Microbial

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 intact skin . While most proteins themselves are poor immunogens on the skin, we have shown that the addition of cholera toxin (CT), a mucosal adjuvant, results in cellular and humoral immune responses to the adjuvant and coadministered antigens . The present study explores the breadth of adjuvants that have activity on the skin, using diphtheria toxoid (DTx) and tetanus toxoid as model antigens . Heat-labile enterotoxin (LT) displayed adjuvant properties similar to those of CT when used on the skin and induced protective immune responses against tetanus toxin challenge when applied topically at doses as low as 1 microg . Interestingly, enterotoxin derivatives LTR192G, LTK63, and LTR72 and the recombinant CT B subunit also exhibited adjuvant properties on the skin . Consistent with the latter finding, non-ADP-ribosylating exotoxins, including an oligonucleotide DNA sequence, as well as several cytokines (interleukin-1beta {IL-1beta} fragment, IL-2, IL-12, and tumor necrosis factor alpha) and lipopolysaccharide also elicited detectable anti-DTx immunoglobulin G titers in the immunized mice . These results indicate that enhancement of the immune response to topical immunization is not restricted to CT or the ADP-ribosylating exotoxins as adjuvants . This study also reinforces earlier findings that addition of an adjuvant is important for the induction of robust immune responses to vaccine antigens delivered by topical application.

Genomics, 2000 Jul 1, 67(1), 78 - 82
Isolation and preliminary characterization of the human and mouse homologues of the bacterial cell cycle gene era; Britton RA et al.; Era is an essential GTPase that is required for proper cell cycle progression and cell division in Escherichia coli and is found in nearly all bacteria sequenced to date . To determine whether Era is also present in eukaryotic organisms, we searched the dbEST database and found EST clones coding for proteins that were similar to Era . Full sequencing of these ESTs from human and mouse identified a conserved homologue, ERAL1 (Era-like 1) . ERAL1 maps to 17q11.2 in human and is located in the syntenic region of mouse chromosome 11 . ERAL1 may be an attractive candidate for a tumor suppressor gene since ERAL1 is located in a chromosomal region where loss of heterozygosity is often associated with various types of cancer.

Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9419 - 24
Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays; Khodursky AB et al.; We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells . We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression . When both enzymes were inhibited, the replication fork stopped rapidly . The elongation rate with topo IV alone was 1/3 of normal . Genetic data confirmed and extended these results . Inactivation of gyrase alone caused a slow stop of replication . Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork.

EMBO J, 2000 Aug 15, 19(16), 4292 - 7
The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin; Goh KC et al.; Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity . Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress . The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear . Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines . This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress . Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR . We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS . Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.

Acta Paediatr, 2000 Jul, 89(7), 803 - 5
Cerebrospinal fluid concentrations of alpha-melanocyte-stimulating hormone in bacterial and aseptic meningitis; Ichiyama T et al.; alpha-Melanocyte-stimulating hormone (alpha-MSH) has potent anti-inflammatory effects in several experimental models of inflammation . It inhibits both the actions and production of proinflammatory cytokines and neutrophil migration . We investigated whether alpha-MSH in cerebrospinal fluid (CSF) increases during the acute stage in patients with bacterial and aseptic meningitis by measuring alpha-MSH in CSF via radioimmunoassay . The alpha-MSH concentrations in CSF from the children with bacterial meningitis who survived (n = 8), those with aseptic meningitis (n = 16), and the control subjects (n = 23) were all below the detection limit . However, CSF alpha-MSH was elevated in four of the five children with bacterial meningitis who had neurological sequelae . We speculate that elevated alpha-MSH levels in CSF during acute bacterial meningitis reflect negative feedback in response to severe inflammation associated with neurological sequelae induced by proinflammatory cytokines . CONCLUSION: CSF alpha-MSH is elevated in children with severe bacterial meningitis who had neurological sequelae.

Postepy Hig Med Dosw, 2000, 54(3), 317 - 27
{Bacterial ion canals}; Kubalski A et al.; After an overview of potassium and chloride ion channels found in bacterial inner membrane this review focuses on mechanosensitive ion channels from the inner membrane of Escherichia coli . Mechanosensitive channels, MscL and MscS, have major roles in managing the transition from high to low environments . Biochemical and genetic studies of MscL, combined with structural information derived from X-ray crystalography, have brought the knowledge of how a mechanosensitive channel senses membrane tension . Physiological studies on MscL and on MscS have demonstrated how the mechanosensitive channels contribute to the bacterial membrane response upon hypo-osmotic stress.

Respiration, 2000, 67(4), 426 - 32
Diagnostic accuracy of pleural fluid polymorphonuclear elastase in the differentiation between pyogenic bacterial infectious and non-infectious pleural effusions; Alegre J et al.; BACKGROUND AND OBJECTIVES: To establish the diagnostic accuracy of the markers of neutrophil activity (elastase and lysozyme) determined in pleural fluid, for differentiating between pyogenic bacterial infectious and non-infectious pleural effusions . PATIENTS AND METHODS: At our tertiary referral teaching hospital, 160 patients over 14 years with pleural effusion (PE), classified as pyogenic bacterial infectious (41 parapneumonic complicated, 32 parapneumonic non-complicated) and non-infectious (32 neoplasm and 55 undiagnosed pleural exudates) were examined in a prospective study . Polymorphonuclear elastase (PMN-E) was determined by an immunoactivation method and lysozyme by a turbidimetric method . Receiver operating characteristic (ROC) curves were used to evaluate diagnostic accuracy . RESULTS: Pleural fluid PMN-E was the biochemical marker that best differentiated between pyogenic bacterial infectious and non-infectious PE . The ROC area under the curve (AUC) for PMN-E was 0.8276 . A PMN-E value over 230 microg/l diagnosed infectious PE with a specificity of 0.81 and a sensitivity of 0.74 . The ROC AUC for proteins plus lactate dehydrogenase was 0 . 7430 . Differences between the two ROC curves were significant (p = 0 . 032) . After excluding purulent parapneumonic complicated PE, the sensitivity of a pleural fluid PMN-E value equal to or greater than 230 microg/l was 0.64 and the specificity 0.81 . CONCLUSIONS: Pleural fluid PMN-E was the marker that best differentiated infectious from non-infectious PE, and PMN-E values lower than 230 microg/l suggest non-infectious PE .

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1463 - 9
A rapid method for determining the G+C content of bacterial chromosomes by monitoring fluorescence intensity during DNA denaturation in a capillary tube; Xu HX et al.; A simple and rapid method to determine the G+C content of bacterial chromosomal DNA was developed . It involves determination of Tm by a Light Cycler and calculation of the G+C content by an empirical formula relating Tm to G+C content . Instead of a conventional thermal denaturation method, which monitors the increase of absorbance at 260 nm, thermal denaturation was monitored by the decrease of fluorescence intensity in the presence of SYBR Green 1 . In this method, the apparent Tm of DNA was influenced by the concentration of SYBR Green 1, DNA and salt . In addition, when the G+C content was calculated from a linear equation {(mol% G+Cx = mol% G+Cr+1.99(Tmx-Tmr), where x is the unknown organism and y is the reference organism}, an error value was introduced among strains with extremely low or high G+C content . Based upon five standards (G+C contents in the range 33-66 mol%), a suitable equation was formulated for the capillary method: mol% G+Cx = mol% G+Cr+1.4652(Tmx-Tmr)+0.0063(Tmx2-Tmr2) . To determine the Tm of organisms within this range of G+C contents, Escherichia coli ATCC 11775T was used as a DNA standard and fixed concentrations of SYBR Green 1, sodium citrate and DNA were used . The data from 37 bacterial strains indicated that this equation behaved well . Because it is rapid and simple, it may prove useful for bacterial identification.

Mol Plant Microbe Interact, 2000 Aug, 13(8), 869 - 76
Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight; Yin Z et al.; Many plant mutants develop spontaneous lesions that resemble disease symptoms in the absence of pathogen attack . In several pathosystems, lesion mimic mutations have been shown to be involved in programmed cell death, which in some instances leads to enhanced disease resistance to multiple pathogens . We investigated the relationship between spontaneous cell death and disease resistance in rice with nine mutants with a range of lesion mimic phenotypes . All nine mutations are controlled by recessive genes and some of these mutants have stunted growth and other abnormal characteristics . The lesion mimics that appeared on the leaves of these mutants were caused by cell death as measured by trypan blue staining . Activation of six defense-related genes was observed in most of the mutants when the mimic lesions developed . Four mutants exhibited significant enhanced resistance to rice blast . One of the mutants, spl11, confers non-race-specific resistance not only to blast but also to bacterial blight . The level of resistance in the spl11 mutant to the two pathogens correlates with the defense-related gene expression and lesion development on the leaves . The results suggest that some lesion mimic mutations in rice may be involved in disease resistance, and cloning of these genes may provide a clue to developing broad-spectrum resistance to diverse pathogens.

J Mol Microbiol Biotechnol, 2000 Apr, 2(2), 179 - 89
Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism; Wu LF et al.; The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the Sec machinery . It is structurally and mechanistically similar to the delta pH-dependent pathway used for importing chloroplast proteins into the thylakoid . The tat genes are not ubiquitously present and are absent from half of the completely sequenced bacterial genomes . The presence of the tat genes seems to correlate with genome size and with the presence of important enzymes with a twin-arginine signal peptide . A minimal Tat system requires a copy of tatA and a copy of tatC . The composition and gene order of a tat locus are generally conserved within the same taxonomy group but vary considerably to other groups, which would exclude an acquisition of the Tat system by recent horizontal gene transfer . The tat genes are also found in the genomes of chloroplasts and plant mitochondria but are absent from animal mitochondrial genomes . The topology of evolution trees suggests a bacterial origin of the Tat system . In general, the twin-arginine signal peptide is capable of targeting any passenger protein to the Tat pathway . However, a structural signal carried by the mature part of a passenger protein can override targeting information in a signal peptide under certain circumstances . Tat systems show a substrate-Tat component specificity and a species specificity . The pore size of the Tat channel is estimated as being between 5 and 9 nm . Operational models of the Tat system are proposed.

Evolution Int J Org Evolution, 2000 Apr, 54(2), 517 - 25
Cospeciation between bacterial endosymbionts (Buchnera) and a recent radiation of aphids (Uroleucon) and pitfalls of testing for phylogenetic congruence; Clark MA et al.; Previous studies of phylogenetic congruence between aphids and their symbiotic bacteria (Buchnera) supported long-term vertical transmission of symbionts . However, those studies were based on distantly related aphids and would not have revealed horizontal transfer of symbionts among closely related hosts . Aphid species of the genus Uroleucon are closely related phylogenetically and overlap in geographic ranges, habitats, and parasitoids . To examine support for congruence of phylogenies of Buchnera and Uroleucon, sequences from four mitochondrial, one nuclear, and one endosymbiont gene (trpB) were obtained . Congruence of phylogenies based on pooled aphid genes with phylogenies based on trpB was highly significant: Most nodes resolved by trpB corresponded to nodes resolved by the pooled aphid genes . Furthermore, no nodes were both inconsistent between the trees and strongly supported in both trees . Two kinds of analyses testing the null hypothesis of perfect congruence between pairwise combinations of datasets and tree topologies were performed: the Kishino-Hasegawa test and the likelihood-ratio test . Both tests indicated significant disagreement among most pairwise combinations of mitochondrial, nuclear, and symbiont datasets . Because rampant recombination among mitochondrial genomes of different aphid species is unlikely, inaccurate assumptions in the evolutionary models underlying these tests appear to be causing the hypothesis of a shared history to be incorrectly rejected . Moreover, trpB was more consistent with the aphid genes as a set than any single aphid gene was with the others, suggesting that the symbionts show the same phylogeny as the aphids . Overall, analyses support the interpretation that symbionts and aphids have undergone strict cospeciation, with no horizontal transmission of symbionts even among closely related, ecologically similar aphid hosts.

J Am Med Womens Assoc, 2000 Summer, 55(4), 220 - 4
Bacterial vaginosis: a public health problem for women; Rauh VA et al.; Bacterial vaginosis (BV) remains a moderately prevalent condition with clearly observed links to adverse reproductive, gynecological, and other outcomes in women, including human immunodeficiency virus infection . Because of inconsistent findings from clinical studies concerning BV's etiologic role, no definitive policies with respect to screening and treatment have yet been established . Of concern is the high, unexplained prevalence of BV among African-American women, who are also at extremely high risk for preterm birth . The complexity of the sociodemographic picture challenges the field of public health to continue to explore the role of BV and its relationship to a whole host of social and biomedical conditions that may contribute to adverse health outcomes among society's most vulnerable members . Future decisions about screening and treatment, currently based on the biomedical model, may need to take into consideration issues of social context and expanded views of causality if we are to better understand and eliminate those factors that place individual women at risk of adverse outcomes, as well as the conditions that underlie racial and ethnic disparities in health.

Anal Biochem, 2000 Aug 15, 284(1), 70 - 6
An easy and accurate agarose gel assay for quantitation of bacterial plasmid copy numbers; Pushnova EA et al.; An assay for quantitation of plasmid copy numbers in bacterial cell cultures has been developed and validated . The method combines isolation of total bacterial DNA (including both plasmid and genomic DNA), running a series of twofold dilutions of total DNA in an agarose gel followed by ethidium bromide staining, and subsequent scanning of the gel picture negatives . We have developed a novel set of rules for integration of the scan data that allows us to achieve high assay precision, accuracy, and sensitivity . The assay validation results were as follows: intra- and interassay precision with %CV of 8.2-9.9 and 7.1-9.8%, respectively; ruggedness with %CV of 9.3-17.5%; spike recovery of 80-102%; and sensitivity of 1 plasmid copy per genome .

J Invest Surg, 2000 May-Jun, 13(3), 169 - 73
Effects of somatostatin analogues and vitamin C on bacterial translocation in an experimental intestinal obstruction model of rats; Akyildiz M et al.; The passage of viable endogenous bacteria and their products across the intact intestinal mucosal barrier, disseminating to the mesenteric lymph nodes, peritoneal cavity, spleen, liver, and circulation, is defined as bacterial translocation . Intestinal obstruction induces bacterial translocation due to mucosal disruption, motility dysfunction, and increased intestinal volume, leading to bacterial overgrowth . In a rat model of intestinal obstruction, the effects of both high-dose vitamin C (350 microg/kg), an antioxidant agent known to have a cytoprotective effect in ischemia-reperfusion injury, and somatostatin (20 microg/kg), a gastrointestinal antisecretory agent, in preventing bacterial translocation were studied . Both intestinal and liver samples from the rats was observed, and it was found that the rate of bacterial translocation was 100% in the control group, and only 43% for the rats who were given intraperitoneal vitamin C and somatostatin . The difference was statistically significant . In conclusion, we are convinced that vitamin C and somatostatin analogues may have protective effects against bacterial translocation in mechanical bowel obstruction.

Rev Latinoam Microbiol, 1998 Jan-Jun, 40(1-2), 53 - 71
{Bacterial systems for expelling toxic metals}; Vargas E et al.; Bacteria have developed diverse resistance strategies towards toxic metals with which they interact in the environment . The mechanisms of tolerance include extracellular precipitation, sequestration by cell envelopes, intracellular accumulation, redox transformations and membrane efflux systems . Genes responsible for these processes may be encoded by the chromosome or by plasmids . Since some toxic metals are also essential micronutrients (i.e . copper, cobalt, zinc, nickel), bacteria must precisely adjust the functioning of uptake and efflux systems to maintain their adequate intracellular levels . In the case of metals with no biological function (i.e . cadmium, silver), transport systems must be oriented only to the extrusion of the toxic ions . In the last few years, several bacterial systems devoted to the efflux of toxic metals were analyzed at the molecular level resulting in a detailed understanding of the biochemical mechanisms of resistance . Among these are the membrane pathways that extrude cations derived from copper, cadmium, zinc, nickel, cobalt and silver . Two general mechanisms have been found: those involving P-type ATPases, and some using proton antiporter systems.

Can J Microbiol, 2000 Jul, 46(7), 618 - 22
Phenotypic characterization of the Xenorhabdus bacterial symbiont of a Texas strain of the entomopathogenic nematode Steinernema riobrave, and characterization of the Xenorhabdus bovienii bacterial symbiont of a Newfoundland strain of Steinernema feltiae; He H et al.; Two bacterial symbionts of entomopathogenic nematodes, one of which originated from Texas, U.S.A., and the other from Newfoundland, Canada, were characterized phenotypically . These strains belonged to the genus Xenorhabdus . The Newfoundland (NF) strain was shown to be X . bovienii but the Texas (TX) strain was not identified at the species level . Four additional cultures of Xenorhabdus were included in the study . These were a strain of X . bovienii (Umea), which was from a nematode of European origin, and strains of X . nematophilus, X . beddingii, and X . poinarii . The tests used in this study indicated identical properties for the NF (North American) and Umea (European) strains of X . bovienii . These could be differentiated from the other strains studied by their failure to grow at 34 degrees C and resistance to low concentrations of a mixture of amoxilline and clavulanic acid . The Xenorhabdus TX strain could be differentiated from the other strains studied by its failure to grow at 10 degrees C . Of the tests done, approximately 30 were useful in distinguishing between the strains and species studied.

Gastroenterology, 2000 Aug, 119(2), 339 - 47
Omeprazole, Helicobacter pylori status, and alterations in the intragastric milieu facilitating bacterial N-nitrosation; Mowat C et al.; BACKGROUND & AIMS: Omeprazole produces greater acid inhibition in Helicobacter pylori-positive than -negative subjects . We investigated whether this is accompanied by more profound changes in the intragastric milieu that facilitates bacterial synthesis of N-nitroso compounds . METHODS: Gastric juice pH; nitrite, ascorbic acid, and total vitamin C concentrations; and colonization by other bacteria were examined before and during omeprazole treatment in subjects with and without H . pylori infection . Studies were performed in the fasting state and after consumption of 2 mmol nitrate (equivalent to a salad meal) . RESULTS: Before omeprazole, H . pylori-positive and -negative subjects were similar for all parameters . During omeprazole, H . pylori-positive subjects had a higher intragastric pH (7.8 vs . 3.0; P < 0.00001) and greater colonization with non-H . pylori species (5 x 10(7) vs . 5 x 10(5) CFU/mL; P < 0.05) . These bacteria included nitrosating species . During omeprazole treatment, H . pylori-positive subjects had higher intragastric nitrite levels after the nitrate meal (median area under the concentration/time curve, 12,450 vs . 4708 micromol/L . min; P = 0.04) . Omeprazole lowered intragastric vitamin C levels in H . pylori-positive but not -negative subjects (1.8 vs . 3.4 microg/mL, respectively; P = 0.02) . CONCLUSIONS: In H . pylori-positive subjects, omeprazole produces disturbances in intragastric nitrite, vitamin C, and bacterial colonization that facilitate bacterial N-nitrosation . This may place them at increased risk of mutagenesis and carcinogenesis.

Am J Perinatol, 2000, 17(1), 41 - 5
Preterm prediction study: is socioeconomic status a risk factor for bacterial vaginosis in Black or in White women?
Meis PJ, Goldenberg RL, Mercer BM, Iams JD, Moawad AH, Miodovnik M, Menard MK, Caritis SN, Thurnau GR, Dombrowski MP, Das A, Roberts JM, McNellis D.
Bacterial vaginosis (BV), an important risk factor for preterm birth, is a more common infection in Black compared with White pregnant women . Because Black women in the United States are more likely to have lower measures of socioeconomic status (SES), this study examined the hypothesis that BV is associated with low SES . The project evaluated data from the Preterm Prediction Study of 2,929 women prospectively followed during their pregnancies . The women, who were screened for BV at 24 and 28 weeks of gestation, underwent a structured interview to evaluate demographic factors, SES, home and work environment, drug or alcohol use, and prior medical history . Black women in the study had many measures of lower SES compared with the White women, and reported less use of tobacco, alcohol and drugs . In neither the Black nor White women was an association found between BV and measures of SES (with the sole exception of "absence of a home telephone") . Most measures of SES do not explain the difference in rates of BV in Black and in White pregnant women.

Z Naturforsch {C}, 2000 May-Jun, 55(5-6), 485 - 8
The in vitro anti-fungal and anti-bacterial activities of beta-sitosterol from Senecio lyratus (Asteraceae); Kiprono PC et al.; From a methanol extract of dried-ground aerial parts of Senecio lyratus, an anti-fungal and anti-bacterial active compound was isolated and identified as beta-sitosterol by spectroscopic analysis.

Rev Esp Enferm Dig, 2000 May, 92(5), 301 - 15
Pathogenic implications of interleukin-8 activity and bacterial phenotype in antral gastritis associated with Helicobacter pylori; Martin Guerrero JM et al.; OBJECTIVE: Helicobacter pylori (Hp) infection is characterized by an intense inflammatory infiltrate in the gastric mucosa, which is chemoattracted by different cytokines . Interleukin-8 (IL-8) seems to play an important role in the recruitment of circulating neutrophils, and modulation of IL-8 secretion seems to be a strain marker . This study was designed to examine IL-8 concentrations in the gastric mucosa and their relationship with H . pylori phenotype and histologic findings . METHODS: Gastric biopsies were obtained from the antrum and corpus in 106 patients (69 Hp-positive and 37 Hp-negative) . IL-8 levels in the gastric mucosa were analyzed by ELISA and Hp phenotype was determined with a western blot test . RESULTS: 75% of H . pylori strains were CagA+ and 54.2% were VacA+ . The Houston classification was used for histologic findings . No association between gastric atrophy or intestinal metaplasia and Hp phenotype was found . The highest IL-8 levels were found in CagA+ infected gastric mucosa, but the difference with respect to infection by a VacA+ strain was not statistically significant . IL-8 levels were highest when neutrophils were the predominant cell in the gastric inflammatory infiltrate (p < 0.05) . IL-8 levels were higher in patients with atrophic gastritis than in patients with nonatrophic gastritis (p < 0.05) . CONCLUSIONS: In patients with H . pylori infection, IL-8 levels are higher than in Hp-negative patients regardless of Hp phenotype . There is an association between IL-8 and a neutrophilic infiltrate . Perpetuation of a chronic infiltrate could lead to more severe lesions such as atrophic gastritis or intestinal metaplasia, as deduced from the IL-8 levels found in these types of lesion.

Exp Clin Endocrinol Diabetes, 2000, 108(3), 220 - 7
Age-related differences in the effects of bacterial endotoxin (LPS) upon the release of LHRH, gonadotropins and hypothalamic inhibitory amino acid neurotransmitters measured in tissues explanted from intact male rats; Feleder C et al.; In adult rats, bacterial endotoxin (lipopolysaccharide or LPS) is known to diminish the activity of the reproductive axis, mainly by inhibiting luteinizing hormone-releasing hormone (LHRH) secretion; until now, this effect has not been studied in immature rats . The aim of the present study was to evaluate the effect of LPS 1) on LHRH output (and associated changes in the release of inhibitory amino acid neurotransmitters such as gamma-aminobutyric acid (GABA) and taurine) by superfused hypothalamic fragments, and 2) on gonadotropin secretion by incubated hemipituitaries, obtained from young adult (60-day-old) and peripubertal (30-day-old) intact male rats . In adult animals, LPS induced a significant inhibition (50% of basal values) of LHRH release, accompanied by an increase in GABA and taurine output . In juvenile rats the inhibition of LHRH secretion by LPS attained 90% of basal values (p<0.0001 versus adult rats), and the concurrent increase in GABA release was significantly greater (p<0.0001 versus adult rats) . LPS did not affect in vitro gonadotropin secretion in adult animals . Conversely, the release of these hormones was significantly (p<0.001 and <0.02 for LH and FSH, respectively) reduced in 30-day-old rats . Our results demonstrate the existence of age-related differences in the effect of LPS on LHRH and gonadotropin secretion . These differences might well be attributed to an increased activity of the hypothalamic GABAergic system . Furthermore, the participation of other factors known to play a role in immune-neuroendocrine relationships (e.g., corticotropin-releasing hormone, testosterone) is discussed.

J Immunol, 2000 Aug 15, 165(4), 2173 - 82
Colitis induced by enteric bacterial antigen-specific CD4+ T cells requires CD40-CD40 ligand interactions for a sustained increase in mucosal IL-12; Cong Y et al.; C3H/HeJBir is a mouse substrain that is highly susceptible to colitis . Their CD4+ T cells react to Ags of the commensal enteric bacteria, and the latter can mediate colitis when activated by these Ags and transferred to histocompatible scid recipients . In this study, multiple long-term C3H/HeJBir CD4+ T cell (Bir) lines reactive to commensal enteric bacterial Ags have been generated . All these were Ag specific, pauciclonal, and Th1 predominant; most induced colitis uniformly after transfer to scid recipients . Lesions were focal and marked by increased expression of IL-12p40 and IFN-gamma mRNA and protein . Pathogenic Bir T cell lines expressed CD40 ligand (CD40L) when cultured with Ag-pulsed APCs in vitro . Production of IL-12 was also increased in such cultures, an effect that was Ag- and T cell-dependent and required costimulation by CD40, but not by B7 . The two Bir T cell lines that did not induce lesions after transfer failed to significantly express CD40L or increase IL-12 when cultured with Ag-pulsed APCs . Administration of anti-CD40L blocked disease expression induced by pathogenic T cells . We conclude that interactions in the colon mucosa between CD40L-expressing Bir Th1 cells with APCs endogenously loaded with commensal bacterial Ags are critical for sustained increases in local IL-12 production and progression to colitis.

Transfus Sci, 2000 Aug, 23(1), 21 - 7
Detection of nucleic acid sequences from bacterial species with molecular genetic methods; Petershofen EK et al.; While blood products become more safe in terms of viral contamination, the risk of transfusion-related bacterial infection has re-emerged to one of the major hazards in transfusion medicine . In recent prospective studies the rate of contaminated platelets ranged from 0.04 to 0.5%, and a rate of transfusion reactions between 0.007% and 0.046%.It is generally agreed that most of the organisms isolated from donated blood originate from the normal skin flora or from the environment . As it is unlikely that antiseptic methods can achieve absolute sterilization of the skin before venepuncture, blood banks have to rely on laboratory tests to detect contaminated blood products before release . But most of the currently available methods detecting bacterial contaminations do not have the potential to be sensitive and fast enough for a routine contamination screening in transfusion services.Here we present two alternative strategies based on molecular genetic techniques (Real-Time-PCR and Haystack processing) that detect or semi-quantify bacterial rRNA gene sequences for the majority of bacterial species . In addition we discuss some aspects on target selection, routine preparation and residual 16S-rDNA-contamination of enzymes.

Biochim Biophys Acta, 2000 Jul 20, 1459(1), 69 - 76
The natural product capsaicin inhibits photosynthetic electron transport at the reducing side of photosystem II and purple bacterial reaction center: structural details of capsaicin binding; Spyridaki A et al.; Capsaicin, a natural quinone analog, was found to block electron transport, in both plant photosystem II (PSII) and bacterial reaction center (RC) from Rhodobacter sphaeroides, at the QB site . The mode of action of capsaicin was investigated by O2 evolution measurements and fluoresence induction studies in the case of PSII, and flash-induced absorbance spectroscopy in the case of the bacterial RC . Structural details of capsaicin binding to the bacterial RC complex were determined by X-ray crystallographic analysis.

Genetics, 2000 Aug, 155(4), 1505 - 19
Natural selection, infectious transfer and the existence conditions for bacterial plasmids; Bergstrom CT et al.; Despite the near-ubiquity of plasmids in bacterial populations and the profound contribution of infectious gene transfer to the adaptation and evolution of bacteria, the mechanisms responsible for the maintenance of plasmids in bacterial populations are poorly understood . In this article, we address the question of how plasmids manage to persist over evolutionary time . Empirical studies suggest that plasmids are not infectiously transmitted at a rate high enough to be maintained as genetic parasites . In part i, we present a general mathematical proof that if this is the case, then plasmids will not be able to persist indefinitely solely by carrying genes that are beneficial or sometimes beneficial to their host bacteria . Instead, such genes should, in the long run, be incorporated into the bacterial chromosome . If the mobility of host-adaptive genes imposes a cost, that mobility will eventually be lost . In part ii, we illustrate a pair of mechanisms by which plasmids can be maintained indefinitely even when their rates of transmission are too low for them to be genetic parasites . First, plasmids may persist because they can transfer locally adapted genes to newly arriving strains bearing evolutionary innovations, and thereby preserve the local adaptations in the face of background selective sweeps . Second, plasmids may persist because of their ability to shuttle intermittently favored genes back and forth between various (noncompeting) bacterial strains, ecotypes, or even species.

Transfus Clin Biol, 2000 Jun, 7 Suppl 1, 55s - 62s
{Blood transfusion and bacterial risk}; Morel P et al.; Initial hemovigilance data confirm the incidence and severity of transfusion reactions due to the bacterial contamination of blood components (TRBC) . With 18 deaths reported through the French hemovigilance network over the past 5 years, bacterial risks represent one of the major immediate complications of blood components (BC) transfusion . BC contamination may lead to more or less severe TRBC, depending on their origin: bacteria growth, the BC itself or unknown origin . Although the rate of donated blood or BC contamination is known (0.5% and 0.05%, respectively) it is still difficult to assess the actual incidence of TRBC, as it is difficult to identify them and relate them to transfusion . Likewise, better knowledge of bacteria, symptoms and outcome is required to improve prevention methods . Better prevention can reduce BC contamination and proliferation of bacteria at each stage of blood transfusion . Methods to detect BC contamination are still under investigation . Through continuous education of hemovigilance actors in identifying and dealing with TRBC, as well as drawing up procedures to perform inquiries and specific bacterial analyses, case reporting can be further improved in order to achieve more efficient prevention.

Environ Mol Mutagen, 2000, 36(1), 72 - 7
Comparison of the results of a modified miniscreen and the standard bacterial reverse mutation assays; Diehl MS et al.; The bacterial reverse mutation assay (Ames test) provides a rapid assessment of the mutagenic potential of chemicals . The assay is widely used in the pharmaceutical industry for early assessment during candidate compound selection and for regulatory drug submissions . Early in development, many candidate compounds are available in only very small quantities . The use of the standard plate incorporation bacterial reverse mutation assay for screening, using only a single petri plate per concentration, requires the use of approximately 140 mg of test compound to test up to a stock concentration of 100 mg/ml (5000 microg/plate) in five strains of bacteria . A modification of the existing Ames Miniscreen assay has been developed using six-well cell-culture dishes that requires only 21 mg of compound to test a stock concentration of up to 100 mg/ml (2000 microg/well) in three strains of bacteria . The standard plate incorporation assay and the modified Miniscreen assays conducted on proprietary compounds without and with metabolic activation have yielded a high degree of concordance in findings .

Pediatr Infect Dis J, 2000 Jul, 19(7), 598 - 602
Serum procalcitonin, C-reactive protein and interleukin-6 for distinguishing bacterial and viral pneumonia in children; Toikka P et al.; OBJECTIVE: Serum procalcitonin (PCT), C-reactive protein (CRP) and interleukin-6 (IL-6) concentrations were measured in 126 children hospitalized for community-acquired, radiologically confirmed pneumonia to assess whether these host response values could be used to distinguish bacterial from viral pneumonia . METHODS: The samples for PCT, CRP and IL-6 measurements were obtained on admission or the first day of hospitalization . The etiology of pneumonia was studied with an extensive panel of methods that detected 6 bacteria and 11 viruses . RESULTS: In all, 54% had evidence of bacterial pneumonia, and 32% had evidence of sole viral pneumonia . In 14% of the cases the etiology could not be determined . Children with bacterial pneumonia had significantly higher PCT (median 2.09 ng/ml vs . 0.56 ng/ml, P = 0.019) and CRP concentrations (96 mg/l vs . 54 mg/l, P = 0.008) than those with sole viral etiology . However, the values markedly overlapped . No significant difference in IL-6 concentrations was seen between the two patient groups . Using PCT > or = 2.0 ng/ml, CRP > or = 150 mg/l or IL-6 > or = 40 pg/ml, the specificity was > or =80% for bacterial pneumonia . The sensitivities with these cutoff values were 50% for PCT, 31% for CRP and 34% for IL-6 . CONCLUSIONS: The results indicate that the measurement of serum PCT, CRP and IL-6 has little value in the differentiation of bacterial and viral pneumonia in children . However, in some patients with very high serum PCT, CRP or IL-6 values, bacterial pneumonia is probable.

Am J Physiol Heart Circ Physiol, 2000 Aug, 279(2), H619 - 29
CD14-independent activation of cardiomyocyte signal transduction by bacterial endotoxin; Cowan DB et al.; In the heart, lipopolysaccharide (LPS) induces the production of proinflammatory cytokines that cause myocardial dysfunction; however, the signaling pathways involved in cardiomyocyte responses are poorly understood . We studied LPS-induced signaling by treating cardiomyocyte cultures with 0.01-10 microgram/ml LPS for 0-24 h in the presence or absence of 2.5% serum . Cytosolic and nuclear proteins were analyzed for expression and activation of protein kinases . Members of the extracellular signal-regulated kinase (ERK) and signal transducer and activators of transcription (STAT) protein families were uniformly expressed and specifically phosphorylated in response to LPS . Activation was biphasic; peaking at 5-10 min and 24 h after treatment . Inhibitor experiments provided evidence that ERK proteins may regulate STAT activity . Serum did not augment endotoxin-induced phosphorylation . Although cardiomyocytes expressed low levels of CD14 and LPS-binding protein, specific enzymatic removal of glycosyl phosphatidylinositol-linked receptors or incubation with an anti-CD14 antibody had no effect on kinase activation . Treatment of cells with an excess of detoxified LPS attenuated endotoxin-induced signaling . In addition, endotoxin stimulated specific binding of nuclear factors to AP-1, nuclear factor-kappaB (NF-kappaB), STAT1 (SIE, sis-inducible element), and STAT3 consensus-binding sequences . Finally, inhibition of ERK phosphorylation reduced, and NF-kappaB nuclear translocation prevented, tumor necrosis factor-alpha production . Our results indicate that LPS-induced activation of signal transduction in cardiomyocytes occurs by a CD14-independent mechanism.

Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1302 - 4
Effects of bacterial glyceroglycolipid M874B on growth and TPA-induced differentiation of HL60 cells; Matsufuji M et al.; Bacterial monogalactosyldiacylglycerol M874B (MGDAG), which protects against oxygen radicals, was found to increase the growth of the human promyelocytic leukemia cell HL60 when added to the cell culture, but suppresses the 12-O-tetradecanoyl phorbol-13-acetate-induced differentiation . Analogous MGDAG, S365B had weak, but similar effects . These activities were not observed with analogous plant glyceroglycolipids and diacylglycerol.

J Clin Microbiol, 2000 Aug, 38(8), 3100 - 2
Lactoferrin and eosinophilic cationic protein in nasal secretions of patients with experimental rhinovirus colds, natural colds, and presumed acute community-acquired bacterial sinusitis; Niehaus MD et al.; To distinguish sinusitis from uncomplicated "colds," we examined lactoferrin and eosinophilic cationic protein (ECP) in nasal secretions . Lactoferrin titers were >/=1:400 in 4% of persons with uncomplicated colds and controls but in 79% of persons with sinusitis or purulent sputa . ECP levels were >200 ng/ml in 61% of persons with colds and >3,000 ng/ml in 62% of persons with sinusitis . Nasal lactoferrin helps distinguish sinusitis from colds.

J Clin Microbiol, 2000 Aug, 38(8), 3096 - 7
Use of 5-bromo-4-chloro-3-indolyl-alpha-D-N-acetylneuraminic acid in a novel spot test To identify sialidase activity in vaginal swabs from women with bacterial vaginosis; Wiggins R et al.; The validity of measuring vaginal sialidase activity to identify bacterial vaginosis (BV) was determined by using 5-bromo-4-chloro-3-indolyl-alpha-D-N-acetylneuraminic acid in a near-patient test . The sensitivity and specificity of the test for prediction of BV were 95.6 and 96.3%, respectively . Positive and negative predictive values were 95.6 and 96.3%, respectively . This test may be an alternative to Gram staining.

Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9701 - 5
Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington's disease; Carmichael J et al.; Huntington's disease (HD) is an autosomal dominant neurodegenerative condition caused by expansions of more than 35 uninterrupted CAG repeats in exon 1 of the huntingtin gene . The CAG repeats in HD and the other seven known diseases caused by CAG codon expansions are translated into long polyglutamine tracts that confer a deleterious gain of function on the mutant proteins . Intraneuronal inclusions comprising aggregates of the relevant mutant proteins are found in the brains of patients with HD and related diseases . It is crucial to determine whether the formation of inclusions is directly pathogenic, because a number of studies have suggested that aggregates may be epiphenomena or even protective . Here, we show that fragments of the bacterial chaperone GroEL and the full-length yeast heat shock protein Hsp104 reduce both aggregate formation and cell death in mammalian cell models of HD, consistent with a causal link between aggregation and pathology.

Am J Respir Cell Mol Biol, 2000 Aug, 23(2), 146 - 53
Production of the acute-phase protein lipopolysaccharide-binding protein by respiratory type II epithelial cells: implications for local defense to bacterial endotoxins; Dentener MA et al.; This study demonstrates for the first time that respiratory epithelial cells are able to produce the acute phase protein lipopolysaccharide (LPS)-binding protein (LBP), which is known to play a central role in the defense to bacterial endotoxins (or LPS) . Indications for local presence of LBP in human lung was obtained via reverse transcriptase/polymerase chain reaction that showed LBP messenger RNA (mRNA) expression . Therefore, LBP production by the human lung epithelial cell line A549, a human adenocarcinoma with features of type II pneumocytes, was studied . These cells produced LBP in response to interleukin (IL)-1beta, IL-6, and tumor necrosis factor- alpha, a response that was strongly enhanced by dexamethasone . In addition, LBP mRNA was detected in A549 cells, in increasing amounts as a result of stimulation . The pattern of cytokine-induced LBP production in A549 cells was similar to the pattern in the human liver epithelial cell line HuH-7 . Moreover, the molecular weight of A549-derived LBP was approximately 60 kD, which is similar to HuH-7-derived LBP . Biologic activity of LBP produced by A549 cells was evaluated on the basis of its ability to interact with LPS . Further indications that type II alveolar epithelial cells are able to produce LBP were obtained from the observations that the murine lung type II epithelial cell line C10 produced murine LBP, and that isolated human primary type II pneumocytes expressed LBP mRNA, which was enhanced after stimulation of cells . The local production of this endotoxin binding protein by lung epithelial cells might contribute to a highly specific response at the site of exposure to bacteria and bacterial endotoxins.

Hum Mol Genet, 2000 Jul 22, 9(12), 1853 - 64
Mice trisomic for a bacterial artificial chromosome with the single-minded 2 gene (Sim2) show phenotypes similar to some of those present in the partial trisomy 16 mouse models of Down syndrome; Chrast R et al.; The Drosophila single-minded (sim) transcription factor, is a master regulator of fruitfly neurogenesis . Recently, we have cloned and mapped a human homolog of sim, SIM2, to chromosome 21 in the so-called 'Down syndrome chromosomal region' . Three copies of SIM2 may contribute to some Down syndrome (DS) phenotypes because of the mapping position function as transcriptional repressor, temporal and spatial expression pattern of mouse Sim2, and the potentially analogous role of human SIM2 to that of Drosophila sim during neurogenesis . In order to validate this hypothesis in vivo, we have created the first bacterial artificial chromosome transgenic mice overexpressing a gene possibly involved in DS with only one or two additional copies of mouse Sim2 . The transgene was shown to be expressed in the same spatial pattern as the endogenous gene . The mice develop normally, are fertile and do not show detectable histopathological abnormalities . However, detailed analysis of their behavior revealed anxiety-related/reduced exploratory behaviour and sensitivity to pain, phenotypes similar to those also present in other partial trisomy 16 mouse models of DS . Our data therefore suggest that overexpression of SIM2 contributes to some of the complex DS phenotypes.

Colloids Surf B Biointerfaces, 2000 Oct 1, 18(3-4), 355 - 370
Platelet and bacterial repellence on sulfonated poly(ethylene glycol)-acrylate copolymer surfaces; Lee HJ et al.; Novel poly(ethylene glycol) (PEG) and sulfonated PEG (PEG-SO(3)) acrylate copolymers have been prepared and characterized to apply as coating and blending materials for biomedical applications . The modified surfaces using acrylate copolymers demonstrated increased hydrophilicity, possibly due to the hypothesized reorientation of PEG/PEG-SO(3) chains into water phase . All copolymer surfaces demonstrated less platelet adhesion than control . In addition, platelet adhesion on copolymer surfaces decreased as the chain length of PEG and sulfonated PEG in copolymers increases . All copolymer surfaces reduced bacterial adhesion significantly and the adhesion level differs depending on surfaces as well as media . The obtained results attest to the usefulness of these copolymers as a coating or additive material to improve the blood compatibility of blood contacting devices.

J Infect Dis, 2000 Aug, 182(2), 467 - 73 Epub 2000 Jul 18.
High levels of tumor necrosis factor-alpha and interleukin-1beta in bacterial vaginosis may increase susceptibility to human immunodeficiency virus; Sturm-Ramirez K et al.; Bacterial vaginosis (BV) was identified recently as a cofactor that promotes sexual transmission of human immunodeficiency virus (HIV) . This study was done to determine if interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha could be measured consistently in cervical secretions and if high levels of these cytokines were associated with BV . Secretions were obtained from 209 study subjects; most samples had detectable levels of TNF-alpha (84.2%) and IL-1beta (79.8%) . BV was detected in 53 (27.0%) of 196 women . High cytokine levels were significantly associated with BV (adjusted odds ratio {AOR}, 4.17; 95% confidence interval {CI}, 1.69-10.30), oral contraceptive use (AOR, 2.78; 95% CI, 1.04-7.48), and high leukocyte counts on vaginal smear (AOR, 1.18; 95% CI, 1.03-1.36) . Since these cytokines could up-regulate local HIV replication through activation of the long terminal repeat promoter region, the association of BV with high levels of IL-1beta or TNF-alpha may partly explain the mechanism by which this risk factor enhances HIV transmission.

Clin Infect Dis, 2000 Jul, 31(1), 80 - 4 Epub 2000 Jul 14.
Matrix metalloproteinase (MMP)-8 and MMP-9 in cerebrospinal fluid during bacterial meningitis: association with blood-brain barrier damage and neurological sequelae; Leppert D et al.; To evaluate the spectrum and regulation of matrix metalloproteinases (MMPs) in bacterial meningitis (BM), concentrations of MMP-2, MMP-3, MMP-8, and MMP-9 and endogenous inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were measured in the cerebrospinal fluid (CSF) of 27 children with BM . MMP-8 and MMP-9 were detected in 91% and 97%, respectively, of CSF specimens from patients but were not detected in control patients . CSF levels of MMP-9 were higher (P<.05) in 5 patients who developed hearing impairment or secondary epilepsy than in those who recovered without neurological deficits . Levels of MMP-9 correlated with concentrations of TIMP-1 (P<.001) and tumor necrosis factor-alpha (P=.03) . Repeated lumbar punctures showed that levels of MMP-8 and MMP-9 were regulated independently and did not correlate with the CSF cell count . Therefore, MMPs may derive not only from granulocytes infiltrating the CSF space but also from parenchymal cells of the meninges and brain . High concentrations of MMP-9 are a risk factor for the development of postmeningitidal neurological sequelae.

Biochemistry, 2000 Jul 25, 39(29), 8353 - 61
Electrochromic detection of a coherent component in the formation of the charge pair P(+)H(L)(-) in bacterial reaction centers; Vos MH et al.; We demonstrate coupling of an intraprotein electron transfer reaction to coherent vibrational motions . The kinetics of charge separation toward the radical pair state P(+)H(L)(-) were studied in reaction centers of Rhodobacter sphaeroides at 15 K . The electrochromic shift of the bacteriochlorophyll monomers is the most prominent spectral feature associated with this charge displacement . The newly reported absolute absorption spectrum of the P(+)H(L)(-) state is discussed in terms of this shift . In wild-type reaction centers, the rise kinetics of the electrochromic shift display a small but significant 30 cm(-)(1) periodic modulation (period of approximately 1 ps) . This modulation is also present in FL181Y mutant reaction centers, where overall charge separation is somewhat more rapid than in the wild-type reaction center . In contrast, in YM210L mutant reaction centers, where the charge separation is much slower, the modulation is absent . The conclusion that the motion along the reaction coordinate has a 30 cm(-)(1) coherent component is discussed in light of possible mechanisms of electron transfer.

Mol Cell, 2000 Jun, 5(6), 1003 - 11
FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis; Gomez-Gomez L et al.; Flagellin, the main protein of the bacterial flagella, elicits defence responses and alters growth in Arabidopsis seedlings . Previously, we identified the FLS1 locus, which confers flagellin insensitivity in Ws-0 . To identify additional components involved in flagellin perception, we screened for flagellin insensitivity mutants in the flagellin-sensitive accession La-er . Here, we describe the identification of a new locus, FLS2, by a map-based strategy . The FLS2 gene is ubiquitously expressed and encodes a putative receptor kinase . FLS2 shares structural and functional homologies with known plant resistance genes and with components involved in the innate immune system of mammals and insects.

Protein Expr Purif, 2000 Aug, 19(3), 403 - 10
Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale; Schnurr K et al.; 15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function . Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon . Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply . There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far . To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli . The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein) . Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme . Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen . The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials .

Shock, 2000 Jul, 14(1), 53 - 9
Interruption of hepatic gap junctional communication in the rat during inflammation induced by bacterial lipopolysaccharide; De Maio A et al.; Gap junctional cellular communication is important in the propagation of signals that coordinate hepatic metabolism . Hepatocytes express two different connexin (Cx) genes, Cx32 and Cx26, which encode for the subunit component of gap junction channels . Previous studies have shown that the expression of hepatic Cx32 is reduced during inflammatory conditions . The objective of this study was to evaluate whether this decrease in Cx32 expression results in a decrease in hepatic gap junctional communication . Transfer of the dye Lucifer Yellow between hepatocytes was measured after microinjection of single cells in an isolated perfused liver . Livers were harvested from rats subjected to an inflammatory condition induced by administration of bacterial lipopolysaccharide (LPS) . A decrease in gap junctional cellular communication was observed within 6 h of the LPS treatment . This decrease in dye coupling was reversible, because gap junctional communication returned to control levels within 48 h of the LPS injection . The inhibition of hepatic gap junctional communication was associated with the disappearance of Cx32 and Cx26 from the hepatocyte plasma membrane as detected by indirect immunostaining . Cx32 mRNA levels were also reduced during inflammation as previously reported . However, Cx26 mRNA levels were unaffected or even transiently increased after the injection of LPS without significant increase in the polypeptide level . Thus, the down-regulation of Cx32 and Cx26 from the hepatocyte surface is apparently due to a rapid degradation of the polypeptide from the cell surface . We hypothesize that this loss of gap junctional cellular communication within the liver may contribute to the disordered hepatic metabolic that occurs during inflammatory states.

Schweiz Med Wochenschr, 2000 Jun 17, 130(24), 928 - 35
{In search of strategies for preventing brain damage as a sequela of bacterial meningitis}; Leib SL et al.; Multiplication of bacteria within the central nervous system compartment triggers a host response with an overshooting inflammatory reaction which leads to brain parenchyma damage . Some of the inflammatory and neurotoxic mediators involved in the processes leading to neuronal injury during bacterial meningitis have been identified in recent years . As a result, the therapeutic approach to the disease has widened from eradication of the bacterial pathogen with antibiotics to attenuation of the detrimental effects of host defences . Corticosteroids represent an example of the adjuvant therapeutic strategies aimed at downmodulating excessive inflammation in the infected central nervous system . Pathophysiological concepts derived from an experimental rat model of bacterial meningitis revealed possible therapeutic strategies for prevention of brain damage . The insights gained led to the evaluation of new therapeutic modalities such as anticytokine agents, matrix metalloproteinase inhibitors, antioxidants, and antagonists of endothelin and glutamate . Bacterial meningitis is still associated with persistent neurological sequelae in approximately one third of surviving patients . Future research in the model will evaluate whether the neuroprotective agents identified so far have the potential to attenuate learning disabilities as a long-term consequence of bacterial meningitis.

Obstet Gynecol, 2000 Aug, 96(2), 256 - 60
Vaginal clindamycin and oral metronidazole for bacterial vaginosis: a randomized trial; Paavonen J et al.; OBJECTIVE: To compare the efficacy and safety of clindamycin vaginal ovules with oral metronidazole for treatment of bacterial vaginosis . METHODS: Women with bacterial vaginosis received either 100-mg ovules of clindamycin (intravaginally for 3 consecutive days) plus placebo capsules (orally twice daily for 7 days) or metronidazole 500 mg (two 250-mg capsules orally twice daily for 7 days) plus placebo ovules (intravaginally for 3 consecutive days) . The sample was determined prospectively to provide a probability of.84 of correctly concluding that the rate of success for clindamycin is not more than 15% less than the expected 75% success rate for metronidazole . Clinical outcome was determined on the basis of vaginal fluid amine odor and clue cells . RESULTS: Of the 399 patients enrolled, 233 could be evaluated for efficacy . Of those, 77 (68.1%) of 113 patients were cured with clindamycin, compared with 80 (66 . 7%) of 120 who were cured with metronidazole (95% confidence interval -10.6%, 13.4%; P =.810) . Treatment-related adverse events were reported more frequently in the metronidazole treatment group . Systemic symptoms, such as nausea and taste perversion, accounted for most of the difference between groups . CONCLUSION: A 3-day regimen of clindamycin, given as intravaginal ovules, was as effective as and better tolerated than a 7-day regimen of oral metronidazole 500 mg, given twice daily, for treatment of bacterial vaginosis.

J Fam Pract, 1999 Nov, 48(11), 885 - 92
Bacterial vaginosis in pregnancy and the risk of prematurity: a meta-analysis; Flynn CA et al.; OBJECTIVE: We conducted this meta-analysis to determine the magnitude of risk conferred by bacterial vaginosis during pregnancy on preterm delivery . SEARCH STRATEGY: We selected articles from a combination of the results of a MEDLINE search (1966-1996), a manual search of bibliographies, and contact with leading researchers . SELECTION CRITERIA: We included case control and cohort studies evaluating the risk of preterm delivery, low birth weight, preterm premature rupture of membranes, or preterm labor for pregnant women who had bacterial vaginosis and those who did not . DATA COLLECTION AND ANALYSIS . Two investigators independently conducted literature searches, applied inclusion criteria, performed data extraction, and critically appraised included studies . Summary estimates of risk were calculated as odds ratios (ORs) using the fixed and random effects models . MAIN RESULTS: We included 19 studies in the final analysis . Bacterial vaginosis during pregnancy was associated with a statistically significant increased risk for all outcomes evaluated . In the subanalyses for preterm delivery, bacterial vaginosis remained a significant risk factor . Pooling adjusted ORs yielded a 60% increased risk of preterm delivery given the presence of bacterial vaginosis . CONCLUSIONS: Bacterial vaginosis is an important risk factor for prematurity and pregnancy morbidity . Further studies will help clarify the benefits of treating bacterial vaginosis and the potential role of screening during pregnancy.

Arch Dis Child, 2000 Aug, 83(2), 111 - 6
Twelve year outcomes following bacterial meningitis: further evidence for persisting effects; Grimwood K et al.; AIM: To determine whether intellectual and cognitive impairments observed seven years following early childhood bacterial meningitis persist into adolescence . METHODS: Blinded neuropsychological, auditory, and behaviour assessments were conducted in 109 (69%) subjects from an original cohort of 158 children, seven and 12 years after their meningitis, and in 96 controls . RESULTS: Meningitis subjects remained at greater risk than controls for any disability (odds ratio OR 4.7, confidence interval 2.2 to 9.6) . Those with acute neurological complications had more sequelae than children with uncomplicated meningitis or controls (47% v 30% v 11.5% respectively; p < 0.001) . Differences in intellectual, academic, and high level cognitive function between subjects and controls were maintained at the seven and 12 year assessments . In contrast, lower order skills improved, while behaviour scores deteriorated significantly (p = 0.033) . CONCLUSIONS: Many of the deficits identified at the seven year follow up persist 12 years after an episode of bacterial meningitis.

Comp Biochem Physiol B Biochem Mol Biol, 2000 Apr, 125(4), 563 - 9
Expression and characterization of human tyrosinase from a bacterial expression system; Kong KH et al.; To carry out biochemical characterizations of human tyrosinase and to provide an unlimited source of the enzyme for further study, an expression plasmid, pHis-Tyrosinase, which contains the entire coding sequence except the signal sequence of a human tyrosinase was constructed and expressed in Escherichia coli . The expressed enzyme was simply purified by an immobilized metal affinity chromatography . The recombinant enzyme had the same electrophoretic mobility as the native enzyme from human melanoma cell and cross-reacted with the polyclonal antibody raised against the native enzyme . The recombinant enzyme retained its catalytic function with both hydroxylating and oxidative activities . Km values for L-tyrosine and L-3,4-dihydroxy-phenylalanine of the recombinant enzyme were 0.17 and 0.36 mM, respectively . The activity of the recombinant enzyme was optimal at pH 7.5 . Glutathione notably inhibited the enzymatic activity . This work is a further enzymatic characterization of human tyrosinase.

Herz, 2000 May, 25(3), 233 - 9
Detection of viral and bacterial protein in endomyocardial biopsies of patients with inflammatory heart muscle disease?
Davydova J, Pankuweit S, Crombach M, Eckhardt H, Strache D, Faulhammer P, Maisch B.
The development of highly sensitive molecular biological methods such as in-situ hybridization and polymerase chain reaction (PCR) made it possible to detect viral/bacterial nucleic acid in human endomyocardial biopsies . However, only a few investigations addressed the problem of latent persistence of viral and bacterial genome and the detection of the corresponding proteins, which could have important consequences for the clinical course of the disease . The purpose of this study was to determine whether protein of various viruses (adenovirus, enterovirus, cytomegalovirus, influenza A and B virus, herpes simplex virus 1 and 2) and bacteria (chlamydia pneumonia) can be detected in endomyocardial biopsies of patients with myocarditis and dilated cardiomyopathy with and without inflammation by use of an immunofluorescence assay and to compare the frequency of its detection with the results of PCR, immunohistology and serology . Thirty-nine patients with myocarditis and dilated cardiomyopathy with and without inflammation were examined by a direct immunofluorescence assay using the endomyocardial biopsy as antigen . Each of the samples was additionally studied by immunohistological methods and PCR for the detection of infiltrating cells and the genome of cardiotropic viruses or bacteria . Fourteen of patients were considered to have myocarditis (group 1), 9 dilated cardiomyopathy with inflammation (group 2), 10 dilated cardiomyopathy (group 3), 6 to have no myocarditis or dilated cardiomyopathy (group 4) . Using a direct immunofluorescence assay we could show only that 1 patient without histological myocarditis or dilated cardiomyopathy (group 4) was positive for influenza B and chlamydia pneumonia antigens in the endomyocardial biopsy . In addition we have determined influenza B-specific antibodies, such as IgG (marginal titer) and IgA (high titer) and chlamydia pneumonia-specific antibodies, such as IgG (marginal titer) in serum of this patient . A second patient with dilated cardiomyopathy was found to be positive for protein of chlamydia pneumonia, who was shown to have chlamydia pneumonia-specific antibodies, such as IgG (high titer) in serum . There was no correlation with PCR results, but good correlation with influenza B and chlamydia pneumonia-specific antibodies in sera of these patients . In this investigation we have determined viral/bacterial-specific antibodies using serological methods and proteins of these agents using immunoflourescence . Despite the detection of virus or bacteria-specific antibodies in the sera and detection of viral and/or bacterial protein in the biopsies of some of the patients viral and/or bacterial genome was not found in the biopsy . This may be explained by the focal character of myocarditis and sampling error, because for technical reasons we use different biopsies for immunohistochemical and molecular biological investigations.

J Immunol, 2000 Aug 1, 165(3), 1171 - 4
Cutting edge: cell autonomous rather than environmental factors control bacterial superantigen-induced T cell anergy in vivo; Attinger A et al.; Anergic T cells display a marked decrease in their ability to produce IL-2 and to proliferate in the presence of an appropriate antigenic signal . Two nonmutually exclusive classes of models have been proposed to explain the persistence of T cell anergy in vivo . While some reports indicate that anergic T cells have intrinsic defects in signaling pathways or transcriptional activities, other studies suggest that anergy is maintained by environmental "suppressor" factors such as cytokines or Abs . To distinguish between these conflicting hypotheses, we employed the well-characterized bacterial superantigen model system to evaluate in vivo the ability of a trace population of adoptively transferred naive or anergized T cells to proliferate in a naive vs anergic environment upon subsequent challenge . Our data clearly demonstrate that bacterial superantigen-induced T cell anergy is cell autonomous and independent of environmental factors.

Am J Respir Crit Care Med, 2000 Jul, 162(1), 64 - 7
Effect of antiretroviral therapy on the incidence of bacterial pneumonia in patients with advanced HIV infection; Sullivan JH et al.; To determine the relationship of combination antiretroviral therapy and bacterial pneumonia, we assessed incidence of and risk factors for bacterial pneumonia in 1,898 human immunodeficiency virus (HIV)-infected patients with CD4 cell counts < 200/mm(3) followed in the Johns Hopkins HIV clinic between 1993 and 1998 . A total of 352 episodes of bacterial pneumonia occurred during 2,310 patient-years of follow-up . Incidence of bacterial pneumonia decreased from 22.7 episodes/100 person-years (py) in the first half of 1993 to 12.3 episodes/100 py in the first half of 1996, reaching a nadir of 9.1 episodes/100 py in the second half of 1997 (p < 0.05) . The use of protease inhibitor-containing regimens was associated with a decreased risk of bacterial pneumonia (risk ratio {RR} 0.55, 95% CI 0.31 to 0.94) . Lower CD4 cell counts (RR 2.22, 95% CI 1.54 to 3.18), injection drug use as HIV transmission category (RR2.0, 95% CI 1.43 to 2.76), and prior Pneumocystis carinii pneumonia (RR 3.88, 95% CI 1.65 to 9.16) were also significantly associated with bacterial pneumonia . Trimethoprim-sulfamethoxazole and macrolide use were not significantly associated with risk of bacterial pneumonia . There has been a dramatic decline in the incidence of bacterial pneumonia resulting from the use of combination antiretroviral therapy containing protease inhibitors.

Infect Immun, 2000 Aug, 68(8), 4752 - 8
Interleukin-10 gene therapy-mediated amelioration of bacterial pneumonia; Morrison DF et al.; Respiratory infection by Actinobacillus pleuropneumoniae causes a highly pathogenic necrotizing pleuropneumonia with severe edema, hemorrhage and fever . Acute infection is characterized by expression of inflammatory cytokines, including interleukin-1 (IL-1), IL-6 and IL-8 . To determine if high level production of inflammatory cytokines contributed to disease pathogenesis, we investigated if inhibiting macrophage activation with adenovirus type 5-expressed IL-10 (Ad-5/IL-10) reduced the severity of acute disease . Porcine tracheal epithelial cells infected with Ad-5/IL-10 produced bioactive human IL-10 . When pigs were intratracheally infected with A . pleuropneumoniae, pigs pretreated with Ad-5/IL-10 showed a significant reduction in the amount of lung damage when compared to adenovirus type 5-expressing beta-galactosidase (Ad-5/beta-Gal)-treated and untreated pigs . In addition, serum zinc levels were unchanged, the lung weight/body weight ratio (an indicator of vascular leakage) was significantly reduced, and lung pathology scores were reduced . Myeloperoxidase activity in lung lavage fluid samples, an indicator of neutrophil invasion, was decreased to levels similar to that seen in pigs not infected with A . pleuropneumoniae . Reduction in inflammatory cytokine levels in lung lavage fluid samples correlated with the clinical observations in that pigs pretreated with Ad-5/IL-10 showed a corresponding reduction of IL-1 and tumor necrosis factor (TNF) compared with untreated and Ad-5/beta-Gal-treated pigs . IL-6 levels were unaffected by pretreatment with Ad-5/IL-10, consistent with observations that IL-6 was not derived from alveolar macrophages . Since inflammatory cytokines are expressed at high levels in acute bacterial pleuropneumonia, these results indicate that macrophage activation, involving overproduction of IL-1 and TNF, is a prime factor in infection-related cases of massive lung injury.

J Biol Chem, 2000 Oct 13, 275(41), 32098 - 105
A CDC6 protein-binding peptide selected using a bacterial two-hybrid-like system is a cell cycle inhibitor; Zhu W et al.; Peptides or small molecules able to modulate protein-protein interactions hold promise as tools with which to probe and manipulate biological pathways . An important issue in this nascent field is to evaluate different methods with which to search libraries for molecules that modulate the function of specific target proteins . One strategy is to screen libraries for molecules that bind specifically to a protein known to be critical in the pathway of interest, with the expectation that the molecules isolated will recognize regions of the target protein important for its function and thereby exhibit biological activity . Here, a peptide library was screened using a two-hybrid-like system for molecules able to bind human CDC6 protein (CDC6p), required for the initiation of DNA replication in eukaryotic cells . From a collection of over a million peptides, a single species that exhibited good affinity and specificity for binding CDC6p was obtained . When expressed in human cells, the peptide inhibited cell cycle progression and exhibited other properties expected of a CDC6p inhibitor . This approach, which does not require detailed knowledge of the mechanism of action of a protein target, may be generally useful for isolating peptides capable of manipulating biological pathways.

Arch Microbiol, 2000 May-Jun, 173(5-6), 319 - 24
Membrane targeting and translocation of bacterial hydrogenases; Wu LF et al.; Periplasmic or membrane-bound bacterial hydrogenases are generally composed of a small subunit and a large subunit . The small subunit contains a peculiar N-terminal twin-arginine signal peptide, whereas the large subunit lacks any known targeting signal for export . Genetic and biochemistry data support the assumption that the large subunit is cotranslocated with the small subunit across the cytoplasmic membrane . Indeed, the signal peptide carried by the small subunit directs both the small and the large subunits to the recently identified Mtt/Tat pathway, independently of the Sec machinery . In addition, the twin-arginine signal peptide of hydrogenase is capable of directing protein import into the thylakoidal lumen of chloroplasts via the homologous deltapH-driven pathway, which is independent of the Sec machinery . Therefore, the translocation of hydrogenase shares characteristics with the deltapH-driven import pathway in terms of Sec-independence and requirement for the twin-arginine signal peptide, and with protein import into peroxisomes in a "piggyback" fashion.

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8926 - 31
Bacterial-type DNA holliday junction resolvases in eukaryotic viruses; Garcia AD et al.; Homologous DNA recombination promotes genetic diversity and the maintenance of genome integrity, yet no enzymes with specificity for the Holliday junction (HJ)-a key DNA recombination intermediate-have been purified and characterized from metazoa or their viruses . Here we identify critical structural elements of RuvC, a bacterial HJ resolvase, in uncharacterized open reading frames from poxviruses and an iridovirus . The putative vaccinia virus resolvase was expressed as a recombinant protein, affinity purified, and shown to specifically bind and cleave a synthetic HJ to yield nicked duplex molecules . Mutation of either of two conserved acidic amino acids abrogated the catalytic activity of the A22R protein without affecting HJ binding . The presence of bacterial-type enzymes in metazoan viruses raises evolutionary questions.

Crit Care Med, 2000 Jun, 28(6), 1828 - 32
Serum procalcitonin levels in bacterial and abacterial meningitis; Schwarz S et al.; OBJECTIVES: To test the hypothesis that serum procalcitonin (PCT) levels are elevated in patients with bacterial meningitis and remain within normal limits in patients with abacterial meningitis . DESIGN: Prospective case series . SETTING: Tertiary care center . PATIENTS: A total of 30 patients (13 men and 17 women) with a mean age of 52 yrs, having acute bacterial (n = 16) or abacterial (n = 14) meningitis . INTERVENTIONS: Blood and cerebrospinal fluid samples . MEASUREMENTS AND MAIN RESULTS: Patients with abacterial meningitis were younger and had a shorter hospital stay . Of 16 patients with bacterial meningitis, 14 were in a septic condition at admission, but only 5 of 14 patients with abacterial meningitis were in a septic condition at admission . At discharge, 12 patients were without symptoms, 9 patients were moderately disabled, and 9 were severely disabled . No patient died . At admission, PCT, C-reactive protein, white blood cell and cerebrospinal fluid leukocyte counts, and cerebrospinal fluid protein and lactate levels were higher and the serum/cerebrospinal fluid glucose quotient was lower in patients with bacterial meningitis as compared with those with abacterial meningitis (p < .001) . PCT was the variable with the highest specificity for bacterial infections (100%), but there were false-negative findings in five patients with bacterial meningitis (a sensitivity of 69%) . Persistently elevated or increasing PCT levels after 2 days were associated with an unfavorable clinical course . CONCLUSIONS: Our results indicate that PCT is a useful additional variable for distinguishing bacterial from abacterial meningitis . In patients with abacterial meningitis, PCT levels do not increase even in cases of viral sepsis . Elevated PCT levels indicate a bacterial origin with high specificity, but false-negative results can occur.

J Biol Chem, 2000 Nov 10, 275(45), 34909 - 21
Understanding glucose transport by the bacterial phosphoenolpyruvate:glycose phosphotransferase system on the basis of kinetic measurements in vitro; Rohwer JM et al.; The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected . To address the issue of whether the behavior in vivo of the PTS can be understood in terms of these enzyme kinetics, a detailed kinetic model was constructed . Each overall phosphotransfer reaction was separated into two elementary reactions, the first entailing association of the phosphoryl donor and acceptor into a complex and the second entailing dissociation of the complex into dephosphorylated donor and phosphorylated acceptor . Literature data on the K(m) values and association constants of PTS proteins for their substrates, as well as equilibrium and rate constants for the overall phosphotransfer reactions, were related to the rate constants of the elementary steps in a set of equations; the rate constants could be calculated by solving these equations simultaneously . No kinetic parameters were fitted . As calculated by the model, the kinetic parameter values in vitro could describe experimental results in vivo when varying each of the PTS protein concentrations individually while keeping the other protein concentrations constant . Using the same kinetic constants, but adjusting the protein concentrations in the model to those present in cell-free extracts, the model could reproduce experiments in vitro analyzing the dependence of the flux on the total PTS protein concentration . For modeling conditions in vivo it was crucial that the PTS protein concentrations be implemented at their high in vivo values . The model suggests a new interpretation of results hitherto not understood; in vivo, the major fraction of the PTS proteins may exist as complexes with other PTS proteins or boundary metabolites, whereas in vitro, the fraction of complexed proteins is much smaller.

Org Lett, 2000 Jun 29, 2(13), 1839 - 1842
beta-Glycosides of 2,3-Diazido-2,3-dideoxy-D-mannose, a Synthetic Precursor of a Rare Bacterial Cell-Wall Building Unit; Szurmai Z et al.; 2,3-Diazido-2,3-dideoxy-beta-D-mannopyranoside derivatives were synthesized in order to prepare beta-glycosides of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, a rare moiety of bacterial oligosaccharides . A direct glycosyl donor, 4,6-di-O-acetyl-2,3-diazido-2,3-dideoxy-alpha-D-mannopyranosyl bromide, was prepared, and its synthetic capacity was tested in glycosylation reactions . An indirect route was also elaborated: 3-azido-3-deoxy-beta-D-glucopyranosides were converted into beta-D-mannopyranosides . The cis vicinal diazido function successfully tolerated the conditions of mild acidic hydrolysis, tritylation, Jones oxidation, TEMPO oxidation, acetolysis, and bromination with TiBr(4).

Nat Biotechnol, 2000 Jul, 18(7), 779 - 83
Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance; Verberne MC et al.; After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens . There is compelling evidence that SA plays a crucial role in triggering SAR . We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process . When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants . Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants . This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.

J Virol, 2000 Aug, 74(15), 6964 - 74
Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome; Adler H et al.; Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients . Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis . Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model . To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) in Escherichia coli . During propagation in E . coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed . The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells . BAC vector sequences were flanked by loxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties . Infectious virus was reconstituted from the BAC-cloned MHV-68 . Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68 . To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated . Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.

Immunology, 2000 Jun, 100(2), 252 - 8
Activation of human Vgamma9Vdelta2 T cells by a Brucella suis non-peptidic fraction impairs bacterial intracellular multiplication in monocytic infected cells; Ottones F et al.; Human gamma delta T cells are considered to play an important role in the early response to infection with intracellular pathogens . Evidence has been presented that the percentage of gamma delta T cells with Vgamma9Vdelta2 phenotype is dramatically increased in the peripheral blood of patients with acute brucellosis . This specific gd T-cell subpopulation is known to be activated by small non-peptidic molecules that can either be produced by the pathogen itself or released from damaged cells after infection . In the present work we provide evidence that Vgamma9Vdelta2 T lymphocytes from peripheral blood mononuclear cells of healthy donors can be specifically activated by non-peptidic low-molecular-weight compound(s) from Brucella suis lysate . Moreover, we show that Vgamma9Vdelta2 T cells activated by this B . suis fraction produce tumour necrosis factor-alpha and interferon-gamma, which reduce bacterial multiplication inside infected cells.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7778 - 83
tmRNAs that encode proteolysis-inducing tags are found in all known bacterial genomes: A two-piece tmRNA functions in Caulobacter; Keiler KC et al.; A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA . This RNA encodes a peptide tag that is incorporated at the end of the aberrant polypeptide and targets it for proteolysis . We have identified a circularly permuted version of the tmRNA gene in alpha-proteobacteria as well as in a lineage of cyanobacteria . The genes in these two groups seem to have arisen from two independent permutation events . As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules . The mature two-piece tmRNAs are predicted to have a tRNA-like domain and an mRNA-like domain similar to those of standard one-piece tmRNAs, with a break located in the loop containing the tag reading frame . A related sequence was found in the mitochondrial genome of Reclinomonas americana, but only the tRNA-like portion is retained . Although several sequence and structural motifs that are conserved among one-piece tmRNAs have been lost, the alpha-proteobacterium Caulobacter crescentus produces a functional two-piece tmRNA.

Infection, 1999, 27(4-5), 239 - 43
Usefulness of clinical scores to predict outcome in bacterial meningitis; Merkelbach S et al.; The predictive usefulness of clinical scores in patients with acute bacterial meningitis was investigated . Fifty-one consecutive patients with acute bacterial meningitis were scored on days 1, 3, 5, 8, and 14 after admission according to the Sandinavian Stroke Scale (SSS), Glasgow Coma-Scale (GCS) and Hunt & Hess Scale (HH) . As an index of their usefulness to predict the outcome, the scales were correlated with short-term outcome on day 21 assessed by the Glasgow Outcome Scale (GOS).The scores of all three scales correlated highly significantly with short-term outcome . Depending on the day of assessment, Spearman correlation coefficients ranged between 0.52 and 0.88 for SSS, between 0.50 and 0.84 for GCS, and between -0.47 and -0.82 for HH . The scales differed in their ability to predict outcome on and after day 1: mortality was best predicted by GCS, and complete recovery was best predicted by SSS . The use of scales in bacterial meningitis provides a rational quantitative basis to predict outcome more graduated than in dead or alive . Because the scales accentuate different aspects of outcome (e.g . mortality, restitution), the selection of a scale to be used in clinical trials should take into consideration the main focus of the study.

Arq Gastroenterol, 1999 Oct-Dec, 36(4), 169 - 76
{Breath hydrogen test to evaluate lactose absorption and small bowel bacterial overgrowth in children}; dos Reis JC et al.; The aim of this study was to determine the lactose absorption capacity and possible existence of bacterial overgrowth in the small bowel in asymptomatic school children of low social economic level in Marilia, a city located in the interior of Sao Paulo state . Eighty three children aging 7 to 15 years old without any gastrointestinal manifestations at least 30 days prior to the tests were studied . All the patients had fasted for at least 8 hours before the tests were performed . Lactose absorption was evaluated by breath hidrogen test after an overload of lactose 18 g in 10% aquous solution . Lactose intolerance was determined by the occurrence of clinical symptoms, such as diarrhea, abdominal pain, flatulence, etc in the following 24 hours after the test was performed . Bacterial overgrowth was evaluated by the breath hidrogen test after a 10 g lactulose load in aqueous solution . Lactose malabsorption was detected in 19 (22.9%) children and lactose intolerance was observed in 10 (12%) children . Lactose intolerance was more frequently observed in children who showed lactose malabsorption (6/19; 31.6%) than in those who presented a normal test (4/64; 6.3%) (P = 0.008) . Bacterial overgrowth was detected in six (7.2%) children and showed no statistical relationship with lactose malabsorption . Ontogenetic lactose malabsorption verified in this group of school children is similar to the reported for Caucasian populations . Presence of bacterial overgrowth confirms the existence of asymptomatic environmental enteropathy in children of low social economic level.

J Med Microbiol, 2000 Jul, 49(7), 613 - 20
A potential role for tumour necrosis factor-alpha in synergy between porcine respiratory coronavirus and bacterial lipopolysaccharide in the induction of respiratory disease in pigs; Van Reeth K et al.; This study examined whether exposure of pigs to both porcine respiratory coronavirus (PRCV) and bacterial lipopolysaccharide (LPS) can potentiate respiratory disease and lung secretion of tumour necrosis factor-a (TNF-alpha) and interleukin-1 (IL-1) . Caesarian-derived colostrum-deprived pigs were inoculated intratracheally with PRCV, with LPS from Escherichia coli O111:B4 (20 microg/kg), or with a combination of the two, and killed at set times after inoculation . Clinical signs, virus replication and (histo)pathological changes in the lungs, percentage of neutrophils and bioactive TNF-alpha and IL-1 in broncho-alveolar lavage (BAL) fluids were examined . The effects of separate virus or LPS inoculations were subclinical and failed to induce high and sustained cytokine levels . In a preliminary study, pigs were inoculated with PRCV and then with LPS 24 h later and killed sequentially . Severe respiratory disease and significantly enhanced TNF-alpha titres (208-3601 U/ml versus 40-89 U/ml after LPS only) were seen during the first 12 h after LPS inoculation . IL-1 levels (106-1631 U/ml versus 28-654 U/ml after LPS only) were also increased, but persisted for longer after clinical recovery than TNF-alpha . In a second study, pigs were inoculated with PRCV and subsequently with LPS at various time intervals ranging from 0 to 24 h, and killed 5 h after inoculation with LPS . A time interval of at least 12 h between inoculations was necessary for prominent respiratory signs to develop . Production of TNF-alpha, but not IL-1, was also dependent on the time interval between inoculations and was tightly correlated with disease . Lung neutrophil infiltration and pathological changes were comparable after combined PRCV-LPS and single LPS inoculations, and were not associated with disease . These data show that exposure to high endotoxin concentrations in swine buildings can precipitate respiratory disease in PRCV-infected pigs, and that TNF-alpha is probably an important mediator of these effects . This is the first in-vivo demonstration of synergy between respiratory viruses and LPS.

Eur J Surg, 2000 May, 166(5), 367 - 74
Bacterial translocation in obstructive jaundice in rats: role of mucosal lacteals; Kordzaya DJ et al.; OBJECTIVE: To study the ultrastructure of the ileal wall in rats with obstructive jaundice alone and after passive external biliary drainage to see if we could discover the reason for the increased risk of infective complications and multisystem failure in the presence of obstructive jaundice and after external biliary drainage . DESIGN: Histological examination of the wall of the terminal ileum using light microscopy, as well as scanning and transmission electron microscopy (EM) . SETTING: Experimental laboratory, Republic of Georgia . ANIMALS: 56 adult male Wistar rats . Interventions: Rats were divided into 7 groups: controls (not operated on, n = 6); sham-operated and studied after 6 and 12 days (n = 6 in each); bile duct ligation alone studied after 6 and 12 days (n = 10 in each); and bile duct ligation followed 6 and 12 days later by one-day of external biliary drainage (n = 9 in each) . MAIN OUTCOME MEASURES: Percentage of destroyed villi . RESULTS: The extent of oedema of villi, necrosis of neurons, and disturbances in the secretory capacity of enterocytes correlated well with the duration of cholestasis . After 6 and 12 days ligation alone 7.2% and 17.3%, respectively, of villi were found to be destroyed; their connective tissue framework including lymphatics was in direct contact with the intestinal contents . The changes were not reversed by one day of external biliary drainage . CONCLUSION: The gaps in the ileal mucosa caused by obstructive jaundice (and not relieved by one day of external biliary drainage) may enable gut bacteria and their endotoxin to reach the systemic circulation through the lymphatic-system . This could increase the risk of infective complications.

EMBO J, 2000 Jul 3, 19(13), 3179 - 91
The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography; Mosyak L et al.; In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, a membrane-anchored protein that binds to FtsZ, are two essential components of the septal ring structure that mediates cell division . Recent data indicate that ZipA is involved in the assembly of the ring by linking FtsZ to the cytoplasmic membrane and that the ZipA-FtsZ interaction is mediated by their C-terminal domains . We present the X-ray crystal structures of the C-terminal FtsZ-binding domain of ZipA and a complex between this domain and a C-terminal fragment of FtsZ . The ZipA domain is a six-stranded beta-sheet packed against three alpha-helices and contains the split beta-alpha-beta motif found in many RNA-binding proteins . The uncovered side of the sheet incorporates a shallow hydrophobic cavity exposed to solvent . In the complex, the 17-residue FtsZ fragment occupies this entire cavity of ZipA and binds as an extended beta-strand followed by alpha-helix . An alanine-scanning mutagenesis analysis of the FtsZ fragment was also performed, which shows that only a small cluster of the buried FtsZ side chains is critical in binding to ZipA.

Trends Microbiol, 2000 Jul, 8(7), 306 - 13
The modulation of host cell apoptosis by intracellular bacterial pathogens; Gao LY et al.; Recent years have witnessed significant advances in unraveling the elegant mechanisms by which intracellular bacterial pathogens induce and/or block apoptosis, which can influence disease progression . This intriguing aspect of the host-pathogen interaction adds another fascinating dimension to our understanding of the exploitation of host cell biology by intracellular bacterial pathogens.

FEBS Lett, 2000 Jun 30, 476(1-2), 18 - 21
Bacterial protein translocase: a unique molecular machine with an army of substrates; Economou A; Secretion of most polypeptides across the bacterial plasma membrane is catalyzed by the Sec protein translocase . This complex molecular machine comprises a flexible transmembrane conduit coupled to a motor-like component and displays four activities: (a) it is a specific receptor at its cytoplasmic side for all secretory polypeptides, (b) it converts metabolic energy from ATP and proton gradients into mechanical motion, (c) it prevents substrates from folding in statu translocanti and (d) it binds and releases short segments of the polymeric substrate sequentially . Combination of these activities allows translocase to move processively along the length of the substrate . Substrates are thus gradually expelled from the membrane and are released for subsequent extracytoplasmic folding.

Appl Environ Microbiol, 2000 Jul, 66(7), 3024 - 30
Molecular and ecological evidence for species specificity and coevolution in a group of marine algal-bacterial symbioses; Ashen JB et al.; The phylogenetic relationships of bacterial symbionts from three gall-bearing species in the marine red algal genus Prionitis (Rhodophyta) were inferred from 16S rDNA sequence analysis and compared to host phylogeny also inferred from sequence comparisons (nuclear ribosomal internal-transcribed-spacer region) . Gall formation has been described previously on two species of Prionitis, P . lanceolata (from central California) and P . decipiens (from Peru) . This investigation reports gall formation on a third related host, Prionitis filiformis . Phylogenetic analyses based on sequence comparisons place the bacteria as a single lineage within the Roseobacter grouping of the alpha subclass of the division Proteobacteria (99.4 to 98.25% sequence identity among phylotypes) . Comparison of symbiont and host molecular phylogenies confirms the presence of three gall-bearing algal lineages and is consistent with the hypothesis that these red seaweeds and their bacterial symbionts are coevolving . The species specificity of these associations was investigated in nature by whole-cell hybridization of gall bacteria and in the laboratory by using cross-inoculation trials . Whole-cell in situ hybridization confirmed that a single bacterial symbiont phylotype is present in galls on each host . In laboratory trials, bacterial symbionts were incapable of inducing galls on alternate hosts (including two non-gall-bearing species) . Symbiont-host specificity in Prionitis gall formation indicates an effective ecological separation between these closely related symbiont phylotypes and provides an example of a biological context in which to consider the organismic significance of 16S rDNA sequence variation.

Appl Environ Microbiol, 2000 Jul, 66(7), 2797 - 803
Bacterial growth stimulation with exogenous siderophore and synthetic N-acyl homoserine lactone autoinducers under iron-limited and low-nutrient conditions; Guan LL et al.; The growth of marine bacteria under iron-limited conditions was investigated . Neither siderophore production nor bacterial growth was detected for Pelagiobacter sp . strain V0110 when Fe(III) was present in the culture medium at a concentration of <1.0 microM . However, the growth of V0110 was strongly stimulated by the presence of trace amounts of exogenous siderophore from an alpha proteobacterium, V0902, and 1 nM N-acyl-octanoylhomoserine lactone (C(8)-HSL), which is known as a quorum-sensing chemical signal . Even though the iron-binding functionality of a hydroxamate siderophore was undetected in the supernatant of V0902, a hydroxamate siderophore was detected in the supernatant of V0110 under the above conditions . These results indicated that hydroxamate siderophore biosynthesis by V0110 began in response to the exogenous siderophore from V0902 when in the presence of C(8)-HSL; however, C(8)-HSL production by V0110 and V0902 was not detected . Direct interaction between V0902 and V0110 through siderophore from V0902 was observed in the dialyzing culture . Similar stimulated growth by exogenous siderophore and HSL was also observed in other non-siderophore-producing bacteria isolated from marine sponges and seawater . The requirement of an exogenous siderophore and an HSL for heterologous siderophore production indicated the possibility that cell-cell communication between different species was occurring.

Nat Struct Biol, 2000 Jul, 7(7), 565 - 9
Crystal structure of the bacterial conjugation repressor finO; Ghetu AF et al.; The conjugative transfer of F-like plasmids is repressed by FinO, an RNA binding protein . FinO interacts with the F-plasmid encoded traJ mRNA and its antisense RNA, FinP, stabilizing FinP against endonucleolytic degradation and facilitating sense-antisense RNA recognition . Here we present the 2.0 A resolution X-ray crystal structure of FinO, lacking its flexible N-terminal extension . FinO adopts a novel, elongated, largely helical conformation . An N-terminal region, previously shown to contact RNA, forms a positively charged alpha-helix (helix 1) that protrudes 45 A from the central core of FinO . A C-terminal region of FinO that is implicated in RNA interactions also extends out from the central body of the protein, adopting a helical conformation and packing against the base of the N-terminal helix . A highly positively charged patch on the surface of the FinO core may present another RNA binding surface . The results of an in vitro RNA duplexing assay demonstrate that the flexible N-terminal region of FinO plays a key role in FinP-traJ RNA recognition, and supports our proposal that this region and the N-terminus of helix 1 interact with and stabilize paired, complementary RNA loops in a kissing complex.

Microb Ecol, 2000 May, 39(4), 263 - 272
Heterogeneous Cell Density and Genetic Structure of Bacterial Pools Associated with Various Soil Microenvironments as Determined by Enumeration and DNA Fingerprinting Approach (RISA); Ranjard L et al.; The cell density and the genetic structure of bacterial subcommunities (further named pools) present in the various microenvironments of a silt loam soil were investigated . The microenvironments were isolated first using a procedure of soil washes that separated bacteria located outside aggregates (outer part) from those located inside aggregates (inner part) . A nondestructive physical fractionation was then applied to the inner part in order to separate bacteria located inside stable aggregates of different size (size fractions, i.e., two macroaggregate fractions, two microaggregate fractions, and the dispersible day fraction) . Bacterial densities measured by acridine orange direct counts (AODC) and viable heterotrophic (VH) cell enumerations showed the heterogeneous quantitative distribution of cells in soil . Bacteria were preferentially located in the inner part with 87.6% and 95.4% of the whole AODC and VH bacteria, respectively, and in the microaggregate and dispersible clay fractions of this part with more than 70% and 80% of the whole AODC and VH bacteria, respectively . The rRNA intergenic spacer analysis (RISA) was used to study the genetic structure of the bacterial pools . Different fingerprints and consequently different genetic structures were observed between the unfractionated soil and the microenvironments, and also among the various microenvironments, giving evidence that some populations were specific to a given location in addition to the common populations of all the microenvironments . Cluster and multivariate analysis of RISA profiles showed the weak contribution of the pools located in the macroaggregate fractions to the whole soil community structure, as well as the clear distinction between the pool associated to the macroaggregate fractions and the pools associated to the microaggregate ones . Furthermore, these statistical analyses allowed us to ascertain the influence of the clay and organic matter content of microenvironments on the genetic structure relatedness between pools.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2000 Mar, 17(1), 84 - 6
{Researches on surface modification for prevention of bacterial adhesion to implanting biomaterials}; Wu G et al.; The failure of operation caused by biomaterials centered infections (BCI) has seriously restricted the clinical application of biomaterials . Two mechanisms of bacterial adhesion and the relationship between surface free energy of bimaterials and bacterial adhesion are introduced and discussed in this paper . Increasing the surface free energy can improve (decrease) the adhesion of some kinds of bacteria . At last, some methods of the surface modification are reviewed.

Klin Lab Diagn, 2000 Jan, (1), 9 - 11
{The significance of detecting medium-weight molecules in the plasma of patients with viral and bacterial infectious}; Nagoev BS et al.; The content of medium-weight molecules (MWM) in the plasma was evaluated by the screening method in 280 patients with prevalent infectious diseases (viral hepatitides A, B, and C, influenza, acute dysentery, alimentary toxicoinfections, and tonsillitis) . Control group consisted of 70 donors . The maximum levels of MWM were observed at the peak of disease . In viral infections the increase in the level of MWM was longer . The values of MWM depended on the disease severity and presence of the intoxication syndrome . Plasma concentration of MWM correlated with the presence of the leading clinical syndromes in infectious diseases . Measurement of MWM level in the plasma of infectious patients can be used as a criterion of recovery and a prognostic and additional diagnostic test.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 705 - 12
The immune responses to bacterial antigens encountered in vivo at mucosal surfaces; Dougan G et al.; Mammals have evolved a sophisticated immune system for handling antigens encountered at their mucosal surfaces . The way in which mucosally delivered antigens are handled influences our ability to design effective mucosal vaccines . Live attenuated derivatives of pathogens are one route towards the development of mucosal vaccines . However, some molecules, described as mucosal immunogens, are inherently immunogenic at mucosal surfaces . Studies on mucosal immunogens may facilitate the identification of common characteristics that contribute to mucosal immunogenicity and aid the development of novel, non-living mucosal vaccines and immunostimulators.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 601 - 11
Measurement of bacterial gene expression in vivo; Hautefort I et al.; The complexities of bacterial gene expression during mammalian infection cannot be addressed by in vitro experiments . We know that the infected host represents a complex and dynamic environment, which is modified during the infection process, presenting a variety of stimuli to which the pathogen must respond if it is to be successful . This response involves hundreds of ivi (in vivo-induced) genes which have recently been identified in animal and cell culture models using a variety of technologies including in vivo expression technology, differential fluorescence induction, subtractive hybridization and differential display . Proteomic analysis is beginning to be used to identify IVI proteins, and has benefited from the availability of genome sequences for increasing numbers of bacterial pathogens . The patterns of bacterial gene expression during infection remain to be investigated . Are ivi genes expressed in an organ-specific or cell-type-specific fashion? New approaches are required to answer these questions . The uses of the immunologically based in vivo antigen technology system, in situ PCR and DNA microarray analysis are considered . This review considers existing methods for examining bacterial gene expression in vivo, and describes emerging approaches that should further our understanding in the future.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 551 - 64
Questions about the behaviour of bacterial pathogens in vivo; Smith H; Bacterial pathogens cause disease in man and animals . They have unique biological properties, which enable them to colonize mucous surfaces, penetrate them, grow in the environment of the host, inhibit or avoid host defences and damage the host . The bacterial products responsible for these five biological requirements are the determinants of pathogenicity (virulence determinants) . Current knowledge comes from studies in vitro, but now interest is increasing in how bacteria behave and produce virulence determinants within the infected host . There are three aspects to elucidate: bacterial activities, the host factors that affect them and the metabolic interactions between the two . The first is relatively easy to accomplish and, recently, new methods for doing this have been devised . The second is not easy because of the complexity of the environment in vivo and its ever-changing face . Nevertheless, some information can be gained from the literature and by new methodology . The third aspect is very difficult to study effectively unless some events in vivo can be simulated in vitro . The objectives of the Discussion Meeting were to describe the new methods and to show how they, and conventional studies, are revealing the activities of bacterial pathogens in vivo . This paper sets the scene by raising some questions and suggesting, with examples, how they might be answered . Bacterial growth in vivo is the primary requirement for pathogenicity . Without growth, determinants of the other four requirements are not formed . Results from the new methods are underlining this point . The important questions are as follows . What is the pattern of a developing infection and the growth rates and population sizes of the bacteria at different stages? What nutrients are present in vivo and how do they change as infection progresses and relate to growth rates and population sizes? How are these nutrients metabolized and by what bacterial mechanisms? Which bacterial processes handle nutrient deficiencies and antagonistic conditions that may arise? Conventional and new methods can answer the first question and part of the second; examples are described . The difficulties of trying to answer the last two are discussed . Turning to production in vivo of determinants of mucosal colonization, penetration, interference with host defence and damage to the host, here are the crucial questions . Are putative determinants, which have been recognized by studies in vitro, produced in vivo and are they relevant to virulence? Can hitherto unknown virulence determinants be recognized by examining bacteria grown in vivo? Does the complement of virulence determinants change as infection proceeds? Are regulatory processes recognized in vitro, such as ToxR/ToxS, PhoP/PhoQ, quorum sensing and type III secretion, operative in vivo? What environmental factors affect virulence determinant production in vivo and by what metabolic processes? Examples indicate that the answers to the first four questions are 'yes' in most but not all cases . Attempts to answer the last, and most difficult, question are also described . Finally, sialylation of the lipopolysaccharide of gonococci in vivo by host-derived cytidine 5'-mono-phospho-N-acetyl neuraminic acid, and the effect of host lactate are described . This investigation revealed a new bacterial component important in pathogenicity, the host factors responsible for its production and the metabolism involved.

Curr Opin Cell Biol, 2000 Aug, 12(4), 420 - 30
Multiple pathways allow protein secretion across the bacterial outer membrane; Thanassi DG et al.; Secretion of proteins across the bacterial outer membrane takes place via a variety of mechanisms from simple one-component systems to complex multicomponent pathways . Secretion pathways can be organized into evolutionarily and functionally related groups, which highlight their relationship with organelle biogenesis . Recent work is beginning to reveal the structure and function of various secretion components and the molecular mechanisms of secretion.

Biochem Biophys Res Commun, 2000 May 27, 272(1), 290 - 2
Phytanyl-pyrophosphate-linked substrate for a bacterial alpha-mannosyltransferase; Lellouch AC et al.; The biochemical characterization of bacterial glycosyltransferases involved in the assembly of cell-wall-associated polysaccharides is often hindered by the lack of the appropriate undecaprenyl-pyrophosphate-linked acceptor substrate . In order to find a suitable synthetic substrate for the alpha1,3-mannosyltransferase AceA from Acetobacter xylinum, phytanyl-pyrophosphate-linked cellobiose was prepared . In the presence of GDP-{14C}mannose and recombinant AceA, the phytanyl-pyrophosphate-linked cellobiose afforded a 14C-labeled trisaccharide that was sensitive to alpha-mannosidase degradation in a fashion analogous to the natural undecaprenyl-pyrophosphate-linked cellobiose substrate . These results suggest that phytanyl-pyrophosphate-linked oligosaccharides may be useful substrates for other important bacterial glycosyltransferases.

Toxicol Sci, 2000 Jul, 56(1), 203 - 10
Pentoxifylline attenuates bacterial lipopolysaccharide-induced enhancement of allyl alcohol hepatotoxicity; Sneed RA et al.; Small amounts of exogenous lipopolysaccharide (LPS) (10 ng/kg-100 microg/kg) enhance the hepatotoxicity of allyl alcohol in male Sprague-Dawley rats . This augmentation of allyl alcohol hepatotoxicity appears to be linked to Kupffer cell function, but the mechanism of Kupffer cell involvement is unknown . Since Kupffer cells produce tumor necrosis factor-alpha (TNF alpha) upon exposure to LPS, and this cytokine has been implicated in liver injury from large doses of LPS, we tested the hypothesis that TNF alpha contributes to LPS enhancement of allyl alcohol hepatotoxicity . Rats were treated with LPS (10-100 microg/kg iv) 2 h before allyl alcohol (30 mg/kg ip) . Co-treatment with LPS and allyl alcohol caused liver injury as assessed by an increase in activity of alanine aminotransferase in plasma . Treatment with LPS caused an increase in plasma TNF alpha concentration, which was prevented by administration of either pentoxifylline (PTX) (100 mg/kg iv) or anti-TNF alpha serum (1 ml/rat iv) one h prior to LPS . Only PTX protected rats from LPS-induced enhancement of allyl alcohol hepatotoxicity; anti-TNF alpha serum had no effect . Exposure of cultured hepatocytes to LPS (1-10 microg/ml) or to TNF alpha (15-150 ng/ml) for 2 h did not increase the cytotoxicity of allyl alcohol (0.01-200 microM) . These data suggest that neither LPS nor TNF alpha alone was sufficient to increase the sensitivity of isolated hepatocytes to allyl alcohol . Furthermore, hepatocytes isolated from rats treated 2 h earlier with LPS (i.e., hepatocytes which were exposed in vivo to TNF alpha and other inflammatory mediators) were no more sensitive to allyl alcohol-induced cytotoxicity than hepatocytes from naive rats . These data suggest that circulating TNF alpha is not involved in the mechanism by which LPS enhances hepatotoxicity of allyl alcohol and that the protective effect of PTX may be due to another of its biological effects.

Indian Pediatr, 2000 Jun, 37(6), 608 - 14
CSF interleukin-1 beta, tumor necrosis factor-alpha and free radicals production in relation to clinical outcome in acute bacterial meningitis; Jain M et al.; OBJECTIVE: To study the relationship of CSF IL-1 beta and TNF-alpha with free radicals in acute bacterial meningitis (ABM) and to evaluate the clinical outcome in relation to the levels of these cytokines and free radicals in CSF . DESIGN: Prospective with controls . SETTING: Referral unit of a teaching hospital . METHODS: 32 children between 3m-12 yrs of age with proven acute bacterial meningitis comprised the study group . In the control group, 20 children with febrile seizures were included . CSF cytokines- Interleukin Ib and tumour necrosis factor a,free radicals O(2)-, H(2)O(2) and enzymes SOD and CPK were measured in all the children . RESULTS: CSF IL-Ib and TNF-a concentration were markedly elevated in children with ABM (441.5 +/- 216.1 pg/ml, and 1009 +/- 529.1 pg/ml, respectively) as compared to controls (52.67 +/- 6.92 pg/ml, and 86.42 +/- 16.24 pg/ml) (p <0.0001) . Free radicals viz., superoxide anion, hydrogen peroxide production and enzymes creatinine phosphokinase and superoxide dismutase were also significantly elevated in ABM as compared to controls . There was direct correlation of CSF cytokines with CSF cytology, protein and free radicals production in ABM . Patients who expired or had neurological sequelae had markedly elevated concentrations of cytokines and free radicals . CONCLUSION: IL-I beta, TNF-alpha and free radicals are significantly elevated in CSF of patients with ABM . The concentration of these cytokines correlated well with free radical production, and with routinely measured CSF parameters and had a direct bearing on outcome of ABM

FEMS Microbiol Lett, 2000 Jul 1, 188(1), 1 - 6
Bacterial protein toxins targeting rho GTPases; Lerm M et al.; Several bacterial protein toxins target eukaryotic cells by modulating the functions of Rho GTPases that are involved in various signal processes and in the regulation of the actin cytoskeleton . The toxins inhibit Rho functions by ADP-ribosylation or glucosylation and activate them by deamidation and transglutamination . New findings indicate that the GTPases are also targeted by various 'injected' toxins which are introduced into the eukaryotic cells by the type-III secretion system . The injected toxins do not covalently modify Rho GTPases, but manipulate their regulatory GTPase cycle by acting as GTPase-activating proteins or guanine nucleotide exchange factors.

Biochimie, 2000 Mar, 82(3), 237 - 44
Bacterial luminescence: luminescence mechanism with cyclic peroxide participation and dependence on reactive oxygen species (a hypothesis); Dmitriev LF; Chemically initiated exchange (CIEE) luminescence reactions were reviewed and a new mechanism of luminescence with peracid as an intermediate is proposed; bacterial luminescence is generally considered to be a case of dioxetane luminescence, or, to be more precise, CIEE-luminescence which includes the generation of a cyclic peroxide . In the hypothesis the monooxygenase reaction (aldehyde -->fatty acid) should not be coupled with emitter generation as is usually believed, but only with the generation of peracid . As to the generation of the emitter, excited flavin, it is likely to occur later, during the interaction of flavin with cyclic peroxide . Its consequence is the breaking of two chemical bonds (O-O and C-C) in the cyclic peroxide and simultaneous generation of 4alpha-hydroxyflavin in exited state . In general, the generation of light includes three stages: 1) the monooxygenase reaction and the concurrent production of peracid; 2) the conversion of peracid to cyclic peroxide; and 3) the interaction of cyclic peroxide with flavin (through the CIEE mechanism).

Biotechnol Bioeng, 2000 Jul 20, 69(2), 196 - 203
Effect of anions on selective solubilization of zinc and copper in bacterial leaching of sulfide ores; Harahuc L et al.; Bacterial leaching of sulfide ores using Thiobacillus ferrooxidans, Thiobacillus thiooxidans, or a combination of the two was studied at various concentrations of specific anions . Selective zinc and copper solubilization was obtained by inhibiting iron oxidation without affecting sulfur/sulfide oxidation . Phosphate reduced iron solubilization from a pyrite (FeS(2))-sphalerite (ZnS) mixture without significantly affecting zinc solubilization . Copper leaching from a chalcopyrite (CuFeS(2))-sphalerite mixture was stimulated by phosphate, whereas chloride accelerated zinc extraction . In a complex sulfide ore containing pyrite, chalcopyrite, and sphalerite, both phosphate and chloride reduced iron solubilization and increased copper extraction, whereas only chloride stimulated zinc extraction . Maximum leaching obtained was 100% zinc and 50% copper . Time-course studies of copper and zinc solubilization suggest the possibility of selective metal recovery following treatment with specific anions .

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7090 - 5
Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals; Artsimovitch I et al.; Transcript elongation by RNA polymerase is discontinuous and interrupted by pauses that play key regulatory roles . We show here that two different classes of pause signals punctuate elongation . Class I pauses, discovered in enteric bacteria, depend on interaction of a nascent RNA structure with RNA polymerase to displace the 3' OH away from the catalytic center . Class II pauses, which may predominate in eukaryotes, cause RNA polymerase to slide backwards along DNA and RNA and to occlude the active site with nascent RNA . These pauses differ in their responses to antisense oligonucleotides, pyrophosphate, GreA, and general elongation factors NusA and NusG . In contrast, substitutions in RNA polymerase that increase or decrease the rate of RNA synthesis affect both pause classes similarly . We propose that both pause classes, as well as arrest and termination, arise from a common intermediate that itself binds NTP substrate weakly.

Kansenshogaku Zasshi, 2000 May, 74(5), 431 - 40
{Basic study on anti-bacterial urethral catheter . I . Development of a new anti-bacterial coating material for silicon catheters}; Hashimoto H et al.; In order to develop a new anti-bacterial urethral catheter, we studied anti-bacterial and anti-adherent coating material suitable for silicon catheters . Several aspects of various silver compounds were examined, including anti-bacterial activity, chemical property and toxicity . Among silver citrate, silver phosphate and silver oxide, which were found to have excellent anti-bacterial activities, silver citrate was regarded as the material of choice for anti-bacterial coating in terms of durable activity and biological safety . It was also found that several surfactants inhibited bacterial adherence to the surface of silicon catheters . Among them soybean lecithin exhibited excellent anti-adherent activity in a dose dependent manner . Finally, a mixture of silver citrate, soybean lecithin and liquid silicon at the ratio of 2:2:8 was regarded as an ideal anti-bacterial coating material for silicon catheters.

Med Hypotheses, 2000 May, 54(5), 723 - 5
Reactive arthritis: the result of an anti-idiotypic immune response to a bacterial lipopolysaccharide antigen where the idiotype has the immunological appearance of a synovial antigen; Kennedy JR; The term reactive arthritis (ReA) was first used in 1969 to describe sterile joint disease that follows infection elsewhere in the body . This is an attempt to explain the immunological basis of this disease, give a rationale for the presence of a single bacterial antigen in the involved joints, explain why the class I MHC molecule HLA-B27 is necessary and to suggest possible therapy . This paper proposes an anti-idiotypic (anti-id) model for this disease where a bacterial lipopolysaccharide (LPS) epitope is recognized by idiotypic (Id) T cell receptors and antibody Fab immune recognition surfaces (IRS) which have the immunologic appearance of an antigen on the synovial surface . These Id immune effectors utilize an HLA-B27 molecule to present their IRS on their surface, which results in an anti-id response that can also target the synovial antigen . The anti-id IRS have the immunologic appearance of LPS and their detection in the arthritic joint falsely suggests the presence of bacterial LPS . Evidence is presented which supports this reactive arthritis model in which there is a synovial antigen that is attacked by an anti-id response against the LPS of arthritogenic bacteria . Therapeutic vaccination is supported by this hypothesis .

Biochem J, 2000 Jul 1, 349(Pt 1), 99 - 104
Indirect induction of suppressor of cytokine signalling-1 in macrophages stimulated with bacterial lipopolysaccharide: partial role of autocrine/paracrine interferon-alpha/beta; Crespo A et al.; It has previously been reported by us that a brief prior exposure of mouse bone marrow culture-derived macrophages to bacterial lipopolysaccharide (LPS) resulted in a dramatic reduction in their ability to produce NO in response to a subsequent stimulus with either interferon-gamma (IFN-gamma) or IFN-gamma plus LPS . We show here that this brief exposure to LPS results in an impaired response to subsequently added IFN-gamma . A 2--4 h pretreatment with LPS leads to a dramatic reduction in the IFN-gamma-induced DNA-binding of the transcription factor, signal transducer and activator of transcription 1 alpha (STAT1 alpha) . This loss in ability to activate STAT1 alpha temporally correlates with the LPS-induced accumulation of mRNA encoding the suppressor of cytokine signalling-1 (SOCS-1) . However, LPS does not directly induce the synthesis of SOCS-1 . Rather, LPS induces the synthesis of autocrine/paracrine factors that are the true mediators of SOCS-1 induction . IFN-alpha/beta is one of these mediators, but plays only a partial role in the induction of SOCS-1 because neutralization of LPS-induced IFN-alpha/beta production incompletely inhibits the induction of SOCS-1 . We show that mouse IFN-beta directly induces the synthesis of SOCS-1, without the need for prior protein synthesis, and does so with faster kinetics than does LPS . Our results are consistent with the non-specific nature of LPS-induced tolerance and provide a mechanistic insight into nonspecificity; LPS indirectly induces the synthesis of a protein mediator, SOCS-1, which inhibits the signalling that is induced by IFN-gamma.

Chest, 2000 Jun, 117(6), 1679 - 84
Decreased apoptosis and increased activation of alveolar neutrophils in bacterial pneumonia; Droemann D et al.; STUDY OBJECTIVES: The central role of apoptosis in the regulation of lung inflammation is increasingly recognized . The aim of this study was to determine the parameters of cell activation and apoptosis on neutrophils from the circulation and the pulmonary compartment in patients with community-acquired pneumonia (CAP), and to assess the role of the Fas system and of complement-regulating molecules in this context . DESIGN AND METHODS: The study population consisted of nine patients with CAP (group 1) and six age-matched control patients without evidence of bronchopulmonary inflammation (group 2) . Apoptosis rate and expression of CD11b, CD16, CD55, CD59, CD95, and CD114 surface molecules on systemic and bronchoalveolar neutrophils were assessed ex vivo using fluorescence-activated cell sorter analysis . RESULTS: In patients with CAP, we found a significant decrease of the mean apoptosis rate in pulmonary neutrophils compared to systemic neutrophils, without concomitant changes in Fas expression . In contrast, cell activation markers were significantly increased on pulmonary cells (CD11b, 288 +/- 98.2 relative mean fluorescence intensity {rMFI} vs 53.8 +/- 10.8 rMFI on peripheral cells), and similar changes were observed with respect to the expression of complement-regulating molecules . Pulmonary polymorphonuclear neutrophils of the control group showed analogous changes, compared to systemic neutrophils, but a significantly higher rate of apoptosis and a lower increase of activation-marker expression were found, compared to pulmonary neutrophils of patients with pneumonia . CONCLUSIONS: Pulmonary neutrophils from patients with CAP show a decreased rate of apoptosis and increased activation status in the alveolar compartment, which may be important for effective control of pulmonary inflammation.

Adv Biochem Eng Biotechnol, 2000, 67, 1 - 33
Function and regulation of temperature-inducible bacterial proteins on the cellular metabolism; Schumann W; Temperature is an important environmental factor which, when altered, requires adaptive responses from bacterial cells . While a sudden increase in the growth temperature induces a heat shock response, a decrease results in a cold shock response . Both responses involve a transient increase in a set of genes called heat and cold shock genes, respectively, and the transient enhanced synthesis of their proteins allows the stressed cells to adapt to the new situation . A sudden increase in the growth temperature results in the unfolding of proteins, and hydrophobic amino acid residues normally buried within the interior of the proteins become exposed on their surface . Via these hydrophobic residues which often form hydrophobic surfaces proteins can interact and form aggregates which may become life-threatening . Here, molecular chaperones bind to these exposed hydrophobic surfaces to prevent the formation of protein aggregates . Some chaperones, the foldases, allow refolding of these denatured proteins into their native conformation, while ATP-dependent proteases degrade these non-native proteins which fail to fold . Most chaperones and energy-dependent proteases are heat shock proteins, and their genes are either regulated by alternate sigma factors or by repressors . The cold shock response evokes two major threats to the cells, namely a drastic reduction in membrane fluidity and a transient complete stop of translation at least in E . coli . Membrane fluidity is restored by increasing the amount of unsaturated fatty acids and translation resumes after adaptation of the ribosomes to cold . Neither an alternative sigma factor nor a repressor seems to be involved in the regulation of the cold shock genes in E . coli, the only species studied so far in this respect.

EMBO J, 2000 Jun 15, 19(12), 2803 - 12
Type 1 pilus-mediated bacterial invasion of bladder epithelial cells; Martinez JJ et al.; Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili . We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells . In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization . FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases . Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between alpha-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion . Inhibitors that prevented bacterial invasion also blocked the formation of these complexes . Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7382 - 7
A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions; Joung JK et al.; We have developed a bacterial "two-hybrid" system that readily allows selection from libraries larger than 10(8) in size . Our bacterial system may be used to study either protein-DNA or protein-protein interactions, and it offers a number of potentially significant advantages over existing yeast-based one-hybrid and two-hybrid methods . We tested our system by selecting zinc finger variants (from a large randomized library) that bind tightly and specifically to desired DNA target sites . Our method allows sequence-specific zinc fingers to be isolated in a single selection step, and thus it should be more rapid than phage display strategies that typically require multiple enrichment/amplification cycles . Given the large library sizes our bacterial-based selection system can handle, this method should provide a powerful tool for identifying and optimizing protein-DNA and protein-protein interactions.

Acta Paediatr, 2000 May, 89(5), 519 - 22
Leukocyte adhesiveness/aggregation test (LAAT) to discriminate between viral and bacterial infections in children; Urbach J et al.; OBJECTIVES: We previously noted that white blood cells (WBC) have increased adhesive properties during bacterial infections . Here, we aim to explore the possibility of using the different adhesive properties of WBC as a means of differentiating between viral and bacterial infections, a common problem in paediatrics . METHODS: The adhesive properties of WBC in the peripheral blood of 25 children with documented bacterial infections, 15 with documented viral infections and 36 with probable viral infections, were studied by means of a leukocyte adhesiveness/aggregation slide test (LAAT) . The results of the LAAT were compared with those of the other acute phase reactants, namely WBC, differential count and erythrocyte sedimentation rate (ESR), which were taken in the same blood sample in each patient . RESULTS: The sensitivity, specificity and positive predictive value were 92%, 96%, and 92%, respectively for the LAAT; 83%, 87% and 80% for the ESR; 56%, 78% and 56% for the white blood cell count; and 54%, 74% and 50% for the differential count . CONCLUSIONS: The presence of bacterial infections in children can be tested using a simple slide test to reveal the increased state of leukocyte adhesiveness/aggregation in the peripheral blood . The LAAT is a reliable, rapid and inexpensive test, and it can be a useful laboratory tool for the paediatrician treating a child with acute febrile illness.

Int J Mol Med, 2000 Jul, 6(1), 29 - 33
The role of bacterial DNA in septic arthritis; Deng GM et al.; Unmethylated CpG motifs are frequently found in bacterial DNA and have recently been shown to exert immunostimulatory effects on leukocytes . Bacteria produce severe septic arthritis; bacterial DNA may be involved in this process . We injected intraarticularly bacterial DNA and oligonucleotides containing unmethylated CpG motifs into knee joints of mice . Arthritis was seen by histopathology within 2 h and lasted for at least 14 days, and was characterized by an influx of monocytic, Mac-1+ cells and by a lack of T lymphocytes . Macrophages and their products such as tumor necrosis factor (TNF) alpha are essential for development of arthritis triggered by bacterial DNA containing CpG motifs . In contrast, neurophils, NK cells, and T/B cells were not instrumental in this condition . This review demonstrates that bacterial DNA containing unmethylated CpG motifs induces arthritis and indicates an important pathogenic role for bacterial DNA in septic arthritis.

Curr Opin Microbiol, 2000 Jun, 3(3), 270 - 5
Bacterial endosymbionts in animals; Moran NA et al.; Molecular phylogenetic studies reveal that many endosymbioses between bacteria and invertebrate hosts result from ancient infections followed by strict vertical transmission within host lineages . Endosymbionts display a distinctive constellation of genetic properties including AT-biased base composition, accelerated sequence evolution, and, at least sometimes, small genome size; these features suggest increased genetic drift . Molecular genetic characterization also has revealed adaptive, host-beneficial traits such as amplification of genes underlying nutrient provision.

J Bacteriol, 2000 Jul, 182(13), 3661 - 72
Purification and characterization of Sa-lrp, a DNA-binding protein from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius homologous to the bacterial global transcriptional regulator Lrp; Enoru-Eta J et al.; Archaea, constituting the third primary domain of life, harbor a basal transcription apparatus of the eukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear to be of the bacterial type . To date, little information is available on these predicted regulators and on the intriguing interplay that necessarily has to occur with the transcription machinery . Here, we focus on Sa-lrp of the extremely thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, encoding an archaeal homologue of the Escherichia coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer . Sa-lrp was shown to produce a monocistronic mRNA that was more abundant in the stationary-growth phase and produced in smaller amounts in complex medium, this down regulation being leucine independent . We report on Sa-Lrp protein purification from S . acidocaldarius and from recombinant E . coli, both identified by N-terminal amino acid sequence determination . Recombinant Sa-Lrp was shown to be homotetrameric and to bind to its own control region; this binding proved to be leucine independent and was stimulated at high temperatures . Interference binding experiments suggested an important role for minor groove recognition in the Sa-Lrp-DNA complex formation, and mutant analysis indicated the importance for DNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp . The DNA-binding capacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence of L-leucine, suggesting a potential physiological role of the amino acid as a cofactor.

Cancer Res, 2000 Jun 1, 60(11), 2955 - 63
Cationic lipid:bacterial DNA complexes elicit adaptive cellular immunity in murine intraperitoneal tumor models; Lanuti M et al.; Previous studies with a mycobacterial heat shock protein (hsp-65) have demonstrated some efficacy using cationic liposome-mediated gene transfer in murine i.p . sarcoma models . To further analyze the efficacy of hsp-65 immunotherapy in clinically relevant models of localized cancer, immunocompetent mice bearing i.p . murine mesothelioma were treated with four i.p . doses of a cationic lipid complexed with plasmid DNA (pDNA) containing hsp65, LacZ, or a null plasmid . We observed >90% long-term survival (median survival, 150 days versus approximately 25 days, treated versus saline control, respectively) in a syngeneic, i.p . murine mesothelioma model (AC29) . Long-term survivors were observed in all groups treated with lipid complexed with any pDNA . Lipid alone or DNA alone provided no demonstrable survival advantage . In a more aggressive i.p . model of mesothelioma (AB12), we observed >40% long-term survival in groups treated with lipid:pDNA complexes, again irrespective of the transgene . To ask whether these antitumor effects had led to an adaptive immune response against the tumor cell, we rechallenged long-term survivors in both murine models s.c . with the parental tumor cell line . Specific, long-lasting systemic immunity against the tumor was readily demonstrated in both models (AB12 and AC29) . Consistent with these results, splenocytes from long-term survivors specifically lysed the parental tumor cell lines . Depleting the CD8+ T-cells from the splenocyte pool eliminated this lytic activity . Lipid:pDNA treatment of athymic, SCID, and SCID/Beige mice bearing a murine i.p . mesothelioma (AC29) resulted in only a slight survival advantage, but there were no long-term survivors . Treatment of immunocompetent mice depleted of specific immune effector cells demonstrated roles for CD8+ and natural killer cells . Although the exact mechanism(s) responsible for these antitumor effects is unclear, the results are consistent with roles for both innate and adaptive immune responses . An initial tumor cell killing stimulated by cationic lipid:pDNA complexes appears to be translated into long-term, systemic immunity against the tumor cell . These results are the first to demonstrate that adaptive immunity against a tumor cell can be induced by the administration of lipid:pDNA complexes . Multiple administrations of cationic lipid complexed with pDNA lacking an expressed transgene could provide a promising generalized immune-mediated modality for treating cancer.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 441 - 6
Two-dimensional analysis of proteins specific to the bacterial magnetic particle membrane from Magnetospirillum sp . AMB-1; Okamura Y et al.; We report the identification of five proteins expressed specifically on the bacterial magnetic particle (BMP) membrane of Magnetospirillum sp . AMB-1 . These proteins are major components of the BMP membrane . The molecular weights were determined to be 12.0, 16.0, 24.8, 35.6, and 66.2 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis . Of these five, the 16.0-kDa protein was the most abundant in the BMP membrane . Furthermore, the 16.0-kDa protein consisted of two components each of differing pI . The 35.6-kDa protein was the second most abundant protein of the five detected.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 431 - 40
Effect of light/dark cycle on bacterial hydrogen production by Rhodobacter sphaeroides RV . From hour to second range; Wakayama T et al.; Hydrogen production by photosynthetic bacteria provides an efficient energy conversion method under low light intensity . However, under strong illumination, such as midday sunlight, the efficiency drops . This prevents the method from being applied industrially . To overcome this problem, we examined a method to thin out the excessive illumination . Light was given intermittently to reduce the total energy flux . The on/off ratio was set at 1/1 throughout the study, so that the time average of the light energy flux became half the continuous illumination . By keeping the time-average light flux constant (0.6 kW.m-2), the effects of the cycle period were examined in the range of hours to seconds . The hydrogen production rate was greatly affected by the cycle period, but cell growth and substrate consumption rates remained almost constant . The 30-min light/dark cycle (30 min on and 30 min off) provided the highest rate of hydrogen production (22 L.m-2.24 h-1) . At the shorter cycles, the rate decreased except that there was a suboptimum at about 40 s . Under excessive light intensity (1.2 kW.m-2), the light-to-hydrogen conversion efficiency was greatly enhanced . The hydrogen production rate during the 30-min cycle was twice as high as during a 12-h cycle under the same conditions.

Adv Exp Med Biol, 2000, 477, 455 - 65
The role of bacterial and host proteinases in periodontal disease; Travis J et al.; It is abundantly obvious that the uncontrolled degradation and/or activation of host defense pathways is the major pathway by which the periodontal pathogen P . gingivalis promotes its growth and proliferation . By being able to shed host receptors, degrade cytokines, and activate coagulation, complement, and kallikrein/kinin pathways it is clear that this organism has found a mechanism(s) to evade host defense and at the same time develop a system for cannibalizing host proteins for its own nutritional usage (Fig 2) . Thus, it seems only logical that the development of inhibitors against these bacterial proteinases would be a useful method for negating their activities and making such pathogens more susceptible to attack by host phagocyte cells . In this respect, the structure of the truncated form of RGP has just been elucidated . Thus, it should only be a question of time before inhibitors to this enzyme will be developed and, hopefully, be used to reduce the pathologies associated with the development of periodontitis and/or eliminate the disease altogether.

Plant J, 2000 May, 22(4), 335 - 43
Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione; Harms K et al.; The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1 . 30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter . In order to target the protein into the chloroplast, cysE was translationally fused to the 5'-signal sequence of rbcS from Arabidopsis thaliana . Transgenic plants showed a high accumulation of the cysE mRNA . The chloroplastic localisation of the E . coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions . Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants . The transgenic potato plants expressing the E . coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves . Both were up to twofold higher than in control plants . However, the thiol content in tubers of transgenic lines was unaffected . The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine . Only a minor effect on its enzymatic activity was observed . In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.

Ultrasound Obstet Gynecol, 2000 Mar, 15(3), 242 - 5
Morphology assessed by transvaginal ultrasonography differs in patients in preterm labor with vs . without bacterial vaginosis; Surbek DV et al.; OBJECTIVE: To determine whether cervical morphology in preterm labor patients differs in the presence or absence of bacterial vaginosis . DESIGN: Observational study . SUBJECTS: One hundred and twelve consecutive patients with objectively confirmed preterm labor admitted to a tertiary care centre were included in the study . Patients with placenta previa, active uterine bleeding or indication for an immediate delivery (e.g . severe pre-eclampsia or suspected fetal asphyxia), or severe fetal anomalies were excluded . METHODS: Transvaginal ultrasonography was used to measure cervical length and internal os width . Bacterial vaginosis was diagnosed by Gram stain of a vaginal smear . RESULTS: A total of 36 patients (32%) had bacterial vaginosis . Cervical length in this group was shorter than in patients with normal flora (mean 20.4 +/- 7.2 mm vs . 26.4 +/- 6.7 mm; P = 0.0002), and more patients with bacterial vaginosis had a dilated internal cervical os > or = 5 mm (67% vs . 30%, P = 0.001) . There were no significant differences, however, in preterm delivery rate and birth weight between the two groups; the overall preterm delivery rate was 40% . A cervical length < 25 mm was predictive of preterm delivery (P = 0.001, RR 4.2, 95% CI 1.8-9.7) . CONCLUSIONS: These data suggest that cervical change in preterm labor is more pronounced in patients with bacterial vaginosis but without a concomitant increase in the risk for preterm delivery . Despite this association, the cervical length measured by transvaginal ultrasonography alone is a useful predictor of preterm delivery, independent of the presence or absence of bacterial vaginosis.

J Biol Chem, 2000 Oct 6, 275(40), 31121 - 7
Correlating a protein structure with function of a bacterial mechanosensitive channel; Moe PC et al.; MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes . First isolated from Escherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension . Recently the structure of a functionally uncharacterized MscL homologue from Mycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 A . Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E . coli membranes and azolectin proteoliposomes . Furthermore, we show that M . tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization . Finally, we apply functional clues acquired from the E . coli MscL to the M . tuberculosis channel and show a mechanistic correlation between these channels . However, the inability of the M . tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E . coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.

Mol Microbiol, 2000 May, 36(4), 796 - 805
Molecular structure of bacterial endotoxin (Escherichia coli Re lipopolysaccharide): implications for formation of a novel heterogeneous lattice structure; Kato N et al.; Analyses of crystals of Escherichia coli Re lipopolysaccharide (LPS) formed after storage in 1% triethylamine indicate that the LPS molecules are assembled to form a monolayered structure consisting of a novel heterogeneous lattice structure, the greater part of which is occupied by one kind of lattice (lattice I), corresponding to the acyl chain portion of lipid A, and the remainder is occupied by the other kind of lattice (lattice II), corresponding to the 3-deoxy-Dmanno-octulosonic acid (dOclA) dimer and the N-acetylglucosamine disaccharide of lipid A . X-ray diffraction reveals that the type of cell is monoclinic (a = 5.53 A, b = 27.2 A, c = 6.47 A, alpha = 90 degrees, beta = 125.8 degrees, gamma = 90 degrees ) . Atomic force microscopy shows that crystals consist of multiple layers; the thickness of a layer corresponds to the b-axis value, and two types of surface topographies are visualized . One, regarded as the view onto the acyl chain ends, is two-dimensional arrays of oval bodies that constitute the lattice, with the lattice constants corresponding to the a- and c-axes and the angle of beta (lattice I) . The other, regarded as the view onto the dOclA dimers, is two-dimensional arrays of dromedary-back-like bodies that constitute the lattice with axes of 9.0 and 10.7 A and the angle of 65 degrees formed by both axes (lattice II) . Based on these results, we present the molecular model of E . coli Re LPS.

Neuroscience, 2000, 97(4), 757 - 64
High concentrations of extracellular potassium enhance bacterial endotoxin lipopolysaccharide-induced neurotoxicity in glia-neuron mixed cultures; Chang RC et al.; A sudden increase in extracellular potassium ions (K(+)) often occurs in cerebral ischemia and after brain trauma . This increase of extracellular K(+) constitutes the basis for spreading depression across the cerebral cortex, resulting in the expansion of neuronal death after ischemic and traumatic brain injuries . Besides spreading depression, it has become clear that cerebral inflammation also is a key factor contributing to secondary brain injury in acute neurological disorders . Experiments to validate the relationship between elevated levels of extracellular K(+) and inflammation have not been studied . This study aims to elucidate the roles of high concentrations of extracellular K(+) in bacterial endotoxin lipopolysaccharide-induced production of inflammatory factors . Increased concentration of KCl in the medium (20mM) significantly enhanced neurotoxicity by lipopolysaccharide in glia-neuron mixed cultures . To delineate the underlying mechanisms of increased neurotoxicity, the effects of high extracellular K(+) were examined by using mixed glial cultures . KCl at 20mM significantly enhanced nitrite, an index for nitric oxide, production by about twofold, and was pronounced from 24 to 48h, depending on the concentration of KCl . Besides nitric oxide production of tumor necrosis factor-alpha was also enhanced . The augmentative effects of high KCl on the production of inflammatory factors were probably due to the further activation of microglia, since high KCl also enhanced the production of tumor necrosis factor-alpha in microglia-enriched cultures . The increased production of nitrite by high K(+) was eliminated through use of a K(+)-blocker.Taken together, the results show that increases of extracellular K(+) concentrations in spreading depression augment lipopolysaccharide-elicited neurotoxicity, because production of inflammatory factors such as nitric oxide and tumor necrosis factor-alpha are potentiated . Since spreading depression and cerebral inflammation are important in acute neurological disorders, the present results suggest a biochemical mechanism: elevated extracellular K(+) concentrations augment glial inflammatory responses, and thus the neurotoxicity.

Biochim Biophys Acta, 2000 May 31, 1458(2-3), 263 - 9
The epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases . Structure, arrangement, and role of the epsilon subunit in energy coupling within the complex; Capaldi RA et al.; Recent studies show that the epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases is a component of the central stalk that links the F(1) and F(0) parts . This subunit interacts with alpha, beta and gamma subunits of F(1) and the c subunit ring of F(0) . Along with the gamma subunit, epsilon is a part of the rotor that couples events at the three catalytic sites sequentially with proton translocation through the F(0) part . Structural data on the epsilon subunit when separated from the complex and in situ are reviewed, and the functioning of this polypeptide in coupling within the ATP synthase is considered.

J Infect Dis, 2000 Jun, 181(6), 2092 - 4 Epub 2000 May 26.
Hepatocyte growth factor levels in cerebrospinal fluid: a comparison between acute bacterial and nonbacterial meningitis; Nayeri F et al.; The organotrophic functions of the hepatocyte growth factor (HGF) have been the subject of several studies . In the more recent studies, this function has been reported in the brain . In the present study, we have measured the levels of HGF in cerebrospinal fluid (CSF) and sera from 78 patients divided into 6 different groups according to central nervous system (CNS) infection and control . Quantitative measurements of HGF in the CSF and serum were performed by an enzyme-linked immunosorbent assay . Elevated values of CSF HGF were found in the patients with acute bacterial/probable bacterial meningitis (P<.001), compared with nonbacterial CNS infections and facial palsy, as well as with a control group without signs of CNS involvement . The values of CSF HGF were not correlated to blood-brain-barrier disruption in the groups . These observations might indicate an intrathecal production of HGF in acute bacterial/probable bacterial meningitis.

Nihon Arukoru Yakubutsu Igakkai Zasshi, 2000 Apr, 35(2), 61 - 8
Ethanol suppresses L-arginine-induced relaxation response of rat aorta stimulated with bacterial lipopolysaccharide; Hatake K et al.; Using isolated rat aortic strips without endothelium, we investigated the effect of ethanol on L-arginine-induced relaxation . L-Arginine (10(-5)-10(-3) M) produced a relaxation response in vascular strips incubated for 6 hr with bacterial lipopolysaccharide (1 microgram/ml) . The relaxation was either abolished by cycloheximide (10(-5) M) and actinomycin D (10(-5) M) or diminished by polymyxin B (10 micrograms/ml), dexamethasone (10(-5) M) and nitro-L-arginine (10(-4) M) under conditions in which these inhibitors were coincubated with lipopolysaccharide . Also, L-arginine-induced relaxation was significantly inhibited when ethanol (200, 400 mM) was present together with lipopolysaccharide during the 6-hr incubation . However, when ethanol was added to the organ bath after 6-hr incubation with lipopolysaccharide, it did not inhibit the relaxation . These results suggest that the relaxation response to L-arginine is mediated by an inducible type of nitric oxide synthase, which can be induced by lipopolysaccharide, and that ethanol may attenuate this nitric oxide-mediated relaxation by inhibiting expression of inducible nitric oxide synthase.

Nat Biotechnol, 2000 Jun, 18(6), 679 - 82
Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis; Talaat AM et al.; DNA microarrays have the ability to analyze the expression of thousands of the same set of genes under at least two different experimental conditions . However, DNA microarrays require substantial amounts of RNA to generate the probes, especially when bacterial RNA is used for hybridization (50 microg of bacterial total RNA contains approximately 2 microg of mRNA) . We have developed a computer-based algorithm for prediction of the minimal number of primers to specifically anneal to all genes in a given genome . The algorithm predicts, for example, that 37 oligonucleotides should prime all genes in the Mycobacterium tuberculosis genome . We tested the usefulness of the genome-directed primers (GDPs) in comparison to random primers for gene expression profiling using DNA microarrays . Both types of primers were used to generate fluorescent-labeled probes and to hybridize to an array of 960 mycobacterial genes . Compared to random-primer probes, the GDP probes were more sensitive and more specific, especially when mammalian RNA samples were spiked with mycobacterial RNA . The GDPs were used for gene expression profiling of mycobacterial cultures grown to early log or stationary growth phases . This approach could be useful for accurate genome-wide expression analysis, especially for in vivo gene expression profiling, as well as directed amplification of sequenced genomes.

Genetics, 2000 Jun, 155(2), 499 - 508
Methods for estimating gene frequencies and detecting selection in bacterial populations; Rannala B et al.; Recent breakthroughs in molecular technology, most significantly the polymerase chain reaction (PCR) and in situ hybridization, have allowed the detection of genetic variation in bacterial communities without prior cultivation . These methods often produce data in the form of the presence or absence of alleles or genotypes, however, rather than counts of alleles . Using relative allele frequencies from presence-absence data as estimates of population allele frequencies tends to underestimate the frequencies of common alleles and overestimate those of rare ones, potentially biasing the results of a test of neutrality in favor of balancing selection . In this study, a maximum-likelihood estimator (MLE) of bacterial allele frequencies designed for use with presence-absence data is derived using an explicit stochastic model of the host infection (or bacterial sampling) process . The performance of the MLE is evaluated using computer simulation and a method is presented for evaluating the fit of estimated allele frequencies to the neutral infinite alleles model (IAM) . The methods are applied to estimate allele frequencies at two outer surface protein loci (ospA and ospC) of the Lyme disease spirochete, Borrelia burgdorferi, infecting local populations of deer ticks (Ixodes scapularis) and to test the fit to a neutral IAM.

EMBO J, 2000 Jun 1, 19(11), 2751 - 62
Partial suppression of the fission yeast rqh1(-) phenotype by expression of a bacterial Holliday junction resolvase; Doe CL et al.; A key stage during homologous recombination is the processing of the Holliday junction, which determines the outcome of the recombination reaction . To dissect the pathways of Holliday junction processing in a eukaryote, we have targeted an Escherichia coli Holliday junction resolvase to the nuclei of fission yeast recombination-deficient mutants and analysed their phenotypes . The resolvase partially complements the UV and hydroxyurea hypersensitivity and associated aberrant mitoses of an rqh1(-) mutant . Rqh1 is a member of the RecQ subfamily of DNA helicases that control recombination particularly during S-phase . Significantly, overexpression of the resolvase in wild-type cells partly mimics the loss of viability, hyper-recombination and 'cut' phenotype of an rqh1(-) mutant . These results indicate that Holliday junctions form in wild-type cells that are normally removed in a non-recombinogenic way, possibly by Rqh1 catalysing their reverse branch migration . We propose that in the absence of Rqh1, replication fork arrest results in the accumulation of Holliday junctions, which can either impede sister chromatid segregation or lead to the formation of recombinants through Holliday junction resolution.

EMBO J, 2000 Jun 1, 19(11), 2701 - 9
Origins of minigene-dependent growth inhibition in bacterial cells; Heurgue-Hamard V et al.; The expression of very short open reading frames in Escherichia coli can lead to the inhibition of translation and an arrest in cell growth . Inhibition occurs because peptidyl-tRNA hydrolase fails to recycle sufficiently rapidly peptidyl-tRNA released from ribosomes at the stop signal in competition with normal termination, causing starvation for essential species of tRNA . Previous studies have shown that the last sense codon, the strength of the Shine-Dalgarno sequence and the nature and context of the stop codon affect the toxicity associated with mini-gene expression . Here, several important parameters are studied as a function of the length of the mini-gene coding sequence . The rate of peptidyl-tRNA drop-off catalysed by translation factors decreases dramatically for peptides longer than a hexamer . The probability that ribosomes recycle without dissociation of the mini-gene mRNA varies strongly with the length of the coding sequence . The peptidyl-tRNA hydrolase rap mutant, unlike the wild-type enzyme, is highly sensitive to the length and sequence of the peptide . Together, these parameters explain the length dependence of mini-gene toxicity.

Science, 2000 Jun 2, 288(5471), 1640 - 3
Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor; Peluso P et al.; The mechanism by which a signal recognition particle (SRP) and its receptor mediate protein targeting to the endoplasmic reticulum or to the bacterial plasma membrane is evolutionarily conserved . In Escherichia coli, this reaction is mediated by the Ffh/4.5S RNA ribonucleoprotein complex (Ffh/4.5S RNP; the SRP) and the FtsY protein (the SRP receptor) . We have quantified the effects of 4.5S RNA on Ffh-FtsY complex formation by monitoring changes in tryptophan fluorescence . Surprisingly, 4.5S RNA facilitates both assembly and disassembly of the Ffh-FtsY complex to a similar extent . These results provide an example of an RNA molecule facilitating protein-protein interactions in a catalytic fashion.

Eur J Clin Microbiol Infect Dis, 2000 Apr, 19(4), 312 - 6
Role of bacterial virulence factors and host factors in the outcome of Escherichia coli bacteraemia; Hekker TA et al.; In a study of the role of virulence factors in the outcome of Escherichia coli bacteraemia, blood isolates from 30 hospitalised patients were characterised with regard to O and K antigens, P and type 1 fimbriae, haemolysin production, cytonecrotising factor 1 production, serum resistance, ability to activate neutrophils and resistance to killing . Patients were analysed to identify host factors contributing to morbidity and mortality . In univariate analyses the presence of a K antigen, type 1 fimbriae, absence of haemolysin production, serum resistance and resistance to killing were associated with morbidity and mortality . In multivariate analyses only the absence of haemolysin production was associated with morbidity and mortality, after taking host factors into account . These preliminary findings suggest that host factors override bacterial virulence factors in determining the course of Escherichia coli bacteraemia . The negative association between haemolysin production and clinical deterioration during Escherichia coli bacteraemia might indicate predominance of less virulent strains in patients with other risk factors for morbidity and mortality or inactivation of neutrophil products needed for host defence.

Crit Care Med, 2000 May, 28(5), 1550 - 5
Neutrophil depletion in rats reduces burn-injury induced intestinal bacterial translocation; Fazal N et al.; OBJECTIVE: To determine whether neutrophil depletion could eradicate intestinal bacterial translocation in bum-injured rats . DESIGN: Prospective, randomized, controlled study . SETTING: University research laboratory . SUBJECTS: Adult male Sprague-Dawley rats . INTERVENTIONS: The rats were intravenously administered a rabbit anti-rat neutrophil antibody causing profound neutropenia and subjected to a 30% total body surface area scald burn . MEASUREMENTS AND MAIN RESULTS: The depletion of neutrophils from the intestine was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates . In addition, the presence of activated/extravasated neutrophils in intact intestines was determined via immunohistochemical localization of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component protein p47phox . Bacterial translocation was measured using agar cultures and by determining Escherichia coli beta-galactosidase gene via polymerase chain reaction/Southern blot analyses of mesenteric lymph node and spleen, liver, lung, and blood . MPO measurements demonstrated a six-fold increase above the control value in the intestinal tissue in rats on day 1 postburn . The presence of activated neutrophils (expression of p47phox protein) was also markedly increased in the intestines of these rats . The increased MPO activity and p47phox expression accompanied a translocation of indigenous E . coli into the mesenteric lymph node without a spread to other organs . The administration of anti-neutrophil antibody to burn animals prevented an increase in MPO activity and bacterial translocation . CONCLUSION: These studies indicate that enhanced intestinal bacterial translocation caused by burn injury could be related to the increased infiltration of activated neutrophils into the intestinal tissue after bum . The release of neutrophil products such as superoxide anion may effect intestinal tissue damage leading to bacterial translocation of indigenous E . coli.

Transpl Immunol, 2000 Mar, 8(1), 17 - 29
Chemokine and chemokine receptor expression by liver-derived dendritic cells: MIP-1alpha production is induced by bacterial lipopolysaccharide and interaction with allogeneic T cells; Drakes ML et al.; Dendritic cells (DC) are highly-specialized antigen-presenting cells (APC), that initiate and modulate immune responses . Their specialized migratory and tissue-homing properties are regulated by small molecular weight proteins (chemokines) that govern leukocyte migration and activation . Little is known about the capacity of liver DC to produce or respond to chemokines . Here we examined chemokine and chemokine receptor (CR) gene expression in both immature DC progenitors (DCp) and comparatively mature DC generated from mouse liver . Factors affecting production of the chemokine macrophage inflammatory protein (MIP)-1alpha, and the influence of MIP-1alpha on liver DC migration were also investigated . Dendritic cells were propagated in response to granulocyte-macrophage colony stimulating factor (GM-CSF) +/- interleukin (IL)-4 from bone marrow (BM) cells or liver non-parenchymal cells (NPC) isolated from normal mice, or from mice treated with the hematopoietic growth factor Flt3 ligand (FL) . Their phenotype and allostimulatory function were assessed by monoclonal antibody (mAb) staining and flow cytometry, and by the capacity to induce mixed leukocyte reactions, respectively . Specific chemokine and CR gene expression was studied using the RNase protection assay (RPA) . Production of MIP-1alpha was determined by enzyme-linked immunoabsorbent assay (ELISA), and the migratory activity of liver DC induced by MIP-1alpha quantitated using microchemotaxis chambers . Like DC generated simultaneously from BM, liver-derived DC expressed mRNA for a variety of CC and CXC chemokines . RANTES (regulated upon activation, normal T cell expressed and secreted) transcripts were the most strongly expressed . Gene transcripts for the receptor CCR1, that binds RANTES and MIP-1alpha were also readily detected, as was CCR2, the receptor for the monocyte chemotactic proteins (MCP)1-4 . No major differences in chemokine or CR mRNA expression were detected between immature and more mature liver DC . MIP-1alpha production by liver-derived DC was stimulated by bacterial lipopolysaccharide (LPS), and high levels were also detected in co-cultures of hepatic DC and allogeneic T cells . Chemotactic migration of liver-derived DC was stimulated by MIP-1alpha . Thus, liver-derived DC express mRNA for several CC and CXC chemokines and their receptors that may play key roles in the regulation of hepatic inflammatory responses . Production of MIP-1alpha by liver DC, and their migratory responses to this chemokine, suggest that MIP-1alpha and other chemokines may play significant roles in the regulation of liver DC function and in interactions of liver DC with other leukocytes, under normal and inflammatory conditions.

Dev Comp Immunol, 2000 Sep-Oct, 24(6-7), 553 - 63
Serum amyloid A transcription in Atlantic salmon (Salmo salar L.) hepatocytes is enhanced by stimulation with macrophage factors, recombinant human IL-1 beta, IL-6 and TNF alpha or bacterial lipopolysaccharide; Jorgensen JB et al.; Serum amyloid A (A-SAA) has previously been reported to be an acute-phase protein in salmonids . Hepatocytes represent a major source of A-SAA in salmonids, but nothing is known about hepatocyte SAA synthesis in fish . In the present work, the expression of A-SAA transcripts in primary cultures of Atlantic salmon hepatocytes in response to macrophage derived cytokines, human recombinant cytokines and bacterial lipopolysaccharide (LPS) was studied by Northern blot analysis . The macrophage supernatants were prepared by stimulating Atlantic salmon head kidney macrophages with LPS, yeast glucan or a leukocyte derived macrophage activating factor (MAF) . The supernatants from glucan- or MAF-stimulated macrophages had no effect on A-SAA expression of the hepatocytes, while supernatants from LPS-stimulated macrophages gave about a 2-fold increase in expression . The combination of either glucan and MAF, or LPS and MAF were more effective and these supernatants gave a 3.4- and 5.2-fold increase in A-SAA expression, respectively . The hepatocytes were also treated with the human recombinant cytokines TNFalpha, IL-1beta and IL-6, alone or in combination . The A-SAA response to each of them alone was modest, but TNFalpha and IL-6 or IL-1beta and IL-6 in combination gave a higher response than each cytokine alone . These data suggest that the expression of A-SAA by hepatocytes from Atlantic salmon is induced by cytokine-like molecules . Interestingly, hepatocytes treated directly with LPS gave a more than 10-fold increase in SAA mRNA expression, but it is not known if this is a direct effect of LPS on the hepatocytes or if it is mediated by other contaminating cell types.

Nature, 2000 May 18, 405(6784), 299 - 304
Lateral gene transfer and the nature of bacterial innovation; Ochman H et al.; Unlike eukaryotes, which evolve principally through the modification of existing genetic information, bacteria have obtained a significant proportion of their genetic diversity through the acquisition of sequences from distantly related organisms . Horizontal gene transfer produces extremely dynamic genomes in which substantial amounts of DNA are introduced into and deleted from the chromosome . These lateral transfers have effectively changed the ecological and pathogenic character of bacterial species.

J Inorg Biochem, 2000 Apr, 79(1-4), 381 - 5
Bacterial cytochrome c nitrite reductase: new structural and functional aspects; Stach P et al.; Cytochrome c nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia as a key step within the biological nitrogen cycle . Most recently, the crystal structure of the soluble enzyme from Sulfurospirillum deleyianum could be solved to 1.9 A resolution . This set the basis for new experiments on structural and functional aspects of the pentaheme protein which carries a Ca(2+) ion close to the active site heme . In the crystal, the protein was a homodimer with ten hemes in very close packing . The strong interaction between the nitrite reductase monomers also occurred in solution according to the dependence of the activity on the protein concentration . Addition of Ca(2+) to the enzyme as isolated had a stimulating effect on the activity . Ca(2+) could be removed from the enzyme by treatment with chelating agents such as EGTA or EDTA which led to a decrease in activity . In addition to nitrite, the enzyme converted NO, hydroxylamine and O-methyl hydroxylamine to ammonia at considerable rates . With N2O the activity was much lower; most likely dinitrogen was the product in this case . Cytochrome c nitrite reductase exhibited a remarkably high sulfite reductase activity, with hydrogen sulfide as the product . A paramagnetic Fe(II)-NO, S = 1/2 adduct was identified by rapid freeze EPR spectroscopy under turnover conditions with nitrite . This potential reaction intermediate of the reduction of nitrite to ammonia was also observed with PAPA NONOate and Spermine NONOate.

J Inorg Biochem, 2000 Apr, 79(1-4), 225 - 9
Bacterial metal-resistance proteins and their use in biosensors for the detection of bioavailable heavy metals; Bontidean I et al.; We have expressed and purified metal-resistance and metal regulatory proteins from the bacterial determinants of resistance to heavy metals and utilised these in the development of biosensors for heavy metals . Both the metallothionein from the cyanobacterium Synechococcus PCC 7942 and the MerR regulatory protein from transposon Tn501 allow the detection of non-specific metal binding down to 10(-15) M concentrations of Hg(II), Cu(II), Zn(II) and Cd(II) in pure solution . Differential effects of the metals can be detected at both low and high concentrations, and the shape of the capacitance curves may reflect biologically relevant responses of the proteins to metals . Further work is required to establish the relationship between the detected binding of metal and the biological response of the protein, or to provide biosensors of use in the natural environment.

Indian J Pediatr, 1996 Mar-Apr, 63(2), 217 - 25
Brainstem auditory evoked response (BAER) in childhood bacterial meningitis; Kapoor RK et al.; Brainstem auditory responses were recorded in 50 children of bacterial meningitis and age matched 50 normal children . Abnormal BAER was found in 32 (64%) patients of bacterial meningitis . These abnormalities included prolonged latency (56.2%); unilateral absent response (25%); bilateral absent response (25%) and prolonged interwave interval (25%) . Follow-up could be done in 23 patients of 46 survivors . All the patients with prolonged latency either became normal or improved . In majority of the patients having absent response, the abnormality persisted . Abnormal BAER was significantly associated with age < 2 years (p < 0.02), Modified GCS Score < or = 8 (p < 0.001), Seizures (p < 0.02), raised Intracranial Pressure (ICP) (p < 0.02) and CSF sugar < 20 mg% (p < 0.05).

Indian J Pediatr, 1996 Mar-Apr, 63(2), 210 - 6
Auditory profile in children recovering from bacterial meningitis; Singh K et al.; In the present study BERA profile of 30 post-meningitic children was compared with 15 normal children of the same age and it was observed that 36.6% children in the age range of 6 months to 36 months were found to have varying degree of sensorineural deafness . Severe bilateral sensorineural hearing loss (> 80 dB) was observed in 6.6% children and moderate (40-80 dB) hearing loss in 30% of children . Abnormalities were bilateral in both the samples of children with severe hearing loss (> 80 dB) whereas among 9 children who had moderate hearing loss abnormalities were bilateral in one patient and unilateral in the remaining 8 children . A relationship between higher incidence of sensorineural deafness and younger age of children, and occurrence of seizures during meningitis were noted . But no relationship was observed with either sex, hydrocephalus, subdural effusion or with low CSF sugar and high CSF proteins.

Toxicol Sci, 2000 Jun, 55(2), 444 - 52
Bacterial lipopolysaccharide exposure augments aflatoxin B(1)-induced liver injury; Barton CC et al.; Bacterial endotoxin (lipopolysaccharide; LPS) given to animals in large doses results in pronounced, midzonal liver injury . Exposure to smaller, non-injurious doses of LPS augments the toxicity of certain hepatotoxicants . This study was conducted to delineate the development of injury in a rat model of augmentation of aflatoxin B(1) (AFB(1)) hepatotoxicity by LPS . At large doses (i.e., > 1 mg/kg, ip), AFB(1) administration resulted in pronounced injury to the periportal regions of the liver . Male, Sprague-Dawley rats (250-350 g) were treated with 1 mg AFB(1)/kg, ip or its vehicle (0.5% DMSO/saline) and 4 h later with either E . coli LPS (7.4 x 106 EU/kg, iv) or its saline vehicle . Liver injury was assessed 6, 12, 24, 48, 72, or 96 h after AFB(1) administration . Hepatic parenchymal cell injury was evaluated as increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum and from histologic examination of liver sections . Biliary tract alterations were evaluated as increased concentration of serum bile acids and activities of gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), and 5'-nucleotidase (5'-ND) in serum . At all times and for all markers, injury in rats treated with either AFB(1) or LPS alone was absent or modest . In the AFB(1)/LPS cotreated group, hepatic parenchymal cell injury was pronounced by 24 h and had returned to control values by 72 h . The injury began in the periportal region and spread midzonally with time . Furthermore, changes in serum markers indicative of biliary tract alterations were evident by 12 h and had returned to control values by 72 h . Thus, the nature of the hepatic lesions suggested that LPS potentiated the effects of AFB(1) on both parenchymal and bile duct epithelial cells.

Microb Ecol, 2000 Feb, 39(2), 101 - 115
Bacterioplankton Production in Humic Lake Örträsket in Relation to Input of Bacterial Cells and Input of Allochthonous Organic Carbon; Bergstrom A et al.; In order to compare riverine bacteria input with lake water bacterial production and grazing loss with output loss, a bacterial cell budget was constructed for humic Lake Ortrasket in northern Sweden . The riverine input of bacterial cells in 1997 represented 29% of the number of bacterial cells produced within the layer of the lake affected by inlet water . A large share of the in situ lake bacterial production was consumed by grazers, mainly flagellates, which stresses the importance of bacteria as energy mobilizers for the pelagic food web in the lake . The bacterial production in Lake Ortrasket, which is almost entirely dependent on humic material as an energy source, was clearly stimulated by high flow episodes which brought high amounts of little degraded material into the lake . During base flow condition the bacterial production in the inlet rivers was high, which led to an input of more degraded material to the lake . This material did not stimulate the lake bacterial production . Internal factors that determined the utilization of the allochthonous DOC in the lake were the retention time and the exposure to light and high temperatures . Thus, the potential for in situ production of bacteria in Lake Ortrasket was to a large extent a function of how precipitation and runoff conditions affected terrestrial losses and river transport of humic material.

J Am Anim Hosp Assoc, 2000 May-Jun, 36(3), 239 - 43
Meningoencephalitis secondary to bacterial otitis media/interna in a dog; Spangler EA et al.; Central nervous system (CNS) complications of bacterial otitis media/interna are an infrequent occurrence in human patients and have rarely been reported in the veterinary literature . Early recognition of CNS involvement and the use of appropriate diagnostic tests to characterize the nature of the lesion(s) are crucial in determining the best course of treatment . In this paper, the authors describe a dog with bacterial meningoencephalitis secondary to otitis media/interna.

Pediatr Infect Dis J, 2000 May, 19(5 Suppl), S9 - 15; discussion S15-6
Bacterial otitis media: pathogenetic considerations; Murphy TF; A variety of exciting and important new observations regarding the pathogenesis of nontypable H . influenzae infection have been made in the past decade . The interactions between mucin and OMPs show a high degree of specificity . Multiple adhesins have been identified on the bacterial surface . Colonization of the upper respiratory tract is a dynamic process . Immunodominant, antigenically heterogeneous OMPs are the targets of strain-specific immune responses, accounting in part for the recurrent nature of OM in otitis-prone children . The LOS of nontypable H . influenzae displays a remarkable degree of antigenic and phase variation and may be involved in molecular mimicry of host antigens . Finally nontypable H . influenzae not only lives on the mucosal surface but also clearly has been demonstrated to enter epithelial cells and remain viable in intracellular and intercellular locations in the human upper respiratory tract . These areas of investigation have important implications in understanding the pathogenesis of OM . Elucidating mechanisms of pathogenesis will be important in guiding development of novel ways to prevent OM.

Biochemistry, 2000 May 2, 39(17), 5196 - 205
Biochemical characterization of rat P450 2C11 fused to rat or bacterial NADPH-P450 reductase domains; Helvig C et al.; cDNAs coding for rat P450 2C11 fused to either a bacterial (the NADPH-cytochrome P450 BM3 reductase domain of P450 BM3) or a truncated form of rat NADPH-P450 reductases were expressed in Escherichia coli and characterized enzymatically . Measurements of NADPH cytochrome c reductase activity showed fusion-dependent increases in the rates of cytochrome c reduction by the bacterial or the mammalian flavoprotein (21 and 48%, respectively, of the rates observed with nonfused enzymes) . Neither the bacterial flavoprotein nor the truncated rat reductase supported arachidonic acid metabolism by P450 2C11 . In contrast, fusion of P450 2C11 to either reductase yielded proteins that metabolized arachidonic acid to products similar to those obtained with reconstituted systems containing P450 2C11 and native rat P450 reductase . Addition of a 10-fold molar excess of rat P450 reductase markedly increased the rates of metabolism by both fused and nonfused P450s 2C11 . These increases occurred with preservation of the regioselectivity of arachidonic acid metabolism . The fusion-independent reduction of P450 2C11 by bacterial P450 BM3 reductase was shown by measurements of NADPH-dependent H(2)O(2) formation {73 +/- 10 and 10 +/- 1 nmol of H(2)O(2) formed min(-)(1) (nmol of P450)(-)(1) for the reconstituted and fused protein systems, respectively} . These studies demonstrate that (a) a self-sufficient, catalytically active arachidonate epoxygenase can be constructed by fusing P450 2C11 to mammalian or bacterial P450 reductases and (b) the P450 BM3 reductase interacts efficiently with mammalian P450 2C11 and catalyzes the reduction of the heme iron . However, fusion is required for metabolism and product formation.

Br J Gen Pract, 1999 Nov, 49(448), 913 - 8
Should sexual partners of women with bacterial vaginosis receive treatment?
Potter J.
Bacterial vaginosis is the most prevalent infectious cause of vaginitis . It is associated with significant morbidity, particularly in pregnant women and following gynaecological operations . Cure is difficult . There is some controversy over whether treating sexual partners of affected women can improve cure rates . This paper provides a critical appraisal of the evidence for simultaneously treating the male partner of women affected by bacterial vaginosis . Unfortunately, no evidence was found supporting the treatment of partners of women affected by bacterial vaginosis.

Biotechniques, 2000 May, 28(5), 890 - 2, 894-5
Analysis of recombinant protein expression by MALDI-TOF mass spectrometry of bacterial colonies; Winkler MA et al.; E . coli expressing soluble recombinant HIV antigens were analyzed directly by MALDI-TOF mass spectrometry (MS) from bacterial colonies picked from agar plates . An HIV envelope (ENV) antigen construct, penvA, was expressed in E . coli by transformation of the plasmid pPL/penvA-M . The plasmid was co-transformed into E . coli DH5 alpha cells with an equal quantity of the plasmid pKRR826, the parent vector without the penvA insert, and plated at medium density on L-agar plus ampicillin plates . A total of 24 colonies from four agar plates (six colonies per plate) were picked and transferred into 50% acetonitrile--0.1% trifluoroacetic acid aliquots for analysis by MALDI-TOF MS . The MS analysis detected 10 of 24 colonies expressing the recombinant protein; one colony expressed a mutant penvA protein; eleven of 24 colonies showed ions only from E . coli; and two of 24 colonies showed no detectable proteins . When E . coli transformed only with plasmid pPL/penvA-M were examined, all (10 of 10) colonies showed the penv insert by the MALDI-TOF MS method . The method is fast (less than 1.5 h for 24 colonies) and allows identification of colonies expressing intact or mutant proteins directly from culture plates without sample purification.

Int Surg, 2000 Jan-Mar, 85(1), 27 - 9
Effect of plaunotol on bacterial translocation in the rat small intestine; Sakai T et al.; BACKGROUND AND AIMS: Bacterial translocation is precipitated by an increase in bacteria or endotoxin, depression of the membrane barrier, and an increase in mucosal permeability . Plaunotol is a mucosal protective agent, and observed to have a strong suppressive effect on superoxide production . In this study, the effect of plaunotol on bacterial translocation was examined using the model of ischemia and reperfusion . METHOD: Male Sprague Dawley rats were used to create the following model for evaluation of bacterial translocation: (i) the control group; (ii) the preventive dose group (plaunotol 30 mg/kg/day one week before surgery); (iii) the therapeutic dose group (plaunotol 30 mg/kg/day one week after surgery); and (iv) the full dose group (plaunotol 30 mg/kg/day one week before surgery and one week after surgery) . Bacterial translocation was assessed as the blood concentration of the endotoxin . RESULTS: In the control group, the endotoxin increased significantly 3 days postsurgery (13.7+/-5.6 pg/ml) compared with before surgery (1.1+/-0.1 pg/ml) . In the preventive and full-dose groups, the erndotoxin decreased significantly 3 days postsurgery (4.4+/-2.8 pg/ml, 5.7+/-2.7 pg/ml, respectively) compared with that of the control group . CONCLUSION: Plaunotol in the preventive and full-dose groups decreased the endotoxin . This suggests that plaunotol is one of the protectors for bacterial translocation.

Infect Immun, 2000 Jun, 68(6), 3108 - 15
Enterohemorrhagic Escherichia coli induces apoptosis which augments bacterial binding and phosphatidylethanolamine exposure on the plasma membrane outer leaflet; Barnett Foster D et al.; Enterohemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome . We investigated the ability of EHEC to kill host cells using three human epithelial cell lines . Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death . The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenic E . coli (EPEC) . The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone . Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression . Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding . Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding . These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.

FEMS Microbiol Ecol, 2000 Apr 1, 32(2), 129 - 141
Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE; Iwamoto T et al.; Changes in bacterial diversity during the field experiment on biostimulation were monitored by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments . The results revealed that the bacterial community was disturbed after the start of treatment, continued to change for 45 days or 60 days and then formed a relatively stable community different from the original community structure . DGGE analysis of soluble methane monooxygenase (sMMO) hydroxylase gene fragments, mmoX, was performed to monitor the shifts in the numerically dominant sMMO-containing methanotrophs during the field experiment . Sequence analysis on the mmoX gene fragments from the DGGE bands implied that the biostimulation treatment caused a shift of potential dominant sMMO-containing methanotrophs from type I methanotrophs to type II methanotrophs.

Cell Biochem Funct, 2000 Jun, 18(2), 103 - 8
Salivary biomass assessed by bioluminescence ATP assay related to (bacterial and somatic) cell counts; Gallez F et al.; The present work aimed (1) to evaluate ATP content in saliva by the bioluminescent luciferin-luciferase method, (2) to evaluate the relationships between ATP content, bacterial count and epithelial cell numbers in saliva, (3) to study the effect of two different antiseptics (peroxidase system producing hypothiocyanite and chlorhexidine) on the salivary biomass . In 45 young adults, the salivary ATP content ranged from 8 to 1515 nM . Salivary ATP content was significantly and directly correlated to bacterial count and epithelial cell numbers (Spearman-Rank correlation, P< or =0.001) . Regression analysis allowed the inference of a mean epithelial cell and bacterial ATP content of 152.7 fg and 8.3 fg per cell, respectively . The salivary ATP content decreased significantly to 38 . 8+/-12.3 per cent (mean+/-SEM, N=6) of its initial value after a 30-min incubation in the presence of a peroxidase system producing hypothiocyanite (OSCN(-)) . Chlorhexidine (CHX) reduced salivary ATP content to 52.0+/-16.7 per cent . OSCN(-) did not affect the transformed logarithm of bacterial count but CHX reduced it from 7 . 02+/-0.26 to 0.52+/-0.33 . No effect of OSCN(-) was seen on the ratio of epithelial cell viability while CHX reduced it from 46.7+/-5.1 to 3.9+/-1.1 per cent . It is concluded that the combination of the evaluations of the ATP content and cell numbers in saliva can provide reliable data about the effects of oral antiseptics on salivary biomass .

J Pediatr Surg, 2000 May, 35(5), 692 - 5
Effect of growth hormone, epidermal growth factor, and insulin on bacterial translocation in experimental short bowel syndrome; Eizaguirre I et al.; BACKGROUND/PURPOSE: An adaptive process starts in the remaining intestine after massive resection, and several trophic factors including growth hormone (GH), epidermal growth factor (EGF), and insulin (INS) have been shown to have a positive effect on it . Bacterial translocation (BT) is frequent after extensive small bowel resection, but the effects of GH, EGF, or INS have not been investigated in experimental short bowel syndrome (SBS) . This study tests the hypothesis that GH, EGF, or INS decrease BT in SBS in rats with parenteral nutrition (PN) . METHODS: Thirty-eight adult Wistar rats underwent central venous cannulation and were assigned randomly to 1 of 4 groups receiving for 10 days 4 treatment regimes: (1) PN group (n = 10): fasting, all-in-one PN solution (300 mL/kg/24 h, 280 kcal/kg/24 h), 80% gut resection including ileo-cecal valve; (2) GH group (n = 9): fasting, same PN regime and resection, GH (1 mg/kg/d, subcutaneously); (3) EGF group (n = 9): fasting, PN, resection, EGF (150 microg/24 h intravenously); (4) INS group (n = 9): fasting, PN, resection, INS (1 UI/100 g/24 h subcutaneously) . At the end of the experiment they were killed, and mesenteric lymph nodes (MLN) and peripheral and portal blood samples were recovered and cultured . Several fragments of intestine were taken to determine cell proliferation (PCNA index) and morphometric parameters (villous height, crypt depth) . RESULTS: GH, EGF, and INS groups showed a 28%, 29%, and 30% increase in gut mucosal thickness, and PCNA index rose 21%, 20%, and 25%, respectively in comparison with PN controls . Bacterial translocation to peripheral blood was detected in 0% of PN animals and in 44%, 40%, and 28% of GH, EGF, or INS rats, respectively (P < .05) . No differences were found in BT in MLN or portal blood among groups . CONCLUSION: Administration of GH, EGF, or INS improves gut mucosal structure in rats with SBS under PN, but, surprisingly, the incidence of BT detected in peripheral blood was increased rather than decreased in animals receiving these treatments.

Biochim Biophys Acta, 2000 May 12, 1458(1), 148 - 63
Proton and electron transfer in bacterial reaction centers; Okamura MY et al.; The bacterial reaction center couples light-induced electron transfer to proton pumping across the membrane by reactions of a quinone molecule Q(B) that binds two electrons and two protons at the active site . This article reviews recent experimental work on the mechanism of the proton-coupled electron transfer and the pathways for proton transfer to the Q(B) site . The mechanism of the first electron transfer, k((1))(AB), Q(-)(A)Q(B)-->Q(A)Q(-)(B), was shown to be rate limited by conformational gating . The mechanism of the second electron transfer, k((2))(AB), was shown to involve rapid reversible proton transfer to the semiquinone followed by rate-limiting electron transfer, H(+)+Q(-)(A)Q(-)(B) ifQ(-)(A)Q(B)H-->Q(A)(Q(B)H)(-) . The pathways for transfer of the first and second protons were elucidated by high-resolution X-ray crystallography as well as kinetic studies showing changes in the rate of proton transfer due to site directed mutations and metal ion binding.

Anticancer Res, 2000 Mar-Apr, 20(2B), 1221 - 8
Impact of bacterial eradication on the cell proliferation and p53 protein accumulation in Helicobacter pylori-associated gastritis; Hsu PI et al.; BACKGROUND: This study investigates the cell proliferation and the expression of p53 protein in Helicobacter pylori (H . pylori)-associated gastritis and assesses the effect of bacterial eradication on these epithelial factors . MATERIAL AND METHODS: Seventy-nine patients with H . pylori-associated gastritis were randomized into the control group (n = 38) and anti-H . pylori group (n = 41) . Each patient received endoscopic examinations with gastric biopsy before and 8 weeks after the treatment . The specimens from gastric antrum were immunostained for monoclonal antibodies against the proliferating cell nuclear antigen (PCNA) and p53 protein . RESULTS: In the control group, the total labeling index (L.I.) of PCNA and the positive index (P.I.) of p53 in the whole foveolar epithelium were unchanged after treatment . In the anti-H . pylori group, 35 of 41 cases (85.3%) achieved eradication of H . pylori . Amongst the H . pylori-eradicated cases, the total L.I . of PCNA in the whole foveolar epithelium did not meaningfully alter after H . pylori elimination (p > 0.05) . However, a significant reduction of L.I . was observed in the middle compartments of the gastric pits (before vs . after treatment: 14.0 vs . 7.3, p < 0.05) . With regard to the p53 expression, the P.I.s were significantly decreased in the whole foveolar epithelium (before vs . after treatment: 0.57 vs . 0.17, p < 0.05) and in each compartment of the gastric pits (before vs . after treatment: {upper compartment}: 0.34 vs . 0.15, p < 0.05; {middle compartment}: 0.67 vs . 0.23, p < 0.05; {lower compartment}: 0.71 vs . 0.20, p < 0.05) after eradication of H . pylori . CONCLUSIONS: Bacterial eradication reverses the hyperproliferating status of the foveolar epithelium in patients with H . pylori gastritis and leads to a decrease in p53 accumulation in the epithelial cells.

J Bacteriol, 2000 Jun, 182(11), 3037 - 44
mRNA composition and control of bacterial gene expression; Liang ST et al.; The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA . This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes . To characterize the effects of mRNA competition on gene expression, the specific activity of beta-galactosidase expressed from three different promoter-lacZ fusions (P(spc)-lacZ, P(RNAI)-lacZ, and P(RNAII)-lacZ) was measured (i) in a relA(+) background during exponential growth at different rates and (ii) in relA(+) and DeltarelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp . Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp . From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp . These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp.

Crit Care Med, 2000 Apr, 28(4), 1027 - 32
Dependency of cerebral blood flow on mean arterial pressure in patients with acute bacterial meningitis; Moller K et al.; OBJECTIVE: Patients with acute bacterial meningitis are often treated with sympathomimetics to maintain an adequate mean arterial pressure (MAP) . We studied the influence of such therapy on cerebral blood flow (CBF) . DESIGN: Prospective physiologic trial . SETTING: The Department of Infectious Diseases, Copenhagen University Hospital, Denmark . PATIENTS: Sixteen adult patients with acute bacterial meningitis . INTERVENTION: Infusion of norepinephrine to increase MAP . MEASUREMENTS: During a rise in MAP induced by norepinephrine infusion, we measured relative changes in CBF by transcranial Doppler ultrasonography of the middle cerebral artery, recording mean flow velocity (Vmean), and by the arterial to jugular oxygen saturation difference . In 10 out of 16 patients, serial measurements were performed until recovery or death . Individual autoregulation curves were analyzed by a computer program . Autoregulation was classified as impaired if Vmean increased by >10% per 30 mm Hg increase in MAP and if no lower limit of autoregulation was identified by the computer program; otherwise, autoregulation was classified as preserved . MAIN RESULTS: Initially, Vmean increased from a median value of 46 cm/sec (range, 30-87 cm/sec) to 63 cm/sec (33-105 cm/sec) (p < .0001), and arterial to jugular oxygen saturation difference decreased from 0.28 (0.16-0.51) to 0.21 (0.08-0.39) (p < .001) when MAP was raised from 69 mm Hg (55-102 mm Hg) to 110 mm Hg (93-129 mm Hg) . CBF autoregulation was restored in eight of ten patients undergoing serial examination after 7 (range, 2-10) days . Six of these patients had an uncomplicated course, one had a protracted recovery, and one died . Autoregulation was not restored in two patients; one died and one had a protracted recovery . CONCLUSION: In patients in the early phase of acute bacterial meningitis, CBF autoregulation is impaired . With recovery from meningitis, the cerebral vasculature regains the ability to maintain cerebral perfusion at a constant level despite variations in MAP.

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5516 - 21
Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome; Almazan F et al.; The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria . Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules . A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome . The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus . The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm . Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed . The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine . During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate . The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified . This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Anal Biochem, 2000 Apr 10, 280(1), 94 - 102
Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin--biotin binding; Yang Q et al.; In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis . The immobilized amount was up to 80 nmol of RC and 33 micromol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel . By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated . The dissociation constant for the RC-cyt c interaction was determined to be 0.20-0.57 microM . QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC . It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.

J Biol Chem, 2000 Jul 21, 275(29), 22090 - 7
Interaction between RNA polymerase and RapA, a bacterial homolog of the SWI/SNF protein family; Sukhodolets MV et al.; Recently, we identified a novel Escherichia coli RNA polymerase (RNAP)-associated protein, an ATPase, called RapA (Sukhodolets, M . V . , and Jin, D . J . (1998) J . Biol . Chem . 273, 7018-7023) . RapA is a bacterial homolog of SWI2/SNF2 . We showed that RapA forms a stable complex with RNAP holoenzyme and that binding to RNAP holoenzyme stimulates the ATPase activity of RapA . We have further analyzed the interactions between purified RapA and the two forms of RNAP: core RNAP and RNAP holoenzyme . We found that RapA interacts with either form of RNAP . However, RapA exhibits higher affinity for core RNAP than for RNAP holoenzyme . Chemical cross-linking of the RNAP-RapA complex indicated that the RapA-binding sites are located at the interface between the alpha and beta' subunits of RNAP . Contrary to previously reported results (Muzzin, O., Campbell, E., A., Xia, L., Severinova, E., Darst, S . A., and Severinov, K . (1998) J . Biol . Chem . 273, 15157-15161), our in vivo analysis of a rapA null mutant suggested that RapA is not likely to be directly involved in DNA repair.

Curr Biol, 2000 May 4, 10(9), 507 - 16
A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus; Colletti KS et al.; BACKGROUND: Chloroplast division in plant cells occurs by binary fission, yielding two daughter plastids of equal size . Previously, we reported that two Arabidopsis homologues of FtsZ, a bacterial protein that forms a cytokinetic ring during cell division, are essential for plastid division in plants, and may be involved in the formation of plastid-dividing rings on both the stromal and cytosolic surfaces of the chloroplast envelope membranes . In bacteria, positioning of the FtsZ ring at the center of the cell is mediated in part by the protein MinD . Here, we identified AtMinD1, an Arabidopsis homologue of MinD, and investigated whether positioning of the plastid-division apparatus at the plastid midpoint might involve a mechanism similar to that in bacteria . RESULTS: Sequence analysis and in vitro chloroplast import experiments indicated that AtMinD1 contains a transit peptide that targets it to the chloroplast . Transgenic Arabidopsis plants with reduced AtMinD1 expression exhibited variability in chloroplast size and number and asymmetrically constricted chloroplasts, strongly suggesting that the plastid-division machinery is misplaced . Overexpression of AtMinD1 inhibited chloroplast division . These phenotypes resemble those of bacterial mutants with altered minD expression . CONCLUSIONS: Placement of the plastid-division machinery at the organelle midpoint requires a plastid-targeted form of MinD . The results are consistent with a model whereby assembly of the division apparatus is initiated inside the chloroplast by the plastidic form of FtsZ, and suggest that positioning of the cytosolic components of the apparatus is specified by the position of the plastidic components.

J Mol Biol, 2000 May 19, 298(5), 737 - 48
The bacterial DNA-binding protein H-NS represses ribosomal RNA transcription by trapping RNA polymerase in the initiation complex; Schroder O et al.; The interaction of the bacterial regulatory protein H-NS with RNA polymerase and the ribosomal RNA P1 promoter was analyzed to better understand the mechanism of H-NS-dependent transcriptional repression . We could show that initial binding of RNA polymerase to the promoter was not inhibited by the simultaneous interaction of H-NS, although H-NS binding sites extend into the core promoter region . Binding of sigma(70)-saturated RNA polymerase and H-NS to the promoter DNA occurs cooperatively and results in a stable complex of slower gel electrophoretic mobility as compared to complexes formed with the single proteins . The presence of the upstream curved H-NS binding site contributes strongly to the cooperative RNA polymerase-promoter interaction . By KMnO(4) modification of single-stranded template nucleotides we could show that open complex formation at the rrnB P1 promoter was not inhibited by H-NS binding . An increased KMnO(4) reactivity of several positions within the open complex rather supports the view that open complex formation is stimulated in presence of H-NS . Moreover, subtle changes in the modification pattern indicate that the open complex formed in the presence of H-NS are structurally distinct from the H-NS-free complex . In vitro transcriptional analysis of the abortive and productive yields revealed that the formation of transcription products longer than three nucleotides is dramatically reduced in the presence of H-NS, while the amount of shorter abortive products remained unaffected . Together the results demonstrate that H-NS inhibits transcription at the rrnB P1 promoter not by interfering with initial RNA polymerase binding but by blocking chain elongation steps subsequent to the first (two) phosphodiester bond formations . The mechanism of H-NS dependent repression at rRNA promoters can thus be explained as a trap which inhibits substrate NTP incorporation beyond template position +3 into the initial transcribing complex .

Trends Ecol Evol, 2000 Jun, 15(6), 243 - 247
The emergence and maintenance of diversity: insights from experimental bacterial populations; Rainey PB et al.; Mechanisms maintaining genetic and phenotypic variation in natural populations are central issues in ecology and evolution . However, the long generation times of most organisms and the complexity of natural environments have made elucidation of ecological and evolutionary mechanisms difficult . Experiments using bacterial populations propagated in controlled environments reduce ecosystem complexity to the point where understanding simple processes in isolation becomes possible . Recent studies reveal the circumstances and mechanisms that promote the emergence of stable polymorphisms.

Biochem Pharmacol, 2000 Jun 15, 59(12), 1583 - 8
Expression of human mitochondrial thymidine kinase in Escherichia coli: correlation between the enzymatic activity of pyrimidine nucleoside analogues and their inhibitory effect on bacterial growth; Wang J et al.; Mitochondrial thymidine kinase (TK2) phosphorylates pyrimidine nucleosides to monophosphates and is expressed constitutively through the cell cycle in all cells . Because of the overlap of its substrate specificity with that of the cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), it has been difficult to determine the role of TK2 in activating nucleosides used in chemotherapy . In this report, we described the construction of a recombinant Escherichia coli strain which could be used to test if TK2 activity is limiting for the toxicity of nucleosides . Enzymes of bacterial origin which are involved in thymidine and deoxyuridine anabolism and catabolism were eliminated, and the cDNA for human TK2 was introduced . In the crude extract of the engineered E . coli, the level of thymidine kinase was, after induction of TK2 expression, several hundred fold higher than in the control strain . Several pharmacologically interesting nucleoside analogues, including 3'-azidothymidine, 2',3'-didehydro-2',3'-dideoxythymidine, and 2', 3'-dideoxy-beta-L-3'-thiacytidine, were tested for their effects on the growth of this recombinant strain . For a comparison, the phosphorylation of these compounds was determined with purified recombinant TK1, TK2, and dCK . A correlation was observed between the phosphorylation of several of these compounds by TK2 and their effects on bacterial growth . These results demonstrate that activation of growth-inhibiting pyrimidine nucleosides can be catalyzed by TK2, and together with recombinant E . coli strains expressing other cellular nucleoside kinases, this whole-cell bacterial system may serve as a tool to predict the efficacy and side effects of chemotherapeutic nucleosides.

Indian J Pediatr, 1999 Jan-Feb, 66(1), 7 - 10
Effect of handwashing agents on bacterial contamination; Anuradha P et al.; In order to assess the effect of handwashing agents like soap and ash on the control of bacterial contamination, the study was carried out in two villages of Rajendranagar mandal of Rangareddy district, Andhra Pradesh . Twenty households belonging to high income group and twenty households belonging to low income group having 1-2 years age children were randomly selected for the study handwash samples . Before feeding the child handwash samples after washing with different agents were collected and analysed for bacterial contamination . The study revealed that use of soap or ash for washing hands before feeding the child reduced hand contamination significantly.

Stroke, 2000 May, 31(5), 1116 - 22
Effect of short-term hyperventilation on cerebral blood flow autoregulation in patients with acute bacterial meningitis; Moller K et al.; BACKGROUND AND PURPOSE: Cerebral blood flow (CBF) autoregulation is impaired in patients with acute bacterial meningitis: this may be caused by cerebral arteriolar dilatation . We tested the hypothesis that CBF autoregulation is recovered by acute mechanical hyperventilation in 9 adult patients with acute bacterial meningitis . METHODS: Norepinephrine was infused to increase mean arterial pressure (MAP) 30 mm Hg from baseline . Relative changes in CBF were concomitantly recorded by transcranial Doppler ultrasonography of the middle cerebral artery, measuring mean flow velocity (V(mean)), and by measurement of arterial to jugular oxygen content difference (a-v DO(2)) . The slope of the regression line between MAP and V(mean) was calculated . Measurements were performed during normoventilation and repeated after 30 minutes of mechanical hyperventilation . RESULTS: At normoventilation (median PaCO(2) 4.4 kPa, range 3.5 to 4.9), MAP was increased from 68 mm Hg (60 to 101) to 109 mm Hg (95 to 126) . V(mean) increased with MAP from 48 cm/s (30 to 61) to 65 cm/s(33 to 86) (P<0.01), and a-v DO(2) decreased from 2.2 mmol/L (1.0 to 2.7) to 1.4 mmol/L (0.8 to 1.8) (P<0.05) . During hyperventilation (PaCO(2) 3.5 kPa, range 3.3 to 4.1), MAP was increased from 76 mm Hg (58 to 92) to 109 mm Hg (95 to 121) . V(mean) increased from 45 cm/s (29 to 55) to 53 cm/s (33 to 78) (P<0.01), and a-v DO(2) decreased from 2.5 mmol/L (1.8 to 3.0) to 1.8 mmol/L (1.2 to 2.4) (P<0.05) . Four patients recovered autoregulation completely during hyperventilation . The slope of the autoregulation curve decreased during hyperventilation compared with normoventilation (P<0.05) . CONCLUSIONS: CBF autoregulation is partially recovered during short-term mechanical hyperventilation in patients with acute bacterial meningitis, indicating that cerebral arteriolar dilation in part accounts for the regulatory impairment of CBF in these patients.

J Mol Evol, 2000 Apr, 50(4), 366 - 80
Phylogenetic depth of the bacterial genera Aquifex and Thermotoga inferred from analysis of ribosomal protein, elongation factor, and RNA polymerase subunit sequences; Bocchetta M et al.; The phylogenetic placement of the Aquifex and Thermotoga lineages has been inferred from (i) the concatenated ribosomal proteins S10, L3, L4, L23, L2, S19, L22, and S3 encoded in the S10 operon (833 aa positions); (ii) the joint sequences of the elongation factors Tu(1alpha) and G(2) coded by the str operon tuf and fus genes (733 aa positions); and (iii) the joint RNA polymerase beta- and beta'-type subunits encoded in the rpoBC operon (1130 aa positions) . Phylogenies of r-protein and EF sequences support with moderate (r-proteins) to high statistical confidence (EFs) the placement of the two hyperthermophiles at the base of the bacterial clade in agreement with phylogenies of rRNA sequences . In the more robust EF-based phylogenies, the branching of Aquifex and Thermotoga below the successive bacterial lineages is given at bootstrap proportions of 82% (maximum likelihood; ML) and 85% (maximum parsimony; MP), in contrast to the trees inferred from the separate EF-Tu(1alpha) and EF-G(2) data sets, which lack both resolution and statistical robustness . In the EF analysis MP outperforms ML in discriminating (at the 0.05 level) trees having A . pyrophilus and T . maritima as the most basal lineages from competing alternatives that have (i) mesophiles, or the Thermus genus, as the deepest bacterial radiation and (ii) a monophyletic A . pyrophilus-T . maritima cluster situated at the base of the bacterial clade . RNAP-based phylogenies are equivocal with respect to the Aquifex and Thermotoga placements . The two hyperthermophiles fall basal to all other bacterial phyla when potential artifacts contributed by the compositionally biased and fast-evolving Mycoplasma genitalium and Mycoplasma pneumoniae sequences are eschewed . However, the branching order of the phyla is tenuously supported in ML trees inferred by the exhaustive search method and is unresolved in ML trees inferred by the quartet puzzling algorithm . A rooting of the RNA polymerase-subunit tree at the mycoplasma level seen in both the MP trees and the ML trees reconstructed with suboptimal amino acid substitution models is not supported by the EF-based phylogenies which robustly affiliate mycoplasmas with low-G+C gram-positives and, most probably, reflects a "long branch attraction" artifact.

Arch Microbiol, 2000 Feb, 173(2), 146 - 53
Isolation and characterization of two new methanesulfonic acid-degrading bacterial isolates from a Portuguese soil sample; De Marco P et al.; Two novel bacterial strains that can utilize methanesulfonic acid as a source of carbon and energy were isolated from a soil sample collected in northern Portugal . Morphological, physiological, biochemical and molecular biological characterization of the two isolates indicate that strain P1 is a pink-pigmented facultative methylotroph belonging to the genus Methylobacterium, while strain P2 is a restricted methylotroph belonging to the genus Hyphomicrobium . Both strains are strictly aerobic, degrade methanesulfonate, and release small quantities of sulfite into the medium . Growth on methanesulfonate induces a specific polypeptide profile in each strain . This, together with the positive hybridization to a DNA probe that carries the msm genes of Methylosulfonomonas methylovora strain M2, strongly endorses the contention that a methanesulfonic acid monooxygenase related to that found in the previously known methanesulfonate-utilizing bacteria is present in strains P1 and P2 . The isolation of bacteria containing conserved msm genes from diverse environments and geographical locations supports the hypothesis that a common enzyme may be globally responsible for the oxidation of methanesulfonate by natural methylotrophic communities.

Arch Microbiol, 2000 Feb, 173(2), 138 - 45
Successional changes in the genetic diversity of a marine bacterial assemblage during confinement; Schafer H et al.; The successional changes in the genetic diversity of Mediterranean bacterioplankton subjected to confinement were studied in an experimental 300 1 seawater enclosure . Five samples were taken at different times and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting to rapidly monitor changes in the bacterial genetic diversity . DGGE analysis clearly showed variations between the samples . Three of the five samples, with different DGGE banding patterns, were further analyzed by cloning and sequencing of 16S rRNA genes . Comparative sequence analysis indicated a shift from a mixed bacterial assemblage to a community dominated by bacteria closely affiliated to a single genus, Alteromonas . Sequences obtained at the start of the experiment were affiliated with two alpha-proteobacterial and three gamma-proteobacterial lineages known from other studies of marine picoplankton . One sequence was affiliated with the Verrucomicrobiales . After 161 h of incubation two sequences represented a gamma-proteobacterial lineage also present at 0 h, but the majority of sequences clustered around that of Alteromonas macleodii . After 281 h only the dominant Alteromonas-like bacteria and bacteria distantly related to Legionella were found by cloning and sequencing . Mortality rates of bacteria indicated that grazing was the dominant mortality process when heterotrophic protozoa were abundant . Hence, changes in the genetic diversity of bacteria were partly influenced by the differential mortality of bacterial populations during the course of incubation.

Int J Oral Maxillofac Implants, 2000 Mar-Apr, 15(2), 283 - 6
Tissue reactions, fluids, and bacterial infiltration in implants retrieved at autopsy: a case report; Orsini G et al.; A 72-year-old patient underwent the placement of 2 screw-type implants . After 5 months the patient died of a massive stroke, and a block section of the portion of the mandible containing the implants was done . The specimen was treated to obtain thin ground sections . A 1- to 5-micron gap was present between the implant and the healing cover screw, and this space was filled by bacteria and calculus; bacteria were also present in the most apical portion of the hollow part of the implant . An inflammatory infiltrate was present in the connective peri-implant tissues . The spaces between all implant components (implant, abutment, and healing screw) can act as conduits and reservoirs for bacteria, which could cause inflammation of the peri-implant soft tissues . In conclusion, the histologic data from this autopsy case may help to confirm the penetration by fluids and bacteria into the internal portion of the implants, obtained from previous in vitro and in vivo studies.

Lett Appl Microbiol, 2000 May, 30(5), 390 - 5
The relationship between hide cleanliness and bacterial numbers on beef carcasses at a commercial abattoir; McEvoy JM et al.; Cattle were visually inspected in the lairage of a commercial abattoir and assigned to a category ranging from 1 (very clean) to 5 (very dirty) depending on the observed cleanliness of the hide . Animals from categories 2, 3 and 5 were slaughtered and total viable counts (TVCs) enumerated at five sites (hock, brisket, cranial back, bung and inside round) on the subsequent carcasses . The TVCs at the hock were significantly higher on category 5 than on category 2 carcasses (P < 0.05) . Similarly, TVCs at the brisket were significantly higher on categories 3 and 5 than on category 2 carcasses (P < 0.05) . There were no differences in counts among the categories at any of the other sites . The TVCs averaged over the five carcass sites were higher for category 5 than for category 2 carcasses (P < 0.05) . The TVCs at the brisket were significantly higher than all other sites (P < 0.01) . In general, carcasses from category 2 animals slaughtered in a batch with dirtier animals (categories 3 and 5) did not have higher TVCs than carcasses of category 2 animals slaughtered at the beginning of the day in the absence of dirtier animals . The introduction of improved hygienic practices during the dehiding of category 4 animals resulted in reduced TVCs at the brisket (P < 0.001).

Lett Appl Microbiol, 2000 Apr, 30(4), 312 - 6
Rapid detection of malto-oligosaccharide-forming bacterial amylases by high performance anion-exchange chromatography; Duedahl-Olesen L et al.; High performance anion-exchange chromatography with pulsed amperometric detection was applied for the rapid analysis of malto-oligosaccharides formed by extracellular enzyme preparations from 49 starch-degrading bacterial strains isolated from soil and compost samples . Malto-oligosaccharide-forming amylases, indicated by a predominant formation of maltohexaose from starch, were produced by enzyme preparations from four of the isolates growing at pH 7.0 and 10.

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5197 - 202
The long-range supraorganization of the bacterial photosynthetic unit: A key role for PufX; Frese RN et al.; Bacterial photosynthesis relies on the interplay between light harvesting and electron transfer complexes, all of which are located within the intracytoplasmic membrane . These complexes capture and transfer solar energy, which is used to generate a proton gradient . In this study, we identify one of the factors that determines the organization of these complexes . We undertook a comparison of the organization of the light-harvesting complex 1 (LH1)/reaction center (RC) cores in the LH2(-) mutant of Rhodobacter sphaeroides in the presence or absence of the PufX protein . From polarized absorption spectra on oriented membranes, we conclude that PufX induces a specific orientation of the reaction center in the LH1 ring, as well as the formation of a long-range regular array of LH1-RC cores in the photosynthetic membrane . From our data, we have constructed a precise model of how the RC is positioned within the LH1 ring relative to the long (orientation) axis of the photosynthetic membrane.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 171 - 4
Is the accumulation of osmoprotectant the unique mechanism involved in bacterial osmoprotection?
Gouffi K, Blanco C.
Sucrose, trehalose, maltose, cellobiose, gentiobiose, turanose and palatinose are very unusual osmoprotectants for Sinorhizobium meliloti, because these compounds, unlike other bacterial osmoprotectants, do not accumulate as cytosolic osmolytes in salt-stressed S . meliloti cells . Rather, these compounds were catabolized during early exponential growth, and contributed to enhance the cytosolic levels of the two endogenously-synthesized osmolytes: glutamate and the dipeptide N-acetylglutaminylglutamine amide (NAGGN) . Furthermore, all of the disaccharides that acted as powerful osmoprotectants shared the same uptake routes in S . meliloti . Here, we show that these disaccharides, in fact, belong to a new family of non-accumulated sinorhizobial osmoprotectants and that two mechanisms of osmoprotection coexist in S . meliloti.

Microb Ecol, 2000 Jan, 39(1), 32 - 40
Stability in Natural Bacterial Communities: I . Nutrient Addition Effects on Rhizosphere Diazotroph Assemblage Composition; Piceno YM et al.; The ability of rhizosphere diazotrophs to remain competitive during increased nitrogen availability in situ was tested in a salt marsh grass stand . Nitrogen (16.3 g m(-2)) or nitrogen (16.3 g m(-2)) and phosphorus (18.0 g m(-2)) were added to plots of short form Spartina alterniflora for either 2 weeks or 8 weeks . The diazotroph assemblage composition was monitored via the polymerase chain reaction using nifH specific primers followed by denaturing gradient gel electrophoresis (DGGE) analysis . DGGE profiles from the short-term experiments (2 and 8 weeks) were compared to profiles from control (no additions) and from long-term (>10 y) nutrient addition plots . Nitrogen fixation activity was assayed in each short-term treatment and control plot using an acetylene reduction technique . The control and nutrient addition DGGE profiles were very similar throughout the short-term experiments . One DGGE band that was prominent in the control plots was not found in the long-term nutrient addition plots . Diazotrophy may provide a competitive advantage for some species in this system, as indicated by results from the long-term nutrient amended plots . However, the rhizosphere environment in situ appears to limit the immediate impacts of increased nutrient availability on the diazotroph assemblage composition . Results from the short-term nutrient amended plots showed no measurable effect on the diazotroph assemblage . These results indicate substantial short-term stability of the diazotroph assemblage composition, but the potential for change in the face of long-term changes in nutrient availability . </hea

AJR Am J Roentgenol, 2000 May, 174(5), 1385 - 90
Dynamic contrast-enhanced MR imaging of bacterial abscess and VX2 carcinoma in rabbits: comparison of gadopentetate dimeglumine and a macromolecular contrast agent; Moon WK et al.; OBJECTIVE: The objective of this study was to compare enhancement patterns of a blood-pool contrast agent, Gadomer-17, with those of gadopentetate dimeglumine in bacterial abscesses and VX2 carcinoma in rabbits . MATERIALS AND METHODS: Fourteen rabbits with experimentally induced bacterial abscesses and VX2 carcinoma in both thighs underwent dynamic contrast-enhanced MR imaging with Gadomer-17 and gadopentetate dimeglumine at a 24-hr interval . The enhancement ratios (postcontrast to precontrast signal intensities) of lesions in the same animal were assessed and correlated with microvessel density . RESULTS: For Gadomer-17, the enhancement ratio of the abscesses (1.66 +/- 0.39) peaked 15 min after the injection, while that of the carcinoma (2.05 +/- 0.16) peaked at 10 min . The enhancement ratios of the carcinoma were consistently higher than those of the abscesses up to 30 min . For gadopentetate dimeglumine, peak enhancement ratio of the abscesses (2.30 +/- 0.75) was seen 5 min after the injection, while that of the carcinoma (2.32 +/- 0.51) was seen at 3 min . The enhancement ratios of the carcinomas were significantly higher at 1 min, but significantly lower at 20-30 min, compared with those of the abscesses, as a result of rapid decrease of enhancement ratios in the carcinomas . The microvessel density was 9.8 +/- 5.2 vessels per field of view for the abscesses and 36.3 +/- 9.5 vessels per field of view for the carcinoma (p < 0.001) . CONCLUSION: Delayed peak enhancement and slow decay were found in both bacterial abscess and VX2 carcinoma with Gadomer-17, whereas early peak enhancement and rapid decay were found especially in VX2 carcinoma with gadopentetate dimeglumine . Enhancement ratios on MR imaging with a blood-pool contrast agent correlated well with the microvessel density in bacterial abscess and VX2 carcinoma.

Appl Environ Microbiol, 2000 May, 66(5), 1947 - 52
A novel amidase (half-amidase) for half-amide hydrolysis involved in the bacterial metabolism of cyclic imides; Soong CL et al.; A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp . strain A17p-4 . The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia . Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases . The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K(m) = 6.2 mM; k(cat) = 5.76 s(-1)) and glutaramic acid (K(m) = 2.8 mM; k(cat) = 2.23 s(-1)) . However, the substrates of known amidases such as short-chain (C(2) to C(4)) aliphatic amides, long-chain (above C(16)) aliphatic amides, amino acid amides, aliphatic diamides, alpha-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme . Based on its high specificity toward half-amides, the enzyme was named half-amidase . This half-amidase exists as a monomer with an M(r) of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.

Proc Int Conf Intell Syst Mol Biol . 1999;:223-33.
Building dictionaries of 1D and 3D motifs by mining the Unaligned 1D sequences of 17 archaeal and bacterial genomes; Rigoutsos I et al.; We have used the Teiresias algorithm to carry out unsupervised pattern discovery in a database containing the unaligned ORFs from the 17 publicly available complete archaeal and bacterial genomes and build a 1D dictionary of motifs . These motifs which we refer to as seqlets account for and cover 97.88% of this genomic input at the level of amino acid positions . Each of the seqlets in this 1D dictionary was located among the sequences in Release 38.0 of the Protein Data Bank and the structural fragments corresponding to each seqlet's instances were identified and aligned in three dimensions: those of the seqlets that resulted in RMSD errors below a pre-selected threshold of 2.5 Angstroms were entered in a 3D dictionary of structurally conserved seqlets . These two dictionaries can be thought of as cross-indices that facilitate the tackling of tasks such as automated functional annotation of genomic sequences, local homology identification, local structure characterization, comparative genomics, etc.

Neurol Med Chir (Tokyo), 2000 Feb, 40(2), 98 - 104; discussion 104-5
Treatment of bacterial brain abscess by repeated aspiration--follow up by serial computed tomography; Yamamoto M et al.; Bacterial brain abscess often requires repeated aspiration before the abscess finally resolves . However, there are no guidelines for treatment by aspiration; for example, when should the abscess be tapped again, or when can an abscess be treated by antibiotics alone without further aspiration . Eleven patients with bacterial brain abscess treated by aspiration were evaluated to establish treatment guidelines for brain abscess, in particular the abscess size on serial computed tomography (CT) after aspiration . CT was performed about 24 hours after aspiration to evaluate the size of the abscess, and almost weekly during follow up . The diameter of the brain abscess before and after the initial and last aspirations were reviewed . In eight of the 11 patients, abscesses were aspirated repeatedly: two to three times in most patients . The diameter of the abscesses was 2.5-4.5 cm (mean 3.5 cm) before the last aspiration, and 1.4-3.4 cm (mean 2.3 cm) after the last aspiration, or when continuous drainage was discontinued . Perifocal edema was moderately decreased within 3 weeks after the last aspiration by medical treatment alone, with a concomitant decrease in the volume of the abscess . There were no deaths, and most patients had a favorable outcome . These results suggest that after the diameter of the abscess becomes less than 2 to 3 cm and does not increase anymore on serial CT, medical treatment alone can be anticipated to give satisfactory results without further aspiration.

Nurs Stand, 1999 Sep 29-Oct 5, 14(2), 33 - 5
Bacterial contamination of curtains in clinical areas; Palmer R; A small-scale study carried out by student nurses revealed ward curtains to be a source of contaminants and bacteria, including MRSA . Patients and medical staff can contaminate and be contaminated by bacteria which may be a source of cross-infection . Recommendations from the study included the promotion of further research in this area and more frequent laundering of curtains.

Eur J Biochem, 2000 May, 267(9), 2599 - 608
Bacterial-injection-induced syntheses of N-beta-alanyldopamine and Dopa decarboxylase in the hemolymph of coleopteran insect, Tenebrio molitor larvae; Kim MH et al.; Injection of Escherichia coli into larvae of the coleopteran Tenebrio molitor resulted in the appearance of a dopamine-like substance on the electrochemical detector . To characterize this dopamine-like substance, we purified it to homogeneity from the immunized hemolymph and determined its molecular structure to be N-beta-alanyldopamine using the liquid chromatographic/tandem mass spectrometric method . Chemically synthesized N-beta-alanyldopamine showed the same retention time on HPLC as the purified N-beta-alanyldopamine from immunized larvae . To elucidate the molecular mechanism of N-beta-alanyldopamine synthesis in vivo, we examined the enzyme activity of Dopa decarboxylase against E . coli-injected hemolymph of T . molitor larvae . The enzyme activity of Dopa decarboxylase increased dramatically approximately 8 h after injection; Dopa decarboxylase activity of injected larvae being 10-times higher than naive larvae after 24 h . To evaluate the extent of quantitative changes of Dopa decarboxylase in response to bacterial challenge, Tenebrio Dopa decarboxylase was purified to homogeneity from the whole larvae and a cDNA clone for Tenebrio Dopa decarboxylase was isolated . RNA blot hybridization revealed that expression of the Dopa decarboxylase gene was activated transiently 3-8 h after E . coli challenge . Immunoprecipitation experiments showed that Tenebrio Dopa decarboxylase was detected from 8 to 24 h in E . coli-injected larval extract . Thus, bacterial injection into T . molitor larvae might induce transcriptional activation of a Dopa decarboxylase gene, and then synthesis of N-beta-alanyldopamine . The synthesized N-beta-alanyldopamine might be used as a substrate by phenoloxidase during melanin synthesis in the humoral defense response or the melanotic encapsulation reaction of the cellular defense response.

J Soc Biol, 1999, 193(6), 451 - 6
{Bacterial toxins: useful for studying G-proteins implicated in the mechanism of exocytosis in neuroendocrine cells}; Gasman S et al.; In neuroendocrine cells, regulated exocytosis is a multistep process that comprises the recruitment and priming of secretory granules, their docking to the exocytotic sites, and the subsequent fusion of granules with the plasma membrane leading to the release of secretory products into the extracellular space . Using bacterial toxins which specially inactivate subsets of G proteins, we were able to demonstrate that both trimeric and monomeric G proteins directly control the late stages of exocytosis in chromaffin cells . Indeed, in secretagogue-stimulated chromaffin cells, the subplasmalemmal actin cytoskeleton undergoes a specific reorganization that is a prerequisite for exocytosis . Our results suggest that a granule-bound trimeric Go protein controls the actin network surrounding secretory granules through a pathway involving the GTPase RhoA and a downstream phosphatidylinositol 4-kinase . Furthermore, the GTPase Cdc42 plays a active role in exocytosis, most likely by providing specific actin structures to the late docking and/or fusion steps . We propose that G proteins tightly control secretion in neuroendocrine cells by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis . Our data highlight the use of bacterial toxins, which proved to be powerful tools to dissect the exocytotic machinery at the molecular level.

MMW Fortschr Med, 2000 Jan 20, 142(3 Suppl), 187 - 94
{Amoxicillin/clavulanic acid in therapy of bacterial infections of the diabetic foot . Results of an observational study}; Gerards V et al.; The results of an observational study of amoxicillin/clavulanic acid (Augmentan) in 191 patients are presented . The average duration of treatment was 15 days (median) . Healing or improvement was observed in 76% of the cases . In view of the fact that the condition often has a multifactorial genesis and neural and vascular changes impair healing, this result must be considered a clinical success . Variables with an influence on the efficacy of the treatment were the incidence of manifestation and pathogenesis of the ulcer, renal function, blood pressure and smoking . Amoxicillin/clavulanic acid was well tolerated, with adverse events being observed in only 9 out of 193 patients (4.7%) . Treatment was abandoned because of adverse events in 3 out of 193 patients (1.6%) . Overall, treatment of the infected diabetic foot with amoxicillin/clavulanic acid was well tolerated and effective.

Genomics, 2000 Apr 15, 65(2), 87 - 94
Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library; Le Paslier MC et al.; A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni . This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector . The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage . Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library . End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S . mansoni sequences (ESTs, genes, or repetitive sequences) . A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones) . This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen .

Am J Respir Cell Mol Biol, 2000 May, 22(5), 604 - 12
Modification of the inflammatory response to allergen challenge after exposure to bacterial lipopolysaccharide; Tulic MK et al.; The potential role of respiratory infections in altering the development of atopy and asthma is complex . Infections have been suggested to be effective in preventing the induction of T-helper 2-polarized allergen-specific immunity in early life, but also to exacerbate asthma in older, sensitized individuals . The mechanism(s) underlying these effects are poorly defined . The aim of this work was to determine the influence of lipopolysaccharide (LPS) exposure on the development of sensitization to allergen and the response to allergen challenge in vivo . Piebald-Virol-Glaxo rats were exposed to a single aerosol of LPS 1 d before or 1, 2, 4, 6, 8, or 10 d after sensitization with ovalbumin (OVA) . On Day 11 animals were exposed to 1% OVA and responses to allergen were measured 24 h later, monitoring inflammatory cell influx and microvascular leakage into bronchoalveolar lavage (BAL) fluid as well as pulmonary responses to methacholine using the forced oscillation technique . Histologic analysis was included to complement the BAL results . Single aerosol exposure to LPS 1 d before and up to 4 d after intraperitoneal injection of OVA protected against the development of OVA-specific immunoglobulin (Ig) E . LPS exposure 6, 8, or 10 d after sensitization further exacerbated the OVA-induced cellular influx, resulting in neutrophilia and increased Evans Blue dye leakage with no effect on serum IgE levels . In addition, LPS abolished the OVA-induced hyperresponsiveness in sensitized animals when given 18 h after OVA challenge . This study demonstrates that exposure to LPS can modify the development of allergic inflammation in vivo by two independent mechanisms . Exposure early in the sensitization process, up to Day 6 after exposure to allergen, prevented allergen sensitization . Exposure to LPS after allergen challenge in sensitized animals abolished the hyperresponsiveness and modified the inflammatory cell influx characteristic of late-phase response to allergen.

Bioorg Med Chem Lett, 2000 Apr 17, 10(8), 715 - 7
4-Thiazolidinones: novel inhibitors of the bacterial enzyme MurB; Andres CJ et al.; 4-Thiazolidinones were synthesized and evaluated for their ability to inhibit the bacterial enzyme MurB . Selected 4-thiazolidinones displayed activity against the enzyme in vitro . This activity, coupled with the design principles of the thiazolidinones, supports the postulate that 4-thiazolidinones may be recognized as diphosphate mimics by a biological selector.

J Chem Neuroanat, 2000 Apr, 18(4), 167 - 79
Temporal, regional, and cell-specific changes of iNOS expression after intrastriatal microinjection of interferon gamma and bacterial lipopolysaccharide; Heneka MT et al.; Here we study expression of the inducible isoform of nitric oxide synthases after intrastriatal microinjection of interferon-gamma and bacterial lipopolysaccharide in the rat at different time points to detect time- and localisation-dependent changes of iNOS expression . Three different areas in the striatum and the corpus callosum were evaluated . Antibodies against the glial fibrillary acidic protein and the microglia/brain macrophage epitope ED1 were used to detect colocalization of inducible nitric oxide synthase with astrocytes or activated microglia/brain macrophages, respectively . Inducible nitric oxide synthase-positive cells occurred first in intravascular and perivascular cells at 4 h . Perivascular and parenchymal inducible nitric oxide synthase expression increased up to 24 h in the striatum, whereas in the corpus callosum inducible nitric oxide synthase expression was maximal after 16 h . Inducible nitric oxide synthase was still present in perivascular cells 7 days after immunostimulation . At all time points, inducible nitric oxide synthase was predominantly detected in ED1-positive microglia/brain . Nitrotyrosine immunohistochemistry was performed to detect NO-mediated nitration of proteins at all time points . Nitrotyrosine-positive neurons and microglial cells were detected from 24 h until 7 days after immunostimulation and were absent in controls . Detailed knowledge of the changes in the time course and cellular source of inducible nitric oxide synthase expression following brain immunostimulation provide a basis for establishing treatment strategies and windows of therapeutic intervention during neuroinflammation.

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