Gram negative

FEBS J, 2005 Jan, 272(2), 327 - 40
Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity; Seydel U et al.; Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system . Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506) . Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition . The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups . We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A . To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties . We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively . In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-d-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed . These data allow a better understanding of endotoxic activity and its structural prerequisites.

J Pediatr Hematol Oncol, 2005 Jan, 27(1), 37 - 38
Two Cases of Ralstonia pickettii Bacteremias in a Pediatric Oncology Unit Requiring Removal of the Port-A-Caths; Kismet E et al.; Ralstonia pickettii is an aerobic, gram-negative bacterium causing bacteremia following the use of contaminated saline vials, respiratory therapy solutions, skin disinfectants, blood culture mediums, and water supplies . It is rarely associated with human infections . The authors report two cases of R . pickettii bacteremia in patients with Port-A-Caths that could be treated only by removal of the ports.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 479 - 486
Brevundimonas mediterranea sp . nov., a non-stalked species from the Mediterranean Sea; Fritz I et al.; Six strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from the Mediterranean Sea . 16S rRNA gene sequence analysis indicated that the strains were affiliated within the alphaproteobacterial genus Brevundimonas, with Brevundimonas intermedia (99.4 %) and Brevundimonas vesicularis (99.2 %) as their closest relatives . This affiliation was supported by chemotaxonomic data (major polar lipids: phosphatidyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidyl glucopyranosyl diacylglycerol; major fatty acids: C(18 : 1), C(16 : 0), C(16 : 1), C(15 : 0), C(17 : 1)omega8c, 11-Me-C(18 : 1)omega5t) . The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the strains from all recognized Brevundimonas species . The strains therefore represent a novel species, for which the name Brevundimonas mediterranea sp . nov . is proposed, with the type strain V4.BO.10(T) (=LMG 21911(T)=CIP 107934(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 423 - 6
Chryseobacterium formosense sp . nov., isolated from the rhizosphere of Lactuca sativa L . (garden lettuce); Young CC et al.; A yellow-pigmented bacterial strain (CC-H3-2(T)), isolated from the rhizosphere of Lactuca sativa L . (garden lettuce) in Taiwan, was investigated using a polyphasic taxonomic approach . The cells were Gram-negative, rod-shaped and non-spore-forming . Phylogenetic analyses using the 16S rRNA gene sequence of the isolate indicated that the organism belongs to the genus Chryseobacterium, with the highest sequence similarity to the type strains of Chryseobacterium indoltheticum (97.7 %), Chryseobacterium scophthalmum (97.5 %), Chryseobacterium joostei (97.2 %) and Chryseobacterium defluvii (97.2 %) . The major whole-cell fatty acids were iso-C(15 : 0) (52.2 %) and iso-C(17 : 0) 3-OH . DNA-DNA hybridization experiments revealed levels of only 27.4 % to C . scophthalmum, 27.1 % to C . indoltheticum, 14.1 % to C . joostei and 7.8 % to C . defluvii . DNA-DNA relatedness and biochemical and chemotaxonomic properties demonstrate that strain CC-H3-2 (T) represents a novel species, for which the name Chryseobacterium formosense sp . nov . is proposed . The type strain is CC-H3-2(T) (=CCUG 49271(T)=CIP 108367(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 309 - 313
Marinomonas ushuaiensis sp . nov., isolated from coastal sea water in Ushuaia, Argentina, sub-Antarctica; Prabagaran SR et al.; A Gram-negative, rod-shaped, psychrophilic, motile, non-spore-forming bacterium, strain U1(T), was isolated from Ushuaia located at the southernmost tip of Argentina . On the basis of 16S rRNA gene sequence similarity, strain U1(T) was found to be closely related to Marinomonas communis (DSM 5604(T)) and Marinomonas primoryensis (IAM 15010(T)) . At the DNA-DNA level, however, the values for similarity were 41 and 25 %, respectively . The major fatty acids present were iso-C(16 : 0), C(16 : 1)omega7c, iso-C(17 : 1) and C(18 : 1)omega7c and the G+C content of the DNA was 43.6 mol% . All of the above characteristics support the affiliation of strain U1(T) to the genus Marinomonas . Furthermore, on the basis of phenotypic features, chemotaxonomic characteristics and phylogenetic analysis of the 16S rRNA gene sequence, it appears that strain U1(T) is distinct from the four Marinomonas species with validly published names . Strain U1(T), therefore, represents a novel species, for which the name Marinomonas ushuaiensis sp . nov . is proposed . The type strain of M . ushuaiensis is U1(T) (=MTCC 6143(T)=DSM 15871(T)=JCM 12170(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 275 - 9
Marinomonas pontica sp . nov., isolated from the Black Sea; Ivanova EP et al.; A Gram-negative, polarly flagellated bacterium was isolated from a sea-water sample collected from the Karadag Natural Reserve of the Eastern Crimea and characterized to clarify its taxonomic position . 16S rRNA gene sequence-based phylogenetic analysis of this novel organism revealed Marinomonas vaga, Marinomonas communis, Marinomonas mediterranea, Marinomonas primoryensis and 'Marinomonas protea' as its closest relatives (similarity 95-97 %) . The G+C content of the DNA was 46.5 mol% . The organism grew between 4 and 33 degrees C, tolerated 10 % NaCl, was slightly alkaliphilic and was not able to degrade starch, gelatin, agar or Tween 80 . Phosphatidylethanolamine (53.4 %) and phosphatidylglycerol (46.6 %) were the predominant phospholipids . The major fatty acids were 16 : 0 (15.5 %), 16 : 1omega7 (26.7 %) and 18 : 1omega7 (47.1 %) . The phylogenetic, genetic and physiological properties of the organism placed it within a novel species, proposed as Marinomonas pontica sp . nov., the type strain of which is 46-16(T) (=LMG 22531(T)=KMM 3492(T)=UCM 11075(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 235 - 8
Salegentibacter mishustinae sp . nov., isolated from the sea urchin Strongylocentrotus intermedius; Nedashkovskaya OI et al.; A bacterial strain, designated KMM 6049(T), was isolated from the sea urchin Strongylocentrotus intermedius inhabiting the Sea of Japan . The bacterium studied was strictly aerobic, heterotrophic, yellow-pigmented, non-motile, Gram-negative and oxidase-, catalase-, beta-galactosidase- and alkaline phosphatase-positive . 16S rRNA gene sequence analysis indicated that strain KMM 3524(T) was closely related to Salegentibacter holothuriorum and Salegentibacter salegens (sharing 97.7 and 98 % sequence similarity, respectively) . DNA-DNA relatedness levels between strains KMM 6049(T) and S . holothuriorum KMM 3524(T) and S . salegens DSM 5424(T) were 24 and 45 %, respectively, indicating that KMM 6049(T) belongs to a novel species of the genus Salegentibacter, for which the name Salegentibacter mishustinae sp . nov . is proposed . The type strain is KMM 6049(T) (=KCTC 12263(T)=LMG 22584(T)=NBRC 100592(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 231 - 4
Roseivirga ehrenbergii gen . nov., sp . nov., a novel marine bacterium of the phylum 'Bacteroidetes', isolated from the green alga Ulva fenestrata; Nedashkovskaya OI et al.; The taxonomic position of a novel marine bacterium isolated from the green alga Ulva fenestrata collected in the Sea of Japan was established . Cells of the strain studied, designated KMM 6017(T), were strictly aerobic, heterotrophic, pink-pigmented, non-motile by gliding, Gram-negative and oxidase-, catalase-, beta-galactosidase- and alkaline phosphatase-positive . 16S rRNA gene sequence analysis indicated that the strain occupied a distinct lineage within the phylum 'Bacteroidetes' and formed a cluster with {Flexibacter} tractuosus and Reichenbachia agariperforans . The G+C content of the DNA of KMM 6017(T) was 40.2 mol% . The major respiratory quinone was MK-7 . The predominant fatty acids were i15 : 1, i15 : 0 and i17 : 0 3-OH (34.2, 24 and 7.7 %, respectively) . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium was assigned to the genus Roseivirga gen . nov., as Roseivirga ehrenbergii gen . nov., sp . nov . The type strain is KMM 6017(T) (=KCTC 12282(T)=LMG 22567(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 133 - 8
Chryseobacterium daecheongense sp . nov., isolated from freshwater lake sediment; Kim KK et al.; A novel nitrate-reducing bacterium, CPW406(T), was isolated from the sediment of a shallow, freshwater lake . The strain was a Gram-negative, non-motile, non-spore-forming rod, which formed yellow-pigmented colonies on nutrient agar and contained a polyamine pattern with sym-homospermidine as the major compound, MK-6 as the predominant menaquinone, 15 : 0 iso and 17 : 0 iso 3-OH as the major fatty acids and phosphatidylethanolamine and several unknown lipids in the polar lipid profile . The 16S rRNA gene sequence of strain CPW406(T) was found to be most similar to that of the type strain of Chryseobacterium defluvii (DSM 14219(T); 97.9 % similarity) . However, DNA-DNA relatedness data and its phenotypic properties showed that strain CPW406(T) could be distinguished from all known Chryseobacterium species and thus represented a novel species, for which the name Chryseobacterium daecheongense sp . nov . is proposed; the type strain is CPW406(T) (=DSM 15235(T)=KCTC 12088(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 41 - 7
Roseisalinus antarcticus gen . nov., sp . nov., a novel aerobic bacteriochlorophyll a-producing {alpha}-proteobacterium isolated from hypersaline Ekho Lake, Antarctica; Labrenz M et al.; A Gram-negative, aerobic to microaerophilic rod was isolated from 10 m depths of the hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica) . The strain was oxidase- and catalase-positive, metabolized a variety of carboxylic acids and sugars and produced lipase . Cells had an absolute requirement for artificial sea water, which could not be replaced by NaCl . A large in vivo absorption band at 870 nm indicated production of bacteriochlorophyll a . The predominant fatty acids of this organism were 16 : 0 and 18 : 1omega7c, with 3-OH 10 : 0, 16 : 1omega7c and 18 : 0 in lower amounts . The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine . Ubiquinone 10 was produced . The DNA G+C content was 67 mol% . 16S rRNA gene sequence comparisons indicated that the isolate represents a member of the Roseobacter clade within the alpha-Proteobacteria . The organism showed no particular relationship to any members of this clade but clustered on the periphery of the genera Jannaschia, Octadecabacter and 'Marinosulfonomonas' and the species Ruegeria gelatinovorans . Distinct morphological, physiological and genotypic differences to these previously described taxa supported the description of a new genus and a novel species, for which the name Roseisalinus antarcticus gen . nov., sp . nov . is proposed . The type strain is EL-88(T) (=DSM 11466(T)=CECT 7023(T)).

FEMS Microbiol Rev, 2005 Jan, 29(1), 83 - 98
Enteropathogenic Escherichia coli: unravelling pathogenesis; Deborah Chen H et al.; Enteropathogenic Escherichia coli (EPEC) is a gram-negative bacterial pathogen that adheres to intestinal epithelial cells, causing diarrhoea . It constitutes a significant risk to human health and remains an important cause of infant mortality in developing countries . Although EPEC was the first E . coli strain to be implicated in human disease in the 1940s and 1950s, the mechanisms by which this pathogen induced diarrhoea remained a complete mystery throughout most of the 40 years since its description . It was only during the late 1980s that major advances were made in unravelling the mechanisms behind EPEC pathogenesis . Ever since, progress has been made at a stunning pace and there have been major breakthroughs in identifying the bacterial factors involved in attaching and effacing (A/E) lesion formation, host signal transduction pathways in response to EPEC infection and the genetic basis of EPEC pathogenesis . The rapid pace of discovery is a result of intensive research by investigators in this field and portends that EPEC will soon be among one of the most understood diarrhoea-causing infectious agents . This review aims to trace the progress of EPEC research since its existence was first reported by John Bray in 1945, highlighting the major findings that have revolutionised our understanding of EPEC pathogenesis.

J Microbiol Methods, 2005 Mar, 60(3), 383 - 93
Isolation of Brucella abortus total RNA from B . abortus-infected murine RAW macrophages; Covert J et al.; Brucella is a Gram-negative facultative bacterium that persists intracellularly in macrophages . However, the intracellular survival mechanisms used by Brucella are not fully understood . Isolation of Brucella RNA from infected macrophages has been challenging, and the inability to isolate sufficient Brucella RNA from infected macrophages has contributed to the failure in understanding bacterial transcriptional events . We describe the isolation of sufficient Brucella abortus RNA from its infective host cell environment using osmotic lysis and RNase and DNase digestion . This method takes advantage of the B . abortus cell envelope 1that protects bacterial RNA and DNA . The cell envelope of B . abortus was digested using SDS/proteinase K (PK) that, importantly, inhibits any residual RNase after digesting macrophage RNA permitting the extraction of B . abortus RNA . In our experiments, 4.5 mug of RNA was routinely isolated from 1 ml bacterial culture and 2-9 mug of bacterial RNA from infected macrophages without detectable host cell RNA or DNA contamination . The method is rapid and uses inexpensive, commonly available reagents . Total bacterial RNA was isolated in quantities sufficient for RT-PCR and microarray analysis.

BMC Bioinformatics . 2005 Jan 12;6(1):7 {Epub ahead of print}
Evaluation of methods for predicting the topology of beta-barrel outer membrane proteins and a consensus prediction method; Bagos PG et al.; BACKGROUND: Prediction of the transmembrane strands and topology of beta-barrel outer membrane proteins is of interest in current bioinformatics research . Several methods have been applied so far for this task, utilizing different algorithmic techniques and a number of freely available predictors exist . The methods can be grossly divided to those based on Hidden Markov Models (HMMs), on Neural Networks (NNs) and on Support Vector Machines (SVMs) . In this work, we compare the different available methods for topology prediction of beta-barrel outer membrane proteins . We evaluate their performance on a non-redundant dataset of 20 beta-barrel outer membrane proteins of gram-negative bacteria, with structures known at atomic resolution . Also, we describe, for the first time, an effective way to combine the individual predictors, at will, to a single consensus prediction method . RESULTS: We assess the statistical significance of the performance of each prediction scheme and conclude that Hidden Markov Model based methods, HMM-B2TMR, ProfTMB and PRED-TMBB, are currently the best predictors, according to either the per-residue accuracy, the segments overlap measure (SOV) or the total number of proteins with correctly predicted topologies in the test set . Furthermore, we show that the available predictors perform better when only transmembrane beta-barrel domains are used for prediction, rather than the precursor full-length sequences, even though the HMM-based predictors are not influenced significantly . The consensus prediction method performs significantly better than each individual available predictor, since it increases the accuracy up to 4% regarding SOV and up to 15% in correctly predicted topologies . CONCLUSIONS: The consensus prediction method described in this work, optimizes the predicted topology with a dynamic programming algorithm and is implemented in a web-based application freely available to non-commercial users at http://bioinformatics.biol.uoa.gr/ConBBPRED.

Crit Rev Microbiol, 2004, 30(4), 275 - 86
Autotransporter and two-partner secretion: delivery of large-size virulence factors by gram-negative bacterial pathogens; Newman CL et al.; A number of protein secretion mechanisms have been identified in gram-negative pathogens . Many of these secretion systems are dependent upon the Sec translocase for protein export from the cytoplasm into the periplasm and then utilize other mechanisms for transport from the periplasm through the outer membrane . In this article, we review secretion similarities between autotransporter and two-partner secretion systems, and we report similarities between the autotransporter secretion mechanism with that of intimin/invasins . Considering that many secreted proteins are virulence factors, a better understanding of their secretion mechanisms will aid in the development of disease treatments and new bacterial vaccines.

J Biol Chem . 2005 Jan 10; {Epub ahead of print}
Crystal structure of CD14 and its implications for lipopolysaccharide signaling; Kim JI et al.; Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome . The core receptors recognizing lipopolysaccharide are CD14, TLR4 and MD-2 . CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling . In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products . Here, we present the first crystal structure of CD14 to 2.5 A resolution . A large hydrophobic pocket was found on the N-terminal side of the horseshoe-like structure . Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide binding site . Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket . Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket and the multiplicity of grooves available for ligand binding.

J Mol Biol, 2005 Feb 4, 345(5), 1185 - 97 Epub 2004 Dec 15.
The Solution Structure of the C-terminal Domain of TonB and Interaction Studies with TonB Box Peptides; Sean Peacock R et al.; The TonB protein transduces energy from the proton gradient across the cytoplasmic membrane of Gram-negative bacteria to TonB-dependent outer membrane receptors . It is a critically important protein in iron uptake, and deletion of this protein is known to decrease virulence of bacteria in animal models . This system has been used for Trojan horse antibiotic delivery . Here, we describe the high-resolution solution structure of Escherichia coli TonB residues 103-239 (TonB-CTD) . TonB-CTD is monomeric with an unstructured N terminus (103-151) and a well structured C terminus (152-239) . The structure contains a four-stranded antiparallel beta-sheet packed against two alpha-helices and an extended strand in a configuration homologous to the C-terminal domain of the TolA protein . Chemical shift perturbations to the TonB-CTD (1)H-(15)N HSCQ spectrum titrated with TonB box peptides modeled from the E.coli FhuA, FepA and BtuB proteins were all equivalent, indicating that all three peptides bind to the same region of TonB . Isothermal titration calorimetry measurements demonstrate that TonB-CTD interacts with the FhuA-derived peptide with a K(D)=36(+/-7)muM . On the basis of chemical shift data, the position of Gln160, and comparison to the TolA gp3 N1 complex crystal structure, we propose that the TonB box binds to TonB-CTD along the beta3-strand.

Lett Appl Microbiol, 2005, 40(2), 146 - 51
Copper amendment of agricultural soil selects for bacterial antibiotic resistance in the field; Berg J et al.; Abstract j . berg, a . tom-petersen and o . nybroe . 2004.Aims: The objective of this study was to determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance patterns in indigenous soil bacteria . Methods and Results: Soil bacteria were isolated from untreated and Cu-amended field plots . Cu-amendment significantly increased the frequency of Cu-resistant isolates . A panel of isolates were characterized by Gram-reaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics . More than 95% of the Cu-resistant isolates were Gram-negative . Cu-resistant Gram-negative isolates had significantly higher incidence of resistance to ampicillin, sulphanilamide and multiple (>/=3) antibiotics than Cu-sensitive Gram-negative isolates . Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher incidence of resistance to chloramphenicol and multiple (>/=2) antibiotics than corresponding isolates from control plots . Significance and Impact of the Study: The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu-resistant bacteria . Hence, the widespread accumulation of Cu in agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance.

Cardiovasc Res, 2005 Feb 1, 65(2), 317 - 27
Chlamydia pneumoniae infections in mouse models: relevance for atherosclerosis research; de Kruif MD et al.; Mouse models have been frequently used in the study of Chlamydia pneumoniae (also known as Chlamydophila pneumoniae) infections . This gram-negative obligate intracellular bacterium causes respiratory infections, followed by dissemination of the bacterium to various organs throughout the body, including cardiovascular tissues, supporting the current hypothesis of a relationship between C . pneumoniae and atherosclerosis . Recently, clinical trials evaluated the effect of antichlamydial antibiotics on secondary cardiovascular events . Although small studies showed some effect, the large WIZARD study did not confirm these results, and the role of antichlamydial antibiotics in prevention of secondary events was questioned . To address these issues, data obtained from mouse models were systematically reviewed here . C . pneumoniae infections showed atherogenic properties in mice that were reproducible and confirmed by different research groups . However, antibiotic therapy was of limited value in these mouse models . Antibiotic therapy effectively cleared the acute infection, but did not influence the atherogenic properties of C . pneumoniae unless the therapy was started early during the acute infection . The results summarized here may help to better understand the results of the clinical antibiotic trials.

J Mol Evol, 2004 Oct, 59(4), 498 - 506
Molecular evolution of daphnia immunity genes: polymorphism in a gram-negative binding protein gene and an alpha-2-macroglobulin gene; Little TJ et al.; Studies of DNA polymorphism have shown that some immune system genes of mammals and plants are exceptionally diverse, indicating that coevolution between these taxa and their parasites mediates positive selective sweeps and/or balancing selection . The genes of the arthropod immune system remain comparatively unstudied . We isolated two putative immune system genes from the cladoceran crustacean Daphnia and examined DNA sequence diversity . For one gene, encoding a putative gram-negative binding protein, we found evidence of only purifying selection, indicating that this gene is under strong functional constraint and that selection acts to eliminate amino acid variation . For another gene, encoding a putative alpha-2-macroglobulin, we found evidence of positive selection, indicating the possible involvement of this gene in a host-parasite arms race . We discuss the assumed function of these genes and offer speculation regarding which components of the arthropod immune system might experience diversifying adaptive evolution.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
Identification of a new membrane-associated protein which influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis; Sato K et al.; The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of the organisms . The organisms have developed several systems for transport across the inner and outer membranes . The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors . Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB and hagA on the chromosome . In this study, we isolated a P . gingivalis mutant (porT) which showed very weak activities of gingipains in the cell lysates and culture supernatants . Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells . Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm . The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum . These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.

Yi Chuan Xue Bao, 2004 Dec, 31(12), 1448 - 54
{Sequence of Escherichia coli O141 O-antigen gene cluster and analyses of its evolutionary history}; Kong QK et al.; Lipopolysaccharide (LPS) is one of the major components of the outer membrane of gram-negative bacteria . It is an amphipathic molecule compose of lipid A, a core oligosaccharide and an O-specific antigen . O-antigen, which is a repeat-unit polysaccharide, is a major contribution to the antigenic variability of the bacterial cell surface . The genes of O-antigen gene cluster are responsible for the synthesis of the O-antigen . The O-antigen gene cluster of E . coli O141 was sequenced and found to contain the genes rmlBDAC and manBC for the biosynthesis of nucleotide sugars dTDP-rhamnose and GDP-mannose, respectively, encoding genes for Ounit flippase (wzx), O-antigen polymerase (wzy) and potential transferase genes . The possible biosynthesis pathway for O-antigen of E . coli O141 was proposed . Two genes specific to E . coli O141 were identified . This work provides the basis for a sensitive test by PCR for the rapid detection of E . coli O141 . Phylogenetic trees for the rmlB, rmlD, rmlA, and rmlC genes and manB, manC genes were generated and the comparisons were made among different strains . We find that these genes are typical E . coli genes and might have been involved in recombination events between O-antigen gene clusters.

Microbiology, 2005 Jan, 151(Pt 1), 259 - 68
Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA; Baumgarth B et al.; The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa . The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions . ExpG is a member of the MarR family of regulatory proteins . Analysis of target sequences of an ExpG(His)(6) fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs . Association and dissociation kinetics of the specific binding of ExpG(His)(6) to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM) . Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG-DNA interaction.

Eur J Pediatr Surg, 2004 Dec, 14(6), 418 - 21
{In Process Citation}; Ameh EA et al.; BACKGROUND: Fournier's gangrene is uncommon in childhood and little is known about the disease in this age group . METHOD: A retrospective review was carried out of neonates and infants treated for Fournier's gangrene over a period of 16 years . RESULTS: Twelve neonates and infants aged 5 days - 3 months (median 3 weeks) were treated in our hospital . The precipitating cause was omphalitis in 7 babies, strangulated inguinal hernia in 2 and in 3 babies there was no identifiable cause . Gram-negative bacteria were cultured in 3 patients, but in most the culture was sterile . Treatment consisted of debridement of devitalised tissue and administration of broad-spectrum antibiotics . Primary closure was achieved in 1 baby and secondary closure in 2 others . In 7 babies the wound contracted rapidly and healed . There was no mortality . CONCLUSION: Fournier's gangrene in neonates and infants in our environment is largely preventable . Early debridement and appropriate antibiotics give good results.

East Afr Med J, 2004 Sep, 81(9), 474 - 9
Clinical and laboratory features of spontaneous bacterial peritonitis; Filik L et al.; BACKGROUND: Spontaneous Bacterial Peritonitis (SBP) is a complication of cirrhosis . The mortality rate is approximately 30-50% . SBP is defined as an ascitic fluid infection in the absence of any obvious intraabdominal infectious foci . While earlier reports of SBP emphasized high mortality rates, recently lower mortality rates have been reported . OBJECTIVE: To evaluate the clinical and laboratory features and prognostic indicators of SBP . DESIGN: Retrospective study . SETTING: Hacettepe University Hospitals . SUBJECTS: A total of 281 SBP episodes of 214 patients between 3rd march 1981 and 3rd August 1999, in Hacettepe University Hospital were evaluated . Statistical analysis was performed in the group of patients having chronic liver diseases . RESULTS: One hundred and forty nine of the patients 214 (69.6%) were males and 65(30.4%) were females . The mean age of all patients were 49.91+/-15.01 years (17 to 90 years) . All spontaneous ascites infection episodes were symptomatic . In all of the episodes, most common clinical features were as follows: icterus (54.5%), abdominal tenderness (54.5%), hepatic encephalopathy (50.7%), fatigue (46.7%), abdominal pain (44.4%) and fever (38.8%) . The culture of the ascitic fluid resulted in isolation of a bacteria in 25.4% of all episodes . The most frequently isolated microorganisms turned out to be gram negative enteric bacterias (76.2%) . Sixty seven patients in 179 cases with liver disease passed away (37.4%) . The use of cefotaxime and newer cephalosporins seemed to have less mortality (31.7%) as compared with that (42.2%) observed in patients treated with other antibiotic regimens . CONCLUSIONS: Of all the factors analysed in patients with chronic liver diseases, being Child-Pugh class C, having fatigue, hepatic encephalopathy, hypotension, higher peripheral blood leukocyte count (> or =12000/mm3), renal dysfunction (serum creatinine level > or = 2mg/dl), longer prothrombin time (INR > or = 2.5), lower ascites protein level (<Igr/ dl) and, liver disease for longer time, developing superinfection (an infection other than SBP starting during SBP treatment) were statistically correlated with a higher death rate (p<0.05).

Invest Ophthalmol Vis Sci, 2005 Jan, 46(1), 114 - 20
Lipopolysaccharide-induced expression of intercellular adhesion molecule-1 and chemokines in cultured human corneal fibroblasts; Kumagai N et al.; PURPOSE: Invasion of bacteria into the corneal stroma induces the infiltration of leukocytes and subsequent corneal ulceration . The role of corneal fibroblasts in the detection of bacterial invasion into the stroma was investigated by examining the in vitro expression of the receptor complex for lipopolysaccharide (LPS), a common component of Gram-negative bacteria, as well as the possible effects of LPS on both the expression of adhesion molecules and the release of chemokines in cultured human corneal fibroblasts . METHODS: Expression of the LPS receptor complex, intercellular adhesion molecule (ICAM)-1, and the chemokines interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 was examined by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, or immunofluorescence analysis . RESULTS: Corneal fibroblasts were found to contain transcripts encoding toll-like receptor-4, CD14, and MD-2, all of which are components of the LPS receptor complex . The expression of ICAM-1 at the surface of corneal fibroblasts and the amount of ICAM-1 mRNA in the cells were both increased by LPS . Similarly, LPS increased both the release of IL-8 and MCP-1 by corneal fibroblasts as well as the amounts of the corresponding mRNAs in these cells . These various effects of LPS were potentiated by the presence of a low concentration of human serum . CONCLUSIONS: Corneal fibroblasts may play an important role in the defense system of the cornea by recognizing the presence of LPS and subsequently expressing adhesion molecules and chemokines that promote leukocyte infiltration.

Nat Struct Mol Biol . 2004 Dec 26; {Epub ahead of print}
Structural characterization of a type III secretion system filament protein in complex with its chaperone; Yip CK et al.; The type III secretion system (TTSS) mediates the specific translocation of bacterial proteins into the cytoplasm of eukaryotic cells, a process essential for the virulence of many Gram-negative pathogens . The enteropathogenic Escherichia coli TTSS protein EspA forms a hollow extracellular filament believed to be a molecular conduit for type III protein translocation . Structural analysis of EspA has been hampered by its polymeric nature . We show that EspA alone is sufficient to form filamentous structures in the absence of other pathogenicity island-encoded proteins . CesA is the recently proposed chaperone of EspA, and we demonstrate that CesA traps EspA in a monomeric state and inhibits its polymerization . Crystallographic analysis of the heterodimeric CesA-EspA complex at a resolution of 2.8 A reveals that EspA contains two long a-helices, which are involved in extensive coiled-coil interactions with CesA.

Infect Immun, 2005 Jan, 73(1), 352 - 61
Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila; Akamine M et al.; The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2(-/-)), TLR4(-/-), and wild-type (WT) littermate (C57BL/6 x 129Sv) mice . Intracellular growth of L . pneumophila was enhanced within TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages . There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice . Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L . pneumophila was attenuated in TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages . Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L . pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain . There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria . An L . pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2(-/-) macrophages was significantly lower than those of WT and TLR4(-/-) macrophages . Treatment of L . pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production . Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L . pneumophila, although other TLRs may also contribute to innate immunity against this organism.

Infect Immun, 2005 Jan, 73(1), 62 - 9
An immunoreactive 38-kilodalton protein of Ehrlichia canis shares structural homology and iron-binding capacity with the ferric ion-binding protein family; Doyle CK et al.; Ehrlichiae are tick-transmitted, gram-negative, obligately intracellular bacteria that live and replicate in cytoplasmic vacuoles, but little is known about iron acquisition mechanisms necessary for their survival . In this study, a genus-conserved immunoreactive ferric ion-binding protein (Fbp) of Ehrlichia canis was identified and its iron-binding capability was investigated . E . canis Fbp was homologous to a family of periplasmic Fbp's involved in iron acquisition and transport in gram-negative bacteria . E . canis Fbp had a molecular mass (38 kDa) consistent with those of Fbp's in other bacteria and exhibited substantial immunoreactivity in its native conformation . The predicted three-dimensional structure of E . canis Fbp demonstrated conservation of important Fbp family structural motifs: two domains linked with a polypeptide "hinge" region . Under iron-binding conditions, the recombinant Fbp exhibited an intense red color and an absorbance spectrum indicative of iron binding, and it bound Fe(III) but not Fe(II) . Fbp was observed primarily in the cytoplasm of the reticulate forms of E . canis and Ehrlichia chaffeensis but was notably found on extracellular morula fibers in morulae containing dense-cored organisms . Although expression of Fbp is regulated through an operon of three functionally linked genes in other gram-negative bacteria, the absence of an intact fbp operon in Ehrlichia spp . suggests that genes involved in ehrlichial iron acquisition have been subject to reductive evolution.

J Periodontal Res, 2005 Feb, 40(1), 28 - 35
Chronic treatment with the glutamate receptor antagonist MK-801 alters periodontal disease susceptibility; Breivik T et al.; Breivik T, Gundersen Y, Osmundsen H, Opstad PK, Fonnum F . Chronic treatment with the glutamate receptor antagonist MK-801 alters periodontal disease susceptibility . J Periodont Res 2004; doi: 10.1111/j.1600-0765.2004.00765.x (c) Blackwell Munksgaard 2004Objective: Previous experiments in rats suggest that hypothalamic-pituitary-adrenal (HPA) axis over-responsiveness, which leads to increased secretion of immunoregulatory glucocorticoid hormones, increases periodontal disease susceptibility, whereas HPA axis under-responsiveness is associated with increased resistance to the disease . The present study was designed to investigate whether MK-801 (dizocilipine malate), an antagonist of the glutamate receptor N-methyl-d-aspartate (NMDA) in the brain, which has been found to play an important role in the regulation of the HPA axis, would influence the outcome of experimental ligature-induced periodontal disease in a rat model . Methods: Experimental periodontal disease was induced in periodontal disease susceptible and HPA axis high-responding Fischer 344 rats 2 days before chronic treatment with MK-801(1 mg/kg intraperitoneally) . The periodontal breakdown was assessed after the ligatures had been in place for 23 days . Following intraperitoneal Gram-negative bacterial lipopolysaccharide stimulation (Escherichia coli, 250 microg/kg), concentrations of glucocorticoid receptors (GRs) in the hippocampus, and levels of the cytokine tumour necrosis factor alpha (TNF-alpha), as well as the HPA axis-derived hormone corticosterone, were measured in serum . Results: Compared to vehicle-treated controls, MK-801-treated rats had significantly increased periodontal tissue destruction (p < 0.01) . MK-801-treated rats also showed significantly increased expression of GRs in the hippocampus (p < 0.05), elevated levels of corticosterone (p < 0.001) and reduced levels of TNF-alpha (p < 0.01) in serum 2 h after lipopolysaccharide stimulation . Conclusion: These findings may implicate glutamate receptor-dependent mechanisms in periodontal disease, and support the concept of a bidirectional immune-brain-immune regulatory network with importance for periodontal health and disease.

Oral Microbiol Immunol, 2005 Feb, 20(1), 1 - 9
A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis; Yamane K et al.; Yamane K, Yamanaka T, Yamamoto N, Furukawa T, Fukushima H, Walker CB, Leung K-P . A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis . Oral Microbiol Immunol 2005: 20: 1-9 . (c) Blackwell Munksgaard, 2005 . Prevotella nigrescens, a gram-negative black-pigmented anaerobic rod, has frequently been isolated from periodontitis and periapical periodontitis lesions . We have isolated an exopolysaccharide-producing P . nigrescens, strain 22, from a chronic periodontitis lesion . The purpose of this study was to determine the chemical composition and function of the exopolysaccharide associated with this clinical isolate . The chemical composition and structure of the purified exopolysaccharide from strain 22 were determined by high performance liquid chromatography and methylation analysis . To define the biological function of this exopolysaccharide, a chemically induced exopolysaccharide nonproducing mutant, strain 328, which was derived from strain 22, was established . The biological effects of exopolysaccharide were determined by comparing the ability of strain 22, strain 328 or heat-killed strain 22 to form abscesses in mice and to interfere with the phagocytic activity of peripheral blood polymorphonuclear leukocytes . Chemical analysis showed that isolated exopolysaccharide consisted of mannose (521.6 mug/mg), glucose (25.6 mug/mg), fructose (65.8 mug/mg), galactose (12.5 mug/mg), arabinose (6.2 mug/mg), xylose (3.2 mug/mg), rhamnose (6.1 mug/mg), and ribose (0.6 mug/mg) . Methylation analysis of exopolysaccharide indicated that the linkages of mannose were primarily (1-->2, 1-->6) (1-->2) (1-->6), and (1-->3) . Strain 22 and, to a lesser extent, its heat-killed counterpart induced greater abscess formation in mice than strain 328, even though the enzymatic profile of strain 22 was similar to that of strain 328 . The ability of strain 328 to induce abscess formation was restored by adding the purified exopolysaccharide isolated from strain 22 to the cell suspension of strain 328 . Exopolysaccharide alone failed to induce abscess formation in mice . Further, strain 328 but not the untreated or heat-killed strain 22, was phagocytosed by polymorphonuclear leukocytes both in the presence and in the absence of opsonic factors . The results suggest that these polysaccharides isolated from strain 22, which primarily consisted of mannose, may play a key role in the development of the chronic inflammatory lesion from which this strain was isolated.

J Physiol Pharmacol, 2004 Jul, 55 Suppl 2, 105 - 15
Association of the presence the Helicobacter pylori in the oral cavity and in the stomach; Czesnikiewicz-Guzik M et al.; Helicobacter pylori is a gram-negative, microaerophilic rod-shaped bacteria that lives beneath the gastric mucous layer, on the surface of epithelial cells . Stomach infection with this organism causes inflammation of the gastric mucosa, which can lead to gastritis, duodenal or gastric ulcer and even in rare cases to gastric carcinoma or MALT lymphoma . Approximately 50% of the world's population is believed to be infected with H . pylori . Most infections is probably acquired in childhood, but the exact route of transmission is unknown . It has been speculated that dental plaque might harbour Helicobacter pylori and, therefore, might be a source of gastric infection . In order to address this issue we studied the relationships between oral and gastric infections with H . pylori in 100 subjects . Methods: Gastric H . pylori infection was determined by (13)C-urea breath test (UBT) and the presence of the bacteria in oral cavity was monitored by the culture from the saliva and from dental plaque . Results: H . pylori was found in the stomach in 51% of studied individuals, while oral H . pylori was found in 54% (in saliva) and in 48.3% (in gingival pockets), the difference was not statistical significant (p=NS) . Interestingly, anti-Hp IgA was found in 84% of studied individuals . No relationship was found between the presence of the bacteria in the oral cavity and the H . pylori gastric infection . 54.9% of subjects with stomach infection showed concomitant presence of H . pylori in saliva . 52.3% of examined subjects with negative UBT-test revealed the presence of H . pylori in culture from the saliva . The X(2) value of relationship between UBT and culture H pylori in saliva was 0.029 (p=0.9) . Similarly, no relationship was found between the presence of H . pylori in the stomach and in the dental plaque (X2=0.6); p=0.4) . As expected, the presence of H . pylori in the dental plaque was significantly correlated with the presence of bacteria in the saliva (X2=18.4; p=0.0002) . We also compared the presence of H . pylori in the saliva of patients with and without teeth . The cultured H . pylori was found in 63.7% of patients without teeth and in 52.9% of patients with teeth . This indicates that the presence of teeth does not seem to affect the occurence of H . pylori in saliva . We conclude that oral activity contamination with of H . pylori occurs at similar degree to that in the stomach . However, there was no significant correlation between the occurence of H . pylori in the stomach and in the oral cavity indicating that other factors, like susceptibility to infection due to acid environment in the stomach may be the major factor in gastric infection with that bacteria, while oral cavity may serve only as transient food-related contamination without clear relation to gastric infection.

Expert Rev Vaccines, 2004 Dec, 3(6), 681 - 691
Antigen discovery and delivery of subunit vaccines by nonliving bacterial ghost vectors; Walcher P et al.; The bacterial ghost (BG) platform system is a novel vaccine delivery system endowed with intrinsic adjuvant properties . BGs are nonliving Gram-negative bacterial cell envelopes which are devoid of their cytoplasmic contents, yet maintain their cellular morphology and antigenic structures, including bioadhesive properties . The main advantages of BGs as carriers of subunit vaccines include their ability to stimulate a high immune response and to target the carrier itself to primary antigen-presenting cells . The intrinsic adjuvant properties of BGs enhance the immune response to target antigens, including T-cell activation and mucosal immunity . Since native and foreign antigens can be carried in the envelope complex of BGs, combination vaccines with multiple antigens of diverse origin can be presented to the immune system simultaneously . Beside the capacity of BGs to function as carriers of protein antigens, they also have a high loading capacity for DNA . Thus, loading BGs with recombinant DNA takes advantage of the excellent bioavailability for DNA-based vaccines and the high expression rates of the DNA-encoded antigens in target cell types such as macrophages and dendritic cells . There are many spaces within BGs including the inner and outer membranes, the periplasmic space and the internal lumen which can carry antigens, DNA or mediators of the immune response . All can be used for subunit antigen to design new vaccine candidates with particle presentation technology . In addition, the fact that BGs can also carry piggyback large-size foreign antigen particles, increases the technologic usefulness of BGs as combination vaccines against viral and bacterial pathogens . Furthermore, the BG antigen carriers can be stored as freeze-dried preparations at room temperature for extended periods without loss of efficacy . The potency, safety and relatively low production cost of BGs offer a significant technical advantage over currently utilized vaccine technologies.

Arch Gynecol Obstet . 2004 Dec 17; {Epub ahead of print}
Chemoprophylactic and bactericidal efficacy of 80 mg gentamicin in a single and once-daily dosing; Sifakis S et al.; OBJECTIVE: The objective was to examine the biodistribution, the chemoprophylactic, and the bactericide efficacy of 80-mg gentamicin single or once-daily dosing.STUDY DESIGN: Ninety-six patients who had had cesarean section or gynecological surgery received 80 mg gentamicin for chemoprophylaxis . A second group of 92 patients with Gram-negative infection received once-daily 80-mg gentamicin intramuscularly, combined with cefoxitin or ceforanide, for 5 days . Gentamicin serum and tissue concentration was determined 1 h after the first administration.RESULTS: The chemoprophylactic efficacy of gentamicin was 93.7% . The treatment efficacy was high in patients with chorioamnionitis and endometritis (92.9%), moderate in those with wound infection (69.5%), and less effective in those with septicemia (55.6%) . Twenty-six percent of patients continued with antibiotics for infection control . The mean serum level was 4.48+/-0.49 and 5.56+/-0.66 mug/ml in obstetrical and gynecological patients respectively (p>0.05) . Serum levels >4 mug/ml were achieved in 91% of patients.CONCLUSIONS: A single dose of 80 mg gentamicin offers chemoprophylaxis and achieves therapeutic serum-concentrations 1 h after administration . The 5-day combination of once-daily 80 mg gentamicin with a second-generation cephalosporin is effective in patients with chorioamnionitis and endometritis, but only moderately effective in those with wound infections and septicemia.

APMIS, 2004 Oct, 112(10), 674 - 85
Cell cycle arrest of human gingival fibroblasts and periodontal ligament cells by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin; Belibasakis GN et al.; The cytolethal distending toxin (Cdt) is produced by several Gram-negative bacterial species and causes growth arrest and morphological alterations in mammalian cells . Actinobacillus actinomycetemcomitans, which is involved in the pathogenesis of localized aggressive periodontitis, also produces a Cdt that affects periodontal connective tissue cells . The aim of this study was to investigate in which phase of the cell cycle these cells are arrested and enlarged when challenged with A . actinomycetemcomitans, and to evaluate the involvement of its Cdt . Human gingival fibroblasts and periodontal ligament cells were challenged with A . actinomycetemcomitans extract, or with purified Cdt, and cell cycle analysis was performed by propidium iodide staining and flow cytometry . Cells exposed to an A . actinomycetemcomitans wild-type strain, or to purified Cdt, were arrested in both G1 and G2/M phases, and appeared enlarged compared to the corresponding controls . The cellular enlargement occurred in both G1 and G2/M arrested cells . In contrast, cells exposed to an A . actinomycetemcomitans cdt-knockout mutant strain showed cell cycle phase distribution and size similar to the controls . In conclusion, A . actinomycetemcomitans causes a combined G1 and G2/M growth arrest and enlargement in periodontal connective tissue cells, which is attributed to its Cdt.

Mol Plant Microbe Interact, 2004 Dec, 17(12), 1328 - 36
Pseudomonas Type III effector AvrPto suppresses the programmed cell death induced by two nonhost pathogens in Nicotiana benthamiana and tomato; Kang L et al.; Many gram-negative bacterial pathogens rely on a type III secretion system to deliver a number of effector proteins into the host cell . Though a number of these effectors have been shown to contribute to bacterial pathogenicity, their functions remain elusive . Here we report that AvrPto, an effector known for its ability to interact with Pto and induce Pto-mediated disease resistance, inhibited the hypersensitive response (HR) induced by nonhost pathogen interactions . Pseudomonas syringae pv . tomato T1 causes an HR-like cell death on Nicotiana benthamiana . This rapid cell death was delayed significantly in plants inoculated with P . syringae pv . tomato expressing avrPto . In addition, P . syringae pv . tabaci expressing avrPto suppressed nonhost HR on tomato prf3 and ptoS lines . Transient expression of avrPto in both N . benthamiana and tomato prf3 plants also was able to suppress nonhost HR . Interestingly, AvrPto failed to suppress cell death caused by other elicitors and nonhost pathogens . AvrPto also failed to suppress cell death caused by certain gene-for-gene disease resistance interactions . Experiments with avrPto mutants revealed several residues important for the suppression effects . AvrPto mutants G2A, G99V, P146L, and a 12-amino-acid C-terminal deletion mutant partially lost the suppression ability, whereas S94P and 196T enhanced suppression of cell death in N . benthamiana . These results, together with other discoveries, demonstrated that suppression of host-programmed cell death may serve as one of the strategies bacterial pathoens use for successful invasion.

Protein Expr Purif, 2005 Jan, 39(1), 27 - 34
Cloning, expression, and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp . 22b; Cieslinski H et al.; The gram-negative antarctic bacterium Pseudoalteromonas sp . 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase . The gene encoding this beta-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized . The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues . The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG-Sepharose . Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp . 22b beta-galactosidase . The enzyme is cold-adapted and at 10 degrees C retains 20% of maximum activity . The purified enzyme displayed maximum activity close to 40 degrees C and at pH of 6.0-8.0 . PNPG was its preferred substrate (58% higher activity than against ONPG) . The enzyme was particularly thermolabile, losing all activities within 10min at 50 degrees C . The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6h at 30 degrees C and during 28h at 15 degrees C . Because of its attributes, the recombinant Pseudoalteromonas sp . 22b beta-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products.

Biochemistry, 2004 Dec 21, 43(50), 15767 - 74
SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange) analysis of the thermodynamics of synergistic anion binding by ferric-binding protein (FbpA), a bacterial transferrin; Roulhac PL et al.; SUPREX (stability of unpurified proteins from rates of H/D exchange) is a H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for characterizing the equilibrium unfolding/refolding properties of proteins and protein-ligand complexes . Here, we describe the application of SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-binding protein (Fe(3+)FbpA-X, X = synergistic anion) . The in vivo function of FbpA is to transport unchelated Fe(3+) across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneous binding of a synergistic anion . Our results indicate that Fe(3+)FbpA-X is not a so-called "ideal" protein system for SUPREX analyses because it does not exhibit two-state folding properties and it does not exhibit EX2 H/D exchange behavior . However, despite these nonideal properties of the Fe(3+)FbpA-X protein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to the quantitative thermodynamic analysis of synergistic anion binding to Fe(3+)FbpA . As part of this work, the SUPREX technique was used to evaluate the DeltaDeltaG(f) values of four synergistic anion-containing complexes of Fe(3+)FbpA (i.e., Fe(3+)FbpA-PO(4), Fe(3+)FbpA-citrate, Fe(3+)FbpA-AsO(4), and Fe(3+)FbpA-SO(4)) . The DeltaDeltaG(f) value obtained for Fe(3+)FbpA-citrate relative to Fe(3+)FbpA-PO(4) (1.45 +/- 0.44 kcal/mol), is in good agreement with that reported previously (1.98 kcal/mol) . The value obtained for Fe(3+)FbpA-AsO(4) (0.58 +/- 0.45 kcal/mol) was also consistent with that reported previously (0.68 kcal/mol), but the measurement error is very close to the magnitude of the value . This work (i) demonstrates the utility of the SUPREX method for studying anion binding by FbpA, (ii) provides the first evaluation of a DeltaDeltaG(f) value for Fe(3+)FbpA-SO(4), -1.43 +/- 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anion plays a role in controlling the lability of iron bound to FbpA in the transport process.

Microbiol Mol Biol Rev, 2004 Dec, 68(4), 771 - 95
Process of protein transport by the type III secretion system; Ghosh P; The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells . The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells . While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation . This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport . The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum . It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane . Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells . A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS . Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells . Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.

Microbiol Mol Biol Rev, 2004 Dec, 68(4), 692 - 744
Type V protein secretion pathway: the autotransporter story; Henderson IR et al.; Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides . To overcome this barrier, bacteria have developed several systems for protein secretion . The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins . Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria . Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis . However, most of the autotransporter proteins remain to be characterized . In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function.

J Med Microbiol, 2004 Dec, 53(Pt 12), 1187 - 93
Chlamydophila pneumoniae induces p44/p42 mitogen-activated protein kinase activation in human fibroblasts through Toll-like receptor 4; Haralambieva IH et al.; Chlamydophila pneumoniae, an obligately intracellular Gram-negative bacterium and a common causative agent of respiratory tract infections, has been implicated in the induction and progression of atherosclerosis and coronary artery disease . In this study, the signalling mechanism of C . pneumoniae in human fibroblasts, a prominent cell population in chronic inflammation and persistent infection, contributing to plaque formation, was investigated . C . pneumoniae elementary bodies were demonstrated to up-regulate the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) in human fibroblasts . The effect was independent of the chlamydial lipopolysaccharide and was likely to be mediated by a heat-labile chlamydial protein . Furthermore, an anti-Toll-like receptor 4 (TLR4) antibody was shown to abolish C . pneumoniae-induced cell activation, whereas an anti-TLR2 antibody had no effect, indicating the role of TLR4 in p44/p42 MAPK activation . Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 and phosphodiesterase 4 (PDE 4) inhibitor Rolipram enhanced C . pneumoniae-induced MAPK phosphorylation and attenuated C . pneumoniae infectivity in vitro . Together the results indicate that C . pneumoniae triggers rapid TLR4-mediated p44/p42 MAPK activation in human fibroblasts and chemical enhancement of MAPK phosphorylation modulates in vitro infection at the molecular level.

Immunol Lett, 2005 Jan 15, 96(1), 73 - 83
Polycationic lipids inhibit the pro-inflammatory response to LPS; Leon-Ponte M et al.; Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria . As such, it signals monocytes, macrophages and neutrophils to up-regulate phagocytic functions and to release pro-inflammatory cytokines . Despite the established role of CD14 as the main LPS receptor, the precise nature of the LPS signalling complex and its compartmentalization remain unknown . Interactions of LPS with other cell surface molecules such as TLR-4 and MD-2, and its subsequent internalization are required for LPS signalling . Here, we show that the polycationic lipid LipoFectaminetrade mark causes inhibition of the LPS-induced MAPK activation and lack of pro-inflammatory cytokine production, despite proper localization of CD14 within lipid rafts and massive LPS internalization . The ability of LipoFectaminetrade mark to inhibit LPS induced pro-inflammatory responses may be due to uncoupling of CD14 from TLR-4/MD-2 in the LPS signalling complex of mouse macrophages/microglial cells, as suggested by inhibition of LPS-induced concomitant internalization of these surface molecules . Thus, LipoFectaminetrade mark may be a useful tool to dissect the molecular interactions leading to LPS signalling, and identifies a potential therapeutic strategy for LPS clearance.

Comb Chem High Throughput Screen, 2004 Dec, 7(8), 733 - 47
Anti-endotoxin agents . 2 . Pilot high-throughput screening for novel lipopolysaccharide-recognizing motifs in small molecules; Wood SJ et al.; Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are an integral part of the outer leaflet of the outer-membrane of Gram-negative bacteria . Lipopolysaccharides play a pivotal role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient, worldwide . The sequestration of circulatory endotoxin may be a viable therapeutic strategy for the prophylaxis and treatment of Gram-negative sepsis . We have earlier shown that the pharmacophore necessary for small molecules to bind LPS is simple, comprising of two protonatable cationic functions separated by about 15 A, permitting the simultaneous interaction with the negatively charged phosphates on lipid A, the toxically active center of endotoxin . In this report, we employ high-throughput screening methods, using a novel fluorescent probe displacement method . Searches in three-dimensional structure databases yielded about approximately 4000 commercially available small molecules, each possessing two cationic functions spaced approximately 15 A apart . Approximately 400 such compounds have been screened in an effort to validate the method by which high-affinity endotoxin binders can be identified . We show that the IC50 values that are obtained from the fluorescence-based primary screen are correlated both to the enthalpy of binding, as measured by isothermal titration calorimetry, as well as to biological potency in vitro assays . By performing rapid toxicity screens in tandem with the bioassays, lead compounds of interest can be easily identified for further systematic structural modifications and SAR studies.

J Biol Chem . 2004 Dec 2; {Epub ahead of print}
Lipopolysaccharide transport to the bacterial outer membrane in spheroplasts; Tefsen B et al.; The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown . Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins . Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released . We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane . The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP) . We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes . In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts . Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1757 - 65
Effect of process variables on supercritical fluid disruption of Ralstonia eutropha cells for poly(R-hydroxybutyrate) recovery; Khosravi-Darani K et al.; This research focuses on the disruption of the gram-negative bacterium Ralstonia eutropha cells by supercritical CO2 for poly(R-hydroxybutyrate) (PHB) recovery . The variables affecting cell disruption such as drying strategy, type of modifier, and cultivation time, as well as operating pressure, temperature, and repeated release of supercritical CO2 pressure, have been studied . Effect of this disruption technique on PHB molecular mass was also investigated . PHB recovery was examined using a combination of this method and chemical pretreatments . For salt pretreatment, the cells were exposed to 140 mM NaCl and heat (60 degrees C, 1 h) . The cells were also exposed to 0.2-0.8% (w/w) NaOH to examine the effect of alkaline pretreatment . Bacterial cells treated in growth phase exhibited less resistance to disruption than nutrient-limited cells in the stationary phase . It was also found that the wet cells could be utilized to recover PHB, but purity of the product was lower than that obtained from freeze-dried cells . Pretreatment with a minimum of 0.4% (w/w) NaOH was necessary to enable complete disruption with two times pressure release . Salt pretreatment was less effective; however, disruption was improved by the application of alkaline shock . The proposed method is economic and comparable with other recovery methods in terms of the percentage of PHB recovery and energy consumption, while it is environmentally more benign.

Ambio, 2004 Nov, 33(7), 436 - 47
Effects on the structure of Arctic ecosystems in the short- and long-term perspectives; Callaghan TV et al.; Species individualistic responses to warming and increased UV-B radiation are moderated by the responses of neighbors within communities, and trophic interactions within ecosystems . All of these responses lead to changes in ecosystem structure . Experimental manipulation of environmental factors expected to change at high latitudes showed that summer warming of tundra vegetation has generally led to smaller changes than fertilizer addition . Some of the factors manipulated have strong effects on the structure of Arctic ecosystems but the effects vary regionally, with the greatest response of plant and invertebrate communities being observed at the coldest locations . Arctic invertebrate communities are very likely to respond rapidly to warming whereas microbial biomass and nutrient stocks are more stable . Experimentally enhanced UV-B radiation altered the community composition of gram-negative bacteria and fungi, but not that of plants . Increased plant productivity due to warmer summers may dominate food-web dynamics . Trophic interactions of tundra and sub-Arctic forest plant-based food webs are centered on a few dominant animal species which often have cyclic population fluctuations that lead to extremely high peak abundances in some years . Population cycles of small rodents and insect defoliators such as the autumn moth affect the structure and diversity of tundra and forest-tundra vegetation and the viability of a number of specialist predators and parasites . Ice crusting in warmer winters is likely to reduce the accessibility of plant food to lemmings, while deep snow may protect them from snow-surface predators . In Fennoscandia, there is evidence already for a pronounced shift in small rodent community structure and dynamics that have resulted in a decline of predators that specialize in feeding on small rodents . Climate is also likely to alter the role of insect pests in the birch forest system: warmer winters may increase survival of eggs and expand the range of the insects . Insects that harass reindeer in the summer are also likely to become more widespread, abundant and active during warmer summers while refuges for reindeer/caribou on glaciers and late snow patches will probably disappear.

Dermatol Ther, 2004, 17(6), 499 - 504
Treatment and prevention of rickettsial and ehrlichial infections; Maender JL et al.; Rickettsial and ehrlichial infections are both carried by arthropod vectors . Both Rickettsia and Ehrlichia are small intracellular gram-negative coccobacilli . Clinical manifestations of Rickettsia range from spotted fevers to various forms of typhus . Human ehrlichiosis can present as monocytic ehrlichiosis or granulocytic anaplasmosis . Prevention is by avoidance of the responsible vectors . Therapy is usually with doxycycline, but chloramphenicol can also be used.

Acta Microbiol Immunol Hung, 2004, 51(3), 351 - 8
The antimotility action of a trifluoromethyl ketone on some gram-negative bacteria; Spengler G et al.; The inhibition of bacterial motility was studied by a trifluoro methyl ketone derivative on two Escherichia coli strains (wild strain having a proton pump system and the proton pump-deficient mutant strain) and two Helicobacter pylori strains (clarithromycin susceptible and clarithromycin resistant) . Evidence is presented of the inhibitory action of 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18) on the proton motive forces of the two bacterial strains by affecting the action of biological motor and proton efflux in the membranes . The swimming, the forward motion was more sensitive than the vibration or tumbling to the inhibition . We suppose that the inhibiton of bacterial motility is related to the virulence of bacteria: consequently the pathogenicity can be reduced in the presence of TF18.

Photochem Photobiol Sci, 2004 Nov-Dec, 3(11-12), 992 - 8 Epub 2004 Nov-Dec.
Photodynamic activity of cationic and non-charged Zn(ii) tetrapyridinoporphyrazine derivatives: biological consequences in human erythrocytes and Escherichia coli; Dupouy EA et al.; The photodynamic activity of a cationic Zn(ii) tetramethyltetrapyridinoporphyrazinium salt (ZnPc ) was compared with that of a non-charged Zn(ii) tetrapyridinoporphyrazine (ZnPc ), both in vitro using human red blood (HRB) cells and a typical Gram-negative bacterium Escherichia coli . Absorption and fluorescence spectroscopic studies were analyzed in different media . Fluorescence quantum yields ({small phi}(F)) of 0.35 for ZnPc and 0.30 for ZnPc were calculated in N,N-dimethylformamide (DMF) . The singlet molecular oxygen, O(2)((1){capital Delta}(g)), production was evaluated using 9,10-dimethylanthracene (DMA) in DMF yielding values of {capital Phi}({capital Delta})= 0.56 for ZnPc and 0.50 for ZnPc . In biological medium, the photodynamic effect was first evaluated in HRB cells . Both phthalocyanines produce similar photohemolysis of HRB cells, reaching values >90% of lysis after 5 min of irradiation with visible light . The photodynamic effect is accompanied by an increase in the membrane fluidity of HRB cells . However, these studies on E . coli cells showed that the cationic ZnPc produces a higher photoinactivation of Gram-negative bacteria than ZnPc . Also, these results were established by stopped of growth curves for E . coli . Therefore the studies show that cationic ZnPc is an efficient phototherapeutic agent with potential applications in tumor cell and Gram-negative bacteria inactivation by photodynamic therapy.

Histopathology, 2004 Dec, 45(6), 633 - 7
Presence of nanobacteria in psammoma bodies of ovarian cancer: evidence for pathogenetic role in intratumoral biomineralization; Hudelist G et al.; AIMS: The presence of laminated, calcified extracellular debris known as psammoma bodies is a well-known histomorphological feature of ovarian adenocarcinomas and other human malignancies . Biomineralization has recently been found to be associated with a group of extremely small Gram-negative bacteria capable of precipitating calcium salts . The aim of the present study was to evaluate a possible pathogenic link between the development of psammoma bodies and nanobacteria infection . MATERIAL AND RESULTS: Immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyse nanobacterial protein and gene expression in eight psammona body-containing adenocarcinomas and in 10 malignant ovarian tumours without signs of biomineralization . Nanobacterial proteins were detected in eight out of eight (100%) psammoma-positive tumour samples . Conversely, none of the 10 psammoma-negative tissues (0%) was positive for nanobacterial antigens . Furthermore, nanobacterial mRNA was detectable in all of the four tissues (100%) that contained psammoma bodies, but was absent in all 10 ovarian cystadenocarcinomas (0%) that were psammoma negative . CONCLUSIONS: We found a 100% concordance between the expression of nanobacteria and the presence of psammoma bodies in malignant ovarian tumours . Several lines of evidence suggest the involvement of these organisms in the process of biomineralization . We therefore conclude that nanobacterial infection of malignant ovarian tissue contributes to mechanisms leading to the formation of calcified deposits known as psammoma bodies.

Genes Immun . 2004 Nov 25; {Epub ahead of print}
The TLR4 +896 polymorphism is not associated with lipopolysaccharide hypo-responsiveness in leukocytes; Imahara SD et al.; Toll-like receptor 4 (TLR-4) is required for detection of Gram negative bacterial infections by binding lipopolysaccharide (LPS) and for the initiation of inflammatory signaling . Recent studies have demonstrated that a nonsynonymous single-nucleotide polymorphism (Asp299Gly, A+896G) is associated with decreased endotoxin responsiveness and poor outcomes from sepsis . We show that human carriers of this polymorphism show no deficit in LPS induced peripheral blood mononuclear cell (PBMC) mitogen-activated protein kinase (MAPK) activity, no reduction in sensitivity to endotoxin, and variable differences in whole-blood inflammatory cytokine production . These results indicate that this mutation is not a primary determinant of human endotoxin sensitivity.Genes and Immunity advance online publication, 25 November 2004; doi:10.1038/sj.gene.6364147.

Clin Chim Acta, 2005 Jan, 351(1-2), 17 - 29
Clinical laboratory differentiation of infectious versus non-infectious systemic inflammatory response syndrome; Mitaka C; OBJECTIVE: To evaluate the accuracy of C-reactive protein (CRP), procalcitonin (PCT), neopterin, and endotoxin in the differential diagnosis of sepsis and non-infectious systemic inflammatory response syndrome (SIRS) . METHODS: A Medline database and references from identified articles were used to perform a literature search relating to the differential diagnosis of sepsis versus non-infectious SIRS . RESULTS: CRP, PCT, and neopterin are released both in sepsis and in non-infectious inflammatory disease . CRP and PCT are equally effective, although not perfect, in differentiating between sepsis and non-infectious SIRS . However, CRP and PCT have different kinetics and profiles . The kinetics of CRP is slower than that of PCT, and CRP levels may not further increase during more severe stages of sepsis . On the contrary, PCT rises in proportion to the severity of sepsis and reaches its highest levels in septic shock . PCT tends to be higher in nonsurvivor than in survivor . Therefore, PCT demonstrated a closer correlation with the severity of sepsis and outcome than CRP . Unlike CRP and PCT, neopterin is increased in viral infection as well as bacterial infection, and neopterin is also a useful indicator of sepsis . Endotoxemia was detected in no more than half of patients with Gram-negative bacteremia, and Gram-negative bacteremia was detected in half of patients with endotoxemia . CONCLUSIONS: The diagnostic capacity of PCT is superior to that of CRP due to the close correlation between PCT levels and the severity of sepsis and outcome . Neopterin is very useful in the diagnosis of viral infection . The endotoxin assay in combination with CRP, PCT, or neopterin may help as a diagnostic marker for Gram-negative bacterial infection.

J Biol Chem . 2004 Nov 23; {Epub ahead of print}
NF-kappa B-and C/EBP beta-driven interleukin-1beta gene expression and PAK1-mediated caspase 1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharide (LPS)-stimulated macrophages; Basak C et al.; Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer and gastric carcinoma . Interleukin 1 beta (IL-1 beta) is one of the potent proinflammatory cytokines elicited by H . pylori infection . We have dissected the signaling pathways leading to H . pylori LPS-induced IL-1 beta secretion . Both the NF- kappa B and the C/EBP beta binding elements of the IL-1 beta promoter drive LPS-induced IL-1 beta gene expression . NF-kappa B activation requires the classical TLR4-initiated signaling cascade leading to IB phosphorylaiton as well as PI3-K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBP beta activation requires PI3-K/Akt/p38 mitogen activated protein (MAP) kinase signaling . We observed a direct interaction between activated p38 MAP kinase and C/EBP beta, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBP beta . Most importantly, we observed a role of Rac1/PAK1 signaling in activation of caspase 1 which is necessary for maturation of pro- IL-1 beta . H . pylori LPS induced direct interaction between PAK1 and caspase 1 which was inhibited in cells transfected with dominant-negative Rac1 . PAK1 immunoprecipitated from lysates of H . pylori LPS-challenged cells was able to phosphorylate recombinant caspase 1, but not its S376A mutant . LPS-induced caspase 1 activation was abrogated in cells transfected with caspase 1(S376A) . These results suggested a role of PAK1-induced phosphorylation of caspase 1 at S376 in activation of caspase 1 . Our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase 1 phosphorylation, is crucial for caspase 1 activation . These studies also provide detailed insight into the regulation of IL-1 beta gene expression by H . pylori LPS, and are particularly important in the light of the observations that IL-1 beta gene polymorphisms are associated with increased risk of H . pylori-associated gastric cancer.

Klin Mikrobiol Infekc Lek, 2004 Oct, 10(5), 207 - 13
{Bartonelloses.}; Medkova Z; Bartonellae belong to less known causal agents of many human diseases . They are gram-negative bacteria growing slowly on culture media enriched with hemin or bovine serum . The genus Bartonella, which currently involves more than 15 species, is present worldwide . Bartonellae live in natural foci in dependence on the occurrence of natural host (rodents, felines, canidae, human) and insect vector (flea, tick, louse) . By reservoir animals they usually cause permanent intraerythrocytic bacteraemia without system inflammation symptoms . A classical example of a human disease is cat scratch disease (CSD) caused by Bartonella henselae and characterised by regional lymphagoitis and lymphadenitis . Increasing interest is being devoted to the ability of Bartonella sp . (e.i . B . quintana) to cause the opportune infections with diverse clinical manifestation: bacillary angiomatosis, specific liver and spleen vasculitis (peliosis hepatis, splenis), endocarditis and others . The issue of Bartonella infections is relatively new and its importance is still growing with increasing knowledge in this field.

Infect Immun, 2004 Dec, 72(12), 7315 - 7
In vitro model of Bartonella henselae-induced angiogenesis; Kirby JE; Bartonella henselae is a gram-negative pathogen that causes angiogenesis . Here, I establish in vitro models to study Bartonella-induced blood vessel formation . I found that B . henselae induces long-term endothelial survival and tubular differentiation within type I collagen matrix.

Infect Immun, 2004 Dec, 72(12), 7231 - 9
Differential regulation of inflammatory cytokine secretion by human dendritic cells upon Chlamydia trachomatis infection; Gervassi A et al.; Chlamydia trachomatis is an obligate intracellular gram-negative bacterium responsible for a wide spectrum of diseases in humans . Both genital and ocular C . trachomatis infections are associated with tissue inflammation and pathology . Dendritic cells (DC) play an important role in both innate and adaptive immune responses to microbial pathogens and are a source of inflammatory cytokines . To determine the potential contribution of DC to the inflammatory process, human DC were infected with C . trachomatis serovar E or L2 . Both C . trachomatis serovars were found to infect and replicate in DC . Upon infection, DC up-regulated the expression of costimulatory (B7-1) and cell adhesion (ICAM-1) molecules . Furthermore, chlamydial infection induced the secretion of interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12p70, IL-18, and tumor necrosis factor alpha (TNF-alpha) . The mechanisms involved in Chlamydia-induced IL-1beta and IL-18 secretion differed from those of the other cytokines . Chlamydia-induced IL-1beta and IL-18 secretion required infection with viable bacteria and was associated with the Chlamydia-induced activation of caspase-1 in infected host cells . In contrast, TNF-alpha and IL-6 secretion did not require that the Chlamydia be viable, suggesting that there are at least two mechanisms involved in the Chlamydia-induced cytokine secretion in DC . Interestingly, an antibody to Toll-like receptor 4 inhibited Chlamydia-induced IL-1beta, IL-6, and TNF-alpha secretion . The data herein demonstrate that DC can be infected by human C . trachomatis serovars and that chlamydial components regulate the secretion of various cytokines in DC . Collectively, these data suggest that DC play a role in the inflammatory processes caused by chlamydial infections.

Infect Immun, 2004 Dec, 72(12), 7202 - 11
Toll-like receptor 9 can be expressed at the cell surface of distinct populations of tonsils and human peripheral blood mononuclear cells; Eaton-Bassiri A et al.; Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9) . However, the nature and cellular localization of TLR9 in primary human cells remain controversial . Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface . The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed . Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9(+) cells were confined to the major histocompatibility complex (MHC) class II(+) CD19(-) populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II(+) CD19(+) cells . Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10% . In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression . Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization . Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.

Am J Gastroenterol, 2004 Nov, 99(11), 2147 - 9
Novosphingobium aromaticivorans: a potential initiator of primary biliary cirrhosis; Kaplan MM; Primary biliary cirrhosis (PBC) is characterized by a T-cell-mediated destruction of bile duct epithelial cells that line the small intrahepatic bile ducts . The targets of activated T-lymphocytes are the dihydrolipoamide acetyltransferase components of the 2 oxo acid dehydrogenases, enzyme complexes that are important in oxidative energy metabolism . Pyruvate dehydrogenase is the best known of these . Its dihydrolipoamide acetyltransferase component is referred to as PDC-E2 . A major question in understanding the pathogenesis of PBC is why PBC patients lose their tolerance to antigens that are found in virtually every cell in the body . A possible cause is molecular mimicry between microbial agents and self-antigens . Infection with or exposure to a microorganism whose PDC-E2 bears close homology with human PDC-E2 could act as an immunological trigger that initiates the development of PBC . Emerging data suggest that there is a microorganism that may initiate the onset of PBC . Novosphingobium aromaticivorans is a gram negative strictly aerobic bacteria that is found worldwide in soil, water, and coastal plain sediments . Its PDC-E2-like proteins have a higher degree of homology with the immunodominant region of human PDC-E2 than any microorganism thus far studied (100-1,000 times greater than that of Escherichia coli) . In addition, N . aromaticivorans can metabolize xenobiotics that are similar to the chemical compounds that react with sera from PBC patients . Some of these xenobiotics are immunologically related to lipoic acid, the cofactor that is at the active center of PDC-E2 . Thus, N . aromaticivorans can theoretically break down self-tolerance in two ways: by molecular mimicry due to subclinical infection and by the metabolism of xenobiotics that are present in the environment . In an initial study, investigators found that antibodies against N . aromaticivorans were found in 77 of 77 PBC patients from Milan, Italy, who had antibodies to PDC-E2 and that the titers to N . aromaticivorans proteins were similar to those to human PDC-E2 . The report in this issue of The American Journal of Gastroenterology confirms these earlier findings and demonstrates that exposure to N . aromaticivorans occurs in genetically different PBC patients from other regions . Thirteen of 14 Icelandic PBC patients who were AMA positive reacted against at least one of the 2 oxo acid dehydrogenase-E2 complexes . These observations provide additional evidence that exposure to N . aromaticivorans may trigger the development of PBC.

Clin Exp Pharmacol Physiol, 2004 Oct, 31(10), 723 - 31
Role of endogenous macrophage inflammatory protein-2 in regulating fever induced by bacterial endotoxin in normal and immunosuppressed rats; Minano FJ et al.; During myelosuppressive chemotherapy, Gram-negative bacterial infection with consequent exposure to lipopolysaccharide (LPS) is one of the most important causes of persistent fever . The classical model of the pathogenesis of fever suggests that pro-inflammatory cytokines, produced by leucocytes in the bloodstream in response to exogenous pyrogens such as bacterial LPS, represent the distal mediators of the febrile response . Neutrophils are the first effectors cells and the most prominent leucocyte population involved in acute bacterial infection . Macrophage inflammatory protein (MIP)-2 plays a crucial role in influencing early cell trafficking and neutrophil activation during pathophysiological processes and serves the same chemotactic function as human interleukin-8 . In the present study, we investigated the role of MIP-2 in the development of a febrile response induced by LPS in immunocompetent and leukopenic rats . Intraperitoneal injection of LPS in leukopenic rats elicited a biphasic febrile response of rapid onset, the magnitude and duration of which were significantly greater than in immunocompetent animals . The febrile responses to LPS were accompanied by a pronounced induction of serum MIP-2 levels at 1, 2 and 4 h compared with their respective controls . In both normal and leukopenic rats, neutralization of endogenous MIP-2 bioactivity by systemic administration of antirat MIP-2 antibody caused a significant attenuation of the early phase of LPS fever . However, in contrast with normal rats, the second phase of fever was unimpaired by anti-MIP-2 in leukopenic rats . These findings suggest that circulating MIP-2 is involved in the generation of the early phase of LPS fever that contributes to the maintenance of the later phase of fever in immunocompetent, but not leukopenic, rats . Our data support a regulatory role for endogenous MIP-2 in initiating the fever responses to LPS . Furthermore, these results provide evidence that different cellular and humoral mechanisms are implicated in the development of a febrile response triggered by Gram-negative bacterial infections in leukopenic hosts.

Drug Des Discov, 2003, 18(4), 109 - 16
Conformational analysis of globomycin with a signal peptidase II inhibitory activity using molecular dynamics simulation; Kiho T et al.; Globomycin (1), a 19-membered cyclic depsipeptide, exhibited an antibiotic activity against gram-negative bacteria by inhibiting signal peptidase II in the cytoplasmic membrane . Although only one conformation of 1 was observed for the crystal structure, it was revealed by 1H NMR spectroscopic analysis that 1 exists as a mixture of two rotational isomers in solution (CDCl3 and CD3OD) . A conformational analysis of 1 was, therefore, performed by high-temperature molecular dynamics simulation in combination with 1H NMR analysis to elucidate the conformations in solution . The relative ratio of the major and minor isomers present, which differs depending on the solvent, was then derived from their relative energy differences obtained in the conformational analysis . The difference in the relative ratios corresponded with that calculated from the 1H NMR analysis . Finally, the predicted conformations in solution were compared with that of the X-ray crystal structure to find local and global differences that characterize these conformations.

Mol Plant Microbe Interact, 2004 Nov, 17(11), 1259 - 68
NopB, a soybean cultivar-specificity protein from Sinorhizobium fredii USDA257, is a type III secreted protein; Lorio JC et al.; The type III secretion system (TTSS) of plant- and animal-pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes . Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max (L.) Merr.) . This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction . Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium . We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257 . The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NoIB) . Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB . Similarly, a nonpolar nopB deletion mutant was compromised in its ability to secrete several Nops . A construct containing the coding region of nopB under control of a T7 promoter was expressed successfully in Escherichia coli and, subsequently, the recombinant NopB was purified by nickel-affinity column chromatography . Polyclonal antibodies raised against purified recombinant NopB were used in Western blot analysis to demonstrate the association of NopB with pilus-like surface appendages . Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent protein reporter . Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB . We also show that USDA257 and its nonpolar nopB deletion mutant exhibited discernible differences in their ability to nodulate legume hosts.

Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 2003, 14(2), 133 - 41
{Comparison of a rapid antigen test and cultures for diagnosis of meningitis in children}; Hashikita G et al.; Fourteen pediatric patients diagnosed as bacterial meningitis between August 1997 and April 2002 were enrolled in this study . Both rapid antigen detection test, Slidex Meningite 5 Kit (Biomerieux) and culture were performed using cerebrospinal fluids (CSF) . H . influenzae was isolated from 11 samples and was the most frequently isolated bacteria, followed by S . pneumoniae from 4 samples and enteric bacteriae from 2 samples . Five out of six samples with positive result by culture were also positive by the rapid antigen test . Gram-negative rod was identified in smear specimens of CSF from all these 5 samples . Significance of the rapid antigen test should be recognized under drug resistance of those bacteriae are increasing.

J Infect Dis, 2004 Dec 15, 190(12), 2121 - 8 Epub 2004 Dec 15.
Virulence factor profiles and phylogenetic background of Escherichia coli isolates from veterans with bacteremia and uninfected control subjects; Sannes MR et al.; BACKGROUND: Escherichia coli is the most common cause of gram-negative bloodstream infections, causing an estimated 40,000 deaths from sepsis each year in the United States . The present study sought to determine specifically which virulence factors (VFs) and phylogenetic groups of E . coli are epidemiologically associated with bacteremia . METHODS: E . coli isolates from 63 veterans with bacteremia and rectal isolates from 71 matched uninfected control subjects were compared both for phylogenetic group and for the presence of VFs and O antigens . RESULTS: Bacteremia isolates exhibited a significantly greater prevalence of most VFs studied . In multivariate logistic regression analysis, ompT (outer membrane protein T) was the strongest VF predictor of bacteremia (P<.001) . Despite the concentration of most individual VFs within group B2, bacteremia and rectal isolates differed little by phylogenetic distribution, a finding explained by the greater prevalence of VFs among bacteremia isolates than rectal isolates within groups B2 and D . CONCLUSIONS: Although phylogenetic group partially corresponds with virulence potential in E . coli bacteremia, VFs are more-powerful predictors of pathogenic potential . Bacteremia isolates exhibit an arsenal of VFs that distinguishes them from rectal isolates from uninfected hosts, which makes these differences attractive potential targets in vaccine or drug development.

Neoplasia, 2004 Sep-Oct, 6(5), 423 - 31
Chemotherapy-induced and/or radiation therapy-induced oral mucositis--complicating the treatment of cancer; Naidu MU et al.; The term mucositis is coined to describe the adverse effects of radiation and chemotherapy treatments . Mucositis is one of the most common adverse reactions encountered in radiation therapy for head and neck cancers, as well as in chemotherapy, in particular with drugs affecting DNA synthesis (S-phase-specific agents such as fluorouracil, methotrexate, and cytarabine) . Mucositis may limit the patient's ability to tolerate chemotherapy or radiation therapy, and nutritional status is compromised . It may drastically affect cancer treatment as well as the patient's quality of life . The incidence and severity of mucositis will vary from patient to patient . It will also vary from treatment to treatment . It is estimated that there is 40% incidence of mucositis in patients treated with standard chemotherapy and this will not only increase with the number of treatment cycles but also with previous episodes . Similarly, patients who undergo bone marrow transplantation and who receive high doses of chemotherapy have a 76% chance of getting mucositis . Patients receiving radiation, in particular to head and neck cancers, have a 30% to 60% chance . The exact pathophysiology of development is not known, but it is thought to be divided into direct and indirect mucositis . Chemotherapy and/or radiation therapy will interfere with the normal turnover of epithelial, cells leading to mucosal injury; subsequently, it can also occur due to indirect invasion of Gram-negative bacteria and fungal species because most of the cancer drugs will cause changes in blood counts . With the advancement in cytology, a more precise mechanism has been established . With this understanding, we can select and target particular mediators responsible for the mucositis . Risk factors such as age, nutritional status, type of malignancy, and oral care during treatment will play important roles in the development of mucositis . Many treatment options are available to prevent and treat this condition, but none of them can completely prevent or treat mucositis . More and more pathological methods are being developed to understand this condition so that better therapeutic regimens can be selected . Emphasis also should be made in assessing the patient's psychologic condition, particular depressive disorders . This is important because treatment with antidepressants will not only contribute in lifting depression but also reduces pain somatization . Although mucositis is rarely life-threatening, it will interfere with treatment of cancer to a great extent.

Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 235 - 57
Protein secretion through autotransporter and two-partner pathways; Jacob-Dubuisson F et al.; Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity . Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains . As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter . In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters . The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria . Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane . Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface . A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter . In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules . In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.

Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 181 - 206
Type III protein secretion mechanism in mammalian and plant pathogens; He SY et al.; The type III protein secretion system (TTSS) is a complex organelle in the envelope of many Gram-negative bacteria; it delivers potentially hundreds of structurally diverse bacterial virulence proteins into plant and animal cells to modulate host cellular functions . Recent studies have revealed several basic features of this secretion system, including assembly of needle/pilus-like secretion structures, formation of putative translocation pores in the host membrane, recognition of N-terminal/5' mRNA-based secretion signals, and requirement of small chaperone proteins for optimal delivery and/or expression of effector proteins . Although most of our knowledge about the TTSS is derived from studies of mammalian pathogenic bacteria, similar and unique features are learned from studies of plant pathogenic bacteria . Here, we summarize the most salient aspects of the TTSS, with special emphasis on recent findings.

Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 163 - 79
The underlying mechanisms of type II protein secretion; Filloux A; The cell envelope of Gram-negative bacteria is composed of two membranes, which are separated by the peptidoglycan-containing periplasm . Whereas the envelope forms an essential barrier against harmful substances, it is nevertheless a compartment of intense traffic for large proteins such as enzymes and toxins . Numerous studies dealing with the molecular mechanism of protein secretion have revealed that Gram-negative bacteria evolved different strategies to achieve this process . Among them, the type II secretion mechanism is part of a two-step process . Exoproteins following this pathway are synthesized as signal peptide-containing precursors . After cleavage of the signal peptide, the mature exoproteins are released into the periplasm, where they fold . The type II machinery, also known as the secreton, is responsible for the translocation of the periplasmic intermediates across the OM . The type II system is broadly conserved in Gram-negative bacteria and involves a set of 12-16 different proteins named GspC-M, GspAB, GspN, GspO, and GspS . The type II secretion system is highly reminiscent of the type IV piliation assembly system . Based on findings about the subcellular localisation of the Gsp components, protein-protein interactions between Gsps and their multimerisation status, structural data and electron microscopy observation, it could be proposed a working model that strikingly runs both systems in parallel.

Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 149 - 61
Type I secretion in gram-negative bacteria; Delepelaire P; In gram-negative bacteria, type I secretion is carried out by a translocator made up of three proteins that span the cell envelope . One of these proteins is a specific outer membrane protein (OMP) and the other two are cytoplasmic membrane proteins: an ATP-binding cassette (ABC) and the so-called membrane fusion or adaptor protein (MFP) . Type I secretion is sec-independent and bypasses the periplasm . This widespread pathway allows the secretion of proteins of diverse sizes and functions via a C-terminal uncleaved secretion signal . This C-terminal secretion signal specifically recognizes the ABC protein, triggering the assembly of the functional trans-envelope complex . This report will mainly deal will recent data concerning the structure and assembly of the secretion complex as well as the effects and role of substrate folding on secretion by this pathway.

Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 121 - 34
Quality control in the bacterial periplasm; Duguay AR et al.; Studies of the mechanisms that Gram-negative bacteria use to sense and respond to stress have led to a greater understanding of protein folding in both cytoplasmic and extracytoplasmic locations . In response to stressful conditions, bacteria induce a variety of stress response systems, examples of which are the sigma(E) and Cpx systems in Escherichia coli . Induction of these stress response systems results in upregulation of several gene targets that have been shown to be important for protein folding under normal conditions . Here we review the identification of stress response systems and their corresponding gene targets in E . coli . In addition, we discuss the apparent redundancy of the folding factors in the periplasm, and we consider the potential importance of the functional overlap that exists.

Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 5 - 16
Translocation of bacterial proteins-an overview; Holland IB; Recent progress in the understanding of the nature of the extraordinary variety of protein translocation systems, mainly in Gram negative bacteria, is reviewed . This takes us from the insertion of proteins into the inner membrane via the sophisticated Sec apparatus, the lethal injection of Type III proteins into host cells and on to the beautiful machine that assembles the flagellum . Attempts are made to establish some order, some common principles that might explain the variety and the complexity of some systems . The fundamentals considered are the nature of different transport signals, the nature of translocons (a wide variety of inner membrane types, outer membrane translocons are more conserved), the process of docking to translocons, the role of chaperones and the folding of transported proteins, the energetics of translocation, and prospects for future advances.

Mar Biotechnol (NY), 2004 Jul-Aug, 6(4), 335 - 46 Epub 2004 Jun 23.
Isolation and characterization of a fucoidan-degrading marine bacterial strain and its fucoidanase; Sakai T et al.; A marine bacterial strain that degraded fucoidan from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) was isolated in our laboratory . The strain was gram-negative, ubiquinone 8 was the predominant respiratory quinone, and the GC-content of its genomic DNA was 36% . The cells of the strain were rod-shaped (2.0 microm long x 1.0 microm wide), and each cell was motile by means of one polar flagellum . Phylogenetic analysis of its 16S ribosomal DNA sequence indicated that it was a member of the family Alteromonadaceae . It produced a type of extracellular fucoidanase, an endosulfated fucan-digesting enzyme . The enzyme was purified with 3500-fold purity at 12.0% yield . Optimum conditions for the enzyme reaction were approximately pH 6.5 to 8.0 and temperature 30 degrees to 35 degrees C . The enzyme was activated by calcium ions, and maximum activity was observed in the presence of greater than 30 mM calcium ion.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2263 - 7
Saccharibacter floricola gen . nov., sp . nov., a novel osmophilic acetic acid bacterium isolated from pollen; Jojima Y et al.; Three Gram-negative, aerobic, rod-shaped bacterial strains were isolated, from the pollen of Japanese flowers, as producers of xylitol; these strains were subjected to a polyphasic taxonomic study . Phylogenetic analyses of the 16S rRNA gene sequences demonstrated that these three isolates formed a new cluster within a group of acetic acid bacteria in the alpha-Proteobacteria . The characteristics of the three isolates were as follows: (i) their predominant quinone was Q-10; (ii) their cellular fatty acid profile contained major amounts of 2-hydroxy acids and an unsaturated straight-chain acid (C(18 : 1)omega7c); and (iii) their DNA G+C contents were in the range 51.9-52.3 mol%, which is around the lower limit of the reported range for the genera of acetic acid bacteria . The negligible or very weak productivity of acetic acid from ethanol and the osmophilic growth properties distinguished these strains from other acetic acid bacteria . The unique phylogenetic and phenotypic characteristics suggest that the three isolates should be classified within a novel genus and species with the proposed name Saccharibacter floricola gen . nov., sp . nov . The type strain is strain S-877(T) (=AJ 13480(T)=JCM 12116(T)=DSM 15669(T)).

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2223 - 30
Proposals of Curvibacter gracilis gen . nov., sp . nov . and Herbaspirillum putei sp . nov . for bacterial strains isolated from well water and reclassification of {Pseudomonas} huttiensis, {Pseudomonas} lanceolata, {Aquaspirillum} delicatum and {Aquaspirillum} autotrophicum as Herbaspirillum huttiense comb . nov., Curvibacter lanceolatus comb . nov., Curvibacter delicatus comb . nov . and Herbaspirillum autotrophicum comb . nov; Ding L et al.; Two strains of curved bacteria, 7-1(T) and 7-2(T), isolated from well water, were phylogenetically examined to determine their taxonomic position . Strain 7-1(T) is a Gram-negative, slightly curved rod . Analysis of the 16S rRNA gene sequence showed that strain 7-1(T) formed a cluster with {Aquaspirillum} delicatum and {Pseudomonas} lanceolata . It has some similar characteristics to {A.} delicatum and {P.} lanceolata, but has sufficient distance to separate it from other genera . DNA-DNA hybridization analysis, as well as chemotaxonomic and morphological studies, demonstrated that strain 7-1(T), {A.} delicatum and {P.} lanceolata belong to a new genus, Curvibacter gen . nov . Strain 7-1(T) (=IAM 15033(T)=ATCC BAA-807(T)) is classified as the type strain of Curvibacter gracilis gen . nov., sp . nov., and {A.} delicatum and {P.} lanceolata are classified as Curvibacter delicatus comb . nov . and Curvibacter lanceolatus comb . nov., respectively . Strain 7-2(T) is a Gram-negative spirillum . Phylogenetic study based on the 16S rRNA gene sequences showed that it formed a cluster with the members of the genus Herbaspirillum, {Pseudomonas} huttiensis and {Aquaspirillum} autotrophicum . The classification is therefore proposed of strain 7-2(T) (=IAM 15032(T)=ATCC BAA-806(T)) as the type strain of Herbaspirillum putei sp . nov., and {P.} huttiensis and {A.} autotrophicum are transferred to the genus Herbaspirillum as Herbaspirillum huttiense comb . nov . and Herbaspirillum autotrophicum comb . nov., respectively.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2185 - 90
Sphingomonas oligophenolica sp . nov., a halo- and organo-sensitive oligotrophic bacterium from paddy soil that degrades phenolic acids at low concentrations; Ohta H et al.; The taxonomic position of a halo- and organo-sensitive, oligotrophic soil bacterium, strain S213(T), was investigated . Cells were Gram-negative, non-motile, strictly aerobic, yellow-pigmented rods of short to medium length on diluted nutrient broth . When 0.1-0.4 % (w/v) NaCl was added to diluted media composed of peptone and meat extract, growth was inhibited with increasing NaCl concentration and the cells became long aberrant forms . When 6 mM CaCl(2) was added, the cells grew quite normally and aberrant cells were no longer found at 0.1-0.5 % (w/v) NaCl . Chemotaxonomically, strain S213(T) contains chemical markers that indicate its assignment to the Sphingomonadaceae: the presence of ubiquinone Q-10 as the predominant respiratory quinone, C(18 : 1) and C(16 : 0) as major fatty acids, C(14 : 0) 2-OH as the major 2-hydroxy fatty acid and sphingoglycolipids . 16S rRNA gene sequence analysis indicated that strain S213(T) belongs to the genus Sphingomonas, exhibiting high sequence similarity to the 16S rRNA gene sequences of Sphingomonas mali IFO 15500(T) (98.3 %), Sphingomonas pruni IFO 15498(T) (98.0 %), Sphingomonas asaccharolytica IFO 15499(T) (97.9 %) and Sphingomonas echinoides DSM 1805(T) (97.8 %) . The results of DNA-DNA hybridization experiments and its phenotypic characteristics clearly distinguished the strain from its nearest neighbours and demonstrate that strain S213(T) represents a novel Sphingomonas species, for which the name Sphingomonas oligophenolica sp . nov . is proposed . The type strain is S213(T) (=JCM 12082(T)=CIP 107926(T)).

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2147 - 50
Sphingomonas phyllosphaerae sp . nov., from the phyllosphere of Acacia caven in Argentina; Rivas R et al.; Two bacterial strains (FA1 and FA2(T)) were isolated from the phyllosphere of a leguminous tree, Acacia caven, in central Argentina . The strains were Gram-negative, strictly aerobic, rod-shaped, motile and formed yellow-pigmented colonies on nutrient agar . The two-primer RAPD patterns of the two strains were identical, suggesting that they belong to the same species . The complete 16S rRNA gene sequences of the two strains were obtained and comparisons demonstrated that they cluster phylogenetically with the species of the genus Sphingomonas sensu stricto . Strain FA2(T) was most closely related (97.6 %) to Sphingomonas adhaesiva . 16S rRNA gene sequence similarities to all other established Sphingomonas species ranged from 94.4 % (to Sphingomonas echinoides) to 97.6 % (to S . adhaesiva) . Strains FA1 and FA2(T) were catalase-positive and oxidase-negative . Aesculin was hydrolysed, gelatin and urea were not . beta-Galactosidase was produced . From 51 compounds tested 21 were used as single sources of carbon . The major respiratory lipoquinone was ubiquinone-10 . The predominant cellular fatty acids were 16 : 0, 18 : 1omega7c and 16 : 1omega7c (from summed feature 3) . Hydroxy fatty acids 14 : 0 2-OH and 15 : 0 iso 2-OH were present as well (from summed feature 4) . The polar lipids detected in strain FA2(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and two unidentified phospholipids . The DNA G+C content of strain FA2(T) was 61 mol% . DNA-DNA hybridization experiments showed 27.6 % relatedness between strain FA2(T) and S . adhaesiva DSM 7418(T) . Based upon phenotypic and molecular evidence, a novel species of the genus Sphingomonas is proposed, Sphingomonas phyllosphaerae sp . nov., with strain FA2(T) (=LMG 21958(T)=CECT 5832(T)) as the type strain.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 1969 - 73
Algoriphagus antarcticus sp . nov., a novel psychrophile from microbial mats in Antarctic lakes; Van Trappen S et al.; A taxonomic study was performed on six strains isolated from microbial mats of lakes Reid, Fryxell and Ace in Antarctica . Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belonged to the family 'Flexibacteraceae' and were closely related to the recently described genera Algoriphagus and Hongiella . The isolates were Gram-negative, chemoheterotrophic, aerobic, psychrophilic, orange-red-pigmented bacteria and their DNA G+C content ranged from 39.9 to 41.0 mol% . Whole-cell fatty acid profiles included mainly branched fatty acids and summed feature 3, comprising 15 : 0 iso 2OH, 16 : 1omega7c or both . On the basis of genotypic, phenotypic, chemotaxonomic and phylogenetic results, the novel strains were classified as Algoriphagus antarcticus sp . nov . The type strain is LMG 21980(T) (=DSM 15986(T)=R-10710(T)).

J Dairy Sci, 2004 Dec, 87(12), 4132 - 44
Increase of Escherichia coli inoculum doses induces faster innate immune response in primiparous cows; Vangroenweghe F et al.; The objective of the current study was to evaluate the dynamics of infection and the immunological response to varying numbers of Escherichia coli injected into the mammary glands of primiparous cows during the periparturient period . Primiparous cows have been shown to be more resistant to intramammary E . coli challenge, and an increase of the inoculum dose by 2 log10 units induced a more rapid clinical response and clearance of the organisms . Recognition of lipopolysaccharide (LPS) is a key event in the innate immunity response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP) . Primiparous cows were inoculated with 1 x 10(4) (Group A; n=8) or 1 x 10(6) (Group B; n=8) cfu E . coli P4:O32 in their 2 left quarters during the periparturient period . Clinical examination and analysis of blood and milk parameters, including IL-8, complement fragment 5a (C5a), LBP, and soluble CD14 (sCD14), were performed from d -4 to d +3 relative to infection . Primiparous cows in Group B initiated a more rapid clinical response following intramammary infection (IMI), resulting in typical clinical signs and changes in blood and milk parameters approximately 3 h earlier compared with primiparous cows in Group A . Based on average milk production in the noninfected quarters on d +2 postinoculation, all heifers reacted as moderate responders . Distinct differences in the kinetic patterns of rectal temperature, somatic cell count (SCC), IL-8, C5a, LBP, and sCD14 were observed between both groups during the early phase of inflammation . Both C5a and IL-8 increased before cellular influx into the infected glands, followed by increases in sCD14 and LBP . In conclusion, primiparous cows are able to clear an intramammary E . coli infection efficiently . Moreover, increasing the inoculum dose induces a more rapid inflammatory reaction, mainly because of early activation of the innate host immune response.

Vaccine, 2004 Dec 16, 23(5), 709 - 17
Efficacy of an Escherichia coli J-5 mutant strain bacterin in the protection of calves from endotoxin disease caused by subcutaneous challenge with endotoxins from Escherichia coli; Deluyker HA et al.; The purpose of this trial was to examine the potential of a new Escherichia (E) coli J-5 mutant strain bacterin to reduce the severity of clinical disease caused by subcutaneous challenge with endotoxins of Gram-negative bacteria in calves . Day-old to 3-day old calves (n = 40 per study phase) were randomly assigned to either of two treatment groups, i.e . a vaccinated or a placebo group . Calves in the vaccinated group received an inactivated bacterin containing a J-5 mutant strain of E . coli via subcutaneous route at 2-4 days of age and at 14 days thereafter . The placebo contained only adjuvant and saline in lieu of the antigen . Lipopolysaccharides (LPS) originating from E . coli were administered subcutaneously 3 weeks after the booster dose . The LPS challenge dosages were 1 and 8 microg/kg in study phases I and II, respectively . Various clinical, physiological, hematological, and serological parameters were measured at specific time intervals after challenge . The data were mostly analysed using peak changes from baseline recorded during the observation period . By the time of challenge the titers in vaccinated calves had increased significantly more than in the unvaccinated controls . Disease severity following subcutaneous challenge was dose dependent . In phase I, placebo calves were only mildly challenged whereas in phase II placebo calves showed a moderate challenge . After a mild challenge, there was little evidence of protection due to vaccination as only attitude was significantly improved in the vaccinates . In contrast, after a moderate challenge rectal temperature, hematocrit, blood glucose concentrations, and leukocyte changes were significantly better in the vaccinated group . In conclusion, the results of this study show that following a subcutaneous endotoxin challenge that induces a moderate clinical response, calves that were previously vaccinated with the E . coli J-5 bacterin were better protected than those in the placebo group.

Clin Diagn Lab Immunol, 2004 Nov, 11(6), 1060 - 3
Monoclonal antibodies for specific detection of Encephalitozoon cuniculi; Mo L et al.; Seven species-specific monoclonal antibodies (MAbs) were produced against Encephalitozoon cuniculi and characterized . The MAbs were immunoglobulin G, and when used for indirect microimmunofluorescence microscopy and Western immunoblot assays, they detected E . cuniculi originating from clinical samples . They did not cross-react with other Encephalitozoon species (E . intestinalis and E . hellem) or with a collection of gram-negative bacteria, yeast, and other parasites . The MAbs reacted primarily with 121-, 56-, 45-, 43-, and 41-kDa protein epitopes of E . cuniculi . These epitopes were demonstrated to be E . cuniculi species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . We developed MAbs to strains of E . cuniculi that can be used successfully to distinguish E . cuniculi from other microsporidial species in cultures established from clinical specimens . These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification and diagnosis of infections due to microsporidia.

Biochem J . 2004 Nov 10; {Epub ahead of print}
Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression; Roger T et al.; Toll-like receptor 4 (TLR4) is essential for sensing endotoxin of Gram-negative bacteria . Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis . Herein we studied the expression and the regulation of the mouse Tlr4 gene . In vivo, TLR4 levels were higher in macrophages than in B, T or NK cells . High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector . Analysis of truncated and mutated promoter constructs identified several positive (two Ets and one AP-1 sites) and negative (a GATA-like and an octamer sites) regulatory elements within 350 bp upstream of the transcriptional start site . The myeloid and B cell specific transcription factor PU.1 bound to the proximal Ets site . By contrast, neither PU.1 nor Ets-1, Ets-2 or Elk-1, but possibly a member of the ESE sub-family of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription . Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady state mRNA levels by increasing TLR4 transcripts turnover . TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin that strongly increased promoter activity and TLR4 mRNA levels . The mouse and human TLR4 genes are highly conserved . Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

Avian Dis, 2004 Sep, 48(3), 522 - 30
A prospective study of management and litter variables associated with cellulitis in California broiler flocks; Schrader JS et al.; Cellulitis has emerged as an economically important disease of broiler chickens . The impact of environmental risk factors on the incidence of cellulitis has not been evaluated in the United States . Escherichia coli (E . coli), the causative agent, is introduced through skin scratches during the grow out . Our previous work suggested that the litter was an important reservoir for cellulitis-associated E . coli . We hypothesized that factors contributing to a positive environment for E . coli growth would increase the opportunity for exposure of a broiler to an infectious dose of E . coli, capable of initiating a cellulitis lesion . This prospective study of 304 flocks on five farms from two integrated broiler companies was conducted to determine the effect of environmental factors on the prevalence of cellulitis in California broiler flocks . Environmental variables included temperature, wind velocity, and relative humidity (RH) at the litter surface . Litter variables measured included E . coli and total gram-negative bacteria load (colony forming units/g dry matter), water activity, and pH . Management variables such as clean out, the number of flocks reared on the same litter (litter run, LR), and downtime (DT) between flocks were also evaluated . Cellulitis ranged from 0.197% to 6.04% . Significant associations were identified using linear regression between farm, LR, DT, ambient temperature during the brooding period, gram-negative bacteria load in the litter during the brooding period, RH mid-grow out, and E . coli load late in the grow out . The significant variation in the rate of cellulitis between farms combined with the strong association of LR and DT with cellulitis demonstrated that management choices were highly influential in this disease syndrome . Based on these data and our previous findings, managers would be advised to increase DT between flocks and perform a total clean out of the house when a flock processes with a high incidence of cellulitis.

J Clin Microbiol, 2004 Nov, 42(11), 5286 - 97
Characterization of "Candidatus piscichlamydia salmonis" (order Chlamydiales), a chlamydia-like bacterium associated with epitheliocystis in farmed Atlantic salmon (Salmo salar); Draghi A 2nd et al.; To characterize intracellular gram-negative bacteria associated with epitheliocystis in farmed Atlantic salmon (Salmo salar), gills with proliferative lesions were collected for histopathology, conventional transmission and immunoelectron microscopy, in situ hybridization, and DNA extraction during epitheliocystis outbreaks in Ireland and Norway in 1999 and 2000, respectively, and compared by ultrastructure and immunoreactivity to nonproliferative gills from Ireland archived in 1995 . Genomic DNA from proliferative gills was used to amplify 16S ribosomal DNA (rDNA) for molecular phylogenetic analyses . Epitheliocystis inclusions from proliferative gills possessed variably elongate reticulate bodies, examples of binary fission, and vacuolated and nonvacuolated intermediate bodies, whereas inclusions in nonproliferative gills had typical chlamydial developmental stages plus distinctive head-and-tail cells . Immunogold processing using anti-chlamydial lipopolysaccharide antibody labeled reticulate bodies from proliferative and nonproliferative gills . 16S rDNA amplified directly from Irish (1999) and Norwegian (2000) gill samples demonstrated 99% nucleotide identity, and riboprobes transcribed from cloned near-full-length 16S rDNA amplicons from Norwegian gills hybridized with inclusions in proliferative lesions from Irish (1999) and Norwegian (2000) sections . A 1,487-bp consensus 16S rRNA gene sequence representing the chlamydia-like bacterium (CLB) from proliferative gills had the highest percent nucleotide identity with endosymbionts of Acanthamoeba spp . (order Chlamydiales) . Molecular phylogenetic relationships inferred from 16S rRNA gene sequences using distance and parsimony indicated that the CLB from proliferative gills branched with members of the order Chlamydiales . "Candidatus Piscichlamydia salmonis" is proposed for the CLB associated with epitheliocystis from proliferative gills of Atlantic salmon, which exhibits developmental stages different from those identified in nonproliferative gills.

J Clin Microbiol, 2004 Nov, 42(11), 5238 - 44
Carious dentine provides a habitat for a complex array of novel Prevotella-like bacteria; Nadkarni MA et al.; Previous analysis of the microbiology of advanced caries by culture and real-time PCR emphasized the high incidence and abundance of gram-negative anaerobic species, particularly Prevotella-like bacteria . The diversity of Prevotella-like bacteria was further explored by analyzing pooled bacterial DNA from lesions of carious dentine . This was achieved by amplification of a region of the 16S ribosomal DNA with a Prevotella genus-specific forward primer and a universal bacterial reverse primer, followed by cloning and sequencing . Cultured Prevotella species commonly associated with oral tissues constituted only 12% of the Prevotella clones isolated from advanced carious lesions . The remaining 88% consisted of a diverse range of phylotypes . These included five clusters of previously recognized but uncultured oral Prevotella spp . and a major cluster containing Prevotella-like bacteria most closely related to uncharacterized rumen bacteria . Cluster-specific primers were designed, and the numbers of bacteria within clusters were quantified by real-time PCR, confirming the abundance of these organisms . The data indicated that advanced dental caries provides a unique environment for a complex array of novel and uncultured Prevotella and Prevotella-like bacteria which, in some cases, may dominate the diverse polymicrobial community associated with the disease.

Blood . 2004 Nov 4; {Epub ahead of print}
A cardioprotective role for the endothelial protein C receptor in lipopolysaccharide-induced endotoxemia in the mouse; Iwaki T et al.; A model of gram-negative lethal endotoxin shock, involving continuous peritoneal infusion of lipopolysaccharide (LPS), has been applied to wild-type (WT) mice and mice with a severe deficiency of endothelial protein C receptor (EPCR(delta/delta)) . The survival of EPCR(delta/delta) mice was significantly diminished as compared to WT mice after administration of LPS via this route . Heart rates and central blood pressures were also significantly more depressed in EPCR(delta/delta) mice, indicating that the receptor-based Protein C (PC) pathway functions in regulation of hemodynamic properties in the mouse . Further, heart muscle damage was more severe in EPCR(delta/delta) mice as compared to WT mice after endotoxin administration, as revealed by the more elevated plasma myoglobin levels in EPCR(delta/delta) mice and by microscopic examination of stained heart sections . Neutrophil infiltration was more pronounced in heart tissue of EPCR(delta/delta) mice, perhaps in response to the greatly increased expression level of the chemokine, MIP-2, which was also significantly more upregulated in the LPS-treated EPCR(delta/delta) mouse cohort . In conclusion, a severe deficiency of EPCR adversely affects survival of mice subjected to continuous infusion of endotoxin, via contributions of more responsive hemodynamic and cardiac alterations, thus suggesting that, among its other functions, the PC-based receptor system has a cardioprotective role after acute inflammatory challenge.

Gene, 2004 Nov 10, 342(1), 179 - 87
Identification of an operon involved in tyrosinase activity and melanin synthesis in Marinomonas mediterranea; Lopez-Serrano D et al.; The genomic region of Marinomonas mediterranea containing the genes required for tyrosinase activity and melanin synthesis has been cloned by marker rescue using the transposon-generated, amelanogenic strain T105 . Five ORFs, two incomplete and three complete, have been sequenced in the genomic region where the transposon was inserted . RT-PCR analysis indicates that ORF 3, coding for tyrosinase, and ORF4, coding for a protein of 250 amino acids, are in the same transcriptional unit, constituting an operon whose promoter region has been determined by 5'-RACE . This operon has been sequenced in the wild-type and several mutant strains, indicating that both ORFs are required for expression of tyrosinase activity and melanin synthesis . The nitrosoguanidine generated, amelanogenic mutant ng56, shows a nonsense mutation in ORF3 coding for the tyrosinase . On the other hand, in the strain T105 the transposon is inserted in ORF4 . The product of this gene is related to copper metabolism, since the addition of this metal ion to cell extracts or culture media partially restores melanin synthesis and tyrosinase activity in the strain T105 . However, it does not show significant sequence similarity to previously characterized metallochaperones and hence may be an example of a new kind of those proteins . The operon has been denoted as ppoB, taking into consideration that ppoA denotes the M . mediterranea gene coding for the previously cloned polyphenol oxidase with laccase activity . This is the first demonstration of the tyrosinase gene forming part of an operon in a Gram-negative bacterium.

Biopolymers, 2004 Dec 15, 75(6), 480 - 90
Polymyxin B-lipid interactions in Langmuir-Blodgett monolayers of Escherichia coli lipids: a thermodynamic and atomic force microscopy study; Clausell A et al.; The dramatically increased frequency of antibiotic resistance has led to intensive efforts towards developing new families of antibiotics . Membrane-active antibiotic peptides such as polymyxin B (PxB) hold promise as the next generation of antibiotics, since they rarely spur the evolution of resistance . At low concentrations in the membrane, PxB forms vesicle-vesicle contacts and induces lipid exchange without leakage or fusion, a phenomenon that can explain its specificity towards gram-negative bacteria by contact formation between the two phospholipids interfaces in the periplasmatic space . In this work, the interaction of PxB and the nonantibiotic derivative polymyxin B nonapeptide (PxB-NP) with monolayers of Escherichia coli membrane lipids (ECL) has been studied by thermodynamic and structural methods . PxB inserts itself into ECL monolayers as a conformation that forms intermembrane contacts with vesicles injected underneath, and induces lipid exchange when the monolayer surface pressure is set at 32 mN/m (membrane equivalence pressure) or net transfer vesicle-to-monolayer at lower surface pressures . Thermodynamic analysis of the compression isotherms of mixed monolayers indicates that PxB inserts into the monolayer with an expansion of the mean molecular area, implying that peptide and lipids form nonideal mixtures . At low concentrations, corresponding to the membrane-membrane contact form of PxB, the mixed monolayers present positive excess energy values (deltaGm(Ex)), and atomic force microscopy (AFM) imaging reveals structures of approximately 120-nm diameter that protrude from the lipid surface approximately 0.7 nm . At concentrations of PxB above 4 mol %, thermodynamic analysis gives a very high deltaGm(Ex), corresponding to nonfavorable interactions, and AFM images show round structures of 20-30 nm diameter . PxB-NP behaves in a totally different way, in agreement with its inability to form vesicle-vesicle contacts and its lack of antibiotic effect . These results are discussed in the light of the mechanism of action of PxB on the membrane of gram-negative bacteria . 2004 Wiley Periodicals, Inc.

Exp Lung Res, 2004 Sep, 30(6), 479 - 93
Direct visual instillation as a method for efficient delivery of fluid into the distal airspaces of anesthetized mice; Su X et al.; Although several methods have been used to deliver fluid into the distal airspaces of the lung, the efficiency of these methods has been variable . Therefore, the authors have modified prior techniques to design a better method for direct visual instillation (DVI) of fluid into the trachea and compared its efficiency with two commonly used methods: nasal inhalation and invasive intratracheal instillation (delivery of the instillate by needle puncture of the trachea) . The results showed that this method (DVI) can deliver fluid efficiently into either both lungs or into a single lung . Using an 131I-albumin labeling technique, DVI resulted in 92 +/- 1% retention of the labeled albumin in the lungs 1 hour after instillation, significantly greater than nasal inhalation (48 +/- 3%, P < .01) and invasive intratracheal instillation (77 +/- 3%, P < .05) . Also, when bacteria (Escherichia coli) were instilled with the DVI method, the severity of gram-negative pneumonia was greater (6.5 +/- 0.5 g water/g dry weight) compared to delivery by nasal inhalation (5.5 +/- 0.4 g water/g dry weight, P < .05) or by invasive intratracheal instillation (5.9 +/- 0.4g water/g dry weight, P < .05) . The authors conclude that DVI is more efficient than nasal inhalation and invasive intratracheal instillation for delivering experimental fluids into the distal airspaces of anesthetized mice . This method should be valuable for experimental lung studies in mice.

Med Dosw Mikrobiol, 2004, 56(1), 49 - 55
{Comparison of the results of disc diffusion methods applied for detection of ESBL-positive strains of gram-negative rods}; Rokosz A et al.; Examinations were undertaken to compare the results of disc diffusion tests applied for detection of strains producing extended-spectrum beta-lactamases (ESBLs) . A total of 120 clinical strains were used in experiments . These strains were determined as ESBL-positive on the basis of consistent results of two methods: the double disc synergy test (DDST) according to Jarlier et al . (1988) and the diagnostic disc test (DD, version CPD/CD 01) according to Appleton (1999) . In the next step examined strains were analysed in two further tests, which are variants of DD method: CAZ/CD 02 test with discs containing ceftazidime and ceftazidime/clavulanic acid, and CTX/CD 03 test with the use of cefotaxime and cefotaxime/clavulanic acid discs . ESBL-positive strains first of all belonged to the species E . coli and K . pneumoniae . In the case of seven analysed strains consistent results of determinations were not obtained with the use of different disc diffusion methods . Application of several disc diffusion methods to determine ESBL-positive strains of gram-negative rods increases the probability of their proper identification.

Am J Vet Res, 2004 Oct, 65(10), 1421 - 6
Effect of proinflammatory mediators and glucocorticoids on L-selectin expression in peripheral blood neutrophils from dairy cows in various stages of lactation; Diez-Fraile A et al.; OBJECTIVE: To determine whether proinflammatory mediators and glucocorticoids affect CD62L(L-selectin) expression on peripheral blood neutrophils from cows in various stages of lactation . ANIMALS: 100 healthy dairy cows during early (13.1 +/- 0.79 days after parturition; n = 31), peak (58.7 +/- 1.64 days after parturition; 31), and mid (137.2 +/- 2.59 days after parturition; 38) lactation . PROCEDURE: In vitro effects of relevant proinflammatory mediators that are released in response to mastitis caused by gram-negative bacteria such as lipopolysaccharide (endotoxin), tumor necrosis factor-alpha, and platelet-activating factor (PAF) on CD62L expression on bovine neutrophils were assessed by flow cytometry . Influences of cortisol and dexamethasone on CD62L expression on bovine neutrophils were also investigated . RESULTS: Basal CD62L expression on neutrophils from cows during early, peak, and mid lactation were similar . Lipopolysaccharide and tumor necrosis factor-alpha had no effect on CD62L expression on neutrophils from cows at any stage of lactation . Conversely, PAF elicited a time- and dose-dependent, down regulatory effect on CD62L expression . However, no differential shedding of CD62L from neutrophils of cows at any stage of lactation were detected . In addition, no effects on CD62L expression on bovine neutrophils after whole blood incubation with cortisol or dexamethasone were observed . Incubation with glucocorticoids did not prevent the down regulatory effect of PAF on CD62L expression . CONCLUSIONS AND CLINICAL RELEVANCE: Comparable basal CD62L expression on bovine neutrophils and equal amounts of CD62L shedding from bovine neutrophils during all stages of lactation suggest that variations in CD62L density are not a likely cause of susceptibility of cows to coliform-induced mastitis during early lactation.

Blood . 2004 Nov 2; {Epub ahead of print}
C1 inhibitor prevents gram negative bacterial lipopolysaccharide-induced vascular permeability; Liu D et al.; Gram negative bacterial endotoxemia may lead to the pathological increase of vascular permeability with systemic vascular collapse, a vascular leak syndrome, multiple organ failure (MOF), and/or shock . Previous studies demonstrated that C1 inhibitor (C1INH) protects mice from LPS-induced lethal septic shock via a direct interaction with lipopolysaccharide (LPS) . Here, we report that C1INH blocked the LPS-induced increase in transendothelial flux through an endothelial monolayer . In addition, LPS-mediated detachment of cultured endothelial cells was prevented with C1INH . C1INH also inhibited LPS-induced endothelial cell apoptosis as demonstrated by suppression of DNA fragmentation and annexin V expression . As illustrated by laser scanning confocal microscopy, C1INH completely blocked the binding of FITC-LPS to human umbilical vein endothelial cells (HUVEC) . C1INH protected from localized LPS-induced increased plasma leakage in C57BL/6J mice and in C1INH deficient mice . Local vascular permeability in response to LPS was increased to a greater extent in C1INH deficient mice in comparison with wild type littermate controls and was reversed by treatment with C1INH . Systemic administration of LPS to mice resulted in increased vascular permeability, which was reduced by C1INH . Therefore, these studies demonstrate that C1INH, in addition to its role in suppression of LPS-mediated macrophage activation, may play an important role in the prevention of LPS-mediated increased vascular permeability, endothelial cell injury, and multiple organ failure.

J Inorg Biochem, 2004 Nov, 98(11), 1975 - 7
Phosphate ester hydrolysis is catalyzed by a bacterial transferrin: potential implications for in vivo iron transport mechanisms; Dhungana S et al.; Two synergistic anions, p-nitrophenyl phosphate ester (NPP) and SO(4)(2-), were found to form new stable assemblies with Fe(3+) and a bacterial transferrin, FbpA (FbpA=ferric binding protein) . Fe(3+)FbpA-SO(4) undergoes rapid anion exchange in the presence of NPP to form Fe(3+)FbpA-NPP . Formation of Fe(3+)FbpA-NPP was found to accelerate the rate of hydrolysis of the bound phosphate ester (k(hyd)=1.6 x 10(-6) s(-1) at 25 degrees C and pH 6.5) by >10(3) fold over the uncatalyzed reaction . These findings suggest a dual function for FbpA in vivo: transport of Fe(3+) across the periplasmic space to the inner membrane in certain gram-negative bacteria and hydrolysis of periplasmic polyphosphates.

Biochem Biophys Res Commun, 2004 Dec 3, 325(1), 91 - 6
Toll-like receptor 4 mutation impairs the macrophage TNFalpha response to peptidoglycan; Li Q et al.; Macrophages produce TNFalpha when infected by bacteria, a response that follows recognition of microbial components by members of the Toll-like receptor (TLR) family . Cells that lack functional TLR4 are known to have markedly diminished responses to Gram-negative lipopolysaccharide . We demonstrate in the present work that peritoneal macrophages derived from strains of mice that carry a spontaneous, inactivating mutation in TLR4 also have impaired production of TNFalpha in response to peptidoglycan, a ligand for TLR2 . This impairment is at a step of biosynthesis subsequent to the generation of mRNA . TLR4-activated signals act at this step to enhance peptidoglycan-induced TNFalpha production in wild-type mice . Based on these observations, we conclude that macrophages from wild-type mice are primed by chronically acting TLR4 signals, probably resulting from exposure to environmental lipopolysaccharide . These signals are required for optimal production of TNFalpha in response to TLR2 stimulation, and are absent in macrophages from TLR4 mutant animals.

Mol Microbiol, 2004 Nov, 54(4), 863 - 75
Genetic screening of Hrp type III-related pathogenicity genes controlled by the HrpB transcriptional activator in Ralstonia solanacearum; Mukaihara T et al.; As in many other Gram-negative phytopathogenic bacteria, the Hrp type III secretion system is essential for the pathogenicity of Ralstonia solanacearum on host plants . The expression of most of the type III effector genes previously isolated from R . solanacearum is co-regulated with those of hrp genes by an AraC-type transcriptional activator, HrpB . In order to isolate type III-related pathogenicity genes, we screened hrpB-regulated genes in R . solanacearum . Using a transposon-based system, we isolated 30 novel hpx (hrpB-dependent expression) genes outside the hrp gene cluster . Most of the hpx genes contain a PIP (plant-inducible promoter) box-like motif in their putative promoter regions . Seven hpx genes encoded homologues of known type III effectors and type III-related proteins found in other animal and plant pathogens . Four encoded known enzymes, namely, glyoxalase I, Nudix hydrolase, spermidine synthase and transposase . Interestingly, six hpx genes encoded two types of leucine-rich repeat (LRR) protein . Products of the remaining genes did not show any significant homology to known proteins . We also identified two novel hrpB-regulated genes, hpaZ and hpaB, downstream of hrpY in the hrp cluster . The hpaB gene of R . solanacearum, but not hpaZ, was required for both the pathogenicity and ability to induce hypersensitive reaction on plants . We show that a hpaB null mutant still produces Hrp pili on the cell surface although it shows a typical Hrp-defective phenotype on plants.

Harefuah, 2004 Sep, 143(9), 669 - 75, 693
{Early events in chlamydial infection of host cells}; Israeli E et al.; Chlamydiae, gram-negative, obligate intracellular bacteria, are major pathogens worldwide, causing several diseases including trachoma, respiratory diseases and sexually transmitted disease . Penetration of chlamydiae to epithelial cells, the environment which supports their growth and survival, leads to various events that begin with changes to the bacteria-containing vacuole, allowing for its progression from the endosomal to the exocytic pathway . The changes include fusion with vesicles carrying glycerophospholipids and sphingolipids and originating in the endoplasmic reticulum (ER) and the Golgi apparatus . The bacteria then reproduce in the inclusion vesicles . In this survey we describe the chlamydial life cycle and review recent reports on early intracellular events in chlamydial infection . While antibiotics currently recommended for treatment of chlamydial infections interfere with bacterial macromolecular synthesis, newer forms of treatment may be developed based on our increasing understanding of chlamydial manipulation of intracellular processes . This manipulation, described in this article on early intracellular events in chlamydial infection, enables these pathogens to escape destruction in the endosomal compartment and begin replication in the target cell.

Biochim Biophys Acta, 2004 Nov 3, 1666(1-2), 250 - 63
Folding and assembly of beta-barrel membrane proteins; Tamm LK et al.; Beta-barrel membrane proteins occur in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts . The membrane-spanning sequences of beta-barrel membrane proteins are less hydrophobic than those of alpha-helical membrane proteins, which is probably the main reason why completely different folding and membrane assembly pathways have evolved for these two classes of membrane proteins . Some beta-barrel membrane proteins can be spontaneously refolded into lipid bilayer model membranes in vitro . They may also have this ability in vivo although lipid and protein chaperones likely assist with their assembly in appropriate target membranes . This review summarizes recent work on the thermodynamic stability and the mechanism of membrane insertion of beta-barrel membrane proteins in lipid model and biological membranes . How lipid compositions affect folding and assembly of beta-barrel membrane proteins is also reviewed . The stability of these proteins in membranes is not as large as previously thought (<10 kcal/mol) and is modulated by elastic forces of the lipid bilayer . Detailed kinetic studies indicate that beta-barrel membrane proteins fold in distinct steps with several intermediates that can be characterized in vitro . Formation of the barrel is synchronized with membrane insertion and all beta-hairpins insert simultaneously in a concerted pathway.

Transplant Proc, 2004 Sep, 36(7), 1975 - 6
Effects of pirfenidone on endotoxin-induced liver injury after partial hepatectomy in rats; Kaibori M et al.; BACKGROUND: In living donor liver transplantation, restrictions on graft size are a serious obstacle to expand indications for adult recipients . The sequence of gram-negative infection, septicemia, and multiple-organ failure is a common cause of early mortality after liver transplantation . An effective therapy has not been established for endotoxemia following extended hepatectomy in donors or small-for-size grafts in recipients . Pirfenidone (PFD), a new experimental antifibrotic agent, was used to ameliorate on endotoxin-induced liver injury following partial hepatectomy . METHODS: Male Sprague-Dawley rats were intravenously administered lipopolysaccharide (LPS) 48 hours after 70% hepatectomy . Prior to LPS administration, PFD (300 mg/kg) or its vehicle (0.5% carboxymethylcellulose) was given orally twice . RESULTS: The survival rate of the PFD-treated group was markedly improved compared with that of the controls . PFD prevented the increases in the activities of serum enzymes (aspartate transaminase {AST}, alanine transaminase {ALT}, and lactate dehydrogenase {LDH}) and total bilirubin . The serum and liver tissue levels of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma, and interleukin-6, were significantly lower among the PFD than the control group . Furthermore, the degree of necrosis in the remnant liver was significantly decreased in the PFD-treated rats compared with controls . CONCLUSION: These results indicate that PFD alleviates endotoxin-induced liver injury after partial hepatectomy through the inhibition of production of inflammatory cytokines in the residual liver . PFD may be useful to prevent endotoxin-induced liver injury after hepatectomy.

J Biol Chem, 2005 Jan 14, 280(2), 1360 - 8 Epub 2004 Oct 29.
67-kDa Laminin Receptor Promotes Internalization of Cytotoxic Necrotizing Factor 1-expressing Escherichia coli K1 into Human Brain Microvascular Endothelial Cells; Kim KJ et al.; Escherichia coli K1 is the most common Gram-negative organism causing meningitis, and its invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for penetration into the central nervous system . We have reported previously that cytotoxic necrotizing factor 1 (CNF1) contributes to E . coli K1 invasion of HBMEC and interacts with 37-kDa laminin receptor precursor (37LRP) of HBMEC, which is a precursor of 67-kDa laminin receptor (67LR) . In the present study, we examined the role of 67LR in the CNF1-expressing E . coli K1 invasion of HBMEC . Immunofluorescence microscopy and ligand overlay assays showed that 67LR is present on the HBMEC membrane and interacts with CNF1 protein as well as the CDPGYIGSR laminin peptide . 67LR was up-regulated and clustered at the sites of E . coli K1 on HBMEC in a CNF1-dependent manner . Pretreatment of CNF1+ E . coli K1 with recombinant 37-kDa laminin receptor precursor reduced the invasion rate to the level of Deltacnf1 mutant, and the invasion rate of CNF1+ E . coli K1 was enhanced in 67LR-overexpressing HBMEC, indicating 67LR is involved in the CNF1+ E . coli K1 invasion of HBMEC . Coimmunoprecipitation analysis showed that, upon incubation with CNF1+ E . coli K1 but not with Deltacnf1 mutant, focal adhesion kinase and paxillin were recruited and associated with 67LR . When immobilized onto polystyrene beads, CNF1 was sufficient to induce internalization of coupled beads into HBMEC through interaction with 67LR . Taken together, this is the first demonstration that E . coli K1 invasion of HBMEC occurs through the ligand-receptor (CNF1-67LR) interaction, and 67LR promotes CNF1-expressing E . coli K1 internalization of HBMEC.

Lipids, 2004 May, 39(5), 491 - 505
Effects of hydrocarbon structure on fatty acid, fatty alcohol, and beta-hydroxy acid composition in the hydrocarbon-degrading bacterium Marinobacter hydrocarbonoclasticus; Soltani M et al.; The lipids of the gram-negative bacterium Marinobacter hydrocarbonoclasticus grown in a synthetic seawater medium supplemented with various hydrocarbons as the sole carbon source were isolated, purified, and their structures determined . The hydrocarbons were normal, iso, anteiso, and mid-chain branched alkanes, phenylalkanes, cyclohexylalkanes, and a terminal olefin . According to the sequential procedure used for lipid extraction, three pools were isolated: unbound lipids extracted with organic solvents (corresponding to metabolic lipids and to the main part of membrane lipids), OH- labile lipids {mainly ester-bound in the lipopolysaccharides (LPS)}, and H+ labile lipids (mainly amide-bound in the LPS) . Each pool contained FA, fatty alcohols, and beta-hydroxy acids . The proportions of these lipids in the unbound lipid pools were 84-98%, 1.1-11.6%, and 0.1-3.6% (w/w), respectively . The chemical structures of the lipids were strongly correlated with those of the hydrocarbons fed; analytical data suggested a metabolism essentially through oxidation into primary alcohol, then into FA and degradation via the beta-oxidation pathway . Sub-terminal oxidation of the hydrocarbon chains, alpha-oxidation of FA or double-bond oxidation in the case of the terminal olefin, were minor, although sometimes substantial, routes of hydrocarbon degradation . Cyclohexyldodecane did not support growth, likely because of the toxicity of cyclohexylacetic acid formed in the oxidation of the alkyl side chain . In the OH- and H+ labile lipid pools, beta-hydroxy acids, the lipophilic moiety of LPS, generally dominated (28-72% and 64-98%, w/w, respectively) . The most remarkable feature of these cultures on hydrocarbons was the incorporation in LPS of beta-hydroxy acids with Codd, omega-unsaturated, iso, or anteiso alkyl chains in addition to the specific beta-hydroxy acid of M . hydrocarbonoclasticus, 3-OH-n-12:0 . These beta-hydroxy acids were tolerated insofar as their geometry and steric hindrance were close to those of the 3-OH-n-12:0 acid.

Infect Immun, 2004 Nov, 72(11), 6650 - 8
Toll-like receptor 4-dependent early elicited tumor necrosis factor alpha expression is critical for innate host defense against Bordetella bronchiseptica; Mann PB et al.; Toll-like receptor 4 (TLR4) mediates the response to lipopolysaccharide, and its activation induces the expression of a large number of inflammatory genes, many of which are also induced by other pathogen-associated molecular patterns . Interestingly, the subset of genes that are dependent on TLR4 for optimal expression during gram-negative bacterial infection has not been determined . We have previously shown that TLR4-deficient mice rapidly develop acute pneumonia after inoculation with Bordetella bronchiseptica, suggesting that TLR4 is required for expression of early elicited gene products in this model . Microarray analysis with macrophages derived from wild-type and TLR4-deficient mice was used to identify genes whose expression, within 1 h of bacterial exposure, is dependent on TLR4 . The results of this investigation suggest that TLR4 is not required for the majority of the transcriptional response to B . bronchiseptica . However, early tumor necrosis factor alpha (TNF-alpha) mRNA expression is primarily dependent on TLR4 and in vitro and in vivo protein levels substantiate this finding . TLR4-deficient mice and TNF-alpha-/- mice are similarly susceptible to infection with relatively low doses of B . bronchiseptica and in vivo neutralization studies indicate that it is the TLR4-dependent early elicited TNF-alpha response that is critical for preventing severe pneumonia and limiting bacterial growth . These results suggest that one critical role for TLR4 is the generation of a robust but transient TNF-alpha response that is critical to innate host defense during acute gram-negative respiratory infection.

Infect Immun, 2004 Nov, 72(11), 6455 - 62
Role of protein tyrosine kinase p53/56lyn in diminished lipopolysaccharide priming of formylmethionylleucyl- phenylalanine-induced superoxide production in human newborn neutrophils; Yan SR et al.; Human newborns are more susceptible than adults to bacterial infection . With gram-negative bacteria, this may be due to a diminished response of newborn leukocytes to lipopolysaccharide (LPS) . Since protein tyrosine kinase inhibition abolishes LPS priming in adult cells, we hypothesized that protein tyrosine kinases may have a critical role in LPS priming of polymorphonuclear neutrophils (PMNs) and that newborn PMNs may have altered protein tyrosine kinase activities . In the present study, we investigated the role of src family protein tyrosine kinases in the LPS response of newborn PMNs compared to adult cells . In a respiratory assay, the LPS-primed increase in formylmethionylleucylphenylalanine (fMLP)-triggered O2- release by adult PMNs was greatly decreased by PP1 {4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo{3,4-d}pyrimidine}, a src kinase inhibitor, to the level of untreated newborn PMNs, in which LPS failed to prime . LPS activated the src-like kinases p59hck (HCK) and p58fgr (FGR) in both adult and newborn PMNs but increased the activation of p53/56lyn (LYN) only in adult cells . In newborn PMNs, LYN was highly phosphorylated independent of LPS . We evaluated subcellular fractions of PMNs and found that the phosphorylated form of LYN was mainly in the Triton-extractable, cytosolic fraction in adult PMNs, while in newborn cells it was located mainly in Triton-insoluble, granule- and membrane-associated fractions . In contrast, the phosphorylated mitogen-activated protein kinases ERK1/2 and p38 were mainly detected in the cytosol in both adult and newborn PMNs . These data indicate a role for LYN in the regulation of LPS priming . The trapping of phosphorylated LYN in the membrane-granule fraction in newborn PMNs may contribute to the deficiency of newborn cells in responding to LPS stimulation.

Infect Immun, 2004 Nov, 72(11), 6446 - 54
Intact gram-negative Helicobacter pylori, Helicobacter felis, and Helicobacter hepaticus bacteria activate innate immunity via toll-like receptor 2 but not toll-like receptor 4; Mandell L et al.; Molecular and genetic studies have demonstrated that members of the Toll-like receptor (TLR) family are critical innate immune receptors . TLRs are recognition receptors for a diverse group of microbial ligands including bacteria, fungi, and viruses . This study demonstrates that distinct TLRs are responsible for the recognition of Helicobacter lipopolysaccharide (LPS) versus intact Helicobacter bacteria . We show that the cytokine-inducing activity of Helicobacter LPS was mediated by TLR4; i.e., TLR4-deficient macrophages were unresponsive to Helicobacter pylori LPS . Surprisingly, the cytokine response to whole Helicobacter bacteria (H . pylori, H . hepaticus, and H . felis) was mediated not by TLR4 but rather by TLR2 . Studies of HEK293 transfectants revealed that expression of human TLR2 was sufficient to confer responsiveness to intact Helicobacter bacteria, but TLR4 transfection was not sufficient . Our studies further suggest that cag pathogenicity island genes may modulate the TLR2 agonist activity of H . pylori as cagA+ bacteria were more active on a per-cell basis compared to cagA mutant bacteria for interleukin-8 (IL-8) cytokine secretion . Consistent with the transfection studies, analysis of knockout mice demonstrated that TLR2 was required for the cytokine response to intact Helicobacter bacteria . Macrophages from both wild-type and TLR4-deficient mice produced a robust cytokine secretion response (IL-6 and MCP-1) when stimulated with intact Helicobacter bacteria . In contrast, macrophages from TLR2-deficient mice were profoundly unresponsive to intact Helicobacter stimulation, failing to secrete cytokines even at high (100:1) bacterium-to-macrophage ratios . Our studies suggest that TLR2 may be the dominant innate immune receptor for recognition of gastrointestinal Helicobacter species.

Res Microbiol, 2004 Nov, 155(9), 703 - 9
Nitrogen fixation in acidophile iron-oxidizing bacteria: the nif regulon of Leptospirillum ferrooxidans; Parro V et al.; The Gram-negative iron-oxidizing bacterium Leptospirillum ferrooxidans contains all genes necessary for nitrogen fixation, from genes encoding the Mo-Fe nitrogenase, the specific regulator (nifA), global regulators like glnB and ntrC like genes, to other sensors and transport systems somehow related to nitrogen assimilation . We review current knowledge about the nif regulon and its connection with other metabolic functions in L . ferrooxidans.

Drug Metab Pharmacokinet, 2004 Jun, 19(3), 159 - 70
Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity; Nagai J et al.; Aminoglycosides such as gentamicin and amikacin are the most commonly used antibiotics worldwide in the treatment of Gram-negative bacterial infections . However, serious complications like nephrotoxicity and ototoxicity are dose-limiting factors in the use of aminoglycosides . A relatively large amount of the intravenously administered dose is accumulated in the kidney (about 10% of dose), whereas little distribution of aminoglycosides to other tissues is observed . Aminoglycosides are taken up in the epithelial cells of the renal proximal tubules and stay there for a long time, resulting in nephrotoxicity . Acidic phospholipids are considered as a binding site for aminoglycosides in the brush-border membrane of the proximal tubular cells . More recently, it has been reported that megalin, a giant endocytic receptor abundantly expressed at the apical membrane of renal proximal tubules, plays an important role in binding and endocytosis of aminoglycosides in the proximal tubular cells . The elucidation of the aminoglycoside-binding receptor would help design a strategy to prevent against aminoglycoside-induced nephrotoxicity . In this review, we summarize recent advances in the understandings of the molecular mechanisms responsible for renal accumulation of aminoglycosides, especially megalin-mediated endocytosis . In addition, approaches toward prevention of aminoglycoside-induced nephrotoxicity are discussed, based on the molecular mechanisms of the renal accumulation of aminoglycosides.

DNA Seq, 2004 Jun, 15(3), 225 - 7
Genetic and physical map of the pLAFR1 vector; Vanbleu E et al.; This paper presents the complete sequencing and annotation of the pLAFR1 vector . pLAFR is a tetracycline-resistant "cosmid" cloning vector, which is derived from the 20 kb plasmid pRK290, a RK2-derivative . Due to its broad host range, the pLAFR1 vector has been widely used in the genetic analysis of a broad number of gram-negative bacterial species . The availability of the complete pLAFR1 sequence will most definitely help in the construction and analysis of clone librares based on pRK290 or pLAFR vectors.

Cochrane Database Syst Rev . 2004 Oct 18;(4):CD004496.
Intraventricular antibiotics for bacterial meningitis in neonates; Shah S et al.; BACKGROUND: Neonatal meningitis may be caused by bacteria, especially gram-negative bacteria, which are difficult to eradicate from the cerebrospinal fluid (CSF) using safe doses of antibiotics . In theory, intraventricular administration of antibiotics would produce higher antibiotic concentrations in the CSF than intravenous administration alone, and eliminate the bacteria more quickly . However, ventricular taps may cause harm . OBJECTIVES: To assess the effectiveness and safety of intraventricular antibiotics (with or without intravenous antibiotics) in neonates with meningitis (with or without ventriculitis) as compared to treatment with intravenous antibiotics alone . SEARCH STRATEGY: MEDLINE, EMBASE, The Cochrane Library, Issue 2, 2004, Science Citation Index, and the Oxford Database of Perinatal Trials were searched in June 2004 . Pediatric Research (abstracts of proceedings) were searched (1990 - April 2004) as were reference lists of identified trials and personal files . No language restrictions were applied . SELECTION CRITERIA: Selection criteria for study inclusion were: Randomized or quasi-randomized controlled trials in which intraventricular antibiotics with or without intravenous antibiotics were compared with intravenous antibiotics alone in neonates (< 28 days old) with meningitis . One of the following outcomes was required to be reported: mortality during initial hospitalization, neonatal and/or infant mortality, neurodevelopmental outcome, duration of hospitalization, duration of culture positivity of CSF and side effects . DATA COLLECTION AND ANALYSIS: All reviewers abstracted information for outcomes reported and one reviewer checked for discrepancies and entered data into RevMan 4.2 . Relative risk (RR), risk difference (RD), number needed to treat (NNT) or number needed to harm (NNH), and mean difference (MD), using the fixed effects model are reported with 95% confidence intervals (CI) . The fixed effect model was used for meta-analysis . MAIN RESULTS: One study was included in the review . This study assessed the effect of intraventricular gentamicin in a mixed population of neonates (69%) and older infants (31%) with gram negative meningitis and ventriculitis . Mortality was statistically significantly higher in the group that received intraventricular gentamicin in addition to intravenous antibiotics compared to the group receiving intravenous antibiotics alone {RR 3.43 (95% CI, 1.09, 10.74; RD 0.30 (95% CI, 0.08, 0.53); NNH was 3 (95% CI; 2 ,13)} . Duration of CSF culture positivity did not differ significantly (MD -1.20 days (95% CI, -2.67, 0.27) . REVIEWERS' CONCLUSIONS: In one trial, enrolling infants with gram negative meningitis and ventriculitis, the use of intraventricular antibiotics in addition to intravenous antibiotics resulted in a 3 fold increased RR for mortality compared to standard treatment with intravenous antibiotics alone . Based on this result, intraventricular antibiotics as tested in this trial should be avoided . Further trials comparing these interventions are not justified in this population.

J Mol Biol, 2004 Nov 5, 343(5), 1379 - 89
Rapid analysis of large protein-protein complexes using NMR-derived orientational constraints: the 95 kDa complex of LpxA with acyl carrier protein; Jain NU et al.; Characterization of protein-protein interactions that are critical to the specific function of many biological systems has become a primary goal of structural biology research . Analysis of these interactions by structural techniques is, however, challenging due to inherent limitations of the techniques and because many of the interactions are transient, and suitable complexes are difficult to isolate . In particular, structural studies of large protein complexes by traditional solution NMR methods are difficult due to a priori requirement of extensive assignments and a large number of intermolecular restraints for the complex . An approach overcoming some of these challenges by utilizing orientational restraints from residual dipolar couplings collected on solution NMR samples is presented . The approach exploits existing structures of individual components, including the symmetry properties of some of these structures, to assemble rapidly models for relatively large protein-protein complexes . An application is illustrated with a 95 kDa homotrimeric complex of the acyltransferase protein, LpxA (UDP-N-acetylglucosamine acyltransferase), and acyl carrier protein . LpxA catalyzes the first step in the biosynthesis of the lipid A component of lipopolysaccharide in Gram-negative bacteria . The structural model generated for this complex can be useful in the design of new anti-bacterial agents that inhibit the biosynthesis of lipid A.

Nat Immunol, 2004 Nov, 5(11), 1166 - 74 Epub 2004 Oct 17.
Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island; Viala J et al.; Epithelial cells can respond to conserved bacterial products that are internalized after either bacterial invasion or liposome treatment of cells . We report here that the noninvasive Gram-negative pathogen Helicobacter pylori was recognized by epithelial cells via Nod1, an intracellular pathogen-recognition molecule with specificity for Gram-negative peptidoglycan . Nod1 detection of H . pylori depended on the delivery of peptidoglycan to host cells by a bacterial type IV secretion system, encoded by the H . pylori cag pathogenicity island . Consistent with involvement of Nod1 in host defense, Nod1-deficient mice were more susceptible to infection by cag pathogenicity island-positive H . pylori than were wild-type mice . We propose that sensing of H . pylori by Nod1 represents a model for host recognition of noninvasive pathogens.

Shock, 2004 Nov, 22(5), 423 - 30
Infection-induced modulation of m1 and m2 phenotypes in circulating monocytes: role in immune monitoring and early prognosis of sepsis; Mehta A et al.; To monitor and better understand the immunoinflammatory sequelae in sepsis and septic shock, systemic and monocyte-related cytokine responses were evaluated in baboons with experimental peritonitis induced by an E . coli-laden fibrin clot . Despite similar bacterial inocula, considerable interindividual variability in clinical manifestation and outcome of infection was observed . Because monocytes and macrophages are a key component of innate immunity, we hypothesized that early polarization of distinct activation programs in circulating monocytes that culminates in the emergence of either classically (M1) or alternatively (M2) activated monocytes may underlie the observed susceptibility or resistance to infection . To test our hypothesis, we analyzed infection-induced expression of cytokine mRNAs in monocytes isolated from surviving and dead animals . Our data show that resistance to E . coli sepsis may well be associated with a mixed M1/M2 activation state of circulating monocytes, whereas M1 phenotype appeared to be prevailing in monocytes from animals that died . Together with data on systemic cytokine responses, the latter findings indicate that morbidity and mortality of animals with gram-negative sepsis may well result from an overwhelming proinflammatory response . Collectively, our data contribute to a better understanding of cytokine networking in the immunoinflammatory response to microbial infection and suggest M1/M2 immunophenotypic profiling of readily available circulatory monocytes for early prognosis of severe infections.

Med Hypotheses, 2004, 63(5), 905 - 10
Did an epidemic of tularemia in Ancient Egypt affect the course of world history?
Trevisanato SI.
The dynamics of the spreading, and the identity of a virulent epidemic, similar to bubonic plague or typhus, which hit Ancient Egypt in the middle of the Bronze Age, are hereby presented . Documented in medical papyri as well as archaeological findings, and re-echoed in biblical texts, a plague entered Egypt's main harbor, Avaris, around 1715 BC . As a result, the country was severely weakened at a time when it was already facing serious sociopolitical issues: crumbling central government, immigration, foreign influence, and the rise in power of the army and of warlords . The Hebrews, one of the ethnic groups within the Avaris area, appeared immune amidst the devastation of the plague . This immunity was key to identify the etiological agent of the epidemic: Francisella tularensis, the Gram-negative bacterium passed by ticks, and that causes tularemia . The disease manifests itself as either an ulceroglandular or a typhoid syndrome, accounting for its similarity with bubonic plague and typhus . Cellular immunity provides the main defense against F . tularensis, and is achieved through continuous contact with avirulent strains of the bacterium . Urban dwellers of Avaris, who had a limited contact to animals, would have been defenseless against the virulent strains, unlike the Hebrews, who lived off sheep and other animals . Attempting to consolidate Egypt's central government in the aftermath of the tularemia epidemic, led among others to the internment of the Hebrews in labor camps . The repressive measures against the Hebrews left traces in the subsequent archaeological layer (stratum F) at Avaris . As a corollary, the enslavement would set the stage for the exodus of the Hebrew community from Egypt at a later time.

Scand J Work Environ Health, 2004, 30 Suppl 2, 80 - 90
Chemical and in vitro toxicologic characterization of wintertime and springtime urban-air particles with an aerodynamic diameter below 10 microm in Helsinki; Salonen RO et al.; OBJECTIVES: The chemical composition and toxicity of wintertime urban-air particulate matter with an aerodynamic diameter of <10 microm (PM10), derived mostly from long-range transport and local combustion sources, were compared with those of springtime PM10 derived mostly from the resuspension of road dust . METHODS: Water-soluble ions and elements and polycyclic aromatic hydrocarbons (PAH) were analyzed from seasonally pooled PM10 samples collected at a busy traffic site in Helsinki in 1999 . These PM10 samples were also tested for cytotoxicity {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test} and the production of proinflammatory cytokines {tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6)} and nitric oxide (NO) in the mouse macrophage cell line RAW 264.7 . Their oxidative capacity and the associated DNA (deoxyribonucleic acid) damage were investigated by electron paramagnetic resonance and the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-DG) in isolated calf thymus DNA, respectively . RESULTS: The late wintertime and springtime PM10 had similar compositions of water-soluble ions and elements, but the winter PM10 had a higher content of PAH . The spring PM10 was a much more potent inducer of TNF-alpha and IL-6 production than the winter PM10 was, but there were no consistent differences in cytotoxic potency . In contrast, the winter PM10 was a significantly more potent inducer of NO production and 8-OH-DG formation . The large cytokine responses to the spring PM10 were caused by its insoluble fraction and largely inhibited by the endotoxin antagonist polymyxin B . The transition metal chelator deferoxamine did not modify the proinflammatory or cytotoxic responses to the PM10 samples . CONCLUSIONS: The toxicity profile of urban-air PM10 changed with season in a subarctic climate . Particulate-bound endotoxin from soil gram-negative bacteria is suggested as a highly proinflammatory constituent of springtime resuspended road dust.

J Gen Appl Microbiol, 2004 Jun, 50(3), 143 - 7
Rhodanobacter fulvus sp . nov., a beta-galactosidase-producing gammaproteobacterium; Im WT et al.; A taxonomic study was carried out on a bacterial strain designated as Jip2T isolated from a soil sample mixed with rotten rice straw . It was a Gram-negative, aerobic, motile, and rod-shaped bacterium . It grew well on nutrient agar medium and utilized a fairly narrow spectrum of carbon source . The G+C content of the genomic DNA was 65.3 mol% . The major ubiquinone was Q-8 . The major fatty acids were branched fatty acids, especially large amounts of iso C15:0 and iso C17:1 w9c were detected in the cells grown on TSA agar for 24 h . Comparative 16S rDNA study showed a clear affiliation of this bacterium to the genus Rhodanobacter . The 16S rDNA sequence of strain Jip2T showed 96.4% sequence similarity to that of Rhodanobacter lindaniclasticus RP5575T . On the basis of phenotypic characteristics and 16S rDNA sequence analysis, strain Jip2T is clearly distinct from Rhodanobacter lindaniclasticus . We propose the name Rhodanobacter fulvus sp . nov . for strain Jip2T (=IAM 15025T=KCTC 12098T).

Infect Control Hosp Epidemiol, 2004 Sep, 25(9), 747 - 52
Survey of knowledge, beliefs, and practices of neonatal intensive care unit healthcare workers regarding nosocomial infections, central venous catheter care, and hand hygiene; Kennedy AM et al.; OBJECTIVE: To assess the knowledge, beliefs, and practices of neonatal intensive care unit (NICU) healthcare workers (HCWs) . DESIGN: Self-administered survey . SETTING: A 55-bed NICU . PARTICIPANTS: NICU HCWs (N = 215) . RESULTS: The response rate was 68% . Ninety-two percent knew central venous catheters (CVCs) should be capped, clamped, or connected to running fluids at all times . Ninety-five percent knew when to change gloves . Thirty-one percent knew the recommended duration for handwashing . Most HCWs believed sterile technique in CVC care (96%), gloves (91%), and handwashing (99%) prevent nosocomial infection (NI) . Sixty-seven percent used sterile barriers to insert CVCs, 76% reported wearing gloves, 81% reported routine handwashing, 35% knew that bacterial hand counts are higher with rings, 30% knew that long fingernails are associated with higher gram-negative bacterial hand contamination, and 35% knew that artificial fingernails are associated with higher gram-negative bacterial hand contamination . Most (93%) believed HCWs can affect outcomes of patients with NIs . Fewer believed rings (40%), artificial fingernails (61%), and long fingernails (48%) play a role in NIs, or that policies concerning number of rings (50%), cutting fingernails (35%), or prohibiting artificial fingernails (47%) would prevent NIs . Sixty-one percent of HCWs regularly wore at least one ring to work, 56% wore their fingernails shorter than the fingertip, and 8% wore artificial fingernails . CONCLUSIONS: A disconnect existed between CVC knowledge and beliefs and practice . HCWs did not know the relationship between bacterial hand counts and rings and fingernails, and did not believe rings or long or artificial fingernails increased the risk of NIs.

Hong Kong Med J, 2004 Oct, 10(5), 337 - 43
Update of the management of postoperative endophthalmitis; Yam JC et al.; Postoperative endophthalmitis is a rare but potentially devastating complication of intra-ocular surgery . Most cases are caused by bacterial infection . The clinical presentation of endophthalmitis can be classified as acute postoperative endophthalmitis, chronic postoperative endophthalmitis, and bleb-associated endophthalmitis . They each have a different aetiology, treatment, and prognosis . This review discusses these features, as well as the microbiology, diagnosis, and treatment outcomes of these three types of inflammation, with particular emphasis on recent advances in their management . The role of steroids, systemic fluoroquinolones, and the choice of antibiotic against gram-negative bacteria are still controversial and need further study . Moreover, the management of chronic postoperative and bleb-associated endophthalmitis has not been standardised, and no conclusive findings on the efficacy of the various prophylactic measures are available . Thus, the treatment and prevention of postoperative endophthalmitis remain a clinical challenge.

J Bone Miner Res, 2004 Nov, 19(11), 1905 - 16 Epub 2004 Sep 27.
Sesquiterpene lactone parthenolide blocks lipopolysaccharide-induced osteolysis through the suppression of NF-kappaB activity; Yip KH et al.; Effective treatment for bacteria-induced bone lytic diseases is not yet available . In this study, we showed that PAR, an NF-kappaB inhibitor found in medicinal herbs, can block LPS-induced osteolysis . PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-kappaB activity . INTRODUCTION: Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis . Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases . In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-kappaB activity . MATERIALS AND METHODS: The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo . RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro . Assays for NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors . Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis . Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6 . RESULTS: We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model . In vitro studies showed that low concentrations of PAR (<1 microM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 microM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner . Furthermore, PAR inhibited LPS-induced NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner . In addition, PAR inhibited NF-kappaB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1beta, or TNF-alpha to varying degrees and reduced the gene expression of RANK and TRAF6 . CONCLUSION: The NF-kappaB pathway is known to mediate both osteoclast differentiation and survival . These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-kappaB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.

Biochemistry, 2004 Oct 19, 43(41), 12979 - 89
Overexpression, purification, and characterization of ProQ, a posttranslational regulator for osmoregulatory transporter ProP of Escherichia coli; Smith MN et al.; ProP is an osmosensor and osmoregulatory transporter in Escherichia coli . Osmotic activation of ProP is attenuated 5-fold in the absence of soluble protein ProQ, but proQ lesions do not influence proP transcription or ProP levels . The mechanism by which ProQ amplifies ProP activity is unknown . Putative proQ orthologues are found in Gram-negative bacteria (only), but none have known functions . ProQ was overexpressed to low and high levels with and without a C-terminal histidine tag (His(6)) . Plasmid-encoded ProQ or ProQ-His(6) complemented in-frame chromosomal deletion DeltaproQ676, restoring ProP activity . After overexpression, both proteins were poorly soluble unless cells were lysed in media of high salinity . ProQ copurified with DNA binding proteins of similar size (HU and a histone-like protein) by ion exchange and exclusion chromatographies, whereas ProQ-His(6) could be purified to homogeneity by nickel chelate affinity chromatography . Sequence-based analysis and modeling suggest that ProQ includes distinct N- and C-terminal domains linked by an unstructured sequence . The N-terminal domain can be modeled on the crystal structure of alpha-helical RNA binding protein FinO, whereas the C-terminal domain can be modeled on an SH3-like domain (beta-structure) . Both ProQ and ProQ-His(6) appeared to be monomeric, though the higher Stokes radius of ProQ-His(6) may reflect altered domain interactions . The measured secondary structure content of ProQ (circular dichroism (CD) spectroscopy) contrasted with sequence-based prediction but was as expected if the spectrum of the C-terminal domain is analogous to those reported for SH3 domains . The CD spectrum of ProQ was pH- but not NaCl-sensitive.

Braz J Infect Dis, 2004 Jun, 8(3), 263 - 6 Epub 2004 Sep 29.
Acute hemiplegia associated with cat-scratch disease; Rocha JL et al.; Cat scratch disease (CSD) is an infectious illness caused by a Gram-negative rod named Bartonella henselae . Typical CSD is characterized by a small skin lesion at the site of a scratch or a bite, followed by regional lymphadenopathy, one to two weeks later . Atypical forms may present as ocular manifestations, neurological manifestations, hepatosplenic involvement and vertebral osteomyelitis . Among neurological complications, encephalopathy is by far the most common . Other neurological manifestations are very rare . We report a case of an 11-year-old boy, with a posterior cervical lymphadenopathy and fever . Cat scratch disease was diagnosed and treated after a positive "Whartin-Starry" stain on lymph node biopsy . Two weeks after treatment, the patient was readmitted presenting an acute episode of left hemiplegia . A brain MRI demonstrated a right subcortical fronto-parietal lesion with no contrast enhancement . Complete recovery was observed after corticosteroid treatment.

Int Arch Allergy Immunol, 2004 Nov, 135(3), 216 - 20 Epub 2004 Nov.
Lipopolysaccharide contamination of beta-lactoglobulin affects the immune response against intraperitoneally and orally administered antigen; Brix S et al.; BACKGROUND: Microbial components in the environment are potent activators of the immune system with capacity to shift the active immune response towards priming of Th1 and/or Th2 cells . Lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria, is extensively present in food products like cow's milk . It is not well established, however, how this presence of LPS affects oral tolerance induction . METHODS: We studied the effect of LPS contamination in a commercial preparation of the cow milk protein beta-lactoglobulin (beta-LG) on antigen-specific immune responses . IgG1/IgG2a production upon intraperitoneal immunization without adjuvant was measured, and oral tolerance induction against beta-LG after administration of either an aqueous solution or water-in-oil (w/o) emulsion of beta-LG was evaluated . RESULTS: LPS contamination of beta-LG provoked a beta-LG-specific IgG2a response, as well as an enhanced beta-LG-specific IgG1 response upon intraperitoneal immunization . Oral tolerance induction to beta-LG was induced by aqueous solutions of beta-LG with and without LPS administration . Conversely, oral administration of w/o-emulsified beta-LG prevented oral tolerance to beta-LG only when the beta-LG was contaminated with LPS . CONCLUSIONS: LPS contamination of an aqueous protein solution does not affect oral tolerance induction, whereas LPS present in emulsion prevents oral tolerance induction towards the food protein.

Emerg Med Clin North Am, 2004 Nov, 22(4), 1099 - 115
Rapid infectious killers; Schneider JI; Central to the practice of emergency medicine is the ability to identify patients in whom immediate intervention is needed to prevent long-term morbidity and mortality . This article has highlighted some of the characteristics of several infectious diseases that may become fatal quickly if not treated quickly and appropriately by physicians . Bacterial meningitis,necrotizing soft tissue infections, invasive gram-negative disease, pneumo-coccal pneumonia, and West Nile encephalitis all require prompt recognition and treatment by emergency care providers.

Diagn Microbiol Infect Dis, 2004 Oct, 50(2), 81 - 7
Bacteremia in a patient with colonic carcinoma caused by a novel Sedimentibacter species: Sedimentibacter hongkongensis sp . nov; Woo PC et al.; A bacterium was isolated from the blood culture of a 91-year-old patient with colonic carcinoma . The cells were strict anaerobic, motile, Gram-negative, sporulating, straight, or slightly curved rods . The bacterium grew on agar using the BACTEC anaerobic blood culture broth or buffered charcoal yeast extract agar as pinpoint colonies after 72 h of incubation at 37 degrees C in anaerobic conditions . It did not grow on blood agar, chocolate agar, MacConkey agar, nutrient agar or broth, brain heart infusion agar or broth, Brucella agar, or cooked meat medium . It produces catalase but not cytochrome oxidase . 16S rRNA gene sequencing and phylogenetic analysis showed that it is closely related to Sedimentibacter hydroxybenzoicus and Sedimentibacter saalensis, with 10.5% and 11.9% differences between the 16S rRNA gene sequence of the bacterium and those of S . hydroxybenzoicus and S . saalensis respectively . A new species, Sedimentibacter hongkongenesis sp . nov., is proposed, for which HKU2(T) is the type strain.

Gene, 2004 Oct 27, 341, 1 - 17
Molecular biology of gastric cancer: Helicobacter infection and gastric adenocarcinoma: bacterial and host factors responsible for altered growth signaling; Stoicov C et al.; Gastric cancer remains the second most common cause of cancer-related mortality worldwide . The single most common cause of gastric cancer is chronic infection with the gram-negative microaerophilic spiral bacterium: Helicobacter pylori . Recent advances in this field have identified host factors which predispose to gastric cancer formation via modulation of the host immune response . In addition, recent work has explored bacterial virulence factors which may directly cause tissue damage, and lead to gastric carcinogenesis, as well as factors responsible for enhanced immune response . Environmental factors, long associated with a predilection for gastric cancer, are recognized as modifiers of key growth signalling pathways within the gastric mucosa and as such lead to growth alterations . This review focuses on exploring new advances in our understanding of bacterial factors, host genetic polymorphisms and the interaction between the bacterium and host at the level of the immune response and the regulation of proliferative and apoptotic signal transduction cascades . Modulation of the pivotal balance between cell growth and cell death leads to the formation of gastric adenocarcinoma.

Arterioscler Thromb Vasc Biol, 2004 Dec, 24(12), 2227 - 36 Epub 2004 Dec.
Potential role of endotoxin as a proinflammatory mediator of atherosclerosis; Stoll LL et al.; Atherosclerosis is increasingly recognized as a chronic inflammatory disease . Although a variety of inflammatory markers (ie, C-reactive protein) have been associated with atherosclerosis and its consequences, it is important to identify principal mediators of the inflammatory responses . One potentially important source of vascular inflammation in atherosclerosis is bacterial endotoxin . Mutations in Toll-like receptor 4 (TLR-4), an integral component of the endotoxin signaling complex, are fairly common in the Caucasian population and have recently been associated with reduced incidence of atherosclerosis and other cardiovascular diseases in some studies . Moreover, epidemiological studies suggest that endotoxemia at levels as low as 50 pg/mL constitutes a strong risk factor for the development of atherosclerosis . Endotoxin concentrations in this range may be produced by a variety of common subclinical Gram-negative infections . In this article, we outline the main elements of the endotoxin signaling receptor complex that initiates proinflammatory signaling (lipopolysaccharide binding protein {LBP}, CD14, TLR-4, and MD-2) and discuss how changes in expression of these molecules may affect proatherogenic responses in the vessel wall . We also describe some of the proinflammatory effects of endotoxin that may be relevant to atherosclerosis, and discuss how serum lipoproteins, especially high-density lipoprotein, may modulate endotoxin-induced inflammatory responses . Further, we discuss recent findings suggesting that the lipid-lowering statins may have an additional protective role in blocking at least some of these proinflammatory signaling pathways . Finally, we discuss species diversity with regard to endotoxin signaling that should be considered when extrapolating experimental data from animal models to humans.

Bioorg Khim, 2004 Jul-Aug, 30(4), 409 - 16
{Elucidation of structure of lipid A from the marine Gram-negative bacterium Pseudoalteromonas haloplanktis ATCC 14393T}; Potential role of a novel psychrotolerant member of the family Geobacteraceae et al.; Department of Microbiology, 106N Morrill IV N, University of Massachusetts, Amherst, MA 01003, USAPrevious studies have shown that members of the family Geobacteraceae that attach to the anodes of sediment fuel cells are directly involved in harvesting electricity by oxidizing organic compounds to carbon dioxide and transferring the electrons to the anode . In order to learn more about this process, microorganisms from the anode surface of a marine sediment fuel cell were enriched and isolated with Fe(III) oxide . Two unique marine isolates were recovered, strains A1(T) and A2 . They are gram-negative, nonmotile rods, with abundant c-type cytochromes . Phylogenetic analysis of the 16S rRNA, recA, gyrB, fusA, rpoB, and nifD genes indicated that strains A1(T) and A2 represent a unique phylogenetic cluster within the Geobacteraceae . Both strains were able to grow with an electrode serving as the sole electron acceptor and transferred ca . 90% of the electrons available in their organic electron donors to the electrodes . These organisms are the first psychrotolerant members of the Geobacteraceae reported thus far and can grow at temperatures between 4 and 30 degrees C, with an optimum temperature of 22 degrees C . Strains A1(T) and A2 can utilize a wide range of traditional electron acceptors, including all forms of soluble and insoluble Fe(III) tested, anthraquinone 2,6-disulfonate, and S(0) . In addition to acetate, both strains can utilize a number of other organic acids, amino acids, long-chain fatty acids, and aromatic compounds to support growth with Fe(III) nitrilotriacetic acid as an electron acceptor . The metabolism of these organisms differs in that only strain A1(T) can use acetoin, ethanol, and hydrogen as electron donors, whereas only strain A2 can use lactate, propionate, and butyrate . The name Geopsychrobacter electrodiphilus gen . nov., sp . nov., is proposed for strains A1(T) and A2, with strain A1(T) (ATCC BAA-880(T); DSM 16401(T); JCM 12469) as the type strain . Strains A1(T) and A2 (ATCC BAA-770; JCM 12470) represent the first organisms recovered from anodes that can effectively couple the oxidation of organic compounds to an electrode . Thus, they may serve as important model organisms for further elucidation of the mechanisms of microbe-electrode electron transfer in sediment fuel cells.

Am J Pathol, 2004 Oct, 165(4), 1423 - 31
FLICE-like inhibitory protein (FLIP) protects against apoptosis and suppresses NF-kappaB activation induced by bacterial lipopolysaccharide; Bannerman DD et al.; Bacterial lipopolysaccharide (LPS) via its activation of Toll-like receptor-4 contributes to much of the vascular injury/dysfunction associated with gram-negative sepsis . Inhibition of de novo gene expression has been shown to sensitize endothelial cells (EC) to LPS-induced apoptosis, the onset of which correlates with decreased expression of FLICE-like inhibitory protein (FLIP) . We now have data that conclusively establish a role for FLIP in protecting EC against LPS-induced apoptosis . Overexpression of FLIP protected against LPS-induced apoptosis, whereas down-regulation of FLIP using antisense oligonucleotides sensitized EC to direct LPS killing . Interestingly, FLIP overexpression suppressed NF-kappaB activation induced by LPS, but not by phorbol ester, suggesting a specific role for FLIP in mediating LPS activation . Conversely, mouse embryo fibroblasts (MEF) obtained from FLIP -/- mice showed enhanced LPS-induced NF-kappaB activation relative to those obtained from wild-type mice . Reconstitution of FLIP-/- MEF with full-length FLIP reversed the enhanced NF-kappaB activity elicited by LPS in the FLIP -/- cells . Changes in the expression of FLIP had no demonstrable effect on other known LPS/Tlr-4-activated signaling pathways including the p38, Akt, and Jnk pathways . Together, these data support a dual role for FLIP in mediating LPS-induced apoptosis and NF-kappaB activation.

J Bacteriol, 2004 Oct, 186(20), 6876 - 84
Isolation, free-living capacities, and genome structure of "Candidatus Glomeribacter gigasporarum," the endocellular bacterium of the mycorrhizal fungus Gigaspora margarita; Jargeat P et al.; "Candidatus Glomeribacter gigasporarum" is an endocellular beta-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita . We established a protocol to isolate "Ca . Glomeribacter gigasporarum" from its host which allowed us to carry out morphological, physiological, and genomic investigations on purified bacteria . They are rod shaped, with a cell wall typical of gram-negative bacteria and a cytoplasm rich in ribosomes, and they present no flagella or pili . Isolated bacteria could not be grown in any of the 19 culture media tested, but they could be kept alive for up to 4 weeks . PCR-based investigations of purified DNA from isolated bacteria did not confirm the presence of all genes previously assigned to "Ca . Glomeribacter gigasporarum." In particular, the presence of nif genes could not be detected . Pulsed-field gel electrophoresis analyses allowed us to estimate the genome size of "Ca . Glomeribacter gigasporarum" to approximately 1.4 Mb with a ca . 750-kb chromosome and a 600- to 650-kb plasmid . This is the smallest genome known for a beta-proteobacterium . Such small genome sizes are typically found in endocellular bacteria living permanently in their host . Altogether, our data suggest that "Ca . Glomeribacter gigasporarum" is an ancient obligate endocellular bacterium of the AM fungus G . margarita.

J Vet Diagn Invest, 2004 Sep, 16(5), 374 - 81
Characterization of a wild-type strain of Francisella tularensis isolated from a cat; Inzana TJ et al.; Francisella tularensis type A is the primary cause of tularemia in animals and humans in North America . The majority of research on F . tularensis has been done with the attenuated live vaccine strain (LVS), which is a type B, but very few wild-type F . tularensis strains have been characterized . A gram-negative coccobacillus that was isolated in pure culture from the lungs of a cat that died after being lost for 5 days was received for identification at the Virginia-Maryland Regional College of Veterinary Medicine Teaching hospital . The isolate (strain TI0902) was not identified (or was misidentified) by commercial identification systems; however, it was identified as F . tularensis subspecies tularensis (type A) by sequencing a portion of the 16S ribosomal RNA gene . Furthermore, repetitive extragenic palindromic sequences-polymerase chain reaction amplified a 4-kb DNA fragment from TI0902 that was characteristic of F . tularensis type A but not type B . The electrophoretic profile of the lipopolysaccharide of strain TI0902 was identical to that of the LVS by Western blotting with antiserum to LVS . The protein-enriched outer membrane of strain TI0902 contained 6-8 proteins, which were similar in molecular size to those from the LVS . Electron microscopy of negatively stained and alcian blue-stained LVS and TI0902 cells showed that both strains were coccobacillary in shape and may be encapsulated . However, after mouse challenge, the TI0902 strain was clearly more virulent than the LVS strain . Results of this study indicate that the genotype and phenotype of wild-type F . tularensis type A strain TI0902 is similar, but not identical, to that of the LVS strain . Further studies will help determine whether pathogenesis and host-pathogen interactions are also similar between the 2 strains.

Postepy Hig Med Dosw (Online), 2004 Sep 20, 58, 333 - 42
{Chemical structure and biosynthesis of lipopolysaccharide--important component of the cell envelope of Gram-negative bacteria}; Kaszowska M; The outer membrane is the first layer of the cell envelope of Gram-negative bacteria . This fragment of the cell envelope is built of phospholipids in the internal part and mainly of lipopolysaccharides in the external part . Lipopolysaccharide (LPS, endotoxin) is a thermostabile component consisting of three parts which differ in chemical structure and biological activity . LPS contributes greatly to the structural integrity of bacteria and protects them from host immune defenses.This review explains what makes this LPS leaflet an effective barrier to permeability and describes the pathways of biosynthesis and the assembly of the hydrophobic domain, known as lipid A, a core oligosaccharide, and the distal polysaccharide (O-antigen).

Microb Pathog, 2004 Oct, 37(4), 185 - 91
Toll-like receptor 4 (TLR4) does not confer a resistance advantage on mice against low-dose aerosol infection with virulent type A Francisella tularensis; Chen W et al.; Francisella tularensis, the causative agent of tularemia, is a gram-negative facultative intracellular bacterium . Toll-like receptor (TLR) 4 is considered to be critical for inducing host innate immunity against many gram-negative bacteria including many respiratory pathogens . To determine the role of TLR4 in host defense against airborne F . tularensis infection, TLR4-defective C3H/HeJ (TLR4(d)) or wild-type C3H/HeOuJ (WT) mice were challenged by low-dose aerosol with type A F . tularensis, and the course of the infection and host responses were compared at day 2 and 4 post-inoculation (dpi) . At dpi 2, bacterial burdens in the lungs were similar between TLR4(d) and WT mice, but TLR4(d) mice surprisingly harbored approximately 10-fold fewer bacteria in their spleens and livers . However, the bacterial burdens at dpi 4, the mortality and median time to irreversible moribundity were indistinguishable between the two mouse strains . In addition, the inflammatory responses to the infection, as reflected by the cytokine levels and leukocyte influx in the bronchoalveolar lavage fluid and histopathological analysis, were similar between both mouse strains . Additionally, as with C3H mice, we found no difference in either the median time to death or the survival rate between TLR4-deleted C57BL/10ScNJ mice and WT C57BL/10 mice . Combined, these data suggest that TLR4 does not contribute to resistance of mice to airborne type A F . tularensis infection.

J Vet Med B Infect Dis Vet Public Health, 2004 Aug, 51(6), 285 - 7
Virulence genes of Escherichia coli strains isolated from mastitic milk; Bean A et al.; Escherichia coli, a Gram-negative environmental pathogen associated with bovine mastitis was isolated from the milk of 34 symptomatic cows that had been diagnosed with clinical mastitis . Eighty isolates were obtained over a 17-month period and these isolates were screened by DNA amplification for the following E . coli virulence genes: cnf1, cnf2, eaeA, eagg, einv, ltx1, stx1, stx2 and vt2e . Thirty of the bacterial isolates, obtained from 23 different cows, had toxin genes identified in their DNA . The most common virulence gene detected was stx1, with a prevalence of 31%, followed by cnf2 (7.5%), vt2e (6.25%) and eaeA (4%) . The possession of different virulence genes by the bacterial isolates had no discernable impact on the health status of the cows as there was no correlation between the potential for toxin production by the E . coli isolates and the systemic clinical condition of the respective infected cows.

Mol Microbiol, 2004 Oct, 54(1), 14 - 22
Recognition of iron-free siderophores by TonB-dependent iron transporters; Schalk IJ et al.; TonB-dependent iron transporters reside in the outer membranes of Gram-negative bacteria, transporting ferric-complexes into the periplasm by a mechanism requiring proton motive force and an integral inner membrane complex, TonB-ExbB-ExbD . Certain TonB-dependent transporters contain an additional domain at the N-terminus, which interacts with an inner membrane regulatory protein and a cytoplasmic sigma factor to induce transcription of iron transport genes when a ferric-ligand is bound at the extracellular surface of the transporter . Transport of the ferric-ligand is apparently not necessary for transcription induction . Recent biophysical and crystallographic experiments have shown that this subclass of TonB-dependent iron transporters can bind iron-free ligands, whereas only the ferric-ligands are transported into the periplasm . This review focuses on the ligand binding properties of these transporters and includes a discussion of the biological function of the additional domain, the mechanism of transcription induction and the mechanism of ferric-ligand transport.

Biotechnol Prog, 2004 Sep-Oct, 20(5), 1528 - 33
Extracellular release of recombinant alpha-amylase from Escherichia coli using pulsed electric field; Shiina S et al.; It is difficult for Escherichia coli to secrete products such as recombinant enzymes, because the Gram-negative bacterium has a double membrane structure and so some of the products are accumulated in a periplasmic space . In this study, we demonstrated that recombinant alpha-amylase can be released from recombinant E . coli HB101/pHI301A during cultivation by applying a pulsed electric field (PEF) . When a PEF (12 kV, 2 Hz) was applied for 30 min with an interval of 30 min from the point of OD660=0.7, the amount of released alpha-amylase was about 30% of the total amount of alpha-amylase produced in the cells . As a result of SDS-PAGE and activity staining analyses, it was confirmed that the released proteins were not all of the intracellular proteins, and the alpha-amylase, which was identical with intracellular alpha-amylase, was released by applied PEF cultivation . PEF treatment could be useful for easy release of periplasmic protein with selectivity.

Med Sci Monit, 2004 Oct, 10(10), BR362 - 70 Epub 2004 Sep 23.
T cell-specific immune response induced by bacterial ghosts; Felnerova D et al.; BACKGROUND: Bacterial ghosts, genetically inactivated Gram-negative bacterial pathogens, possess significant advantages over commonly used vaccination technologies . The autolysis of the bacteria, by the expression of a cloned viral gene, results in empty bacterial envelopes through the expulsion of cytoplasmic content . Immunostimulatory properties are generally presented through the targeting of professional antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs) . MATERIAL/METHODS: This study investigated the interactions between porcine antigen-presenting cells and bacterial ghosts derived from the bacterial pathogen Actinobacillus pleuropneumoniae . The maturation process of DCs and their generation of immune responses to bacterial ghosts was shown by the expression of activation markers on their surface, as well as in the functional tests . RESULTS: A population of porcine APCs was generated from PBS by incubation with rpo-GMCSF and rh-IL-4 . The cells expressed SWC3, MIL-2, CD80/86 molecules, as well as a high level of MSA3 molecules . The internalization of bacterial ghosts by the cells resulted in increased expression of MSA3 molecules . The capacity of T cells to proliferate when induced by bacterial ghosts was 4 times higher in the cultures including APCs than in cultures stimulated with bacterial ghosts only . CONCLUSIONS: We found that antigen-presenting cells have the capacity to stimulate specific T cells after the internalization and processing of Actinobacillus ghosts, as demonstrated by a strong specific T-cell response generated against the ghost antigens.

Pediatr Blood Cancer, 2004 Nov, 43(6), 637 - 43
A pilot study of prophylactic ciprofloxacin during delayed intensification in children with acute lymphoblastic leukemia; Yousef AA et al.; BACKGROUND: We hypothesized that prophylactic administration of an appropriate antibiotic following each delayed intensification (DI) in children with acute lymphoblastic leukemia (ALL) would reduce the episodes of fever and bacteremia associated with neutropenia, and hence reduce both the rate and duration of hospitalization . PROCEDURE: All patients in the study were treated according to a modified Medical Research Council United Kingdom ALL XI (MRC UKALL XI) protocol utilizing three DI courses . Between June and December 2000 patients received prophylactic ciprofloxacin following DI courses . The rates of hospitalization and bacteremias were compared to ALL patients who had received between one and three DI courses prior to June 2000 . RESULTS: There were 69 patients who received a total of 194 DIs (controls 130; study group 64) . The rate of hospitalization was 90% in the controls and 58% in the study group (P < 0.001) . The median hospital stay was 10.1 days for controls and 6.0 for the study group (P < 0.001) . Intensive care unit admissions were reduced from 12 to 1.5% (P = 0.02) . The overall rate of proven bacteremia was reduced from 22 to 9% (P = 0.028) . There were no Gram-negative bacteremias in the study group compared to 10 (7.7%) in the controls (P < 0.001) . CONCLUSIONS: Compared to historical controls, patients in this study receiving prophylactic ciprofloxacin had a reduced rate and duration of hospitalization and incidence of Gram-negative bacteremia . (c) 2004 Wiley-Liss, Inc.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1829 - 35
Kangiella koreensis gen . nov., sp . nov . and Kangiella aquimarina sp . nov., isolated from a tidal flat of the Yellow Sea in Korea; Yoon JH et al.; Two Gram-negative, non-motile, non-spore-forming, rod-shaped organisms, strains SW-125T and SW-154T, were isolated from tidal flat sediment of the Yellow Sea in Korea, and subjected to a polyphasic taxonomic study . Strains SW-125T and SW-154T grew optimally at 30-37 degrees C and in the presence of 2-3 % (w/v) NaCl . They contained ubiquinone-8 (Q-8) as the predominant respiratory lipoquinone and iso-C(15 : 0) as the major fatty acid . The DNA G + C contents of strains SW-125T and SW-154T were 44 mol% . Phylogenetic trees based on 16S rRNA gene sequences revealed that the two strains form deep evolutionary lineages of descent within the gamma-Proteobacteria . Strains SW-125T and SW-154T exhibited 16S rRNA gene sequence similarity levels of less than 90 % to members of the gamma-Proteobacteria used in this analysis . Strains SW-125T and SW-154T showed a 16S rRNA gene sequence similarity level of 98.5 % and a mean DNA-DNA relatedness level of 9.4 % . Therefore, on the basis of phenotypic, phylogenetic and genomic data, a new genus, Kangiella gen . nov., is proposed to accommodate the novel strains, comprising two novel species, Kangiella koreensis sp . nov . (type strain, SW-125T = KCTC 12182T = DSM 16069T) and Kangiella aquimarina sp . nov . (type strain, SW-154T = KCTC 12183T = DSM 16071T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1811 - 4
Duganella violaceinigra sp . nov., a novel mesophilic bacterium isolated from forest soil; Li WJ et al.; A mesophilic bacterium, designated strain YIM 31327T, was isolated from a forest soil sample collected from Yunnan Province, China, and was then investigated using a polyphasic approach . The strain grew optimally at 28-30 degrees C and pH 7.2 . The cells were Gram-negative, short, rod-shaped, motile and non-spore-forming with flagella . The major ubiquinone was Q-8 and the cellular fatty acids were C(16 : 0) and C(12 : 0) . The DNA G + C content of strain YIM 31327T was 62.8 mol% . Phylogenetic analysis revealed that strain YIM 31327T was a member of the beta-Proteobacteria, being most closely related to Duganella zoogloeoides, with which it exhibited less than 96 % 16S rRNA gene sequence similarity . On the basis of the phenotypic and genotypic differences between strain YIM 31327T and D . zoogloeoides, a novel species, Duganella violaceinigra sp . nov., is proposed, with YIM 31327T (= CIP 108077T = KCTC 12193T) as the type strain.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1741 - 5
Psychrobacter maritimus sp . nov . and Psychrobacter arenosus sp . nov., isolated from coastal sea ice and sediments of the Sea of Japan; Romanenko LA et al.; Four strains of Gram-negative, aerobic, psychrotolerant, non-motile, non-pigmented bacteria were isolated from coastal sea-ice and sediment samples . These strains displayed the general chemotaxonomic and phenotypic features of members of the genus Psychrobacter . 16S rRNA gene sequencing positioned the three isolates KMM 3646T, KMM 3643 and KMM 3645 and isolate KMM 3659T in two distinct lineages within the genus Psychrobacter, displaying less than 98.5 % sequence similarity to the type strains of other Psychrobacter species . Genomic distinctness was supported by phenotypic differences in growth temperatures, salinity range for growth and metabolic properties . Based on a combination of phenotypic and biochemical characteristics and phylogenetic position, it is proposed that the members of these two distinct lineages represent two novel species, for which the names Psychrobacter maritimus sp . nov . (type strain Pi2-20T = KMM 3646T = DSM 15387T) and Psychrobacter arenosus sp . nov . (type strain R7T = KMM 3659T = DSM 15389T) are proposed.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1687 - 92
Jannaschia cystaugens sp . nov., an Alexandrium (Dinophyceae) cyst formation-promoting bacterium from Hiroshima Bay, Japan; Adachi M et al.; Heterotrophic bacteria isolated from water samples taken from Hiroshima Bay, Japan, and referred to as Alexandrium (Dinophyceae) cyst formation-promoting bacteria, were assigned to the Roseobacter-Sulfitobacter-Silicibacter group within the alpha-Proteobacteria on the basis of nearly complete 16S rRNA gene sequences . Phylogenetic analyses showed that two strains, CFPB-A9T and CFPB-A5, are closely related to each other and that their closest relative was Jannaschia helgolandensis (95.9 % sequence similarity) . These strains were Gram-negative, motile, obligately aerobic rods that required sodium ions and 2-7 % sea salts for growth and did not produce bacteriochlorophyll a . Their optimal growth temperature was 25-30 degrees C . The strains had Q-10 as the dominant respiratory quinone . Primary cellular fatty acid in both strains was 18 : 1omega7c . The DNA G + C contents of strains CFPB-A9T and CFPB-A5 were 59.1 and 59.2 mol%, respectively . Based on physiological, biological, chemotaxonomic and phylogenetic data, the strains are considered to represent a novel species, Jannaschia cystaugens sp . nov., with type strain CFPB-A9T (= LMG 22015T = NBRC 100362T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1639 - 42
Desulfatibacillum alkenivorans sp . nov., a novel n-alkene-degrading, sulfate-reducing bacterium, and emended description of the genus Desulfatibacillum; Cravo-Laureau C et al.; An alkene-degrading, sulfate-reducing bacterium, strain PF2803T, was isolated from oil-polluted sediments (Fos Harbour, France) . The cells were found to be Gram-negative, non-sporulating, non-motile and to have a slightly curved rod shape . Optimum growth occurred at 1 % (w/v) NaCl, pH 6.8 and 28-30 degrees C . Strain PF2803T oxidized alkenes (from C8 to C23) . The G + C content of the genomic DNA was 57.8 mol% (HPLC) . On the basis of 16S rRNA gene sequence analyses, strain PF2803T belongs to the family 'Desulfobacteraceae' in the class 'Deltaproteobacteria', with Desulfatibacillum aliphaticivorans as its closest relative (99.6 % identity) . Comparative sequence analyses of the dissimilatory sulfite reductase (dsrAB) gene supported the affiliation of strain PF2803T to the genus Desulfatibacillum . DNA-DNA hybridization with its closest taxon demonstrated 48.4 % similarity . On the basis of the results of physiological and genetic analyses, strain PF2803T is identified as a novel species of the genus Desulfatibacillum, for which the name Desulfatibacillum alkenivorans sp . nov . is proposed . The type strain is PF2803T (= DSM 16219T = ATCC BAA-924T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1633 - 7
Pseudomonas psychrotolerans sp . nov; Hauser E et al.; Three yellow-pigmented, Gram-negative, rod-shaped, non-spore-forming bacterial strains, C36T, C37 and C39, were isolated in the Medical Clinic for Small Animals and Ungulates at the University for Veterinary Medicine in Vienna, Austria . On the basis of 16S rRNA gene sequence similarity, strain C36T was shown to belong to the genus Pseudomonas; Pseudomonas oleovorans DSM 1045T was the nearest relative (99.5 % sequence similarity) . Other Pseudomonas species shared <97 % sequence similarity with strain C36T . The presence of Q-9 as the major ubiquinone, the predominance of putrescine and spermidine in its polyamine patterns and its fatty acid profile {i.e . the predominance of C(16 : 0), summed feature 3 (C(16 : 1)omega7c and/or 2-OH C(15 : 0) iso), C(18 : 1)omega7c and the presence of 3-OH C(10 : 0), 3-OH C(12 : 0) and 2-OH C(12 : 0)} were in agreement with identification of this strain as a member of the genus Pseudomonas . Physiological and biochemical characteristics and the results of genomic fingerprinting clearly differentiated strain C36T from its phylogenetic relative P . oleovorans DSM 1045T . Results from DNA-DNA hybridization showed that strain C36T represents a species that is distinct from P . oleovorans DSM 1045T . These data demonstrate that strain C36T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas psychrotolerans sp . nov . is proposed . The type strain is C36T (= LMG 21977T = DSM 15758T) . Additionally, physiological, biochemical, chemotaxonomic and genomic fingerprints indicate that P . oleovorans ATCC 29347 may not be a member of the species P . oleovorans sensu stricto.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1627 - 31
Colwellia piezophila sp . nov., a novel piezophilic species from deep-sea sediments of the Japan Trench; Nogi Y et al.; Two strains of obligately piezophilic bacteria were isolated from sediment collected from the bottom surface of a small canyon on the seaward slope of the Japan Trench at a depth of 6278 m . The isolated strains, Y223GT and Y251E, are closely affiliated with members of the genus Colwellia on the basis of 16S rRNA gene sequence analysis . The G + C contents of both strains were about 39 mol% . DNA-DNA hybridization values between these strains and Colwellia reference strains were significantly lower than those accepted as the phylogenetic definition of a species . The novel strains are Gram-negative, polarly flagellated and facultatively anaerobic . The optimal pressure for growth was 60 MPa at both 4 and 10 degrees C; the most rapid growth rate was observed at 10 degrees C and 60 MPa . No growth occurred at 15 degrees C under any pressure studied . The major isoprenoid quinone is Q-8 . The predominant cellular fatty acids are C16 : 0 and C16 : 1 . Based on the taxonomic differences observed, the isolated strains appear to represent a novel obligately piezophilic Colwellia species . The name Colwellia piezophila sp . nov . (type strain Y223GT = JCM 11831T = ATCC BAA-637T) is proposed.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1581 - 4
Sutterella stercoricanis sp . nov., isolated from canine faeces; Greetham HL et al.; Morphological, biochemical and molecular genetic studies were carried out on an unknown non-spore-forming, Gram-negative, rod-shaped bacterium which was isolated from dog faeces . The bacterium grew under anaerobic conditions, was asaccharolytic, resistant to 20 % (v/v) bile and was oxidase- and urease-negative . Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that the unidentified bacterium clustered with Sutterella wadsworthensis, although a sequence divergence of >5 % indicated that the bacterium from dog faeces represented a previously unrecognized subline within the genus . On the basis of the presented findings, a novel species, Sutterella stercoricanis sp . nov., is described . The type strain of Sutterella stercoricanis is 5BAC4T (= CCUG 47620T = CIP 108024T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1477 - 82
Sulfurovum lithotrophicum gen . nov., sp . nov., a novel sulfur-oxidizing chemolithoautotroph within the epsilon-Proteobacteria isolated from Okinawa Trough hydrothermal sediments; Inagaki F et al.; A novel mesophilic sulfur- and thiosulfate-oxidizing bacterium, strain 42BKTT, was isolated from the gas-bubbling sediment at the Iheya North hydrothermal system in the mid-Okinawa Trough, Japan . The isolate was a Gram-negative, non-motile and coccoid to oval-shaped bacterium . Growth was observed at 10-40 degrees C (optimum 28-30 degrees C) and in the pH range 5.0-9.0 (optimum 6.5-7.0) . Strain 42BKTT grew chemolithoautotrophically with elemental sulfur or thiosulfate as a sole electron donor and oxygen (optimum 5 % in gas phase) or nitrate as an electron acceptor . The G + C content of the genomic DNA was 48.0 mol% . Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate belonged to the previously uncultivated Group F within the epsilon-Proteobacteria, which includes phylotypes of vent epibiont and environmental sequences from global deep-sea cold seep and hydrothermal vent fields . On the basis of the physiological and molecular characteristics of this isolate, the type species of a novel genus, Sulfurovum lithotrophicum gen . nov., sp . nov., is proposed . The type strain is 42BKTT (= ATCC BAA-797T = JCM 12117T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1469 - 76
Akkermansia muciniphila gen . nov., sp . nov., a human intestinal mucin-degrading bacterium; Derrien M et al.; The diversity of mucin-degrading bacteria in the human intestine was investigated by combining culture and 16S rRNA-dependent approaches . A dominant bacterium, strain MucT, was isolated by dilution to extinction of faeces in anaerobic medium containing gastric mucin as the sole carbon and nitrogen source . A pure culture was obtained using the anaerobic soft agar technique . Strain MucT was a Gram-negative, strictly anaerobic, non-motile, non-spore-forming, oval-shaped bacterium that could grow singly and in pairs . When grown on mucin medium, cells produced a capsule and were found to aggregate . Strain MucT could grow on a limited number of sugars, including N-acetylglucosamine, N-acetylgalactosamine and glucose, but only when a protein source was provided and with a lower growth rate and final density than on mucin . The G + C content of DNA from strain MucT was 47.6 mol% . 16S rRNA gene sequence analysis revealed that the isolate was part of the division Verrucomicrobia . The closest described relative of strain MucT was Verrucomicrobium spinosum (92 % sequence similarity) . Remarkably, the 16S rRNA gene sequence of strain MucT showed 99 % similarity to three uncultured colonic bacteria . According to the data obtained in this work, strain MucT represents a novel bacterium belonging to a new genus in subdivision 1 of the Verrucomicrobia; the name Akkermansia muciniphila gen . nov., sp . nov . is proposed; the type strain is MucT (= ATCC BAA-835T = CIP 107961T).

Arterioscler Thromb Vasc Biol, 2004 Nov, 24(11), 2174 - 80 Epub 2004 Sep 23.
Severe periodontitis enhances macrophage activation via increased serum lipopolysaccharide; Pussinen PJ et al.; OBJECTIVE: In periodontitis, overgrowth of Gram-negative bacteria and access of lipopolysaccharide (LPS) to circulation may activate macrophages leading to foam cell formation . We investigated whether periodontal treatment affects proatherogenic properties of low-density lipoprotein (LDL) and, thus, macrophage activation . METHODS AND RESULTS: LDL was isolated and characterized before and after treatment from 30 systemically healthy patients with periodontitis . Production of cytokines and LDL cholesteryl ester (LDL-CE) uptake by macrophages (RAW 264.7) was determined . Baseline periodontal variables correlated positively with serum LPS and C-reactive protein concentrations, as well as macrophage cytokine production and LDL-CE uptake . LPS concentration correlated positively with serum concentration of oxidized LDL and cytokine production . Higher cytokine production and LDL-CE uptake were induced by LDL isolated from patients with elevated number of affected teeth before treatment . Patients with serum LPS concentrations above the median (0.87 ng/mL) at baseline had higher serum high-density lipoprotein (HDL) cholesterol (baseline versus after treatment, 1.30+/-0.19 versus 1.48+/-0.28 mmol/L; P=0.002) and HDL/LDL ratio (0.31+/-0.01 versus 0.34+/-0.10; P=0.048), but lower serum LPS concentration (1.70+/-0.49 versus 0.98+/-0.50 ng/mL; P=0.004) and autoantibodies to beta2-glycoprotein I (0.11+/-0.06 versus 0.09+/-0.04 ELISA units; P=0.022) after treatment . CONCLUSIONS: Our results suggest that in systemically healthy patients, the infected/inflamed area in periodontitis is associated with macrophage activation via increased serum LPS concentration.

Genet Eng (N Y), 2004, 26, 255 - 77
Gene regulation by tetracyclines; Berens C et al.; Gene regulation by tetracyclines has become a widely-used tool to study gene functions in pro- and eukaryotes . This regulatory system originates from Gram-negative bacteria, in which it fine-tunes expression of a tetracycline-specific export protein mediating resistance against this antibiotic . This review attempts to describe briefly the selective pressures governing the evolution of tetracycline regulation, which have led to the unique regulatory properties underlying its success in manifold applications . After discussing the basic mechanisms we will present the large variety of designed alterations of activities which have contributed to the still growing tool-box of components available for adjusting the regulatory properties to study gene functions in different organisms or tissues . Finally, we provide an overview of the various experimental setups available for pro- and eukaryotes, and touch upon some highlights discovered by the use of tetracycline-dependent gene regulation.

Infect Immun, 2004 Oct, 72(10), 6040 - 9
Bacterial overgrowth in the cystic fibrosis transmembrane conductance regulator null mouse small intestine; Norkina O et al.; We recently reported the inflammation of the cystic fibrosis (CF) mouse small intestine, and we hypothesized bacterial overgrowth as a possible cause . Quantitative PCR of bacterial 16S genomic DNA in the CF mouse small intestine revealed an increase of greater than 40-fold compared to controls . Sequencing of 16S PCR products and Gram staining showed that the majority of bacteria in the CF mouse intestine were gram negative . Bacteria were observed to colonize the mucus that accumulates in the intestinal lumen of mice with CF . Impaired Paneth cell defenses were suggested by observation of partially dispersed Paneth granules in the mucus plugs of CF mouse intestinal crypts, and this mucus was strongly immunoreactive for Paneth cell bactericidal products . The role of bacterial overgrowth in intestinal inflammation in CF was tested by treating mice with oral antibiotics (ciprofloxacin and metronidazole) for 3 weeks, which reduced bacterial load in the CF mouse small intestine over 400-fold . Antibiotic treatment decreased the expression of the inflammation-related genes mast cell protease 2, leucine-rich alpha2 glycoprotein/leucine-rich high endothelial venule glycoprotein, suppressor of cytokine signaling 3, hematopoietic cell transcript 1, and resistin-like molecule beta/found in inflammatory zone 2, all of which were no longer expressed at levels significantly different from control levels . The reduction of intestinal bacteria also significantly improved the growth of CF mice but had no effect on the growth of wild-type mice . These data suggest that bacterial overgrowth in the CF mouse small intestine has a role in inflammation and contributes to the failure to thrive in this mouse model of CF.

Infect Immun, 2004 Oct, 72(10), 5983 - 92
Legionella pneumophila DotU and IcmF are required for stability of the Dot/Icm complex; Sexton JA et al.; Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease . In contrast to all other L . pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS . Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure . We show here that dotU is partially required for L . pneumophila intracellular growth, similar to the known requirement for icmF . In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex . We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L . pneumophila becomes virulent . Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a DeltadotU DeltaicmF strain . Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the DeltadotU DeltaicmF mutant . This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L . pneumophila T4SS . Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

Infect Immun, 2004 Oct, 72(10), 5972 - 82
IcmF and DotU are required for optimal effector translocation and trafficking of the Legionella pneumophila vacuole; VanRheenen SM et al.; The gram-negative bacterium Legionella pneumophila causes a severe form of pneumonia called Legionnaires' disease, characterized by bacterial replication within alveolar macrophages . Prior to intracellular replication, the vacuole harboring the bacterium must first escape trafficking to the host lysosome, a process that is dependent on the Dot/Icm type IV secretion system . To identify genes required for intracellular growth, bacterial mutants were isolated that were delayed in escape from the macrophage but which retain a minimally functional Dot/Icm machinery . The mutations were found in eight distinct genes, including three genes known to be required for optimal intracellular growth . Two of these genes, icmF and dotU, are located at one end of a cluster of genes that encode the type IV secretion system, yet both icmF and dotU lack orthologs in other type IV translocons . DotU protein is degraded in the early postexponential phase in wild-type L . pneumophila and at all growth phases in an icmF mutant . IcmF contains an extracytoplasmic domain(s) based on accessibility to a membrane-impermeant amine-reactive reagent . In the absence of either gene, L . pneumophila targets inappropriately to LAMP-1-positive compartments during macrophage infection, is defective in the formation of replicative vacuoles, and is impaired in the translocation of the effector protein SidC . Therefore, although IcmF and DotU do not appear to be part of the core type IV secretion system, these proteins are necessary for an efficiently functioning secretion apparatus.

Infect Immun, 2004 Oct, 72(10), 5783 - 90
The Ton system, an ABC transporter, and a universally conserved GTPase are involved in iron utilization by Brucella melitensis 16M; Danese I et al.; Brucella spp . are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural host . The mechanism of DHBA entry into Brucella and other gram-negative bacteria is poorly understood . Using mini-Tn5Kmcat mutagenesis, we created a transposon library of Brucella melitensis 16M and isolated 32 mutants with a defect in iron acquisition or assimilation . Three of these transposon mutants are deficient in utilization of DHBA . Analysis of these three mutants indicated that the ExbB, DstC, and DugA proteins are required for optimal assimilation of DHBA and/or citrate . ExbB is part of the Ton complex, and DstC is a permease homologue of an iron(III) ABC transporter; in gram-negative bacteria these two complexes are involved in the uptake of iron through the outer and inner membranes, respectively . DugA is a new partner in iron utilization that exhibits homology with the bacterial conserved GTPase YchF . Based on this homology, DugA could have a putative regulatory function in iron assimilation in Brucella . None of the three mutants was attenuated in cellular models or in the mouse model of infection, which is consistent with the previous suggestion that DHBA utilization is not required in these models.

J Biol Chem, 2004 Dec 3, 279(49), 51647 - 53 Epub 2004 Dec 3.
Neurotoxic effects of lipopolysaccharide on nigral dopaminergic neurons are mediated by microglial activation, interleukin-1beta, and expression of caspase-11 in mice; Arai H et al.; The endotoxin lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall, selectively induces degeneration of substantia nigral (SN) dopaminergic neurons via activation of microglial cells in rats and mice . Caspase-11 plays a crucial role in LPS-induced septic shock in mice . We examined the mechanism of LPS neurotoxicity on SN dopaminergic neurons in C57BL/6 mice and caspase-11 knockout mice . Mice were stereotaxically injected with LPS into the SN on one side and vehicle into the SN of the other side . Immunohistochemistry, Western blotting analysis, enzyme-linked immunosorbent assay, and reverse transcriptase-PCR were performed to evaluate damage of SN dopaminergic neurons and activation of microglial cells . Intranigral injection of LPS at 1 or 3 microg/microl/site decreased tyrosine hydroxylase-positive neurons and increased microglial cells in the SN compared with the contralateral side injected with vehicle at days 7 and 14 post-injection in C57BL/6 mice . Intranigral injection of LPS at 3 microg/microl/site induced the expression of caspase-11 mRNA in the ventral midbrain at 6, 8, and 12 h post-injection, and the expression of caspase-11-positive cells in the SN at 8 and 12 h post-injection . Moreover, LPS at 3 microg/microl/site increased interleukin-1beta content in the ventral midbrain at 12 and 24 h post-injection . LPS failed to elicit these responses in caspase-11 knockout mice . Our results indicate that the neurotoxic effects of LPS on nigral dopaminergic neurons are mediated by microglial activation, interleukin-1beta, and caspase-11 expression in mice.

Biochem Biophys Res Commun, 2004 Oct 22, 323(3), 1069 - 74
Targeted mutagenesis of five conserved tryptophan residues of LolB involved in membrane localization of Escherichia coli lipoproteins; Wada R et al.; LolB, catalyzing the last step of lipoprotein transfer from the inner to the outer membrane of Escherichia coli, is itself a lipoprotein anchored to the outer membrane . Five Trp residues of LolB are conserved among LolB homologs in Gram-negative bacteria . These Trp residues were mutagenized to obtain defective LolB mutants . Mutation of Trp at position 52 to Pro impaired the receptor activity and caused accumulation of the LolA-lipoprotein complex in the periplasm . Similar mutants were obtained for Trp at position 117 . A mutant with Gly instead of Trp at position 148 retained the receptor activity but inhibited growth upon its overproduction . The outer membrane sorting of this mutant seemed to be defective, lipoprotein transfer thereby being perturbed when it was overproduced . Despite the strong conservation, no defective mutant for Trp at position 183 was obtained, and only weak mutants were isolated for Trp at position 18 . Based on the crystal structure of LolB, the phenotypes of these mutants are discussed.

Am J Med, 2004 Sep 15, 117(6), 420 - 8
Bacterial pyomyositis in the United States; Crum NF; The incidence of reported bacterial pyomyositis is increasing in the United States, especially among immunocompromised persons . This review summarizes all reported cases of pyomyositis among human immunodeficiency virus (HIV)-infected persons worldwide and HIV-negative persons in the United States since 1981 . During the era of combination antiretroviral therapy, bacterial pyomyositis among HIV-infected persons typically occurred in those with end-stage acquired immunodeficiency syndrome . Among non-HIV-infected patients, about half have a serious underlying medical problem, most commonly diabetes mellitus, malignancy, or a rheumatologic condition . These patients are more likely to have a gram-negative infection, a normal white blood cell count, multifocal involvement, or higher mortality than those without an underlying medical condition . The characteristics of cases in temperate areas are similar to tropical cases, except that the former occurs more often in immunocompromised persons; this may change with the HIV epidemic in tropical regions .

Curr Drug Targets Inflamm Allergy, 2004 Sep, 3(3), 291 - 7
Endotoxin recognition molecules MD-2 and toll-like receptor 4 as potential targets for therapeutic intervention of endotoxin shock; Miyake K; Gram-negative sepsis is the major cause of deaths in intensive care units of hospitals and continues to increase worldwide due to the increased frequency of invasive procedures and therapy leading to immunosuppression . This syndrome is characterized by endothelial damage, coagulopathy, loss of vascular tone, tissue hypoperfusion, and multiple-organ failure . They are caused by uncontrolled, overwhelming inflammatory responses, which are triggered by microbial products . Amongst these products, endotoxin also called LPS (lipopolysaccharide), a constituent of the outer membrane of Gram-negative bacteria, is known to play a central role by eliciting immune responses leading to production of proinflammatory cytokines . Our understanding of LPS recognition has increased dramatically over the last several years by identification of Toll-like receptor 4 (TLR4) and MD-2 as LPS recognition molecules . TLR4 is a mammalian homologue of drosophila Toll . The extracellular domain of TLR4 is associated with a molecule called MD-2 . Mice lacking either TLR4 or MD-2 do not respond to LPS and are resistant to endotoxin shock . Here, the potential for TLR4-MD-2 as target molecules for therapeutic intervention is discussed.

Langmuir, 2004 Sep 28, 20(20), 8817 - 22
Analysis of bacterial adhesion using a gradient force analysis method and colloid probe atomic force microscopy; Li X et al.; The atomic force microscope (AFM) has been used to examine the stickiness of bacteria on the basis of the analysis of approach and retraction force curves between the AFM tip and the bacterial surface . One difficulty in analyzing approach curve data is that the distance between the AFM tip and the surface of the bacterium is difficult to define . The exact distances are difficult to determine because the surface of the bacterium deforms during force imaging, producing a highly nonlinear region in the approach curve . In this study, AFM approach and retraction curves were obtained using a colloid probe AFM for three strains of Escherichia coli (D21, D21f2, and JM109) . These strains differed in their relative adhesion to glass surfaces, on the basis of measurements of sticking coefficients in packed bed flow through column tests . A gradient force curve analysis method was developed to model the interactions between the colloid probe and a surface . Gradient analysis of the approach curve revealed four different regions of colloid-surface interactions during the approach and contact of the probe with the bacterial surface: a noninteraction region, a noncontact phase, a contact phase, and a constant compliance region . The noncontact phase, which ranged from 28 to 59 nm for the three bacterial strains, was hypothesized to arise primarily from steric repulsion of the colloid by extracellular polymers on the bacterial surface . The contact phase, spanning 59-113 nm, was believed to arise from the initial pressure of the colloid on the outer membrane of the cell . The constant compliance region likely reflected the response of the colloid probe to the stiff peptidoglycan layer that confers strength and rigidity to gram negative bacteria . It was shown that the sticking coefficients reported for the three E . coli strains were correlated with the length of the noncontact phase but not the properties of the other phases . Sticking coefficients were also not correlated with any parameters determined from retraction force curves such as pull-off distances or separation energies . These results show that gradient analysis is useful for studying the contribution of the length of the exopolymers on the cell surface to bacterial adhesion to glass surfaces.

Nat Biotechnol, 2004 Oct, 22(10), 1275 - 81 Epub 2004 Sep 19.
The genome sequence of the capnophilic rumen bacterium Mannheimia succiniciproducens; Hong SH et al.; The rumen represents the first section of a ruminant animal's stomach, where feed is collected and mixed with microorganisms for initial digestion . The major gas produced in the rumen is CO(2) (65.5 mol%), yet the metabolic characteristics of capnophilic (CO(2)-loving) microorganisms are not well understood . Here we report the 2,314,078 base pair genome sequence of Mannheimia succiniciproducens MBEL55E, a recently isolated capnophilic Gram-negative bacterium from bovine rumen, and analyze its genome contents and metabolic characteristics . The metabolism of M . succiniciproducens was found to be well adapted to the oxygen-free rumen by using fumarate as a major electron acceptor . Genome-scale metabolic flux analysis indicated that CO(2) is important for the carboxylation of phosphoenolpyruvate to oxaloacetate, which is converted to succinic acid by the reductive tricarboxylic acid cycle and menaquinone systems . This characteristic metabolism allows highly efficient production of succinic acid, an important four-carbon industrial chemical.

J Gastroenterol Hepatol, 2004 Oct, 19(10), 1179 - 86
Effect of mesalazine, metronidazole and gentamicin on bacterial translocation in experimental colitis; Yigitler C et al.; BACKGROUND AND AIM: In inflammatory bowel disease it has been established that enteric microorganisms are present in the final stage of the active inflammatory process . The purpose of the present study was to investigate the effects of mesalazine, and metronidazole-gentamicin combination, on bacterial translocation in an animal colitis model . METHODS: Fifty rats were stratified into five groups . The control group (group NC) was given only 2 mL saline enema and the remaining four groups were given 2 mL acetic acid enema . Group CC was the diseased control group . The treatment regimens started on the fifth day: mesalazine enema in group MesC, metronidazole-gentamicin in group MGC, and mesalazine + metronidazole + gentamicin in group MesMGC . After death on day 10, 2.5-cm colonic segments from all groups were weighed separately . In all rats, histopathological scoring was done, and samples from feces, blood, liver and spleen underwent microbiological analyses . RESULTS: For all diseased rats, both mean weight loss and colonic segment weight/bodyweight ratio was significantly higher than that in the sham group . As compared with other groups, body and colonic segment changes as well as histopathological scoring in rats receiving mesalazine enema either solely or in combination with the antibiotics were lower . No bacterial growth was found in the blood, liver and spleen of the rats in the control group while enteric bacteria, mainly Escherichia coli (35%) were the most common bacteria translocated to that in the latter . Antibiotic combination, alone or in combination with mesalazine was effective in reducing the bacterial translocation while mesalazine administration did not properly influence its regression . CONCLUSIONS: Gram-negative enteric bacteria, predominantly E . coli, was the most common bacteria isolated in bacterial translocation occurring in acetic acid-induced colitis . This trial showed that mesalazine alone did not incorporate the reduction of infectious events, despite its beneficial effect on inflammatory changes in experimental colitis . Metronidazole and gentamicin combination given intraperitoneally was more effective than topical mesalazine in decreasing bacterial translocation.

Histol Histopathol, 2004 Oct, 19(4), 1201 - 8
Toll-like receptor 4 in normal and inflamed lungs and other organs of pig, dog and cattle; Wassef A et al.; Bacterial diseases, especially those of the lung caused by Gram-negative bacteria, inflict significant economic loss associated with mortality and morbidity in domestic animals . Toll-like receptor 4 (TLR4) has recently been recognized as a major receptor for cellular interactions with lipopolysaccharides derived from Gram-negative bacteria . However, there are no data on the expression of TLR4 in various organs of domestic animals . We performed immunohistochemistry and immuno-gold electron microscopy to localize TLR4 in lung and seven other organs from normal pig, dog and calf (n=2 each) and in inflamed lungs from calves (n=4) challenged with Mannheimia hemolytica . The data show TLR4 in macrophages in lung, small intestine, liver and spleen in all the species and pulmonary intravascular macrophages in calves and pigs . Epithelium in lung, small intestine, cornea and convoluted and straight renal tubules was stained for TLR4 . Vascular endothelium of large blood vessels only in lungs and skin was positive, and skeletal muscles were negative for TLR4 . In inflamed lungs, airway epithelium showed reduced staining for TLR4 while staining in macrophages remained unaltered . These are the first immunocytochemical data on TLR4 expression in domestic animal species and show similarity in TLR4 staining in macrophages, epithelium and vascular endothelium among dog, pig and cattle.

J Bacteriol, 2004 Oct, 186(19), 6430 - 6
A Francisella tularensis pathogenicity island required for intramacrophage growth; Nano FE et al.; Francisella tularensis is a gram-negative, facultative intracellular pathogen that causes the highly infectious zoonotic disease tularemia . We have discovered a ca . 30-kb pathogenicity island of F . tularensis (FPI) that includes four large open reading frames (ORFs) of 2.5 to 3.9 kb and 13 ORFs of 1.5 kb or smaller . Previously, two small genes located near the center of the FPI were shown to be needed for intramacrophage growth . In this work we show that two of the large ORFs, located toward the ends of the FPI, are needed for virulence . Although most genes in the FPI encode proteins with amino acid sequences that are highly conserved between high- and low-virulence strains, one of the FPI genes is present in highly virulent type A F . tularensis, absent in moderately virulent type B F . tularensis, and altered in F . tularensis subsp . novicida, which is highly virulent for mice but avirulent for humans . The G+C content of a 17.7-kb stretch of the FPI is 26.6%, which is 6.6% below the average G+C content of the F . tularensis genome . This extremely low G+C content suggests that the DNA was imported from a microbe with a very low G+C-containing chromosome.

J Endotoxin Res, 2004, 10(4), 223 - 8
Detection of endotoxin in sera from children hospitalized for treatment of diarrhea in Bangladesh; Ahmed T et al.; The level of circulating endotoxin was determined in the sera from children hospitalized for treatment of diarrhea at the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh . A significant level of endotoxin was detected in the sera from 23 (54%) of 42 children . On the other hand, in 32 normal controls, endotoxin was below the limits of detection of the assay . Antibiotic and fluid therapy markedly reduced the level of serum endotoxin and improved the general condition of most patients . Non-survivors (n = 5) had higher levels of circulating endotoxin before treatment than survivors (n = 37), suggesting a significant correlation between the serum endotoxin level before treatment and mortality . Malnutrition did not affect the serum endotoxin level in the patients with diarrhea . It was suggested that infection of Gram-negative bacteria might be involved in a significant number of patients with diarrhea in Bangladesh and that endotoxin might play a pathogenic role in those patients.

J Agric Food Chem, 2004 Sep 8, 52(18), 5627 - 34
Bacterial ghost technology for pesticide delivery; Hatfaludi T et al.; Bacterial ghosts are nondenaturated empty cell envelopes of Gram-negative bacteria produced by E-mediated lysis . Such envelopes from the plant-adhering bacterium Pectobacterium cypripedii were tested for their ability to adhere to plant material and to be used as carriers for pesticide delivery . We show, using fluorescence-labeled P . cypripedii ghosts, that depending on the target plants 55 or 10% (rice or soya, respectively) of the applied bacterial ghosts was retained on the leaves after heavy simulated rain (84 mm) . Furthermore, the bacterial ghosts could be loaded with the lipophilic triazole fungicide tebuconazole . In subsequent plant experiments in the glass house, the efficacy of the loaded bacterial ghost for resistance to rainfall and the protective and curative effects against the pathogens Erysiphe graminis, Leptosphaeria nodorum, and Pyrenophora teres on barley and wheat and against Sphaerotheca fuliginea on cucumber were tested . The bacterial ghosts were compared primarily with a commercial tebuconazole formulation, a wettable powder, as it has similar physical characteristics . The comparison revealed similar effects and showed consistently higher or comparable efficacy against the pathogens . The standard operational comparison with the most protective, cereal specific emulsion of oil in water displayed that the bacterial ghosts had equal to or lower efficacy than the emulsion . This study confirmed the potential of bacterial ghost platform technology as a new alternative carrier system for pesticides.

Leuk Lymphoma, 2004 Aug, 45(8), 1617 - 21
Infectious complications in adult acute lymphoblastic leukemia (ALL): experience at one single center; Offidani M et al.; Literature provides no specific data concerning the type and the risk factors for infection in adult patients with acute lymphoblastic leukemia (ALL) . We retrospectively analyzed 97 adult ALL patients who underwent conventional chemotherapy during a 14-year period with the aim to assess the incidence and the factors affecting onset and outcome of infections . We found that during induction therapy 50% of patients developed infection, mainly caused by gram-negative bacteria and with a mortality rate of 11% . In multivariate analysis age > 60 years was significantly associated with more infections (P = 0.04) and higher related mortality (P = 0.03) . Moreover, in 22% of patients infectious complications occurred during consolidation or maintenance therapy . Mortality rate of these infections, mostly due to opportunistic pathogens, was 16% . Factors affecting mortality was the cumulative dose of methylprednisolone given during induction therapy ( < or = 2600 mg = 31% vs . > 2600 mg = 69%; P = 0.03) . Among neutropenic patients, adults with ALL represent a peculiar population since they frequently develop gram negative infections during induction and opportunistic infections during post-remission treatments . Advanced age and high-dose methylprednisolone result the major risk factors for infection related mortality in the former therapeutic phase and in the latter one, respectively.

Syst Appl Microbiol, 2004 Aug, 27(4), 407 - 13
Fusobacterium canifelinum sp . nov., from the oral cavity of cats and dogs; Conrads G et al.; Fourteen strains of Gram-negative, anaerobic, fluoroquinolone-resistant, non-sporulating rods were isolated from various infections in cats and dogs, as well as from wounds in humans after cat- or dog-bites . These strains were characterized by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) regions, 16S rDNA, DNA-DNA hybridization, phylogenetic analysis, and phenotypic tests . The results indicate that the novel strains belong to a distinct species, closely related to Fusobacterium nucleatum . The species Fusobacterium canifelinum sp . nov . is proposed, with strain ATCC BAA 689T as the type strain.

Syst Appl Microbiol, 2004 Aug, 27(4), 399 - 406
Psychrobacter nivimaris sp . nov., a heterotrophic bacterium attached to organic particles isolated from the South Atlantic (Antarctica); Heuchert A et al.; An aggregate-attached bacterium, strain 88/2-7, was isolated from samples of the Southern Ocean and investigated in a polyphasic approach . The novel marine isolate is an aerobic, Gram-negative, oxidase- and catalase-positive, non-motile short rod and grows in form of cream-colored colonies . Growth was observed at 5-35 degrees C . The bacterium tolerated concentrations of 0-13% (w/v) NaCl and utilized a relatively restricted spectrum of carbon sources . The analysis of the fatty acids revealed 18:1 cis 9 (18:1omega9c) as main fatty acid . The G+C content of the DNA was approximately 42 mol% . The sequence of the 16S rDNA assigned strain 88/2-7 to the gamma-subclass of Proteobacteria with a similarity of 99.65% to Psychrobacter proteolyticus (DSM 13887T) . A DNA-DNA-hybridization study showed only 26.8% renaturation to the respective strain . Based on the morphological, physiological and molecular properties of the new isolate, the name Psychrobacter nivimaris sp . nov . (type strain 88/2-7T) is proposed.

J Infect Chemother, 2004 Aug, 10(4), 200 - 7
Opsonophagocytic dysfunction in patients with liver cirrhosis and low responses to tumor necrosis factor-alpha and lipopolysaccharide in patients' blood; Ono Y et al.; To evaluate their defense level against bacterial infection of patients with liver cirrhosis, we compared the luminol-dependent chemiluminescence (CL) response of peripheral blood from 40 patients with that from 40 healthy volunteers . Small quantities of heparinized whole blood (100 microl; final dilution, 1:10) were used for phagocytes, and CL was measured on addition of nonopsonized zymosan or Escherichia coli without special opsonization . Whole blood CL in cirrhotic patients was significantly lower than that in the healthy controls . The incidence of lower CL response in patients increased as disease stage advanced . Polymorphonuclear leukocytes (PMN) from cirrhotic patients exhibited a slightly lower CL response than those from controls, but this was not statistically significant . In contrast, the CL response of monocytes in patients was significantly lower than that of controls . The opsonizing capacity of the patients' sera and ascitic fluid was also decreased . In fact, the levels of opsonins such as complement in the patients' sera and both immunoglobulins and complement in the ascitic fluids were found to be lower in cirrhotic patients . On the basis of these findings, defect of opsonophagocytic function seems to participate in the increased susceptibility to infection in cirrhotic patients . Furthermore, whole blood CL induced by nonopsonized zymosan at the onset of relatively severe bacterial infections such as sepsis, pneumonia, or spontaneous bacterial infection was less augmented in the blood of cirrhotic patients than that in noncirrhotic patients . To clarify the reason why whole blood exhibits a lower CL response in the acute phase of bacterial infections, we investigated the priming effects of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha), well-known CL activators, on the CL response of whole blood obtained from cirrhotic patients in comparison with that from healthy persons . The priming effects were significantly decreased in patients' blood when compared with that of healthy persons . These low responses of patients' blood to LPS or TNF-alpha support our finding that phagocytes are not fully activated when gram-negative bacterial infections occur.

J Clin Microbiol, 2004 Sep, 42(9), 4393 - 5
Bacteremic infection with Pantoea ananatis; De Baere T et al.; A 73-year-old man was hospitalized for dyspnea and bilateral ankle edema . During his hospital stay he presented anal hemorrhage and developed a high fever after colonoscopy . A set of aerobic and anaerobic blood culture bottles yielded a pure culture of gram-negative rods, susceptible to all antibiotics tested . The API20E code was 1005133, resulting in a very good identification as Pantoea sp . Subsequent sequencing of the 16S rRNA gene revealed a final identification as Pantoea ananatis . The patient was given intravenous and oral therapy with piperacillin-tazobactam and ofloxacin and recovered completely from his infection.

Photochem Photobiol, 2004 Sep-Oct, 80(2), 286 - 93
Photodynamic studies and photoinactivation of Escherichia coli using meso-substituted cationic porphyrin derivatives with asymmetric charge distribution; Lazzeri D et al.; The photodynamic activities of novel asymmetrically meso-substituted cationic porphyrins, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 1 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 2 and its metal complex with Pd(II) 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli . The amphiphilic character of porphyrin 2 was increased by the presence of a high-lipophilic trifluoromethyl group and its photophysical properties changed by forming a complex with Pd(II) . Absorption and fluorescence spectroscopic studies were compared in different media . Fluorescence quantum yields (phi(F)) of 0.16 for 1 in tetrahydrofuran and 0.08 for 2 in N, N-dimethylformamide (DMF) were calculated, whereas no significant emission was detected for Pd(II) porphyrin 3 . The singlet molecular oxygen, O(2)((1)Delta(g)), production was evaluated using 9,10-dimethylanthracene in DMF yielding relative values of 1, 0.55 and 0.47 for porphyrins 3, 2 and 1, respectively . A faster decomposition of l-tryptophan was obtained using Pd(II) porphyrin 3 as sensitizer with respect to the free-base porphyrins 1 and 2 . In biological medium, the behavior of cationic porphyrins 1-3 were compared with that of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl)porphyrin 4, which was used as a noncationic sensitizer . These porphyrins are rapidly bound to E . coli cells in 5 min and the amount of cell-bound sensitizer is not appreciably changed incubating the cultures for longer times . The recovered porphyrin 2 after one washing step reaches a value of approximately 2.9 nmol/10(6) cells and this amount remains high even after three washes, indicating that this sensitizer is tightly bound to cells . Photosensitized inactivation of E . coli was analyzed using cells without and with one washing step . In both cases, a higher photoinactivation of cells was found for tricationic porphyrin 2 and 3, causing a approximately 5.5 log (99.999%) decrease of cell survival, when treated with 10 microM of sensitizer . Under these conditions, a lower effect was found for porphyrin 1 (approximately 4 log) whereas sensitizer 4 did not produce appreciable photodamage . The results were also confirmed by growth delay experiments . These studies show that the amphiphilic tricationic porphyrin 2 and 3 bearing a trifluoromethyl group can be a promising model for phototherapeutic agents with potential applications in inactivation of bacteria by photodynamic therapy.

J Immunol, 2004 Sep 15, 173(6), 3589 - 93
Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B; Park HS et al.; LPS, the primary constituent of the outer membrane of Gram-negative bacteria, is recognized by TLR4 . Binding of TLR4 to LPS triggers various cell signaling pathways including NF-kappaB activation and reactive oxygen species (ROS) production . In this study, we present the data that LPS-induced ROS generation and NF-kappaB activation are mediated by a direct interaction of TLR4 with (NAD(P)H oxidase 4 (Nox) 4), a protein related to gp91phox (Nox2) of phagocytic cells, in HEK293T cells . Yeast two hybrid and GST pull-down assays indicated that the COOH-terminal region of Nox4 interacted with the cytoplasmic tail of TLR4 . Knockdown of Nox4 by transfection of small interference RNA specific to the Nox4 isozyme in HEK293T cells expressing TLR4 along with MD2 and CD14 resulted in inhibition of LPS-induced ROS generation and NF-kappaB activation . Taken together, these results indicate that direct interaction of TLR4 with Nox4 is involved in LPS-mediated ROS generation and NF-kappaB activation .

J Biol Chem, 2004 Nov 26, 279(48), 50391 - 400 Epub 2004 Nov 26.
Structural and biochemical identification of a novel bacterial oxidoreductase; Loschi L et al.; By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria . The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ . YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E . coli characterized by the presence of this cofactor form . We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis . YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase . However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase . Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.

Vet Immunol Immunopathol, 2004 Oct, 101(3-4), 235 - 42
Surfactant protein D expression in normal and pneumonic ovine lung; Grubor B et al.; Surfactant protein D (SP-D) is a collagenous calcium-dependent lectin constitutively expressed by alveolar type II pneumocytes and non-ciliated bronchiolar epithelial (Clara) cells . It binds to surface glycoconjugates expressed by a wide variety of microorganisms such as Gram-negative bacteria, influenza A virus, and various fungi, leading to pathogen inactivation or enhanced neutrophil and macrophage activity . Since a hallmark of bronchopneumonia is the initiation of inflammation in the bronchi and bronchoalveolar junction, we chose a classic ruminant model of bronchopneumonia caused by Mannheimia haemolytica to study the expression of SP-D within the bronchioles of infected lambs . Healthy weaned lambs were inoculated with either pyrogen-free saline (controls) or M . haemolytica intrabronchially using a fiber-optic bronchoscope . SP-D protein and mRNA expression in lung was detected by immunohistochemistry (IHC) and fluorogenic real-time relative quantitative reverse transcriptase polymerase chain reaction (real-time RT-PCR), respectively, during acute (1 day), subacute (15 days), and chronic (45 days) bronchopneumonia . At 15 and 45 days post-inoculation, areas of lung had peribronchiolar inflammatory cell infiltrate, epithelial cell hyperplasia, tortuosity of the airway lumens, and decreased intensity of SP-D protein staining and number of positive cells . The levels of SP-D mRNA were not increased or significantly altered by M . haemolytica infection when compared to control animals . In conclusion, cell-associated SP-D protein expression significantly decreases within hyperplastic epithelium of lungs from infected animals during chronic bronchopneumonia . Exhaustion of SP-D protein reserves and absence of SP-D gene upregulation during the progression of bacterial pneumonia into chronicity may result in failure to clear the pathogen from the lung and/or cause animals to be more susceptible to re-infection.

Mol Microbiol, 2004 Sep, 53(6), 1771 - 83
Complex spatial distribution and dynamics of an abundant Escherichia coli outer membrane protein, LamB; Gibbs KA et al.; Advanced techniques for observing protein localization in live bacteria show that the distributions are dynamic . For technical reasons, most such techniques have not been applied to outer membrane proteins in Gram-negative bacteria . We have developed two novel live-cell imaging techniques to observe the surface distribution of LamB, an abundant integral outer membrane protein in Escherichia coli responsible for maltose uptake and for attachment of bacteriophage lambda . Using fluorescently labelled bacteriophage lambda tails, we quantitatively described the spatial distribution and dynamic movement of LamB in the outer membrane . LamB accumulated in spiral patterns . The distribution depended on cell length and changed rapidly . The majority of the protein diffused along spirals extending across the cell body . Tracking single particles, we found that there are two populations of LamB--one shows very restricted diffusion and the other shows greater mobility . The presence of two populations recalls the partitioning of eukaryotic membrane proteins between 'mobile' and 'immobile' populations . In this study, we have demonstrated that LamB moves along the bacterial surface and that these movements are restricted by an underlying dynamic spiral pattern.

J Periodontol, 2004 Jul, 75(7), 939 - 48
Inhibition of osteogenesis in vitro by a cigarette smoke-associated hydrocarbon combined with Porphyromonas gingivalis lipopolysaccharide: reversal by resveratrol; Andreou V et al.; BACKGROUND: Smoking and infection with Gram-negative bacterial pathogens are risk factors for alveolar bone loss . The aims of this study were: 1) to examine the combined effects of an aryl hydrocarbon, benzo{a}pyrene (BaP), that is concentrated in cigarette smoke, and lipopolysaccharide (LPS) extracted from Porphyromonas gingivalis on osteogenesis in a rat bone marrow cell (RBMC) model of osteogenesis; and 2) to determine whether resveratrol (Res), an aryl hydrocarbon receptor antagonist, could reverse the putative inhibitory effects of BaP + LPS on osteogenesis . METHODS: LPS of P . gingivalis strain 2561 was introduced in various concentrations to the RBMC in 96-well plates and kept in culture for 8 to 12 days . The same protocol was used for studying BaP and LPS + BaP combinations . Following the incubation periods, parameters of osteogenesis were measured, including formation of mineralized bone nodules, alkaline phosphatase activity, and total cell protein . Transcription of the pro-inflammatory cytokine interleukin (IL)-1beta in the cultures was determined by reverse transcriptase polymerase chain reaction (RT-PCR) . RESULTS: Bone nodule formation generally decreased significantly with increasing LPS concentrations (P<0.05), whereas total cell protein decreased only slightly (P>0.05) . BaP in previously high concentrations alone also caused a significant dose-dependent decrease in bone nodule formation (P<0.05) but when half maximal doses were used, significant decreases were most often seen when LPS was added . Hence, in combination, the inhibitory effects of LPS + BaP on osteogenesis were additive, inhibiting bone nodule formation up to 9-fold . Resveratrol partially reversed the inhibitory effects of low concentrations of LPS alone, and completely reversed the inhibition of nodule formation when low concentrations of LPS were combined with BaP . IL-1beta expression generally fluctuated inversely to the inhibitory activity of LPS, LPS + BaP, and LPS + BaP + Res combinations . CONCLUSIONS: Smoke-derived aryl hydrocarbons and bacterial LPS may act additively to inhibit bone formation . The findings may explain, in part, why net periodontal bone loss is greater and bone healing is less successful in smokers than non-smokers with periodontal infections . Reversal of the inhibitory effects in vitro by resveratrol suggests that this phytoalexin should be studied further for its potential therapeutic value, given its aryl hydrocarbon receptor antagonism and apparent anti-inflammatory activity.

Rev Physiol Biochem Pharmacol, 2004, 152, 111 - 33 Epub 2004.
Cytolethal distending toxins; Thelestam M et al.; The cytolethal distending toxins (CDTs) constitute the most recently discovered family of bacterial protein toxins . CDTs are unique among bacterial toxins as they have the ability to induce DNA double strand breaks (DSBs) in both proliferating and nonproliferating cells, thereby causing irreversible cell cycle arrest or death of the target cells . CDTs are encoded by three linked genes ( cdtA, cdtB and cdtC) which have been identified among a variety of Gram-negative pathogenic bacteria . All three of these gene products are required to constitute the fully active holotoxin, and this is in agreement with the recently determined crystal structure of CDT . The CdtB component has functional homology with mammalian deoxyribonuclease I (DNase I) . Mutation of the conserved sites necessary for this catalytic activity prevents the induction of DSBs as well as all subsequent intoxication responses of target cells . CDT is endocytosed via clathrin-coated pits and requires an intact Golgi complex to exert the cytotoxic activity . Several issues remain to be elucidated regarding CDT biology, such as the detailed function(s) of the CdtA and CdtC subunits, the identity of the cell surface receptor(s) for CDT, the final steps in the cellular internalization pathway, and a molecular understanding of how CDT interacts with DNA . Moreover, the role of CDTs in the pathogenesis of diseases still remains unclear.

Bone, 2004 Sep, 35(3), 629 - 35
Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF-kappa B ligand in periodontal ligament fibroblasts through the induction of interleukin-1 beta and tumor necrosis factor-alpha; Wada N et al.; Our recent work showed that human periodontal ligament fibroblasts (HPLF) secrete bioactive osteoprotegerin (OPG), which inhibits osteoclastic differentiation and activity . However, it is unknown how HPLF regulate bone metabolism in the presence of lipopolysaccharide (LPS), which is a cell component of gram-negative bacteria and a pathogen in inflammatory bone diseases such as periodontitis . The present study examined the effects of Escherichia coli LPS on the gene expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), OPG, and receptor activator of NF-kappa B ligand (RANKL) in HPLF using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis . In HPLF cultured with LPS for 48 h, expression of both OPG and RANKL mRNA was up-regulated, whereas for up to 24 h of stimulation, such up-regulation was not observed . However, LPS increased expression of IL-1 beta and TNF-alpha mRNA within 6 h of treatment . Moreover, in HPLF cultured with IL-1 beta or TNF-alpha, OPG and RANKL expression was induced within 12 h of culture . The administration of neutralizing antibodies against human IL-1 beta or TNF-alpha to LPS-treated cultures of HPLF inhibited the induction of OPG and RANKL expression . These suggest that LPS stimulates both OPG and RANKL expression in HPLF by up-regulating IL-1 beta and TNF-alpha . In addition, administration of conditioned medium (CM) from HPLF (HPLF-CM) stimulated with LPS for 48 h to mouse bone marrow culture failed to induce osteoclast-like cell (OCL) formation . When mouse spleen cells were cocultured with HPLF in the presence of LPS, OCL formation was completely blocked . Taken together, our results indicate that human periodontal ligament cells stimulated with LPS inhibit osteoclastogenesis by producing more effective OPG than RANKL via the induction of IL-1 beta and TNF-alpha.

Plasmid, 2004 Sep, 52(2), 69 - 83
Twenty years of the pPS10 replicon: insights on the molecular mechanism for the activation of DNA replication in iteron-containing bacterial plasmids; Giraldo R et al.; This review focuses on the contributions of the Pseudomonas replicon pPS10 to understanding the initiation of DNA replication in iteron-containing plasmids from Gram-negative bacteria . Dimers of the pPS10 initiator protein (RepA) repress repA transcription by binding to the two halves of an inverted repeat operator . RepA monomers are the active initiator species that bind to four directly repeated sequences (iterons) . pPS10 initiator was the first Rep protein whose domains were defined (two "winged-helix," WH modules) and their binding sites were identified at each half of the iteron repeat . This was confirmed by the crystal structure of the monomer of a homologous initiator (RepE from F plasmid) bound to iteron DNA . The recently solved structure of the dimeric N-terminal domain (WH1) of pPS10 RepA, when compared to the RepE monomer, shows that upon dimer dissociation an alpha-helix at WH1 C-terminus becomes part of an interdomain beta-sheet . In solution, the iteron sequence, by itself, can induce the same kind of structural transformation in RepA . This seems to alter the package of both WH domains to adapt their DNA reading heads (HTH motifs) to the distinct spacing between half repeats in iterons and operator . Based on biochemical and spectroscopic work, structural and functional similarities were proposed between RepA and archaeal/eukaryal initiators . This was independently confirmed by the crystal structure of the archaeal initiator Cdc6 . Characterization of mutants, either in pPS10 or in the Escherichia coli chromosome, has provided some evidence on a WH1-mediated interaction between RepA and the chromosomal initiator DnaA that results in a broadened-host range.

Pathol Biol (Paris), 2004 Sep, 52(7), 415 - 22
{Pathophysiological role of endotoxins, a common denominator to various diseases}; Amoureux MC; A growing number of investigations point to endotoxin or lipopolysaccharide as a central player in many pathophysiological states and diseases . Endotoxins are one of the most toxic biological contaminants continuously shed by both dead and live Gram negative bacteria . Endotoxins induce the primitive form of defense called innate immunity . Endotoxins have been related to inflammatory reactions observed in patients suffering from respiratory distress syndrome, multiorgan failure and septic shock, hepatic diseases, or in subjects affected by graft versus host disease after allogeneic transplantation . As our understanding of the molecular mechanisms underlying pathologies progresses, more diseases involving endotoxins emerge . Although these illnesses are multifactorial, the objective of this article is to review some of the common and distinct processes involving endotoxins in various disease states.

Ceylon Med J, 2004 Jun, 49(2), 57 - 60
Chryseobacterium meningosepticum infections in a dialysis unit; Perera S et al.; BACKGROUND: Chryseobacterium species are Gram-negative bacteria with an unusual antibiotic profile . Chryseobacterium meningosepticum is the species most commonly encountered as a human pathogen . OBJECTIVES: To study the microbiological, clinical and therapeutic features of C . meningosepticum infections in patients on dialysis, at Sri Jayewardenepura General Hospital (Teaching) (SJGH), and to trace the source of infections . DESIGN: A retrospective descriptive study . SETTING: Dialysis unit of SJGH . PATIENT: population Patients who underwent long term haemodialysis (HD) and manual intermittent peritoneal dialysis (IPD) in the dialysis unit . METHODS: Clinical and microbiological records of patients with C . meningosepticum infections over a period of 2 years were reviewed retrospectively . Environmental screening was carried out to detect a possible source of infection . RESULTS: Thirty five episodes of infection due to C . meningosepticum in 33 patients on HD and IPD were detected . There were 30 episodes of peritonitis, four of bacteraemia and one of asymptomatic colonization of a PD catheter . Isolates were resistant to aminoglycosides, chephalosporins and aztreonam, and sensitive to cotrimoxazole, vancomycin and rifampicin . They showed variable sensitivity to imipenem and ciprofloxacin . All except one patient had a favourable outcome . C . meningosepticum was cultured from a sink in the dialysis unit, but the original source of the organism was not known . CONCLUSION: C . meningosepticum could be an important pathogen in a dialysis unit, and fluoroquinolones and vancomycin are effective as empiric therapy.

Am J Otolaryngol, 2004 Sep-Oct, 25(5), 313 - 7
Effects of gentamicin on sensorineural elements of the cochlea in human temporal bones; Kusunoki T et al.; PURPOSE: Gentamicin is commonly prescribed for treatment of gram-negative bacterial infections . It has, however, been known to have vestibulotoxic and ototoxic effects in humans . Previous studies were confounded by the use of other types of ototoxic drugs, higher than therapeutic doses, and prolonged time periods between gentamicin administration and death . The purpose of this study is to observe the effects of parenteral gentamicin administration on the sensorineural elements of the cochlea in which the previously described factors did not exist . MATERIALS AND METHODS: Temporal bone specimens were divided into 2 groups . Group 1 included 16 "normal" temporal bones with no history or histopathologic findings of otologic disease or ototoxic drug administration, and group 2 included 17 temporal bones that received gentamicin treatment within 6 months before death . Temporal bones were excluded from group 2 if patients had histopathologic findings or history of otologic disease and/or other ototoxic drugs . All temporal bones were examined under light microscopy . RESULTS: The percentages of intact outer hair cells in the basal turn was significantly greater in group 1 compared with group 2 . There was no significant difference in hair cell number between the groups in the other turns . There was no significant difference in the number of spiral ganglion cells between groups in any turn . CONCLUSION: Although gentamicin is primarily considered to be vestibulotoxic, this study showed that gentamicin can damage sensorineural elements of the cochlea, even in younger patients . During the administration of this drug, hearing of the patient should be carefully monitored.

Tohoku J Exp Med, 2004 Sep, 204(1), 27 - 36
Activation of eosinophils by rice-husk dust exposure: a possible mechanism for the aggravation of asthma during rice harvest; Kayaba H et al.; Grain dust and other irritants affect the airway of allergic patients in rice-growing area during the harvest . The aim of this study was to elucidate the mechanism of airway hypersensitivity in rice-growing areas during the harvest . Firstly, the effect of rice-husk dust on eosinophil activation was studied . Secondary, the concentration of lipopolysaccharides (LPS), a potent activator of inflammatory cells, in rice-husk dust was measured . Since it is possible for LPS, a component of gram-negative bacterial cell wall, to adhere to the particle of smoke generated from rice-husk dust, LPS contained in the smoke was also measured . Furthermore, chemical irritants contained in the smoke generated from the rice-husk dust were analyzed . Microscopically, the dust contained fine thorns dropped off from the outer sheath of the rice, and irritated the skin, throat and eyes . The grain dust extract increased the expressions of eosinophil activation markers . These up-regulatory effects were largely dependent on LPS . The smoke contained LPS and several chemical irritants such as formaldehyde and acetaldehyde . Rice-husk dust and its smoke, hazardous air pollutants, probably play a major role in the aggravation of airway diseases in agricultural areas.

Clin Lab Med, 2004 Sep, 24(3), 605 - 26, vi
Emerging gram-negative enteric infections; Pitout JD et al.; This is an overview describing the microbiology, epidemiology, clinical presentations, laboratory diagnosis, and treatment of emerging gram-negative enteric infections . This review illustrates the shifts in epidemiology of infections caused by new and reemerging bacteria and highlights the role of the clinical microbiology laboratory in detecting these pathogens . It concludes with suggestions for the implementation of molecular techniques to ensure rapid surveillance and enhanced patient care.

J Periodontal Res, 2004 Oct, 39(5), 300 - 7
Inhibitory effects of green tea polyphenol (-)-epigallocatechin gallate on the expression of matrix metalloproteinase-9 and on the formation of osteoclasts; Yun JH et al.; BACKGROUND: Alveolar bone resorption is a characteristic feature of periodontal diseases and involves the removal of both the mineral and organic constituents of the bone matrix, which is caused by either multinucleated osteoclast cells or matrix metalloproteinases (MMPs) . The gram-negative bacterium, Porphyromonas gingivalis has been reported to stimulate the activity and expression of several groups of MMPs, whereas (-)-epigallocatechin gallate (EGCG), the main constituent of green tea polyphenols, has been reported to have inhibitory effects on the activity and expression of MMPs . OBJECTIVES: In the present study, we investigated the effects of the green tea polyphenol, EGCG, on the gene expression of osteoblast-derived MMP-2, -9 and -13, stimulated by P . gingivalis, and on the formation of osteoclasts . METHODS: The effect of EGCG on the gene expression of MMPs was examined by treating mouse calvarial primary osteoblastic cells with EGCG (20 microM) in the presence of sonicated P . gingivalis extracts . The transcription levels of MMP-2, -9 and -13 were assessed by reverse transcription-polymerase chain reaction (RT-PCR) . The effect of EGCG on osteoclast formation was confirmed by tartrate-resistant acid phosphatase (TRAP) staining in a co-culture system of mouse bone marrow cells and calvarial primary osteoblastic cells . RESULTS: Treatment with the sonicated P . gingivalis extracts stimulated the expression of MMP-9 mRNA and this effect was significantly reduced by EGCG, whereas the transcription levels of MMP-2 and MMP-13 were not affected by either the sonicated P . gingivalis extracts or EGCG . In addition, EGCG significantly inhibited osteoclast formation in the co-culture system at a concentration of 20 microM . CONCLUSIONS: These findings suggest that EGCG may prevent the alveolar bone resorption that occurs in periodontal diseases by inhibiting the expression of MMP-9 in osteoblasts and the formation of osteoclasts.

Microbiol Immunol, 2004, 48(8), 607 - 15
The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum; Kato C et al.; Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection . Fn is a gram-negative rod periodontal pathogen . NO could not be induced by heat-killed Fn or in untreated mice . This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr . We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo . The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal) . PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups . In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed . The apoptotic rate was very low and did not increase at 24 hr in vivo . Therefore, apoptosis was seldom relevant to the NO . In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time . Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death . Therefore, the NO derived from the iNOS is involved in the cytotoxicity . These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.

Shock, 2004 Sep, 22(3), 204 - 12
Abnormal PGE(2) regulation of monocyte TNF-alpha levels in trauma patients parallels development of a more macrophage-like phenotype; Laudanski K et al.; Some trauma patients' monocytes (MO) increase TNF-alpha levels concomitant to augmenting production of the TNF-alpha inhibitor prostaglandin E2 (PGE2), suggesting posttrauma MO insensitivity to PGE2 effects . This study assesses additional posttrauma MO PGE2 insensitivity effects on altering TNF-alpha form (membrane versus secreted), down-regulating MO receptor expression, and depressing MO APC function . Posttrauma MO TNF-alpha insensitivity to exogenous and autocrine PGE2 correlated to accumulation of TNF-alpha primarily as a membrane-bound cytokine (mTNF-alpha) . MO retention of mTNF-alpha correlated with unfavorable clinical outcomes and loss of antigen-presenting cell (APC) function as assessed by depressed MLR and dendritic cell (DC) differentiation . MO TNF-alpha sensitivity to down-regulation by IL-10 was retained, suggesting that PGE2-related functions are specifically altered in these patients' MO . Freshly isolated MO from all trauma patients had decreased expression of Toll-like receptor 4 (TLR4) for gram-negative bacteria . Exogenous PGE2 at high (10 (-6) M) or low (10 (-8) M) concentrations decreased normals' and further decreased APC-competent patients' MO TLR4 expression but had no effect on TLR2 . Patients' APC-dysfunctional MO failed to further down-regulate their TLR4 expression in response to additional PGE2, demonstrating another form of PGE2 insensitivity . One of the primary MO prostaglandin receptors, eicosanoid receptor 4 (EP4), was decreased on patients' APC dysfunctional MO, suggesting that depressed EP4 expression could contribute to PGE2 insensitivity in patients' MO . The APC dysfunctional MO's dysregulation of TLR4 expression paralleled increased macrophage-like characteristics such as increased CD64 expression density, elevated mTNF-alpha production, and increased PGE2 levels . Increased PGE2 levels still decreased patients' MO APC functions but failed to depress either MO TLR4 expression or mTNF-alpha levels, suggesting differential involvement of EP receptors in postinjury PGE2-mediated effects.

Mikrobiologiia, 2004 May-Jun, 73(3), 320 - 5
{The effect of the extracellular bacteriolytic enzymes of Lysobacter sp . on gram-negative bacteria}; Begunova EA et al.; The effect of the extracellular bacteriolytic enzymes of Lysobacter sp . on gram-negative bacteria was studied . These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria . The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells had been altered by treating them with polymyxin B.

Ann Univ Mariae Curie Sklodowska {Med}, 2003, 58(1), 459 - 65
Health effects of inhalation exposure to organic dust in hops farmers; Skorska C et al.; Medical examinations were performed in a group of 23 hops farmers exposed to organic dust from hop (Humulus lupulus) . The examinations took place in individual farms during harvesting, sorting and transporting of hop cones . As a reference group, 50 urban dwellers not exposed to organic dust were examined . There were conducted physical examinations, interviews concerning the occurrence of respiratory disorders and work-related symptoms, lung function tests, determination of cytokines concentrations, and allergological tests comprising skin prick test with 4 microbial antigens associated with organic dust, precipitin test with 12 microbial antigens, and a test for inhibition of leukocyte migration . Five farmers (21.7%) reported occurrence of work-related symptoms, including dry cough and dyspnoea . Eight farmers (34.8%) reported symptoms of chronic bronchitis . Mean spirometric values were within normal ranges . The farmers showed positive responses in precipitin test and test for inhibition of leukocyte migration to antigens of environmental microbes, mainly to the antigen of Gram-negative bacterium Pantoea agglomerans . The results showed a potential risk of occupational respiratory diseases in the population of hops farmers.

Mol Cell Biol, 2004 Sep, 24(17), 7359 - 69
Sphingosine kinase protects lipopolysaccharide-activated macrophages from apoptosis; Wu W et al.; Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria . Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs) . LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1 . Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA) . Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment . Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation . The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-kappaB reporters in RAW 264.7 cells . Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis . These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria .

Int J Biochem Cell Biol, 2004 Nov, 36(11), 2144 - 52
Host defense in oral and airway epithelia: chromosome 20 contributes a new protein family; Bingle CD et al.; The innate immune response is of pivotal importance in defending the mucosal barriers of the body against pathogenic attack . The list of proteins that contribute to this defense mechanism is constantly being updated . In this review we introduce a novel family of secreted proteins, palate, lung, and nasal epithelium clones (PLUNCs), that are expressed in the mouth, nose and upper airways of humans, mice, rats and cows . In humans, PLUNC genes are located in a compact cluster on chromosome 20, with similar loci being found in synteneic locations in other species . The protein products of this gene cluster are predicted to be structural homologues of the human lipopolysaccharide binding proteins, lipopolysaccharide binding-protein (LBP) and bacterial permeability-increasing protein (BPI), which are known mediators of host defense against Gram-negative bacteria . On the basis of these observations we outline why we believe PLUNC proteins mediate host defense functions in the oral, nasal and respiratory epithelia.

J Surg Res, 2004 Sep, 121(1), 76 - 83
PKC-zeta is essential for endotoxin-induced macrophage activation; Cuschieri J et al.; BACKGROUND: A critical element in sepsis-induced tissue injury is the release of pro-inflammatory mediators from LPS-activated macrophages . The cellular mechanisms involved in this process remain incompletely understood . The aim of the current study was to further clarify the mechanism of LPS activation through the TLR4 receptor complex by examining the roles of the various isoforms of PKC . MATERIALS AND METHODS: Differentiated THP-1 cells were subjected to LPS stimulation . Selected cells were pretreated with various concentrations of Go6983 to inhibit conventional, novel, and atypical PKC isoforms . Lipid raft, cellular, and nuclear proteins were then extracted and analyzed by Western blot and EMSA for components of the TLR4 pathway . Supernatants harvested under the various conditions were analyzed by ELISA for the production of TNF-alpha . RESULTS: LPS stimulation led to the mobilization of TLR4 to lipid rafts followed by phosphorylation and activation of IRAK, ERK 1/2, p38, and JNK/SAPK . Subsequently, LPS induced the activation of NF-kappaB and AP-1 . Activation of these TLR4-signaling components resulted in the production of TNF-alpha . Inhibition of conventional and novel PKC isoforms had no significant effect on macrophage activation . Inhibition of the atypical PKC, PKC-zeta, was associated with significant attenuation in the mobilization of TLR4 to lipid rafts, the activation of all TLR4-signaling components, and the production of TNF-alpha . CONCLUSION: This study demonstrates that the atypical PKC isoform, PKC-zeta, is critical to regulation of LPS-induced TLR4 lipid raft mobilization within macrophages, TLR4-signaling, and TNF-alpha production . Although the mechanism of its activation remains unresolved, it appears that modulation of PKC-zeta activity during Gram-negative infections may limit associated inflammatory-induced morbidity.

Heart, 2004 Sep, 90(9), 983 - 8
CD14 and toll-like receptor 4: a link between infection and acute coronary events?
Arroyo-Espliguero R, Avanzas P, Jeffery S, Kaski JC.
The CD14 receptor is a pattern recognition molecule in the innate immune response against microorganisms and other exogenous and endogenous stress factors . The most important CD14 signalling co-receptor is toll-like receptor 4 (TLR4), which activates, among others, the nuclear factor kappaB (NF-kappaB) inflammatory pathway . Besides its role in innate immunity and host defence, the proinflammatory cytokines expressed upon TLR4/NF-kappaB pathway activation exert proatherogenic effects . The CD14 C(-260)T promoter and TLR4 Asp299Gly functional polymorphisms have been recently implicated in the development of cardiovascular events, suggesting that the genetically determined inflammatory response against pathogens or their antigens may have a major role in atherogenesis and subsequent acute events . Is the association of these polymorphisms with cardiovascular disease more evidence for the implication of infection, especially by Gram negative bacteria, in the development of acute coronary events? This article reviews the molecular basis, biological functions, and clinical implications of the CD14/TLR4 polymorphisms in the development of cardiovascular events.

Anaesthesia, 2004 Sep, 59(9), 885 - 90
Antibiotic-treated infections in intensive care patients in the UK; Cuthbertson BH et al.; The purpose of this audit was to study reasons for starting antibiotic therapy, duration of antibiotic treatment, reasons for changing antibiotics and the agreement between clinical suspicion and microbiological results in intensive care practice . We conducted a multicentre observational audit of 316 patients . Data on demographic details, site, treatment and nature of infection were collected . The median duration of antibiotic therapy was 7 days . Infections were community-acquired in 160 patients (55%) . Antibiotics were started on clinical suspicion of infection in 237 patients (75%) . Pulmonary infections were the most common, representing 52% of all proven infections . Gram-negative organisms were the most common cause of proven infections (n = 90 (50%)) . The antibiotic spectrum was narrowed in light of microbiology results in 78 patients (43%) and changed due to antibiotic resistance in 38 patients (21%) . We conclude that the mean duration of treatment contrasts with existing published guidelines, highlighting the need for further studies on duration and efficacy of treatment in intensive care.

Crit Rev Clin Lab Sci, 2004, 41(3), 313 - 37
H . pylori infection: bacterial virulence factors and cytokine gene polymorphisms as determinants of infection outcome; Basso D et al.; The gram negative bacterium H . pylori infects the human stomach worldwide, invariably causing mucosal inflammation . In the majority of cases, H . pylori-associated gastritis remains the only clinical manifestation of the infection, which might cause, otherwise, peptic ulcer, gastric adenocarcinoma . or MALToma . The balance between the bacterial virulence machinery and the host response to the infection determines the different clinical outcomes . The main bacterial virulence factors comprise adhesins (BabA, SabA), the vacuolating cytotoxin VacA, and the products of cag pathogenicity island . The pattern of cytokine production in response to the infection is one of the main host determinants involved in limiting the infection outcome to gastritis or in favoring peptic ulcer or cancer onset . The polymorphisms of some cytokine genes (IL-1beta IL-1RN, TNF-alpha, IFN-gamma) have been correlated with H . pylori-associated gastric adenocarcinoma or peptic ulcer, possibly because they influence the amount of cytokine production in response to H . pylori infection . This review focuses on the role of H . pylori virulence genes and on host cytokines' genes polymorphisms in determining clinical outcome to H . pylori infection.

Curr Microbiol, 2004 Jul, 49(1), 13 - 21
Cell surface display of the chlamydial glycolipid exoantigen (GLXA) demonstrated by antibody-dependent complement-mediated cytotoxicity; Webley WC et al.; The chlamydial species are Gram-negative bacterial pathogens critical to human health . Their developmental cycle is associated with the formation and release of the broadly conserved glycolipid exoantigen (GLXA), which has been implicated in the chlamydial elementary body-host cell interaction . This study examines the potential surface display of this glycolipid by chlamydiae-infected cells and the ability of the GLXA they secrete to associate with the plasma membranes of uninfected cells, a prerequisite for exerting influence on them . The sequential incubation of anti-GLXA antibody and complement with Chlamydia trachomatis serovar K or C . pneumoniae AR-39-infected HeLa 229 or macrophage cells resulted in significant cellular cytotoxicity, which preceded the formation of mature elementary bodies . For uninfected cells, co-incubation of GLXA, purified from supernatants of either C . trachomatis or C . pneumoniae-infected HeLa 229 cells, followed by the successive addition of mouse anti-GLXA antibody and complement, yielded similar levels of cellular cytotoxicity . Thus, GLXA indeed is displayed on the surface of infected cells and, therefore, if antibody of appropriate specificity were present, this GLXA could serve to target these infected cells for elimination . Furthermore, released GLXA can associate with uninfected cells and therefore would be positioned to influence their behavior, especially in the context of infection.

J Clin Microbiol, 2004 Aug, 42(8), 3873 - 6
Distribution of Porphyromonas gingivalis biotypes defined by alleles of the kgp (Lys-gingipain) gene; Nadkarni MA et al.; Paired subgingival plaque samples representing the most-diseased and least-diseased sites were collected from 34 adult patients with diagnosed chronic periodontitis . The percentage of Porphyromonas gingivalis relative to the total anaerobic and gram-negative bacterial load at each site was determined by real-time PCR . Based on variations in the noncatalytic C terminus of the Lys-gingipain (Kgp), it was reasoned that DNA sequence variation in the 3'-coding region of the kgp gene might determine functional biotypes . Perusal of the available sequence information in GenBank indicated three such forms of the kgp gene corresponding to P . gingivalis strains HG66, 381, and W83 . Analysis of patient samples revealed the presence of a fourth genotype (W83v) that showed duplication of a sequence recognized by the W83 reverse primer . The four biotypes, HG66, 381, W83, and W83v, were present in the study group in the ratio 8:11:6:5, respectively . Each subject was colonized by one predominant biotype, and only three patients were colonized by a trace amount of a second biotype.

Toxicology, 2004 Sep 1, 201(1-3), 197 - 207
How does peripheral lipopolysaccharide induce gene expression in the brain of rats?
Singh AK, Jiang Y.
Lipopolysaccharide (LPS), the principal cell-wall component of gram-negative bacteria, is responsible for alterations in the central and peripheral tissues associated with gram-negative infections . However, the mechanism by which peripheral LPS cause central effects is not fully known . This study showed that peripheral LPS sequentially increased IL-1beta and iNOS mRNA levels, NO2 level, and CRF mRNA level in the hypothalamic PVN, and corticosterone concentration in blood . Brain-endothelium, but not hypothalamic PVN samples, from LPS injected rats contained ions for LPS lipids, bound BODIPY-LPS (bLPS), and expressed TLR-4, TLP-2 and CD14 mRNAs . This suggests that (1) LPS does not cross the blood-brain barrier, and (2) brain-endothelial cells contain LPS binding sites, TLR-4, TLR-2 and CD14 . Systemic LPS injection increased {14C}sucrose uptake, but did not affect {14C}dextran uptake into the brain . Thus, when injected systemically, LPS binds to its receptor and enter the endothelial cells where it increase BBB permeation in a mass-selective manner and triggers a series of signaling events leading to the development of inflammatory response in the brain.

Resuscitation, 2004 Aug, 62(2), 237 - 42
Ketamine improves survival and suppresses IL-6 and TNFalpha production in a model of Gram-negative bacterial sepsis in rats; Shaked G et al.; OBJECTIVE: In a previous study, ketamine suppressed Escherichia coli-induced production of the cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF) . In other previous studies ketamine improved survival after E . coli inoculation . However, the relationship between cytokines and survival following ketamine treatment is uncertain because no study has examined both cytokines and survival after E . coli inoculation . METHODS: Rats were given E . coli (0.4 x 10(9) colony forming unit (CFU)) at time 0, followed by ketamine (50 mg/kg, n=30) or saline (n=30) at 5 min or 2 h . IL-6 and TNF were measured in serum at 6 h, and mortality was recorded for 7 days . RESULTS: Survival rate with ketamine was 57% (17/30) and was significantly increased compared to saline (27%, 8/30, P=0.01) . IL-6 and TNF were lower with ketamine than saline (15,197 +/- 3444 versus 30,725 +/- 4623 pg/ml {mean +/- S.E.M.}, P=0.013 and 38.5 +/- 9.5 versus 122.5 +/- 14.0 pg/ml, P=0.001, respectively) . With ketamine, IL-6 (but not TNF) concentrations were lower in the survivors (10,900 +/- 776 pg/ml) as compared to the non-survivors (P=0.01) . IL-6 in ketamine-treated survivors was not different from that in saline-treated survivors . Conclusion: We conclude that ketamine given 5 min or 2 h after induction of E . coli sepsis significantly improves survival, possibly by interfering with the inflammatory cascade (as evidenced by attenuation of cytokine production).

Int J Med Microbiol, 2004 Jul, 294(1), 15 - 25
Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects virulence-associated regulators and growth in Acanthamoeba castellanii; Forsbach-Birk V et al.; Legionella bacteria have a developmental cycle in which they go from existing in the aquatic environment to replicating inside eukaryotic host cells . The adaptation to the new environment requires an efficient regulatory system . Overexpression of CsrA, a global regulatory protein found in a variety of gram-negative bacteria has been shown to suppress virulence-associated traits in Legionella pneumophila . Since evidence resulting only from overproduction may not be sufficient to validate the role of a regulatory protein, a csrA mutant strain, CsrA(-), with a drastically reduced production of CsrA, was created . Using RNA slot blots and Western blotting it was shown that fliA and flaA, genes which contribute to flagellation, were expressed early in the mutant . Additionally, in CsrA(-) the levels of the stationary-phase sigma factor, RpoS, and a recently described regulator of virulence traits, LetE, were increased . Growth curves of CsrA(-) bacteria were delayed with pigment production occurring at the same OD578 but at reduced levels in the mutant . Replication ability of the CsrA(-) mutant in amoebae was also affected . Based on these results, we could show that CsrA is involved in the regulation of the bacterial switch from the replicative to the transmissible form.

J Bacteriol, 2004 Aug, 186(16), 5342 - 54
Penetration of membrane-containing double-stranded-DNA bacteriophage PM2 into Pseudoalteromonas hosts; Kivela HM et al.; The icosahedral bacteriophage PM2 has a circular double-stranded DNA (dsDNA) genome and an internal lipid membrane . It is the only representative of the Corticoviridae family . How the circular supercoiled genome residing inside the viral membrane is translocated into the gram-negative marine Pseudoalteromonas host has been an intriguing question . Here we demonstrate that after binding of the virus to an abundant cell surface receptor, the protein coat is most probably dissociated . During the infection process, the host cell outer membrane becomes transiently permeable to lipophilic gramicidin D molecules proposing fusion with the viral membrane . One of the components of the internal viral lipid core particle is the integral membrane protein P7, with muralytic activity that apparently aids the process of peptidoglycan penetration . Entry of the virion also causes a limited depolarization of the cytoplasmic membrane . These phenomena differ considerably from those observed in the entry process of bacteriophage PRD1, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.

J Rheumatol, 2004 Aug, 31(8), 1546 - 50
Association between systemic lupus erythematosus and Helicobacter pylori seronegativity; Sawalha AH et al.; OBJECTIVE: Helicobacter pylori is a gram negative spiral bacterium that is clearly associated with a variety of gastrointestinal pathologies . A number of non-gastrointestinal diseases have also been associated with H . pylori . We investigated the prevalence of H . pylori seropositivity as part of a larger serologic survey in a group of 466 patients with systemic lupus erythematosus (SLE) and 466 controls . METHODS: We studied subjects for seropositivity against 5 antigens including mumps, measles, rubella, varicella zoster, and H . pylori . The 466 SLE patients were taken from a total of 290 pedigrees multiplex for SLE and matched to 466 controls for age (+/- 3 yrs), sex, and ethnicity to non-SLE affected individuals, taken mostly from the same collection of pedigrees multiplex for SLE . Assays for seropositivity were performed using a heterogeneous immunoassay technique . Pearson's chi-square was used to test for association of categorical variables and Student t-test for continuous variables . Logistic regression was used to compute the odds ratio for H . pylori seropositivity in patients and controls . RESULTS: There was a significant difference only in H . pylori seropositivity between SLE cases and their controls . The results were not altered by intrafamilial correlation . Subset analysis by race and sex showed that the differences between the African-American female patients with SLE and their matched controls were responsible for this association . Female African-American patients with SLE had a lower prevalence of H . pylori seropositivity compared to controls (38.1% vs 60.2%, OR 0.41, p = 0.0009, 95% CI 0.24-0.69) . Of the 113 African-American female SLE patients in the study group, 43 were seropositive for H . pylori . The mean age of onset for SLE was older in the seropositive group (34.4 yrs) compared to the seronegative SLE patients (28.0 yrs) (t = 2.11, p = 0.039) . CONCLUSION: Of 5 serologic tests performed, only the frequency of H . pylori seropositivity was different between SLE cases and their controls, and then only in African-Americans . We found an association between being seronegative for H . pylori and the development of SLE in African-American women, who also tend to be younger at the time of disease onset . These findings suggest that there is a possible protective role for H . pylori infection against the development of SLE or that immunoregulatory events leading to H . pylori seropositivity are inversely related to the risk of SLE.

Acad Emerg Med, 2004 Aug, 11(8), 867 - 73
Bench to bedside: HMGB1-a novel proinflammatory cytokine and potential therapeutic target for septic patients in the emergency department; Sama AE et al.; Overwhelming gram-negative bacterial infection and life-threatening systemic inflammation are widespread problems in critically ill emergency department patients . Currently, the treatment of these patients is largely supportive, focusing on antibiotics, fluids, hemodynamic and ventilatory support, and intensive monitoring . The only Food and Drug Administration-approved pharmaceutical agent for the treatment of sepsis is activated protein C, with its use largely relegated to the intensive care unit . The subject thus remains an active area of exploration for emergency medicine research . During sepsis and inflammation, innate immune cells release excessive amounts of proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1beta . If delivered early enough, anti-TNF antibodies can be an effective therapy in experimental models of septic shock . Anti-TNF antibodies have been developed for clinical use in rheumatoid arthritis and Crohn's disease . However, anti-TNF treatment for sepsis has been difficult to achieve in the clinical setting, perhaps because TNF's early release and transient appearance in the serum create a narrow therapeutic window . An alternative strategy would be to identify "late" mediators that may be clinically more accessible . High mobility group box 1 (HMGB1), a protein previously known only as a nuclear transcription factor, is now implicated as a late mediator of sepsis . Targeting late mediators of lethal systemic inflammation represents a novel approach that may widen the therapeutic window and lead to new strategies for inhibiting the deleterious effects of the inflammatory cascade . Here the authors review the studies that led to the discovery of HMGB1 as a late mediator of systemic inflammation and discuss the possibility of HMGB1 as a therapeutic target for septic patients in the emergency department.

Crit Care Med, 2004 Aug, 32(8), 1662 - 8
Open randomized phase II trial of an extracorporeal endotoxin adsorber in suspected Gram-negative sepsis; Reinhart K et al.; OBJECTIVE: An initial phase II trial to investigate the safety and therapeutic effect of the endotoxin adsorber system EN 500 in septic patients suffering from presumed Gram-negative infection . DESIGN: Open, controlled, prospective, randomized, multiple-center, parallel-group clinical trial . SETTING: Intensive care units of 31 university-affiliated and community hospitals in Europe . PATIENTS: One hundred forty-five patients with a clinical diagnosis of severe sepsis or septic shock due to suspected Gram-negative infection . INTERVENTIONS: Patients were randomized to receive either standard therapy alone for sepsis (n = 76) or standard therapy plus extracorporeal endotoxin adsorption (n = 67) daily for the first 4 days following study entry . MEASUREMENTS AND MAIN RESULTS: The primary end point was the proportion of responders (defined as a decrease in Acute Physiology and Chronic Health Evaluation II score by > or =4 points from study entry to day 4) . Secondary outcomes were the Sequential Organ Failure Assessment score and its components, length of intensive care unit stay, survival rate, and safety of the adsorber treatment . Patient characteristics at entry were well balanced between the two treatment groups, except for a higher Sequential Organ Failure Assessment score in the adsorber group . On all-subjects-treated analysis, 65% of the adsorber group were responders vs . 57% for the standard (p =.389) . A planned interim analysis restricted further enrollment to patients with peritonitis, in whom a slightly higher proportion of responders was observed with the adsorber treatment (69%) vs . standard treatment (54%, p =.159) . There were no differences in survival, but adsorption treatment in peritonitis patients was associated with trends toward a reduction in length of intensive care unit stay and a more rapid decline in plasma endotoxin concentrations . There was a significantly greater reduction in platelet count with the adsorber; however, this did not require extra treatment . CONCLUSIONS: The endotoxin adsorber system did not result in a significantly improved primary end point in patients with presumed Gram-negative sepsis . In patients with peritonitis, the adsorber treatment likewise did not result in significantly improved Acute Physiology and Chronic Health Evaluation II scores . There were no clinically important side effects . These results provide encouragement for further study of adsorber treatment in patients with high likelihood of Gram-negative sepsis (e.g., peritonitis).

Arh Hig Rada Toksikol, 2004 Jun, 55(2-3), 175 - 81
Endotoxin measurement in house dust using the end-point Limulus amoebocyte lysate method; Varnai VM et al.; Endotoxin is a lipopolysaccharide, a part of gram-negative bacteria cell membrane commonly present in general and many occupational environments . This paper describes sample preparation and endotoxin measurement in 16 samples of house dust from urban homes (Zagreb, Croatia) using end-point chromogenic Limulus amoebocyte lysate (LAL) bioassay . House dust was collected on cellulose filters by vacuuming bedroom and living room floors, and was kept frozen until assayed . Samples were extracted from filters with a 0.05% solution of Tween-20 in endotoxin-free water . Serial dilutions of samples were measured in duplicates . The linearity of the standard curve was satisfying (r=0.983), as well as the recovery (92 and 110%) and repeatability (coefficient of variation from 0 to 8.5%) . The endotoxin levels found in the house dust samples ranged from 4.8 to 200 EU/mg, with the arithmetic mean of 49.5 EU/mg (standard error of the mean of 12.1 EU/mg), and were in the range of house dust endotoxin values obtained by other authors.

Environ Toxicol Chem, 2004 Mar, 23(3), 591 - 8
Anaerobic transformation of compounds of technical toxaphene . 2 . Fate of compounds lacking geminal chlorine atoms; Ruppe S et al.; The major toxaphene metabolites in sediment and soils (2-exo,3-endo,6-exo,8,9,10-hexachlorobornane {B6-923} and 2-endo,3-exo,5-endo,6-exo,8,9,10-heptachlorobornane {B7-1001}) were incubated with the isolated gram-negative bacterium Dehalospirillum multivorans . Within 14 d, biotransformation of B7-1001 was nearly quantitative, resulting in two penta- and six hexachlorobornanes, as well as one unsaturated hexachloro compound of technical toxaphene . The major transformation product (approximately 50% of all metabolites) was identified as 2-exo,3-endo,5-exo,8,9,10-hexachlorobornane (B6-903) . Abiotic dehydrochlorination of B7-1001 with methanolic KOH resulted in the formation and subsequent identification of the lone unsaturated compound as 2,5-endo,6-exo,8,9,10-hexachloroborn-2-ene . Thus, dehydrochlorination was found to be a minor process of the anaerobic transformation of toxaphene . Biotransformation of 70% of amended B6-923 within 14 d demonstrated that reductive dechlorination was not exclusively associated with geminal Cl atoms, as previously suggested . Three pentachlorobornanes were identified as transformation products, one of which was identical with a transformation product of B7-1001 . This commonality unequivocally proves this metabolite to be 2-exo,3-endo,8,9,10-pentachlorobornane . Fifteen previously unknown metabolites of B6-923, B7-1001, and other toxaphene compounds identified in this study were detected in sediment from Lake Ontario (Canada), underscoring the importance of microbial toxaphene transformation in natural, aquatic environments.

Curr Allergy Asthma Rep, 2004 Sep, 4(5), 359 - 64
Complement-induced impairment of the innate immune system during sepsis; Albrecht EA et al.; The complement system is an integral part of innate immunity and is chiefly responsible for controlling bacterial infections, especially those involving gram- negative organisms . To accomplish this task, serum proteins engage in a series of enzymatic cascades . The cleaved proteins assemble pores on membranous structures, which lead to cell lysis . During this process, powerful inflammatory mediators are produced, including the anaphylatoxins, C5a, C3a, and the membrane attack complex (MAC) . Under systemic inflammatory conditions, an overactive complement system may compromise the effectiveness of innate immunity . We review the detrimental effects that are caused by uncontrolled complement activation during sepsis.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1311 - 6
Veillonella montpellierensis sp . nov., a novel, anaerobic, Gram-negative coccus isolated from human clinical samples; Jumas-Bilak E et al.; Three strains of a hitherto unknown, Gram-negative, anaerobic coccus were isolated from human samples . At the phenotypic level, the isolates displayed all the characteristics of bacteria belonging to the genus Veillonella . Sequence analysis revealed that the three strains shared >99.5% similarity in 16S rDNA sequence and >98.4% similarity in dnaK sequence . The three unknown strains formed a separate subclade that was clearly remote from Veillonella species of human and animal origin . Based on these results, the three strains were considered to represent a novel species within the genus Veillonella, for which the name Veillonella montpellierensis is proposed . The type strain of the species is ADV 281.99T (=CIP 107992T=CCUG 48299T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1263 - 9
Loktanella salsilacus gen . nov., sp . nov., Loktanella fryxellensis sp . nov . and Loktanella vestfoldensis sp . nov., new members of the Rhodobacter group, isolated from microbial mats in Antarctic lakes; Van Trappen S et al.; A taxonomic study was performed on 26 strains isolated from microbial mats in Antarctic lakes of the Vestfold Hills and the McMurdo Dry Valleys . Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the Rhodobacter group of the alpha-subclass of the Proteobacteria . Sequence similarity values for the strains with their nearest phylogenetic neighbours (Jannaschia, Octadecabacter and Ketogulonicigenium) ranged between 94.0 and 95.8% . DNA-DNA hybridizations and comparison of repetitive extragenic palindromic DNA-PCR (rep-PCR) fingerprinting patterns revealed that these strains are members of three distinct species . The isolates are Gram-negative, chemoheterotrophic, non-motile rods and their DNA G+C contents range from 59.4 to 66.4 mol% . Whole-cell fatty acid profiles are similar and the primary fatty acid in all the strains is 18 : 1 omega7c (74.1-87.7% of total) . Genotypic results together with phenotypic characteristics allowed the differentiation of these species from related recognized species of the alpha-Proteobacteria and the strains are assigned to a new genus, Loktanella gen . nov., with three novel species: Loktanella salsilacus sp . nov . (type species), consisting of ten strains with LMG 21507T (=CIP 108322T) as type strain; Loktanella fryxellensis sp . nov., consisting of 12 strains with LMG 22007T (=CIP 108323T) as type strain; and Loktanella vestfoldensis sp . nov., consisting of four strains with LMG 22003T (=CIP 108321TT) as type strain.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1185 - 90
Swaminathania salitolerans gen . nov., sp . nov., a salt-tolerant, nitrogen-fixing and phosphate-solubilizing bacterium from wild rice (Porteresia coarctata Tateoka); Loganathan P et al.; A novel species, Swaminathania salitolerans gen . nov., sp . nov., was isolated from the rhizosphere, roots and stems of salt-tolerant, mangrove-associated wild rice (Porteresia coarctata Tateoka) using nitrogen-free, semi-solid LGI medium at pH 5.5 . Strains were Gram-negative, rod-shaped and motile with peritrichous flagella . The strains grew well in the presence of 0.35% acetic acid, 3% NaCl and 1% KNO3, and produced acid from l-arabinose, d-glucose, glycerol, ethanol, d-mannose, d-galactose and sorbitol . They oxidized ethanol and grew well on mannitol and glutamate agar . The fatty acids 18 : 1omega7c/omega9t/omega12t and 19 : 0cyclo omega8c constituted 30.41 and 11.80% total fatty acids, respectively, whereas 13 : 1 AT 12-13 was found at 0.53% . DNA G+C content was 57.6-59.9 mol% and the major quinone was Q-10 . Phylogenetic analysis based on 16S rRNA gene sequences showed that these strains were related to the genera Acidomonas, Asaia, Acetobacter, Gluconacetobacter, Gluconobacter and Kozakia in the Acetobacteraceae . Isolates were able to fix nitrogen and solubilized phosphate in the presence of NaCl . Based on overall analysis of the tests and comparison with the characteristics of members of the Acetobacteraceae, a novel genus and species is proposed for these isolates, Swaminathania salitolerans gen . nov., sp . nov . The type strain is PA51T (=LMG 21291T=MTCC 3852T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1177 - 84
Oceanibulbus indolifex gen . nov., sp . nov., a North Sea alphaproteobacterium that produces bioactive metabolites; Wagner-Dobler I et al.; A water sample from the North Sea was used to isolate the abundant heterotrophic bacteria that are able to grow on complex marine media . Isolation was by serial dilution and spread plating . Phylogenetic analysis of nearly complete 16S rRNA gene sequences revealed that one of the strains, HEL-45T, had 97.4% sequence similarity to Sulfitobacter mediterraneus and 96.5 % sequence similarity to Staleya guttiformis . Strain HEL-45T is a Gram-negative, non-motile rod and obligate aerobe and requires sodium and 1-7% sea salts for growth . It contains storage granules and does not produce bacteriochlorophyll . Optimal growth temperatures are 25-30 degrees C . The DNA base composition (G+C content) is 60.1 mol% . Strain HEL-45T has Q10 as the dominant respiratory quinone . The major polar lipids are phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine and an aminolipid . The fatty acids comprise 18 : 1omega7c, 18 : 0, 16 : 1omega7c, 16 : 0, 3-OH 10 : 0, 3-OH 12 : 1 (or 3-oxo 12 : 0) and traces of an 18 : 2 fatty acid . Among the hydroxylated fatty acids only 3-OH 12 : 1 (or 3-oxo 12 : 0) appears to be amide linked, whereas 3-OH 10 : 0 appears to be ester linked . The minor fatty acid components (between 1 and 7%) allow three subgroups to be distinguished in the Sulfitobacter/Staleya clade, placing HEL-45T into a separate lineage characterized by the presence of 3-OH 12 : 1 (or 3-oxo 12 : 0) and both ester- and amide-linked 16 : 1omega7c phospholipids . HEL-45T produces indole and derivatives thereof, several cyclic dipeptides and thryptanthrin . Phylogenetic analysis of 16S rRNA gene sequences and chemotaxonomic data support the description of a new genus and species, to include Oceanibulbus indolifex gen . nov., sp . nov., with the type strain HEL-45T (=DSM 14862T=NCIMB 13983T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1129 - 36
Oceanicola granulosus gen . nov., sp . nov . and Oceanicola batsensis sp . nov., poly-beta-hydroxybutyrate-producing marine bacteria in the order 'Rhodobacterales'; Cho JC et al.; Three Gram-negative, chemoheterotrophic, non-motile, rod-shaped bacterial strains that accumulate poly-beta-hydroxybutyrate granules were isolated from the Bermuda Atlantic Time-series Study site by high-throughput culturing methods and characterized by polyphasic approaches . DNA-DNA hybridization, DNA G+C content and phylogenetic analyses based on 16S rRNA gene sequences divided the three isolates into two distinct genospecies that were clearly differentiated by fatty acid profiles, carbon source utilization patterns, antibiotic susceptibility and biochemical characteristics . The strains utilized a wide range of substrates, including pentoses, hexoses, oligosaccharides, sugar alcohols, organic acids and amino acids . DNA G+C contents were 71.5, 70.9 and 67.3 mol% for strains HTCC2516T, HTCC2523 and HTCC2597T, respectively . The most dominant fatty acid was 18 : 1omega7c in strains HTCC2516T and HTCC2523, and cyclo 19 : 0 in strain HTCC2597T . The type strains HTCC2516T and HTCC2597T were clearly differentiated by the presence or absence of 12 : 0, 12 : 1omega11c, 14 : 0, 15 : 0 and methyl 18 : 1 . Phylogenetic analyses indicated that the strains formed a distinct monophyletic lineage within the Roseobacter clade in the order 'Rhodobacterales' of the Alphaproteobacteria, and which did not associate with any of the described genera . Genotypic and phenotypic differences of the isolates from the previously described genera support the description of Oceanicola granulosus gen . nov., sp . nov . with the type strain HTCC2516T (=ATCC BAA-861T=DSM 15982T=KCTC 12143T) and of Oceanicola batsensis sp . nov . with the type strain HTCC2597T (=ATCC BAA-863T=DSM 15984T=KCTC 12145T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1107 - 10
Salegentibacter holothuriorum sp . nov., isolated from the edible holothurian Apostichopus japonicus; Nedashkovskaya OI et al.; Strain KMM 3524T was isolated from the holothurian Apostichopus japonicus living in the Sea of Japan . The bacterial strain was pigmented, non-motile, Gram-negative, strictly aerobic and oxidase-, catalase- and beta-galactosidase-positive . From the results of 16S rDNA sequence analysis, strain KMM 3524T was found to be related closely to Salegentibacter salegens (98.1%) . DNA-DNA homology between strains KMM 3524T and S . salegens DSM 5424T was 38%; this showed clearly that the holothurian isolate KMM 3524T belongs to a novel species of the genus Salegentibacter for which the name Salegentibacter holothuriorum sp . nov . is proposed, with KMM 3524T (=NBRC 100249T=LMG 21968T) as the type strain.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1011 - 6
Reclassification of salt-water Bdellovibrio sp . as Bacteriovorax marinus sp . nov . and Bacteriovorax litoralis sp . nov; Baer ML et al.; Bdellovibrios are unique, predatory bacteria with an intraperiplasmic growth and multiplication phase within their prey, which consists of many Gram-negative bacteria . Until recently, all bacteria that exhibited these traits were included in the genus Bdellovibrio . However, analysis of 16S rDNA sequences and other studies have demonstrated substantial genotypic, phenotypic and ecotypic diversity among the organisms in this genus (Baer et al., 2000; Snyder et al., 2002) . This has resulted in reclassification of Bdellovibrio stolpii and Bdellovibrio starrii into the newly constructed genus Bacteriovorax (Baer et al., 2000) . In this study, examination of marine isolates of Bdellovibrio (designated SJT, AQ and JS5T) has revealed them to be related more closely to the newly designated genus Bacteriovorax . Phylogenetic analysis of 16S rRNA gene sequences revealed that marine isolates SJT, AQ and JS5T clustered in a separate clade from Bdellovibrio bacteriovorus 100T as part of the clade that contains Bacteriovorax spp., indicating a much closer taxonomic relationship to the latter . DNA-DNA hybridization experiments also demonstrated <5 % similarity between Bdellovibrio bacteriovorus 100T and the marine isolates . Distinct differences between the salt-water group and Bdellovibrio spp . were also observed by determination of DNA G+C content, salinity growth testing and antibiotic sensitivity analysis . On the basis of the results from the studies described above, it is proposed that marine isolates SJT (=ATCC BAA-682T=DSM 15412T) and JS5T (=ATCC BAA-684T=DSM 15409T) should be classified within the genus Bacteriovorax as the type strains of Bacteriovorax marinus sp . nov . and Bacteriovorax litoralis sp . nov., respectively.

Genetics, 2004 Jul, 167(3), 1341 - 60
Diverse evolutionary mechanisms shape the type III effector virulence factor repertoire in the plant pathogen Pseudomonas syringae; Rohmer L et al.; Many gram-negative pathogenic bacteria directly translocate effector proteins into eukaryotic host cells via type III delivery systems . Type III effector proteins are determinants of virulence on susceptible plant hosts; they are also the proteins that trigger specific disease resistance in resistant plant hosts . Evolution of type III effectors is dominated by competing forces: the likely requirement for conservation of virulence function, the avoidance of host defenses, and possible adaptation to new hosts . To understand the evolutionary history of type III effectors in Pseudomonas syringae, we searched for homologs to 44 known or candidate P . syringae type III effectors and two effector chaperones . We examined 24 gene families for distribution among bacterial species, amino acid sequence diversity, and features indicative of horizontal transfer . We assessed the role of diversifying and purifying selection in the evolution of these gene families . While some P . syringae type III effectors were acquired recently, others have evolved predominantly by descent . The majority of codons in most of these genes were subjected to purifying selection, suggesting selective pressure to maintain presumed virulence function . However, members of 7 families had domains subject to diversifying selection.

Curr Top Med Chem, 2004, 4(11), 1185 - 201
The search for molecular determinants of LPS inhibition by proteins and peptides; Pristovsek P et al.; Lipo-poly-saccharide (LPS) induced Gram-negative sepsis and septic shock remain lethal in up to 60 % of cases, and LPS antagonists that neutralize its endotoxic action are the subject of intensive research . The molecular motifs of specific binding of LPS by antiendotoxin proteins and peptides may lead to an understanding of LPS action at the atomic level and provide clues for the development of new immunomodulatory compounds for use as therapy in the treatment of Gram-negative bacterial sepsis . The interaction of LPS with its cognate binding proteins has been structurally elucidated in the single case of the X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from E . coli K-12 (Ferguson et al., Science 1999, 282, 2215) . This structure and other known structures of LPS binding proteins have been used to propose a common binding motif of LPS to proteins . Another independent source of structural information are solution structures of peptides in complex with LPS that can be determined using the transferred NOE effect . The molecular mechanisms of biological activity of bacterial endotoxins can additionally be probed by theoretical means . The growing structural knowledge is opening pathways to the design of peptides or peptidomimetics with improved antiendotoxin properties.

Curr Top Med Chem, 2004, 4(11), 1147 - 71
Inhibition of endotoxin response by synthetic TLR4 antagonists; Hawkins LD et al.; Endotoxin, from the outer membrane of Gram-negative bacteria, has been implicated as the etiological agent of a variety of pathologies ranging from relatively mild (fever) to lethal (septic shock, organ failure, and death) . While endotoxin (also known as lipopolysaccharide or LPS) is a complex heterogeneous molecule, the toxic portion of LPS (the lipid A portion) is relatively similar across a wide variety of pathogenic strains of bacteria, making this molecule an attractive target for the development of an LPS antagonist . Research over the past fifteen years focused on the design of various lipid A analogs including monosaccharide, acyclic and disaccharide compounds has lead to the development of E5564, an advanced, unique and highly potent LPS antagonist . E5564 is a stable, pure LPS antagonist that is selective against endotoxin-mediated activation of immune cells in vitro and in animal models . In Phase I clinical trials, we have developed an ex vivo endotoxin antagonism assay that has provided results on pharmacodynamic activity of E5564 in addition to the more typical safety and pharmacokinetic evaluations . Results from these assays have been reinforced by analysis of in vivo antagonistic activity using a human endotoxemia model . Results from all of these studies indicate that E5564 is an effective in vivo antagonist of endotoxin, and may prove to be of benefit in a variety of endotoxin-mediated diseases . This review discusses the evolution of synthetic LPS antagonists with emphasis on the SAR and development of E5564 and its precursors.

Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1467 - 80
Divergent structures of carbazole degradative car operons isolated from gram-negative bacteria; Inoue K et al.; Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10 . Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp . strain K23 are nearly identical to that of strain CA10 . In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp . strain J3 . In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp . strain KA1 possesses a putidaredoxin-type ferredoxin . We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1 . In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.

Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1449 - 60
Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus; Gunji Y et al.; The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase . The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine . In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition . The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative . Comparison of the sequences revealed a point mutation in each of these genes in G49 . The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.

Physiol Behav, 2004 Sep 15, 82(2-3), 251 - 61
Estradiol-mediated increases in the anorexia induced by intraperitoneal injection of bacterial lipopolysaccharide in female rats; Geary N et al.; Lipopolysaccharide (LPS) derived from the cell walls of gram-negative bacteria causes a robust acute phase response (APR) that includes fever, anorexia, and many other elements . Because immune system function, including some models of illness anorexia, is sexually differentiated, we investigated the sexual differentiation of the anorexia induced by intraperitoneal LPS injections in rats . Cycling female Long-Evans rats tested either during diestrus or estrus ate less following 6.25 microg/kg LPS than did intact males . Following 12.5 microg/kg LPS, females in estrus ate less than either females during diestrus or males . Similarly, a more pronounced anorexia occurred following 12.5, 25, and 50 microg/kg LPS in ovariectomized females that received cyclic estradiol treatment and were tested on the day modeling estrus than in untreated ovariectomized rats . LPS also increased the length of the rats' ovarian cycles, usually by a day, especially when injected during diestrus . As in male rats, when LPS injections were repeated in the same rats, both estradiol-treated and untreated rats failed to display any significant anorexia . The inhibitory effects of LPS on eating in intact and ovariectomized rats were expressed solely as decreases in spontaneous meal frequency, without significant alteration of spontaneous meal size . These data indicate that anorexia following peripheral LPS administration is sexually differentiated and that estradiol is sufficient to produce this response . The mechanism of the pathophysiological effect of estradiol on meal frequency appears to be different from the physiological effect of estradiol on food intake because the latter is expressed solely as a change in meal size.

Trends Biochem Sci, 2004 Jun, 29(6), 294 - 300
MD-2: the Toll 'gatekeeper' in endotoxin signalling; Gangloff M et al.; Lipopolysaccharide (LPS) from the outer cell wall of Gram-negative bacteria is a potent stimulator of the mammalian innate immune system . The Toll-like receptor 4 (TLR4) pathway triggers the inflammatory responses induced by LPS in a process that requires the interaction of LPS-bound myeloid differentiation-2 (MD-2) with TLR4 . Here we propose two possible mechanisms for LPS recognition and signalling that take into account both the structural information available for TLR4 and MD-2, and the determinants of endotoxicity, namely, the acylation and phosphorylation patterns of LPS . In our first model, LPS induces the association of two TLR4-MD-2 heterodimers by binding to two different molecules of MD-2 through the acyl chains of lipid A . In our second model, the binding of LPS to a single TLR4-MD-2 complex facilitates the recruitment of a second TLR4-MD-2 heterodimer . These models contrast with the activation of Drosophila Toll, where the receptor is crosslinked by a dimeric protein ligand.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 2861 - 5
Effects of an antibiotic cycling program on antibiotic prescribing practices in an intensive care unit; Merz LR et al.; Various interventions have been proposed to combat the increase of antibiotic resistance and influence antibiotic prescribing practices . A prospective cohort study in a medical intensive care unit was conducted to determine the effect of an antibiotic cycling program on patterns of antibiotic use and to determine patient factors associated with cycling adherence . Four major classes of antibiotics for empirical therapy of suspected gram-negative bacterial infections were rotated at 3- and 4-month intervals . During the study, 1,003 patients received antibiotic therapy with at least one of the study drugs; of the 792 receiving cycle antibiotics during the cycling period, 598 (75.5%) received an on-cycle drug . Compared to the baseline, cycling recommendations increased the use of the target cycle agent: the use of cephalosporins increased during cycle 1 (56 to 64% of total antibiotic days, P < 0.001), fluoroquinolone use increased in cycle 2 (24 to 55%, P < 0.001), carbapenem use increased during cycle 3 (14 to 38%, P < 0.001), and use of extended-spectrum penicillins increased in cycle 4 (5 to 36%, P < 0.001) . Overall, 48% of total cycle antibiotic days were compliant with the cycling protocol . On average, 8.8 days per patient were spent receiving on-cycle drugs (range, 1 to 109) . Cycle periods that specified carbapenem and fluoroquinolone use had the highest number of off-cycle days (62 and 64%) . Predictors of on-cycle antibiotic use were increased severity of illness, as measured by an acute physiology and chronic health evaluation II score, and greater length of intensive care unit stay . In conclusion, the successful implementation of this cycling protocol increased antibiotic heterogeneity over time in the study unit.

Cell Microbiol, 2004 Sep, 6(9), 805 - 16
Conserved features of type III secretion; Tampakaki AP et al.; Type III secretion systems (TTSSs) are essential mediators of the interaction of many Gram-negative bacteria with human, animal or plant hosts . Extensive sequence and functional similarities exist between components of TTSS from bacteria as diverse as animal and plant pathogens . Recent crystal structure determinations of TTSS proteins reveal extensive structural homologies and novel structural motifs and provide a basis on which protein interaction networks start to be drawn within the TTSSs, that are consistent with and help rationalize genetic and biochemical data . Such studies, along with electron microscopy, also established common architectural design and function among the TTSSs of plant and mammalian pathogens, as well as between the TTSS injectisome and the flagellum . Recent comparative genomic analysis, bioinformatic genome mining and genome-wide functional screening have revealed an unsuspected number of newly discovered effectors, especially in plant pathogens and uncovered a wider distribution of TTSS in pathogenic, symbiotic and commensal bacteria . Functional proteomics and analysis further reveals common themes in TTSS effector functions across phylogenetic host and pathogen boundaries . Based on advances in TTSS biology, new diagnostics, crop protection and drug development applications, as well as new cell biology research tools are beginning to emerge.

EMBO J, 2004 Aug 18, 23(16), 3187 - 95 Epub 2004 Jul 22.
Crystal structure of the bacterial nucleoside transporter Tsx; Ye J et al.; Tsx is a nucleoside-specific outer membrane (OM) transporter of Gram-negative bacteria . We present crystal structures of Escherichia coli Tsx in the absence and presence of nucleosides . These structures provide a mechanism for nucleoside transport across the bacterial OM . Tsx forms a monomeric, 12-stranded beta-barrel with a long and narrow channel spanning the outer membrane . The channel, which is shaped like a keyhole, contains several distinct nucleoside-binding sites, two of which are well defined . The base moiety of the nucleoside is located in the narrow part of the keyhole, while the sugar occupies the wider opening . Pairs of aromatic residues and flanking ionizable residues are involved in nucleoside binding . Nucleoside transport presumably occurs by diffusion from one binding site to the next.

Infect Immun, 2004 Aug, 72(8), 4357 - 67
Chlamydia trachomatis-specific human CD8+ T cells show two patterns of antigen recognition; Matyszak MK et al.; Chlamydia trachomatis is an intracellular gram-negative bacteria which causes several clinically important diseases . T-cell-mediated immunity and the production of gamma interferon (IFN-gamma) are known to be essential for the clearance of the bacteria in vivo . Here we have investigated CD8(+)-T-cell responses to C . trachomatis in patients with previous episodes of chlamydia infection . To isolate C . trachomatis-specific CD8(+)-T-cell lines, dendritic cells (DC) were infected with C . trachomatis and cocultured with purified CD8(+) T cells to generate C . trachomatis-specific CD8(+)-T-cell lines which were then cloned . Two patterns of recognition of C . trachomatis-infected cells by CD8(+)-T-cell clones were identified . In the first, C . trachomatis antigens were recognized in association with classical class I HLA antigens, and responses were inhibited by class I HLA-specific monoclonal antibodies . The second set of clones was unrestricted by classical HLA class I, and further studies showed that CD1 molecules were also not the restriction element for those clones . Both types of clones produced IFN-gamma in response to C . trachomatis and were able to lyse C . trachomatis-infected target cells . However, unrestricted clones recognized C . trachomatis-infected cells at much earlier time points postinfection than HLA-restricted clones . Coculture of C . trachomatis-infected DC with the C . trachomatis-specific clones induced DC activation and a rapid enhancement of interleukin-12 (IL-12) production . Early production of IL-12 during C . trachomatis infection, facilitated by unrestricted CD8(+)-T-cell clones, may be important in ensuring a subsequent Th1 T-cell-mediated response by classical major histocompatibility complex-restricted CD4(+) and CD8(+) T cells.

Anim Reprod Sci, 2004 Jul, 82-83, 389 - 400
Endocrine aspects in pathogenesis of mastitis in postpartum dairy cows; Huszenicza G et al.; In well-managed dairy herds some environmental pathogens including Gram-negative (GN) strains (E . coli and others) have been recognized recently as the predominant causative microbes of mastitis in the peri-parturient period . In early weeks of lactation hyperketonaemia may predispose the high-producing cows for GN mastitis . In GN mastitis cytokines, eicosanoids and oxygen radicals are released, which are responsible for the local and systemic symptoms . Experimental administration of endotoxin induces a complex endocrine cascade . Similar changes in plasma levels of cortisol, insulin, insulin-like growth factor-I and thyroid hormones are seen also in severe cases of GN mastitis . However, leptin is not responsible for the anorexia associated with severe mastitis in ruminants . Mastitis can postpone the resumption of ovarian cyclic activity in dairy cows when its outbreak occurs between days 15 and 28 after calving (at the expected time of first ovulation) . In cyclic cows severe cases of GN mastitis can induce premature luteolysis or prolong the follicular phase.

Biochem Soc Trans, 2004 Aug, 32(Pt 4), 636 - 9
Heat-shock protein 70 and heat-shock protein 90 associate with Toll-like receptor 4 in response to bacterial lipopolysaccharide; Triantafilou M et al.; Mammalian responses to bacterial LPS (lipopolysaccharide) from the outer membrane of Gram-negative bacteria can lead to an uncontrolled inflammatory response that can be deadly for the host . It has been shown that the innate immune system employs at least three cell surface receptors, CD14, TLR4 (Toll-like receptor 4) and MD-2, in order to recognize bacterial LPS . In our previous work we have found that Hsps (heat-shock proteins) are also involved in the innate recognition of bacterial products . Their presence on the cell surface, as well as their involvement in the innate recognition process, are poorly understood . In the present study we have investigated the association of TLR4 with Hsp70 and Hsp90 following LPS stimulation, both on the cell surface and intracellularly . Our results show that Hsp70 and Hsp90 form a cluster with TLR4 within lipid microdomains following LPS stimulation . In addition, Hsp70 and Hsp90 seem to be involved in TLR4/LPS trafficking and targeting to the Golgi apparatus, since upon LPS stimulation we found that both Hsps are targeted to the Golgi along with TLR4 . The present study sheds new light into the involvement of Hsps in the innate immune response.

J Nat Prod, 2004 Jul, 67(7), 1172 - 4
Further membranolide diterpenes from the antarctic sponge dendrilla membranosa; Ankisetty S et al.; Chemical investigation of the Antarctic sponge Dendrilla membranosa collected from the vicinity of Palmer Station on Anvers Island, Antarctica, yielded three new diterpenes, membranolides B-D (2-4), as well as three previously reported sponge metabolites . Membranolides C and D (3, 4), bearing carboxylic acid functional groups, display Gram-negative antibiotic and antifungal activities.

Methods Mol Biol, 2004, 267, 225 - 37
Recombinant protein production in Antarctic Gram-negative bacteria; Duilio A et al.; This review reports some results from our laboratory on the setting up of a psychrophilic expression system for the homologous/heterologous protein production in cold-adapted bacteria by using natural plasmids as cloning vectors.By screening some Antarctic bacteria for the presence of extrachromosomal elements, we identified three new plasmids, pMtBL from Pseudoalteromonas haloplanktis TAC125, and pTAUp and pTADw, from Psychrobacter sp . TA144.The latter autoreplicating elements were isolated, cloned, and fully sequenced and their molecular characterisation was carried out; however, we focused our attention on the small multicopy plasmid, pMtBL, from the Gram-negative P . haloplanktis TAC125 strain . This episome turned out to be an interesting extrachromosomal element, since it displays unique molecular features as its transcriptional inactivity . Being cryptic, the inheritance of pMtBL totally relied on the efficiency of its replication function . This function was bound to a region of about 850 bp, identified by an in vivo assay based on the possibility to efficiently mobilize plasmidic DNA from a mesophilic donor (Escherichia coli) to psychrophilic recipient by intergeneric conjugation . This information was instrumental in the construction of a shuttle vector, able to replicate either in E . coli or in several cold-adapted hosts (clone Q).Since the conversion of a cloning system into an expression vector requires the insertion of transcription and translation regulative sequences, the corresponding signals from the aspartate aminotransferase gene isolated from P . haloplanktis TAC125 were inserted, generating the pFF vector.To investigate the possibility of obtaining recombinant proteins in this cold-adapted host, we used the psychrophilic alpha-amylase from the Antarctic bacterium P . haloplanktis TAB23 (previously known as Alteromonas haloplanktis A23) as a model enzyme to be produced . Our results demonstrate that the cold-adapted enzyme was not only produced but also efficiently secreted by the recombinant PhTAC125 cells . The described expression system represents the first example of heterologous protein production based on a true cold-adapted replicon.

FEMS Microbiol Lett, 2004 Aug 1, 237(1), 97 - 103
In vivo induced antigenic determinants of Actinobacillus actinomycetemcomitans; Cao SL et al.; Actinobacillus actinomycetemcomitans is a Gram-negative capnophilic rod and the etiological agent of localized aggressive periodontitis . The genome-wide survey of A . actinomycetemcomitans using in vivo induced antigen technology (IVIAT) has previously resulted in the discovery of antigenic determinants expressed specifically in diseased patients . The present study evaluated the potential of these antigens as putative disease markers, and investigating their contribution to the pathogenesis of the microorganism . Sera from patients had a significantly greater antibody titer than sera from healthy controls against six antigens, which supports the in vivo expression of these antigens, and suggests their usefulness as disease markers . A . actinomycetemcomitans invasion of epithelium-derived HeLa cells resulted in the induction of all three genes tested, as evidenced by real-time PCR . Isogenic mutants of these three genes were constructed and the adhesion and intracellular survival of the mutants was assayed in a competition assay with the wild-type strain . A significant defect in the intracellular survival of two of these mutant strains (orf1402 and orf859) was found . This defect could not be attributed to an adhesion defect . In contrast, a mutation in vapA, a homologue of a novel putative transcriptional regulator, out-competed the wild-type strain in the same assay . The virulent phenotype was restored for a mutant strain in orf859 upon complementation . This data provided new insight into the pathogenic personality of A . actinomycetemcomitans in vivo and supported the use of HeLa cells as a valid in vitro host-pathogen interactions model for that microorganism . IVIAT is applicable to most pathogens and will undoubtedly lead to the discovery of novel therapies, antibiotics and diagnostic tools.

J Bacteriol, 2004 Aug, 186(15), 5101 - 15
Nucleotide sequence and evolution of the five-plasmid complement of the phytopathogen Pseudomonas syringae pv . maculicola ES4326; Stavrinides J et al.; Plasmids are transmissible, extrachromosomal genetic elements that are often responsible for environmental or host-specific adaptations . In order to identify the forces driving the evolution of these important molecules, we determined the complete nucleotide sequence of the five-plasmid complement of the radish and Arabidopsis pathogen Pseudomonas syringae pv . maculicola ES4326 and conducted an intraspecific comparative genomic analysis . To date, this is the most complex fully sequenced plasmid complement of any gram-negative bacterium . The plasmid complement comprises two pPT23A-like replicons, pPMA4326A (46,697 bp) and pPMA4326B (40,110 bp); a pPS10-like replicon, pPMA4326C (8,244 bp); and two atypical, replicase-deficient replicons, pPMA4326D (4,833 bp) and pPMA4326E (4,217 bp) . A complete type IV secretion system is found on pPMA4326A, while the type III secreted effector hopPmaA is present on pPMA4326B . The region around hopPmaA includes a shorter hopPmaA homolog, insertion sequence (IS) elements, and a three-element cassette composed of a resolvase, an integrase, and an exeA gene that is also present in several human pathogens . We have also identified a novel genetic element (E622) that is present on all but the smallest plasmid (pPMA4326E) that has features of an IS element but lacks an identifiable transposase . This element is associated with virulence-related genes found in a wide range of P . syringae strains . Comparative genomic analyses of these and other P . syringae plasmids suggest a role for recombination and integrative elements in driving plasmid evolution.

J Bacteriol, 2004 Aug, 186(15), 5031 - 9
A type II protein secretory pathway required for levansucrase secretion by Gluconacetobacter diazotrophicus; Arrieta JG et al.; The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose . LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway . We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane . Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases . The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane . The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking . The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G . diazotrophicus structural gene . Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized . Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose . Replacement of Cys(162) by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins . Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G . diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions . To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae.

Colloids Surf B Biointerfaces, 2004 Apr 15, 34(4), 205 - 12
Correlated atomic force microscopy and fluorescence lifetime imaging of live bacterial cells; Micic M et al.; We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM) . For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces . We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells . Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded . Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images . Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution . We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution . Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.

Acta Vet Scand Suppl, 2003, 98, 141 - 55
Bovine endotoxicosis--some aspects of relevance to production diseases . A review; Andersen PH; This review describes some circumstances where endotoxins of gram negative bacteria may be related to the pathogenesis of some common production diseases . Decisive evidence for the pathogentical role of endotoxins remains scarce, and therefore an interdisciplinary background covering epidemiological, biological, biochemical, clinical and experimental aspects is given . Several authors have suggested that endotoxins play a significant role for the development of diseases such as laminitis, abomasal displacement, sudden death syndrome of feed-lot steers etc . While the biological, biochemical and clinical pictures of bovine endotoxicosis is quite well known, and certainly may resemble the clinical and biochemical pictures seen in some of the before mentioned diseases, it is however still not clear how or when endotoxins would gain parenteral access . This review describes excerpts of the biology of endotoxins, key clinical signs and the biochemistry associated to these . It is described how ruminal acidosis may facilitate the translocation of endotoxin from the intestinal/ruminal contents to the portal and eventually the systemic bloodstream . The function of the liver hence becomes central, and the role of hepatic fatty infiltration around parturition is discussed . The review finally suggest that acute ruminal acidosis may be viewed as an analogue to the human syndrome Gut-Derived Infectious Toxic Shock (GITS), where shock is propagated primarily by the translocation of bacterial endotoxin from the gut.

Shock, 2004 Aug, 22(2), 174 - 9
Protection against an Escherichia coli-induced sepsis by alkaline phosphatase in mice; Verweij WR et al.; Alkaline phosphatase (AP) is a phosphate transferase present in bacteria and eukaryotes . In previous studies, we have shown that AP is able to dephosphorylate lipopolysaccharide (LPS) at physiological pH levels . Because LPS is the causative agent of gram-negative sepsis, we hypothesize that AP might be used as a medication during early stages of LPS-induced septic shock . We have demonstrated protective effects of AP when this enzyme was administered simultaneously with LPS . However, a major question of anti-LPS therapies is whether they are also effective after systemic infiltration of whole bacteria and if they also act in later stages of the disease . To test this, we explored the protective effects of AP from human placenta (plAP) in a bacterial challenge model in Balb/c mice . AP was intravenously administered 20 min after a bacterial intraperitoneal inoculation of 2 to 5 x 10 CFU of Escherichia coli suspended in a 100-microL volume of saline . It was shown that AP attenuated the systemic host response upon E . coli . Body temperature was normalized as compared with untreated septic mice . Also, serum nitric oxide levels 24 h after the injection of bacteria were reduced almost to control levels in mice that received AP . Moreover, survival after 24 h was significantly higher in the AP-treated group compared with the nontreated control group.

Eur J Gastroenterol Hepatol, 2004 Aug, 16(8), 737 - 42
Liver abscess complicating transcatheter arterial embolization: a rare but serious complication . A retrospective study after 3878 procedures; Ong GY et al.; OBJECTIVES: Liver abscess is one of the complications of transcatheter arterial embolization (TAE) for hepatocellular carcinoma . We studied the clinical features and analysed the incidence, risk factors, helpful clinical clues, culture profiles and predictive factors of post-TAE liver abscess . The influence of abscess development on the evolution of the tumour process was also studied . METHODS: We retrospectively reviewed records of 3878 TAE procedures performed over a 6 year period . RESULTS: Ten cases of liver abscess developed in nine patients (eight males and one female) . The incidence was 0.26% (10 episodes/3878 procedures) . The main clinical presentations included fever (91.7%), chills (50%) and abdominal pain (33.3%) . All but one febrile patient presented fever in a recurrent form . The positive culture rates were 41.7% for blood and 83.3% for pus . Gram negative bacteria were found in 80% of blood cultures and 68% of pus cultures . Polymicrobial infections were encountered in 60% of the blood cultures and 70% of pus cultures . Management included antibiotics, drainage and operation . Four patients died due to the direct complications of liver abscess . One patient experienced total tumour resolution after successful treatment for liver abscess . Patients with larger liver abscesses and patients with greater age carried higher mortality rates . CONCLUSIONS: Liver abscess is a rare complication after TAE for hepatocellular carcinoma . Recurrent fevers after an initial symptom free interval should arouse suspicion of an abscess . The mortality is high and a large abscess and higher age predict an unfavourable outcome . Abscess formation can lead to complete tumour resolution.

Microbiology, 2004 Jul, 150(Pt 7), 2229 - 35
Symbionts of the gut flagellate Staurojoenina sp . from Neotermes cubanus represent a novel, termite-associated lineage of Bacteroidales: description of 'Candidatus Vestibaculum illigatum'; Stingl U et al.; The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites . However, little is known about the identity of the symbionts . One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genus Staurojoenina . By using scanning electron microscopy, it was shown that the whole surface of Staurojoenina sp . from the termite Neotermes cubanus is densely covered with long rod-shaped bacteria of uniform size and morphology . PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences . Phylogenetic analysis placed them as a new lineage among the Bacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellate Mixotricha paradoxa . The closest cultivated relative was Tannerella forsythensis (<85 % sequence identity) . Fluorescence in situ hybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts of Staurojoenina sp . Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host . Based on the isolated phylogenetic position and the specific association with the surface of Staurojoenina sp., we propose to classify this new taxon of Bacteroidales under the provisional name 'Candidatus Vestibaculum illigatum'.

J Expo Anal Environ Epidemiol, 2004 Jul, 14(4), 293 - 9
Heterogeneity in microbial exposure in schools in Sweden, Poland and Jordan revealed by analysis of chemical markers; Wady L et al.; We used gas chromatography--tandem mass spectrometry to analyze microbial components in 85 samples of airborne dust from schools in Jordan, Sweden, and Poland . To collect the samples, we allowed dust to settle on plexiglass plates hanging in the breathing zone in school buildings during both summer and winter . In each of the three countries, we conducted such sampling in two schools: one in an urban environment and the other in rural surroundings . The microbial marker profiles differed significantly between the schools and seasons . For example, samples from Jordan contained remarkably low levels of ergosterol (marker of fungal biomass) and high levels of 3-hydroxy acids (markers of lipopolysaccharide) of 10, 12, and 14 carbon chain lengths relative to such acids of 16 and 18 carbons in comparison with samples from Sweden and Poland . This dissimilarity in 3-hydroxy fatty acid distribution indicates significant differences in the populations of Gram-negative bacteria . We also noted that muramic acid (marker of bacterial biomass) exhibited the smallest variation between schools and seasons . In summary, our results demonstrate that exposure to microorganisms in indoor air in school buildings may differ markedly between countries, between seasons, and between urban and rural environments.

J Endourol, 2004 Jun, 18(5), 418 - 24
Gut-inhabiting bacterium Oxalobacter formigenes: role in calcium oxalate urolithiasis; Mittal RD et al.; Oxalate plays a crucial role in the formation of most renal stones . Oxalate is a common constituent of most diets and a byproduct of metabolism, and if it is not sufficiently degraded, it may accumulate . In humans, gut bacteria degrade 70 to 100 mg of oxalate per day . Oxalobacter formigenes is a gram-negative, obligately anaerobic, rod-shaped bacterium with an absolute requirement for oxalate . Although not present in the gut at birth, it quickly colonizes most children, and there is epidemiologic evidence that its absence is a risk factor in calcium oxalate stone formation . We review the metabolism, genetics, and identification of this organism and its possible therapeutic role in recurrent stone-forming patients.

J Immunol Methods, 2004 Jun, 289(1-2), 81 - 7
Single step high-throughput determination of Toll-like receptor 4 polymorphisms; Van Rijn BB et al.; Background: Toll-like receptors are central components of host defence in humans, responsible for recognition of pathogen-associated molecular patterns and activation of innate immunity . Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS) and other microbial components, thereby initiating the expression and release of pro-inflammatory cytokines . The common, frequently co-segregating allelic variants Asp299Gly and Thr399Ile have been related to susceptibility to gram-negative infections and sepsis and may be involved in the development of atherosclerosis . Identification of TLR4 Asp299Gly and Thr399Ile genotypes can be important for examination of genotype/phenotype relationships as well as for individual risk assessment of patients . Methods: TLR4 Asp299Gly and Thr399Ile genotypes were detected by a single tube polymerase chain reaction (PCR), based on exonuclease degradation of dual labelled allele-specific oligonucleotides . The assay results were compared with conventional restriction fragment length polymorphism (RFLP) analysis . Results: Genotypes of 345 individuals were determined simultaneously in a single PCR assay . Allele frequencies for our population were 6.8% for the TLR4 Asp299Gly polymorphism and 6.4% for the Thr399Ile polymorphism . Validation by RFLP analysis revealed a correct detection of all genotypes . Conclusions: We have developed a novel method for the detection of the TLR4 Asp299Gly and Thr399Ile mutations, permitting rapid genotyping which should be useful for large-scale population studies as well as applicable for routine clinical testing.

J Infect Dis, 2004 Aug 1, 190(3), 527 - 34 Epub 2004 Jul 02.
Diagnostic and prognostic implications of endotoxemia in critical illness: results of the MEDIC study; Marshall JC et al.; A novel assay for endotoxin, based on the ability of antigen-antibody complexes to prime neutrophils for an augmented respiratory burst response, was studied in a cohort study of 857 patients admitted to an intensive-care unit (ICU) . On the day of ICU admission, 57.2% of patients had either intermediate (>or=0.40 endotoxin activity {EA} units) or high (>or=0.60 units) EA levels . Gram-negative infection was present in 1.4% of patients with low EA levels, 4.9% with intermediate levels, and 6.9% with high levels; EA had a sensitivity of 85.3% and a specificity of 44.0% for the diagnosis of gram-negative infection . Rates of severe sepsis were 4.9%, 9.2%, and 13.2%, and ICU mortality was 10.9%, 13.2%, and 16.8% for patients with low, intermediate, and high EA levels, respectively . Stepwise logistic regression analysis showed that elevated Acute Physiology and Chronic Health Evaluation II score, gram-negative infection, and emergency admission status were independent predictors of EA.

J Gastroenterol Hepatol, 2004 Aug, 19(8), 884 - 90
Induction of a 72-kDa heat shock protein and protection against lipopolysaccharide-induced liver injury in cirrhotic rats; Mikami K et al.; BACKGROUND AND AIM: A 70-kDa heat shock protein (stress-inducible HSP70, HSP72) has been reported to be a cytoprotectant in a variety of organs . It has been reported that HSP72 protected non-cirrhotic rats against endotoxemia . However, its cytoprotective effect against endotoxemia in cirrhotic rats has not yet been studied . In this study, we investigated the cytoprotective effect of HSP72 on lipopolysaccharide (LPS)-induced liver injury in carbon tetrachloride (CCl(4))-induced cirrhotic rats . METHODS: Liver cirrhosis was produced by an 8-week intraperitoneal injection of CCl(4) in male Sprague-Dawley rats . Expression of HSP72 was investigated using western blot analysis . Cirrhotic rats were given an intraperitoneal injection of LPS (10 mg/kg) with or without hyperthermia (42.5 degrees C, 15 min) preconditioning . Liver injury was assessed biochemically (aspartate transaminase, alanine transaminase, bilirubin, lactate dehydrogenase, creatinine) and histologically . The plasma tumor necrosis factor (TNF)-alpha level was determined . RESULTS: Hyperthermia preconditioning induced a 4-fold increase in HSP72 in the cirrhotic rat liver . Pre-induction of HSP72 prevented LPS-induced liver injury, as evaluated using serum biochemical parameters and histology with reduced TNF-alpha response . CONCLUSION: These findings suggest that pre-induction of HSP72 may provide therapeutic strategies for Gram-negative sepsis-induced liver injury in liver cirrhosis.

Eur Biophys J . 2004 Jul 6; {Epub ahead of print}
Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes; Brandenburg K et al.; Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates . We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane . These investigations comprise functional aspects of the protein-lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface . The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids . The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes . The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups . From this, a dominant role of the protein on the function of the outer membrane can be deduced . OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation . This might be of general importance for infections induced by Gram-negative bacteria.

Cell Mol Life Sci, 2004 Jul, 61(14), 1697 - 713
Lipopolysaccharide-binding molecules: transporters, blockers and sensors; Chaby R; Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, can be beneficial to the host by activating the innate immune system, or harmful, by inducing inflammation, disseminated intravascular coagulation, multiple organ failure, shock and often death . On the bacteria, and in host biological fluids and cells, LPS is never free but constantly attached to cognate-binding proteins . Understanding how LPS is transported and further recognized by sensors able to deliver a signal, or by inactivating molecules able to neutralize its biological effects, is an important goal . This review describes the large panel of peptides and proteins reported to associate with LPS, and provides information on their origin, their structure and the location of amino acid residues involved in their interaction with LPS . A better understanding of the mode of recognition of LPS by cognate proteins prompted many laboratories to design on a rational basis synthetic molecules which can be used to detect low amounts of endotoxin, or to act as efficient blockers of in vitro and in vivo responses to LPS.

Ann Agric Environ Med, 2004, 11(1), 121 - 7
Immunologic reactivity to work-related airborne allergens in people occupationally exposed to dust from herbs; Golec M et al.; A group of 150 people occupationally exposed to dust from herbs were examined . The examined group consisted of 47 thyme farmers, 32 chamomile farmers, 31 sage farmers and 40 workers of herbs processing industry . As a reference group, 50 urban dwellers, not exposed to any kind of organic dust, were examined . Skin prick tests and precipitin tests were conducted with, respectively, 4 and 11 microbial antigens associated with organic dust . Both skin and precipitin tests were also conducted with herbal extracts of chamomile and sage . Precipitin tests were carried out with sera not concentrated and sera 3-fold concentrated . Tests for inhibition of leukocyte migration (MIF) were also conducted with 4 microbial antigens . People occupationally exposed to dust from herbs showed a higher frequency of positive skin reactions to microbial antigens compared to the reference group . The results of precipitin test also revealed greater reactivity to the environmental microbial antigens in the examined group, compared to the reference group . The highest frequency of positive results was noted with the antigen of Pantoea agglomerans (30.6 % with sera not concentrated and 48.3 % with sera 3-fold concentrated) - the difference compared to the reference group (12.0 %) was highly significant (p < 0.01) . The frequencies of positive results of MIF test in the examined group were high with all antigens tested: Arthrobacter globiformis (12.6 %), Pantoea agglomerans (11.1 %), Saccharopolyspora rectivirgula (17.0 %), Aspergillus fumigatus (13.3 %), and, compared to the reference group with no positive result for any antigen, all the differences were significant (p < 0.05) . In conclusion, the frequency of positive allergological reactions to airborne microorganisms was high in people occupationally exposed to dust from herbs and suggests a potential role of microbial allergens in the pathogenesis of work-related health disorders among herb workers . The risk of sensitization seems to be greatest among thyme farmers, who showed the highest positive response . The results confirmed the particular allergenic importance of Gram-negative bacterium Pantoea agglomerans.

Support Care Cancer, 2004 Sep, 12(9), 634 - 9
Prognostic significance of wound infections following major head and neck cancer surgery: an open non-comparative prospective study; Penel N et al.; OBJECTIVE: We evaluated the incidence, risk factors and consequences of wound infection (WI) following major head and neck cancer surgery in an open non-comparative study . PATIENTS AND METHODS: The study group, comprising 95 patients who underwent clean-contaminated procedures with opening of the upper aerodigestive tract for biopsy-proven squamous cell cancer, were studied over a 1-year period . Antibiotic prophylaxis was amoxicillin and clavulanic acid . More than 20 variables were prospectively recorded for each patient . The mean follow-up was 30 months . MAIN RESULTS: The overall WI rate was 50.5% (48/95) . Most pathogens isolated from samples were gram-negative rods . In univariate analysis, we found three risk factors for WI: alcohol consumption (P = 0.07), a hypopharyngeal location (P = 0.02) and laryngectomy stoma (P = 0.01) . WI were associated with postoperative fever (P = l.5 x 10(-11)), postoperative antibiotic therapy (P = 1.5 x 10(-5)) and postoperative death (P = 0.043) . Patients without WI had a median postoperative hospital stay of 15 days compared with 29 days for those with WI (P < 0.001) . Healing of WI was achieved after a median time of 48 days . WI delayed postoperative radiation therapy in 21 out of 33 evaluable patients . But overall survival, and local and metastatic failures were similar with and without WI . CONCLUSIONS: WI are associated with a heavy postoperative morbidity, but have no prognostic impact on cancer control.

J Microbiol Methods, 2004 Aug, 58(2), 243 - 50
Tightly regulated vectors for the cloning and expression of toxic genes; Anthony LC et al.; A series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed . These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter . For additional regulatory control, the vectors utilize low-copy origins of replication . Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria . The resulting plasmids have no detectable leaky expression . To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes . Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin . These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems.

Int J Dermatol, 2004 Jul, 43(7), 494 - 7
Plasma reactive oxygen species activity and antioxidant potential levels in rosacea patients: correlation with seropositivity to Helicobacter pylori; Baz K et al.; BACKGROUND: Recent studies have suggested that there might be an etiologic role for Helicobacter pylori (HP) in rosacea . HP is a Gram-negative bacterium that colonizes the gastric mucosa, increases the generation of reactive oxygen species (ROS), and decreases plasma antioxidants such as ascorbic acid . AIM: To investigate plasma ROS activities and antioxidant status, and their relationship with HP infection, in rosacea patients . METHODS: Twenty-nine rosacea patients and 20 age- and sex-matched healthy controls were examined for specific immunoglobulin G (IgG) and IgM against HP, plasma malondialdehyde (MDA), and antioxidant potential (AOP) levels . RESULTS: Compared with controls, the seropositivity of HP for IgM was significantly higher (P = 0.03) and the seropositivity of HP for IgG was significantly lower (P = 0.0001) in patients with rosacea . Plasma MDA levels were higher (P = 0.0001) and AOP levels were lower (P = 0.019) in patients than in controls, regardless of the severity of the disease . Plasma MDA and AOP levels were not affected by the seropositivity of HP for IgM and/or IgG in either group . CONCLUSION: Our results suggest that rosacea is an oxidative stress condition, as reflected by the increased ROS activity and decreased AOP, regardless of HP infection.

Mol Cell Biol, 2004 Jul, 24(14), 6488 - 500
The IkappaB kinase complex and NF-kappaB act as master regulators of lipopolysaccharide-induced gene expression and control subordinate activation of AP-1; Krappmann D et al.; Toll-like receptors (TLRs) recognize conserved products of microbial pathogens to initiate the innate immune response . TLR4 signaling is triggered upon binding of lipopolysaccharides (LPS) from gram-negative bacteria . Using comparative gene expression profiling, we demonstrate a master regulatory role of IkappaB kinase (IKK)/NF-kappaB signaling for immediate-early gene induction after LPS engagement in precursor B cells . IKK/NF-kappaB signaling controls a large panel of gene products associated with signaling and transcriptional activation and repression . Intriguingly, the induction of AP-1 activity by LPS in precursor B cells and primary dendritic cells fully depends on the IKK/NF-kappaB pathway, which promotes expression of several AP-1 family members, including JunB, JunD, and B-ATF . In pre-B cells, AP-1 augments induction of a subset of primary NF-kappaB targets, as shown for chemokine receptor 7 (CCR7) and immunoglobulin kappa light chain . Thus, our data illustrate that NF-kappaB orchestrates immediate-early effects of LPS signaling and controls secondary AP-1 activation to mount an appropriate biological response.

J Mol Biol, 2004 Jul 16, 340(4), 829 - 41
The crystal structure of Helicobacter cysteine-rich protein C at 2.0 A resolution: similar peptide-binding sites in TPR and SEL1-like repeat proteins; Luthy L et al.; Helicobacter pylori is a Gram-negative human pathogen that infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer . Bacterial cell-surface and secreted proteins often play an important role in pathogen-host interactions and are thought to be selective mediators for the pathology of the infection . The Helicobacter cysteine-rich proteins (Hcp) represent a large family of secreted proteins that seem to be specific for microorganisms from the epsilon-subfamily of proteobacteria . Although significantly elevated levels of anti-Hcp antibodies were observed in many patients infected with H.pylori, details on the biological functions of Hcp proteins are sparse . Hcps belong to a large family of Sel1-like multi-repeat proteins . The crystal structure of HcpC was refined at 2.0 A resolution and revealed a super-helical topology composed of seven disulfide bridged alpha/alpha-repeats, an N-terminal capping helix and an extended C-terminal coil consisting of alternating hydrophobic and hydrophilic residues . In the crystal packing, the C-terminal coil interacts with the concave surface of a symmetry-related HcpC super-helix . A hydrophobic pocket and a cluster of negatively charged residues recognize the side-chains of Val290 and Lys287 from the C-terminal coil, respectively . The peptide nitrogen atom of His291 forms a short hydrogen bond with the side-chain of Asn66 . The interactions seen in this crystal contact are strikingly similar to the peptide-binding modes of the Hsp70/Hsp90 organizing protein and the PEX5 receptor . The conservation of the peptide-binding mode suggests that HcpC might recognize its binding partner in a similar way.

Biophys Chem, 2004 Jul 1, 110(1-2), 83 - 92
Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas; Gavel OY et al.; A novel adenylate kinase (AK) has recently been purified from Desulfovibrio gigas and characterized as a Co(2+)/Zn(2+)-containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these enzymes are normally devoid of metals . Here, we studied the conformational stability of holo- and apo-AK as a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy . The thermal unfolding of AK is a cooperative two-state process, and is sufficiently reversible in the 9-11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic model . The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the highly sensitive but integral DSC-method.

Biochemistry, 2004 Jul 6, 43(26), 8600 - 6
Resting state conformation of the MsbA homodimer as studied by site-directed spin labeling; Buchaklian AH et al.; MsbA is the ABC transporter for lipid A and is found in the inner membranes of Gram-negative bacteria such as Escherichia coli . Without MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes . A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes {Chang, and Roth (2001) Science 293, 1793-1800} . Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport . Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful approach for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions within a protein . The quaternary structure of the resting state of the MsbA homodimer reconstituted into lipid membranes has been evaluated by SDSL EPR spectroscopy and chemical cross-linking techniques . SDSL and cross-linking results are consistent with the controversial resting state conformation of the MsbA homodimer found in the crystal structure, with the tips of the transmembrane helices forming a dimer interface . The position of MsbA in the membrane bilayer along with the relative orientation of the transmembrane helical bundles with respect to one another has been determined . Characterization of the resting state of the MsbA homodimer is essential for future studies on the functional dynamics of this membrane transporter.

Arch Gynecol Obstet, 2004 Nov, 270(3), 133 - 46 Epub 2004 Jun 18.
Comprehending the role of LPS in Gram-negative bacterial vaginosis: ogling into the causes of unfulfilled child-wish; Deb K et al.; INTRODUCTION: Intrauterine infection is frequently associated with pregnancy loss in pregnant women . DISCUSSION: This article reviews the role of Gram-negative bacterial infection in various complications related to early pregnancy and subsequent pregnancy loss . Here we discuss the pathways of ascending intrauterine infection, microbiology and the pathophysiology of such infections . The clinical impact, therapy, consequences, prevention and implications of Gram-negative bacterial infections in women during their reproductive life span is also discussed . This article also makes an attempt to discuss our studies and findings, related to the effect of the LPS component of the Gram-negative bacterial endotoxin on preimplantation stage embryonic development and implantation . This early phase of pregnancy remains mostly unnoticed by the mother as well as the health care provider, and therefore holds more threat to the life of the fetus and the mother . The molecular mechanisms of LPS-induced pregnancy losses through abnormal embryonic development, implantation failure, and preterm labor and birth with specific references to the role of proinflammatory cytokines like IL-1 and TNF are discussed . CONCLUSION: Once these inflammatory mediators have increased in the feto-maternal tissues, it may be too late or harmful to try and prevent the adverse outcomes of pregnancy.

PLoS Biol . 2004 Aug;2(8):E203 . Epub 2004 Jun 22.
Functional dissection of an innate immune response by a genome-wide RNAi screen; Foley E et al.; The innate immune system is ancient and highly conserved . It is the first line of defense and the only recognizable immune system in the vast majority of metazoans . Signaling events that convert pathogen detection into a defense response are central to innate immunity . Drosophila has emerged as an invaluable model organism for studying this regulation . Activation of the NF-kappaB family member Relish by the caspase-8 homolog Dredd is a central, but still poorly understood, signaling module in the response to gram-negative bacteria . To identify the genes contributing to this regulation, we produced double-stranded RNAs corresponding to the conserved genes in the Drosophila genome and used this resource in genome-wide RNA interference screens . We identified numerous inhibitors and activators of immune reporters in a cell culture model . Epistatic interactions and phenotypes defined a hierarchy of gene action and demonstrated that the conserved gene sickie is required for activation of Relish . We also showed that a second gene, defense repressor 1, encodes a product with characteristics of an inhibitor of apoptosis protein that inhibits the Dredd caspase to maintain quiescence of the signaling pathway . Molecular analysis revealed that Defense repressor 1 is upregulated by Dredd in a feedback loop . We propose that interruption of this feedback loop contributes to signal transduction.

J Chemother, 2004 Apr, 16(2), 166 - 71
Safety evaluation of piperacillin/tazobactam in very low birth weight infants; Berger A et al.; An open, non-comparative study was designed to evaluate the safety and tolerance of parenteral piperacillin/tazobactam in very low birth weight infants . Twenty-seven patients were included for nosocomial sepsis with gram-negative bacteria (n = 4), nosocomial sepsis not responding to the empirical antibiotic regimen (n = 3), suspected necrotizing enterocolitis (n = 17), and infection after abdominal surgery for reasons other than necrotizing enterocolitis (n = 3) . No clinical adverse events considered related to the study drug were noted, in particular, no cases of phlebitis, rash or stool changes . Several possibly related, mild and transitory abnormalities of laboratory parameters were observed . No long-lasting effect on the intestinal flora was detected . Seventeen patients (63%) were considered to have a favorable clinical response . This study demonstrates that piperacillin/tazobactam is a safe and well tolerated drug for preterm infants with bacterial infections, particularly those involving the gastrointestinal tract . Comparative clinical trials are warranted to further clarify the microbiological efficacy of piperacillin/tazobactam in this particular patient population.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W400 - 4
PRED-TMBB: a web server for predicting the topology of beta-barrel outer membrane proteins; Bagos PG et al.; The beta-barrel outer membrane proteins constitute one of the two known structural classes of membrane proteins . Whereas there are several different web-based predictors for alpha-helical membrane proteins, currently there is no freely available prediction method for beta-barrel membrane proteins, at least with an acceptable level of accuracy . We present here a web server (PRED-TMBB, which is capable of predicting the transmembrane strands and the topology of beta-barrel outer membrane proteins of Gram-negative bacteria . The method is based on a Hidden Markov Model, trained according to the Conditional Maximum Likelihood criterion . The model was retrained and the training set now includes 16 non-homologous outer membrane proteins with structures known at atomic resolution . The user may submit one sequence at a time and has the option of choosing between three different decoding methods . The server reports the predicted topology of a given protein, a score indicating the probability of the protein being an outer membrane beta-barrel protein, posterior probabilities for the transmembrane strand prediction and a graphical representation of the assumed position of the transmembrane strands with respect to the lipid bilayer.

Turk J Pediatr, 2004 Apr-Jun, 46(2), 177 - 8
Community-acquired pneumonia and empyema caused by Pseudomonas stutzeri: a case report; Kose M et al.; Pseudomonas stutzeri is an aerobic, nonfermentative, gram-negative rod with polar monotrichous flagella . We report the case of a four-year-old boy who developed community-acquired pneumonia and empyema caused by P . stutzeri . To our knowledge, this is the first report on community-acquired pneumonia and empyema caused by this organism in childhood.

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