Gram negative

Exp Neurol, 2001 Nov, 172(1), 137 - 52
Systemic LPS injection leads to granulocyte influx into normal and injured brain: effects of ICAM-1 deficiency; Bohatschek M et al.; The lipopolysaccharide (LPS) constituents of the gram-negative bacterial wall are among the most potent activators of inflammation . In the current study, we examined the effect of subcutaneous injection of Escherichia coli LPS on leukocyte influx into the normal and injured brain using endogenous peroxidase (EP) . Normal brain parenchyma does not contain granulocytes and this does not change after indirect trauma, in facial axotomy . However, systemic injection of 1 mg LPS led to a gradual appearance of EP-positive parenchymal granulocytes within 12 h, with a maximum at 1-4 days after injection . Facial axotomy (day 14) led to a further 50-300% increase in granulocyte number . Of the five mouse strains tested in the current study, four--Balb/C, FVB, C57Bl/6, and C3H/N--showed vigorous granulocyte influx (60-90 cells per 20-microm section in axotomized facial nucleus, 20-40 cells per section on the contralateral side) . The influx was an order of magnitude lower in the SJL mice . The peroxidase-positive cells were immunoreactive for neutrophil antigen 7/4 and alpha M beta 2 integrin, were negative for IBA1 (monocytes) and CD3 (T cells), and could be prelabeled by subcutaneous injection with rhodamine B isothiocyanate (RITC), confirming their origin as blood-borne granulocytes . All RITC-positive cells were IBA1 negative . This influx of granulocytes was accompanied by a disruption of the blood-brain barrier to albumin and induction of the cell adhesion molecule ICAM-1 on affected blood vessels . Transgenic deletion of ICAM-1 led to a more than 50% reduction in the number of infiltrating granulocytes compared to litter-matched wild-type controls, in normal brain as well as in axotomized facial motor nucleus . In summary, systemic injection of LPS leads to invasion of granulocytes into the mouse brain and a breakdown of the blood-brain barrier to blood-borne cells and to soluble molecules . Moreover, this mechanism may play a pathogenic role in the etiology of meningitis and in severe bacterial sepsis .

Int J Med Microbiol, 2001 Sep, 291(4), 299 - 305
Host cell signaling in Helicobacter pylori infection; Naumann M; Helicobacter pylori represents a highly successful human microbial pathogen that has infected approximately half of the world's population . This gram-negative microorganism colonizes the human epithelial layer in the stomach and induces a state of chronic inflammation that does not resolve the underlying infection and often leads to gastric or duodenal ulcers, or more rarely to gastric cancer . Among the reactions in H . pylori-infected epithelial cells the induction of proinflammatory cytokines, cell spreading and movement, as well as a scattered phenotype appear strictly dependent on the expression of pathogenicity island-encoded proteins in H . pylori . This review will discuss the features of the H . pylori-induced signal transduction leading to changes in host cellular function . Topics discussed comprise the signaling and the phenotypes associated with the type IV secretion system, the activation of target genes involved in gastric physiology, and putative mechanisms leading to the development of gastric cancer.

Med Clin North Am, 2001 Nov, 85(6), 1493 - 509
Guidelines for the management of community-acquired pneumonia . Current recommendations and antibiotic selection issues; Niederman MS; Numerous guidelines for CAP have been developed, and although each is different, many principles are common to all recommendations . A guideline should focus on a wide range of issues surrounding the delivery of care, including advice about when to admit patients to the hospital or ICU; which antibiotic regimens to select for specific patient populations; which pathogens to target in empiric therapy; which diagnostic tests to order; how to assess the importance of specific causative pathogens, such as drug-resistant pneumococci, atypical pathogens, and gram-negative pathogens; how to evaluate the response to therapy and when to switch responding patients to oral therapy; and how to prevent CAP effectively through appropriate use of immunization against pneumococcus and influenza . Currently, many new antibiotic choices have emerged in the macrolide, quinolone, beta-lactam, ketolide, and oxazolidinone classes, and specific issues surrounding selection of these agents must be considered . All of the available data can be synthesized into a disease management guideline, and current therapy in the United States generally is consistent with existing recommendations . This consistency not only has led to more uniformity in patient care, but also has led to measurable benefits in patient outcomes, including reduced mortality for hospitalized patients with CAP . Guidelines not only are a useful tool for managing patients with CAP, but also they serve the purpose of defining current issues in patient care and stimulating the search for new tools and management approaches for this important clinical problem.

Appl Environ Microbiol, 2001 Nov, 67(11), 5010 - 6
Pathogenicity of Moraxella osloensis, a bacterium associated with the nematode Phasmarhabditis hermaphrodita, to the slug Deroceras reticulatum; Tan L et al.; Moraxella osloensis, a gram-negative bacterium, is associated with Phasmarhabditis hermaphrodita, a nematode parasite of slugs . This bacterium-feeding nematode has potential for the biological control of slugs, especially the grey garden slug, Deroceras reticulatum . Infective juveniles of P . hermaphrodita invade the shell cavity of the slug, develop into self-fertilizing hermaphrodites, and produce progeny, resulting in host death . However, the role of the associated bacterium in the pathogenicity of the nematode to the slug is unknown . We discovered that M . osloensis alone is pathogenic to D . reticulatum after injection into the shell cavity or hemocoel of the slug . The bacteria from 60-h cultures were more pathogenic than the bacteria from 40-h cultures, as indicated by the higher and more rapid mortality of the slugs injected with the former . Coinjection of penicillin and streptomycin with the 60-h bacterial culture reduced its pathogenicity to the slug . Further work suggested that the reduction and loss of pathogenicity of the aged infective juveniles of P . hermaphrodita to D . reticulatum result from the loss of M . osloensis from the aged nematodes . Also, axenic J1/J2 nematodes were nonpathogenic after injection into the shell cavity . Therefore, we conclude that the bacterium is the sole killing agent of D . reticulatum in the nematode-bacterium complex and that P . hermaphrodita acts only as a vector to transport the bacterium into the shell cavity of the slug . The identification of the toxic metabolites produced by M . osloensis is being pursued.

Int J Dermatol, 2001 Jul, 40(7), 434 - 8
Cutaneous findings encountered in brucellosis and review of the literature; Metin A et al.; BACKGROUND: Human brucellosis is an infectious disease produced by Brucella species: small, coccoid or rod-like, aerobic, Gram-negative bacteria . The infection is common in developing countries, and can also affect the skin . Its prevalence is high in our region of Turkey, where stockbreeding is one of the main economic sources, compared with the industrially developed areas of Turkey, and dermatologic complaints due to brucellosis are fairly common . MATERIALS AND METHODS: One hundred and three patients with serologically and clinically confirmed brucellosis were studied in order to investigate the prevalence of cutaneous findings and their variability in brucellosis . Fifty-two (50.49%) were males and 51 (49.51%) were females with an age range of 4-70 years (mean, 30.45 +/- 15.08 years) . RESULTS: Of these patients, 14 (13.59%) had cutaneous findings probably related to brucellosis . These findings were more frequent in females (11 cases) than males, and most of the females (eight cases) were housewives; three were students . Urticaria-like papules and plaques were the most common findings; they were seen in six (35.3%) patients . One case had livedo reticularis and another palmar erythema, which have not been reported previously . No relationship was observed between the serologic values and the cutaneous findings . CONCLUSIONS: Cutaneous findings in our cases were more prevalent than in other reported studies . It is important to emphasize that cutaneous lesions are not specific to brucellosis and may be seen in a variety of other dermatologic diseases caused by many agents; therefore, these agents should be kept in mind in the differential diagnosis.

Proteomics, 2001 May, 1(5), 705 - 20
Analysis of the outer membrane proteome of Caulobacter crescentus by two-dimensional electrophoresis and mass spectrometry; Phadke ND et al.; Caulobacter crescentus, a Gram negative alpha-purple bacterium that displays an invariant asymmetric cell division pattern, has become a key model system for the study of bacterial development . Membrane proteins play key roles in cell cycle events, both as components of landmark morphological structures and as critical elements in regulation of the cell cycle . Recent advances for the isolation and solubilization of bacterial membrane proteins prior to isoelectric focusing have significantly improved the separation of outer membrane proteins by two-dimensional (2-D) electrophoresis . In this work we describe the analysis of the outer membrane proteome of Caulobacter crescentus . Proteins were identified using 2-D gel electrophoresis and peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight mass spectrometry . We identified 54 unique proteins out of which 41 were outer membrane proteins . Of the outer membrane proteins, 16 were identified as TonB-dependent receptor proteins . These studies were executed simultaneously with the Caulobacter genome sequencing project and advantages and limitations of proteomic analysis of a nonannotated genome are discussed . Finally, protein levels between cells grown in rich and minimal media are compared which demonstrates that many of the TonB-dependent receptor proteins are found at higher levels in minimal medium.

Gastroenterol Clin Biol, 2001 Jun-Jul, 25(6-7), 659 - 63
{UreI: a Helicobacter pylori protein essential for resistance to acidity and for the early steps of murine gastric mucosa infection}; Bury-Mone S et al.; Helicobacter pylori (H . pylori) is a Gram negative microaerophilic bacteria whose only known niche is the human gastric mucosa . The presence of H . pylori is associated with various pathologies ranging from peptic ulcer disease to gastric carcinoma . H . pylori virulence is dependent on its exceptional ability to resist to the stomach acidity by hydrolyzing urea into ammonia . Survival of H . pylori to acidity in the presence of urea relies on the activity of a membrane protein, UreI.AIMS: We decided to better characterize the role of UreI (i) in vitro in ammonia production through the action of urease, and (ii) in vivo in the colonization of the gastric mucosa.METHODS: Ammonia production by a wild type strain of H . pylori or by a UreI-deficient strain was measured as a function of extracellular pH . In addition, the kinetics of elimination of a UreI-deficient mutant in vivo were realized in the mouse model for colonization.RESULTS: UreI was associated with an increase of ammonia production in acidic conditions in vitro and was necessary for the initial steps of the mouse stomach colonization.CONCLUSION: UreI thus behaves as a sensor of extracellular pH . This protein activates urease at acidic pH; thereby, it probably allows H . pylori to resist to acidity in vivo during the first steps of infection.

J Immunol, 2001 Nov 1, 167(9), 5231 - 9
Escherichia coli Braun lipoprotein induces a lipopolysaccharide-like endotoxic response from primary human endothelial cells; Neilsen PO et al.; All bacteria contain proteins in which their amino-terminal cysteine residue is modified with N-acyl S-diacylglycerol functions, and peptides and proteins bearing this modification are immunomodulatory . The major outer membrane lipoprotein of Escherichia coli, the Braun lipoprotein (BLP), is the prototypical triacylated cysteinyl-modified protein . We find it is as active as LPS in stimulating human endothelial cells to an inflammatory phenotype, and a BLP-negative mutant of E . coli was less inflammatory than its parental strain . While the lipid modification was essential, the lipidated protein was more potent than a lipid-modified peptide . BLP associates with CD14, but this interaction, unlike that with LPS, was not required to elicit endothelial cell activation . BLP stimulated endothelial cell E-selectin surface expression, IL-6 secretion, and up-regulation of the same battery of cytokine mRNAs induced by LPS . Quantitative microarray analysis of 4400 genes showed the same 30 genes were induced by BLP and LPS, and that there was near complete concordance in the level of gene induction . We conclude that the lipid modification of at least one abundant Gram-negative protein is essential for endotoxic activity, but that the protein component also influences activity . The equivalent potency of BLP and LPS, and their complete concordance in the nature and extent of endothelial cell activation show that E . coli endotoxic activity is not due to just LPS . The major outer membrane protein of E . coli is a fully active endotoxic agonist for endothelial cells.

J Bacteriol, 2001 Nov, 183(22), 6499 - 508
A 12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation; McClain MS et al.; Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells . Several different families of H . pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2) . Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins . To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein . Purified s1/m1 VacA from H . pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity . Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA . However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5) . When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin . Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27) . Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity . Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H . pylori isolates suggests that type s2 VacA proteins retain an important biological activity.

Immunol Lett, 2001 Sep 3, 78(2), 97 - 101
Toll-like receptor 2 and 4 surface expressions on human monocytes are modulated by interferon-gamma and macrophage colony-stimulating factor; Mita Y et al.; Human Toll-like receptor 2 (TLR2) and TLR4 are recently identified receptors . TLR4 was shown to be the main protein involved in recognizing Gram-negative bacteria, whereas TLR2 is apparently the key factor in responses to other types of microbial pathogens . We examined regulations of TLR2 and TLR4 surface expressions in human peripheral blood monocytes by interferon-gamma (IFN-gamma) and macrophage colony-stimulating factor (M-CSF) . IFN-gamma up-regulated both TLR2 and TLR4, but enhanced the surface expression, on human peripheral blood monocytes of TLR4 more than that of TLR2 . On the other hand, M-CSF up-regulated both TLR2 and TLR4 surface expression on human peripheral blood monocytes, with no change in the ratio of TLR2/TLR4 surface expression . These results indicate that IFN-gamma enhances receptors involved in the response to Gram-negative bacteria more than those involved in responses to other types of microbial pathogens, whereas M-CSF enhances the receptor response to Gram-negative bacteria in the same manner as to other types of microbial pathogens.

Biochemistry, 2001 Oct 30, 40(43), 13041 - 50
Conformational change in the stator of the bacterial flagellar motor; Kojima S et al.; MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor . The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation . MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation . We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor . To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32 . Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments . The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes . It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque . Influences of key residues of MotA on conformation were also examined . Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements . Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes . We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria . ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.

J Med Humanit, 1993 Spring, 14(1), 5 - 14
We have a prejudice against ourselves -- sentiment, ethics, and reason; Rosenberg LT; Briefly stated my point is that the well-being of each person in a community conceived abstractly may be all too easily sacrificed for the sake of the abstraction . Physicians may offer critically ill patients places in programs of experimental treatment, but there is commonly a catch to the offer . To take part in a program of clinical experiment a patient must not only risk a possible failure of a fresh drug and the chance of destructive side effects from the drug, but the patients must risk only getting the traditional treatment along with a placebo rather than the experimental drug . Placebo control, double blind critical protocols for testing effects of fresh drugs on critically ill patients are a commonplace . I question the scientific objectivity of the protocols and the underlying ethic, and suggest use of alternate protocols . Experimental tests in the treatment of gram-negative bacteria blood infections, muscular dystrophy, and AIDS and AIDS-related diseases are examples.

Genetika, 2001 Aug, 37(8), 1160 - 4
{Tn5037-a Tn21-like mercury transposon, detected in Thiobacillus ferrooxidans}; Kaliaeva ES et al.; The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidans was cloned and sequenced . This transposon, named Tn5037, belongs to the Tn21 branch of the Tn21 subgroup, many members of which have been isolated from clinical sources . Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037 is organized similarly to most of the Gram-negative bacteria mer operons and is closest to that of Thiobacillus 3.2 . The operator-promoter region of the mer operon of Tn5037 also has the common (Tn21/Tn501-like) structure . However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501 . In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T . ferrooxidance E-15, and Thiobacillus 3.2, respectively . No inducibility of the Tn5037 mer operon was detected in the in vivo experiments . The transposition system (terminal repeats plus gene tnpA) of Tn5037 was inactive in Escherichia coli K12, in contrast to its resolution system (res site plus gene tnpR) . However, transposition of Tn5037 in this host was provided by the tnpA gene of Tn5036, a member of the Tn21 subgroup . Sequence analysis of the Tn5037 res site suggested its recombinant nature.

Genes Immun, 2001 Oct, 2(6), 349 - 55
IFNs activate toll-like receptor gene expression in viral infections; Miettinen M et al.; Toll-like receptors (TLRs) mediate innate immune responses to microbes . TLR2, TLR5, TLR6, and TLR9 have been implicated in responses to bacterial components, and TLR4 is the receptor for Gram-negative bacteria . Recently, TLR4 was described to function in respiratory syncytial virus-induced NF-kappaB activation . Here we have analyzed TLR1-9 mRNA expression in human primary macrophages infected with influenza A and Sendai viruses . TLR1, TLR2, TLR4, TLR6, and TLR8 mRNAs were expressed at basal levels in macrophages . Viral infection enhanced TLR1, TLR2, TLR3, and TLR7 mRNA expression, and neutralizing anti-IFN-alpha/beta antibodies downregulated gene expression of these TLRs . Exogenously added IFN-alpha upregulated TLR1, TLR2, TLR3, and TLR7 mRNA expression in macrophages, as well as TLR3 mRNA expression in epithelial and endothelial cell lines . IFN-gamma enhanced the expression of TLR1 and TLR2 mRNA in macrophages, and TLR3 in epithelial and endothelial cells . The data suggests a novel role for IFNs in the activation of innate immunity.

Int Immunopharmacol, 2001 Oct, 1(11), 1979 - 87
Immunoferon, a glycoconjugate of natural origin, inhibits LPS-induced TNF-alpha production and inflammatory responses; Brieva A et al.; We have analyzed the effect of a patented glycoconjugate (GC) of natural origin, Inmunoferon, in the development of the response to endotoxemia induced by administration of LPS in rodents . We have observed that oral treatment with the drug reduced the levels of serum TNF-alpha induced by an intravenous pulse of LPS . The serum of pretreated mice blocked TNF-alpha production by peritoneal macrophages . The drug increased the levels of TNF-alpha regulators such as IL-10 and corticosteroids, whereas it inhibited TNF-alpha-dependent IL-6 production . Further TNF-alpha-dependent responses, such as cell extravasation, was decreased in treated mice . According to these results, Inmunoferon is postulated as an inhibitor of the systemic response to LPS . Correlation of the observations made in mice with a rat model suggests the efficacy of this product in reducing TNF-alpha production in a species-independent fashion and opens the possibility of its trial as an adjuvant of antibiotics in treatment against gram-negative bacterial infection.

Anal Chem, 2001 Oct 1, 73(19), 4566 - 73
Bioinformatics and mass spectrometry for microorganism identification: proteome-wide post-translational modifications and database search algorithms for characterization of intact H . pylori; Demirev PA et al.; MALDI-TOF mass spectrometry has been coupled with Internet-based proteome database search algorithms in an approach for direct microorganism identification . This approach is applied here to characterize intact H . pylori (strain 26695) Gram-negative bacteria, the most ubiquitous human pathogen . A procedure for including a specific and common posttranslational modification, N-terminal Met cleavage, in the search algorithm is described . Accounting for posttranslational modifications in putative protein biomarkers improves the identification reliability by at least an order of magnitude . The influence of other factors, such as number of detected biomarker peaks, proteome size, spectral calibration, and mass accuracy, on the microorganism identification success rate is illustrated as well.

J Virol, 2001 Nov, 75(22), 10730 - 7
Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus; Haynes LM et al.; The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection . The role of TLR4 in antiviral immunity has been largely unexplored . In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice . TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4 . These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 736 - 41
The meroperon of a mercury-resistant Pseudoalteromonas haloplanktis strain isolated from Minamata Bay, Japan; Iohara K et al.; A mer operon of mercury-resistant Pseudoalteromonas haloplanktis strain M1, isolated from sea water of Minamata Bay, was cloned and analyzed . The mer genes were located in the chromosome and organized as merR-merT-merP-merC-merA-merD, the same order as that in Tn21 . However, the orientation of the merR gene is the same as that of other mer genes (opposite direction to Tn21), and merR was cotranscribed with other mer genes, a pattern that has not been previously seen with mer determinants from other Gram-negative bacteria . Furthermore, the amino acid similarities of the corresponding mer gene products between those from strain M1 and Tn21 were unusually low.

Am J Physiol Cell Physiol, 2001 Nov, 281(5), C1587 - 95
Human endothelial cell response to gram-negative lipopolysaccharide assessed with cDNA microarrays; Zhao B et al.; To assess the feasibility of using cDNA microarrays to understand the response of endothelial cells to lipopolysaccharide (LPS) and to evaluate potentially beneficial agents in treatment of septic shock, human umbilical vein endothelial cells were exposed to Escherichia coli LPS for 1, 4, 7, 12, or 24 h . Total RNA was isolated and reverse-transcribed into (33)P-labeled cDNA probes that were hybridized to human GeneFilter microarrays containing approximately 4,000 genes . The mRNA levels of several genes known to respond to LPS changed after stimulation . In addition, a number of genes not previously implicated in the response of endothelial cells to LPS also appeared to be altered in expression . Nuclear factor-kappaB (NF-kappaB) was shown to play an important role in regulating genes identified from the microarray studies . Pretreatment of endothelial cells with a specific NF-kappaB translocation inhibitor eliminated most of the alterations in gene expression . Quantitative RT-PCR results independently confirmed the microarray results for monocyte chemotactic protein-1 and interleukin-8, and enzyme-linked immunosorbent assays demonstrated that augmented transcription was followed by translation and secretion.

Infect Immun, 2001 Nov, 69(11), 6942 - 50
Dual role of lipopolysaccharide (LPS)-binding protein in neutralization of LPS and enhancement of LPS-induced activation of mononuclear cells; Gutsmann T et al.; The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation . In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions . In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperature-dependent manner . In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids . This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC . We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.

Clin Infect Dis, 2001 Nov 15, 33(10), 1789 - 91 Epub 2001 Oct 05.
Bacteremia caused by a Helicobacter pullorum-like organism; Tee W et al.; We report a case of bacteremia caused by a Helicobacter pullorum-like organism in a 35-year-old man with pyrexia of unknown origin . Culture of blood samples obtained at admission yielded a motile, spiral-shaped gram-negative rod, and 16S rRNA gene sequencing identified this organism as Helicobacter pullorum-like, showing 23 base differences compared with the recently described "Helicobacter canadensis" (a recently proposed group that had previously been classified within H . pullorum) . We believe that this is the first report of bacteremia caused by this organism.

Toxicon, 2001 Nov, 39(11), 1729 - 36
The cytolethal distending toxins induce DNA damage and cell cycle arrest; Cortes-Bratti X et al.; The cytolethal distending toxins (CDTs) are a newly discovered family of bacterial protein toxins with the unique ability to interfere with the cell cycle, causing irreversible cell cycle arrest and consequently death of the target cells . CDTs are encoded by three linked genes (cdtA, cdtB and cdtC) and are produced by a variety of Gram negative bacteria . The mechanism of action of this toxin family only now begins to be elucidated . CDTs are internalized by endocytosis and require an intact Golgi complex to exert their cytotoxic activity . The CdtB component was shown to have functional homology with the mammalian deoxyribonuclease I (DNase I) and the induction of cell cycle arrest in mammalian cells mimicked that induced by DNA damaging agents, suggesting that DNA is the cellular target . Still there are many issues that need to be clarified, such as identification of the function(s) of CdtA and CdtC, characterization of the receptor(s), understanding of the final steps of the internalization pathway and localization of the active component . This review focuses mainly on the effect of CDTs on mammalian cells, highlighting the questions that remain to be answered regarding their molecular mode of action.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1911 - 6
Desulforegula conservatrix gen . nov., sp . nov., a long-chain fatty acid-oxidizing, sulfate-reducing bacterium isolated from sediments of a freshwater lake; Rees GN et al.; A novel sulfate-reducing bacterium, strain Mb1PaT, was isolated from the sediments of a freshwater floodplain lake . Cells of strain Mb1PaT were rod-shaped, 1-1.3 microm wide and 2.6-3 microm long, motile and Gram-negative . The bacterium grew on straight-chain carboxylic acids with 4-17 carbon atoms . Electron donors with an even number of carbon atoms were oxidized to acetate and electron donors with an odd number of carbon atoms were oxidized to acetate and propionate . No other compounds were found to be used as electron donors . No growth occurred in the absence of sulfate . The optimum temperature for growth was between 25 and 30 degrees C and the maximum temperature for growth was 32 degrees C . Strain Mb1PaT grew very slowly in medium with 5 g NaCl l(-1) with optimum growth occurring with up to 1.0 g NaCl l(-1) . Analysis of the 16S rRNA gene showed that strain Mb1PaT belonged to the delta-subclass of the Proteobacteria, was a member of the family Desulfobacteraceae, but lacked similarity with any currently described representatives . The combined phylogenetic analysis and physiological data indicate that strain Mb1PaT represents a new genus and the name Desulforegula conservatrix is proposed . The type strain is Mb1PaT (= DSM 13527T = ATCC BAA-134T).

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1873 - 80
Methanofollis aquaemaris sp . nov., a methanogen isolated from an aquaculture fish pond; Lai MC et al.; A novel methanogen, designated strain N2F9704T, was isolated from an aquaculture fish pond near Wang-gong, Taiwan . The cells were irregular cocci, non-motile, 1.2-2.0 microm in diameter and stained gram-negative . Cells of strain N2F9704T lysed easily by SDS treatment (0.1 g l(-1)) and the S-layer protein had an Mr of 137000 . The catabolic substrates used included formate and H2+CO2, but not acetate, methanol, trimethylamine or secondary alcohols . The optimal growth parameters for strain N2F9704T were pH 6.5, 37 degrees C with 0.5% NaCl . Trace amounts of tungstate not only promoted growth but also extended the range of growth conditions . Analysis of the 16S rDNA sequence revealed a phylogenetic relationship to Methanofollis species and the name Methanofollis aquaemaris sp . nov . is therefore proposed for strain N2F9704T (= OCM 746T = CCRC 16166T) . Additionally, the strain was infected with a novel coccus-shaped, enveloped virus with a diameter of 200 nm.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1825 - 9
Natronobacterium nitratireducens sp . nov., a aloalkaliphilic archaeon isolated from a soda lake in China; Xin H et al.; Two novel haloalkaliphilic archaea, strains C231T and C42, were isolated from a soda lake in China . Cells of the two strains were rod-shaped and gram-negative and colonies were bright red . They required at least 2.5 M NaCl for growth, with an optimum at 3.5 M NaCl, and grew over a pH range from 8.0 to 10.5, with an optimum at pH 8.5 . Hypotonic treatment with less than 1.5 M NaCl caused cell lysis . They had similar polar lipid compositions, possessing the diphytanyl (C20:C20) and phytanyl-sesterterpanyl (C20:C25) diether derivatives of phosphatidylglycerol and phosphatidylglycerophosphate methyl ester and a minor phospholipid, PL1 . No glycolipids were detected . Comparison of 16S rDNA sequences and morphological features placed them in the genus Natronobacterium . Detailed phenotypic characterization and DNA-DNA hybridization studies revealed that the two strains belong to a new species in the genus Natronobacterium, for which the name Natronobacterium nitratireducens sp . nov . is proposed . The type strain is C231T (= AS 1.1980T = JCM 10879T).

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1663 - 9
Methanoculleus chikugoensis sp . nov., a novel methanogenic archaeon isolated from paddy field soil in Japan, and DNA-DNA hybridization among Methanoculleus species; Dianou D et al.; A strictly anaerobic, irregularly coccoid, methanogenic archaeon, strain MG62T (= JCM 10825T = DSM 13459T), was isolated from paddy field soil in Chikugo, Fukuoka, Japan . The cells stained gram-negative, were 1.0-2.0 microm in diameter, were lysed by SDS and hypotonic solutions and were flagellated . Motility was not observed . The strain was able to use H2/CO2, 2-propanol/CO2, formate, 2-butanol/CO2 and cyclopentanol/CO2 as substrates for methanogenesis, but did not utilize acetate, ethanol, methanol or methylamines . The optimum temperature and pH were 25-30 degrees C and 6.7-7.2 . Analysis of lipid component parts (core lipids, phospholipid polar head groups and glycolipid sugar moieties) showed the characteristic pattern of members of the family Methanomicrobiaceae except for the absence of glucose as a glycolipid sugar moiety . The G+C content of the DNA was 62.2 mol % . Sequence analysis of the 16S rDNA revealed that the strain belonged to the genus Methanoculleus . The strain had DNA-DNA hybridization values of less than 50% with type strains of Methanoculleus species . On the basis of phenotypic, genotypic and phylogenetic characteristics, the name Methanoculleus chikugoensis sp . nov . is proposed for strain MG62T (= JCM 10825T = DSM 13459T) . The DNA hybridization study also revealed the close relationships of three species, Methanoculleus olentangyi, Methanoculleus bourgensis and Methanoculleus oldenburgensis, among Methanoculleus species.

Dis Aquat Organ, 2001 Aug 22, 46(1), 57 - 66
Parasitism by the protozoan Perkinsus atlanticus favours the development of opportunistic infections; Montes JF et al.; It has been suggested that opportunistic pathogens could contribute to the mortality of Perkinsus atlanticus-infected clams . Examination of Tapes semidecussatus clams from the northern Mediterranean coast of Spain revealed that while 86% of the clams heavily infected with P . atlanticus were co-infected by bacteria and/or viruses, neither non-infected nor lightly P . atlanticus-infected specimens had bacterial or viral infections . The bacteria, which had a Gram-negative cell wall, were always located in the apical pole of gill epithelial cells and enclosed within membranous compartments . Bacteria-containing cells were hypertrophied and showed dysplasia with loss of cilia and microvilli . The viruses shared ultrastructural, morphologic and cytopathic characteristics of a polyomavirus . Viral particles with icosahedral symmetry were found in both the cytoplasm and the nucleus of numerous cell types . Virus-infected cells showed severe alterations, including hypertrophy, reduction of the intracellular compartments and extrusion of the nuclear envelope . Moreover, gill epithelial cells showed disorganization and swelling of the apical region, which affected the ciliary structure . Our findings show that P . atlanticus parasitism favours the development of opportunistic infections which have detrimental effects in this clam population.

J Biol Chem, 2001 Dec 7, 276(49), 46568 - 74 Epub 2001 Oct 08.
Amino acid sequence determinants of extended spectrum cephalosporin hydrolysis by the class C P99 beta-lactamase; Zhang Z et al.; Class C beta-lactamases are commonly encoded on the chromosome of Gram-negative bacterial species . Mutations leading to increased expression of these enzymes are a common cause of resistance to many cephalosporins including extended spectrum cephalosporins . Recent reports of plasmid- and integrin-encoded class C beta-lactamases are a cause for concern because these enzymes are likely to spread horizontally to susceptible strains . Because of their increasing clinical significance, it is critical to identify the determinants of catalysis and substrate specificity of these enzymes . For this purpose, the codons of a set of 21 amino acid residues that encompass the active site region of the P99 beta-lactamase were individually randomized to create libraries containing all possible amino acid substitutions . The amino acid sequence requirements for the hydrolysis of ceftazidime, an extended spectrum cephalosporin commonly used to treat serious infections, were determined by selecting resistant mutants from each of the 21 libraries . DNA sequencing identified the residue positions that are critical for ceftazidime hydrolysis . In addition, it was found that certain amino acid substitutions in the omega-loop region of the P99 enzyme result in increased ceftazidime hydrolysis suggesting the loop is an important determinant of substrate specificity.

Trends Plant Sci, 2001 Oct, 6(10), 479 - 85
Molecular secrets of bacterial type III effector proteins; Lahaye T et al.; Most Gram-negative phytopathogenic bacteria are thought to inject effector proteins into the plant cell via a type III secretion system that is essential for pathogenicity . Plant targets and the mode of action of type III effector proteins, which include avirulence (Avr) proteins, are largely unknown . However, recent findings have shed light on the molecular mechanisms of Avr action . Here, we focus on two classes of Avr proteins (the AvrBs3 and AvrRxv/YopJ families) that have been suggested to act as transcription factors and proteases, respectively.

Eur J Biochem, 2001 Oct, 268(19), 5092 - 7
Structural investigation on the lipooligosaccharide fraction of psychrophilic Pseudoalteromonas haloplanktis TAC 125 bacterium; Corsaro MM et al.; The core structure of the cell-wall lipooligosaccharide (LOS) fraction of an Antarctic Gram-negative bacterium, Pseudoalteromonas haloplanktis TAC 125 strain, was determined to be deacetylated alditols . These were obtained from native LOS fraction by O-deacylation, dephosphorylation, reduction and finally N-deacylation . Two novel structures were detected, the more highly represented molecule consisting of the following hexasaccharide chain: alpha-D-ManpNH(2)-(1-->3)-beta-D-Galp-(1-->4)-alpha-L-glycero-D-manno-Hepp-(1-->5)-alpha-D-Kdo-(2-->6)-beta-D-GlcpNH(2)-(1-->6)-D-GlcNH(2)(ol) while the corresponding pentasaccharide, lacking the ManpNH(2) residue, was less abundant . To the best of our knowledge, the structural investigation presented here, mainly performed by NMR and MS methods, is the first report of the lipopolysaccharide fraction of a psychrophilic bacterium.

Cornea, 2001 Oct, 20(7), 765 - 8
An unusual case of fungal keratitis: Metarrhizium anisopliae; Jani BR et al.; PURPOSE: To report a case of fungal keratitis caused by Metarrhizium anisopliae, which to our knowledge is the first reported case in the United States . METHOD: Case report . RESULTS: A 36-year-old female librarian who wore extended-wear soft contact lenses was seen by an ophthalmologist on September 11 for an irritated right eye, and a corneal ulcer was diagnosed . Symptoms increased by September 27, and the patient was referred to another ophthalmologist who cultured the ulcer and had scrapings examined, which were Gram-negative for microorganisms . The patient was referred to one of the authors (W.J.R.) . Her exam on October 1 showed vision corrected to 20/25 OD, a 5-mm epithelial defect with a 2.5-mm anterior stromal grayish-type infiltrate, and a quiet anterior chamber; the eye did not appear to be inflamed . The patient was reexamined on October 4 and was noted to have worsening vision . Because the initial cultures remained negative, the patient underwent a corneal biopsy, Gram stain, and cultures on October 6 . Scrapings at the time of the biopsy revealed septate hyphal elements, as did the biopsy specimen, and on October 7, the patient was started on a treatment of bacitracin ointment once a day and natamycin 5% every hour . The eye gradually quieted down . A mold growing from the biopsy culture, which had been sent to a reference laboratory in San Antonio, Texas, was identified as M . anisopliae var . anisopliae . The patient was subsequently fitted with a rigid gas permeable lens, which resulted in a best-corrected visual acuity of 20/20, although glare remained a major problem . CONCLUSION: Although not previously reported in the United States, M . anisopliae can cause a keratomycosis, and one must consider this common insect pathogen in the differential diagnosis of fungal keratitis.

Curr Opin Microbiol, 2001 Oct, 4(5), 565 - 9
Integrons: natural tools for bacterial genome evolution; Rowe-Magnus DA et al.; Integrons were first identified as the primary mechanism for antibiotic resistance gene capture and dissemination among Gram-negative bacteria . More recently, their role in genome evolution has been extended with the discovery of larger integron structures, the super-integrons, as genuine components of the genomes of many species throughout the gamma-proteobacterial radiation . The functional platforms of these integrons appear to be sedentary, whereas their gene cassette contents are highly variable . Nevertheless, the gene cassettes for which an activity has been experimentally demonstrated encode proteins related to simple adaptive functions and their recruitment is seen as providing the bacterial host with a selective advantage . The widespread occurrence of the integron system among Gram-negative bacteria is discussed, with special focus on the super-integrons . Some of the adaptive functions encoded by these genes are also reviewed, and implications of integron-mediated genome evolution in the emergence of novel bacterial species are highlighted.

Perit Dial Int, 2001 Jul-Aug, 21(4), 405 - 10
Late renal transplant failure: an adverse prognostic factor at initiation of peritoneal dialysis; Sasal J et al.; BACKGROUND: Early renal transplant failure necessitating a return to dialysis has been shown to be a poor prognostic factor for survival . Little is known about the outcome of patients with late transplant failure returning to dialysis . It was our clinical impression that late transplant failure (>2 months) carries an increased morbidity and mortality risk in patients returning to dialysis . OBJECTIVE: To determine whether patients with a failed renal transplant have an outcome different to those on dialysis who have never received a kidney transplant . SETTING: Peritoneal dialysis (PD) unit in a teaching hospital . PATIENTS AND DESIGN: All failed renal transplant patients (fTx) in the Toronto Hospital Peritoneal Dialysis program between 1989 and 1996 were identified . This cohort of 42 fTx patients was compared with a cohort of randomly selected never-transplanted PD patients (non-Tx).The PD program was selected because of the availability of well-documented patient archival material . The non-Tx group was matched for age and presence of diabetes . Data were collected until retransplantation, change of dialysis modality or center, death, or until June 1998 . RESULTS: There was no difference at initiation of PD between groups in serum albumin, residual renal function, or mean serum parathyroid hormone level . The mean low-density lipoprotein level was significantly higher in the fTx cohort . The duration of dialysis before Tx in fTx patients accounted for the increased total length of dialysis in fTx (mean 15 months) . However, post-Tx the duration of PD was similar for both groups (30.7 months for fTx vs 31.6 months for non-Tx) . The fTx group had a considerably worse outcome than the non-Tx group . The time to first peritonitis, subsequent episodes of peritonitis, catheter change, or transfer to hemodialysis occurred at a much faster rate in fTx patients.The most dramatic difference was in survival . There were 3 deaths in the non-Tx group and 12 in the fTx group (p < 0.01) . The mean age at time of death in the fTx group was 47.5 years . Deaths were due mainly to gram-negative peritonitis and cardiovascular disease . CONCLUSIONS: We conclude that late failed renal transplant patients starting dialysis are at increased risk of complications and have strikingly higher mortality rates than non-Tx patients . A previously failed kidney transplant can be considered an adverse prognostic factor for patients commencing PD; these patients need to be closely monitored . Although this study was limited to PD patients, the same principles likely apply to fTx patients returning to any form of renal replacement therapy.

J Nutr, 2001 Oct, 131(10), 2789S - 2793S
Helicobacter pylori: ulcers and more: the beginning of an era; Lacy BE et al.; Helicobacter pylori (H . pylori) may be found in up to 50% of the world's population, making it the most common infectious disease worldwide . H . pylori is a gram-negative, microaerophilic rod with flagella and normally resides in the stomach . It can be diagnosed endoscopically, via breath tests, or through a simple blood test . H . pylori can usually be eradicated with a combination of proton pump inhibitors and antibiotics . Although most people infected with this organism never develop a complication, H . pylori infection in others may produce significant mucosal inflammation leading to ulcers in the stomach and duodenum . H . pylori has also been recognized as a risk factor for the development of both gastric carcinoma and mucosal-associated lymphoid tumor . Although not widely known, H . pylori can also affect organ systems outside of the gastrointestinal tract . It is now apparent that H . pylori can infect the skin, liver and heart and that these infections may produce a number of different disease states . In addition, H . pylori infection can adversely affect the nutritional status of both children and adults . This article discusses the epidemiology and bacteriology of H . pylori, reviews the different methods of diagnosing and treating this common infection, and then focuses on the effects that H . pylori has on different organ systems within the body, including the nutritional status of those affected.

FEMS Microbiol Lett, 2001 Sep 25, 203(2), 141 - 8
Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation?
De Rycke J, Oswald E.
Cytolethal distending toxins (CDT) constitute a family of genetically related bacterial protein toxins able to stop the proliferation of numerous cell lines . This effect is due to their ability to trigger in target cells a signaling pathway that normally prevents the transition between the G2 and the M phase of the cell cycle . Produced by several unrelated Gram-negative mucosa-associated bacterial species, CDTs are determined by a cluster of three adjacent genes (cdtA, cdtB, cdtC) encoding proteins whose respective role is not yet fully elucidated . The CDT-B protein presents sequence homology to several mammalian and bacterial phosphodiesterases, such as DNase I . The putative nuclease activity of CDT-B, together with the activation by CDT of a G2 cell cycle checkpoint, strongly suggests that CDT induces an as yet uncharacterized DNA alteration . However, the effective entry of CDT into cells and subsequent translocation into the nucleus have not yet been demonstrated by direct methods . The relationship between the potential DNA-damaging properties of this original family of toxins and their role as putative virulence factors is discussed.

FEMS Microbiol Lett, 2001 Sep 25, 203(2), 131 - 9
Ubiquinone biosynthesis in microorganisms; Meganathan R; The quinoid nucleus of the benzoquinone, ubiquinone (coenzyme Q; Q), is derived from the shikimate pathway in bacteria and eukaryotic microorganisms . Ubiquinone is not considered a vitamin since mammals synthesize it from the essential amino acid tyrosine . Escherichia coli and other Gram-negative bacteria derive the 4-hydroxybenzoate required for the biosynthesis of Q directly from chorismate . The yeast, Saccharomyces cerevisiae, can either form 4-hydroxybenzoate from chorismate or tyrosine . However, unlike mammals, S . cerevisiae synthesizes tyrosine in vivo by the shikimate pathway . While the reactions of the pathway leading from 4-hydroxybenzoate to Q are the same in both organisms the order in which they occur differs . The 4-hydroxybenzoate undergoes a prenylation, a decarboxylation and three hydroxylations alternating with three methylation reactions, resulting in the formation of Q . The methyl groups for the methylation reactions are derived from S-adenosylmethionine . While the prenyl side chain is formed by the 2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway in E . coli, it is formed by the mevalonate pathway in the yeast.

Arch Pediatr, 2001 Sep, 8 Suppl 4, 726s - 731s
{Virulence factors associated with E . coli neonatal meningitis}; Bonacorsi S et al.; Escherichia coli K1 is the leading cause of gram-negative bacterial meningitis in neonates . It is associated with a mortality rate as high as 40%, and more than half of the survivors have neurologic sequelae . Bacterial meningitis is the result of bacterial translocation from gastrointestinal tract to the blood and from blood to the central nervous system . Successful crossing of the BBB by E . coli K1 requires (a) a high degree of bacteremia and (b) several E . coli determinants contributing to invasion of BMEC such as the K1 capsule, Sfa, Ibe proteins, and CNF1 . A better understanding for the molecular basis of E . coli K1 penetration of the BBB could potentially lead to the development of novel therapeutic and preventative strategies for E . coli K1 meningitis.

J Endotoxin Res, 2001, 7(3), 167 - 202
Bacterial lipopolysaccharides and innate immunity; Alexander C et al.; Bacterial lipopolysaccharides (LPS) are the major outer surface membrane components present in almost all Gram-negative bacteria and act as extremely strong stimulators of innate or natural immunity in diverse eukaryotic species ranging from insects to humans . LPS consist of a poly- or oligosaccharide region that is anchored in the outer bacterial membrane by a specific carbohydrate lipid moiety termed lipid A . The lipid A component is the primary immunostimulatory centre of LPS . With respect to immunoactivation in mammalian systems, the classical group of strongly agonistic (highly endotoxic) forms of LPS has been shown to be comprised of a rather similar set of lipid A types . In addition, several natural or derivatised lipid A structures have been identified that display comparatively low or even no immunostimulation for a given mammalian species . Some members of the latter more heterogeneous group are capable of antagonizing the effects of strongly stimulatory LPS/lipid A forms . Agonistic forms of LPS or lipid A trigger numerous physiological immunostimulatory effects in mammalian organisms, but--in higher doses--can also lead to pathological reactions such as the induction of septic shock . Cells of the myeloid lineage have been shown to be the primary cellular sensors for LPS in the mammalian immune system . During the past decade, enormous progress has been obtained in the elucidation of the central LPS/lipid A recognition and signaling system in mammalian phagocytes . According to the current model, the specific cellular recognition of agonistic LPS/lipid A is initialized by the combined extracellular actions of LPS binding protein (LBP), the membrane-bound or soluble forms of CD14 and the newly identified Toll-like receptor 4 (TLR4)*MD-2 complex, leading to the rapid activation of an intracellular signaling network that is highly homologous to the signaling systems of IL-1 and IL-18 . The elucidation of structure-activity correlations in LPS and lipid A has not only contributed to a molecular understanding of both immunostimulatory and toxic septic processes, but has also re-animated the development of new pharmacological and immunostimulatory strategies for the prevention and therapy of infectious and malignant diseases.

Acta Gastroenterol Latinoam, 2001, 31(3), 137 - 41
{Role of cytokines in chronic gastritis by Helicobacter pylori}; Jimenez FP et al.; Helicobacter pylori is a curved, gram negative bacterium that inhabits only the gastric mucous membrane . Since its discovery and characterization, it has been related to the physiopathology of gastroduodenal diseases, including gastritis, peptic ulcers, gastric carcinoma and MALT lymphoma . This has resulted in numerous hypotheses that try to explain the different events that take place during the inflammation . The bacterium Settler, characterized by a marked infiltration of inflammatory cells (neutrophils, monocytes, linfocytes, etc.) which, after being activated, liberate locally various chemical mediators, which cause tissue damage . Among these, the cytokines are important mediators in this process . We have revised the literature related to the various biological functions of cytokines in tissue damage of the gastric mucosa.

Microbiology, 2001 Oct, 147(Pt 10), 2643 - 9
FindTarget: software for subtractive genome analysis; Chetouani F et al.; In silico subtractive/differential genome analysis is a powerful approach for identifying genus- or species-specific genes, or groups of genes that are responsible for a unique phenotype . By this method, one searches for genes present in one group of bacteria and absent in another group . A software package has been developed, named FindTarget, that has a user-friendly web interface to facilitate differential genome analysis . The user chooses the genomes to compare, the similarity criteria and the thresholds to decide if a gene has a counterpart in another genome . The searches are based on BLASTP comparisons of proteomes . FindTarget also includes access to sequences, coloured multiple alignments, phylogenetic trees of conserved proteins and links to public annotated databases which provide a means for validation of the results . To illustrate this approach, a FindTarget search for genes putatively involved in the specificity of cell envelope synthesis of Gram-negative bacteria is presented . The results show that most of the identified genes are clearly involved in cell wall processes, underlining the power of such an approach in general and that of FindTarget in particular.

J Clin Microbiol, 2001 Oct, 39(10), 3548 - 54
Aortic valve endocarditis in a dog due to Bartonella clarridgeiae; Chomel BB et al.; We report the first documented case of endocarditis associated with Bartonella clarridgeiae in any species . B . clarridgeiae was identified as a possible etiological agent of human cat scratch disease . Infective vegetative valvular aortic endocarditis was diagnosed in a 2.5-year-old male neutered boxer . Historically, the dog had been diagnosed with a systolic murmur at 16 months of age and underwent balloon valvuloplasty for severe valvular aortic stenosis . Six months later, the dog was brought to a veterinary hospital with an acute third-degree atrioventricular block and was diagnosed with infective endocarditis . The dog died of cardiopulmonary arrest prior to pacemaker implantation . Necropsy confirmed severe aortic vegetative endocarditis . Blood culture grew a fastidious, gram-negative organism 8 days after being plated . Phenotypic and genotypic characterization of the isolate, including partial sequencing of the citrate synthase (gltA) and 16S rRNA genes indicated that this organism was B . clarridgeiae . DNA extraction from the deformed aortic valve and the healthy pulmonic valve revealed the presence of B . clarridgeiae DNA only from the diseased valve . No Borrelia burgdorferi or Ehrlichia sp . DNA could be identified . Using indirect immunofluorescence tests, the dog was seropositive for B . clarridgeiae and had antibodies against Ehrlichia phagocytophila but not against Ehrlichia canis, Ehrlichia ewingii, B . burgdorferi, or Coxiella burnetii.

Br J Anaesth, 2001 Apr, 86(4), 581 - 6
Review of management of purpura fulminans and two case reports; Nolan J et al.; Purpura fulminans (PF) is a haemorrhagic condition usually associated with sepsis or previous infection . Features include tissue necrosis, small vessel thrombosis and disseminated intravascular coagulation . Gram-negative organisms are the commonest cause of the acute infectious type, which is often associated with multi-organ failure . An idiopathic variety, however, is often confined to the skin . The mortality rate has decreased with better treatment of secondary infections, supportive care and new treatments, but it remains a disabling condition often requiring major amputations . We describe two cases and review the various treatments for this condition.

J Pediatr Gastroenterol Nutr, 2001 Aug, 33(2), 165 - 70
Age-dependent changes in the regulation of cyclooxygenases in the gastrointestinal tract after gram-negative endotoxemia; Martinez FE et al.; BACKGROUND: Cyclooxygenases (COXs) modulate prostaglandin synthesis in the gastrointestinal tract . Prostaglandins have been shown to have a cytoprotective effect on bowel mucosa in adults, but no similar data are available in neonates . Thus, the purpose of the current study was to evaluate age-dependent changes in gastrointestinal tract COX regulation after Escherichia coli lipopolysaccharide exposure in rats . METHODS: Stomach, small bowel, and large bowel COX-1 and COX-2 mRNA levels (reverse transcription polymerase chain reaction technique) and protein content (Western blot) were obtained from neonates (younger than 3 days old) and adult rats 18 hours after exposure to E . coli O111:B4 lipopolysaccharide toxin . Untreated animals served as controls . RESULTS: Stomach, small bowel, and large bowel tissue COX-1 mRNA levels in the newborn were significantly lower (P < 0.01) than in the adult . No age-dependent differences were found for COX-2 mRNA levels . After lipopolysaccharide exposure, no significant changes in COX-1 levels were seen at either age, whereas COX-2 mRNA levels were increased only in the stomach for both ages . Western blot analysis of small bowel tissue for COX-1 and COX-2 showed no lipopolysaccharide-induced changes in protein content, but the COX-1 content was significantly lower in the newborn (P < 0.01) . CONCLUSIONS: In the rat, COX expression in the gastrointestinal tract is regulated in an age-dependent fashion . Lower COX-1 expression and a lack of observable increase in COX-2 mRNA levels in the newborn small bowel after endotoxemia may render the bowel more susceptible to bowel injury early in life.

Vaccine, 2001 Oct 12, 20(1-2), 235 - 41
Antibodies to BrkA augment killing of Bordetella pertussis; Oliver DC et al.; BrkA is a Bvg-regulated Bordetella pertussis protein that mediates serum resistance and adherence . It shares sequence identity with another B . pertussis virulence factor called pertactin, and it is a member of the diverse group of proteins found in Gram-negative bacteria that are secreted by an autotransporter mechanism . Sera, either from individuals who have been vaccinated with acellular pertussis vaccines, or from individuals who have no re-collection of recent infection with B . pertussis fail to kill wild-type B . pertussis, but kill brkA mutant strains very well . We examined whether BrkA could be neutralised in serum fitting this profile . BrkA is synthesised as a 103kDa precursor that is processed into a surface-associated N-terminal 73kDa passenger domain, and an outer-membrane embedded C-terminal 30kDa transporter moiety . Polyclonal antibodies were raised to a recombinant, re-folded histidine-tagged fusion protein representing the 73kDa passenger region . These anti-BrkA antibodies were shown to boost the existing bactericidal capacity of human serum against B . pertussis by neutralising BrkA.

EMBO J, 2001 Sep 17, 20(18), 5040 - 8
Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway; Coutte L et al.; Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place . The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins) . The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its approximately 130 kDa C-terminal intramolecular chaperone domain . We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA . SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters . SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria . This is reminiscent of pro-protein convertases of eukaryotic cells.

EMBO J, 2001 Sep 17, 20(18), 5033 - 9
Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site; Vandeputte-Rutten L et al.; OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria . Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel beta-barrel that protrudes far from the lipid bilayer into the extracellular space . We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity . The proteolytic site is located in a groove at the extracellular top of the vase-shaped beta-barrel . Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His-Asp dyad and an Asp-Asp couple that activate a putative nucleophilic water molecule . The active site is fully conserved within the omptin family . Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis . Coordinates are in the Protein Data Bank (accession No . 1I78)

J Biotechnol, 2001 Oct 4, 91(2-3), 197 - 209
Detection, purification and characterisation of quorum-sensing signal molecules in plant-associated bacteria; Brelles-Marino G et al.; Quorum sensing (also called autoinduction) is a term that describes an environmental sensing system that allows bacteria to monitor their own population density . Autoinduction relies upon the interaction of a small diffusible signal molecule (the autoinducer) with a transcriptional activator protein to couple gene expression with cell population density . These signal molecules diffuse from bacterial cells and accumulate in the environment as a function of cell growth . Once a threshold concentration is reached, these signals serve as co-inducers to regulate the transcription of (a) set(s) of target genes . In Gram-negative bacteria, most autoinducers belong to the family of N-acylhomoserine lactones (AHLs) . The detection of AHLs (or AHL-like activities) has been greatly facilitated by the development of sensitive bioassays that allow fast screening of microorganisms for diffusible signal molecules . AHL or diketopiperazine-mediated cell-cell signalling play roles in regulating different bacterial functions, such as antibiotic biosynthesis, production of virulence factors, exopolysaccharide biosynthesis, bacterial swarming, plasmid conjugal transfer and transition into the stationary phase . Several bacterial species that interact with plants produce AHL-like compounds . In this review, we will summarise the current knowledge about the detection, characterisation and purification of quorum-sensing molecules from plant-associated bacteria . We will also discuss some of the future prospects and biotechnological applications of autoinducers.

Arzneimittelforschung, 2001, 51(8), 690 - 7
Results of an open, non-placebo controlled pilot study investigating the immunomodulatory potential of autovaccine; Rusch V et al.; Autovaccines are prepared from autologous, human, non-pathogenic, "rough" variants of E . coli derived from the stool flora of individuals according to a highly standardized procedure . As a fundamental concept within microbiological therapy, these autovaccines are mainly used to treat chronic inflammatory disorders associated with impaired immune reactions resistant to standard therapeutic treatments . Generally, immunomodulatory effects of outer membrane components or cell wall fragments of gram-negative bacteria on innate or adaptive immunity are widely accepted but nevertheless mechanisms of actions of these autovaccines remained obscure, despite some recent publication about other autovaccine preparations of different origin . Hence, immunomodulating properties of autovaccine were investigated in a pilot study with 78 outpatients with variable disorders ranging from recurrent respiratory infections to diffuse gastrointestinal complaints . Patients received their autologous bacteria parenterally in increasing doses . Before application and 4 to 6 weeks after application of autovaccine, blood samples of the patients were taken to investigate a range of immunological parameters such as acute phase proteins, serum antibodies and cytokines . The results revealed that autovaccines were able to modulate significantly the release of three potent immunoregulatory cytokines e.g . interferon-gamma, granulocyte-macrophage-colony stimulating factor and interleukin-1 beta, whereas specific humoral immunity remained largely unaffected . From these results it may be concluded that the autovaccine mainly act antigen non-specifically on the cytokine level rather than inducing a specific vaccination . Further studies with more detailed kinetic measurements of cytokines will have to verify these results.

Cytokine, 2001 Aug 7, 15(3), 156 - 65
Induction of proinflammatory and chemokine genes by lipopolysaccharide and paclitaxel (Taxol) in murine and human breast cancer cell lines; Zaks-Zilberman M et al.; In murine macrophages, the anti-tumor agent, paclitaxel, induces expression of a wide variety of inflammatory and anti-inflammatory genes, and causes cytokine secretion via signaling pathways that overlap with those engaged by lipopolysaccharide (LPS), the endotoxic component of Gram-negative bacteria . Using semi-quantitative RT-PCR for detection of gene expression, coupled with ELISA for the detection of secreted gene products, we analyzed the responsiveness of an extensive panel of cytokine and non-cytokine genes to induction by paclitaxel and LPS in the murine DA-3 breast cancer line . A subset of the genes examined (e.g., G-CSF, MIP-2, iNOS, and IL-1 beta, and GM-CSF) was upregulated >3-20-fold by both LPS and paclitaxel in the DA-3 cell line, while IP-10 mRNA was induced by paclitaxel, but not by LPS . In the human MDA-MB-231 breast cancer cell line, LPS also increased mRNA levels for both GM-CSF and IP-10 significantly, while, paclitaxel increased IP-10 mRNA levels with delayed kinetics and failed to induce GM-CSF mRNA . Co-cultures of murine breast cancer cells and macrophages, stimulated with IFN-gamma plus either paclitaxel or LPS, resulted in augmented release of nitric oxide . As both GM-CSF and IP-10 have been implicated in tumor rejection in vivo through either indirect actions on the host immune system or by inhibiting tumor angiogenesis, our data strengthen the hypothesis that tumor cell-derived inflammatory mediators may, in part, underlie the anti-tumor efficacy of paclitaxel in breast cancer .

J Mol Biol, 2001 Sep 7, 312(1), 143 - 55
The structure of CMP:2-keto-3-deoxy-manno-octonic acid synthetase and of its complexes with substrates and substrate analogs; Jelakovic S et al.; The enzyme CMP-Kdo synthetase (CKS) catalyzes the activation of the sugar Kdo (2-keto-3-deoxy-manno-octonic acid) by forming a monophosphate diester . CKS is a pharmaceutical target because CMP-Kdo is used in the biosynthesis of lipopolysaccharides that are vital for Gram-negative bacteria . We have refined the structure of the unligated capsule-specific CKS from Escherichia coli at 1.8 A resolution (1 A=0.1 nm) and we have established the structures of its complexes with the substrate CTP, with CDP and CMP as well as with the product analog CMP-NeuAc (CMP-sialate) by X-ray diffraction analyses at resolutions between 2.1 A and 2.5 A . The N-terminal domains of the dimeric enzyme bind CTP in a peculiar nucleotide-binding fold, whereas the C-terminal domains form the dimer interface . The observed binding geometries together with the amino acid variabilities during evolution and the locations of a putative Mg(2+) and of a very strongly bound water molecule suggest a pathway for the catalysis . The N-terminal domain shows sequence homology with the CMP-NeuAc synthetases . Moreover, the chain fold and the substrate-binding position of CKS resemble those of other enzymes processing nucleotide-sugars .

Infection, 2001 Aug, 29(4), 205 - 8
Infections caused by Stenotrophomonas maltophilia--a prospective study; Schaumann R et al.; BACKGROUND: Stenotrophomonas maltophilia is an opportunistic microorganism, often highly resistant to routinely tested antibiotics . This microorganism is isolated in specimens from patients with nosocomial infections with increasing frequency . PATIENTS AND METHODS: During a 1-year period (1998/1999) S . maltophilia was isolated from 137 specimens (0.26% of all investigated specimens) from 80 patients who were treated in a 1,500 bed major tertiary care teaching hospital in Leipzig . The data of 76 patients (133 specimens) could be collected and analyzed completely . RESULTS: The pathogen was most frequently detected in specimens from the respiratory tract (54%) . In five patients (six cases) S . maltophilia was isolated from blood cultures (0.3% of all positive blood cultures; 1.4% of all gram-negative isolates from blood cultures) . 70 of the infected patients were inpatients and 32 (42%) of them were treated on the internal medicine wards . Of these 32 patients only six (19%) were pretreated with imipenem . The Length of stay at the hospital resulted in an independent increased risk of infection with S . maltophilia . In addition, this organism was detected in six infected outpatients . CONCLUSION: S . maltophilia is not only a nosocomial pathogen . Pretreatment with a carbapenem is no longer an unequivocal risk factor for an infection with S . maltophilia.

Nature, 2001 Sep 6, 413(6851), 78 - 83
Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction; Fitzgerald KA et al.; The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns . Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria . All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction . MyD88 is one such protein that contains a TIR domain . It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete . Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome . Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2 . Mal can form homodimers and can also form heterodimers with MyD88 . Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs . Mal associates with IRAK-2 by means of its TIR domain . A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R . Mal associates with TLR-4 . Mal is therefore an adapter in TLR-4 signal transduction.

J Immunol, 2001 Sep 15, 167(6), 3316 - 23
Predominant role of toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells; Prebeck S et al.; Chlamydia pneumoniae is an obligate intracellular human pathogen causing diseases such as pneumonia, bronchitis, and pharyngitis . Because of its intracellular replication, cell-mediated immune responses are needed to mediate successful defenses of the host . Because dendritic cells play a central role in linking innate immunity and Ag-specific cell-mediated immune responses we asked whether dendritic cells are activated upon contact with C . pneumoniae and whether known Toll like receptors (TLR) are involved in this process . Here we show that C . pneumoniae was taken up by bone marrow-derived murine dendritic cells . Ingested C . pneumoniae appeared to be unable to develop mature inclusion inside dendritic cells . Furthermore, upon contact with C . pneumoniae dendritic cells were potently stimulated because NF-kappaB was activated and translocated to the nucleus, cytokines like IL-12p40 and TNF-alpha were secreted, and expression of MHC class II molecules, CD40, CD80, and CD86 was up-regulated . Importantly, secretion of cytokines as well as translocation of NF-kappaB were dependent on the presence of TLR2 and independent from TLR4 with the exception of IL-12p40 secretion, which was attenuated in the absence of either a functional TLR2 or 4 . In conclusion, we show here that recognition of the Gram-negative bacterium C . pneumoniae depends largely on TLR2 and only to a minor extent on TLR4.

J Bacteriol, 2001 Oct, 183(19), 5639 - 44
Novel macrolide-specific ABC-type efflux transporter in Escherichia coli; Kobayashi N et al.; In the Escherichia coli genome, five putative open reading frame (ORF) clusters, mdlAB, ybjYZ, yddA, yojHI, and yhiH, have been assumed to be possible genes for ABC drug efflux transporters (I . T . Paulsen, M . K . Sliwinski, and M . H . Saier, Jr., J . Mol . Biol . 277:573-592, 1998) . We cloned all of these ORFs in multicopy plasmids and investigated the drug resistance of drug-supersensitive host cells lacking constitutive multidrug efflux transporter genes acrAB . Among them, only ybjYZ gave significant erythromycin resistance and significantly decreased the accumulation of {(14)C}erythromycin . Therefore, ybjYZ was renamed macAB (macrolide-specific ABC-type efflux carrier) . Plasmids carrying both the macA and -B genes conferred resistance against macrolides composed of 14- and 15-membered lactones but no or weak resistance against 16-membered ones . Neither of the two genes produced resistance alone . The DNA sequence suggests that MacB is an integral membrane protein with four transmembrane segments and one nucleotide-binding domain, while MacA belongs to a membrane fusion protein (MFP) family with a signal-like sequence at its N terminus . The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein . In addition, MacAB required TolC for its function in a way similar to that of most of the MFP-dependent transporters in E . coli . MacB is thus a novel ABC-type macrolide efflux transporter which functions by cooperating with the MFP MacA and the multifunctional outer membrane channel TolC . This is the first case of an experimentally identified ABC antibiotic efflux transporter in gram-negative organisms.

JPEN J Parenter Enteral Nutr, 1991 Sep-Oct, 15(5), 503 - 8
Arginine supplementation improves histone and acute-phase protein synthesis during gram-negative sepsis in the rat; Leon P et al.; Mechanisms of nutrient alteration of hepatic protein synthesis during sepsis are unclear . In vitro, arginine downregulates endotoxin-stimulated hepatocyte protein synthesis but in vivo effects are unknown . This study evaluated the effects of supplemental arginine or glycine on fibrinogen (acute-phase protein), histone, albumin, and liver protein synthesis after Gram-negative sepsis in the rat . Adult rats (225 g, n=36) were randomized to receive isonitrogenous isocaloric total parenteral nutrition supplemented with 264 mg of N per kilogram per day as either arginine or glycine . On day 5, each group was further randomized to control or sepsis . Sepsis was induced by injection of 8 x 10(7) Escherichia coli per 100 g body weight, and then a continuous infusion of {1-14C} leucine was started . The rats were sacrificed 4 hours later . The fractional protein synthesis rates (percent per day) of histone, fibrinogen, albumin, and liver were determined . Supplemental arginine led to significantly increased histone (p < 0.05, analysis of variance) and fibrinogen (p < 0.01, analysis of variance) synthesis in the septic rats compared with all other groups . Histone and albumin synthesis were also significantly increased (p < 0.05) in the arginine-supplemented control group compared with the glycine-supplemented control group . Arginine supplementation during sepsis significantly increased (p < 0.05) albumin and liver protein synthesis compared with controls . Histones which are involved in DNA synthesis and are rich in arginine may play a role in the host response to stress and sepsis . These in vivo results appear to contradict hepatocyte-Kupffer cell coculture studies perhaps because of the hormonal and cytokine responses to nutrient substrate and acute septicemia.

Anal Chem, 2001 Aug 15, 73(16), 3804 - 7
Direct microextraction and analysis of rough-type lipopolysaccharides by combined thin-layer chromatography and MALDI mass spectrometry; Therisod H et al.; A rapid method for the microscale extraction of lipopolysaccharides (endotoxins, LPSs) from rough-type Gram-negative bacteria was developed using thin-layer chromatography (TLC) combined with improved conditions for LPS analysis by mass spectrometry . TLC of intact bacteria on silica gel plates in an appropriate solvent selectively extracted and separated their LPS components . The bands of molecular species were scraped from the plates after nondestructive visualization, directly mixed with matrix, and analyzed by laser desorption time-of-flight mass spectrometry . Lipids A and Re-type LPSs were analyzed after transfer to a membrane . Adding citric acid to the matrix gave greatly improved mass spectra . The system allows characterization of bacterial LPS at the microscale level and is equally well applicable to heterogeneous LPS and lipid A preparations (Escherichia coli lipid A and Bordetella lipopolysaccharides were used) . The technique provides a rapid determination of the heterogeneity of unmodified preparations and the determination of the molecular weight of each separated component.

Biol Blood Marrow Transplant, 2001, 7(7), 384 - 7
Association of TLR4 mutations and the risk for acute GVHD after HLA-matched-sibling hematopoietic stem cell transplantation; Lorenz E et al.; Lipopolysaccharide (LPS) has been implicated in the pathogenesis of graft-versus-host disease (GVHD) . The toll-like receptor (TLR)-4 has been recently identified as a major receptor for LPS . Mutations of TLR4 have been associated with LPS hyporesponsiveness . We hypothesized that TLR4 mutations reduce the risk of acute GVHD in allogeneic marrow transplant recipients . In a preliminary study to determine the frequency of TLR4 mutations and their possible association with GVHD, we tested 237 patients and their HLA-identical sibling donors for 2 TLR4 polymorphisms . All patients received methotrexate and cyclosporine for GVHD prophylaxis . One or more mutants were detected in 10.8% of patients and 10.6% of donors . Multivariable logistic regression models were used to analyze the association between TLR4 mutations and probability (1-sided) of GVHD . The odds ratio (adjusted for advanced disease, total body irradiation dose, and patient age) for development of grades II to IV GVHD when a mutation was present in the recipient was 0.63 (95% confidence interval {CI}, 0.25-1.60; P = .16) . When a mutation was present in the donor, the adjusted odds ratio was 0.88 (95% CI, 0.36-2.17; P = .40) . When a mutation was present in both recipient and donor, the odds ratio was 0.72 (95% CI, 0.22-2.32; P = .29) . Among 24 patients with TLR4 mutations in either donor or recipient, 4 (16.7%) developed gram-negative bacteremia . Among 213 patients without mutations, 14 (6.6%) developed gram-negative bacteremia (P = .09) . The data indicate that a reduced risk of acute GVHD is associated with TLR4 mutations and that TLR4 mutations may increase the risk for gram-negative bacteremia . However, these associations are not statistically significant in recipients of HLA-matched sibling marrow transplants who are prophylactically treated for infections and GVHD . A much larger study population would be needed to confirm the role of LPS in the pathogenesis of GVHD in humans.

J Environ Monit, 1999 Apr, 1(2), 163 - 8
Use of quadrupole GC-MS and ion trap GC-MS-MS for determining 3-hydroxy fatty acids in settled house dust: relation to endotoxin activity; Saraf A et al.; Gas chromatography-mass spectrometry (GC-MS) using a quadrupole instrument and GC-tandem MS (GC-MS-MS) using an ion trap instrument were applied to determine 3-hydroxy fatty acids (3-OH FAs) with 10-18 carbon chain lengths, specific components of the endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria, in 30 house dust samples . The two methods provided similar detection sensitivity for methyl ester/trimethylsilyl derivatives of the 3-OH FAs and allowed these acids to be distinguished from co-eluting 2-OH FA derivatives . The correlation coefficients between endotoxin activity (Limulus test) and the combined amounts of 3-OH C10, 3-OH C12, and 3-OH C14 were 0.60 and 0.61 when using GC-MS and GC-MS-MS, respectively . The superior selectivity of GC-MS-MS was illustrated in analyses of sub-milligram amounts of dust, where the chromatograms achieved by GC-MS were difficult to interpret due to a high background and several closely eluting compounds . GC-MS-MS is therefore preferable to GC-MS for determining 3-OH FAs in minute (sub-milligram) amounts of dust.

Genes Immun, 2001 Aug, 2(5), 258 - 62
Polymorphisms of Fc gamma-receptors RIIa, RIIIa, and RIIIb in patients with adult periodontal diseases; Meisel P et al.; Polymorphisms influencing the binding affinity between the Fcgamma receptors and IgG of different subclasses are thought to be of importance in the individual susceptibility to infections with Gram-negative bacteria contributing to periodontal disease . One hundred and fifty-four Caucasian subjects were clinically and radiographically examined for their periodontal status and genotyped for their allelic pattern of FcgammaRIIa, FcgammaRIIIa, and FcgammaIIIb polymorphism . In assessing periodontitis according to mean probing depth and attachment loss, no differences were found in allele frequencies or combined allotypes between the subjects with mild or moderate and those with severe signs of periodontitis . However, the extent and severity of bone loss were significantly associated with the genotype of the receptor FcgammaRIIIa . An increased risk of severe bone destruction was observed in individuals carrying the FcgammaRIIIa-VV genotype (OR = 5.3; 95% CI 1.4-26.2) . FcgammaRIIIb is in linkage disequilibrium with FcgammaRIIIa . Hence it is also related to periodontal disease . There is no indication of an association between the polymorphism of FcgammaRIIa and periodontitis . The results are evidence that the FcgammaRIIIa genotype coding for the high affinity receptor imposes an additional risk of bone loss as does the FcgammaRIIIb genotype coding for the low affinity receptor.

Biochem Cell Biol, 2001, 79(4), 449 - 59
Helicobacter pylori from asymptomatic hosts expressing heptoglycan but lacking Lewis O-chains: Lewis blood-group O-chains may play a role in Helicobacter pylori induced pathology; Monteiro MA et al.; Helicobacter pylori is a widespread Gram-negative bacterium responsible for the onset of various gastric pathologies and cancers in humans . A familiar trait of H . pylori is the production of cell-surface lipopolysaccharides (LPSs; O-chain --> core --> lipid A) with O-chain structures analogous to some mammalian histo-blood-group antigens, those being the Lewis determinants (Lea, Leb, Lex, sialyl Lex, Ley) and blood groups A and linear B . Some of these LPS antigens have been implicated as autoimmune, adhesion, and colonization components of H . pylori pathogenic mechanisms . This article describes the chemical structures of LPSs from H . pylori isolated from subjects with no overt signs of disease . Experimental data from chemical- and spectroscopic-based studies unanimously showed that these H . pylori manufactured extended heptoglycans composed of 2- and 3-linked D-glycero-alpha-D-manno-heptopyranose units and did not express any blood-group O-antigen chains . The fact that another H . pylori isolate with a similar LPS structure was shown to be capable of colonizing mice indicates that H . pylori histo-blood-group structures are not an absolute prerequisite for colonization in the murine model also . The absence of O-chains with histo-blood groups may cause H . pylori to become inept in exciting an immune response . Additionally, the presence of elongated heptoglycans may impede exposure of disease-causing outer-membrane antigens . These factors may render such H . pylori incapable of creating exogenous contacts essential for pathogenesis of severe gastroduodenal diseases and suggest that histo-blood groups in the LPS may indeed play a role in inducing a more severe H . pylori pathology.

Nat Immunol, 2001 Sep, 2(9), 882 - 8
Inducible IL-2 production by dendritic cells revealed by global gene expression analysis; Granucci F et al.; Dendritic cells (DCs) are strong activators of primary T cell responses . Their priming ability is acquired upon encounter with maturation stimuli . To identify the genes that are differentially expressed upon maturation induced by exposure to Gram-negative bacteria, a kinetic study of DC gene expression was done with microarrays representing 11,000 genes and ESTs (expressed sequence tags) . Approximately 3000 differentially expressed transcripts were identified . We found that functional interleukin 2 (IL-2) mRNA, which gave rise to IL-2 production, was transiently up-regulated at early time-points after bacterial encounter . In contrast, macrophages did not produce IL-2 upon bacterial stimulation . Thus, IL-2 is an additional key cytokine that confers unique T cell stimulatory capacity to DCs.

Appl Environ Microbiol, 2001 Sep, 67(9), 4024 - 9
Enzymatic manganese(II) oxidation by a marine alpha-proteobacterium; Francis CA et al.; A yellow-pigmented marine bacterium, designated strain SD-21, was isolated from surface sediments of San Diego Bay, San Diego, Calif., based on its ability to oxidize soluble Mn(II) to insoluble Mn(III, IV) oxides . 16S rRNA analysis revealed that this organism was most closely related to members of the genus Erythrobacter, aerobic anoxygenic phototrophic bacteria within the alpha-4 subgroup of the Proteobacteria (alpha-4 Proteobacteria) . SD-21, however, has a number of distinguishing phenotypic features relative to Erythrobacter species, including the ability to oxidize Mn(II) . During the logarithmic phase of growth, this organism produces Mn(II)-oxidizing factors of approximately 250 and 150 kDa that are heat labile and inhibited by both azide and o-phenanthroline, suggesting the involvement of a metalloenzyme . Although the expression of the Mn(II) oxidase was not dependent on the presence of Mn(II), higher overall growth yields were reached in cultures incubated with Mn(II) in the culture medium . In addition, the rate of Mn(II) oxidation appeared to be slower in cultures grown in the light . This is the first report of Mn(II) oxidation within the alpha-4 Proteobacteria as well as the first Mn(II)-oxidizing proteins identified in a marine gram-negative bacterium.

Mar Pollut Bull, 2001 Aug, 42(8), 689 - 95
The effect of TBT on the structure of a marine sediment comunity--a Boxcosm study; Dahllof I et al.; The effect of tri-n-butyl tin (TBT) on an intact marine sediment community after five months exposure was investigated . Changes in the structure of macro- and meiofauna communities were determined, as well as the functional diversity of the microbial community using BIOLOG microplates for Gram negative bacteria . Development of tolerance in the microbial community was investigated using Pollution Induced Community Tolerance (PICT) experiments with fluxes of nutrients as effect indicators . TBT affected the structure and recruitment of the macro- and meiofauna at nominal additions of 30-137 micromol TBT/m2 sediment . Number of species, diversity, biomass and community similarity was reduced at these concentrations compared to control . Species that molt seemed to be the most tolerant since they were predominant in boxes that had received the highest TBT addition and echinoderms were the most sensitive species . Renewed addition of TBT in PICT experiments with sediment from each boxcosm showed that TBT had an effect on individual nutrient fluxes from all sediments . Analyses of the flux patterns revealed a memory of previous TBT exposure, either due to induced tolerance or other community conditioning.

Extremophiles, 2001 Aug, 5(4), 257 - 64
A novel replication element from an Antarctic plasmid as a tool for the expression of proteins at low temperature; Tutino ML et al.; Genetic manipulation of Antarctic bacteria has been very limited so far . This article reports the isolation and molecular characterization of a novel plasmid, pMtBL, from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAC 125 . This genetic element, 4,081 bp long, appeared to be a multicopy cryptic replicon with no detectable transcriptional activity . By an in vivo assay, the pMtBL autonomous replication sequence was functionally limited to an AluI plasmid fragment of about 850 bp . This novel cold-adapted replication element showed quite a broad host range profile: it was cloned into a mesophilic genetic construction, obtaining a cold-adapted expression vector that was able to promote the production of P . haloplanktis A23 alpha-amylase in a psychrophilic bacterium . This study represents the first report of successful recombinant production of a cold-adapted protein in an Antarctic host.

J Endotoxin Res, 2001, 7(2), 147 - 55
Influence of acyl chain fluidity on the lipopolysaccharide-induced activation of complement; Wiese A et al.; Lipopolysaccharides (LPSs, endotoxins) are the major amphiphilic constituents of the outer leaflet of the outer membrane of Gram-negative bacteria . They are known to activate the complement cascade to form lytic membrane pores . Here, we study the influence of the fluidity of the acyl chains of LPSs and lipid As on the formation of lytic pores . To this end, we have performed electrical measurements on asymmetric planar endotoxin/phospholipid bilayers as a reconstitution model of the outer membrane using two deep rough mutant LPSs (from Escherichia coli strains WBB01 and WBB25) and two lipid As (from E . coli WBB25 and Rhodobacter sphaeroides) . The two LPSs and the two lipid As each differ in their acylation pattern which is correlated with the fluidity . The addition of human serum to the endotoxin side of the bilayers led to the formation of membrane pores, and pore formation correlated in each case with acyl chain fluidity, i.e . time required for the first lytic pore to be formed was shorter for the more fluid endotoxin . Furthermore, in the case of LPSs, the activation rate was higher for the more fluid membrane and the respective bacteria had a higher susceptibility to the growth inhibitory action of serum.

J Endotoxin Res, 2001, 7(1), 69 - 72
Outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP) are released in experimental Gram-negative sepsis; Hellman J et al.; We previously showed that Escherichia coli bacteria incubated in normal human serum release complexes that contain three conserved Gram-negative bacterial outer membrane proteins (OMPs) and LPS . We have identified the OMPs as outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP) . These OMPs are conserved among enteric Gram-negative bacteria and are bound by IgG in antisera raised to heat-killed rough bacteria such as E . coli J5 (J5 IgG) . The present experiments were performed to further analyze the release of these OMPs in a rat wound infection model of sepsis . Plasma was collected from thermally injured rats with E . coli O18 sepsis and filtered . LPS was affinity-purified from plasma filtrates using monoclonal antibody specific for the O-polysaccharide side chain of E . coli O18 LPS . Plasma filtrates were also incubated with J5 IgG conjugated to magnetic beads . Affinity-purified samples were analyzed for the OMPs by immunoblotting . OmpA, PAL, and MLP were released into septic rat blood in complexes with LPS . PAL was consistently present in samples affinity-purified using J5 IgG . The results indicate that OmpA, PAL, and MLP are released and circulate in experimental Gram-negative sepsis and suggest that a proportion of released OMPs are tightly associated with LPS.

J Endotoxin Res, 2000, 6(6), 477 - 82
Plasma constituents regulate LPS binding to, and release from, the monocyte cell surface; Kitchens RL et al.; Innate immunity to Gram-negative bacteria involves regulated mechanisms that allow sensitive but limited responses to LPS . Two important pathways that lead to host cell activation and LPS deactivation involve: (i) LPS interactions with CD14 and Toll-like receptor 4 on cells (activation); and (ii) LPS sequestration by plasma lipoproteins (deactivation) . Whereas these pathways were previously thought to be independent and essentially irreversible, we found that they are connected by a third pathway: (iii) the movement of LPS from host cells to plasma lipoproteins . Our data show that, in the presence of human plasma, LPS binds transiently to monocyte surfaces and then moves from the cell surface to plasma lipoproteins . Soluble CD14 enhances LPS release from cells in the presence of lipoproteins, whereas LPS binding protein and phospholipid transfer protein do not . The transfer of cell-bound LPS to lipoproteins is accompanied by reduced cell responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo . Preliminary data suggest that changes that occur in the plasma after trauma or during sepsis decrease LPS binding to leukocytes while greatly increasing the rate of LPS release from cells.

J Endotoxin Res, 2000, 6(5), 389 - 91
Innate recognition of lipopolysaccharide by Toll-like receptor 4/MD-2 and RP105/MD-1; Miyake K et al.; The Toll family of receptors has been implicated in innate recognition and subsequent activation of defense programs against pathogens such as bacteria and fungi . TLR4, for example, signals the presence of lipopolysaccharide (LPS), a membrane constituent of Gram-negative bacteria . LPS signaling via TLR4 is greatly enhanced by a molecule referred to as MD-2, which is associated with the extracellular domain of TLR4 . The TLR4/MD-2 complex, therefore, recognizes LPS . RP105, another member of the Toll family, has a striking similarity to TLR4 in that it is associated with an MD-2-like molecule MD-1 . B-cells lacking RP105 are severely impaired in LPS-induced proliferation and antibody production . Studies employing transfectants showed that RP105/MD-1, like MD-2, enhances the LPS signaling via TLR4 . RP105/MD-1 thus constitutes an LPS-signaling complex on B-cells . These results suggest that a variety of cell surface molecules regulate LPS recognition/signaling by TLR4.

Immunity, 2001 Aug, 15(2), 313 - 21
Role of Janus kinase 3 in mast cell-mediated innate immunity against gram-negative bacteria; Malaviya R et al.; Mast cells play a pivotal role in innate host immune response to gram-negative bacteria . We report that Janus kinase 3 plays a role in mast cell-mediated bacterial clearance and neutrophil recruitment by regulating the release of tumor necrosis factor from mast cells . The role of JAK3 in mast cell-facilitated neutrophil recruitment and bacterial clearance was investigated by comparing the neutrophil influxes and bacterial clearance in mast cell-deficient W/W(v) mice reconstituted with JAK3(+/+) or JAK(-/-) mast cells . The neutrophil influx, bacterial clearance, and survival outcome in W/W(v) mice reconstituted with JAK3(+/+) mast cells was better than in W/W(v) mice reconstituted with JAK3(-/-) mast cells . These findings provide evidence that JAK3 is a key regulator of mast cell-mediated innate immunity against gram-negative bacteria.

Antonie Van Leeuwenhoek, 2001 Jun, 79(2), 209 - 17
Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences; Fjellbirkeland A et al.; Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil . For this purpose we have used DGGE analysis of functional and phylogenetic markers . Functional markers utilised comprised the pmoA-gene coding for the alpha-subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the alpha-subunit of methanol dehydrogenase (MDH) present in all gram-negative methylotrophs . In addition, we have used 16S rDNA as a phylogenetic marker . DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp . and Methylomicrobium sp . The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs . We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.

Handchir Mikrochir Plast Chir, 2001 Jul, 33(4), 262 - 6
{Plasma endotoxin, procalcitonin, C-reactive protein, and organ functions in patients with major burns}; Ulrich D et al.; Sepsis is one of the most frequent causes of death after major burn injury . Usually, sepsis appears as a consequence of a gram-negative bacteriaemia with release of endotoxins.In this study, the plasma endotoxin levels of seven patients (three female, four male; average age 51.3 +/- 23.8 years) with burns between 43.5 and 78 % Total Body Surface Area (Abbreviated Burn Severity Index 8 - 12) were determined for five days after thermal trauma every three hours by ELISA and compared with the concentration of procalcitonin (PCT) and C-reactive protein (CRP) . A calculation of the Horrowitz-Index (PaO(2)/FiO(2)) and the Pressure-Adjusted Heart Rate (HR x CVP/MAP) took place to show a possible correlation between the endotoxin concentration and the cardiopulmonary organ function . Additionally, we analysed whether operative treatment can influence the level of plasma endotoxin in the early phase after burn injury.At any time after burn trauma, endotoxins could be detected in the plasma of all patients . Between the second and third day, there was a considerable increase in the endotoxin concentration with a maximum after 57 hours of 0.48 +/- 0.32 EU/ml . Two patients with sepsis and death in the further course had a rather distinctive increase . From the fourth day on, occasional episodes of increases in endotoxin concentration were noted . Postoperatively, there was a short increase in plasma endotoxin on the second and fourth day . The plasma endotoxin level showed no correlation with the PCT and CRP or with the oxygenation in the patients' blood . However, a positive correlation could be observed with the Pressure-Adjusted Heart Rate (p = 0.0061; r(2) = 0.212).An explanation for the endotoxin increase after 57 hours could be the translocation of intestinal bacteria, the beginning of bacterial colonisation or decomposition products of the burn wound with protein-protein complexes . Later on, infectious diseases such as pneumonia with gram-negative bacteria are of importance, too.According to the Two-Hit Model, the increase of plasma endotoxin can serve as a trigger and cause a recurrence of systemic inflammation with the changes observed in cardiac organ function, multiple organ dysfunction, and multiple organ failure.

Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 241 - 5
{The new antiendotoxin strategies}; Wang DN et al.; Lipopolysaccharide(LPS) is important in the pathogenesis of sepsis infected by gram-negative bacteria in humans, and part or all of this pathophysiology is mediated by a complex secondary inflammatory response . Administration of purified LPS or a variety of LPS-free recombinant mediators can reproduce much of the pathophysiology of gram-negative sepsis . There has been an explosion of new information over the last several years regarding the interactions of LPS with proteins and cells . Blocking sepsis at the bacterial toxin level is appealing because it is upstream, so that the secondary pathologic inflammatory cascade may be limited or prevented . A variety of antiendotoxin strategies have been proposed . Therapies under investigation include agents that bind and neutralize LPS, agents or systems that enhance LPS clearance, and agents that inhibit LPS interaction with serum elements or cellular receptors.

Scand J Infect Dis, 2001, 33(7), 533 - 7
Risk factors and prognostic indicators of bacterial meningitis in a cohort of 3580 postneurosurgical patients; Federico G et al.; In order to identify the incidence, risk factors and prognostic indicators of postneurosurgical bacterial meningitis, a 9-y retrospective cohort study was performed . The cohort comprised 3580 individuals who underwent neurosurgical operations at the Department of Neurosurgery of Catholic University, Rome, a 1,700-bed university hospital . We observed 52 episodes of postneurosurgical meningitis, with infection rates of 1.4/100 patients, 0.8/100 operations for craniotomies and 2.6/100 operations for internal cerebrospinal fluid (CSF) shunt or ventriculostomy with external drainage . The overall infection rate decreased from 2.5/100 operations in 1989 to 0.9/100 in 1997 (p = 0.03) . Logistic regression analysis indicated that duration of ventriculostomy with external drainage {p < 0.01; odds ratio (OR) = 9.67; 95% confidence interval (CI) = 2.44-38.321 and increasing value of the APACHE III score (p <0.01; OR = 8.51; 95% CI = 2.15-33.68) were independent risk factors for development of meningitis . The overall case fatality rate was 8% . Predictors of mortality were low ( < 1.66 mmol/l) CSF glucose concentration (p = 0.001), increasing value of the APACHE IIl score (p = 0.002) and Gram-negative aetiology (p = 0.003).

Genetics, 2001 Aug, 158(4), 1657 - 64
Excess of rare amino acid polymorphisms in the Toll-like receptor 4 in humans; Smirnova I et al.; The Toll-like receptor 4 protein acts as the transducing subunit of the lipopolysaccharide receptor complex and assists in the detection of Gram-negative pathogens within the mammalian host . Several lines of evidence support the view that variation at the TLR4 locus may alter host susceptibility to Gram-negative infection or the outcome of infection . Here, we surveyed TLR4 sequence variation in the complete coding region (2.4 kb) in 348 individuals from several population samples; in addition, a subset of the individuals was surveyed at 1.1 kb of intronic sequence . More than 90% of the chromosomes examined encoded the same structural isoform of TLR4, while the rest harbored 12 rare amino acid variants . Conversely, the variants at silent sites (intronic and synonymous positions) occur at both low and high frequencies and are consistent with a neutral model of mutation and random drift . The spectrum of allele frequencies for amino acid variants shows a significant skew toward lower frequencies relative to both the neutral model and the pattern observed at linked silent sites . This is consistent with the hypothesis that weak purifying selection acted on TLR4 and that most mutations affecting TLR4 protein structure have at least mildly deleterious phenotypic effects . These results may imply that genetic variants contributing to disease susceptibility occur at low frequencies in the population and suggest strategies for optimizing the design of disease-mapping studies.

Am J Surg, 2001 Jun, 181(6), 571 - 5
The effect of glucagon-like peptide 2 on intestinal permeability and bacterial translocation in acute necrotizing pancreatitis; Kouris GJ et al.; BACKGROUND: Acute pancreatitis (AP) initiates a generalized inflammatory response that increases intestinal permeability and promotes bacterial translocation (BT) . Impairment of the intestinal epithelial barrier is known to promote BT . Glucagon-like peptide 2 (GLP-2), a 33 residue peptide hormone, is a key regulator of the intestinal mucosa by stimulating epithelial growth . The purpose of this study was to determine whether GLP-2 decreases intestinal permeability and BT in AP . METHODS: To examine whether GLP-2 can decrease intestinal permeability and thereby decrease BT in acute necrotizing pancreatitis, 34 male Sprague-Dawley rats (200 to 300 g) were studied . AP was induced in group I and group II by pressure injection of 3% taurocholate and trypsin into the common biliopancreatic duct (1 mg/kg of body weight) . The potent analog to GLP-2 called ALX-0600 was utilized . Group I rats received GLP-2 analog (0.1 mg/kg, SQ, BID) and group II rats received a similar volume of normal saline as a placebo postoperatively for 3 days . Group III and group IV received GLP-2 analog and placebo, respectively . At 72 hours postoperatively, blood was drawn for culture of gram-negative organisms . Specimens from mesenteric lymph nodes (MLN), pancreas and peritoneum were harvested for culture of gram-negative bacteria . Intestinal resistance as defined by Ohm's law was determined using a modified Ussing chamber to measure transepithelial current at a fixed voltage . A point scoring system for five histologic features that include intestinal edema, inflammatory cellular infiltration, fat necrosis, parenchymal necrosis, and hemorrhage was used to evaluate the severity of pancreatitis . Specimens from MLN, pancreas, jejunum, and ileum were taken for pathology . RESULTS: All group I and group II rats had AP . The average transepithelial resistance in group I was 82.8 Omega/cm(2) compared with 55.9 Omega/cm(2) in group II (P <0.01) . Gram-negative BT to MLN, pancreas, and peritoneum was 80%, 0%, and 0%, respectively in group I compared with 100%, 30%, and 20% translocation in group II . CONCLUSION: GLP-2 treatment significantly decreases intestinal permeability in acute pancreatitis.

Clin Transpl . 2000;:297-310.
Sixteen-year experience with 1,000 heart transplants at UCLA; Marelli D et al.; 1 . The consecutive pre- and post-1994 eras have demonstrated improved survival for all age groups . This is linked to improved preservation methods, surgical technique and immunosuppression agents . 2 . The use of marginal donor hearts for Status I and alternate elderly patients has followed the model of matching donor and recipient risk without affecting patient outcome and minimized the use of implantable assist devices . 3 . A donor history of systemic gram-negative infection, hypertension, or traumatic intracranial bleeds was an important marker for risk . Younger age and shorter ischemia time could compensate for other hazards . 4 . Heart transplantation in carefully selected elderly recipients yielded clinical results similar to those of younger patients with less rejection . 5 . An adult alternate recipient list proved useful to prevent diversion of standard donors away from younger recipients . 6 . Retransplantation for TCAD is acceptable but much less satisfactory for acute graft failure . 7 . Trends show an increase in the use of implantable devices; refinement in technology for mechanical assist and replacement is forthcoming.

Intensive Care Med, 2001 Aug, 27(8), 1274 - 80
Extremely low doses of tissue factor pathway inhibitor decrease mortality in a rabbit model of septic shock; Matyal R et al.; OBJECTIVE: We sought to determine the lowest dose of recombinant human tissue factor pathway inhibitor (TFPI) that can provide protection from lethality in a rabbit model of septic shock . METHODS: Sepsis was induced in New Zealand white rabbits by intraperitoneal implantation of 7.0 ml of a solution containing hemoglobin (4.8 g/dl), porcine mucin (6 g/dl), and 0.8-1.4 x 10(4) viable Escherichia coli (strain O:18 K+) . Gentamicin (5 mg/kg) was administered 4 h following surgery, and this dose was repeated every 12 h for 3 days . Beginning 4 h following the induction of sepsis, animals were treated with a bolus (1 ml) plus a continuous infusion (100 ml over 24) of either TFPI (various doses) or its vehicle . Four different doses of TFPI were studied, and each experiment included a contemporaneous control group . The primary outcome parameter was survival time . Results were analyzed using the Wilcoxen log rank test . RESULTS: The average survival time for rabbits treated with the highest dose of TFPI tested (50 microg/kg bolus and 0.5 microg/kg per minute infusion) was 118 h, as compared to 81 h in vehicle-treated controls) . The average survival time for septic rabbits treated with a much lower dose of TFPI (100 ng/kg bolus and 1.0 ng/kg per minute infusion) was 119 h as compared to 57 h in surviving vehicle-treated controls . Treatment with an even lower dose of TFPI (10 ng/kg bolus and 0.1 ng/kg per minute infusion) still produced a marginally significant prolongation of average survival time (80 h) relative to contemporaneously studied controls (47 h) . When the dose of TFPI was decreased still further (1.0 ng/kg bolus and 0.01 ng/kg per minute infusion), average survival times were not significantly different between TFPI-treated and vehicle-treated rabbits (77 and 51 h, respectively) . CONCLUSIONS: Delayed infusion with remarkably low doses of recombinant human TFPI prolongs survival in a rabbit model of antibiotic-treated Gram-negative bacterial sepsis . In planning human trials of TFPI as an adjuvant treatment for sepsis it may be reasonable to use much lower doses of the agent than were heretofore contemplated.

J Immunol, 2001 Sep 1, 167(5), 2895 - 901
Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice; Gao X et al.; The in vivo contributions of CD18 integrin-dependent and -independent mechanisms in mediating the increases in lung neutrophil (polymorphonuclear leukocyte; PMN) sequestration and microvascular permeability are not well understood . We determined the time course of these responses to Gram-negative sepsis in the mouse lung and addressed the specific contributions of CD18 integrins and ICAM-1 . PMN sequestration in the lung was assessed by morphometric analysis, and transalveolar PMN migration was assessed by bronchoalveolar lavage . Lung tissue PMN number increased by 6-fold within 1 h after i.p . Escherichia coli challenge; this value peaked at 3 h (7-fold above control) and decreased at 12 h (3.5-fold above control) . PMN migration into the airspace was delayed; the value peaked at 6 h and remained elevated up to 12 h . Saturating concentrations of anti-CD18 and anti-ICAM-1 mAbs reduced lung tissue PMN sequestration and migration; however, peak responses at 3 and 6 h were inhibited by 40%, indicating that only a small component of PMN sequestration and migration was CD18 dependent at these times . In contrast to the time-dependent decreased role of CD18 integrins in mediating PMN sequestration and migration, CD18 and ICAM-1 blockade prevented the increase in lung microvascular permeability and edema formation at all times after E . coli challenge . Thus, Gram-negative sepsis engages CD18/ICAM-1-independent mechanisms capable of the time-dependent amplification of lung PMN sequestration and migration . The increased pulmonary microvascular permeability induced by E . coli is solely the result of engagement of CD18 integrins even when PMN accumulation and migration responses are significantly CD18 independent.

Biometals, 2001 Jun, 14(2), 153 - 7
The remarkable hydrophobic effect of a fatty acid side chain on the microbial growth promoting activity of a synthetic siderophore; Lin YM et al.; The ability of synthetic derivatives of the siderophore tripeptide of N5-hydroxy-N5-acetyl-L-ornithine to promote the growth of various strains of mycobacteria and Gram negative bacteria was found to depend significantly on the hydrophobic nature of the derivative . Although the tripeptide of N5-hydroxy-N5-acetyl-L-ornithine is not normally utilized by mycobacteria, an N-terminal palmitoyl derivative mimicked natural mycobactin J in all studies.

Postepy Hig Med Dosw, 2001, 55(3), 355 - 67
{Organization of the Helicobacter pylori genome}; Zawilak A et al.; Helicobacter pylori is a Gram-negative, spiral-shaped pathogenic bacterium that was firstly isolated and cultured from biopsy specimens by Marshall and Warren in 1983 . This organism is a human gastric pathogen associated with peptic ulcer disease as well as chronic gastritis . Recent epidemiological studies have demonstrated that H . pylori is a primary risk factor for the development of intestinal type gastric adenocarcinoma . H . pylori is the first bacterium for which the genomes of two unrelated strains (26695 and J99) have been sequenced . The genome of H . pylori is relatively low in size (1.6-1.73 Mb) . In this review, we compare the organization of two sequenced H . pylori genomes . A special emphasis on genetic diversity of H . pylori including plasticity zone and cag pathogenicity island has been placed.

Res Microbiol, 2001 Jul-Aug, 152(6), 523 - 9
The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?
Lloubes R, Cascales E, Walburger A, Bouveret E, Lazdunski C, Bernadac A, Journet L.
The outer membrane of gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins . However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope . The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity . These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities.

Microb Pathog, 2001 Sep, 31(3), 145 - 50
Orientia tsutsugamushi suppresses the production of inflammatory cytokines induced by its own heat-stable component in murine macrophages; Kim MK et al.; Orientia tsutsugamushi is a Gram-negative obligate intracellular bacterium, which causes scrub typhus . To understand the pathogenesis of scrub typhus, we have investigated the induction of tumor necrosis alpha (TNF-alpha) and interleukin-6 (IL-6) by O . tsutsugamushi in two murine macrophage cell lines . Both live and heat-killed orientia stimulated the production of cytokines in J774A.1 cells . Polymyxin B does not affect the secretion of cytokines . These together with the fact that the immature macrophage cell line, P388D1, did not produce TNF-alpha when induced by either live or heat-killed O . tsutsugamushi strongly argue against any roles of lipopolysaccharide (LPS) in cytokine production . Furthermore, the result that the cytokine responses were more brisk when macrophage cell lines had been induced by heat-killed O . tsutsugamushi than by live organisms strongly suggest that a heat-stable molecule might be responsible for the induction of cytokine production and O . tsutsugamushi might have mechanisms suppressing the production of inflammatory cytokines induced by its own heat-stable molecule .

An Med Interna, 2001 May, 18(5), 255 - 8
{Bartonella henselae endocarditis . Report of a case and review of the literature}; Cilla Eguiluz G et al.; Bartonella spp are small Gram-negative rods, aerobic and highly fastidious . They are difficult to culture, in the routine bacterial cultures . They are considered as emergent human pathogens . Since 1993, three species of Bartonella (B . quintana, B . henselae, and B . elizabethae) have been described as causative agents of infectious endocarditis . In this paper we describe the case of a 43 year-old woman with a previous valvular heart disease, probably of rheumatic origin, owner of cats, that suffered an infectious endocarditis by Bartonella henselae in the aortic valve . This patient presented IgG titers against B . henselae of 1/4096 and against B . quintana of 1/256 . She also had low IgM titers against B . henselae and B . quintana: 1/64 and 1/32, respectively . The patient received antibiotics for 12 weeks and suffered a valvular replacement due to the severe lesion on the aortic valve . On the endocardiac tissue of the removed valve DNA of B . henselae was detected (polymerase chain reaction-based assay) . Clinical evolution of the patient was good . Diagnosis of Bartonella spp infection must be considered in every patient with infectious endocarditis and negative blood cultures, and particularly B . henselae in patients with previous valvular heart disease and regular contact with cats.

Microbiology, 2001 Aug, 147(Pt 8), 2379 - 87
The evolution of bacterial LuxI and LuxR quorum sensing regulators; Gray KM et al.; Quorum sensing is a widespread form of bacterial communication in which individual cells produce and respond to specific N-acyl homoserine lactone signal metabolites . The different autoinducer synthases that generate these signals and the receptor/activator proteins that mediate the cell's response to them constitute evolutionarily conserved families of regulatory proteins known as the LuxI and LuxR families, respectively . We have performed a phylogenetic analysis of 76 individual LuxI and LuxR homologues present in diverse members of the Gram-negative Proteobacteria . The results were consistent with an early origin for these regulators during the evolution of the Proteobacteria, with functional pairs of luxI and luxR genes possibly coevolving as regulatory cassettes . In many cases, specific LuxI and LuxR family members appeared to have been inherited horizontally . In particular, those species containing multiple LuxI and/or LuxR homologues usually appeared to have obtained each individual homologue or functional pair of homologues from an independent source . Because multiple homologues interact to form regulatory cascades, this finding suggests that hierarchical signalling pathways can potentially evolve by the sequential integration of pre-existing regulatory circuits acquired from diverse sources.

Microbiology, 2001 Aug, 147(Pt 8), 2065 - 75
Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria; Marx CJ et al.; Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation . One bacterium for which new genetic tools are needed is the methylotroph Methylobacterium extorquens AM1 . IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M . extorquens AM1 . This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors . These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in Escherichia coli; (2) small size (7.2 and 8.0 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue-white screening via lacZalpha; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors . Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M . extorquens AM1 . Specific expression vectors were developed for use in M . extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria . The strong mxaF promoter was used for expression, since E . coli lac-derived promoters were expressed at very low levels . This suite of genetic tools will enable a more sophisticated analysis of the physiology of M . extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1463 - 70
Limnobacter thiooxidans gen . nov., sp . nov., a novel thiosulfate-oxidizing bacterium isolated from freshwater lake sediment; Spring S et al.; Two novel thiosulfate-oxidizing strains were isolated from sediment of the littoral zone of a freshwater lake (Lake Chiemsee, Bavaria, Germany) . The new isolates, designated CS-K1 and CS-K2T, were gram-negative, slightly curved rods with pointed ends that were motile by means of single polar flagella . Both strains were obligately aerobic and grew on a variety of organic substrates, but not autotrophically . The utilization of thiosulfate led to an increase in the growth yield, indicating that these strains were able to grow chemolithoheterotrophically by oxidation of thiosulfate to sulfate . The optimum thiosulfate concentrations for growth were determined to be 10 mM for strain CS-K1 and 20 mM for strain CS-K2T . Phylogenetically, both strains were affiliated to the beta-Proteobacteria . Their characterization by a polyphasic approach resulted in the placement of both strains into a single species that is related only distantly to any known type species . Thus, the creation of a novel taxon is proposed, with the name Limnobacter thiooxidans gen . nov., sp . nov., to include the novel strains . In addition, the phylogenetic position of the chemolithoheterotrophic strain 'Thiobacillus' Q was determined.

Surgery, 2001 Aug, 130(2), 192 - 7
Accelerated internalization and detoxification of endotoxin by anti-lipopolysaccharide antibody is an Fc receptor--mediated process; Lazaron V et al.; BACKGROUND: Interaction between lipopolysaccharide (LPS), LPS-binding protein, and the CD14 receptor at the surface of LPS-responsive cells results in inflammatory cytokine release and internalization and detoxification of LPS . Monoclonal antibodies (mAbs) raised against the deep-core lipid A or the O-linked polysaccharide moieties of LPS accelerate internalization and detoxification of LPS without stimulating cytokine release . This study was conducted to test the hypothesis that the antibody-mediated internalization of LPS is an Fc receptor (FcR)--mediated process . METHODS: Fluoroisothiocyanate (FITC)-conjugated Escherichia coli O111:B4 LPS was incubated with RAW 264.7 cells and allowed to internalize for 2 hours in the presence and absence of anti-LPS, anti-CD14, and isotype control mAbs, and Fab fragments from the anti-CD14, anti--Fc receptor, and control mAbs . Tumor necrosis factor--alpha (TNF-alpha) release was measured by WEHI 164 cell bioassay . FITC-LPS uptake was measured by flow cytometry . Statistical analysis was by analysis of variance and Fisher exact test . RESULTS: Addition of anti-LPS antibodies resulted in a 30- to 40-fold acceleration of LPS internalization (P <.01) in agreement with previous studies . This increase was blunted by anti-CD14 and also by isotype control holo-antibody (P <.01), but not by Fab fragments from anti-CD14 or isotype control antibody . Both anti-FcR antibodies and Fab fragments blocked anti-LPS antibody--stimulated uptake of FITC-LPS . Both intact anti-CD14 holo-antibody and Fab fragments blocked TNF-alpha release (P <.01) . CONCLUSIONS: Clearance and detoxification of LPS are thought to be essential to the host response to endotoxin . It has been shown that antibodies to LPS accelerate its internalization by monocytic cell lines without increasing the elaboration of cytokines . We found that specific blockade of CD14 by Fab fragments could block TNF-alpha release but not alter the accelerated internalization of LPS produced by anti-LPS antibodies . In contrast, a nonspecific blockade of internalization was produced by competing antibody, which suggests a mechanistic role for the FcR . Specific blockade of FcR by either holo-antibody or Fab fragments blocked accelerated internalization, which confirms a FcR mechanism . We conclude that the accelerated internalization of LPS produced by anti-LPS antibody is an Fc receptor--mediated process . These results have significance for the development of adjuvant immunotherapy for gram-negative bacterial sepsis.

J Bacteriol, 2001 Sep, 183(17), 5163 - 70
The molecular weight distribution of succinoglycan produced by Sinorhizobium meliloti is influenced by specific tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of the ExoP protein; Niemeyer D et al.; It is thought that in the gram-negative soil bacterium Sinorhizobium meliloti the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I) . The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed . The cytoplasmic domain of the ExoP protein was shown to have ATPase activity . Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein . Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit . The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity . We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.

Br J Pharmacol, 2001 Aug, 133(7), 1047 - 54
Haemodynamic effects of the bacterial quorum sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, in conscious, normal and endotoxaemic rats; Gardiner SM et al.; N-acylhomoserine lactones (AHLs) are small, diffusible signalling molecules, employed by Gram-negative bacteria to coordinate gene expression with cell population density . Recent in vitro findings indicate that AHLs may function as virulence determinants per se, through modification of cytokine production by eukaryotic cells, and by stimulating the relaxation of blood vessels . In the present study, we assessed the influence of AHLs on cardiovascular function in conscious rats, and draw attention to the ability of the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), a signal molecule produced by P . aeruginosa, to cause marked bradycardia . This bradycardic effect was blocked by atropine and atenolol, and did not occur in vitro . Furthermore, modification of the acyl side chain length resulted in the loss of activity, whereas removal of the homoserine lactone ring, did not . The bradycardic effect of 3-oxo-C12-HSL was also observed in endotoxaemic animals, albeit attenuated . In normal rats, 3-oxo-C12-HSL caused initial mesenteric and hindquarters vasoconstriction, but only slight, and delayed signs of vasodilatation in the renal and mesenteric vascular beds . Furthermore, administration of 3-oxo-C12-HSL (pre-treatment or 2 h post-treatment) together with LPS, did not modify the established regional haemodynamic effects of the LPS, 6 h after the onset of its infusion . Our observations do not provide any clear evidence for an ability of 3-oxo-C12-HSL to modify the haemodynamic responses to LPS infusion . However, they are not inconsistent with the hypothesis that some of the cardiovascular sequelae of bacterial infection may be modulated by an influence of bacterial quorum sensing signalling molecules on the host.

Khirurgiia (Sofiia), 1999, 55(6), 13 - 5
{Omentoplasty in surgical management of postpulmonectomy pleural empyema}; Petrov D et al.; A method of treating postpulmonectomy pleural empyema, practically implemented in this country for the first time, is described . Three patients are subjected to operation . Right pulmonectomy for lung cancer and chronic inflammatory process is done twice, and left pleuropulmonectomy for tuberculosis--once . Postpulmonectomy empyema persists in all three cases regardless of the adequate continuous suction drainage and intrapleural antiseptic management . Anaerobic and gram-negative flora is isolated . The size and location of the intrapleural cavity being cured are assayed by CT, thoracic ultrasonography and fistulography . In none of the patients is clinical and FBS evidence of bronchopleural fistula established . The operative procedure consists in resection of a 4 cm segment from the underlying rib in the drainage zone, and further cavity treatment under thorascopic control . Minor median laparotomy and skeletization of the greater omentum are performed preserving a major nutrient vessel depending on the location of the cavity . The omentum is drived into the pleural cavity through a parietal opening of the diaphragm, measuring 3-4 fingerbreadths . Pleural cavity drainage is carried out according to Redon . Two patients run an uneventful postoperative course . One female patient develops pylorospasm successfully cured by spasmolytic therapy and H2 blockers . CT and thoracic echography do not show presence of residual cavities . The patients are free of any complaints over periods ranging from 6 to 16 months postoperatively . The inference is reached that omentoplasty is a new method promoting successful elimination of both postpulmonectomy empyema, and other residual pleural cavities as well, with or without bronchopleural fistula.

J Biol Chem, 2001 Oct 12, 276(41), 37834 - 8 Epub 2001 Jul 30.
The gene ygdP, associated with the invasiveness of Escherichia coli K1, designates a Nudix hydrolase, Orf176, active on adenosine (5')-pentaphospho-(5')-adenosine (Ap5A); Bessman MJ et al.; ygdP, a gene associated with the invasion of brain microvascular endothelial cells by Escherichia coli K1 (Badger, J . L., Wass, C . A., and Kim, K . S . (2000) Mol . Microbiol . 36, 174-182), the primary Gram-negative bacterium causing meningitis in newborns, has been cloned and expressed in E . coli . The protein, YgdP, was purified to near homogeneity and identified as a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases . It catalyzes the hydrolysis of diadenosine tetra-, penta-, and hexa-phosphates with a preference for diadenosine penta-phosphate, from which it forms ATP and ADP . The enzyme has a requirement for a divalent metal cation that can be met with Mg2+, Zn2+, or Mn2+ and, like most of the Nudix hydrolases, has an alkaline pH optimum between 8.5 and 9 . This is the second identification of a gene associated with the invasiveness of a human pathogen as a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases, and an examination of homologous proteins in other invasive bacteria suggests that this may be a common feature of cellular invasion.

J Vet Diagn Invest, 2001 Jul, 13(4), 308 - 11
Multiplex polymerase chain reaction assay for identification of enterotoxigenic Escherichia coli strains; Osek J; A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria . This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E . coli-specific universal stress protein (uspA) . The specificity of the method was validated by single PCR tests performed with the reference E . coli and non-E . coli strains and with bacteria isolated from pig feces . The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E . coli and may be used as a method for direct determination of ETEC and to differentiate them from other E . coli and gram-negative enteric isolates.

Tissue Cell, 2001 Jun, 33(3), 258 - 61
Ectosymbiotic bacteria on ciliated cells of a rotifer; Selmi G; During the examination of Brachionus plicatilis (Rotifera, Monogononta), ectosymbiotic bacteria were found in the ciliated buccal region . These have about the same dimensions as the cilia and possess a specific apical region that apparently serves to attach the bacterium to its host cell . This apical region resembles a vesicle but is interpreted as a specialized region of the bacterial nucleocytoplasm devoid of ribosomes and chromatin strands but containing a crystalline rod . The bacterium has two membranes (as have other Gram-negative bacteria) also over the apical region . Thin strands join the two membranes and similar strands extend from the bacterium to the cell membrane of the host cell . No intracellular bacteria were found in this study.

J Dairy Sci, 2001 Jul, 84(7), 1632 - 9
An investigation of the impact of intramammary antibiotic dry cow therapy on clinical coliform mastitis; Bradley AJ et al.; The efficacy of an intramammary antibiotic dry cow preparation with significant gram-negative spectrum (product A; Leo Red Dry Cow, Leo Animal Health, UK) was compared with a product with no gram-negative efficacy (product B; Orbenin Extra DC, Pfizer Ltd, UK) as assessed by control of coliform mastitis in the first 100 d of the lactation . The efficacy of both products was also compared for control of noncoliform mastitis and for the ability to control existing and new intramammary infections as measured by individual cow somatic cell counts . Cows treated with product A were significantly less likely to develop clinical Escherichia coli or coliform mastitis during the dry period or the first 100 d of lactation than cows treated with product B . Cows treated with product A were no more likely to develop clinical mastitis due to a noncoliform organism than were cows treated with product B . There was no significant difference between the two groups as measured by individual cow somatic cell count changes across the dry period . This study is the first to have demonstrated the clinical efficacy of an intramammary antibiotic dry cow preparation, as measured by reduction in gram-negative clinical mastitis in the subsequent lactation . These findings demonstrate that selection of a dry cow intramammary preparation with a significant gram-negative spectrum can influence the incidence of clinical coliform mastitis in the subsequent lactation . This finding should be one of the factors taken into account when selecting products.

J Immunol, 2001 Aug 1, 167(3), 1609 - 16
Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide; Abreu MT et al.; The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS . Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types . Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC) . The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS . Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-kappaB reporter gene in response to purified, protein-free LPS . No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS . IEC responded vigorously to IL-1beta in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact . To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling . IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2 . To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines . Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-kappaB and IL-8 reporter genes in response to LPS . We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4 . The remainder of the intracellular LPS signaling pathway is functionally intact.

J Bacteriol, 2001 Aug, 183(16), 4687 - 93
Xenorhabdus nematophilus as a model for host-bacterium interactions: rpoS is necessary for mutualism with nematodes; Vivas EI et al.; Xenorhabdus nematophilus, a gram-negative bacterium, is a mutualist of Steinernema carpocapsae nematodes and a pathogen of larval-stage insects . We use this organism as a model of host-microbe interactions to identify the functions bacteria require for mutualism, pathogenesis, or both . In many gram-negative bacteria, the transcription factor sigma(S) controls regulons that can mediate stress resistance, survival, or host interactions . Therefore, we examined the role of sigma(S) in the ability of X . nematophilus to interact with its hosts . We cloned, sequenced, and disrupted the X . nematophilus rpoS gene that encodes sigma(S) . The X . nematophilus rpoS mutant pathogenized insects as well as its wild-type parent . However, the rpoS mutant could not mutualistically colonize nematode intestines . To our knowledge, this is the first report of a specific allele that affects the ability of X . nematophilus to exist within nematode intestines, an important step in understanding the molecular mechanisms of this association.

Clin Infect Dis, 2001 Aug 15, 33(4), 460 - 5 Epub 2001 Jul 11.
Polyclonal bacteremia due to gram-negative rods; Wendt C et al.; The frequency of, risk factors for, and outcome of polyclonal gram-negative bacteremia are still unknown . We investigated them in a prospective cohort study of patients for whom a blood culture yielded >/=1 species of gram-negative aerobic rod . For each patient, pulsed field gel electrophoresis (PFGE) was performed on 4 colonies of each morphologic type . Episodes of bacteremia were considered polyclonal if caused by >1 PFGE type of the same species . Ten (6.5%) of 153 investigated patients had polyclonal bacteremia . Bacteremia due to nonfermenting rods was the single significant risk factor for polyclonal bacteremia . Complications were equally frequent in all patient groups . However, patients with polyclonal bacteremia received more extensive antibiotic therapy than did patients with monoclonal bacteremia . Nearly 20% of episodes of bacteremia due to nonfermenting rods were polyclonal, but it remains unclear why nonfermenting rods were more likely to cause polyclonal bacteremia than were other gram-negative rods.

Mar Environ Res, 2000 Jul-Dec, 50(1-5), 473 - 7
Etiologies, observations and reporting of estuarine finfish lesions; Kane AS et al.; Lesions in estuarine finfish are associated with a variety of organisms including parasites and bacterial, viral, and fungal infectious agents . In addition, trauma, suboptimal water quality, and other abiotic stress factors may result in the loss of homeostasis . We have observed solitary ulcerative lesions on menhaden sampled from the Chesapeake Bay, Maryland, the Pimlico River, North Carolina, and the St . Johns River, Florida . Histologically, the lesions demonstrated a marked chronic inflammatory infiltrate and granulomas in response to fungal hyphae throughout large areas of exposed necrotic muscle . Gram-negative rod-shaped bacteria were also observed in the lesions, a common finding in ulcers of aquatic organisms . Similar observations in menhaden and other species have been described previously in the literature as ulcerative mycosis, mycotic granulomatosis, red spot disease, and epizootic ulcerative syndrome . Despite the many different known causes of fish lesions, the popular press and the scientific literature have recently emphasized Pfiesteria piscicida and other Pfiesteria-like dinoflagellates (and their bioactive compounds) as the primary causative agent for finfish lesions, particularly mycotic granulomatous ulcers in Atlantic menhaden . While some laboratory data suggest that Pfiesteria may play a role in field-observed lesions, much more cause-and-effect evidence is needed to determine the importance of other risk factors, both alone or and in combination with Pfiesteria . In order to better understand the etiology of lesion initiation and progression in estuarine finfish, accurate assessments of environmental conditions collected on appropriate temporal and spatial scales, and fish morphological indicators consistent with gross and histological pathologic terminology, should be used for reporting fish lesion observations and kills . Further, this outlook will help to avoid bias and may foster a broader perspective for examining the health of estuarine systems in general.

Am J Vet Res, 2001 Jul, 62(7), 1040 - 5
Evaluation of phagocytosis, bactericidal activity, and production of superoxide anion, nitric oxide, and tumor necrosis factor-alpha in Kupffer cells of neonatal pigs; Akunda JK et al.; OBJECTIVE: To evaluate the activity of Kupffer cells (KC) of control neonatal pigs and neonatal pigs treated with endotoxin and to compare activity of KC with that of pulmonary alveolar macrophages (PAM) . SAMPLE POPULATION: Kupffer cells and PAM obtained from 24 neonatal pigs (7 to 10 days old) . PROCEDURE: Pairs (n = 7) of littermates served as treated (lipopolysaccharide {LPS}) or untreated pigs . Pigs were euthanatized 24 hours after treatment, and cells were isolated . Cells were obtained from 10 other neonatal pigs for other assays . Functional activity of cells was evaluated by use of in vitro assays to evaluate bactericidal activity, phagocytosis, and production of superoxide anion (SOA), nitric oxide (NO), and tumor necrosis factor-alpha (TNF-alpha) . Each assay was repeated on cells obtained from 4 to 6 pigs . RESULTS: Phagocytic activity was similar in KC and PAM, but bactericidal activity and production of SDA and TNF-alpha was lower in KC . Neither KC nor PAM produced NO in response to LPS stimulation . Phagocytosis, bactericidal activity, and production of SOA were enhanced for KC obtained from neonatal pigs treated with LPS . The PAM from LPS-treated neonatal pigs had similar bactericidal activity to PAM obtained from untreated pigs . CONCLUSIONS AND CLINICAL RELEVANCE: Functional capacity of KC is affected by endotoxin . This provides additional information of the role the liver plays in immune surveillance . In addition, the response of KC in neonatal pigs exposed to endotoxin is of value for understanding gram-negative bacterial sepsis, which is a major cause of mortality in neonatal pigs.

Transfusion, 2001 Jul, 41(7), 862 - 72
Determinants of transfusion-associated bacterial contamination: results of the French BACTHEM Case-Control Study; Perez P et al.; BACKGROUND: Transfusion-associated bacterial contamination (TABC), probably the most frequent transfusion-transmitted infection, may induce serious adverse events . Systematic information and documentation on determinants are lacking . STUDY DESIGN AND METHODS: The BACTHEM Study is a French matched case-control study assessing TABC determinants . Included were cases of TABC reported in France in a 2-year period, as determined from uniform definitions . Information on recipient-, blood component-, and donor-related potential determinants was collected on site . ORs were estimated by conditional logistic regression . RESULTS: Of the 158 cases of suspected TABC reported, 41 that involved transfusion with 25 RBCs and 16 platelet concentrates were included . Gram-negative rods accounted for nearly half of the bacteria species involved and for all six deaths . In comparison with the risk of TABC for patients receiving RBCs for anemia, the risk was higher for patients receiving RBCs for pancytopenia (OR, 7.3; 95% CI, 1.3-41.0) and for those receiving platelets for thrombocytopenia (OR, 5.3; 95% CI, 1.2-24.1) . Other potential determinants were platelet transfusion for pancytopenia (OR, 4.5; 95% CI, 0.5-40.0), immunosuppressive treatment (OR, 2.8; 95% CI, 0.7-10.6), shelf-life of more than 1 day for platelets or 8 days for RBCs (OR, 2.6; 95% CI, 0.7-9.6), and more than 20 previous donations by donors (OR, 1.9; 95% CI, 0.7-5.3) . CONCLUSION: This first comparative study revealed TABC determinants that suggest approaches for prevention.

FEMS Microbiol Ecol, 2001 Jul, 36(2-3), 175 - 183
Structure and diversity of Gram-negative sulfate-reducing bacteria on rice roots; Scheid D et al.; Specific PCR assays were used to amplify the 16S rRNA genes of the Desulfobacteriaceae and the Desulfovibrionaceae from extracted environmental DNA from rice roots . 16S rDNA-based community patterns of the Desulfobacteriaceae were generated via terminal restriction fragment length polymorphism analysis from rice roots and compared with bulk soil . The molecular fingerprints showed no significant difference between rice roots and bulk soil, but changes during the vegetation period . 16S rDNA clone libraries and sequencing showed that the predominant terminal restriction fragments represented distinct phylogenetic groups . The 16S rDNA clone sequences of the Desulfobacteriaceae fell in the phylogenetic radiation of Desulfonema and Desulfosarcina or grouped within the Desulforhabdus-Syntrophobacter assemblage . Three of the latter sequences were closely affiliated with the MPN isolate EZ-2C2 from rice roots . All Desulfovibrionaceae 16S rDNA clone sequences, with one exception, were affiliated with the MPN isolate F1-7b from rice roots . The clustering of the clone sequences and the close phylogenetic affiliation with isolates from MPN enrichments from the same habitat in two cases indicated that these sequence clusters may represent predominant Gram-negative sulfate reducers on rice roots . Quantification of the bacterial abundances was accomplished by rRNA dot blot hybridization . In total the Gram-negative sulfate reducers accounted for approximately 2-3% of the total rRNA content . The relative rRNA abundance of the Desulfobacteriaceae was, at 1.4%, higher than that of the Desulfovibrionaceae (0.5%).

Mikrobiologiia, 2001 May-Jun, 70(3), 398 - 404
{New aerobic methyltrophic isolates from the Soda lakes of the southern Transbaikal}; Doronina NV et al.; Twenty-one bacterial associations isolated from the soda lakes of the southern Transbaikal region were found to be able to actively grow at pH 9-10 on methanol as the source of carbon and energy . Two alkalitolerant facultatively methylotrophic strains, Bur 3 and Bur 5, were obtained in pure cultures . Both strains represent gram-negative, nonmotile, bean-shaped, encapsulated cells that reproduce by binary fission . The strains are able to grow at temperatures ranging from 6 to 42 degrees C, with an optimum growth temperature of 25-29 degrees C (strain Bur 3) and 35-37 degrees C (strain Bur 5) and at pH between 6.5 and 9.5, with an optimum pH value of 8.0-8.5 . At pH 9.0, strain Bur 3 exhibits an increased content of phosphatidylglycerol and a decreased content of phosphatidylethanolamine . Strains Bur 3 and Bur 5 are similar in the G + C content of their DNAs (66.2 and 65.5 mol %, respectively) and in the type of the dominant ubiquinone (Q10) . Unlike Bur 5, strain Bur 3 is able to grow autotrophically in an atmosphere of CO2 + O2 + H2 . The strains oxidize, by the respective dehydrogenases, methanol to CO2, which is assimilated via the ribulose bisphosphate pathway . Ammonium ions are assimilated in the glutamate cycle and by the reductive amination of alpha-ketoglutarate . The strains are highly homologous to each other (92%) and are much less homologous (at a level of 28-35%) to representatives of the genus Ancylobacter, A . aquaticus ATCC 25396T and A . vacuolatum DSM 1277 . Based on the results obtained, both strains are assigned to a new species, Ancylobacter natronum sp . nov.

Adv Microb Physiol, 2001, 45, 51 - 112
Nitrate reduction in the periplasm of gram-negative bacteria; Potter L et al.; In contrast to the bacterial assimilatory and membrane-associated, respiratory nitrate reductases that have been studied for many years, it is only recently that periplasmic nitrate reductases have attracted growing interest . Recent research has shown that these soluble proteins are widely distributed, but vary greatly between species . All of those so far studied include four essential components: the periplasmic molybdoprotein, NapA, which is associated with a small, di-haem cytochrome, NapB; a putative quinol oxidase, NapC; and a possible pathway-specific chaperone, NapD . At least five other components have been found in different species . Other variations between species include the location of the nap genes on chromosomal or extrachromosomal DNA, and the environmental factors that regulate their expression . Despite the relatively small number of bacteria so far screened, striking correlations are beginning to emerge between the organization of the nap genes, the physiology of the host, the conditions under which the nap genes are expressed, and even the fate of nitrite, the product of Nap activity . Evidence is emerging that Nap fulfills a novel role in nitrate scavenging by some pathogenic bacteria.

J Bacteriol, 2001 Aug, 183(15), 4543 - 50
Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25; Delgado MA et al.; Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues . It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way . A mutation causing resistance to MccJ25 was isolated . Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E . coli genetic map . The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene . The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains . Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases . MccJ25 decreased RNA synthesis both in vivo and in vitro . These results point to the RNA polymerase as the target of microcin action . We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins . As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

J Biomater Sci Polym Ed, 2001, 12(4), 409 - 27
Assembly properties and applications of a new exopolymeric compound excreted by Pseudoalteromonas antarctica NF3; Cocera M et al.; The self assembly properties and applications of an exopolymeric compound (EC) of a glycoprotein character excreted by a new gram-negative species, Pseudoalteromonas antarctica NF3, have been reviewed . This compound exhibited surface-active properties in water, with a concentration of 0.20 mg ml(-1) being the key value associated with its physicochemical properties . Unsonicated EC aqueous dispersions showed the coexistence of concentric multilamellar and small unilamellar aggregates by transmission electron microscopy (TEM) . Sonication of these dispersions revealed that each lamellae of the initial multilamellar structures were made up of various subunits coiled coils . As for the ability of this exopolymeric biomaterial to coat phosphatidylcholine (PC) liposomes and to protect these vesicles against different surfactants, freeze-fracture TEM micrographs of liposome/EC aggregates revealed that the addition of the EC to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that coated very well the PC bilayers . The complete coating was already achieved at a PC:EC weight ratio of about 9:1 . An increasing resistance of PC liposomes to surfactants (in particular sodium dodecyl sulfate) occurred as the proportion of EC in the system rose, although this effect was more effective at low EC proportions (PC:EC weight ratios from 9:1 to 8:2) . Although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by the surfactants, the best protection occurred when this structure was a thin film of about 20-25 nm formed by nine to ten layers of about 2-3 nm.

J Biol Chem, 2001 Aug 31, 276(35), 32729 - 37 Epub 2001 Jul 02.
Identification of novel HLA-B27 ligands derived from polymorphic regions of its own or other class I molecules based on direct generation by 20 S proteasome; Alvarez I et al.; HLA-B27 is strongly associated with ankylosing spondylitis . Natural HLA-B27 ligands derived from polymorphic regions of its own or other class I HLA molecules might be involved in autoimmunity or provide diversity among HLA-B27-bound peptide repertoires from individuals . In particular, an 11-mer spanning HLA-B27 residues 169-179 is a natural HLA-B27 ligand with homology to proteins from Gram-negative bacteria . Proteasomal digestion of synthetic substrates demonstrated direct generation of the B27-(169-179) ligand . Cleavage after residue 181 generated a B27-(169-181) 13-mer that was subsequently found as a natural ligand of B*2705 and B*2704 . Its binding to HLA-B27 subtypes in vivo correlated better than B27-(169-179) with association to spondyloarthropathy . Proteasomal cleavage generated also a peptide spanning B*2705 residues 150-158 . This region is polymorphic among HLA-B27 subtypes and class I HLA antigens . The peptide was a natural B*2704 ligand . Since this subtype differs from B*2705 at residue 152, it was concluded that the ligand arose from HLA-B*3503, synthesized in the cells used as a source for B*2704-bound peptides . Thus, polymorphic HLA-B27 ligands derived from HLA-B27 or other class I molecules are directly produced by the 20 S proteasome in vitro, and this can be used for identification of such ligands in the constitutive HLA-B27-bound peptide pool.

Structure (Camb), 2001 Jun, 9(6), 513 - 25
The kappa-carrageenase of P . carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of Clan-B glycoside hydrolases; Michel G et al.; BACKGROUND: kappa-carrageenans are gel-forming, sulfated 1,3-alpha-1,4-beta-galactans from the cell walls of marine red algae . The kappa-carrageenase from the marine, gram-negative bacterium Pseudoalteromonas carrageenovora degrades kappa-carrageenan both in solution and in solid state by an endoprocessive mechanism . This beta-galactanase belongs to the clan-B of glycoside hydrolases . RESULTS: The structure of P . carrageenovora kappa-carrageenase has been solved to 1.54 A resolution by the multiwavelength anomalous diffraction (MAD) method, using a seleno-methionine-substituted form of the enzyme . The enzyme folds into a curved beta sandwich, with a tunnel-like active site cavity . Another remarkable characteristic is the presence of an arginine residue at subsite -1 . CONCLUSIONS: The crystal structure of P . carrageenovora kappa-carrageenase is the first three-dimensional structure of a carrageenase . Its tunnel-shaped active site, the first to be reported for enzymes other than cellulases, suggests that such tunnels are associated with the degradation of solid polysaccharides . Clan-B glycoside hydrolases fall into two subgroups, one with catalytic machinery held by an ancestral beta bulge, and the other in which it is held by a regular beta strand . At subsite -1, all of these hydrolases exhibit an aromatic amino acid that interacts with the hexopyranose ring of the monosaccharide undergoing catalysis . In addition, in kappa-carrageenases, an arginine residue recognizes the sulfate-ester substituents of the beta-linked kappa-carrageenan monomers . It also appears that, in addition to the nucleophile and acid/base catalysts, two other amino acids are involved with the catalytic cycle, accelerating the deglycosylation step.

J Chemother, 2001 Apr, 13 Suppl 1, 69 - 72
Upper airway obstruction due to rhinoscleroma: case report; Al Jahdali H et al.; Rhinoscleroma is a very rare cause of upper airway obstruction with only isolated reports in the literature of rhinoscleroma with isolated tracheal obstruction . The course is usually chronic with the presentation most often being non-specific . We report a 54-year-old woman with progressive shortness of breath and wheezing over 7 years' duration . She was diagnosed and treated as bronchial asthma without improvement in her symptoms . At the time of referral to our institution, her flow-volume loop revealed fixed upper airway obstruction . Her chest radiography and other laboratory tests were normal . Bronchoscopy revealed a 70-80% irregular concentric stenosis of the trachea beginning immediately below the vocal cords and extending 4 cm distally . Biopsy showed characteristic Mikulicz histiocytes containing numerous gram-negative intracellular coccobacilli consistent with a diagnosis of rhinoscleroma . The patient was treated with laser resection of the stenosis followed by a course of ciprofloxcin and trimethoprim-sulfamethoxazole . She has remained asymptomatic over a year follow-up period and repeated biopsies have shown no evidence of recurrence.

Isr Med Assoc J, 2001 Jun, 3(6), 439 - 42
New approaches in the diagnosis of sepsis; von Landenberg P et al.; In diagnosing sepsis the rapid identification of bacteremia at an early stage of the disease is critical for a favorable outcome . Furthermore, it is important that exact information on the stage of the disease be obtained rapidly in order to choose and initiate the appropriate therapy . In recent years many new techniques have been added to the diagnostic tools . In this review we will focus on three new methods for the early diagnosis of sepsis . These are: polymerase chain reaction, which offers the possibility to attain detailed information about the involved bacterial (or viral) species, and the laboratory markers procalcitonin and hypophosphatemia, which are indicators of the presence of infection with gram-negative bacteria . The approaches reviewed here were developed to expedite the diagnosis of especially early sepsis and might be a further step towards the improvement of therapy for sepsis.

Lipids, 2001 May, 36(5), 513 - 9
A novel sphingophosphonolipid head group 1-hydroxy-2-aminoethyl phosphonate in Bdellovibrio stolpii; Watanabe Y et al.; Members of the bacterial genus Bdellovibrio include strains that are free-living, whereas others are known to invade and parasitize larger Gram-negative bacteria . The bacterium can synthesize several sphingophospholipid compounds including those with phosphoryl bonds as well as phosphonyl bonds . In the present study, the dominant sphingophosphonolipid component was isolated by column chromatography, and the long-chain bases, fatty acids, and polar head groups were identified by thin-layer and gas-liquid chromatographic procedures . The definitive structural identity of the sphingolipid was established by nuclear magnetic resonance and mass spectrometry of hydrolysis products and the intact compound . The compound was identified as N-2'-hydroxypentadecanoyl-2-amino-3,4-dihydroxyheptadecan-1-phosphono-(1-hydroxy-2-aminoethane).

Semin Cell Dev Biol, 2001 Jun, 12(3), 215 - 23
Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria; Nikaido H; Bacteria, being unicellular, are constantly exposed to toxic compounds in their environment . Gram-negative bacteria and mycobacteria are unusually successful in surviving in the presence of toxic compounds because they combine two mechanisms of resistance . They produce effective permeability barriers, comprising the outer membrane and the mycolate-containing cell wall, on the cell surface . Further, they actively pump out drug molecules that trickle through the barrier, often utilizing multidrug efflux pumps . In Gram-negative bacteria, multidrug pumps of exceptionally wide specificity frequently interact with outer membrane channels and accessory proteins, forming multisubunit complexes that extrude drug molecules directly into the medium, bypassing the outer membrane barrier .

Acta Otorrinolaringol Esp, 2001 Mar, 52(2), 163 - 6
{Lemierre's syndrome: septic thrombophlebitis of the internal jugular vein secondary to acute amygdalitis}; Laguia M et al.; Lemierre's syndrome is an uncommon clinical entity . It consists of an acute oropharyngeal infection, with secondary septic thrombophlebitis of the internal jugular vein frequently complicated with multiple metastatic infections . It is generally caused by anaerobic Gram-negative organisms . Although it is rarely reported in the antibiotic era, this disease must be taken into account because it is a potentially life-threatening infection requiring a specific and early treatment . We report a case of Lemierre's syndrome managed in our hospital.

Jpn J Infect Dis, 2001 Apr, 54(2), 69 - 71
Periodontitis and serum interleukin-6 levels in the elderly; Murata T et al.; The elderly lose teeth as a result of dental caries and periodontitis caused by pathogenic oral bacteria . Periodontitis produces inflammatory cytokines due to the presence of lipopolysaccharides from oral gram-negative bacteria . Although the number of circulating inflammatory cytokines is related to the severity of the periodontitis, it is unclear whether the concentrations also correlate with periodontitis in the elderly . We investigated the relationship between periodontitis status and the concentrations of serum interleukin-6 (IL-6) in the serum from 276 subjects of 70- and 80-year-olds . Of the 276 subjects, 227 (82%) were dentate, 149 (54%) were found to be positive for serum IL-6, and 29 (13%) of the dentate subjects had severe periodontitis . However, there were no significant differences between the severity of periodontitis or the number of teeth and the mean serum IL-6 concentrations . These results provided no evidence to support an association between circulating IL-6 and periodontitis in the elderly.

Electrophoresis, 2001 May, 22(9), 1697 - 704
Time-dependent expression and processing of a hypothetical protein of possible importance for regulation of the Chlamydia pneumoniae developmental cycle; Vandahl BB et al.; Chlamydia pneumoniae is an obligate intracellular human pathogen infecting epithelial cells of the upper respiratory tract . It is a Gram-negative bacteria and has a unique biphasic developmental cycle . In this study, we use two-dimensional gel electrophoresis in combination with radioactive labeling to investigate time-dependent expression and processing of C . pneumoniae proteins . We report on (i) the identification of a hypothetical protein which is expressed late in the developmental cycle and subsequently processed; we speculate that this protein may be of importance for the developmental cycle of Chlamydia; (ii) the identification of the major outer membrane protein in three different variants, which may all be present in vivo.

J Ind Microbiol Biotechnol, 1999 Oct, 23(4-5), 408 - 413
Chemical structure and function of glycosphingolipids of Sphingomonas spp and their distribution among members of the alpha-4 subclass of Proteobacteria; Kawahara K et al.; Sphingomonas spp are phylogenetically placed in the alpha-4 subclass of Proteobacteria . They have glycosphingolipids (GSL) in their membranes instead of lipopolysaccharide (LPS) as in other Gram-negative bacteria . S . paucimobilis, the type species of the genus, has GSL-1, which contains only glucuronic acid (GlcA) as a sugar moiety, and GSL-4A, which contains a tetrasaccharide including GlcA . GSL-1 and GSL-4A form the outer membrane of S . paucimobilis with outer membrane proteins and phospholipids . In the outer membrane, GSLs are assumed to locate and function as does the LPS of other Gram-negative bacteria . Sphingomonas spp closely related to the type species contain both GSL-1 and the oligosaccharide-type GSL such as GSL-4A, but other Sphingomonas spp and other genera in the alpha-4 subclass of Proteobacteria contain only GSL-1 . Structural variations of fatty acids and dihydrosphingosines in the GSL-1 are presented.

Behav Brain Res, 2001 Sep 28, 124(1), 47 - 54
Lipopolysaccharide causes deficits in spatial learning in the watermaze but not in BDNF expression in the rat dentate gyrus; Shaw KN et al.; We investigated the effects of a single injection and a daily injection of lipopolysaccharide (LPS) on spatial learning and brain-derived neurotrophic factor (BDNF) expression in the rat dentate gyrus . LPS is derived from the cell wall of Gram-negative bacteria and is a potent endotoxin that causes the release of cytokines such as interleukin-1 and tumour necrosis factor . LPS is thought to activate both the neuroimmune and neuroendocrine systems; it also blocks long-term potentiation in the hippocampus . Here, we examined the effects of LPS on a form of hippocampal-dependent learning-spatial learning in the water maze . Rats were injected with LPS intraperitoneally (100 microg/kg) and trained in the water maze . The first group of rats were injected on day 1 of training, 4 h prior to learning the water maze task . Groups 2 and 3 were injected daily, again 4 h prior to the water-maze task; group 2 with LPS and group 3 with saline . A number of behavioural variables were recorded by a computerised tracking system for each trial . The behavioural results showed a single injection of LPS (group 1) impaired escape latency in both the acquisition and retention phases of the study, whereas a daily injection of LPS did not significantly impair acquisition or retention . BDNF expression was analysed in the dentate gyrus of all animals . No significant differences in BDNF expression were found between the three groups.

Respirology, 2001 Jun, 6(2), 171 - 3
Lemierre's syndrome; Turay UY et al.; This is a case report of Lemierre's syndrome in a 17-year-old male patient . Lemierre's disease consists of suppurative thrombophlebitis of the internal jugular vein (SIJVT) in the presence of oropharyngeal infection and can be complicated by septic pulmonary embolism . Other causes of SIJVT include deep neck infections and central venous catheterization . The disease usually results from Gram-negative anaerobic organisms such as Fusobacterium necrophorum.

Eur J Biochem, 2001 Jun, 268(12), 3566 - 76
Structural analysis of the carbohydrate components of the outer membrane of the lipopolysaccharide-lacking cellulolytic ruminal bacterium Fibrobacter succinogenes S85; Vinogradov E et al.; The polysaccharides from the outer membrane of the Gram-negative ruminal bacterium Fibrobacter succinogenes were isolated by phenol/water extraction and separated by size-exclusion chromatography in the presence of deoxycholate detergent into a lower-molecular-mass fraction designated 'glycolipid' and a high-molecular-mass 'capsular polysaccharide' fraction . Both fractions lacked typical lipopolysaccharide components including 2-keto-3-deoxyoctulosonic acid and 3-hydroxy fatty acids . Carbohydrate components of these fractions were represented by two polysaccharides and one oligosaccharide (possibly glycolipid) with the following structures: : : where HEAEP is N-(2-hydroxyethyl)-2-aminoethylphosphonic acid, found for the first time in natural compounds . The polysaccharides contained pentadecanoic acid and anteisopentadecanoic acid, possibly present as the acyl components . All constituent monosaccharides except L-rhamnose had a D-configuration . In addition to having a structural role in the outer membrane, these polysaccharides may provide protection for this lipopolysaccharide-less bacterium in the highly competitive ruminal environment, as phosphonic acids covalently linked to membrane polymers have in the past been attributed the function of stabilizing membranes in the presence of phosphatases and lipases.

Clin Lab Haematol, 2001 Feb, 23(1), 39 - 42
Quinolone resistance in neutropenic patients: the effect of prescribing policy in the UK and Pakistan; Zaidi Y et al.; Quinolones are increasingly used as prophylaxis in neutropenic patients to prevent serious Gram-negative septicaemias but practice is not uniform because of the controversial evidence as to their effectiveness . It is unclear if they are of real benefit in patients with short episodes of neutropenia such as those resulting from treatment for solid tumours and lymphomas . The concern over the use of ciprofloxacin in such settings is the increasing development of quinolone resistant Gram-negative bacteria . We have retrospectively analysed our bacterial isolate resistance patterns in the Queen Elizabeth Hospital (QE) and in the Haematology Department of the Aga Khan Hospital (AKU), Pakistan where all patients would receive ciprofloxacin prophylaxis when neutropenic . Seven out of 57 (12.2%) and 18 out of 55 (32.7%) Gram-negative organisms isolated from blood cultures at the QE and AKU Haematology Departments, respectively, were resistant to ciprofloxacin (P < 0.01) . In the Birmingham community this was significantly lower (P < 0.01) (55 out of 6423: 0.85%) . We also showed a higher level of E . coli resistance at the AKU (18 out of 31: 58%) where ciprofloxacin use was more widespread than at the QE (1 out of 11, P < 0.01) . We conclude that ciprofloxacin should not be used indiscriminately.

Res Microbiol, 2001 Apr-May, 152(3-4), 357 - 64
ABC transporters and the export of capsular polysaccharides from gram-negative bacteria; Silver RP et al.; In this review, we discuss the kps cluster of Escherichia coli as the paradigm for the ABC capsular polysaccharide exporter (CPSE) family . Components of the cluster form a multimeric protein complex consisting of both biosynthetic and export machinery . We compare the Kps exporter with capsule export systems from other members of the CPSE family.

Harefuah, 2001 Jun, 140(6), 519 - 23, 565
{Sepsis associated cholestasis in adults}; Levinson R et al.; Hepatic injury presenting as jaundice, hypglycemia, encephalopathy, coagulation disturbances and a significant increase in transaminases in a late well recognized complication of sepsis . Less known is a cholestatic liver injury, which can precede sepsis . This type of early liver injury is characterized by a rise of direct bilirubin and less prominent increase in liver enzymes . Experimental work in animal models demonstrates that sepsis associated cholestasis is mediated by a variety of gram negative bacterial endotoxins and cytokines which interfere with the function of bile acid intracellular transporters . Although sepsis associated cholestasis is quite common, its clinical and prognostic characteristics are not widely appreciated . This often results in performance of unnecessary procedures and delayed diagnosis . Here we review the relevant updated literature regarding the pathophysiological basis of this phenomenon and its clinical presentation and implications.

J Immunol, 2001 Jul 1, 167(1), 435 - 41
Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G2 arrest of the cell cycle; Shenker BJ et al.; We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several Gram-negative bacteria . Moreover, we have shown that CdtB impairs lymphocyte function by inducing G(2) arrest of the cell cycle . We now report that both CdtB as well as an extract prepared from an Escherichia coli strain that expresses all three of the A . actinomycetemcomitans cdt genes (rCdtABC) induce apoptosis . Pretreatment of lymphocytes with either CdtB or rCdtABC leads to DNA fragmentation in activated lymphocytes at 72 and 96 h . No DNA fragmentation was induced in nonactivated cells . Flow cytometric analysis of the Cdt-treated lymphocytes demonstrates a reduction in cell size and an increase in nuclear condensation . Mitochondrial function was also perturbed in cells pretreated with either CdtB or rCdtABC . An increase in the expression of the mitochondria Ag, Apo 2.7, was observed along with evidence of the development of a mitochondrial permeability transition state; this includes a decrease in the transmembrane potential and elevated generation of reactive oxygen species . Activation of the caspase cascade, which is an important biochemical feature of the apoptotic process, was also observed in Cdt-treated lymphocytes . Overexpression of the bcl-2 gene in the human B lymphoblastoid cell line, JY, led to a decrease in Cdt-induced apoptosis . Interestingly, Bcl-2 overexpression did not block Cdt-induced G(2) arrest . The implications of our results with respect to the immunosuppressive functions of Cdt proteins are discussed.

FEBS Lett, 2001 Jun 15, 499(1-2), 1 - 5
Helicobacter mustelae lipid A structure differs from that of Helicobacter pylori; Therisod H et al.; The lipid A structure of the Gram-negative bacterium Helicobacter mustelae, a ferret gastric pathogen responsible for the onset of gastric diseases in its host, was investigated . Two variant lipid A structures were found in the same strain . One structure contained a bisphosphorylated beta-(1-->6)-linked D-glucosamine backbone disaccharide with hydroxytetradecanoic acid in amide linkages . Unlike the structure described for the lipid A of the related human Helicobacter pylori gastric pathogen, which contains a C1 phosphate moiety, this lipid A presented phosphate groups at both the C1 and C4' positions, and contained no octadecanoyl fatty acid, which is present in H . pylori . The second lipid A structure had a different fatty acid composition in that 3-OH C(16) replaced most of the amide-linked 3-OH C(14).

Leukemia, 2001 Jun, 15(6), 898 - 902
Daunorubicin continuous infusion induces more toxicity than bolus infusion in acute lymphoblastic leukemia induction regimen: a randomized study; Hunault-Berger M et al.; We report the first randomized study assessing the efficacy and safety of daunorubicin (DNR) continuous infusion (CI) compared to the more conventional 30-min infusion (i.v.) in newly diagnosed adult acute lymphoblastic leukemia (ALL) . Seventy-seven patients were initially randomized to receive either a 24-h CI DNR (60 mg/m2 days 2-4) (40 patients) or bolus DNR at the same dosage (37 patients) with vincristine (2 mg i.v . days 1, 8, 15) and oral prednisone (60 mg/m2 days 1-15), without hematopoietic growth factor support, as an induction regimen . The distribution of adverse prognostic factors was comparable in the two-induction arm . Acute toxicity was more important in the CI arm . Gram negative infection (9 vs 1 gram negative septicemia, P = 0.01) and infection-related deaths (6 vs 1 deaths, P = NS) occurred more frequently in the CI arm during the induction treatment than in the i.v . arm, leading to the study interruption . Neutropenia but not thrombopenia duration was significantly longer in the CI arm than in the i.v . arm (18 days vs 14 days, P > 0.05 and 16 days vs 12 days, P > 0.05, respectively) . Despite a similar CR rate according to the method of DNR administration (68% in the CI DNR arm vs 76% in the i.v . arm after the first course), there was a trend toward higher freedom from relapse (FFR) after DNR CI (48% vs 28% in the i.v . arm at 5 years, P = NS), suggesting that despite this high toxicity, DNR CI may improve the CR quality and decrease further the residual disease.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 827 - 41
Sphingomonas pituitosa sp . nov., an exopolysaccharide-producing bacterium that secretes an unusual type of sphingan; Denner EB et al.; Strain EDIVT, an exopolysaccharide-producing bacterium, was subjected to polyphasic characterization . The bacterium produced copious amounts of an extracellular polysaccharide, forming slimy, viscous, intensely yellow-pigmented colonies on Czapek-Dox (CZD) agar . The culture fluids of the liquid version of CZD medium were highly viscous after cultivation for 5 d . Cells of strain EDIVT were Gram-negative, catalase-positive, oxidase-negative, nonspore-forming, rod-shaped and motile . Comparisons of 16S rDNA gene sequences demonstrated that EDIVT clusters phylogenetically with the species of the genus Sphingomonas sensu stricto . The G+C content of the DNA (64.5 mol%), the presence of ubiquinone Q-10, the presence of 2-hydroxymyristic acid (14:0 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the detection of sym-homospermidine as the major component in the polyamine pattern, together with the presence of sphingoglycolipid, supported this delineation . 16S rDNA sequence analysis indicated that strain EDIVT is most closely related (99.4% similarity) to Sphingomonas trueperi LMG 2142T . DNA-DNA hybridization showed that the level of relatedness to S . trueperi is only 45.5% . Further differences were apparent in the cellular fatty acid profile, the polar lipid pattern, the Fourier-transform infrared spectrum and whole-cell proteins and in a number of biochemical characteristics . On the basis of the estimated phylogenetic position derived from 16S rDNA sequence data, DNA-DNA reassociation and phenotypic differences, strain EDIVT (= CIP 106154T = DSM 13101T) was recognized as a new species of Sphingomonas, for which the name Sphingomonas pituitosa sp . nov . is proposed . A component analysis of the exopolysaccharide (named PS-EDIV) suggested that it represents a novel type of sphingan composed of glucose, rhamnose and an unidentified sugar . Glucuronic acid, which is commonly found in sphingans, was absent . The mean molecular mass of PS-EDIV was approximately 3 x 10(6) Da.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1179 - 89
Porphyromonas gulae sp . nov., an anaerobic, gram-negative coccobacillus from the gingival sulcus of various animal hosts; Fournier D et al.; A new species, Porphyromonas gulae sp . nov., is proposed to include strains isolated from the gingival sulcus of various animal hosts which are distinct from related strains of Porphyromonas gingivalis of human origin . This bacterium exhibits the following characteristics: black-pigmented colonies; asaccharolytic, obligate anaerobic growth; and Gram-negative, non-motile and non-spore-forming, rod-shaped cells . Colonies do not fluoresce under UV light . Vitamin K1 and haemin are required for growth . Cells haemagglutinate sheep erythrocytes . Major fatty acid end products are butyric acid, isovaleric acid, succinic acid and phenylacetic acid . Strains are catalase-positive and indole is produced . Alkaline phosphatase, trypsin-like and N-acetyl-beta-glucosaminidase activities are strong . A beta-galactosidase and a glutamylglutamic acid arylamidase are also present . The G+C content of the chromosomal DNA is 51 mol% . DNA-DNA homology data and 16S rRNA gene sequence analysis provide strong evidence that strains from the animal biotype of P . gingivalis represent a Porphyromonas species that is distinct from P . gingivalis . The type strain of P . gulae is Loup 1T (= ATCC 51700T = NCTC 13180T).

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1059 - 70
Taxonomic characterization of Ketogulonigenium vulgare gen . nov., sp . nov . and Ketogulonigenium robustum sp . nov., which oxidize L-sorbose to 2-keto-L-gulonic acid; Urbance JW et al.; Four bacterial strains that oxidize L-sorbose to 2-keto-L-gulonic acid, a key intermediate in the synthesis of vitamin C, were isolated from soils of geographically distinct locations . All were Gram-negative, facultatively anaerobic, chemoheterotrophic rods . Comparative analysis revealed nearly identical 16S rDNA sequences amongst them (99.7-100% identical) and identified them as members of the alpha-subclass of the Proteobacteria . Phylogenetic analysis identified the closest taxonomically defined genus as Roseobacter (92.1-92.8% identical) . On the basis of phylogenetic, phenotypic and genotypic analyses, a new genus is proposed, Ketogulonigenium gen . nov . Based upon these analyses, we also propose the reclassification of strain DSM 4025TP, originally identified as Gluconobacter oxydans, to the genus Ketogulonigenium . Two species are proposed: the type species Ketogulonigenium vulgare gen . nov., sp . nov., consisting of strains 62A-12APP, 266-13BPP and the type strain K . vulgare DSM 4025TP, and Ketogulonigenium robustum gen . nov., sp . nov., consisting of the type strain K . robustum X6LTP (= NRRL B-21627 = KCTC 0858BP) . The species affiliation of the fifth strain (291-19PP) remains unresolved.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1027 - 33
Shewanella japonica sp . nov; Ivanova EP et al.; Two strains of agar-digesting bacteria, KMM 3299T and KMM 3300, respectively isolated from sea water and the mussel Protothaca jedoensis, have been characterized . Based on sequencing of the 16S rRNA gene, KMM 3299T showed the highest similarity (93-95%) to members of the genus Shewanella . The G+C contents of the DNAs of these strains were 43-44 mol% . The level of DNA homology between the two strains was conspecific (95%), indicating that they represent a distinct genospecies . These organisms were non-pigmented, Gram-negative, polarly flagellated, facultatively anaerobic, mesophilic, neutrophilic and able to degrade a wide range of high molecular mass polymers, including alginate, carrageenan, laminaran and agar . The novel organisms were susceptible to gentamycin, carbenicillin, lincomycin and oleandomycin . The predominant cellular fatty acids were i-15:0, 16:0, 16:1(n-7), 18:1(n-7) . Eicosapentaenoic acid, 20:5(n-3), was detected in the two isolates at levels of 1-8%, depending on the temperature of cultivation . Phylogenetic evidence, together with phenotypic characteristics, showed that the two isolates studied constitute a novel species of the genus Shewanella . The name Shewanella japonica is proposed; the type strain is KMM 3299T(= LMG 19691T = CIP 106860T).

Alcohol Clin Exp Res, 2001 Jun, 25(6), 935 - 43
Inhibition of caspases in vivo protects the rat liver against alcohol-induced sensitization to bacterial lipopolysaccharide; Deaciuc IV et al.; BACKGROUND: The mechanisms of liver sensitization by alcohol to Gram-negative bacterial lipopolysaccharide (LPS) remain elusive . The purpose of this study was two-fold: (1) to test the hypothesis that alcohol-enhanced liver apoptosis may be a sensitizing mechanism for LPS and (2) to further characterize the liver apoptotic response to alcohol . METHODS: Rats were fed a high-fat, alcohol-containing liquid diet for 14 weeks, treated with LPS (1.0 mg/kg of body weight, intravenously) or saline, followed by injection of a pan-caspase inhibitor IDN1965; N-{(1,3-dimethylindole-2-carbonyl)-valinyl}-3-amino-4-oxo-5-fluoropentanoic acid; 10 mg/kg of body weight, intraperitoneally or vehicle, and killed . The following parameters were assessed: plasma aspartate: 2-oxoglutarate aminotransferase activity (AST); liver histology and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) response; caspase-3, -8, and -9 activity; and mRNA and protein expression for two apoptosis-signaling molecules: Fas receptor and Fas ligand; and three apoptosis adaptors: Bax, Bcl-XL, and Bcl-2 . RESULTS: Alcohol-feeding-induced liver steatosis, slightly increased caspases' activity, the number of TUNEL-positive nuclei, and facilitated the LPS necrotic effect without affecting mRNA expression of apoptosis signals and adaptors . LPS induced a significant increase in AST and the number of TUNEL-positive nuclei, both effects being more pronounced in alcohol-treated rats . LPS produced hepatic necrosis only in alcohol-treated rats . LPS effects were associated with up-regulation of mRNA expression for both apoptosis adaptors and signaling molecules . IDN1965 administration 3 hr after LPS injection strongly inhibited caspases' activity, particularly that of caspase-3 . IDN1965 also abolished the increase in TUNEL-positive nuclei, reversed the effect of LPS on plasma AST in alcohol-treated rats, and prevented LPS-induced necrosis . CONCLUSIONS: (1) Alcohol-enhanced liver apoptosis may not involve regulatory steps at the transcriptional level . LPS-induced liver apoptosis seems to involve transcriptional regulation of several apoptosis adaptors . Therefore, alcohol and LPS may enhance liver apoptosis through different mechanisms . (2) Alcohol-enhanced liver apoptosis precedes and may facilitate the hepatic effects of LPS . LPS superimposed on alcohol further elevates the rate of apoptosis in the liver . This may exceed the phagocytosing capacity of the liver so that all the apoptotic cells are not phagocytosed, but rather die of necrosis.

Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7271 - 6 Epub 2001 Jun 12.
Afipia felis induces uptake by macrophages directly into a nonendocytic compartment; Luhrmann A et al.; Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease . A . felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A . felis-containing phagosomes is unknown . Here, we demonstrate that, in murine macrophages, most A . felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection . Established Afipia-containing phagosomes possess neither early endosomal marker proteins {early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)} nor late endosomal or lysosomal proteins {cathepsin D, beta-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2} . Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1 . The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1 . Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A . felis-containing phagosomes that interact with the endocytic system . We conclude that most phagosomes containing A . felis are disconnected from the endosome-lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.

Gene, 2001 May 30, 270(1-2), 231 - 6
Sequence of the E . coli O104 antigen gene cluster and identification of O104 specific genes; Wang L et al.; The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones . Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity . We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes . The E . coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104 . It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster . By PCR testing against representative strains for the 166 E . coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9 . This work provides the basis for a sensitive test for rapid detection of O104 E . coli . This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.

Antimicrob Agents Chemother, 2001 Jul, 45(7), 2098 - 105
DNA gyrase-mediated natural resistance to fluoroquinolones in Ehrlichia spp; Maurin M et al.; Fluoroquinolone susceptibility heterogeneity between various Ehrlichia species has been previously demonstrated . In gram-negative bacteria, resistance to fluoroquinolones most often corresponds to specific amino acid variations in a portion of the protein sequence of the A subunit of DNA gyrase (GyrA), referred to as the quinolone resistance-determining region (QRDR) . We suspected a similar mechanism to be responsible for natural resistance in some Ehrlichia species . To verify this hypothesis, we sequenced the entire gyrA gene of the quinolone-susceptible species Ehrlichia sennetsu and designed specific primers to amplify and sequence the QRDR of four other Ehrlichia species as well as the closely related species Cowdria ruminantium . We identified in the fluoroquinolone-resistant species Ehrlichia chaffeensis and Ehrlichia canis a specific GyrA QRDR amino acid sequence, also present in C . ruminantium (whose susceptibility to fluoroquinolones remains unknown) . These three species belong to a single phylogenetic cluster referred to as the E . canis genogroup . A different GyrA QRDR pattern, shared by the Ehrlichia species representatives of the E . sennetsu and Ehrlichia phagocytophila genogroups, was identified . Three of the four species tested are known to be susceptible to fluoroquinolones . A serine residue in position 83 (Escherichia coli numbering) in the susceptible species is replaced by an alanine residue in fluoroquinolone-resistant species . These results are consistent with the current knowledge on fluoroquinolone resistance in other gram-negative bacteria . They are indicative of a natural gyrase-mediated resistance to fluoroquinolones in the E . canis genogroup.

Curr Microbiol, 2001 Sep, 43(3), 187 - 91
Desulfurization of benzothiophene by the Gram-negative bacterium, Sinorhizobium sp . KT55; Tanaka Y et al.; Sinorhizobium sp . KT55 was the first Gram-negative isolate to be capable of utilizing benzothiophene as the sole source of sulfur . By GC-MS analysis of metabolites of benzothiophene by this strain, benzothiophene sulfone, benzo{e}{1,2}oxathiin S-oxide and o-hydroxystyrene were detected, suggesting that the benzothiophene desulfurization pathway of this strain is benzothiophene-->benzothiophene sulfoxide-->benzothiophene sulfone-->benzo{e}{1,2}oxathiin S-oxide-->o-hydroxystyrene . Desulfurization activity of this strain was significantly repressed by methionine, cysteine, sulfate, dimethyl sulfoxide, and Casamino acids.

Best Pract Res Clin Gastroenterol, 2001 Jun, 15(3), 447 - 61
Role of acid suppressants in intensive care medicine; Tryba M; Bleeding from stress-induced mucosal lesions continues to be a potential problem in critically ill patients, although its incidence has decreased dramatically over the past decade . Patients considered to be at risk are those with respiratory failure, coagulopathy, severe burns or tetraplegia . The most important cause of stress ulcer bleeding is tissue hypoxia . Provided that appropriate dosage regimens are administered, all agents approved for stress ulcer prophylaxis may reduce the incidence of overt as well as clinically important bleeding . However, the efficacy of stress ulcer prophylaxis does not correlate with the efficacy of gastric acid inhibition . Although numerous studies have demonstrated that an alkaline gastric juice is associated with gastric Gram-negative bacterial overgrowth, controversy remains over whether the pharmacological suppression of gastric acid in critically ill patients facilitates nosocomial pneumonia . The reasons for these divergent results are discussed, as is a possible association between gastric acid suppression and other systemic infections . Finally, several cost-effectiveness analyses performed over recent years have demonstrated that, in properly selected critically ill patients, stress ulcer prophylaxis is cost-effective.

Syst Appl Microbiol, 2001 Apr, 24(1), 44 - 53
Psychrobacter proteolyticus sp . nov., a psychrotrophic, halotolerant bacterium isolated from the Antarctic krill Euphausia superba Dana, excreting a cold-adapted metalloprotease; Denner EB et al.; An Antarctic marine bacterium (strain 116) excreting an extracellular cold-adapted metalloprotease was subjected to a detailed polyphasic taxonomic investigation . Strain 116 was previously isolated from the stomach of a specimen of the Antarctic krill Euphasia superba Dana and tentatively characterized as Sphingomonas paucimobilis 116 . The 16S rDNA sequence analysis showed that the strain is in fact related to species of the genus Psychrobacter, next to Psychrobacter glacincola (97.4% similarity) . Sequence similarities between strain 116 and other Psychrobacter species ranged from 96.9% (with P . urativorans) to 95.4% (with P . immobilis) . Key phenotypic characteristics as well as chemotaxonomic features of the bacterium were congruent with the description of the genus Psychrobacter i.e . cells were strictly aerobic, strongly oxidase-positive, psychrotrophic, halotolerant, gram-negative non-motile coccobacilli, with ubiquinone-8 as the main respiratory lipoquinone and 18:1 cis 9, 16:1 cis and 17:1 (omega8c being the predominant cellular fatty acids . The G+C content of the DNA was 43.6 mol% . DNA-DNA hybridization studies showed that the relatedness between strain 116 and Psychrobacter glacinola is only 62.2% . Further differences were apparent in whole-cell SDS-PAGE protein pattern, cellular fatty acid profile and in a number of physiological and biochemical characteristics as well as in enzymatic activities . Tolerance to 5% bile salts, nitrate reduction, citrate utilization, acid production from carbohydrates, alkaline phosphatase, acid phosphatase, C4 esterase, C14 lipase and valine arylamidase were found to differentiate strain 116 from Psychrobacter glacincola . On the basis of this phenotypic and molecular evidences, strain 116, previously known as Sphingomonas paucimobilis 116, was recognized as a new species of the genus Psychrobacter for which the name Psychrobacter proteolyticus is proposed . Strain 116 has been deposited in the Collection de l'Institut Pasteur, France, as CIP106830T and in the Deutsche Sammlung von Mikroorganismen and Zellkulturen, as DSM13887.

Infect Immun, 2001 Jul, 69(7), 4407 - 16
Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis; Fernandez-Prada CM et al.; Brucella spp . are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts . Smooth organisms present O polysaccharides (OPS) on their surface . These OPS help the bacteria avoid the bactericidal action of serum . The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella . In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B . abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B . abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS) . The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry . Rough B . abortus mutants were more sensitive to the bactericidal action of NHS than were rough B . melitensis mutants . Complement components were deposited on smooth strains at a slower rate compared to rough strains . Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA . These results indicate that (i) OPS-deficient strains derived from B . melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B . abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.

Microbiology, 2001 Jun, 147(Pt 6), 1525 - 33
Substrate analysis and molecular cloning of the extracellular alkaline phosphatase of Streptomyces griseus; Moura RS et al.; Streptomyces species secrete large amounts of alkaline phosphatase (AP) enzymes that have not been characterized so far . An AP has been purified to homogeneity from cultures of Streptomyces griseus IMRU 3570 . The enzyme has a monomer size of 62 kDa and is processed in the culture to a 33 kDa protein as shown by immunoblotting . The enzyme was purified by ammonium sulfate precipitation, CM-Sephadex cationic exchange, chromatofocusing and HPLC Sphaerogel 3000SW filtration . The pure enzyme uses a variety of organic phosphorylated compounds as substrates . The N-terminal end of the mature protein was found to be RLREDPFTLGVASGDPHP . The gene phoA has been cloned using as probe an oligomer based on the N-terminal sequence of the S . griseus AP . phoA encodes a protein of 62678 Da with low homology to the AP of Escherichia coli . The phoA gene was found to be homologous to three alkaline-phosphatase-encoding genes previously identified in the Streptomyces coelicolor genome . On the basis of the optimal pH, substrate specificity and differences in amino acid sequence of motifs defining the active centre of APs, the S . griseus AP uses a wide range of organic phosphate substrates and is different from the phosphatases of Gram-negative bacteria.

Biochim Biophys Acta, 2001 May 28, 1539(1-2), 140 - 6
Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction; Vadiveloo PK et al.; Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII) . We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM) . The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx . 80% . However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice . Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea . Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types . Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression . Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.

Clin Infect Dis, 2001 Jul 1, 33(1), e8 - 11 Epub 2001 May 23.
"Helicobacter rappini" isolates from 2 homosexual men; Tee W et al.; We report 2 cases of bacteremia due to "Helicobacter rappini" in 2 young, homosexual men, including the first report of H . rappini in a human immunodeficiency virus-positive patient . Blood cultures showed a spiral, fusiform, gram-negative bacterium with bipolar sheathed flagella.

Clin Infect Dis, 2001 Jul 1, 33(1), 54 - 61 Epub 2001 May 23.
Eikenella corrodens infections in children and adolescents: case reports and review of the literature; Paul K et al.; Eikenella corrodens is a slow-growing, gram-negative, nonmotile, facultative rod that can cause infection in humans . Although the clinical characteristics of Eikenella infections in adults are well described, the literature regarding Eikenella infections in children is lacking . Thirteen cases of Eikenella infection in children and adolescents reported from a hospital and an additional 41 cases from the literature were reviewed . Eikenella species can be serious pediatric pathogens, particularly when there is an exposure to human oral secretions . Empirical therapy used to manage most oropharyngeal flora may be ineffective against Eikenella species . The treatment of choice for children and adolescents who are infected with Eikenella species includes a combination of surgical management and antibiotics.

Presse Med, 2001 Apr 28, 30(16), 813 - 8
{Disseminated strongyloidiasis}; Nozais JP et al.; ENDEMIC ZONES: Strongyloidiasis is an intestinal parasitosis which is frequently found in tropical and subtropical regions . RISK: The "autoinfection" cycle during this helminthiasis explains why the infection can be perpetuated without further exposure to exogenous, infective larvae . Hyperinfection may occur by dissemination of Strongyloides stercoralis in immuno-compromised patients, particularly those under corticotherapy . DISSEMINATATED STRONGYLOIDIASIS: Disseminated strongyloidiasis is characterized by severe gastrointestinal and respiratory tract involvement, meningitis, skin rash, or Gram-negative bacteremia . Since disseminated stronglyoidiasis is fatal in 80% of cases it is imperative to diagnose and treat this condition before long-term corticotherapy . Ivermectin is currently recommended because it is effective and well tolerated.

Shock, 2001 Jun, 15(6), 461 - 6
Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro; Power C et al.; Bacterial lipoprotein (BLP) is the most abundant protein in gram-negative bacterial cell walls, heavily outweighing lipopolysaccharide (LPS) . Herein we present findings demonstrating the potent in vitro effects of BLP on neutrophil (PMN) activation status, function, and capacity to transmigrate an endothelial monolayer . PMNs are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens . The systemic inflammatory response syndrome (SIRS) is characterised by significant host tissue injury mediated, in part, by uncontrolled regulation of PMN cytotoxic activity . We found that BLP-activated human PMN as evidenced by increased CD11b/CD18 (Mac-1) expression . Up-regulation of PMN Mac-1 in response to BLP occurred independently of membrane-bound CD14 (mCD14) . A similar up-regulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was observed whilst E-Selectin expression was unaffected . PMN transmigration across a human umbilical vein endothelial cell (HUVEC) monolayer was markedly increased after treating either PMN's or HUVEC independently with BLP . This increased transmigration did not occur as a result of any direct effect of BLP on HUVEC monolayer permeability, assessed objectively using the passage of FITC-labeled Dextran-70 . BLP primed PMN for enhanced respiratory burst and superoxide anion production in response to PMA, but did not influence phagocytosis of opsonized Escherichia coli . BLP far exceeds LPS as a gram-negative bacterial wall component, these findings therefore implicate BLP as an additional putative mediator of SIRS arising from gram-negative infection.

Mol Plant Microbe Interact, 2001 Jun, 14(6), 701 - 6
Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes; Guilhabert MR et al.; Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects . Strains of X . fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus . Although the complete genome sequence of X . fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X . fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions . We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X . fastidiosa . The four suicide vectors failed to produce any detectable transposition events . Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X . fastidiosa . Molecular analysis showed that the transposition insertions were single, independent, stable events . Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X . fastidiosa genome . Transposome-mediated mutagenesis should facilitate the identification of X . fastidiosa genes that mediate plant pathogenicity and insect transmission.

Clin Exp Pharmacol Physiol, 2001 May-Jun, 28(5-6), 376 - 80
Effects in humans of intravenously administered endotoxin on soluble cell-adhesion molecule and inflammatory markers: a model of human diseases; Wilson M et al.; 1 . Endotoxin, a component of the cell wall of Gram-negative bacteria, could be a predisposing mediator of many pathological disorders . The present study was undertaken to determine the effects and time-course of acute endotoxin challenge on inflammatory and cell-adhesion molecule markers shedding in the plasma as potential surrogates . 2 . Six normal male subjects per group (age range 21-35 years) were injected with 4 ng/kg, i.v., reference standard Escherichia coli (0113:h10:k) endotoxin or physiological saline . 3 . Plasma inflammatory markers (tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and TNF-receptor I (RI)) and cell-adhesion molecule markers (soluble L-selectin, soluble P-selectin, soluble vascular cell adhesion molecule (VCAM)-1) were determined using sensitive and specific ELISA . 4 . Tumour necrosis factor-alpha increased from a basal level of 2.8 pg/mL to approximately 800 pg/mL at 90 min after endotoxin . Similarly, IL-6 peaked 2-3 h after endotoxin injection, with a rapid decline by 6-8 h, and levels returned to basal values by 24 h . 5 . In contrast, TNF-RI peaked at 2 h (increasing from basal levels of 900-3300 pg/mL) with a much slower decline and without return to basal levels at 24 h (1400 pg/mL) . 6 . Endotoxin resulted in a rapid rise in soluble L-selectin within 1 h, which increased from a basal of 150-425 ng/mL . This rapid rise in soluble L-selectin was sustained for up to 2.5 h and then rapidly declined to basal levels by 3.5 h . 7 . In contrast, plasma soluble P-selectin levels showed a delayed and progressive increase up to 8 h (increasing from a basal level of 50-95 ng/mL), with a partial decline at 24 h (80 ng/mL) . 8 . Similarly, soluble VCAM-1 levels showed a progressive rise up to 24 h (increasing from basal values of 600-1000 ng/mL) . 9 . This acute human model of endotoxin exposure demonstrated an upregulation of inflammatory stimuli leading to a short-term hyperactivation of leucocytes and a more sustained activation of platelets and endothelium . 10 . This model provides a non-invasive method for studying the complex effects of endotoxin-like pathogens on different cellular events using soluble plasma surrogate markers.

Int J Radiat Oncol Biol Phys, 2001 Jun 1, 50(2), 343 - 52
Mucositis reduction by selective elimination of oral flora in irradiated cancers of the head and neck: a placebo-controlled double-blind randomized study; Wijers OB et al.; PURPOSE: The aim of the study was to test the hypothesis that aerobic Gram-negative bacteria (AGNB) play a crucial role in the pathogenesis of radiation-induced mucositis; consequently, selective elimination of these bacteria from the oral flora should result in a reduction of the mucositis . METHODS AND MATERIALS: Head-and-neck cancer patients, when scheduled for treatment by external beam radiation therapy (EBRT), were randomized for prophylactic treatment with an oral paste containing either a placebo or a combination of the antibiotics polymyxin E, tobramycin, and amphotericin B (PTA group) . Weekly, the objective and subjective mucositis scores and microbiologic counts of the oral flora were noted . The primary study endpoint was the mucositis grade after 3 weeks of EBRT . RESULTS: Seventy-seven patients were evaluable . No statistically significant difference for the objective and subjective mucositis scores was observed between the two study arms (p = 0.33) . The percentage of patients with positive cultures of AGNB was significantly reduced in the PTA group (p = 0.01) . However, complete eradication of AGNB was not achieved . CONCLUSIONS: Selective elimination of AGNB of the oral flora did not result in a reduction of radiation-induced mucositis and therefore does not support the hypothesis that these bacteria play a crucial role in the pathogenesis of mucositis.

J Clin Microbiol, 2001 Jun, 39(6), 2300 - 5
Prevalence of iron transport gene on pathogenicity-associated island of uropathogenic Escherichia coli in E . coli O157:H7 containing Shiga toxin gene; Ye C et al.; Uropathogenc Escherichia coli (UPEC) CFT073 has a pathogenicity-associated island (PAI(CFT073)), which causes pyelonephritis and cystitis . Using PCR method, we found the prrA gene of PAI(CFT073) in E . coli O157:H7 EDL933 . Further detailed PCR screening of 38 open reading frames, the right and left junction sequences of PAI(CFT073), revealed that it is the prrA-modD-yc73-fepC gene cluster but not the PAI(CFT073) present in E . coli O157:H7 EDL933 . A rapid preliminary analysis suggested that the prrA-modD-yc73-fepC gene cluster of the PAI(CFT073), is present in 43 strains of E . coli O157:H7 containing Shiga toxin (Stx) gene but absent in 19 strains of E . coli O157:H7 without Stx gene . A strict co-occurrence of the prrA-modD-yc73-fepC gene cluster and Stx genes was observed, regardless of their origin . The prrA-modD-yc73-fepC gene cluster encode proteins probably involved in iron uptake system, which strongly suggests the importance of iron metabolism in the Stx-mediated virulence . In addition, the prrA-modD-yc73-fepC gene cluster may be used as a diagnostic marker to distinguish E . coli O157:H7 strains containing Stx gene from that without Stx gene, and possibly to quickly detect other pathogenic gram-negative bacteria containing the Stx gene.

Nat Struct Biol, 2001 Jun, 8(6), 492 - 8
The structural basis of protein targeting and translocation in bacteria; Driessen AJ et al.; In Gram-negative bacteria, two distinct targeting routes assist in the proper localization of secreted and membrane proteins . Signal recognition particle (SRP) mainly targets ribosome-bound nascent membrane proteins, whereas SecB facilitates the targeting of periplasmic and outer membrane proteins . These routes converge at the translocase, a protein-conducting pore in the membrane that consists of the SecYEG complex associated with the peripheral ATPase, SecA . Recent structural studies of the targeting and the translocating components provide insights into how substrates are recognized and suggest a mechanism by which proteins are transported through an aqueous pore in the cytoplasmic membrane.

Crit Care Med, 2001 Mar, 29(3), 557 - 61
Gene variants of the bactericidal/permeability increasing protein and lipopolysaccharide binding protein in sepsis patients: gender-specific genetic predisposition to sepsis; Hubacek JA et al.; OBJECTIVES: To determine whether the genotype frequencies of the five bi-allelic polymorphisms in the bactericidal/permeability increasing protein (BPI) (Lys216 --> Glu; PstI polymorphism in intron 5; silent mutation G545 --> C) and the lipopolysaccharide binding protein (LBP) (Cys98 --> Gly; Pro436 --> Leu) are associated with the incidence and lethality of sepsis . DESIGN: Case control study of patients with sepsis . SETTING: Intensive care units within university hospitals . PATIENTS: A total of 204 patients diagnosed with sepsis and 250 healthy blood donors . INTERVENTIONS: None . MEASUREMENTS AND MAIN RESULTS: Short DNA fragments containing the polymorphic sites of the LBP and BPI locus were amplified by the polymerase chain reaction or mismatched polymerase chain reaction . The individual polymorphisms were determined with the appropriate restriction enzyme digestions and subsequent agarose gel electrophoresis . The presence of LBP genotypes with the less frequent Gly98 allele was found to be associated with sepsis (p < .02) in male patients, but not in females . Patients which were homozygote for either of the rare Gly98 (n = 6) and/or Leu436 (n = 5) LBP alleles, furthermore, exclusively were nonsurvivors of sepsis . The genotype frequencies in the BPI gene did not differ between patients and control individuals . CONCLUSIONS: Our findings suggest that common polymorphisms in the gene for LBP in combination with male gender are associated with an increased risk for the development of sepsis and, furthermore, may be linked to an unfavorable outcome . These data support the important immunomodulatory role of LBP in Gram-negative sepsis and suggest that genetic testing may be helpful for the identification of patients with an unfavorable response to Gram-negative infection.

J Am Soc Nephrol, 2001 Jun, 12(6), 1204 - 10
Countervailing influence of tumor necrosis factor-alpha and nitric oxide in endotoxemia; Jaimes EA et al.; Tumor necrosis factor-alpha (TNF-alpha), a crucial mediator in sepsis, elicits multiple biologic effects, including intravascular thrombosis and circulatory shock . TNF-alpha exerts its biologic effects through two distinct cell surface receptors, TNF-R1 and TNF-R2 . The pathophysiologic interaction between TNF-alpha and nitric oxide (NO) in glomerular thrombosis caused by endotoxemia in rats and wild-type mice (C57BL6) as well as in knockout mice that are deficient in TNF-R1 (R1 -/-), TNF-R2 (R2 -/-), or both receptors (R1R2 -/-) was studied . Administration of lipopolysaccharide (LPS; Escherichia coli endotoxin) resulted in increased NO and TNF-alpha production but failed to induce glomerular thrombosis . Concomitant administration of LPS + NG-nitro-L-arginine methyl ester (L-NAME; an NO synthesis inhibitor) resulted in glomerular thrombosis in rats and in wild-type mice . Intraperitoneal administration of pentoxifylline before LPS inhibited TNF-alpha synthesis and prevented glomerular thrombosis in rats given LPS + L-NAME . In contrast to the results observed in rats and wild-type mice, administration of LPS + L-NAME did not result in glomerular thrombosis in knockout mice with either single or double TNF-alpha receptor deletion . Thus, during endotoxemia, (1) TNF-alpha fosters glomerular thrombosis if there is deficiency of NO synthesis and (2) both TNF-alpha receptors are necessary for TNF-alpha's prothrombogenic action . Clinically, these novel studies suggest that in gram-negative endotoxemia, inhibition of NO synthesis and selective blockade of TNF-alpha receptors may provide unique therapeutic approaches for mitigation of glomerular thrombosis and restitution of vascular tone.

J Infect Dis, 2001 Jun 15, 183(12), 1815 - 8 Epub 2001 May 11.
Tissue factor pathway inhibitor does not influence inflammatory pathways during human endotoxemia; de Jonge E et al.; Activation of coagulation induces a proinflammatory response in in vitro and animal experiments . Inhibition of the tissue factor-dependent pathway of coagulation inhibits cytokine release and prevents death in gram-negative sepsis models in primates . This study investigated the influence of blocking the coagulation system by tissue factor pathway inhibitor (TFPI) on endotoxin-induced inflammatory responses in healthy humans . Eight men were studied in a double-blind, randomized, placebo-controlled cross-over study . They received a bolus intravenous injection of 4 ng/kg of endotoxin, followed by a 6-h continuous infusion of either TFPI (0.2 mg/kg/h after a bolus of 0.05 mg/kg) or placebo . Endotoxin induced-activation of coagulation was prevented completely by TFPI . In contrast, TFPI did not influence leukocyte activation, chemokine release, endothelial cell activation, or the acute phase response . Thus, complete prevention of coagulation activation by TFPI does not influence activation of inflammatory pathways during human endotoxemia.

Thromb Res, 2001 May 1, 102(3), 273 - 83
Non-mannose-capped lipoarabinomannan stimulates human peripheral monocytes to expression of the "early immediate genes" tissue factor and tumor necrosis factor-alpha; Moller AW et al.; In the present study, we have shown that stimulation of cryopreserved, human peripheral blood monocytes with the cell wall components from Gram-negative bacteria, lipopolysaccharide (LPS), and from rapid-growing Mycobacterium sp., non-mannose-capped lipoarabinomannan (AraLAM), both induce expression of the "early immediate genes" tissue factor (TF) and tumor necrosis factor-alpha (TNF-alpha) . This was demonstrated both at the protein and the mRNA levels . Antibodies against the CD14 receptor could block the stimulating effects . AraLAM was a significantly weaker inducer than LPS, and we speculate that this may reside in the number of the fatty acids in the part of the molecule that interacts with the CD14/Toll-like receptors (TLR) . Finally, both LPS and AraLAM activated the "early immediate genes" through translocation of the transcription factor proteins NF-kappaB/Rel and increasing the binding activity of AP-1.

Biometals, 2001 Mar, 14(1), 1 - 11
New artificial siderophores based on a monosaccharide scaffold; Heggemann S et al.; New artificial catecholate siderophores with methyl alpha-D-glucopyranoside as scaffold were synthesized . The dihydroxy- or di(acetoxy)benzoyl moieties were attached either directly or via aminopropyl spacer groups, to the carbohydrate scaffold . The siderophore activity of the prepared siderophore analogs was examined by a growth promotion assay using various Gram-negative bacteria and mycobacteria and by the CAS-assay.

Mol Microbiol, 2001 May, 40(3), 542 - 54
flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans; Kachlany SC et al.; Actinobacillus actinomycetemcomitans, a Gram-negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non-specific, tenacious adherence to surfaces . Previous investigations have implicated an abundant, small ( approximately 6.5 kDa), fibril-associated protein (Flp/Fap) as the primary fibril subunit . Here, we report studies on fibril structure and on the function and evolution of Flp . High-resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5- to 7-nm-diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria . Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles . A modified version of the CU1000N Flp1 polypeptide with the T7-TAG epitope fused to its C-terminus was expressed in the wild-type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7-TAG . To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903phikan to isolate insertion mutations in the flp-1 gene (formerly designated flp) . Mutants with insertions early in flp-1 fail to produce fibrils and do not adhere to surfaces . Both fibril production and adherence were restored by cloned flp-1 in trans, thus providing the first evidence that flp-1 is required for fibril formation and tight, non-specific adherence . One mutant was found to have an insertion near the 3' end of flp-1 that results in the expression of a truncated and altered C-terminus of Flp1 . This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild-type bacteria . These findings and related observations with the Flp1-T7-TAG protein indicate that the C-terminus of Flp1 is important for the bundling and adherence properties of pili . Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp-1 to be a member of a novel and widespread subfamily of type IV prepilin genes . Thus, Flp pili are likely to be expressed by diverse bacterial species . Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp-like genes, including the open reading frame (flp-2, previously designated orfA) immediately downstream of flp-1 in A . actinomycetemcomitans . The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.

FEMS Microbiol Lett, 2001 May 15, 199(1), 1 - 7
Multiple facets of bacterial porins; Achouak W et al.; Porins form channels allowing the transport of molecules across lipid bilayer membranes . Their structure, location and large number on the bacterial surface lend them multiple functions . Porin loops are potential targets for adhesion to other cells and binding of bactericidal compounds to the surface of Gram-negative bacteria . Variation of the loop structure as a mechanism to escape immune pressure, or modulation of the porin expression in response to the presence of antibiotics, are survival strategies developed by some pathogenic bacteria . Porins may play a significant role as pathogenesis effectors.

Mol Pharmacol, 2001 Jun, 59(6), 1441 - 5
Insights into the mechanism of azithromycin interaction with an Escherichia coli functional ribosomal complex; Dinos GP et al.; Azithromycin, a derivative of erythromycin with improved activity against Gram-negative bacteria, exhibits a marginal inhibition effect in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes . This renders the study of azithromycin interaction with Ac{(3)H}Phe-tRNA . poly(U) . 70S ribosome complex (complex C) impossible, if we analyze its effect on peptide bond formation . To overcome this problem, we have used an alternative approach to investigate kinetically the azithromycin interaction with complex C and to compare the azithromycin binding properties with those of erythromycin . This approach was based on the ability of azithromycin to compete with tylosin, a macrolide antibiotic strongly inhibiting the puromycin reaction . Detailed kinetic analysis revealed that the encounter complex CA between complex C and azithromycin (A) undergoes a slow isomerization to a tighter complex C*A, which remains active toward puromycin . The determination of inhibition and isomerization rate constants enabled us to classify azithromycin as a slow-binding ligand of ribosomes . Compared with erythromycin, azithromycin is a better inducer and stabilizer of the C*A complex . This finding may explain the superiority of azithromycin as inhibitor of translation in E . coli cells and many other Gram-negative bacteria.

Infect Immun, 2001 Jun, 69(6), 3800 - 8
Helicobacter pylori lipopolysaccharides preferentially induce CXC chemokine production in human monocytes; Innocenti M et al.; Helicobacter pylori infection can cause duodenal ulcers and may also induce gastric adenocarcinoma . The bacteria colonize the gastric mucosa and areas of gastric metaplasia in the duodenum for decades, resulting in active chronic inflammation in the infected areas . A characteristic feature of the infection is the ongoing recruitment of neutrophils to the infected sites . To evaluate the role of H . pylori lipopolysaccharides (LPS) in the recruitment of leukocytes to the gastric mucosa, we have examined the cytokine and chemokine production from human monocytes stimulated with LPS isolated from different H . pylori strains, as well as from several other gram-negative bacteria . Our results show that H . pylori LPS induce a large production of neutrophil-recruiting CXC chemokines (interleukin-8 and growth-related oncogene alpha) from purified human monocytes, to almost the same extent as Escherichia coli LPS . However, and in agreement with previous studies, H . pylori LPS was much less potent in inducing production of proinflammatory cytokines by purified human monocytes and was also a weak inducer of the CC chemokine RANTES . There was no difference between LPS preparations from different H . pylori strains in their ability to induce cytokines and chemokines . The preferential production of CXC chemokines after stimulation with H . pylori LPS indicates an important contribution of this molecule in maintaining neutrophil recruitment during the infection, irrespective of the infecting strain.

J Microbiol Methods, 2001 Jul, 45(3), 197 - 205
Construction of improved vectors and cassettes containing gusA and antibiotic resistance genes for studies of transcriptional activity and bacterial localization; Wielbo J et al.; Broad-host-range, conjugative vectors with a constitutively expressed gusA gene combined with different antibiotic resistance (tetracycline, gentamicin, kanamycin) genes have been constructed . These plasmids are designed for tracking Gram-negative bacterial strains without the risk of random mutagenesis . We also constructed a set of cassettes containing a promoterless gusA gene linked with different antibiotic resistance genes for making transcriptional fusions and for cassette mutagenesis . New plasmids and cassettes can be useful tools for studying gene expression, interaction of bacteria with plants and monitoring bacteria in the environment.

Crit Rev Oral Biol Med, 2001, 12(2), 125 - 35
Involvement of T-lymphocytes in periodontal disease and in direct and indirect induction of bone resorption; Taubman MA et al.; Periodontal disease is a peripheral infection involving species of gram-negative organisms . T-lymphocytes can be found in the dense inflammatory infiltrate in this disease . CD4+ and CD8+ T-cells are present in periodontal lesions, as are memory/activated T-lymphocytes . In addition, Th1- and Th2-type T-lymphocytes and their associated cytokines with a subtle polarization to Th1 may be present . Th1-type T-cells up-regulate the production of pro-inflammatory cytokines IL-1 and TNF-alpha, which can induce bone resorption indirectly by promoting differentiation of osteoclast precursors and subsequently by activating osteoclasts . Such osteoclast differentiation is dependent on stimulation of osteoprotegerin ligand (OPG-L) production by osteoblastic cells . By contrast, activated T-cells, by virtue of direct production and expression of OPG-L, can directly promote osteoclast differentiation . OPG-L appears to be predominantly expressed on Th1-type cells . The direct and indirect T-cell involvement in periodontal bone resorption appears to be dependent on the degree of Th1-type T-cell recruitment into inflamed gingival tissues . This T-cell recruitment is regulated by adhesion molecules and chemokines/chemokine receptors . The adhesion molecules involved include alpha4 and alpha6 integrins, LFA-1, and ICAM-1 . The Th1-type T-cells preferentially express CCR5 and CXCR3, which are found prominently in diseased gingivae . By contrast, little CCR4, expressed by Th2-type T-cells, can be detected . Also, the chemokine ligands RANTES, MIP1-alpha (both CCR5), and IP-10 (CXCR3 ligand) were elevated in inflamed periodontal tissues . The T-cell features in diseased periodontal tissues can be compared with those in rheumatoid arthritis, wherein bone resorption often attributed to Th1-type T-cell involvement has also been demonstrated.

Pediatr Pulmonol, 2001 May, 31(5), 331 - 8
Bronchoalveolar inflammation following airway infection in preterm infants with chronic lung disease; Groneck P et al.; Chronic lung disease (CLD) of the newborn is associated with pulmonary inflammation . However, the origin of this inflammation is not known . We evaluated the impact of airway infection on bronchoalveolar inflammation in mechanically ventilated preterm infant at risk for CLD (n = 68) . Mean and maximum concentrations of the inflammatory mediators (IM) interleukin-1 and interleukin-8 were assayed in the tracheobronchial aspirate fluid (TAF) of neonates with perinatal airway infection (Ureaplasma urealyticum, or bacteria), postnatal nosocomial airway infection, or respiratory disease without airway infection from days 1-10 of postnatal age . Patients with CLD (n = 23;) exhibited increased levels of IM in TAF compared to neonates without CLD . Within the three subgroups, concentrations of IM were increased in CLD patients with perinatal infection and in CLD patients with respiratory disease without airway infection, but not in CLD patients with nosocomial airway infection . Although airway colonization with Gram-negative bacteria was more frequently found in CLD patients within the first month of life, there were no differences between levels of IM in patients colonized with Gram-negative bacteria or coagulase-negative staphyloccoci . We conclude that perinatal infections with Ureaplasma urealyticum or bacteria and respiratory disease without infection, but not nosocomial airway infection, contribute to the bronchopulmonary inflammatory response in neonates with CLD .

Trends Microbiol, 2001 May, 9(5), 209 - 14
How and when are substrates selected for type III secretion?
Aldridge P, Hughes KT.
Many Gram-negative bacteria use type III secretion systems to secrete virulence factors as well as the structural components of the flagellum . Some bacterial secretion systems use a secretion signal contained in the amino acid sequence of the secreted substrate . However, substrates of type III systems lack a single, defined secretion signal . There is evidence for the existence of three independent secretion signals - the 5' region of the mRNA, the amino terminus of the substrate and the ability of a secretion chaperone to bind the substrate before secretion - that direct substrates for secretion through the type III pathways . One or more of these signals might be used for a given substrate . A recent study of flagellar assembly presented evidence for a role of translation in the type III secretion mechanism . We present a unifying model for type III secretion that can be applied to flagellar assembly, needle assembly and the secretion of virulence factors . The potential role of translation in regulating the timing of substrate secretion is also discussed.

Int J Biochem Cell Biol, 2001 May, 33(5), 475 - 82
Protective effect of vitamin E, beta-carotene and N-acetylcysteine from the brain oxidative stress induced in rats by lipopolysaccharide; Kheir-Eldin AA et al.; The major goal of this study was to examine the ability of several antioxidants namely, vitamin E, beta-carotene and N-acetylcysteine, to protect the brain from oxidative stress induced by lipopolysaccharide (LPS, endotoxin) . LPS, a component of the bacterial wall of gram-negative bacteria, has been recognized as one of the most potent bacterial products in the induction of host inflammatory responses and tissue injury and was used in this study to mimic infections . LPS injection resulted in a significant increase in the stress indices, plasma corticosterone and glucose concentration, a significant alteration of the brain oxidative status observed as elevation of the level of malondialdehyde (MDA, index of lipid peroxidation) and reduction of reduced glutathione (GSH), and a disturbance in the brain energy metabolism presented as a reduction in the ATP/ADP ratio and an increase in the mitochondrial/cytosolic hexokinase ratio . However, the activities of brain superoxide dismutase and Na+, K+-ATPase and contents of cholesterol and phospholipids were not altered . Administration of the aforementioned antioxidants prior to LPS injection ameliorated the oxidative stress by reducing levels of MDA, restoring GSH content and normalizing the mitochondrial/cytosolic hexokinase ratio in the brain in addition to lowering levels of plasma corticosterone and glucose . In conclusion, this study showed the increased free radical generation during infections and LPS-induced stress . It also suggests that brain oxidative status and energy is disturbed.

Cochrane Database Syst Rev . 2001;(1):CD001236.
Routine perineal shaving on admission in labour; Basevi V et al.; BACKGROUND: Pubic or perineal shaving is a procedure performed before birth in order to lessen the risk of infection if there is a spontaneous perineal tear or if an episiotomy is performed . OBJECTIVES: To assess the effects of routine perineal shaving on admission in labour on maternal and neonatal outcomes, according to the best available evidence . SEARCH STRATEGY: The register of clinical trials maintained and updated by the Cochrane Pregnancy and Childbirth Group . In addition, the Cochrane Controlled Trials Register was searched . Date of last search: July 2000 . SELECTION CRITERIA: All controlled trials (including quasi randomised) which compared perineal shaving versus no perineal shaving were included in the review . DATA COLLECTION AND ANALYSIS: Trials under consideration were evaluated for methodological quality and appropriateness for inclusion, without consideration of their results . MAIN RESULTS: Only two trials fulfilled the prespecified criteria . In the earlier trial, 389 women were alternately allocated to receive either skin preparation and perineal shaving (control) or clipping of vulval hair only (experimental) . In the second trial, which included 150 participants, perineal shaving was compared with the cutting of long hairs for procedures only . The primary outcome for both trials was maternal febrile morbidity . No differences were found (combined odds ratio (OR) 1.26, 95% confidence interval (CI) 0.75, 2.12) . In the smaller trial, fewer women who had not been shaved had gram negative bacterial colonisation compared with women who had been shaved (OR 0.43, 95% CI 0.20, 0.92) . REVIEWER'S CONCLUSIONS: There is insufficient evidence to recommend perineal shaving for women on admission in labour.

J Biol Chem, 2001 Jul 20, 276(29), 27535 - 40 Epub 2001 Apr 27.
Crystal structure of the dimeric C-terminal domain of TonB reveals a novel fold; Chang C et al.; The TonB-dependent complex of Gram-negative bacteria couples the inner membrane proton motive force to the active transport of iron.siderophore and vitamin B(12) across the outer membrane . The structural basis of that process has not been described so far in full detail . The crystal structure of the C-terminal domain of TonB from Escherichia coli has now been solved by multiwavelength anomalous diffraction and refined at 1.55-A resolution, providing the first evidence that this region of TonB (residues 164-239) dimerizes . Moreover, the structure shows a novel architecture that has no structural homologs among any known proteins . The dimer of the C-terminal domain of TonB is cylinder-shaped with a length of 65 A and a diameter of 25 A . Each monomer contains three beta strands and a single alpha helix . The two monomers are intertwined with each other, and all six beta-strands of the dimer make a large antiparallel beta-sheet . We propose a plausible model of binding of TonB to FhuA and FepA, two TonB-dependent outer-membrane receptors.

Lett Appl Microbiol, 2001 May, 32(5), 316 - 20
Methods for the isolation of water-borne Escherichia coli O157; Lejeune JT et al.; AIMS: To develop improved methods for the detection of Escherichia coli O157 from water and sediments . METHODS AND RESULTS: The effects of different broth enrichment media (unsupplemented tryptic soya broth, tryptic soya broth with antibiotics, and gram-negative broth), incubation durations (5 and 24 hrs), incubation temperatures (37 and 44.5 degrees C) and the use of immunomagnetic separation (IMS) on the sensitivity of E . coli O157 detection were evaluated on artificially and naturally-contaminated water and sediment samples . The sensitivity of recovery of E . coli O157 from samples was dependent upon the media composition, temperature duration of incubation and the use of IMS . CONCLUSION: Use of high temperature (44.5 degrees C) incubation for 24 hrs in unsupplemented tryptic soya broth and the use of IMS improved the sensitivity of E . coli O157 culture from water and sediment samples . SIGNIFICANCE AND IMPACT OF THE STUDY: The methods described can be used to increase the sensitivity of E . coli O157 detection from water and sediments.

J Clin Microbiol, 2001 May, 39(5), 1716 - 20
Helicobacter sp . flexispira bacteremia in an immunocompetent young adult; Iten A et al.; A young immunocompetent patient was admitted for a febrile illness with malaise, arthralgias, painful leg swelling, and polyserositis . Shortly prior to becoming ill, the patient had traveled to the Northern African desert . The symptoms disappeared during treatment with antibiotics (doxycycline and ceftriaxone) but recurred twice after stopping therapy . A motile gram-negative fusiform rod was isolated from a blood culture taken on the first admission . Analysis of the 16S rRNA gene of the blood culture isolate revealed close similarity with Helicobacter sp . flexispira taxon 8 (99.9% identity), a species that was previously reported as "Flexispira rappini." This is the first reported case of a recurrent Helicobacter sp . flexispira bacteremia in an adult, immunocompetent patient.

FEMS Microbiol Lett, 2001 Apr 20, 198(1), 49 - 55
Molecular characterization of a recombinant replication protein (Rep) from the Antarctic bacterium Psychrobacter sp . TA144; Duilio A et al.; The Antarctic Gram-negative bacterium Psychrobacter sp . TA144 contains two small cryptic plasmids, called pTAUp and pTADw . pTAUp encodes a replication enzyme (PsyRep) whose activity is responsible for plasmid replication via the rolling circle replication pathway . Several attempts to produce the wild-type biologically active PsyRep in Escherichia coli failed, possibly due to auto-regulation of the protein population . However, the serendipitous occurrence of a frameshift mutation during the preparation of an expression vector resulted in the over-production of a recombinant protein, changed in its last 14 amino acid residues (PsyRep*), that precipitates in insoluble form . The purification of PsyRep* inclusion bodies and the successful refolding of the cold adapted enzyme allowed us to carry out its functional characterization . The mutated protein still displays a double stranded DNA nicking activity, while the change at the C-terminus impairs the enzyme specificity for the pTAUp cognate Ori+ sequence.

J Physiol Pharmacol, 2001 Mar, 52(1), 145 - 52
Studies on the effects of lipopolysaccharide on lipid peroxidation of erythrocyte and its reversal by mannitol and glycerol; Bhattacharyya J et al.; Gram-negative sepsis often produces endotoxin (LPS) which causes infection . Reduction in tissue perfusion due to microcirculatory failure may lead to septic shock . We studied the effect of LPS on lipid peroxidation of erythrocyte . In vitro studies using 50 microg to 250 microg LPS/ml blood showed increased lipid peroxidation of erythrocyte in a dose-dependent manner . The increased effect of lipid peroxidation does not occur with LPS when erythrocytes were washed to remove plasma and leukocytes . Mannitol and glycerol, known scavengers of hydroxyl radical, arrest the elevation in lipid peroxidation of erythrocytes after LPS treatment . Hemolysis of erythrocytes was reduced with low doses of LPS . Plasma lipid peroxidation was elevated after treatment of blood with LPS . From the results we suggest that the peroxidation of erythrocyte lipid caused by LPS may probably play a role in the production of septic shock.

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 661 - 6
Hahella chejuensis gen . nov., sp . nov., an extracellular-polysaccharide-producing marine bacterium; Lee HK et al.; A bacterial strain, designated 96CJ10356T, which produced abundant extracellular polysaccharides and red pigment was isolated from marine sediment collected from Marado, Cheju Island, Republic of Korea . The organism is Gram-negative, aerobic, rod-shaped and motile . Growth was not observed in the absence of NaCl, and was optimal at an NaCl concentration of 2% . The strain contained oxidase and catalase, and was able to hydrolyse aesculin and gelatin . The major cellular fatty acids were saturated or monounsaturated straight-chain fatty acids . An almost complete 16S rDNA sequence of the test strain was determined . Phylogenetic analysis based on the neighbour-joining and Fitch-Margoliash methods indicated that the organism formed a distinct phyletic line within the gamma Proteobacteria . This relationship was also supported by sequence comparison, as no valid bacterial species showed more than 90% sequence homology with the isolate . It is clear from polyphasic evidence that the isolate merits the status of genus in the gamma subclass of the Proteobacteria, and the name Hahella chejuensis gen . nov., sp . nov . is proposed for the marine isolate 96CJ10356T (= KCTC 2396T = IMSNU 11157T).

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 349 - 55
Pseudomonas alcaliphila sp . nov., a novel facultatively psychrophilic alkaliphile isolated from seawater; Yumoto I et al.; Facultatively psychrophilic alkaliphilic strains were isolated from seawater obtained off the coast of Rumoi, Hokkaido, Japan . They were Gram-negative, aerobic straight rods with polar flagella . The isolates were catalase- and oxidase-positive and able to grow at 4 degrees C, but not at 40 degrees C . They produced acid from D-glucose under aerobic conditions . The isolates reduced nitrate to nitrite and hydrolysed casein and gelatin, but not starch or DNA . NaCl was required for growth at pH 10 but was not required at neutral pH . The major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 62.3-63.2 mol% . The whole-cell fatty acids mainly consisted of C16:0, C16:1(9c) and C18:1(9c), with 3-OH C10:0 and 3-OH C12:0 as the hydroxyl fatty acids . A larger amount of trans-unsaturated fatty acid, C16:1(9t) was observed when the cells were grown at pH 7 compared to when cells were grown at pH 10 . Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacteria are members of the genus Pseudomonas . Analysis of DNA-DNA relatedness data with several close phylogenetic neighbours revealed a low level of hybridization (less than 61%) . On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, it is concluded that these isolates represent a separate new species . Accordingly, the name Pseudomonas alcaliphila is proposed . The type strain is AL15-21T (= JCM 10630T = IAM 14884T).

Microbiology, 2001 May, 147(Pt 5), 1161 - 9
Insertion of fluorescent fatty acid probes into the outer membranes of the pathogenic spirochaetes Treponema pallidum and Borrelia burgdorferi; Cox DL et al.; The authors examined the ability of octadecanoyl (C(18)), hexadecanoyl (C(16)) and dodecanoyl (C(12)) fatty acid (FA) conjugates of 5-aminofluorescein (OAF, HAF and DAF, respectively) to insert into the outer membranes (OMs) of Treponema pallidum, Borrelia burgdorferi and Escherichia coli . Biophysical studies have demonstrated that these compounds stably insert into phospholipid bilayers with the acyl chain within the hydrophobic interior of the apical leaflet and the hydrophilic fluorescein moiety near the phospholipid head groups . Consistent with the known poor intrinsic permeability of the E . coli OM to hydrophobic compounds and surfactants, E . coli was not labelled with any of the FA probes . OAF inserted more readily into OMs of B . burgdorferi than into those of T . pallidum, although both organisms were completely labelled at concentrations at or below 2 microg ml(-1) . Intact spirochaetes were labelled with OAF but not with antibodies against known periplasmic antigens, thereby confirming that the probe interacted exclusively with the spirochaetal OMs . Separate experiments in which organisms were cooled to 4 degrees C (i.e . below the OM phase-transition temperatures) indicated that labelling with OAF was due to insertion of the probe into the OMs . B . burgdorferi, but not T . pallidum, was labelled by relatively high concentrations of HAF and DAF . Taken as a whole, these findings support the prediction that the lack of lipopolysaccharide renders T . pallidum and B . burgdorferi OMs markedly more permeable to lipophilic compounds than their Gram-negative bacterial counterparts . The data also raise the intriguing possibility that these two pathogenic spirochaetes obtain long-chain FAs, nutrients they are unable to synthesize, by direct permeation of their OMs.

Int J Colorectal Dis, 2001 Feb, 16(1), 55 - 7
Nonocclusive ischemic colitis in a 12-year-old girl: value of unenhanced spiral computed tomography; Wiesner W et al.; A 12-year-old girl was hit by a car and arrived in the emergency room in hemorrhagic shock . Contrast-enhanced computed tomography of the abdomen showed traumatic rupture of the liver and large amounts of intraperitoneal hemorrhage . Unenhanced computed tomography showed a hyperdense thickening of the wall of the descending colon . This finding was consistent with a nonocclusive ischemic colitis, which was confirmed some days later by endoscopy, at a time when the patient had already developed Gram-negative bacteremia . We discuss the pathogenesis of nonocclusive ischemic colitis, computed tomography findings, and the value of unenhanced computed tomography.

Mikrobiologiia, 2000 Sep-Oct, 69(5), 712 - 6
{Methylovorus mays--novel species of aerobic, obligatory methylotrophic bacteria associated with plants}; Doronina NV et al.; A bacterial strain utilizing methanol as the sole source of carbon and energy was isolated from the maize phyllosphere . Cells are nonpigmented gram-negative motile rods that do not form spores or prosthecae and reproduce by binary fission . The strain does not require vitamins or supplementary growth factors . It is obligately aerobic and urease-, oxidase-, and catalase-positive . The optimum growth temperature is 35-40 degrees C; the optimum pH is 7.0-7.5 . The doubling time is 2 h . The bacterium implements the ribulose monophosphate pathway and possesses NAD(+)-dependent 6-phosphogluconate dehydrogenase and enzymes of the glutamate cycle . alpha-Ketoglutarate dehydrogenase and enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) are absent . Fatty acids are dominated by palmitic (C16:0) and palmitoleic (C16:1) acids . The major phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine . Cardiolipin is present in minor amounts . The dominant ubiquinone is Q8 . The bacterial genome contains genes controlling the synthesis and secretion of cytokinins . The G + C content of DNA is 57.2 mol %, as determined from the DNA thermal denaturation temperature (Tm) . The bacterium shows low DNA homology (< 10%) with restricted facultative methylotrophic bacteria of the genus Methylophilus (M . methylotrophus NCIMB 10515T and M . leisingerii VKM B-2013T) and with the obligate methylotrophic bacterium (Methylobacillus glycogenes ATCC 29475T) . DNA homology with the type representative of the genus Methylovorus, M . glucosetrophus VKM B-1745T, is high (58%) . The new isolate was classified as a new species, Methylovorus mays sp . now.

J Immunol, 2001 May 1, 166(9), 5688 - 94
Endotoxin-induced maturation of MyD88-deficient dendritic cells; Kaisho T et al.; LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock . All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling . However, MyD88-deficient cells still show activation of NF-kappaB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear . In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice . LPS-induced cytokine production from DCs was dependent on MyD88 . However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity . MyD88-deficient DCs could not mature in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling . MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region . Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c(+) DCs could up-regulate their costimulatory molecule expression in response to LPS . Collectively, the present study provides the first evidence that the MyD88-independent pathway downstream of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.

J Biol Chem, 2001 Jul 20, 276(29), 27329 - 34 Epub 2001 Apr 19.
Evidence that NADP+ is the physiological cofactor of ADP-L-glycero-D-mannoheptose 6-epimerase; Ni Y et al.; ADP-L-glycero-D-mannoheptose 6-epimerase is required for lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria . The enzyme contains both fingerprint sequences Gly-X-Gly-X-X-Gly and Gly-X-X-Gly-X-X-Gly near its N terminus, which is indicative of an ADP binding fold . Previous studies of this ADP-l-glycero-D-mannoheptose 6-epimerase (ADP-hep 6-epimerase) were consistent with an NAD(+) cofactor . However, the crystal structure of this ADP-hep 6-epimerase showed bound NADP (Deacon, A . M., Ni, Y . S., Coleman, W . G., Jr., and Ealick, S . E . (2000) Structure 5, 453-462) . In present studies, apo-ADP-hep 6-epimerase was reconstituted with NAD(+), NADP(+), and FAD . In this report we provide data that shows NAD(+) and NADP(+) both restored enzymatic activity, but FAD could not . Furthermore, ADP-hep 6-epimerase exhibited a preference for binding of NADP(+) over NAD(+) . The K(d) value for NADP(+) was 26 microm whereas that for NAD(+) was 45 microm . Ultraviolet circular dichroism spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP(+) had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD(+) . Perchloric acid extracts of the purified enzyme were assayed with NAD(+)-specific alcohol dehydrogenase and NADP(+)-specific isocitric dehydrogenase . A sample of the same perchloric acid extract was analyzed in chromatographic studies, which demonstrated that ADP-hep 6-epimerase binds NADP(+) in vivo . A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD(+) cofactor, has identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP(+).

J Leukoc Biol, 2001 Apr, 69(4), 598 - 604
Requirement for STAT1 in LPS-induced gene expression in macrophages; Ohmori Y et al.; This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo . LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared from Stat1-/- mice, whereas levels of tumor necrosis factor alpha and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected . A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1-/- mice . The reduced LPS-stimulated gene expression seen in Stat1-/- macrophages was not the result of reduced activation of nuclear factor kappaB . LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-gamma-activated sequence binding activity in macrophages from wild-type mice . Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages from IFN-alpha/betaR-/- mice and blocked by cotreatment with antibodies against type I IFN . These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.

J Assoc Physicians India, 2000 Nov, 48(11), 1070 - 3
Nosocomial pneumonia in medical intensive care unit; Trivedi TH et al.; OBJECTIVES: This study was undertaken to determine incidence, clinical features, microbiological flora and prognosis of patients with nosocomial pneumonia (NP) in medical intensive care unit (ICU) . Also to study impact of comorbid illnesses, intubation and mechanical ventilation on outcome . METHODS: In this prospective study over a period of one year 89 patients were detected to have nosocomial pneumonia amongst 948 total admissions in ICU . All these 89 patients were investigated radiologically, microbiologically and biochemically . Mortality in these patients was correlated with various factors using chi-square test . RESULTS: Incidence of NP was 9.38% . Enteric gram-negative organisms were commonest isolates (61.9%), followed by Staph aureus (29.8%) . Sixty five (73%) patients had severe pneumonia, 47 (52.8%) required intubation and 42 (47.2%) mechanical ventilation . Total mortality was 19 out of 89 (21.3%) . High mortality was associated with habits like smoking (33.3%), age group over 60 years (27.3%), presence of comorbid illness like DM and COAD (38.5%), complications like ARDS (61.3%, p < 0.001) or sepsis with end organ failure (73.7%, p < 0.001) and need of intubation (36.2%) or mechanical ventilation (40.5%, p < 0.005) . CONCLUSION: Nosocomial pneumonias constitute important problem in medical ICU and are associated with high mortality . Patients with advanced age, co-morbid illness, ARDS and end organ failure are at high risk for mortality . However with intensive supportive care and appropriate antibiotics many such lives can be saved.

PDA J Pharm Sci Technol, 2001 Mar-Apr, 55(2), 87 - 113
Retention of water-borne bacteria by membrane filters . Part II: Scanning electron microscopy (SEM) and fatty acid methyl ester (FAME) characterization of bacterial species recovered downstream of 0.2/0.22 micron rated filters; Sundaram S et al.; The results of scanning electron microscopic (SEM) and fatty acid methyl ester (FAME) characterization of the bacterial species shown to penetrate conventional 0.2/0.22 micron rated "sterilizing grade" filters are presented . SEM data suggest that retention of bacteria by these filters appears to be strongly influenced by the morphology, and especially the width of bacteria and less so by length . When the bacterial cell width is small, less than 0.3 micron or so, the cell length does not appear to limit the ability to penetrate 0.2/0.22 micron rated filters . As the bacterial width increases, there is also a strong, almost exponential, decrease in the allowable length for penetration, with most penetrative cells tending to be coccoid beyond a width of 0.5 micron . Significant percentages of the bacteria (40-50%) that were observed downstream of these filters were larger than B . diminuta, the standard organism used to qualify 0.2/0.22 micron rated filters . The average sizes of natural waterborne bacteria that penetrated the filters tested were 20-40% larger in width, and 40-70% larger in length, compared to B . diminuta . These results indicate that size exclusion is not the sole mechanism governing bacterial retention . All isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp . and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems . Most of the bacteria recovered downstream of 0.2/0.22 micron rated filters were gram negative, oxidase positive, motile, nonfermentors.

Mol Microbiol, 2001 Apr, 40(2), 306 - 13
Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins; Jacob-Dubuisson F et al.; A collection of large virulence exoproteins, including Ca2+-independent cytolysins, an iron acquisition protein and several adhesins, are secreted by the two-partner secretion (TPS) pathway in various Gram-negative bacteria . The hallmarks of the TPS pathway are the presence of an N-proximal module called the 'secretion domain' in the exoproteins that we have named the TpsA family, and the channel-forming beta-barrel transporter proteins we refer to as the TpsB family . The genes for cognate exoprotein and transporter protein are usually organized in an operon . Specific secretion signals are present in a highly conserved region of the secretion domain of TpsAs . TpsBs probably serve as specific receptors of the TpsA secretion signals and as channels for the translocation of the exoproteins across the outer membrane . A subfamily of transporters also mediates activation of their cognate cytolysins upon secretion . The exoproteins are synthesized as precursors with an N-terminal cleavable signal peptide, and a subset of them carries an extended signal peptide of unknown function . According to our current model, the exoproteins are probably translocated across the cytoplasmic membrane in a Sec-dependent fashion, and their signal peptide is probably processed by a LepB-type signal peptidase . The N-proximal secretion domain directs the exoproteins towards their transporters early, so that translocation across both membranes is coupled . The exoproteins transit through the periplasm in an extended conformation and fold progressively at the cell surface before eventually being released into the extracellular milieu . Several adhesins also undergo extensive proteolytic processing upon secretion . The genes of many new TpsAs and TpsBs are found in recently sequenced genomes, suggesting that the TPS pathway is widespread.

Mol Microbiol, 2001 Apr, 40(2), 284 - 93
Type III export: new uses for an old pathway; Plano GV et al.; Gram-negative bacteria use type III secretion (TTS) systems to translocate proteins into the extracellular environment or directly into eukaryotic cells . These complex secretory systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway . Secretion substrates are directed to the TTS machinery via mRNA and/or amino acid secretion signals . TTS chaperones bind to select secretion substrates and assist in the export process . Recent progress in the understanding of TTS is reviewed.

J Vet Med Sci, 2001 Mar, 63(3), 275 - 80
Immunization against intestinal bacterial endotoxin prevents alcoholic fatty liver in rats; Nishimura G et al.; Accumulating evidences indicate that an endotoxin originating from intestinal gram-negative bacteria may be involved in alcohol-induced liver injury including fatty liver . Therefore, whether immunization against intestinal bacterial endotoxin blocked fatty liver induced by chronic alcohol and diet including much-unsaturated fatty acid was investigated in rats . The titer of antibody against the endotoxin increased significantly after 13 weeks of continuous immunization . Daily alcohol treatment was initiated at 12 weeks and continued for 4 weeks . Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and triglyceride (TG) levels increased significantly in non-immunized rats receiving alcohol, but not in immunized rats . Continuous alcohol treatment gradually decreased the survival rate to 60% from 13 days after beginning administration in non-immunized, but not immunized, rats . A histochemical study revealed that continuous treatment with alcohol and unsaturated fatty acids caused fatty liver in non-immunized, but not immunized, rats . This study strongly supports the hypothesis that alcohol-induced fatty liver is due to a circulating endotoxin, and suggests that immunization for endotoxin prevent the alcoholic fatty liver.

Int Arch Allergy Immunol, 2001 Jan-Mar, 124(1-3), 151 - 4
Interleukin-4 induces a switch of human intestinal mast cells from proinflammatory cells to Th2-type cells; Bischoff SC et al.; BACKGROUND: During the last years, mast cells have been recognized as a potent cellular source of multiple cytokines . However, little is known about the regulation of cytokine production by mature human mast cells derived from mucosal sites . METHODS: Human mast cells were isolated from intestinal mucosa and cultured for 14 days in the presence of stem cell factor (SCF) alone or in combination with IL-4 . Mast cells were then stimulated by IgE receptor cross-linking or bacterial infection and cytokine production was examined by RT-PCR and ELISA . RESULTS: We found that human intestinal mast cells produce proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-6 without further stimulation . Stimulation of the cells with gram-negative bacteria (Escherichia coli and others) caused an upregulation of TNF-alpha expression . Following IgE receptor cross-linking, we found additional expression of the Th2 cytokines IL-3, IL-5 and IL-13 . Interestingly, mRNA for IL-3, IL-5 and IL-13 was also expressed in unstimulated mast cells provided they were cultured in the presence of SCF and IL-4 . Moreover, IL-4 rendered mast cells capable of releasing IL-5 in response to bacterial challenge . CONCLUSION: In the presence of the mast cell survival factor SCF, mature human mast cells produce predominantly proinflammatory cytokines, whereas in the presence of SCF and IL-4, mast cells produce not only proinflammatory but also Th2 cytokines .

Brain Res Bull, 2001 Mar 1, 54(4), 443 - 53
Pro-inflammatory and anti-inflammatory cytokine mRNA induction in the periphery and brain following intraperitoneal administration of bacterial lipopolysaccharide; Turrin NP et al.; Gram-negative bacteria-derived lipopolysaccharide (LPS or endotoxin) is known to play an important role in immune and neurological manifestations during bacterial infections . LPS exerts its effects through cytokines, and peripheral or brain administration of LPS activates cytokine production in the brain . In this study, we investigated cytokine and neuropeptide mRNA profiles in specific brain regions and peripheral organs, as well as serum tumor necrosis factor (TNF)-alpha protein levels, in response to the intraperitoneal administration of LPS . For the first time, the simultaneous analysis of interleukin (IL)-1beta system components (ligand, signaling receptor, receptor accessory proteins, receptor antagonist), TNF-alpha, transforming growth factor (TGF)-beta1, glycoprotein 130 (IL-6 receptor signal transducer), OB protein (leptin) receptor, neuropeptide Y, and pro-opiomelanocortin (opioid peptide precursor) mRNAs was done in samples from specific brain regions in response to peripherally administered LPS . The same brain region/organ sample was assayed for all cytokine mRNA components . Peripherally administered LPS up-regulated pro-inflammatory cytokine (IL-1beta and/or TNF-alpha) mRNAs within the cerebral cortex, cerebellum, hippocampus, spleen, liver, and adipose tissue . LPS also increased plasma levels of TNF-alpha protein . LPS did not up-regulate inhibitory (anti-inflammatory) cytokine (IL-1 receptor antagonist and TGF-beta1) mRNAs in most brain regions (except for IL-1 receptor antagonist in the cerebral cortex and for TGF-beta1 in the hippocampus), while they were increased in the liver, and IL-1 receptor antagonist was up-regulated in the spleen and adipose tissue . Overall, peripherally administered LPS modulated the levels of IL-1beta system components within the brain and periphery, but did not affect the neuropeptide-related components studied . The data suggest specificity of transcriptional changes induced by LPS and that cytokine component up-regulation in specific brain regions is relevant to the neurological and neuropsychiatric manifestations associated with peripheral LPS challenge.

Nippon Rinsho, 2001 Apr, 59(4), 707 - 11
{AmpC beta-lactamases producing bacteria}; Okamoto R et al.; beta-Lactamases are the principal mechanism of bacterial resistance to beta-lactam antibiotics . In recent years, resistance due to production of beta-lactamases including extended-spectrum beta-lactamses, carbapeneases and AmpC beta-lactamses, has risen at alarming rate in clinical isolates of gram-negative bacteria . AmpC beta-lactamses are classified by whether genes locate on chromosome or plasmid . The purpose of this paper is to address the general mechanism involve in AmpC beta-lactamase production and the clinical importance of the enzymes.

Immunology, 2001 Mar, 102(3), 365 - 72
Complement-mediated lipopolysaccharide release and outer membrane damage in Escherichia coli J5: requirement for C9; O'Hara AM et al.; Lipopolysaccharides (LPS) are major antigenic components of the outer membrane of Gram-negative bacteria and can stimulate activation of the complement system . Such activation leads to formation of the complement membrane attack complex (MAC) on the cell walls, LPS release and, in serum-sensitive strains, to cell death . In this study, Escherichia coli J5 strains, which incorporate exogenous galactose exclusively into LPS, were used to generate target strains with different LPS chemotypes, and the LPS of the strains was labelled with tritium (3H-LPS) . The ability of normal human serum (NHS) and human complement-deficient sera to release LPS was subsequently monitored . NHS-induced release of 64-95.7% of 3H-LPS within 30 min; overall, no significant difference was observed between release of LPS from E . coli J5 strains with different LPS chemotypes . In functional assays, maximum LPS release had occurred by 30 min and before maximum bacterial killing . Electron microscopy revealed NHS-induced outer-membrane disruption in the form of blebs at 15 min; at this time-point the inner membrane remained intact . Background LPS release and no bactericidal activity were detected in heat-inactivated serum or human sera deficient in C6, C7 or C8 . The C9-deficient (C9D) serum had low bactericidal activity and failed to induce LPS release; however, addition of purified human C9 reconstituted its ability to release LPS . This study demonstrated the need for functional C9 molecules for LPS-releasing activities in serum-sensitive E . coli J5 strains.

Mol Microbiol, 2001 Apr, 40(1), 126 - 40
Pph1 from Myxococcus xanthus is a protein phosphatase involved in vegetative growth and development; Treuner-Lange A et al.; Myxococcus xanthus is a Gram-negative bacterium with a complex life cycle that includes vegetative swarming on rich medium and, upon starvation, aggregation to form fruiting bodies containing spores . Both of these behaviours require multiple Ser/Thr protein kinases . In this paper, we report the first Ser/Thr protein phosphatase gene, pph1, from M . xanthus . DNA sequence analysis of pph1 indicates that it encodes a protein of 254 residues (Mr = 28 308) with strong homology to eukaryotic PP2C phosphatases and that it belongs to a new group of bacterial protein phosphatases that are distinct from bacterial PP2C phosphatases such as RsbU, RsbX and SpoIIE . Recombinant His-tagged Pph1 was purified from Escherichia coli and shown to have Mn2+ or Mg2+ dependent, okadaic acid-resistant phosphatase activity on a synthetic phosphorylated peptide, RRA(pT)VA, indicating that Pph1 is a PP2C phosphatase . Pph1-expression was observed under both vegetative and developmental conditions, but peaked during early aggregation . A pph1 null mutant showed defects during late vegetative growth, swarming and glycerol spore formation . Under starvation-induced developmental conditions, the mutant showed reduced aggregation and failure to form fruiting bodies with viable spores . Using the yeast two-hybrid system, we have observed a strong interaction between Pph1 and the M . xanthus protein kinase Pkn5, a negative effector of development . These results suggest a functional link between a Pkn2-type protein kinase and a PP2C phosphatase.

Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 4938 - 43 Epub 2001 Apr 10.
Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p); Giraldo R et al.; The proteins responsible for the initiation of DNA replication are thought to be essentially unrelated in bacteria and archaea/eukaryotes . Here we show that RepA, the initiator from the Pseudomonas plasmid pPS10, and the C-terminal domain of ScOrc4p, a subunit of Saccharomyces cerevisiae (Sc) origin recognition complex (ORC), share sequence similarities . Based on biochemical and spectroscopic evidence, these similarities include common structural elements, such as a winged-helix domain and a leucine-zipper dimerization motif . We have also found that ScOrc4p, as previously described for RepA-type initiators, interacts with chaperones of the Hsp70 family both in vitro and in vivo, most probably to regulate the assembly of active ORC . In evolutionary terms, our results are compatible with the recruitment of the same protein module for initiation of DNA replication by the ancestors of present-day Gram-negative bacteria plasmids, archaea, and eukaryotes.

Genes Immun, 2001 Feb, 2(1), 56 - 9
LPS-hyporesponsiveness of mnd mice is associated with a mutation in Toll-like receptor 4; Bihl F et al.; Toll-like receptors (Tlrs) are transmembrane proteins that have recently been shown to play a critical role in the innate immune recognition of microbial constituents . Among this family, Tlr4 is a crucial signal transducer for lipopolysaccharide (LPS), the major component of the Gram-negative bacteria outer cell membrane . In this paper, we report that C57BL/6.KB2-mnd mice, a model of neuronal ceroid lipofuscinosis, do not respond to LPS . This defect is associated with a spontaneous mutation in Tlr4 consisting of a large insertion within exon 2 predicting a frameshift mutation and a truncated protein.

J Bacteriol, 2001 May, 183(9), 2755 - 64
Characterization of in vitro interactions between a truncated TonB protein from Escherichia coli and the outer membrane receptors FhuA and FepA; Moeck GS et al.; High-affinity iron uptake in gram-negative bacteria depends upon TonB, a protein which couples the proton motive force in the cytoplasmic membrane to iron chelate receptors in the outer membrane . To advance studies on TonB structure and function, we expressed a recombinant form of Escherichia coli TonB lacking the N-terminal cytoplasmic membrane anchor . This protein (H(6)-'TonB; M(r), 24,880) was isolated in a soluble fraction of lysed cells and was purified by virtue of a hexahistidine tag located at its N terminus . Sedimentation experiments indicated that the H(6)-'TonB preparation was almost monodisperse and the protein was essentially monomeric . The value found for the Stokes radius (3.8 nm) is in good agreement with the value calculated by size exclusion chromatography . The frictional ratio (2.0) suggested that H(6)-'TonB adopts a highly asymmetrical form with an axial ratio of 15 . H(6)-'TonB captured both the ferrichrome-iron receptor FhuA and the ferric enterobactin receptor FepA from detergent-solubilized outer membranes in vitro . Capture was enhanced by preincubation of the receptors with their cognate ligands . Cross-linking assays with the purified proteins in vitro demonstrated that there was preferential interaction between TonB and ligand-loaded FhuA . Purified H(6)-'TonB was found to be stable and thus shows promise for high-resolution structural studies.

Infect Immun, 2001 May, 69(5), 3143 - 9
Activation of extracellular signal-related protein kinases 1 and 2 of the mitogen-activated protein kinase family by lipopolysaccharide requires plasma in neutrophils from adults and newborns; Bonner S et al.; Neutrophils exposed to low concentrations of gram-negative lipopolysaccharide (LPS) become primed and have an increased oxidative response to a second stimulus (e.g., formyl-methionyl-leucyl-phenylalanine {fMLP}) . In studies aimed at understanding newborn sepsis, we have shown that neutrophils of newborns are not primed in response to LPS . To further understand the processes involved in LPS-mediated priming of neutrophils, we explored the role of extracellular signal-related protein kinases (ERK 1 and 2) of the mitogen-activated protein kinase family . We found that LPS activated ERK 1 and 2 in cells of both adults and newborns and that activation was plasma dependent (maximal at > or =5%) through LPS-binding protein . Although fibronectin in plasma is required for LPS-mediated priming of neutrophils of adults assessed by fMLP-triggered oxidative burst, it was not required for LPS-mediated activation of ERK 1 and 2 . LPS-mediated activation was dose and time dependent; maximal activation occurred with approximately 5 ng of LPS per ml and at 10 to 40 min . We used the inhibitor PD 98059 to study the role of ERK 1 and 2 in the LPS-primed fMLP-triggered oxidative burst . While Western blotting showed that 100 microM PD 98059 completely inhibited LPS-mediated ERK activation, oxidative response to fMLP by a chemiluminescence assay revealed that the same concentration inhibited the LPS-primed oxidative burst by only 40% . We conclude that in neutrophils, LPS-mediated activation of ERK 1 and 2 requires plasma and that this activation is not dependent on fibronectin . In addition, we found that the ERK pathway is not responsible for the lack of LPS priming in neutrophils of newborns but may be required for 40% of the LPS-primed fMLP-triggered oxidative burst in cells of adults.

Infect Immun, 2001 May, 69(5), 2821 - 8
Distribution and kinetics of lipoprotein-bound endotoxin; Levels JH et al.; Lipopolysaccharide (LPS), the major glycolipid component of gram-negative bacterial outer membranes, is a potent endotoxin responsible for pathophysiological symptoms characteristic of infection . The observation that the majority of LPS is found in association with plasma lipoproteins has prompted the suggestion that sequestering of LPS by lipid particles may form an integral part of a humoral detoxification mechanism . Previous studies on the biological properties of isolated lipoproteins used differential ultracentrifugation to separate the major subclasses . To preserve the integrity of the lipoproteins, we have analyzed the LPS distribution, specificity, binding capacity, and kinetics of binding to lipoproteins in human whole blood or plasma by using high-performance gel permeation chromatography and fluorescent LPS of three different chemotypes . The average distribution of O111:B4, J5, or Re595 LPS in whole blood from 10 human volunteers was 60% (+/-8%) high-density lipoprotein (HDL), 25% (+/-7%) low-density lipoprotein, and 12% (+/-5%) very low density lipoprotein . The saturation capacity of lipoproteins for all three LPS chemotypes was in excess of 200 microg/ml . Kinetic analysis however, revealed a strict chemotype dependence . The binding of Re595 or J5 LPS was essentially complete within 10 min, and subsequent redistribution among the lipoprotein subclasses occurred to attain similar distributions as O111:B4 LPS at 40 min . We conclude that under simulated physiological conditions, the binding of LPS to lipoproteins is highly specific, HDL has the highest binding capacity for LPS, the saturation capacity of lipoproteins for endotoxin far exceeds the LPS concentrations measured in clinical situations, and the kinetics of LPS association with lipoproteins display chemotype-dependent differences.

Immunol Rev, 2001 Feb, 179, 16 - 24
Mast cell modulation of immune responses to bacteria; Malaviya R et al.; Mast cells are key elements of the immune system . These cells release a wide variety of pro-inflammatory mediators which are responsible for the pathophysiology of many allergic diseases . Recent studies, however, have shown that mast cells have the capacity to modulate the host's innate immune response to gram negative bacteria by their ability to phagocytose bacteria, process and present bacterial antigens to T cells and recruit phagocytic help through the release of physiological amounts of pro-inflammatory mediators . Here, current knowledge of mast cell responses to gram negative bacteria and molecular mechanisms associated with mast cell bacteria interaction is reviewed.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 59 - 63
Heat-labile proteases in molecular biology applications; Moran AJ et al.; Thermolabile proteases were identified in three Gram-negative psychrotrophic bacteria . The protease from the psychrotrophic strain A9 was purified and its application to common molecular biology techniques was demonstrated . Heat-stable molecular biology enzymes (Taq polymerase and PvuII) were digested by a heat-labile protease, which was then inactivated by a mild heat treatment . The clear benefit of using heat-labile proteases arises in situations where further reactions may be accomplished without an intermediate purification step, thereby saving time and avoiding the possibility of product loss.

J Lab Clin Med, 2001 Apr, 137(4), 303 - 9
Efficacy of bactericidal/permeability-increasing protein in experimental otitis media with effusion in rats: a new therapy for mucosal infections; Nell MJ et al.; Otitis media with effusion (OME) is characterized by the presence of fluid in the middle ear without signs or symptoms of acute infection and by persistent changes in the middle ear mucosa . These are mainly induced by gram-negative bacterial infection and dysfunction of the eustachian tube (ET) . Gram-negative bacteria (GNB) contain lipopolysaccharide (LPS) in their outer membrane that is responsible for inflammatory reactions in the middle ear . In this study we investigated the therapeutic effect of a recombinant LPS-binding protein, bactericidal/permeability-increasing protein (rBPI21), on the repair of mucosal damage in rats with experimentally induced OME . OME was induced by obstruction of the eustachian tube in combination with LPS injection . Twelve weeks after OME induction, secretory cells in the tympanic orifice of the middle ear were increased from an average of 14 +/- 2 to 31 +/- 5, ciliated cells were decreased from 24 +/- 4 to 6 +/- 4, and the number of macrophages in the subepithelial layer increased from 13 +/- 4 to 27 +/- 3 . A single dose of rBPI21 was administered directly into the middle ear cavity 2 weeks after the induction of OME . Histologic examination of the middle ear mucosa at 4 and 12 weeks after OME induction showed that mucosal changes were restored by rBPI21 treatment . These results demonstrate that the middle ear mucosa recovers from inflammatory changes associated with OME after treatment with rBPI21 . This suggests that rBPI21 may be useful in the treatment of OME and of mucosal infections of the respiratory tract.

Am J Respir Crit Care Med, 2001 Mar, 163(4), 1002 - 9
Liver-lung interactions following Escherichia coli bacteremic sepsis and secondary hepatic ischemia/reperfusion injury; Matuschak GM et al.; We hypothesized that ischemia/reperfusion (I/R) injury of the liver during normotensive gram-negative bacteremic sepsis alters the kinetics of circulating endotoxin, tumor necrosis factor-alpha (TNF-alpha), and coinduced mediators, thereby exacerbating sepsis-induced lung inflammation . Liver and lung dysfunction were studied after hematogenous infection of Sprague-Dawley rats with 10(9) Escherichia coli serotype O55:B5 (EC) and 90 min of secondary hepatic ischemia in EC + I/R and saline-infused (normal saline NS) x I/R rats, followed by brief (1 h) or longer reperfusion (24 h) . TNF- alpha:leukotriene interactions in this model were examined using the 5-lipoxygenase-activating protein inhibitor MK-886 . Compared with sham-operated EC + Sham animals, peak serum endotoxin, TNF-alpha, alanine aminotransferase, interleukin-6 (IL-6), and hepatic neutrophil (PMN) influx were higher in EC + I/R rats through 24 h (p < 0.05) despite comparable arterial pressure . Lung PMN influx and wet/dry weight ratios were likewise enhanced in EC + I/R versus EC + Sham or NS + I/R rats . MK-886 attenuated TNF-alpha concentrations and ischemic liver injury but not mortality . Thus, focal hepatic I/R augments circulating endotoxin, TNF-alpha, and postbacteremic lung inflammation early after normotensive E . coli bacteremic sepsis.

Appl Environ Microbiol, 2001 Apr, 67(4), 1959 - 63
Identification of methyl halide-utilizing genes in the methyl bromide-utilizing bacterial strain IMB-1 suggests a high degree of conservation of methyl halide-specific genes in gram-negative bacteria; Woodall CA et al.; Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source . A single cmu gene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partial metF . CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyltransferase motif . However, cmuB, identified in M . chloromethanicum and H . chloromethanicum, was not detected in IMB-1.

Curr Opin Microbiol, 2001 Apr, 4(2), 186 - 93
Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria; Withers H et al.; Bacterial cell-to-cell communication (quorum sensing) relies upon the interaction of a small diffusible signal molecule with a sensor or transcriptional activator to couple gene expression with cell population density . In Gram-negative bacteria, it is now clear that N-acylhomoserine lactones bind directly to LuxR homologues and can be synthesized via one of three unrelated bacterial protein families and by transgenic plants . New chemical classes of signal molecules have been identified, some of which exhibit crosstalk with N-acylhomoserine-lactone-mediated quorum sensing . As the determinant of cell population density, quorum sensing is emerging as an integral component of bacterial global gene regulatory networks responsible for facilitating bacterial adaptation to environmental stress . N-acylhomoserine lactones are produced during experimental animal and human infections, and a function beyond quorum sensing has been suggested by their intrinsic immunomodulatory and pharmacological activities.

Curr Opin Microbiol, 2001 Apr, 4(2), 166 - 71
Responses of Gram-negative bacteria to certain environmental stressors; Ramos JL et al.; Bacteria in nature are exposed to variations in temperature, and are affected by the availability of nutrients and water and the presence of toxic molecules . Their reactions to these changes require a series of rapid adaptive responses . Although transcriptional regulation is of primary importance in these responses, translational regulation and even activation of 'silenced' enzymes are critical for survival in changing environments . Bacteria have developed a series of mechanisms at the membrane structure level to cope with high concentrations of solvents . In addition, solvent-tolerant strains express highly effective efflux pumps to remove solvents from the cytoplasm . Desiccation tolerance is based on the synthesis and accumulation of osmoprotectants together with changes in fatty acid composition to preserve membrane structure . Both cold shock and heat shock responses are mainly regulated at a post-transcriptional level, translation efficiency in the case of cold shock and mRNA half-life and sigma32 stability in the case of heat shock.

J Biol Chem, 2001 May 25, 276(21), 18249 - 56 Epub 2001 Mar 12.
A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain . Purification, characterization, cloning, and overproduction; Ikushiro H et al.; Serine palmitoyltransferase (SPT, EC ) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl-coenzyme A to 3-ketodihydrosphingosine . We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity and purified SPT to homogeneity . This enzyme is a water-soluble homodimeric protein unlike eukaryotic enzymes, known as heterodimers composed of tightly membrane-bound subunits, named LCB1 and LCB2 . The purified SPT shows an absorption spectrum characteristic of a pyridoxal 5'-phosphate-dependent enzyme . The substrate specificity of the Sphingomonas SPT is less strict than the SPT complex from Chinese hamster ovary cells . We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract . Sphingomonas SPT shows about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved . The recombinant SPT was catalytically and spectrophotometrically indistinguishable from the native enzyme . This is the first successful overproduction of an active enzyme in the sphingolipid biosynthetic pathway . Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.

J Biol Chem, 2001 May 4, 276(18), 14924 - 32 Epub 2001 Feb 14.
A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis; Bannerman DD et al.; Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections . Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival . We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide . Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide . Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation . Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis . Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide . That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway . Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.

J Biol Chem, 2001 Apr 20, 276(16), 13025 - 33 Epub 2001 Jan 19.
Exchangeability of N termini in the ligand-gated porins of Escherichia coli; Scott DC et al.; The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin beta-barrel formed by their C termini . We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively . The resultant chimeric proteins and empty beta-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses . Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane beta-barrel domain . Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound . The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type . The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V . (1999) Mol . Microbiol . 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake . These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.

J Biol Chem, 2001 May 18, 276(20), 16660 - 6 Epub 2001 Feb 15.
In vivo proteolytic degradation of the Escherichia coli acyltransferase HlyC; Guzman-Verri C et al.; Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys564 and Lys690 are able to damage the target cell membrane . Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein . By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated . HlyC was not detected by Western blot in an E . coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC . The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level . HlyC was detected in Western blots of cell lysates from an E . coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted . Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability . We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process . The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.

Nat Immunol, 2001 Apr, 2(4), 346 - 52
Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism; Werts C et al.; Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers . They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins . Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2) . Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L . interrogans . We also show that for intact L . interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway . These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.

Nat Immunol, 2001 Apr, 2(4), 338 - 45
A CD14-independent LPS receptor cluster; Triantafilou K et al.; Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent initiator of an inflammatory response and serves as an indicator of bacterial infection . Although CD14 has been identified as the main LPS receptor, accumulating evidence has suggested the possible existence of other functional receptor(s) . In this study, using affinity chromatography, peptide mass fingerprinting and fluorescence resonance energy transfer, we have identified four new proteins that form an activation cluster after LPS ligation and are involved in LPS signal transduction . Here we present evidence that implicates heat shock proteins 70 and 90, chemokine receptor 4 and growth differentiation factor 5 as the main mediators of activation by bacterial lipopolysaccharide.

Mol Immunol, 2000 Aug-Sep, 37(12-13), 745 - 54
Investigation on interaction of Achatinin, a 9-O-acetyl sialic acid-binding lectin, with lipopolysaccharide in the innate immunity of Achatina fulica snails; Biswas C et al.; Achatinin, a 9-O-acetyl sialic acid (9-O-AcSA) binding lectin, has been demonstrated to be synthesized in amoebocytes of Achatina fulica snails . This lectin was affinity-purified from Achatina amoebocytes lysate (AAL); it appeared as a single band on native polyacrylamide gel electrophoresis (PAGE) and showed 16 identical subunits of M.W . 15 kDa on sodium dodecyl sulphate (SDS)-PAGE . It was found to be homologous with an earlier reported lectin, Achatinin-H, derived from hemolymph of A . fulica snails (Sen, G., Mandal, C., 1995 . The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achantia fulica . Carbohydr . Res., 268, 115-125) . Homology between both lectins was confirmed by their similar electrophoretic mobilities, carbohydrate specificity and cross reactivity on immunodiffusion . Achatinin showed in vitro calcium dependent binding to two 9-O-acetylated sialoglyoconjugates (9-O-AcSG) on lipopolysaccharide (LPS) (Escherichia coli 055: B5) of M.W . 40 kDa and 27.5 kDa, which was abolished following de-O-acetylation . Based on the previously defined narrow sugar specificity of Achatinin towards 9-O-AcSAalpha2-->6GalNAc {Sen, G., Mandal, C., 1995 . The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achatina fulica . Carbohydr . Res., 268, 115-125}, we conclude that LPS contains this lectinogenic epitope at the terminal sugar moiety . The Achatinin-mediated hemagglutination inhibition of rabbit erythrocytes by LPS further confirmed it . The lectin exhibited bacteriostatic effect on Gram-negative bacteria E . coli, DH5alpha and C600 . AAL was earlier reported to undergo coagulation in presence of pg level of LPS (Biswas, C., Mandal, C., 1999 . The role of amoebocytes in the endotoxin-mediated coagulation in the innate immunity of Achatina fulica snail, Scand . J . Immunol . 49, 131-138) . We now demonstrate that Achatinin participates in LPS-mediated coagulation of AAL as indicated by enhanced release of Achatinin from the LPS stimulated amoebocytes and most importantly, by exhibiting a 77% decline in the coagulation of AAL when depleted of Achatinin . Level of Achatinin sharply declined (17-fold) following injection of LPS (20 microg per snail) to the snails, which was reversible by simultaneous injection of LPS and leupeptin implying the presence of LPS-mediated serine protease activity in Achatinin . This was substantiated when purified Achatinin in vitro showed serine protease activity in the presence of LPS followed by its complete blockage in the presence of leupeptin and phenyl methyl sulphonyl fluoride . Therefore, Achatinin, an abundantly available lectin at multiple sites of A . fulica, by virtue of its interaction with LPS, essentially plays a crucial role in the innate immune protection of A . fulica snails.

Immunol Lett, 2001 Mar 1, 76(2), 125 - 31
Tolerance to lipopolysaccharide (LPS) regulates the endotoxin effects on Shiga toxin-2 lethality; Alves-Rosa F et al.; It has been suggested that Shiga toxin (Stx) is necessary but not sufficient for hemolytic uremic syndrome (HUS) development, and pro-inflammatory stimuli such as lipopolysaccharide (LPS) from Gram negative bacteria are needed . Taking into account that LPS is present in the natural infection during HUS development, detoxification or regulation of LPS activity could be crucial to define the course of the disease . The objective of the present study was to investigate whether tolerance to LPS and/or antibodies to LPS, are able to modify the LPS-induced modulation of Stx type-2 (Stx2) lethality in a mouse model . Our results demonstrate that the high levels of IgG anti-LPS antibodies in immunized mice did not modify the dual effects of LPS (enhancement or protection) on Stx2 action . This could be attributed to the fact that antibodies do not recognize the active portion of LPS molecule (lipid A) . However, the enhancement of Stx2 toxicity exerted by LPS was inhibited in tolerant mice . This effect could be ascribed to the inhibition of LPS-induced TNF-alpha and IL-1beta secretion in tolerant animals, two cytokines known to be involved in the overexpression of Stx receptors . The phenomenon of LPS-induced protection on Stx2 toxicity was also inhibited in tolerant animals, although the mechanism involved in this effect is not clear . This is the first description which shows the influence of endotoxin tolerance on the evolution of experimental HUS . However, like in Gram negative infections, further knowledge on tolerance mechanism is necessary in order to achieve a comprehensive view of this phenomenon.

Folia Microbiol (Praha), 2000, 45(3), 211 - 6
Detoxication of the herbicide diuron by Pseudomonas sp; el-Deeb BA et al.; A strain of bacteria able to detoxicate the herbicide diuron in pure culture was isolated from sites contaminated with different urea herbicides . Diuron was used as a sole source of carbon and energy by this isolate which is a Gram-negative, aerobic, rod-shaped bacterium with a single polar flagellum, and grows at 40 degrees C . The strain has been identified as Pseudomonas sp.

Ter Arkh, 2000, 72(11), 18 - 21
{Changes in the levels of acute phase proteins in viral hepatitis}; Volchkova EV et al.; AIM: To investigate changes in the levels of acute phase proteins (APP) in the serum of patients with acute and chronic viral hepatitis of various etiology . MATERIAL AND METHODS: APP (prealbumin, albumin, transferrin, haptoglobin) were measured by automatic kinetic analyzer JCS II TM (Beckman) in 92 patients with acute and 61 patients with chronic viral hepatitis . Synthesis of albumin, prealbumin was depressed, of haptoglobin increased in patients with acute viral hepatitis irrespective of etiology at the height of intoxication . In chronic viral diseases APP content depended on the disease etiology . In chronic hepatitis C, B + C and D albumin, haptoglobin and transferrin were low in clinical exacerbation while in patients with chronic hepatitis B a fall in albumin, prealbumin and transferrin is accompanied with high haptoglobin . CONCLUSION: A complicated multidirectional changes in APP in patients with acute or chronic hepatitis serve, on the one hand, an integral diagnostic indication of hepatic function in conditions of systemic endotoxemia . On the other hand, endotoxemia is caused by lipopolysaccharides of gram-negative bacteria entering blood flow in great amounts in morphofunctional hepatic disorders caused by viral hepatitis of different etiology.

Microbiol Immunol, 2001, 45(1), 69 - 78
Nitric oxide production and iNOS mRNA expression in mice induced by repeated stimulation with live Fusobacterium nucleatum; Kato C et al.; There have been few studies on the detection of direct nitric oxide (NO) production and interferon-gamma (IFN-gamma) in vivo without using animal cell culture . We questioned whether NO and IFN-gamma could be produced at the site of infection . The peritoneal cavity of mice was used as the local infection model . NO and IFN-gamma in abdominal washings from these mice were measured directly at various times after injection of Fusobacterium nucleatum, a gram-negative rod periodontal pathogen . The mice were divided into three groups: those treated with live bacteria (LB), those treated with heat-killed bacteria (HKB) and those untreated: normal (N) . These mice were compared on the basis of cell filtration, NO and IFN-gamma production by injection of live bacteria (LFn) or heat-killed bacteria (HKFn) . In the LB group, the total cell number increased corresponding to an increase in neutrophils after injection of both LFn and HKFn . A low level of NO was constantly produced in abdominal washings, but a significant amount of NO was synthesized in the LB group only 12 hr to 24 hr after injection of LFn . At the same time iNOS enzyme activity and iNOS mRNA expression were detected . IFN-gamma, which may contribute to enhance NO production, was also secreted at a high level from peritoneal exudate cells (PEC) at 12 hr and 24 hr in the LB group by stimulation of LFn . At 12 hr and 24 hr, iNOS positive cells in the LB group by infection of LFn were identified and shown to contain mostly macrophages . These findings indicate that live bacteria play important roles in NO production by macrophages . It is suggested that NO may contribute to the inflammatory response during F . nucleatum infection in periodontitis.

EMBO Rep, 2000 Oct, 1(4), 313 - 8
Chunnel vision . Export and efflux through bacterial channel-tunnels; Andersen C et al.; The Escherichia coli TolC protein is central to toxin export and drug efflux across the inner and outer cell membranes and the intervening periplasmic space . The crystal structure has revealed that TolC assembles into a remarkable alpha-helical trans-periplasmic cylinder (tunnel) embedded in the outer membrane by a contiguous beta-barrel (channel), so providing a large duct open to the outside environment . The channel-tunnel structure is conserved in TolC homologues throughout Gram-negative bacteria, and it is envisaged that they are recruited and opened, through a common mechanism, by substrate-specific inner-membrane complexes.

Minerva Stomatol, 2000 Oct, 49(10), 485 - 500
{Bacterial virulence in the etiology of periodontal diseases}; Sbordone L et al.; Strong relationships have been very often described between various form of periodontal disease (PD) and certain bacterial species, so that nowadays periodontal disease is recognized as an infectious disease . Destruction of periodontal supporting tissues happens as a response to very intricate host-parasite interactions . When the clinician will be able to fully understand and identify such phenomena it would be possible to succeed in a properly diagnosis and control of the active phase of periodontal disease . The first step in such a direction would be to analyze the common characteristic of some bacterial species, the so called suspected periodontopathogens . Such species namely Gram-negative, associated with the outbreak of periodontal disease have in common the capacity to disrupt the integrity of the host defences by means of the so called virulence factors . These factors may enhance the bacterial colonization or may interfere with the host response that ultimately results in periodontal support breakdown . The present review focuses on the virulence factors of the main suspected periodontopathogens evaluating the effects on the host immune response and directly on the periodontal tissues.

Gastroenterology, 2001 Apr, 120(5), 1108 - 16
Complement activation directly induced by Helicobacter pylori; Berstad AE et al.; BACKGROUND AND AIMS: Helicobacter pylori is a frequent gram-negative colonizer of the human stomach . Its interaction with complement may be involved in the pathogenesis of chronic gastritis, and was mechanistically studied in vitro . METHODS: Four H . pylori strains, 2 cytotoxin-associated genes (cag)A+ and 2 cagA-, were isolated from infected patients . Bacteria or purified H . pylori lipopolysaccharides (LPSs) were incubated with nonimmune serum at 37 degrees C; the activation products C3b/iC3b/C3c (C3bc) and terminal complement complex (TCC) were then quantified by immunoassays . The serum sensitivity of 1 strain (L01, cagA+) was tested by counting the numbers of colony-forming units . RESULTS: All strains and LPSs generated large amounts of C3bc and TCC . Blocking of the classic complement pathway by the calcium chelator ethylene glycol tetraacetic acid (EGTA) markedly reduced the complement products, suggesting that H . pylori and its LPSs directly engage the classic activation pathway . H . pylori was shown to be serum sensitive, but 30% or more nonimmune serum was necessary to induce marked killing . After 5 minutes, swelled bacteria coated with C3bc and TCC were shown . CONCLUSIONS: H . pylori is complement sensitive and activates the classic pathway even in the absence of specific antibodies . Released cell wall constituents such as LPSs can activate complement and may explain why this bacterium induces gastric pathology without invading the mucosa.

J Air Waste Manag Assoc, 2001 Mar, 51(3), 352 - 60
Evaluation of health and safety risks in municipal solid waste recycling plants; Lavoie J et al.; Designers and managers of recycling processing plants need to be informed about the inherent occupational health and safety risks; however, there are few studies in the literature describing these risks . Therefore, the objectives of this project were to document the biological, chemical, physical, and ergonomic risks in three household-waste recycling plants . Bioaerosols (molds and bacteria) were measured using the methodology recommended by the American Society for Testing and Materials (ASTM) . Chemical contaminants and physical agents suspected of being present in this type of environment were measured using the standard methods of the Quebec Occupational Health and Safety Research Institute (IRSST) . The ergonomic part of the study identified the work requirements and risk factors causing the workers' physical symptoms . In summer, the average concentrations of total bacteria were greater than the Scandinavian guideline of 10,000 colony forming units per cubic meter of air (CFU/m3) in the receiving areas in plants 1 and 3, in the sorting areas of the three plants, and in shipping in plants 1 and 3 . When the average concentrations of gram-negative bacteria were compared to the Scandinavian guideline of 1000 CFU/m3 of air, only the sorting department in plant 2 in summer exceeded this value . Average indoor concentrations of molds that were statistically significantly greater than those measured in the upwind outdoor air were measured in all departments in plants 1 and 3, regardless of the season, and only in sorting in plant 2 during the summer . The only chemical contaminant measured at average concentrations greater than 50% of the Threshold Limit Values (TLVs) proposed by the American Conference of Governmental Industrial Hygienists (ACGIH) was CO in the sorting departments in plants 1 and 2 during the winter . Noise exceeded the ACGIH TLV in plant 2 . The workers' physical symptoms seem to be caused by the posture and effort required while remaining in a stationary position . Action must be focused on improving aspects such as work organization and personal protection and on informing citizens of the need for cleanliness of the material to be recycled.

Cell Microbiol, 2001 Mar, 3(3), 169 - 80
Formation of stress fibres in human endothelial cells infected with Bartonella bacilliformis is associated with altered morphology, impaired migration and defects in cell morphogenesis; Verma A et al.; Bartonella bacilliformis, a Gram-negative, flagellated bacterium, infects human erythrocytes (haematic phase) and endothelial cells (tissue phase), resulting in a biphasic disease . In the tissue phase of disease (verruga peruana), infection leads to infection of endothelial cells and a pronounced proliferation of these cells, resulting in characteristic skin eruptions of papules and nodules . We have studied the properties of endothelial cells infected in vitro . Extensive cytoskeletal remodelling of endothelial cells occurred after infection in vitro with B . bacilliformis . The cells became spindle shaped and contained arrays of actin stress fibres orientated parallel to the long axis of the cell . Cell-cell contacts were disrupted, along with the distribution of the plasma membrane marker protein, PECAM-1, which participates in cell-cell junctions . The prominent stress fibres terminated in an increased number of focal contacts, which were studied using immunofluorescent staining for paxillin, a cytoplasmic protein that localizes in the focal adhesions . These morphological changes are consistent with activation of intracellular Rho by B . bacilliformis . Formation of stress fibres and the increased number of focal adhesions could be prevented by preincubation of the endothelial cells with C3 exoenzyme, which inactivates intracellular Rho by ADP ribosylation . Endothelial cell motility was greatly diminished in infected cells and the cells did not respond effectively to a stimulus that would evoke motility . In addition, infection of endothelial cells interfered with their ability to form networks of capillary tubes when suspended within three-dimensional collagen matrices . If the properties of infected endothelial cells in vivo are similar, the infected cells will probably not participate effectively in angiogenesis.

Ann Surg Oncol, 2001 Mar, 8(2), 179 - 86
Treatment of nosocomial postoperative pneumonia in cancer patients: a prospective randomized study; Raad I et al.; BACKGROUND: Nosocomial pneumonia continues to be associated with high morbidity and mortality in cancer patients . METHODS: In an attempt to find an optimal treatment for this infection, nonneutropenic cancer patients with postoperative nosocomial pneumonia were randomized to receive either piperacillin/tazobactam (P/T) 4.5 g i.v . every 6 hours (30 patients) or clindamycin (Cl) 900 mg plus aztreonam (Az) 2 g i.v . every 8 hours (22 patients) . Amikacin 500 mg i.v . every 12 hours was given to all patients for the first 48 hours . RESULTS: The two groups were comparable for the characteristics of pneumonia that included gram-negative etiology and duration of intubation . Response rates were 83% for patients who received P/T and 86% for those who received Cl/Az (P > .99) . There were no serious adverse events; however, at our center the cost of the P/T regimen was $73.86 compared with $99.15 for the Cl/Az regimen . CONCLUSIONS: The two regimens had comparable high efficacy, and P/T had a slight cost advantage . Either of these antibiotic regimens combined with an aminoglycoside could lead to favorable outcome in cancer patients at high risk for nosocomial pneumonia.

Ther Apher, 2001 Feb, 5(1), 25 - 30
The severity of hyperdynamic circulation may predict the effects of direct hemoperfusion with the adsorbent column using polymyxin B-immobilized fiber in patients with gram-negative septic shock; Uriu K et al.; It has been reported that direct hemoperfusion with the adsorbent column using polymyxin B-immobilized fiber (DHP with PMX-F column) ameliorates hyperdynamic circulation in septic shock and improves survival rate . However, the clinical characteristics of patients with an improvement of septic shock after DHP with PMX-F column have not been evaluated . To clarify this issue, the clinical profiles of 46 patients who were suggested to have gram-negative septic shock and treated using DHP with PMX-F column were analyzed retrospectively . Of 46 patients, 31 were diagnosed with gram-negative septic shock (G group) . Mean arterial pressure (MAP) just before DHP with PMX-F column was not different between the G and the non-G group . As compared with the non-G group, the G group had a higher cardiac index (CI) and a lower systemic vascular resistance (SVR) . Significant increases in MAP and SVR with a significant decrease in CI were observed after DHP with PMX-F column in the G group . In the non-G group, MAP was significantly increased after the DHP therapy, but systemic hemodynamics were unchanged . Patients in the G group who fulfilled the following criteria were considered as the effective group: MAP was elevated more than 10 mm Hg or 125% of the basal MAP and/or the dose of vasopressors was reduced after DHP with PMX-F column . Twenty-one patients (67.8%) were in the effective group . In comparison with the effective group, the noneffective group was characterized by a significant increase in CI before DHP with PMX-F column . All patients with a CI less than 6 L/min/m2 were in the effective group . These data suggest that DHP with PMX-F column was useful for patients with gram-negative septic shock who did not have severe hyperdynamic circulation.

Cell Signal, 2001 Feb, 13(2), 85 - 94
LPS induction of gene expression in human monocytes; Guha M et al.; Lipopolysaccharide (LPS {endotoxin}) is the principal component of the outer membrane of Gram-negative bacteria . Recent studies have elucidated how LPS is recognized by monocytes and macrophages of the innate immune system . Human monocytes are exquisitely sensitive to LPS and respond by expressing many inflammatory cytokines . LPS binds to LPS-binding protein (LBP) in plasma and is delivered to the cell surface receptor CD14 . Next, LPS is transferred to the transmembrane signaling receptor toll-like receptor 4 (TLR4) and its accessory protein MD2 . LPS stimulation of human monocytes activates several intracellular signaling pathways that include the IkappaB kinase (IKK)-NF-kappaB pathway and three mitogen-activated protein kinase (MAPK) pathways: extracellular signal-regulated kinases (ERK) 1 and 2, c-Jun N-terminal kinase (JNK) and p38 . These signaling pathways in turn activate a variety of transcription factors that include NF-kappaB (p50/p65) and AP-1 (c-Fos/c-Jun), which coordinate the induction of many genes encoding inflammatory mediators.

Cell, 2001 Mar 9, 104(5), 709 - 18
Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae; Levashina EA et al.; We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae . It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin . Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line . Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond . This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.

J Environ Monit, 2000 Feb, 2(1), 73 - 6
Organic dust and gaseous contaminants at wood working shops; Abdel Hameed AA et al.; Airborne dust bioaerosols, ammonia and formaldehyde levels were determined inside two different (ventilated and unventilated) wood working shops . Airborne dust was found at mean values of 4.3 and 3.01 mg m(-3) . These levels were higher than that recommended by Egyptian environmental law {1 mg m(-3) indoor maximum allowable concentration (MAC) for hard wood} . The highest frequency of aerodynamic size distribution of airborne wood dust was detected at a diametre of 4.9 microm which was recorded during a machining operation . Total viable bacteria were recorded at a mean value of 10(4) colony-forming units (cfu) m(-3), whereas Gram-negative bacteria were found at very low counts (10(1) cfu m(-3)) . Fungi levels were recorded at mean values of 10(3) and 10(2) cfu m(-3) in ventilated and unventilated shops, respectively . Penicillium, Aspergillus, Cladosporium and yeast species were dominant isolates . Moreover, actinomycetes were found at a mean value of 10(3) cfu m(-3) at both workshops . Ammonia was detected in relatively low concentrations (mean values of 457 and 623 microg m(-3)), whereas formaldehyde was found in relatively moderate concentrations (mean values of 0.42 and 0.64 ppm).

Infect Immun, 2001 Apr, 69(4), 2621 - 9
Gamma interferon prevents the inhibitory effects of oxidative stress on host responses to Escherichia coli infection; Parmely MJ et al.; Oxidative stress occurs in animals challenged with bacterial endotoxin and can affect the expression of important host inflammatory genes . However, much less is known about the effects of oxidative stress on responses to gram-negative bacteria . The current study compared the effects of redox imbalance on hepatic responses of mice to Escherichia coli bacteria versus purified endotoxic lipopolysaccharide (LPS) . Oxidative stress induced by glutathione depletion virtually eliminated hepatic tumor necrosis factor alpha responses to both E . coli and LPS . Inducible NO synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) expression was also markedly inhibited by glutathione depletion in LPS-challenged mice, but was unaffected in E . coli-infected animals . Three findings suggested that gamma interferon (IFN-gamma) production explained the differences between LPS and bacterial challenge . Glutathione depletion completely inhibited the IFN-gamma response to LPS, but only partially inhibited IFN-gamma production in infected mice . Exogenous IFN-gamma restored iNOS and ICAM-1 responses to LPS in stressed mice . Conversely, IFN-gamma-deficient, glutathione-depleted mice showed a marked decrease in iNOS and ICAM-1 expression when challenged with E . coli . These findings indicate that both the nature of the microbial challenge and the production of IFN-gamma can be important in determining the effects of redox imbalance during gram-negative bacterial infections.

Minerva Cardioangiol, 2000 Dec, 48(12), 467 - 73
{Ischemic cardiovascular diseases . Correlation with Helicobacter pylori infection}; Pellicano R et al.; Coronary heart disease is the primary cause of mortality in western countries . The well-established ("classical") risk factors cannot fully explain epidemiological variations of this disease . From several years infections have been linked to ischemic vascular events and recent studies pointed to the role of Helicobacter pylori (H . pylori), a spiral Gram negative bacterium, that chronically infects human stomach and is involved in the pathogenesis of gastritis and peptic ulceration . Systematic reviews of studies have suggested the existence of a possible weakly positive association between this bacterium and coronary heart disease, but this could be due to confounding bias and influenced by the degree of investigations heterogeneity . Experiments from animal studies demonstrated that H . pylori infection in mice induces the formation of platelet aggregates and in contrast to Chlamydia pneumoniae it has not been found in the plaque: therefore, the role of H . pylori, could be even more important in the acute phase of myocardial infarction . There is the need for extensive prospective studies to evaluate the incidence of these diseases in relation to the presence of H . pylori infection . Appropriately randomized studies employing an antibiotic treatment for patients affected by ischemic vascular disease will answer the question of whether H . pylori has a causal role in the pathogenesis of acute myocardial infarction and ischemic stroke.

Syst Appl Microbiol, 2000 Dec, 23(4), 487 - 93
Hydrogenophaga intermedia sp . nov., a 4-aminobenzenesulfonate degrading organism; Contzen M et al.; The taxonomic status of a gram-negative, oxidase positive rod (strain S1) able to degrade 4-aminobenzenesulfonate was studied using a polyphasic approach . Chemotaxonomic investigations of quinones and polar lipids established the allocation of this strain to the beta-subclass of the Proteobacteria and revealed similarities to Hydrogenophaga palleronii . 16S rRNA sequence comparisons demonstrated that this strain clusters phylogenetically with H . palleronii and H . taeniospiralis, but clearly represents a new species . The fatty acid patterns and substrate utilization profile displayed similarity to the characteristics of the four validly published species of Hydrogenophaga, although clear differentiating characters were also observed . No close similarities between the type strains of H . palleronii and H . taeniospiralis were detected in hybridization experiments with the genomic DNAs . On basis of these results, the new species Hydrogenophaga intermedia sp . nov . is proposed, with the type strain S1T (= DSM 5680).

Annu Rev Immunol, 2001, 19, 523 - 63
The design of vaccines against Helicobacter pylori and their development; Del Giudice G et al.; Helicobacter pylori is a gram negative, spiral, microaerophylic bacterium that infects the stomach of more than 50% of the human population worldwide . It is mostly acquired during childhood and, if not treated, persists chronically, causing chronic gastritis, peptic ulcer disease, and in some individuals, gastric adenocarcinoma and gastric B cell lymphoma . The current therapy, based on the use of a proton-pump inhibitor and antibiotics, is efficacious but faces problems such as patient compliance, antibiotic resistance, and possible recurrence of infection . The development of an efficacious vaccine against H . pylori would thus offer several advantages . Various approaches have been followed in the development of vaccines against H . pylori, most of which have been based on the use of selected antigens known to be involved in the pathogenesis of the infection, such as urease, the vacuolating cytotoxin (VacA), the cytotoxin-associated antigen (CagA), the neutrophil-activating protein (NAP), and others, and intended to confer protection prophylactically and/or therapeutically in animal models of infection . However, very little is known of the natural history of H . pylori infection and of the kinetics of the induced immune responses . Several lines of evidence suggest that H . pylori infection is accompanied by a pronounced Th1-type CD4(+) T cell response . It appears, however, that after immunization, the antigen-specific response is predominantly polarized toward a Th2-type response, with production of cytokines that can inhibit the activation of Th1 cells and of macrophages, and the production of proinflammatory cytokines . The exact effector mechanisms of protection induced after immunization are still poorly understood . The next couple of years will be crucial for the development of vaccines against H . pylori . Several trials are foreseen in humans, and expectations are that most of the questions being asked now on the host-microbe interactions will be answered.

Oral Microbiol Immunol, 2001 Apr, 16(2), 65 - 72
Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell-surface preparations of Porphyromonas gingivalis; Imatani T et al.; Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease . Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases . We therefore investigated the productions of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer-membrane proteins from P . gingivalis ATCC 53977 . Outer-membrane protein from P . gingivalis enhanced the production of IL-6 and IL-8 from the cells of periodontium in vitro as well as lipopolysaccharide did . The IL-8 production activity of polysaccharide from P . gingivalis was higher than that of other cell-surface components . The levels of IL-6 and IL-8 released from the P . gingivalis lipopolysaccharide-treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli . Rabbit antisera against either outer-membrane protein or lipopolysaccharide inhibited the IL-6 and IL-8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P . gingivalis . The present study suggests that, in addition to lipopolysaccharide, outer-membrane protein and polysaccharide of P . gingivalis are also pathological mediators in periodontal diseases.

Am J Pathol, 2001 Mar, 158(3), 1101 - 9
Monocyte/macrophage activation by normal bacteria and bacterial products: implications for altered epithelial function in Crohn's disease; Zareie M et al.; Intestinal immune cells are less reactive than those in the peripheral blood; however, such cells from patients with Crohn's disease may be more responsive to bacterial products . Our study examined if nonpathogenic bacteria or lipopolysaccharide (LPS), can affect epithelial function in the presence of monocytes/macrophages . Lamina propria mononuclear cells (LPMCs) and peripheral blood monocytes (PBMs) were obtained from patients with Crohn's disease and control patients . Filter-grown T84 epithelial monolayers were co-cultured with nonactivated or LPS-activated LPMCs or PBMs for 48 hours . Epithelial secretory {baseline short-circuit current (Isc) and DeltaIsc to forskolin} and barrier (transepithelial electrical resistance) parameters were measured in Ussing chambers . LPS-activated PBMs from both controls and patients with Crohn's disease significantly increased Isc ( approximately 300%) and reduced transepithelial electrical resistance ( approximately 40%) . Epithelial function was not altered after co-culture with control LPMCs +/- LPS . However, LPMCs from patients with Crohn's disease spontaneously secreted tumor necrosis factor-alpha, and induced epithelial changes similar to those produced by LPS-activated PBMs . Co-culture with control Escherichia coli and PBMs induced comparable changes in epithelial physiology, which were abrogated by anti-tumor necrosis factor-alpha antibody . We conclude that LPMCs of patients with Crohn's disease are spontaneously activated, possibly by gram-negative luminal bacteria, and can directly cause significant alterations in epithelial ion transport and barrier functions.

Rev Med Suisse Romande, 2001 Jan, 121(1), 47 - 50
{Endocarditis due to HACEK bacteria . A case report of endocarditis due to Kingella kingae}; Lepori M et al.; Endocarditis is a common disease in hospital practice . Identification of the microorganism responsible for the valvular damage is essential to establish the prognosis and to determine the optimal antibiotic treatment . In some cases of endocarditis the diagnosis is laborious, especially when the responsible microorganism is difficult to detect using standard culture techniques . Here we report a case of native aortic valve endocarditis due to Kingella kingae, a Gram negative organism of the HACEK group . In addition we review 6 other cases of endocarditis caused by organism belonging to this group, treated in our hospital between 1983 and 1999 . Epidemiological studies show that less than 5% of all cases of endocarditis are caused by organisms of the HACEK group . The diagnosis is often delayed because their slow growth on a standard culture medium . We describe clinical and microbiological characteristics of this group of endocarditis.

Clin Infect Dis, 2001 Mar 1, 32(5), 832 - 5 Epub 2001 Feb 23.
Prediction of gram-negative bacteremia in patients with cancer and febrile neutropenia by means of interleukin-8 levels in serum: targeting empirical monotherapy versus combination therapy; Kern WV et al.; In a prospective observational study of 133 neutropenic episodes, interleukin (IL)-8 serum levels > 2000 pg/mL at the onset of fever had a sensitivity of 53% and a specificity of 97% as a predictor of gram-negative bacteremia (GNB; positive predictive value, 73%; negative predictive value, 94%) . The rates of early death differed significantly between patients with high and those with low IL-8 levels (3/11 vs . 1/122; P< .01) . Serum IL-8 levels at the onset of fever define a low-risk subgroup of patients who can safely be treated with monotherapy.

Am J Ophthalmol, 2001 Feb, 131(2), 280 - 1
Orbital cellulitis caused by Fusobacterium necrophorum; Escardo JA et al.; PURPOSE: To report the case of a previously healthy young female who developed orbital cellulitis caused by Fusobacterium necrophorum . We are unaware of previous reports of this condition caused by the same anaerobic, gram-negative, nonsporeforming bacterium and could find no reference to it in a computer search using MEDLINE . METHODS: Case report . RESULTS: In the case of orbital cellulitis presented here, the patient required 3 sinus debridement operations and 30 days of intravenous antibiotics . Despite this, her vision did not fully recover . CONCLUSION: F . necrophorum is capable of causing severe orbital disease that requires aggressive and prompt treatment to preserve sight.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2740 - 5 Epub 2001 Feb 13.
Comparative genomics of the restriction-modification systems in Helicobacter pylori; Lin LF et al.; Helicobacter pylori is a Gram-negative bacterial pathogen with a small genome of 1.64-1.67 Mb . More than 20 putative DNA restriction-modification (R-M) systems, comprising more than 4% of the total genome, have been identified in the two completely sequenced H . pylori strains, 26695 and J99, based on sequence similarities . In this study, we have investigated the biochemical activities of 14 Type II R-M systems in H . pylori 26695 . Less than 30% of the Type II R-M systems in 26695 are fully functional, similar to the results obtained from strain J99 . Although nearly 90% of the R-M genes are shared by the two H . pylori strains, different sets of these R-M genes are functionally active in each strain . Interestingly, all strain-specific R-M genes are active, whereas most shared genes are inactive . This agrees with the notion that strain-specific genes have been acquired more recently through horizontal transfer from other bacteria and selected for function . Thus, they are less likely to be impaired by random mutations . Our results also show that H . pylori has extremely diversified R-M systems in different strains, and that the diversity may be maintained by constantly acquiring new R-M systems and by inactivating and deleting the old ones.

Semin Respir Infect, 2000 Dec, 15(4), 272 - 9
Ventilator-associated pneumonia: incidence, risk factors, and microbiology; Bauer TT et al.; Ventilator-associated pneumonia (VAP) is a pulmonary infection that occurs after at least 48 hours of mechanical ventilation (MV) . The incidence depends on several factors, although the most important are those related to the host and duration of MV . VAP can be differentiated into early-onset (<5 days) and late-onset types (> or =5 days) . The overall incidence of VAP varies between 9% and 70% (average, 20% to 25%), and the majority of episodes occur within the first 5 days . Risk factors for VAP include prolonged MV, older age, supine body position, and type of comorbidity . Oropharyngeal colonization appears to be a risk factor for early-onset pneumonia, whereas prolonged MV and antibiotic pretreatment, especially with broad-spectrum drugs, increase the risk for late-onset VAP Microaspiration of colonized oropharyngeal secretions is a major cause of early-onset VAP, most frequently caused by community-type pathogens . After 5 days of MV, pathological colonization with gram-negative bacteria may occur, and late-onset VAP is more likely to be attributable to this group of microorganism . Incidence, risk factors, and microbiology depend strongly on the time frame in which the episode develops . However, initial and pathological colonization during the intensive care unit stay can modify this concept.

Med Arh, 2000, 54(5-6), 313 - 6
{Helicobacter pylori and gastroduodenal disease in our patients: 2-year experience}; Brigic E et al.; Primary gastritis, duodenitis, peptic ulcer of stomach and duodenum are no longer considered to be disorders of the balance of secretion of acid and immune responses of the gastric mucose but it is thought to be caused by Helicobacter pylori infection . One of the most important factor causign the malignant gastric diseases such as carcinoma and non-Hodgkins MALT Lymphoma is an early infection by this spiral gram-negative bacterium . We performed byopsi of the gastric mucose in 80 of our patients . Endoscopic and histological results showed that 74 patients had gastritis 6 others had normal results . The highest incidence of disease was found in children of 11 to 14 years of age because 67.4% were girls and 22.4% were boys of this age . Ethiological analysis of patients with gastritis showed that Helicobacter pylori was isolated in bioptic material of 46 patients while results of other 28 patients were negative.

Pediatr Infect Dis J, 2001 Feb, 20(2), 171 - 7
Does the empiric use of vancomycin in pediatrics increase the risk for Gram-negative bacteremia?
Van Houten MA, Uiterwaal CS, Heesen GJ, Arends JP, Kimpen JL.
BACKGROUND: Gram-negative bacteremia in children, a major cause of morbidity and mortality, may in part be induced by intensive treatment procedures and nonspecific use of antibiotics . Our primary objective was to study the causal relationship between the use of vancomycin and Gram-negative bacteremia, for which this antibiotic is not specifically indicated . METHODS: The study was conducted in a 105-bed tertiary care children's hospital in the period of 1994 to 1997 . The study pertains to a cohort of children with suspected bacteremia, in whom a blood culture was performed during hospital stay . Using the bacteriologic laboratory registration system, we selected all pediatric cases with bacteriologically proved Gram-negative bacteremia (n = 105) and a random sample of 225 pediatric controls with negative blood cultures . Using logistic regression analysis we examined associations between Gram-negative bacteremia and the following factors: preceding use of antibiotics, antacids, corticosteroids, surgery, mechanical ventilation, parenteral nutrition, and invasive instrumentation; and the intensity of care assessed with the Therapeutic Intensity Scoring System (TISS 28) . RESULTS: Gram-negative bacteremia was positively associated with the use of aminoglycosides, cephalosporins, surgical interventions, central venous catheters, parenteral nutrition, antacids and dexamethasone . The strongest association was with the use of vancomycin (odds ratio, 8.1; 95% confidence interval, 3.1 to 20.9) . In a multiple logistic regression model containing all above-mentioned variables, the use of vancomycin remained positively and strongly associated with Gram-negative bacteremia (odds ratio, 3.88; 95% confidence interval, 1.34 to 11.21) . Further adjustments and restrictions in the analysis did not materially change these findings concerning vancomycin . CONCLUSIONS: Among children suspected of bacteremia there are several drugs and clinical procedures influencing the risk for Gram-negative bacteremia . Empiric use of vancomycin is strongly and independently associated with Gram-negative bacteremia . The safety of using vancomycin solely on the basis of suspicion of bacteremia in children may not be warranted.

Carbohydr Res, 2001 Jan 30, 330(2), 271 - 7
Structural determination of the phytotoxic mannan exopolysaccharide from Pseudomonas syringae pv . ciccaronei; Corsaro MM et al.; The structural determination was performed of a mannan exopolysaccharide from the gram negative bacterium Pseudomonas syringae pv . ciccaronei, which is the pathogenic agent responsible for the leaf spots of carob plants . The structure, obtained by chemical, enzymatic and spectroscopic methods, consisted of a backbone of alpha-(1-->6)-linked mannopyranose units with 80% substituted at C-2 by mono-, di- and trisaccharide side chains . In addition, terminal glucose units and phosphate groups were found to be present . This is, to the best of our knowledge, the first report of a mannan exopolysaccharide structure from a phytopathogenic bacterium . The pure polysaccharide showed phytotoxic effects, i.e., chlorosis and necrosis on tobacco leaves.

J Clin Pathol, 2001 Feb, 54(2), 107 - 10
Diagnosis of gram negative, ventilator associated pneumonia by assaying endotoxin in bronchial lavage fluid; Flanagan PG et al.; AIM: To investigate the usefulness of assaying endotoxin in non-directed bronchial lavage fluid (NBL), bronchoscopic bronchoalveolar lavage fluid (BAL), and sera as a means of diagnosing Gram negative, ventilator associated pneumonia . METHODS: Samples from 64 patients were investigated . Fifty nine BALs and 92 NBLs were assayed in total including specimens taken during 28 episodes of clinical ventilator associated pneumonia (VAP) . RESULTS: The concentration of endotoxin in BAL from patients with VAP developing within four days of commencing ventilation was significantly higher than in those without VAP (p = 0.015) . There was no significant difference in endotoxin concentration in NBL or serum when comparing patients with and without VAP . A BAL endotoxin concentration of 6 EU/ml yielded the optimal operating characteristics (sensitivity, 81%; specificity, 87%; positive predictive value, 67%; negative predictive value, 95%) . However, Gram stain of BAL provided the same information as quickly as the endotoxin assay and is considerably cheaper . CONCLUSIONS: Despite its accuracy and rapidity, the BAL endotoxin assay must be shown to alter clinical management and patient outcome to be cost effective.

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 73 - 9
Sphingomonas alaskensis sp . nov., a dominant bacterium from a marine oligotrophic environment; Vancanneyt M et al.; Seven Gram-negative strains, isolated in 1990 from a 10(6)-fold dilution series of seawater from Resurrection Bay, a deep fjord of the Gulf of Alaska, were identified in a polyphasic taxonomic study . Analysis of 16S rDNA sequences and DNA-homology studies confirmed the phylogenetic position of all strains in the genus Sphingomonas and further indicated that all of the strains constitute a single homogeneous genomic species, distinct from all validly described Sphingomonas species . The ability to differentiate the species, both phenotypically and chemotaxonomically, from its nearest neighbours justifies the proposal of a new species name, Sphingomonas alaskensis sp . nov., for this taxon . Strain LMG 18877T (= RB2256T = DSM 13593T) was selected as the type strain.

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 17 - 26
Azospirillum doebereinerae sp . nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus; Eckert B et al.; A new group of nitrogen-fixing Azospirillum sp . bacteria was isolated from the roots of the C4-gramineous plant Miscanthus . Polyphasic taxonomy was performed, including auxanography using API galleries, physiological tests and 16S rRNA sequence comparison . The ability of the isolates to fix dinitrogen was evaluated by amplification of the nifD gene, immunodetection of the dinitrogenase reductase and acetylene-reduction assay . On the basis of these results, the nitrogen-fixing isolates represent a new species within the genus Azospirillum . Its closest phylogenetic neighbours, as deduced by 16S rDNA-based analysis, are Azospirillum lipoferum, Azospirillum largimobile and Azospirillum brasilense with 96.6, 96.6 and 95.9% sequence similarity, respectively . Two 16S rRNA-targeting oligonucleotide probes were developed which differentiate the new species from the other Azospirillum species by whole-cell fluorescence hybridization . Strains of the new species are curved rods or S-shaped, 1.0-1.5 microm in width and 2,0-3.0 microm in length, Gram-negative and motile with a single polar flagellum . Optimum growth occurs at 30 degrees C and at pH values between 6.0 and 7.0 . No growth takes place at 37 degrees C . They have a respiratory type of metabolism, grow well on arabinose, D-fructose, gluconate, glucose, glycerol, malate, mannitol and sorbitol . They differ from A . largimobile and A . lipoferum by their inability to use N-acetylglucosamine and D-ribose, from A . lipoferum by their ability to grow without biotin supplementation and from A . brasilense by their growth with D-mannitol and D-sorbitol as sole carbon sources . Nitrogen fixation occurs in microaerobic nitrogen-limited conditions . For this species, the name Azospirillum doebereinerae sp . nov . is suggested, with strain GSF71T as the type strain (= DSM 13131T; reference strain Ma4 = DSM 13400) . Its G+C content is 70.7 mol%.

Presse Med . 2001 Jan 13;30(1):28.
{40th ICAAC . Catheter-related septicemia}; el Hajj L; A GROWING PROBLEM: Insertion or use of intravascular material is increasingly associated with septicemia . Septicemia occurs in 1 out of 1000 patients undergoing a non-surgical invasive cardiac procedure . The frequency of septicemia, particularly due to Gram-negative bacteria, is increasingly frequent in stem-cell graft recipients . PREVENTION MEASURES: Educational programs, for both patients and health givers, emphasizing the importance of aseptic can reduce the incidence of Gram-negative bacteria catheter-related septicemias.

Mol Plant Microbe Interact, 2001 Feb, 14(2), 145 - 55
Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system; Guttman DS et al.; Gram-negative phytopathogenic bacteria require a type III secretion apparatus for pathogenesis, presumably to deliver Avr effector proteins directly into plant cells . To extend previous studies of Avr effectors that employed plasmids encoding Avr proteins, we developed a system that permits the integration of any gene into the Pseudomonas syringae genome in single copy . With this system, we confirmed earlier findings showing that P . syringae pv . maculicola strain PsmES4326 expressing the AvrRpt2 effector induces a resistance response in plants with the cognate R gene, RPS2 . Chromosomally located avrRpt2, however, provoked a stronger resistance response than that observed with plasmid-expressed AvrRpt2 in RPS2+ plants . Additionally, chromosomal expression of AvrRpt2 conferred a fitness advantage on P . syringae grown in rps2- plants, aiding in growth within leaves and escape to leaf surfaces that was difficult to detect with plasmid-borne avrRpt2 . Finally, with the use of the genomic integration system, we found that a chimeric protein composed of the N terminus of the heterologous AvrRpml effector and the C-terminal effector region of AvrRpt2 was delivered to plant cells . Because the C terminus of AvrRpt2 cannot translocate into plant cells on its own, this indicates that the N-terminal region can direct secretion and translocation during an infection, which supports the view that Avr proteins have a modular design . This work establishes a readily manipulatable system to study type III effectors in a biologically realistic context.

Int J Med Microbiol, 2000 Dec, 290(7), 579 - 85
Outer membrane proteins as surface display systems; Lang H; Outer membrane proteins (OMPs) of gram-negative bacteria can be used as carrier proteins to present foreign peptide epitopes on the bacterial cell surface . They all have a common structural motif of a beta-barrel that is composed of a variable number of transmembrane beta-strands connected at the periplasmic side with short turns and at the outside with long surface-accessible loops . Outer membrane proteins occur as monomers like OmpA, or assemble into trimers like the porins . Foreign gene products have been fused to surface-accessible regions of several outer membrane proteins including the porins OmpC, PhoE and LamB, lipoproteins as well as the OmpA protein . Short epitopes that are inserted into outer membrane proteins induce epitope-specific antibody responses, and are thus appealing candidates for live recombinant vaccines . Also large insertions, of more than 100 amino acids, are in some cases tolerated and do not affect the overall conformation of the carrier protein . The possible applications for outer membrane display include recombinant vaccines, peptide library screening, development of biocatalysts or whole-cell adsorbents, and adhesin-receptor interaction studies . It is expected that in the near future, development of new display systems will still increase the utilization of this emerging exciting technology.

Arch Microbiol, 2000 Dec, 174(6), 399 - 405
The Myxococcus xanthus wbgB gene encodes a glycosyltransferase homologue required for lipopolysaccharide O-antigen biosynthesis; Yang Z et al.; Myxococcus xanthus is a gram-negative soil bacterium that initiates a complex developmental program in response to starvation . A transposon insertion (Tn5-lac omega109) mutant with developmental deficiencies was isolated and characterized in this study . A strain containing this insertion mutation in an otherwise wild-type background showed delayed developmental aggregation for about 12 h and sporulated at 1-2% of the wild-type level . Tn5-lac omega109 was found to have disrupted the M . xanthus wbgB gene, which is located 2.1 kb downstream of the M . xanthus lipopolysacharide (LPS) O-antigen biosynthesis genes wzm wzt wbgA . The deduced polypeptide sequence of WbgB shares significant similarity with bacterial glycosyltransferases including M . xanthus WbgA . The wbgB::Tn5-lac omega109 mutant was found to be defective in LPS O-antigen synthesis by immunochemical analysis . Further mutational analysis indicated that the defects of the wbgB::Tn5-lac omega109 mutant were not the result of polar effects on downstream genes . Various motility assays demonstrated that the Tn5-lac omega109 mutation affected both social (S) and adventurous (A) gliding motility of M . xanthus cells . The pleiotrophic effects of wbgB mutations indicate the importance of LPS O-antigen biosynthesis for various cellular functions in M . xanthus.

Equine Vet J, 2001 Jan, 33(1), 105 - 9
Acute thrombosis of limb arteries in horses with sepsis: five cases (1988-1998); Brianceau P et al.; A hypercoagulable condition and poor perfusion to distal extremities might occur during equine endotoxaemic or septic shock, which could cause thrombosis of limb arteries . In our review, thrombosis occurred in neonatal foals in association with gram-negative bacteraemia . In 3 older foals and adults, thrombosis was associated with inflammatory bowel disease, diarrhoea and toxaemia . All patients had been treated with broad-spectrum antibiotics, nonsteroidal antiinflammatory drugs and i.v . crystalloid solutions . Two horses received i.v . hyperimmune plasma . A generalised coagulopathy was not suspected prior to clinical signs of distal limb necrosis, although thrombocytopenia occurred in 4 of the 5 cases at the time of, or shortly before, thrombosis . Thrombocytopenia, possibly due to platelets adherence to exposed subendothelial collagen, which induces contact activation of the intrinsic coagulation pathway, has been described in endotoxaemic horses and foals with gastrointestinal infectious or inflammatory diseases and disseminated intravascular coagulation . Activation of procoagulants by endotoxins, decreased blood flow to the limbs and endothelial damage, may have been responsible for a hypercoagulable condition leading to thrombosis in these 5 cases . The 3 enterocolitis patients may have had increased risk of thrombosis because of loss of antithrombin III, haemoconcentration and acidosis.

Environ Microbiol, 1999 Aug, 1(4), 357 - 67
Novel bacterial endosymbionts of Acanthamoeba spp . related to the Paramecium caudatum symbiont Caedibacter caryophilus; Horn M et al.; Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized . In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach . Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp . UWC9 and Acanthamoeba sp . UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans and Holospora obtusa . With overall 16S rRNA sequence similarities to their closest relative, C . caryophilus, of between 87% and 93%, these endosymbionts represent three distinct new species . In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization . We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as 'Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp . strain UWC9 as 'Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp . strain UWE39 as 'Candidatus Paracaedibacter symbiosus' . The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA . Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp . UWC9 and UWE39 exhibit <92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates . Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp . UWC9 and Acanthamoeba sp . UWE39.

Cell Microbiol, 1999 Sep, 1(2), 93 - 9
Molecular response of gastric epithelial cells to Helicobacter pylori-induced cell damage; Zarrilli R et al.; Infection with the Gram-negative bacterium Helicobacter pylori leads to different clinical and pathological outcomes in humans, including chronic gastritis, peptic ulcer disease and adenocarcinoma of the stomach . H . pylori-induced damage to gastric mucosal cells is controlled by bacterial virulence factors encoded by genes of the cag pathogenicity island, which trigger the inflammatory response of the host through the activation of nuclear factor kappaB-dependent gene transcription . Also, H . pylori infection impairs the processes of gastric mucosal healing through inhibition of epidermal growth factor receptor-dependent signal transduction pathways and induction of apoptosis . H . pylori infection may influence the progression from chronic gastritis to gastric adenocarcinoma by stimulating cell proliferation and growth factor expression, inhibiting apoptosis and increasing the DNA mutation rate of infected gastric mucosa.

Vet Immunol Immunopathol, 2001 Jan 10, 78(1), 45 - 56
The plasmid pcDNA3 differentially induces production of interferon-alpha and interleukin-6 in cultures of porcine leukocytes; Magnusson M et al.; An adjuvant effect of invertebrate DNA has been attributed to its relative high frequency of unmethylated CpG dinucleotides . Here we describe the interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) inducing properties of a commonly used eukaryotic expression vector, pcDNA3, in porcine leukocytes . The magnitude of the cytokine response was compared to that induced by the synthetic ds RNA analogue poly(I):poly(C), inactivated preparations of Aujeszky's disease virus (ADV) and the Gram-negative bacteria Actinobacillus pleuropneumoniae . The plasmid, as well as poly(I):poly(C), required lipofectin to induce IFN-alpha production whereas both preparations induced IL-6 irrespective of preincubation with lipofectin . However, the nucleic acid-induced levels of IL-6 were low compared to those induced by A . pleuropneumoniae . The IFN-alpha response elicited by pcDNA3 in the presence of lipofectin was as high as, or higher than that induced by ADV . Interestingly, also A . pleuropneumoniae induced a substantial production of IFN-alpha when preincubated with lipofectin . Plasmid expression was not necessary for induction of IFN-alpha . Furthermore, the IFN-alpha inducing capacity of pcDNA3 was not reduced when the two predicted immunostimulatory sequences 5'AACGTT3' were deleted . Nor did the ability of the plasmid to induce IFN-alpha production decrease when the ampicillin resistance (ampR) gene was replaced with the kanamycin resistance (kanR) gene . However, methylation of all cytidines in CpG dinucleotides of pcDNA3 abolished the IFN-alpha inducing capacity . These in vitro results indicate an immunomodulatory role of bacterial DNA also in the pig . Unmethylated CpG dinucleotides are crucial for induction of IFN-alpha by the plasmid, but other CpG motifs than those within the 5'AACGTT3' sequences of the ampR gene contribute to this induction in porcine cells.

Infect Immun, 2001 Mar, 69(3), 1273 - 9
Bacterial lipopolysaccharide and tumor necrosis factor alpha synergistically increase expression of human endothelial adhesion molecules through activation of NF-kappaB and p38 mitogen-activated protein kinase signaling pathways; Jersmann HP et al.; One of the recognized associations of bacterial infection with cardiovascular events is the activation of endothelium and upregulation of adhesion molecules . The two major proinflammatory mediators implicated in the causation of cardiovascular events, bacterial lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF), were found to cooperate to enhance the adhesive properties of endothelial cells . These caused synergistic upregulation of intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 in human umbilical vein endothelial cells as determined by flow cytometry analysis and enzyme-linked immunosorbent assay . This synergism was not due to TNF causing an upregulation of CD14 expression . Treatment with both LPS and TNF resulted in a marked increase in the translocation of NF-kappaB into the nucleus . The activity of p38 mitogen-activated protein kinase was also synergistically enhanced, while the activity of c-jun N-terminal kinase was increased in an additive manner . The results demonstrate that LPS and TNF act synergistically to upregulate the expression of endothelial cell adhesion molecules, possibly by amplification of signaling pathways upstream of transcription . These findings have implications for the understanding of the acceleration of atherosclerotic events seen in low-grade infections with gram-negative organisms.

ALTEX, 1998, 15(5), 68 - 71
{Cytokine release after administration of endotoxin containing vaccines}; Ecker M et al.; Endotoxins from gram negative bacteria are known to be potent inducers for the synthesis and the release of cytokines such as tumour necrosis factor (TNF) and interleukin 6 (IL-6) . The amount of these proinflammatory mediators in plasma from animals and human patients suffering of an acute infection or sepsis, however, is well correlated with the outcome and the prognosis of such diseases (Hack et al., 1989; Ostermann et al., 1997; Rigato, 1996) . In connection with regular testing of vaccine lots we determine the release and the kinetic of TNF and IL-6 in piglets after immunisation with different vaccines containing endotoxin . The current results suggest that the amounts of both cytokines increased with elevated endotoxin concentration given with the doses . TNF peaked in plasma after one hour, IL-6 peaked between two and four hours p.appl . We did not find any influence of the gender of the animals . In contrast, the body weight seems to affect the cytokine release in different ways . Determination of cytokine changes in plasma is a sensitive tool for the evaluation of systemic reactions and supports data about the clinical and haematological signs.

ALTEX, 1998, 15(5), 65 - 67
{The establishment of endotoxin limits which satisfy animal welfare considerations in the testing of porcine vaccines}; Wegener M et al.; Endotoxin (lipopolysaccharide, LPS) is a constituent of the cell walls of gram-negative bacteria and is found in many vaccines produced from these bacteria . High levels of endotoxin can give rise to a range of pathophysiological reactions, and adverse reactions tend to be seen in animals following vaccination . In this study, pigs of various ages and weights were vaccinated with licensed porcine vaccines and the endotoxin content of the vaccines was determined with the LAL, conducted according to DAB 10 . The experiments followed the DAB guidelines relating to the safety testing of veterinary vaccines . The animals were monitored for 48 h after vaccination, their body temperatures were measured, and blood samples were taken for analysis and for the determination of plasma endotoxin levels . There was a clear relationship between vaccine endotoxin content and changes in blood cell counts and in the clinical picture . Elevated plasma endotoxin levels correlated with the occurrence of initial leucopenia followed by leucocytosis as well as with clinical symptoms ranging from refusal of food and depression to shock-like symptoms . After 24 h, normal physiological values were regained . Young animals weighing between 10 and 40 kg were found to be very sensitive to elevated endotoxin content in vaccines . The differences in individual reactions could be due not only to differences in vaccine endotoxin content, but also to differences in the reactivity of the organism, and in the type of bacteria used or in the composition of the vaccine.

ALTEX, 1998, 15(3), 123 - 128
{Evaluation and further development of a pyrogenicity assay based on human whole blood}; Fennrich S et al.; When cells of the immune system, i.e . primarily blood monocytes and macrophages, come into contact with pyrogens (fever inducing contaminations) they release mediators transmitting the fever reaction within the organism . A new pyrogen test exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample . In case of pyrogen contamination, the formation of interleukin-1 is induced, which is determined by ELISA . According to the various pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample . In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species . In contrast to the well established in vitro alternative, i.e . the limulus amebocyte lysate assay (LAL), the blood assay is not restricted to endotoxins of Gram-negative bacteria and is not to the same extent disturbed by endotoxin-binding blood proteins . Here, interim results of the ongoing optimisation and prevalidation are demonstrated . Preliminary data of the evaluation for biological and pharmaceutical drugs are presented.

ALTEX, 1995, 12(2), 89 - 92
{Determination of Pyrogens: Comparison of Different Methods}; Meisel J; Pyrogens are a group of chemically diverse substances that cause fever and shock in severe cases . The most important pyrogenic substances in pharmaceutical industry are bacterial endotoxins . Bacterial endotoxins are part of the cell wall of gram negative bacteria . There are two methods of detection: the Pyrogen test is based upon the measurement of body temperature of rabbits before and after injection of the specimen, the LAL-test is based upon the clotting reaction of an enzyme complex of cells of the horseshoe crab together with bacterial endotoxins (in vitro test) . Advantages and disadvantages of the two tests have been compared . Although the LAL-test is widely used a total replacement of the Pyrogen test still is not possible since on the one hand the LAL-test will only detect bacterial endotoxins and on the other hand not all specimen can be tested with the LAL-test because of interference with the test . An example has been given by comparing the test results of three related cephalosporins in the Pyrogen test and different methods of the LAL-test (gel clot, turbidimetric kinetic, chromogenic kinetic) . Keywords: pyrogen determination, LAL-tests, gel clot LAL, turbidimetric LAL, chromogenic kinetic LAL, bacterial

ALTEX, 1995, 12(2), 76 - 80
{History of the LAL-test: validation and regulatory acceptance}; Liebsch M; Bacterial endotoxins gram-negative bacteria are the most relevant substances inducing hyperthermia in humans (pyrogens) . Since endotoxins may contaminate pharmaceutical preparations during production process, purity is assured by monitoring the increase in body temperature of rabbits exposed to such preparations . This bioassay can be replaced by the LAL-test in which the clotting reaction of blood cells of the horseshoe crab limulus polyphemus is measured after contact with bacterial endotoxins . Although this reaction is significantly more sensitive to endotoxins than hyperthermia in rabbits, the LAL-test had to undergo 25-30 years of validation to achieve regulatory acceptance . Although endotoxin induced blood clotting in Limulus is quite similar to the same reaction in humans, acceptance of the assay as an alternative to testing in rabbits was delayed, since chemicals present in pharmaceutical preparations may interfere with the LAL clotting reaction . In addition, fever can be induced also by substances other than endotoxins . Therefore it has to be proven for each new preparation that the LAL-test can replace the rabbit pyrogen test in a case by case validation according to guidelines, as e.g . the German guideline, which was released in 1993 . During the past decade some laboratories of the pharmaceutical industry in Germany were able to replace 95% of the rabbit pyrogen tests by the LAL-test without increasing the risk for humans.

Amino Acid, Water Purification, Antibiotic, Microbes, Bioengineering, Bacterium, Botulism, Bacteriology, Staphylococcus Aureus, Microbe, Functional Genomics, Bacteria, Food Microbiology, Microbiology, f, Biofilm, r, Microorganisms, i, Rhizobia, h, Microorganism, n, Nitrification, Pcr, Haemophilus, p, Bacteria, m, Bacteriophage, s, E. coli, b, Escherichia coli, c, Bacillus, s, Streptococcus, m, P. aeruginosa, x, Corynebacteria, s, Staphylococcus, e, E. coli, f, Antibacterial, s, Campylobacter, y, Bacterium, e, Cell suspension, a, Meningococcus, e, Lactobacilli, b, Yeasts, a, Rhodococcus, s, Bacterial, c, Saccharomyces cerevisiae, s, Culture media, b, Bacterium, e, Antibacterial, m, Pasteurella, x, Corynebacteria, t, Microflora, f, Streptococcus, a, E. coli, Escherichia coli,

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005