning, smoking, alcohol and drug habits, sexual behaviour and reproductive history were made . Before and after adjustment for possible confounding factors, smoking, but not alcohol and drug use, was significantly associated with BV . Of the women with BV 52% were smokers versus 32% in the control group . Age-adjusted odds ratio (OR) for smokers was 2.3 before, and 3.0 (95% confidence interval {CI} 1.3-6.9) after adjustment for sexual risk behaviour, reproductive history, and alcohol use . There was also a significant dose-response relationship between BV and smoking habits . The data suggest that there might be a causal association between BV and smoking. Int J Oncol, 2000 Oct, 17(4), 811 - 8 Age-related alterations in 32P-postlabeled DNA adducts in livers of mice infected with the tumorigenic bacterial pathogen, Helicobacter hepaticus; Josyula S et al.; Helicobacter hepaticus causes chronic active hepatitis and liver tumors in mice, with associated increase in reactive oxygen species . Indigenous (I)-compounds are bulky DNA adducts present at low levels and detected by 32P-post-labeling . Some may be caused by reactive oxygen species; others occur normally and decrease during liver tumorigenesis . The identity of most is unknown . We investigated whether mouse liver infection by H . hepaticus and resulting progression of hepatic lesions would be associated with qualitative or quantitative changes in I-compounds . Mice were 3, 6, 9, and 12 months of age; liver disease ranged from minimal through marked . In control A/J mice, up to 20 I-compounds were detected, and the total level of these did not change with age, whereas 11 individual I-compounds showed marked age-related differences . These appeared to be coordinately regulated, as the total of these 11 adducts was constant at 6-12 months . In A/JNCr mice naturally infected with H . hepaticus, up to 12 hepatic I-compounds were found . Total levels varied markedly with age and were high at 6 and 12 months . Neither total adduct levels, nor the amount of any individual adduct, correlated positively with severity of hepatic lesions; in some cases, highest levels were found in livers with least disease . Thus, liver infection and tumorigenesis by H . hepaticus was not associated with an increase in any 32P-postlabeled DNA adduct . Marked, and distinct, age-related changes in total or individual adducts in control and infected mice suggest a role in the physiological alterations of aging and in host response to infection. Nature, 2000 Sep 7, 407(6800), 81 - 6 Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp . APS; Shigenobu S et al.; Almost all aphid species (Homoptera, Insecta) have 60-80 huge cells called bacteriocytes, within which are round-shaped bacteria that are designated Buchnera . These bacteria are maternally transmitted to eggs and embryos through host generations, and the mutualism between the host and the bacteria is so obligate that neither can reproduce independently . Buchnera is a close relative of Escherichia coli, but it contains more than 100 genomic copies per cell, and its genome size is only a seventh of that of E . coli . Here we report the complete genome sequence of Buchnera sp . strain APS, which is composed of one 640,681-base-pair chromosome and two small plasmids . There are genes for the biosyntheses of amino acids essential for the hosts in the genome, but those for non-essential amino acids are missing, indicating complementarity and syntrophy between the host and the symbiont . In addition, Buchnera lacks genes for the biosynthesis of cell-surface components, including lipopolysaccharides and phospholipids, regulator genes and genes involved in defence of the cell . These results indicate that Buchnera is completely symbiotic and viable only in its limited niche, the bacteriocyte. Rev Med Liege, 2000 Jun, 55(6), 581 - 5 {Bacterial sexually transmitted diseases}; Arrese JE et al.; The sexually transmitted diseases (STD) correspond to bacterial, viral, mycotic and parasitic disorders . They are present worldwide . However, their incidences and prevalences are higher and their complications are more severe in the intertropical belt. Rev Med Liege, 2000 Jun, 55(6), 572 - 6 {Tropical bacterial dermatoses}; Pierard-Franchimont C et al.; There is a number of bacterial dermatoses confined to or aggravated by the tropical environment . They are prone to develop due to the lack of hygiene, the difficult access to water, the hostile geoclimatic conditions and malnutrition . Several clinical categories are distinguished according to the nature of the pathogens and the type of disease spread. Infect Immun, 2000 Oct, 68(10), 6012 - 26 Reverse transcriptase-PCR analysis of bacterial rRNA for detection and characterization of bacterial species in arthritis synovial tissue; Kempsell KE et al.; Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents . The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue . Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls . This may be suggestive of the presence of live bacteria . Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled . This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species . These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu . In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients . These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods . In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular . The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation. Infect Immun, 2000 Oct, 68(10), 5673 - 8 alpha(v)beta(3) integrin and bacterial lipopolysaccharide are involved in Coxiella burnetii-stimulated production of tumor necrosis factor by human monocytes; Dellacasagrande J et al.; Coxiella burnetii, the agent of Q fever, enters human monocytes through alpha(v)beta(3) integrin and survives inside host cells . In addition, C . burnetii stimulates the synthesis of inflammatory cytokines including tumor necrosis factor (TNF) by monocytes . We studied the role of the interaction of C . burnetii with THP-1 monocytes in TNF production . TNF transcripts and TNF release reached maximum values within 4 h . Almost all monocytes bound C . burnetii after 4 h, while the percentage of phagocytosing monocytes did not exceed 20% . Cytochalasin D, which prevented the uptake of C . burnetii without interfering with its binding, did not affect the expression of TNF mRNA . Thus, bacterial adherence, but not phagocytosis, is necessary for TNF production by monocytes . The monocyte alpha(v)beta(3) integrin was involved in TNF synthesis since peptides containing RGD sequences and blocking antibodies against alpha(v)beta(3) integrin inhibited TNF transcripts induced by C . burnetii . Nevertheless, the cross-linking of alpha(v)beta(3) integrin by specific antibodies was not sufficient to induce TNF synthesis . The signal delivered by C . burnetii was triggered by bacterial lipopolysaccharide (LPS) . Polymyxin B inhibited the TNF production stimulated by C . burnetii, and soluble LPS isolated from C . burnetii largely mimicked viable bacteria . On the other hand, avirulent variants of C . burnetii induced TNF production through an increased binding to monocytes rather than through the potency of their LPS . We suggest that the adherence of C . burnetii to monocytes via alpha(v)beta(3) integrin enables surface LPS to stimulate TNF production in THP-1 monocytes. Int J STD AIDS, 2000 Aug, 11(8), 495 - 8 Prevalence of bacterial vaginosis in women attending one of three general practices for routine cervical cytology; Lamont RF et al.; A prospective observational study of asymptomatic women from three different general practices was set up to establish the incidence of bacterial vaginosis (BV) . The study group comprised 287 women recalled to their general practitioner's surgery for routine cervical smears . The prevalence of an abnormal vaginal flora was about the same in women attending the 3 practices . Nearly 14% of women had abnormal vaginal flora and about 9% had BV on gram stain examination of vaginal secretions . This was 2-3 times more common than findings consistent with vaginal candidiasis (3.8%) . Significant numbers of women with BV had received antifungal therapy suggesting a misdiagnosis . Because of its potential complications, women should be offered screening for BV in a well-women setting and, if found, should be treated if symptomatic or at risk of adverse obstetric or gynaecological sequelae. Trends Microbiol, 2000 Sep, 8(9), 396 - 401 Intraspecies variation in bacterial genomes: the need for a species genome concept; Lan R et al.; Bacterial populations are clonal . Their evolution involves not only divergence between orthologous genes but also gain of genes from other clones or species, which has only recently been widely appreciated through macrorestriction mapping, genomic subtraction and complete genome sequencing . Genes can also be lost in response to selection or by random mutation after becoming redundant . The bacterial genome is a dynamic structure and intraspecies variation needs to be included in genome analysis if we are to gain insight into the full species genome. Bioorg Med Chem Lett, 2000 Sep 4, 10(17), 1959 - 62 Lipopolyamines incorporating the tetraamine spermine, bound to an alkyl chain, sequester bacterial lipopolysaccharide; Blagbrough IS et al.; Lipopolyamines, with high affinity for calf thymus DNA in an ethidium bromide displacement assay, bind with high affinity to bacterial lipopolysaccharide and neutralise in vitro endotoxic activity as determined by Griess nitric oxide and TNF-alpha ELISA assays. Jpn Heart J, 2000 May, 41(3), 411 - 6 Development of non-bacterial thrombotic endocarditis after percutaneous transvenous mitral commissurotomy for severely calcified mitral stenosis; Tomimoto S et al.; We encountered a case of mitral stenosis, complicated with non-bacterial thrombotic endocarditis, that developed after percutaneous transvenous mitral commissurotomy (PTMC) . A 71-year-old female Japanese patient had severe congestive heart failure and underwent PTMC for critical and severely calcified mitral stenosis . Four weeks later, the echocardiogram demonstrated a highly echoic protrusion in the postero-medial commissure of the mitral valve . There was little evidence of inflammation at that time . She had been anticoagulated adequately since she was admitted . The patient underwent replacement of the mitral valve . She did not show any evidence of systemic embolization . Microscopic evaluation showed only organized thrombus but no evidence of inflammation in the mitral valve . Silent development of non-bacterial thrombotic endocarditis after PTMC should be recognized as a rare but potentially lethal complication of PTMC. J Bacteriol, 2000 Oct, 182(19), 5551 - 5 Sorting out bacterial viability with optical tweezers; Ericsson M et al.; We have developed a method, using laser, optical tweezers and direct microscopic analysis of reproductive potential and membrane integrity, to assess single-cell viability in a stationary-phase Escherichia coli population . It is demonstrated here that a reduction in cell integrity, determined by using the fluorescent nucleic acid stain propidium iodide, correlated well with a reduction in cell proliferating potential during the stationary-phase period studied . Moreover, the same cells that exhibited reduced integrity were found to be the ones that failed to divide upon nutrient addition . A small but significant number of the intact cells (496 of 7,466 {6.6%}) failed to replicate . In other words, we did not find evidence for the existence of a large population of intact but nonculturable cells during the stationary-phase period studied but it is clear that reproductive ability can be lost prior to the loss of membrane integrity . In addition, about 1% of the stationary-phase cells were able to divide only once upon nutrient addition, and in a few cases, only one of the two cells produced by division was able to divide a second time, indicating that localized cell deterioration, inherited by only one of the daughters, may occur . The usefulness of the optical trapping methodology in elucidating the mechanisms involved in stationary-phase-induced bacterial death and population heterogeneity is discussed. Curr Opin Struct Biol, 2000 Aug, 10(4), 462 - 9 Structure of a conserved receptor domain that regulates kinase activity: the cytoplasmic domain of bacterial taxis receptors; Falke JJ et al.; Many bacteria are motile and use a conserved class of transmembrane sensory receptor to regulate cellular taxis toward an optimal living environment . These conserved receptors are typically stimulated by extracellular signals, but also undergo adaptation via covalent modification at specific sites on their cytoplasmic domains . The function of the cytoplasmic domain is to integrate the extracellular and adaptive signals, and to use this integrated information to regulate an associated histidine kinase . The kinase, in turn, triggers a cytoplasmic phosphorylation pathway of the two-component class . The high-resolution structure of a receptor cytoplasmic domain has recently been determined by crystallographic methods and is largely consistent with a structural model independently generated by chemical studies of the domain in the full-length, membrane-bound receptor . These results represent an important step toward a mechanistic understanding of receptor-to-kinase information transfer. Int J Parasitol, 2000 Aug, 30(9), 1013 - 7 Oxidative responses during bacterial phagocytosis of polymorphonuclear leucocytes in primarily and secondarily Fasciola hepatica infected goats; Martinez-Moreno A et al.; The polymorphonuclear leukocyte (PMN) function, in terms of oxidative response during bacterial phagocytosis, was studied using a Luminol-Dependant Chemiluminiscence (LDCL) assay in primarily and secondarily Fasciola hepatica infected goats . Polymorphonuclear leukocytes of F . hepatica infected goats displayed lower LDCL responses than naive goats . The lowest responses were observed in secondarily infected animals that had higher parasitic burdens and more prominent hepatic lesions . The reduced responses were induced by a functional defect of the circulating PMN but also by a direct involvement of serum factors . Both circulating parasite products and the non protective immune response that occurred during secondary F . hepatica infection of goats could be implied in the alteration of PMN function . These findings suggest the existence of an important mechanism for impairment of the host immune system during goat fasciolosis. Bioinformatics, 2000 Jun, 16(6), 560 - 1 Oriloc: prediction of replication boundaries in unannotated bacterial chromosomes; Frank AC et al.; SUMMARY: A program called Oriloc has been developed for the prediction of bacterial replication origins . The method builds on the fact that there are compositional asymmetries between the leading and the lagging strand for replication . The program works with unannotated sequences in fasta format and therefore uses glimmer 2.0 outputs to discriminate between codon positions so as to increase the signal/noise ratio. Biochemistry, 2000 Sep 5, 39(35), 10695 - 701 Identification of the active site acid/base catalyst in a bacterial fumarate reductase: a kinetic and crystallographic study; Doherty MK et al.; The active sites of respiratory fumarate reductases are highly conserved, indicating a common mechanism of action involving hydride and proton transfer . Evidence from the X-ray structures of substrate-bound fumarate reductases, including that for the enzyme from Shewanella frigidimarina {Taylor, P., Pealing, S . L., Reid, G . A., Chapman, S . K., and Walkinshaw, M . D . (1999) Nat . Struct . Biol . 6, 1108-1112}, indicates that the substrate is well positioned to accept a hydride from N5 of the FAD . However, the identity of the proton donor has been the subject of recent debate and has been variously proposed to be (using numbering for the S . frigidimarina enzyme) His365, His504, and Arg402 . We have used site-directed mutagenesis to examine the roles of these residues in the S . frigidimarina enzyme . The H365A and H504A mutant enzymes exhibited lower k(cat) values than the wild-type enzyme but only by factors of 3-15, depending on pH . This, coupled with the increase in K(m) observed for these enzymes, indicates that His365 and His504 are involved in Michaelis complex formation and are not essential catalytic residues . In fact, examination of the crystal structure of S . frigidimarina fumarate reductase has led to the proposal that Arg402 is the only plausible active site acid . Consistent with this proposal, we report that the R402A mutant enzyme has no detectable fumarate reductase activity . The crystal structure of the H365A mutant enzyme shows that, in addition to the replacement at position 365, there have been some adjustments in the positions of active site residues . In particular, the observed change in the orientation of the Arg402 side chain could account for the decrease in k(cat) seen with the H365A enzyme . These results demonstrate that an active site arginine and not a histidine residue is the proton donor for fumarate reduction. Mol Microbiol, 2000 Aug, 37(4), 687 - 95 UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition; Gourse RL et al.; In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the sigma subunit, but also between a DNA element upstream of the core promoter and the alpha subunit . DNA binding by alpha can increase transcription dramatically . Here we review the current state of our understanding of the alpha interaction with DNA during basal transcription initiation (i.e . in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e . when stimulated by transcription factors). J Appl Microbiol, 2000 Aug, 89(2), 370 - 80 Evaluation of ChemChrome V6 for bacterial viability assessment in waters; Parthuisot N et al.; The efficiency of ChemChrome B (CB) and ChemChrome V6 (CV6) dyes to stain viable bacterial cells in water was compared . Both dyes are fluorogenic esters converted to free fluorescein by esterase activity . The dyes were applied to a wide variety of bacterial species, including those poorly stained by CB, and to natural waters . Some species tested gave unacceptable low fluorescence intensities by being inefficiently or non-labelled with the CB . In contrast, CV6-stained bacteria were easily detected by both flow cytometry and solid-phase cytometry . As a consequence, higher viable cell counts were found with CV6 compared with CB in natural waters . Viable counts determined by CV6 staining were always higher than cfu counts . In contrast, respiring cell counts (CTC) were always lower than CV6 counts and, in the case of tap and mineral waters, they were lower than cfu counts. Clin Physiol, 2000 Sep, 20(5), 399 - 410 Regional cerebral blood flow during hyperventilation in patients with acute bacterial meningitis; Moller K et al.; Mechanical hyperventilation is often instituted in patients with acute bacterial meningitis when increased intracranial pressure is suspected . However, the effect on regional cerebral blood flow (CBF) is unknown . In this study, we measured regional CBF (rCBF) in patients with acute bacterial meningitis before and during short-term hyperventilation . In 17 patients with acute bacterial meningitis, absolute rCBF (in ml/100 g min-1) was measured during baseline ventilation and hyperventilation by single-photon emission computed tomography (SPECT) using intravenous 133Xe bolus injection . Intravenous 99mTc-HMPAO (hexamethylpropyleneamine oxime) was subsequently given during hyperventilation . In 12 healthy volunteers, rCBF was measured by SPECT and 99mTc-HMPAO during spontaneous ventilation . Using standard templates to identify regions of interest (ROIs), we calculated rCBF in percentage of cerebellar (99mTc-HMPAO images) or mean hemispheric (133Xe images) flow for each ROI, the degree of side-to-side asymmetry for each ROI, and the anterior-to-posterior flow ratio . On 133Xe images, absolute rCBF decreased significantly during hyperventilation compared to baseline ventilation in all regions, but the relative rCBF did not change significantly from baseline ventilation (n=14) to hyperventilation (n=12), indicating that the perfusion distribution was unchanged . On 99mTc-HMPAO images (n=12), relative rCBF and the anterior-to-posterior flow ratio were significantly lower in patients than in controls in the frontal and parietal cortex as well as in the basal ganglia . Focal perfusion abnormalities were present in 10 of 12 patients . Regional cerebral blood flow abnormalities are frequent in patients with acute bacterial meningitis . Short-term hyperventilation does not enhance these abnormalities. EMBO J, 2000 Sep 1, 19(17), 4601 - 13 Fine tuning bacterial chemotaxis: analysis of Rhodobacter sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and cheY genes; Shah DS et al.; Rhodobacter sphaeroides chemotaxis is significantly more complex than that of enteric bacteria . Rhodobacter sphaeroides has multiple copies of chemotaxis genes (two cheA, one cheB, two cheR, three cheW, five cheY but no cheZ), controlling a single 'stop-start' flagellum . The growth environment controls the level of expression of different groups of genes . Tethered cell analysis of mutants suggests that CheY(4) and CheY(5) are the motor-binding response regulators . The histidine protein kinase CheA(2) mediates an attractant ('normal') response via CheY(4), while CheA(1) and CheY(5) appear to mediate a repellent ('inverted') response . CheY(3) facilitates signal termination, possibly acting as a phosphate sink, although CheY(1) and CheY(2) can substitute . The normal and inverted responses may be initiated by separate sets of chemoreceptors with their relative strength dependent on growth conditions . Rhodobacter sphaeroides may use antagonistic responses through two chemosensory pathways, expressed at different levels in different environments, to maintain their position in a currently optimum environment . Complex chemotaxis systems are increasingly being identified and the strategy adopted by R.sphaeroides may be common in the bacterial kingdom. J Mol Biol, 2000 Sep 15, 302(2), 339 - 58 A chemical phylogeny of group I introns based upon interference mapping of a bacterial ribozyme; Strauss-Soukup JK et al.; Despite its small size, the 205 nt group I intron from Azoarcus tRNA(Ile) is an exceptionally stable self-splicing RNA . This IC3 class intron retains the conserved secondary structural elements common to group I ribozymes, but lacks several peripheral helices . These features make it an ideal system to establish the conserved chemical basis of group I intron activity . We collected nucleotide analog interference mapping (NAIM) data of the Azoarcus intron using 14 analogs that modified the phosphate backbone, the ribose sugar, or the purine base functional groups . In conjunction with a complete interference set collected on the Tetrahymena group I intron (IC1 class), these data define a "chemical phylogeny" of functional groups that are important for the activity of both introns and that may be common chemical features of group I intron catalysts . The data identify the functional moieties most likely to play a conserved role as ligands for catalytic metal ions, the substrate helix, and the guanosine cofactor . These include backbone functional groups whose nucleotide identity is not conserved, and hence are difficult to identify by standard phylogenetic sequence comparisons . The data suggest that both introns utilize an equivalent set of long range tertiary interactions for 5'-splice site selection between the P1 substrate helix and its receptor in the J4/5 asymmetric bulge, as well as an equivalent set of 2'-OH groups for P1 helix docking into most of the single stranded segment J8/7 . However, the Azoarcus intron appears to make an alternative set of interactions at the base of the P1 helix and at the 5'-end of the J8/7 . Extensive differences were observed within the intron peripheral domains, particularly in P2 and P8 where the Azoarcus data strongly support the proposed formation of a tetraloop-tetraloop receptor interaction . This chemical phylogeny for group I intron catalysis helps to refine structural models of the RNA active site and identifies functional groups that should be carefully investigated for their role in transition state stabilization . Biophys J, 2000 Sep, 79(3), 1237 - 52 Self-regulation phenomena in bacterial reaction centers . I . General theory; Goushcha AO et al.; A model for light-induced charge separation in a donor-acceptor system of the reaction center of photosynthetic bacteria is described . This description is predicated on a self-regulation of the flow of photo-activated electrons due to self-consistent, slow structural rearrangements of the macromolecule . Effects of the interaction between the separated charges and the slow structural modes of the biomolecule may accumulate during multiple, sequential charge transfer events . This accumulation produces non-linear dynamic effects on system function, providing a regulation of the charge separation efficiency . For a biomolecule with a finite number of different charge-transfer states, the quasi-stationary populations of these states with a localized electron on different cofactors may deviate from a Lagmuir law dependence with actinic light intensity . Such deviations are predicted by the model to be due to light-induced structural changes . The theory of self-regulation developed here assumes that light-induced changes in the effective adiabatic potential occur along a slow structural coordinate . In this model, a "light-adapted" conformational state appears when bifurcation produces a new minimum in the adiabatic potential . In this state, the lifetime of the charge-separated state may be quite different from that of the "dark-adapted" conformation . The results predicted by this theory agree with previously obtained experimental results on photosynthetic reaction centers. Pediatrics, 2000 Sep, 106(3), 477 - 82 Predicting the outcome of neonatal bacterial meningitis; Klinger G et al.; OBJECTIVE: To build predictive models of severe adverse outcome at various times in the course of neonatal bacterial meningitis . STUDY DESIGN: Retrospective cohort study with follow-up to a minimum age of 1 year of term and near-term infants, admitted between 1979 and 1998 to a regional tertiary care center . Predictors of adverse outcome detectable at 1 year of age (death or moderate or severe neurosensory impairment) were identified by univariate analysis . Independent predictors of adverse outcome were identified by multivariate analysis . Predictive tree models were constructed at 12, 24, 48, and 96 hours after admission and at discharge . RESULTS: Of 101 infants admitted with definitive bacterial meningitis, 13 died and 17 had moderate or severe disability at 1 year of age . Outcomes are known for all patients, to 1 year of age . Twelve hours after admission the important predictors of adverse outcome were presence of seizures, presence of coma, use of inotropes, and leukopenia (sensitivity: 68%; specificity: 100%) . At 96 hours the predictors were seizure duration of >72 hours, presence of coma, use of inotropes, and leukopenia (sensitivity: 88%; specificity: 99%) . CONCLUSIONS: Most infants at risk for adverse outcome can be identified within 12 hours of admission . Duration of seizures for >72 hours, presence of coma, use of inotropes, and leukopenia were the most important predictors of adverse outcome . Although these models have good predictive accuracy, they need to be validated in a contemporary cohort in large multicenter studies. Int J Gynaecol Obstet, 2000 Sep, 70(3), 341 - 6 Bacterial vaginosis and contraceptive methods; Calzolari E et al.; OBJECTIVES The aim of this study was to investigate if bacterial vaginosis is associated with the use of specific contraceptives . METHODS: The study population consisted of 1314 women attending for periodical preventive examinations at our gynecology unit at the II Institute of Obstetrics and Gynecology of the University 'La Sapienza' in Rome . The patient's history and any current genital symptom were recorded on a structured protocol . Current users of contraceptives were compared with non-users . The chi(2) test and the t-test were used in the statistical analysis; a stepwise logistic regression analysis was performed to assess the simultaneous effect of more than one variable and to identify for possible confounding factors . RESULTS: Both oral contraceptive and condom use showed a significant protective effect against bacterial vaginosis . Our results also showed a significant increase of BV among IUD users, either before or after adjustments . CONCLUSIONS: This study showed a significant negative association between BV and OC and condom use, respectively, and a significant positive association between BV and IUD use . Therefore, we suggest that it is advisable to carry out a systematic microscopic evaluation in order to identify BV for IUD users. Cell, 2000 Aug 18, 102(4), 475 - 85 Nucleoid proteins stimulate stringently controlled bacterial promoters: a link between the cAMP-CRP and the (p)ppGpp regulons in Escherichia coli; Johansson J et al.; We report that the H-NS nucleoid protein plays a positive role in the expression of stringently regulated genes in Escherichia coli . Bacteria lacking both H-NS and the paralog StpA show reduced growth rate . Colonies displaying an increased growth rate were isolated, and mapping of a suppressor mutation revealed a base pair substitution in the spoT gene . The spoT(A404E) mutant showed low ppGpp synthesizing ability . The crp gene, which encodes the global regulator CRP, was subject to negative stringent regulation . The stable RNA/protein ratio in an hns, stpA strain was decreased, whereas it was restored in the suppressor strain . Our findings provide evidence of a direct link between the cAMP-CRP modulon and the stringent response. J Trauma, 2000 Aug, 49(2), 306 - 13 Ileal mucosal response to bacterial toxin challenge; Harari Y et al.; BACKGROUND: The cause of postinjury intestinal mucosal barrier disruption remains obscure . The present study examines the hypothesis that the bacterial toxin formyl-methionyl leucyl phenylalanine (FMLP) plays an initial role in this process . METHODS: Mucosal permeability to fluorescein isothiocyanate-labeled dextran (4,400 molecular weight) was measured in perfused distal rat ileum with and without FMLP . Dextran and myeloperoxidase appearance in the lumenal perfusate was assessed in response to surrogates of traumatic stress: ischemia/reperfusion, total abdominal irradiation, and total parenteral nutrition . Recovery of FMLP in the effluent of static closed and perfused ileal loops was determined by mass spectrometry . Release of mast cell mediators in the presence of FMLP was determined in ileal everted sacs . RESULTS: Seventy-five percent of FMLP was recovered in perfusion effluent in contrast to 5% in closed loops . There was a transient increase in ileal permeability in FMLP/perfused, untreated rats, and in ischemia/reperfusion and total parenteral nutrition treated rats that was recorded with a concomitant increment in myeloperoxidase (inflammatory marker) in all experimental models except irradiated rats, which were unresponsive to FMLP . FMLP responsiveness was associ . ated with a significant rise in release of serotonin (mast cell mediator) . CONCLUSION: These results suggest that mast cells and other resident inflammatory cells within the gut wall are involved in FMLP-induced changes in mucosal barrier permeability and raise the possibility that under conditions of traumatic stress, proinflammatory mediators within the gut wall might be activated by toxic factors in the gut lumen. J Biol Chem, 2000 Dec 15, 275(50), 39345 - 53 Structural differences of bacterial and mammalian K+ channels; Wrisch A et al.; Using a peptide toxin, kaliotoxin (KTX), we gained new insight into the topology of the pore region of a voltage-gated potassium channel, mKv1.1 . In order to find new interactions between mKv1.1 and KTX, we investigated the pH dependence of KTX block which was stronger at pH(o) 6.2 compared with pH(o) 7.4 . Using site-directed mutagenesis on the channel and the toxin, we found that protonation of His(34) in KTX caused the pH(o) dependence of KTX block . Glu(350) and Glu(353) in mKv1.1, which interact with His(34) in KTX, were calculated to be 4 and 7 A away from His(34)/KTX, respectively . Docking of KTX into a homology model of mKv1.1 based on the KcsA crystal structure using this and other known interactions as constraints showed structural differences between mKv1.1 and KcsA within the turret (amino acids 348-357) . To satisfy our data, we would have to modify the KcsA crystal structure for the mKv1.1 channel orienting Glu(350) 7 A and Glu(353) 4 A more toward the center of the pore compared with KcsA . This would place Glu(350) 15 A and Glu(353) 11 A away from the center of the pore instead of the distances for the equivalent KcsA residues with 22 A for Gly(53) and 15 A for Gly(56), respectively . Bacterial and mammalian potassium channels may have structural differences regarding the turret of the outer pore vestibule . This topological difference between both channel types may have substantial influence on structure-guided development of new drugs for mammalian potassium channels by rational drug design. Res Microbiol, 2000 Jul-Aug, 151(6), 421 - 7 Phosphocholine of pneumococcal teichoic acids: role in bacterial physiology and pneumococcal infection; Fischer W; Pneumococci have an absolute nutritional requirement for choline . Choline is incorporated as phosphocholine (PCho) into lipoteichoic (LTA) and teichoic acid (TA) . The PCho residues are required for transformability, the activity of autolysins, the separation of daughter cells after cell division and for anchoring a family of surface proteins which play important roles in pneumococcal infection . The genes encoding the enzymes for PCho incorporation are described . Two strains that acquired the ability to grow in the absence of choline are discussed. Biophys Chem, 2000 Jul 15, 85(2-3), 153 - 67 Understanding the function of bacterial outer membrane channels by reconstitution into black lipid membranes Van Gelder P, Dumas F, Winterhalter M. Structural and functional information is obtained by the reconstitution of membrane channel forming proteins into black lipid membranes . Due to this outstanding sensitivity only little material is needed and single molecule detection can be easily achieved . An overview on different types of detection will be given. Curr Biol, 2000 Jul 27-Aug 10, 10(15), 927 - 30 Interactions of the chemotaxis signal protein CheY with bacterial flagellar motors visualized by evanescent wave microscopy; Khan S et al.; The chemotaxis signal protein CheY of enteric bacteria shuttles between transmembrane methyl-accepting chemotaxis protein (MCP) receptor complexes and flagellar basal bodies {1} . The basal body C-rings, composed of the FliM, FliG and FliN proteins, form the rotor of the flagellar motor {2} . Phosphorylated CheY binds to isolated FliM {3} and may also interact with FliG {4}, but its binding to basal bodies has not been measured . Using the chemorepellent acetate to phosphorylate and acetylate CheY {5}, we have measured the covalent-modification-dependent binding of a green fluorescent protein-CheY fusion (GFP-CheY) to motor assemblies in bacteria lacking MCP complexes by evanescent wave microscopy {6} . At acetate concentrations that cause solely clockwise rotation, GFP-CheY molecules bound to native basal bodies or to overproduced rotor complexes with a stoichiometry comparable to the number of C-ring subunits . GFP-CheY did not bind to rotors lacking FIiM/FliN, showing that these subunits are essential for the association . This assay provides a new means of monitoring protein-protein interactions in signal transduction pathways in living cells. Int J Med Microbiol, 2000 Jul, 290(3), 223 - 30 Peptide display on bacterial flagella: principles and applications; Westerlund-Wikstrom B; Expression of foreign peptides as fusions to bacterial cell surface proteins has gained increasing attention in basic, as well as applied research during the last decade . A wide range of heterologous peptides have been expressed, and the spectrum of available carrier proteins is also wide . The choice of carrier protein is frequently ruled by the application of the fusion protein constructed . This review is focused on flagella display, which is based on genetic fusion of foreign peptides into a surface-exposed, dispensable region of flagellin, the flagellar major subunit present in thousands of copies per filament . Expression of these constructs in flagellin-deficient host strains results in hybrid flagella carrying the heterologous peptides in thousands of intimately-associated copies . The first and still most frequent application of flagella display is the construction of novel recombinant vaccines . Flagella display has also been used in peptide display as an alternative to the phage-display technique . One application involves fusion into a disulfide loop of Escherichia coli thioredoxin that has been inserted into flagellin, this system facilitates expression of random peptides in a conformationally constrained manner readily accessible on the flagellar surface . The random peptide library has been applied in antibody epitope mapping and is suitable for biopanning procedures in the study of ligand-receptor interactions . Many bacterial adhesins are of complex nature and thereby difficult to analyse by conventional methods . Direct flagella display has proven to be applicable also in bacterial adhesion technology since large fragments, up to 302 amino acid residues in length, of bacterial adhesins can be functionally expressed as fusions to flagellin . Hybrid flagella are easily purified and can easily be analysed for binding to various targets, such as immobilized proteins, tissue sections, as well as cell cultures. Int J Med Microbiol, 2000 Jul, 290(3), 215 - 21 Fimbriae-assisted bacterial surface display of heterologous peptides; Klemm P et al.; The display of peptide segments on the surface of bacteria offers many new and exciting applications in biotechnology and medical research . Fimbria-assisted display of heterologous sequences is a paradigm for chimeric organelle display on bacteria . Fimbriae are particularly attractive candidates for epitope display for several reasons: (1) they are present in extremely high numbers at the cell surface, (2) they are strong immunogens, (3) they possess inherent adhesive properties, and (4) they can be easily purified . The majority of work dealing with fimbria-assisted peptide display has been focused on the development of recombinant vaccines . A number of different fimbrial types have been used to display immune-relevant sectors of various foreign proteins . Chimeric fimbrial vaccines can be used in the context of purified proteins, however the potential also exists to exploit this technology for the development of live recombinant vaccines . Work has also been performed demonstrating the amenability of fimbriae towards the powerful technology of random peptide display . This review summarises the current state of research in this field. J Microbiol Methods, 2000 Aug, 41(3), 201 - 9 Development of polymerase chain reaction-based assays for bacterial gene detection; Johnson JR; Polymerase chain reaction-based assays provide rapid, simple, and sensitive detection of bacterial genes, but are not without their drawbacks . This review summarizes the principal advantages and disadvantages of PCR-based bacterial gene detection, provides guidelines for the development and validation of new PCR assays, and describes potential pitfalls that may be encountered and how these can be avoided. J Microbiol Methods, 2000 Aug, 41(3), 179 - 84 A fast and simple turbidimetric method for the determination of sulfate in sulfate-reducing bacterial cultures; Kolmert A et al.; A standard turbidimetric assay for the determination of sulfate in water was modified with the objective of achieving a quick and simple method for monitoring the decrease of sulfate in cultures of sulfate-reducing bacteria . The effects of sulfate concentration, mixing time and the ratio of sample to conditioning reagent were optimized using a central composite face-centered response surface model design . The results suggested that a mixing time of 30 s resulted in smaller absorbance variance, the variance in absorbance measurements tended to increase with concentration of sulfate and that the ratio between the amount of conditioning reagent and sample had no significant influence on the absorbance variance . The modified assay thus developed is simple and quick, and covers a comparatively large sulfate concentration range (0-5 mM) compared to the standard turbidimetric assay. Mar Biotechnol (NY), 2000 Jul, 2(4), 309 - 313 Discrimination between Atlantic and Pacific Subspecies of Northern Bluefin Tuna (Thunnus thynnus) by Magnetic-Capture Hybridization Using Bacterial Magnetic Particles; Takeyama H et al.; The previously developed magnetic-capture hybridization technique employing bacterial magnetic particles was applied to discriminate between Atlantic and Pacific subspecies of the northern bluefin tuna (Thunnus thynnus) using specific DNA sequences . Nucleotide sequences of a 925-bp fragment (ATCO) flanking the mitochondrial ATPase and cytochrome oxidase subunit III genes in these two subspecies were compared . Two regions having single-nucleotide and three-nucleotide differences between the subspecies were adopted to design DNA probes (NR1, 21-mer; NR2, 29-mer), and two internal primer sets were designed to amplify DNA fragments containing these regions . The DNA probes were immobilized on bacterial magnetic particles via streptavidin-biotin conjugation and subjected to magnetic-capture hybridization with the digoxigenin-labeled fragments amplified using the internal primers . The luminescence intensities of DNA on bacterial magnetic particles obtained by hybridization between the probes and the complementary fragments were higher than those obtained by hybridization with noncomplementary fragments . These data suggest that this system employing DNA on bacterial magnetic particles may be useful for discrimination of these two subspecies by recognizing a single-nucleotide difference. Acta Pharm Hung, 2000 Feb, 70(1), 11 - 4 {Interaction of immunosuppressive drugs in mice with special regard to bacterial translocation}; Anderlik P et al.; Following intraperitoneally (i.p.) applied treatment with 12.5 mg/mouse prednisolonum (PR) no bacterial translocation (BT) was observed in mice . The PR treatment applied in combination with lymphotropic cytostatics as dianhydrogalactitol (30 mg/kg i.p.) or chlorpromazine (75 mg/kg i.p.) both causing BT, did not increase the mice's drug sensitivity to the used agents . According to our results, RP can be suitable for combined application with other immunosuppressive agents as it can increase immunosuppression without increase of side-effect such as those induced by bacterial translocation. J Exp Med, 2000 Aug 21, 192(4), 595 - 600 Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6; Hacker H et al.; Transition of immature antigen presenting cells (APCs) to the state of professional APCs is essential for initiation of cell-mediated immune responses to pathogens . Signal transduction via molecules of the Toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) pathway is critical for activation of APCs either by pathogen-derived pattern ligands like lipopolysaccharides (LPS) or by CD40 ligation through T helper cells . The capacity of bacterial DNA (CpG-DNA) to induce APCs to differentiate into professional APCs represents an interesting discovery . However, the signaling pathways involved are poorly understood . Here we show that CpG-DNA activates the TLR/IL-1R signaling pathway via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IkappaB kinase complex and the c-jun NH(2)-terminal kinases . Moreover, cells of TLR2- and TLR4-deficient mice are activated by CpG-DNA, whereas cells of MyD88-deficient mice do not respond . The data suggest that CpG-DNA initiates signaling via the TLR/IL-1R pathway in APCs in a manner similar to LPS and to T helper cell-mediated CD40 ligation . Activation of the TLR/IL-1R signaling pathway by foreign bacterial DNA may be one way to initiate innate defense mechanisms against infectious pathogens in vivo. Anal Chem, 2000 Aug 1, 72(15), 3518 - 22 Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes; Tanaka T et al.; We report a fully automated sandwich immunoassay for the determination of human insulin using antibody-protein A-bacterial magnetic particle (BMP) complexes and an alkaline phosphatase-conjugated secondary antibody . BMPs bearing protein A-MagA inserted on the external surface of the membrane were prepared in the Magnetospirillum sp . AMB-1 transconjugant for a protein A-magA fusion gene . MagA protein was used as an anchor to attach protein A onto the membrane . Protein A-BMP complexes harvested from transconjugant AMB-1 were subsequently complexed with anti-human insulin antibodies by specific binding between the Z domain of protein A and the Fc component of IgG to form the antibody-protein A-BMP complexes . The complexes were quite monodisperse after the binding of the antibody . The BMPs' monodispersity resulted in high signal and low noise in the immunoassay . The luminescence intensity ((kilocounts/s)/microg of antibody) from antibody-protein A-BMP complexes after immunoreaction was higher than that from BMPs chemically conjugated to an antibody . This was explained by a difference in dispersion . The fully automated sandwich immunoassay system using antibody-protein A-BMP complexes made possible precise assays of human insulin in serum. Receptors Channels, 2000, 7(2), 109 - 19 Bacterial expression of G-protein-coupled receptors: prediction of expression levels from sequence; Kiefer H et al.; Eleven G-protein-coupled receptors were expressed in Escherichia coli as fusion proteins with N-terminal glutathione-S-transferase and a C-terminal (His)6 tag . Expression levels varied between 0.1% and 10% of the total cellular protein . Low expression levels, as quantified by analytical nickel chelating chromatography, coincided with a toxic effect of protein expression . Multiple linear regression analysis was used to establish a correlation between the occurrence of positively charged amino acid residues in the loop regions and the expression level . Indeed, 44% of the variation in expression levels could be attributed to the positive charge content . Consequently, this sequence feature is the major determinant of expression level . Our results were supported by two mutations where positive charges were introduced into loop regions of two low-expressing receptors: As predicted, these mutations led to a considerably higher expression . A similar mutation of an olfactory receptor described previously increased expression approximately 100-fold and further supports our model . The data are discussed in the context of the "positive inside rule". Transgenic Res, 2000 Apr, 9(2), 137 - 44 Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene; Galili S et al.; Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals . Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids . One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants . Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway . This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants . These plants showed enhanced levels of both free and protein-bound threonine . In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate . Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins. J Biomed Mater Res, 2000 Nov, 52(2), 388 - 94 Influence of surface modifications to titanium on oral bacterial adhesion in vitro; Yoshinari M et al.; The influence of surface modifications to titanium on the initial adherence of Porphyromonas gingivalis ATCC33277 and Actinobacillus actinomycetemcomitans ATCC43718 was evaluated . Surface modifications were performed with dry processes including ion implantation (Ca(+), N(+), F(+)), oxidation (anode oxidation, titania spraying), ion plating (TiN, alumina), and ion beam mixing (Ag, Sn, Zn, Pt) with Ar(+) on polished pure titanium plates . Comparatively large amounts of P . gingivalis and A . actinomycetemcomitans adhered to polished titanium plates . The degree of P . gingivalis adhesion showed a positive correlation with surface energy and the amount of calcium-ion adsorption . Adherence of both P . gingivalis and A . actinomycetemcomitans increased on calcium-implanted surfaces compared with polished titanium surfaces, whereas adherence of P . gingivalis was remarkably decreased on alumina-coated surfaces . These findings indicate that titanium implants exposed to the oral cavity require surface modification to inhibit the adherence of oral bacteria, and that surface modification with a dry process is useful in controlling the adhesion of oral bacteria as well as ensuring resistance against wear. Acta Neurol Scand, 2000 Aug, 102(2), 118 - 23 Cognitive outcome after bacterial meningitis; Merkelbach S et al.; OBJECTIVE: To evaluate long-term cognitive deficits in unselected patients with previously diagnosed meningitis and to compare these deficits to neurologic and psychopathologic impairment . PATIENTS AND METHODS: Twenty-two unselected patients (mean age 52.5 +/- 17.1 years) were examined neurologically, psychiatrically, and psychometrically 30 +/- 11 months after the acute stage of bacterial meningitis . Results of psychometric tests were compared with clinical long-term deficits . Psychometric tests were additionally applied on 17 healthy controls (mean age 49.2 +/- 14.2 years) . RESULTS: Neurologic or psychopathologic symptoms were found in 16 patients . Psychometrically, the speed of cognitive processes and psychomotor performance, concentration, visuoconstructive capacity, and memory functions were reduced significantly in patients as compared to controls . Verbal intelligence was less affected than performance efficiency . Patients with pneumococcal meningitis had significantly lower test results than patients with other pathogens . The psychometric test results were only slightly related with clinical findings of the follow-up examination . CONCLUSION: Psychometric deficits are frequent after bacterial meningitis, and their relation with neurologic and psychopathologic symptoms is loose . The pattern of neuropsychologic impairment accentuates psychomotor slowing combined with memory disturbances, and resembles features observed in subcortical cognitive impairment. Biotechniques, 2000 Aug, 29(2), 271 - 4, 276 Technique for cloning and sequencing the ends of bacterial artificial chromosome inserts; Ripoll PJ et al.; Bacterial artificial chromosome (BAC) libraries are an important tool for positional cloning, gene analysis and physical mapping . During studies using BAC clones, it is often necessary to organize them into contiguous sequences (contigs) . To finalize, join and extend the contigs, both cloning and sequencing of the ends of the inserts are required . Here, we describe a low-cost, accessible, fast and powerful method for the routine isolation of BAC ends . This method allows the isolation of 20 BAC clone ends in one day . The analysis of the ends reveals fragment sizes compatible with sequencing, and the structure of these clones allows the sequencing of both ends using the same plasmid . Moreover, long end fragments can be sequenced in both directions. Infect Immun, 2000 Sep, 68(9), 5306 - 13 Transcutaneous immunization with bacterial ADP-ribosylating exotoxins, subunits, and unrelated adjuvants; Scharton-Kersten T et al.; We have recently described a needle-free method of vaccination, transcutaneous immunization, consisting of the topical application of vaccine antigens to