Gram negative

Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1281 - 3 Epub 2004 Jun 22.
Crystallization and preliminary X-ray analysis of a C-terminal TonB fragment from Escherichia coli; Koedding J et al.; The TonB protein located in the cell wall of Gram-negative bacteria mediates the proton motive force from the cytoplasmic membrane to specific outer membrane transporters . A C-terminal fragment of TonB from Escherichia coli consisting of amino-acid residues 147-239 (TonB-92) has been purified and crystallized . Crystals grew in space group P2(1) to dimensions of about 1.0 x 0.12 x 0.12 mm . A native data set has been obtained to 1.09 A resolution.

Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9618 - 23 Epub 2004 Jun 21.
The integral membrane enzyme PagP alternates between two dynamically distinct states; Hwang PM et al.; PhoPQ-activated gene P (PagP) is an integral membrane enzyme that transfers the sn-1 palmitate chain from phospholipid to lipopolysaccharide in Gram-negative bacteria . A recent x-ray crystallographic study established that the sn-1 palmitate binds within a long cavity at the center of the PagP beta barrel . The high mobility required to permit substrate entry into the central core of the barrel contrasts with the need to assemble a well defined structure in the peripheral loops, where many key catalytic residues are located . To gain insight into how dynamics relate to the function of PagP, the enzyme was reconstituted into CYFOS-7, a detergent that supports enzymatic activity . Under these conditions, PagP exists in equilibrium between two states, relaxed (R) and tense (T) . The kinetics and thermodynamics of the interchange have been investigated by (1)H-(15)N NMR spectroscopy, with Delta H = -10.7 kcal/mol and Delta S = -37.5 cal/mol.K for the R--> T transition . A comparison of chemical shifts between the two states indicates that major structural changes occur in the large extracellular L1 loop and adjacent regions of the beta barrel . In addition to the R,T interconversion, other conformational exchange processes are observed in the R state, showing it to be quite flexible . Thus a picture emerges in which substrate entry is facilitated by the mobility of the R state, whereas the relatively rigid T state adopts a radically different conformation in a region of the protein known to be essential for catalysis . The ability to switch between dynamically distinct states may be a key feature of the catalytic cycle of PagP.

Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9780 - 5 Epub 2004 Jun 21.
Microarray analysis of transposition targets in Escherichia coli: the impact of transcription; Manna D et al.; Transposable elements have influenced the genetic and physical composition of all modern organisms . Defining how different transposons select target sites is critical for understanding the biochemical mechanism of this type of recombination and the impact of mobile genes on chromosome structure and function . Phage Mu replicates in Gram-negative bacteria using an extremely efficient transposition reaction . Replicated copies are excised from the chromosome and packaged into virus particles . Each viral genome plus several hundred base pairs of host DNA covalently attached to the prophage right end is packed into a virion . To study Mu transposition preferences, we used DNA microarray technology to measure the abundance of >4,000 Escherichia coli genes in purified Mu phage DNA . Insertion hot- and cold-spot genes were found throughout the genome, reflecting >1,000-fold variation in utilization frequency . A moderate preference was observed for genes near the origin compared to terminus of replication . Large biases were found at hot and cold spots, which often include several consecutive genes . Efficient transcription of genes had a strong negative influence on transposition . Our results indicate that local chromosome structure is more important than DNA sequence in determining Mu target-site selection.

MedGenMed . 2004 Mar 09;6(1):10.
Causative agents of liver abscess in Thai hepatitis B carriers; Wiwanitkit V et al.; Liver abscess and hepatitis B virus (HBV) infection are two significant tropical gastrointestinal disorders . The concurrence between these two disorders yields poor prognosis, which then often leads to the need for intensive care . The aim of this study was to investigate the causal pathogens of liver abscess in HBV carriers . This retrospective case review was conducted on 35 Thai hospitalized HBV carriers who had diagnosis of liver abscess . A high rate of amoebic liver abscess in this series (37.1%) was demonstrated; Gram-negative aerobes were the major abscess pathogens . The causative pathogens of HBV carriers were similar to those in the overall patient population with abscess . The treatment plan for liver abscess in the general population can also be applied to HBV carriers.

Vet Immunol Immunopathol, 2004 Aug, 100(3-4), 179 - 86
Induction of inflammatory host immune responses by organisms belonging to the genera Chlamydia/Chlamydophila; Entrican G et al.; Chlamydia/Chlamydophila are a family of intracellular gram-negative bacteria that infect their hosts primarily via mucosal epithelia . Chronic disease associated with bacterial persistence, inflammation and tissue damage are common sequelae of infection with these organisms . Human epithelial cell lines respond to infection by releasing pro-inflammatory cytokines and chemokines such as interleukin (IL)-6 and IL-8, and upregulating the expression of mRNA encoding Ikappa-Balpha, the endogenous inhibitor of NF-kappaB . However, Ikappa-Balpha is not upregulated in response to bacterial lipopolysaccharide (LPS) . The failure of epithelial cells to respond to LPS is associated with the absence of surface expression of CD14 . Identification of the components of Chlamydia/Chlamydophila that can induce pro-inflammatory mediators coupled with the mechanisms by which epithelial cells detect infection and respond accordingly will advance the development of preventative strategies.

Eur J Biochem, 2004 Jul, 271(13), 2691 - 704
A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeri OX1 possessing two 4,6-O-(1-carboxy)-ethylidene residues in the outer core region; Leone S et al.; Pseudomonas stutzeri OXI is a Gram-negative microorganism able to grow in media containing aromatic hydrocarbons . A novel lipo-oligosaccharide from P . stutzeri OX1 was isolated and characterized . For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration . The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis . Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-O-acylation) followed by de-N-acylation using hot KOH . The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups . The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy . These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A . All sugars are D-pyranoses and alpha-linked, if not stated otherwise . Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain . This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P . sutzeri OX1 to a hydrocarbon-containing environment.

Urol Res, 2004 Jun, 32(3), 190 - 5 Epub 2004 Feb 06.
Morphological and immunological characteristics of nanobacteria from human renal stones of a north Indian population; Khullar M et al.; The aim of this study was to detect, isolate and characterize the nanobacteria from human renal stones from a north Indian population, and to determine their role in biomineralization . Renal stones retrieved from the kidneys of 65 patients were processed and subjected to mammalian cell culture conditions . The isolated bacteria were examined using scanning (SEM) and transmission electron microscopy (TEM) . They were characterized for the presence of DNA, proteins and antigenicity . The role of these bacteria in biomineralization was studied by using the (14)C-oxalate based calcium oxalate monohydrate (COM) crystallization assay . We observed the presence of apatite forming, ultrafilterable gram negative, coccoid microorganisms in 62% of the renal stones . SEM studies revealed 60-200 nm sized organisms with a distinct cell wall and a capsule . TEM images showed needle like apatite structures both within and surrounding them . They were heat sensitive, showed antibiotic resistance and accelerated COM crystallization . A potent signal corresponding to the presence of DNA was observed in demineralized nanobacterial cells by flow cytometry . The protein profile showed the presence of several peptide bands of which those of 18 kDa and 39kDa were prominent . Apatite forming nanosized bacteria are present in human renal stones and may play a role in the pathophysiology of renal stone formation by facilitating crystallization and biomineralization . However, further studies are required to establish the exact mechanism by which nanobacteria are involved in the causation of renal stones.

J Bacteriol, 2004 Jul, 186(13), 4168 - 76
Sinorhizobium meliloti sulfotransferase that modifies lipopolysaccharide; Cronan GE et al.; Sinorhizobium meliloti is a gram-negative soil bacterium found either in free-living form or as a nitrogen-fixing endosymbiont of a plant structure called the nodule . Symbiosis between S . meliloti and its plant host alfalfa is dependent on bacterial transcription of nod genes, which encode the enzymes responsible for synthesis of Nod factor . S . meliloti Nod factor is a lipochitooligosaccharide that undergoes a sulfate modification essential for its biological activity . Sulfate also modifies the carbohydrate substituents of the bacterial cell surface, including lipopolysaccharide (LPS) and capsular polysaccharide (K-antigen) (R . A . Cedergren, J . Lee, K . L . Ross, and R . I . Hollingsworth, Biochemistry 34:4467-4477, 1995) . We utilized the genomic sequence of S . meliloti to identify an open reading frame, SMc04267 (which we now propose to name lpsS), which encodes an LPS sulfotransferase activity . We expressed LpsS in Escherichia coli and demonstrated that the purified protein functions as an LPS sulfotransferase . Mutants lacking LpsS displayed an 89% reduction in LPS sulfotransferase activity in vitro . However, lpsS mutants retain approximately wild-type levels of sulfated LPS when assayed in vivo, indicating the presence of an additional LPS sulfotransferase activity(ies) in S . meliloti that can compensate for the loss of LpsS . The lpsS mutant did show reduced LPS sulfation, compared to that of the wild type, under conditions that promote nod gene expression, and it elicited a greater number of nodules than did the wild type during symbiosis with alfalfa . These results suggest that sulfation of cell surface polysaccharides and Nod factor may compete for a limiting pool of intracellular sulfate and that LpsS is required for optimal LPS sulfation under these conditions.

J Occup Environ Hyg, 2004 Feb, 1(2), 62 - 8
Microbial air quality in offices at municipal landfills; Lis DO et al.; The purpose of this study was to evaluate the influence of two municipal landfills on the microbiological air quality in offices on landfill sites . The evaluation was based on the concentration levels of airborne bacteria and fungi and the identification of isolated strains . Air samples were collected with a six-stage Andersen impactor . The concentrations of bacterial aerosol ranged from 1.0 x 10(3) to 7.2 x 10(4) colony forming units (CFU)/m(3) indoors, and from 7.0 x 10 to 4.0 x 10(4) CFU/m(3) outdoors . The corresponding fungal aerosol ranges were from 2.3 x 10(2) to 7.3 x 10(3) CFU/m(3) indoors and from 2.0 x 10(2) to 1.2 x 10(4) CFU/m(3) outdoors . The concentration levels were affected by the season of the year . The study showed that both indoor and outdoor air were heavily contaminated with bacteria and fungi . The proximity of the unpaved transport route and the weighing of refuse loads contributed to the increase of bacterial and fungal aerosol concentrations significantly . The air in the offices was characterized not only by elevated concentrations of bacteria and fungi but also by high frequencies of gram-negative bacteria, along with fungal species characteristic of landfills . The quantitative and qualitative changes in the composition of the bacterial and fungal aerosol posed a possible health risk to office workers at municipal waste landfill sites.

Inhal Toxicol, 2004 Apr, 16(4), 217 - 29
The measurement and health impact of endotoxin contamination in organic dusts from multiple sources: focus on the cotton industry; Lane SR et al.; Endotoxin is derived from Gram-negative bacterial membranes, and its inflammatory effects following inhalation are well characterized . The significance of this fact becomes apparent when the wide-ranging environments containing high levels of this microbial product are considered . Endotoxin is present in numerous industrial environments, especially where organic fibers are processed . Microbial contamination of these fibers mainly occurs at the agricultural stage . Materials such as flax and hemp are affected in this way, but the most important product in this context is cotton, from which chronic dust inhalation causes the disease byssinosis . Despite the fact that endotoxin constitutes a significant threat to public health, there are currently no occupational exposure limits for this toxicant . This communication describes the toxicology of endotoxin, and its role in inhalation-induced disease, focusing on measurement of airborne endotoxin in the occupational and domestic environments using the Limulus amebocyte lysate (LAL) enzyme assay . Following the success of the LAL assay for measuring endotoxin in dusts, our laboratory has examined its application to aqueous washes from cotton fibers . Reproducibility of the results was high, and data are presented displaying levels of endotoxin contamination in fibers from different cotton producing countries . Hence, worldwide comparison of industrial endotoxin concentrations can be readily made using this test . It would be highly desirable if the performance of the LAL assay facilitated introduction of industrial endotoxin safety limits, and in spite of minor surmountable shortcomings, the test is accurate, reliable, and well field-tested, so its continued widespread use may achieve this goal.

Inhal Toxicol, 2004 Jun, 16(6-7), 461 - 71
Systemic effects of inhaled ultrafine particles in two compromised, aged rat strains; Elder AC et al.; Epidemiological studies associate morbidity and mortality with exposure to particulate air pollution in elderly individuals with existing cardiopulmonary disease . These associations led to the hypothesis that inhaled particles can exert adverse effects outside of the lung, particularly on the cardiovascular system . We tested this hypothesis by examining the pulmonary and peripheral effects of inhaled ultrafine carbon particles in old rats that were injected with endotoxin (lipopolysaccharide, LPS) to model systemic gram-negative bacterial infection . Fischer 344 rats (23 mo) and spontaneously hypertensive (SH) rats (11-14 mo) were injected with LPS (2 mg/kg, i.p.) immediately before being exposed to inhaled ultrafine carbon particles for 6 h (150 microg/m(3), CMD = 36 nm) . Controls were injected with sterile saline or were sham exposed . Twenty-four hours after LPS injection, bronchoalveolar lavage (BAL) fluid, cells, and blood were obtained to assess endpoints of inflammation, oxidant stress, coagulability, and the acute-phase response . LPS did not cause an influx of neutrophils (PMNs) into the alveolar space, but did increase the number and percentage of circulating PMNs and the concentration of plasma fibrinogen in both rat strains . Inhaled ultrafine particles did not induce lung inflammation in either rat strain . In both strains, ultrafine particles (UFP) were found to decrease the number of blood PMNs, increase the intracellular oxidation of a fluorescent dye (DCFD) in blood PMNs, and affect plasma thrombin-anti-thrombin (TAT) complex and fibrinogen levels . UFP were also found to interact with ip LPS with respect to plasma TAT complex levels and blood PMN DCFD oxidation . Differences between the two rat strains were also found for TAT complex levels, BAL cell reactive oxygen species release, and DCFD oxidation in both BAL macrophages and blood PMNs . These results suggest that inhaled ultrafine carbon particles inhaled at concentrations mimicking high episodic increases in urban air can exert extrapulmonary effects in old rats and that they can change the systemic response to an inflammatory stimulus.

Mol Membr Biol, 2004 May-Jun, 21(3), 151 - 61
The simulation approach to bacterial outer membrane proteins; Bond PJ et al.; The outer membrane of Gram-negative bacteria serves as a protective barrier against the external environment but is rendered selectively permeable to nutrients and waste by proteins called porins . Other outer membrane proteins (OMPs) provide the membrane with a variety of other functions including active transport, catalysis, pathogenesis and signal transduction . A relatively small number of crystal or NMR structures of these proteins are known, and it is therefore essential that the maximum possible information be extracted . In this respect, computational techniques enable extrapolation from time- and space-averaged static structures to dynamic, physiological events . Electrostatics approaches have been used to investigate the structures of porins . The stochastic simulation of ion trajectories through these channels has been possible with Brownian dynamics, which treats the membrane and solvent approximately, enabling the prediction of conduction properties . Molecular dynamics has also been applied, enabling fully atomistic descriptions of 'virtual outer membranes' . This has provided atomic resolution descriptions of solute permeation through porins . It has also yielded insights into the dynamics of gating in active transporters and ion channels, as well as providing clues to catalytic mechanisms in outer membrane enzymes . Additionally, simulations are beginning to reveal the common features of interactions between membrane proteins and lipids, with biological implications for OMP folding, stability and mechanism . Future prospects include the simulation of longer, larger and more complex outer membrane systems, with more accurate descriptions of inter-atomic forces.

Vestn Khir Im I I Grek, 2004, 163(2), 12 - 7
{Septic shock}; Grinev MV et al.; The problem of sepsis and septic shock (SSH) is known to be very actual due to peak-shaped growth of the number of such patients and unsatisfactory results of treatment . The occurrence of SSH is different--from 10% in the structure of infectious complications after polytrauma to 50% in patients with gram-negative sepsis in patients with burn disease . An analysis of treatment of 165 patients with SSH developed against the background of peritonitis has revealed high level of lethality (64%) . In the pathogenesis of SSH an important role is played by immune disorders resulting in the development of generalized inflammation and polyorganic insufficiency . Treatment of patients with SSH must be necessarily supplemented with immuno-correcting techniques: use of medicines of nonspecific anti-cytokine action (pentoxyphillin) and regulators of the immune response (ronkoleukin, blood perfusion through the donor porcine spleen) . The timely performed immunomodulating therapy allowed lethality of patients with SSH to be reduced to 32%.

Arch Surg, 2004 Jun, 139(6), 652 - 4; discussion 655
Endotoxin has an indirect vasodilatory effect on isolated human skeletal muscle arterioles; Campbell M et al.; BACKGROUND: Septic shock and its effects are a major cause of mortality in the intensive care environment . The exact effect and mechanism of endotoxin has yet to be fully described . With a better understanding of this process, better clinical tools could be developed to treat these patients . HYPOTHESIS: Endotoxin has no direct effect on human skeletal muscle microvasculature and requires the release of an endothelial-derived factor to produce the vasodilation seen in gram-negative sepsis . DESIGN: Benchtop research using an isolated arteriole model with controlled exposure to endotoxin . SETTING: University medical center . METHODS: First-order arterioles (approximately 150- micro m diameter) were isolated from human cremasteric muscles and pressurized to physiologic levels before exposure to an endotoxin-rich effluent with and without an upstream conduit vessel (superficial epigastric vein) . The vasodilatory effect was measured with videomicroscopy and compared with control samples . MAIN OUTCOME MEASURES: Mean vessel diameter and percentage of loss in tone . RESULTS: When compared with controls, the isolated arteriole had no significant response when exposed to endotoxin alone (3.5% change in basal tone) . When the endotoxin was allowed to pass over an upstream conduit vessel, the arteriole showed marked dilation (14.2% loss of basal tone) . CONCLUSIONS: This study demonstrates that endotoxin has no direct vasodilatory effect on human skeletal muscle arterioles, but it is the release of an endothelial factor from the upstream conduit vessels that produces the loss of tone in the microvasculature . Further research is ongoing to characterize the factors involved (nuclear factor-kappaB, tumor necrosis factor alpha, and interleuklin 6) for possible clinical intervention (antioxidants, cyclosporine, and nitric oxdide synthase inhibitors).

Res Vet Sci, 2004 Oct, 77(2), 93 - 100
Q fever (coxiellosis): epidemiology and pathogenesis; Woldehiwet Z; Q fever is a widespread zoonosis caused by the Gram-negative bacterium Coxiella burnetii . Aborting domestic ruminants are the main sources of human infection but the reservoir of infection is extremely wide . In humans, Q fever may occur as acute pneumonia, hepatitis or flu-like illness or may take a severe chronic form, characterized by endocarditis, chronic hepatitis and chronic fatigue syndrome . In animals, the main clinical manifestation is late abortion . Infection with C . burnetii can be diagnosed using cultural, serological and genetic methods but because the organism is potentially dangerous and requires specialized skills only specialist laboratories are capable of undertaking diagnostic tests . This paper provides a brief overview of the epidemiology and pathogenesis of Q fever (coxiellosis).

Gut, 2004 Jul, 53(7), 987 - 92
Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis; Franchimont D et al.; BACKGROUND AND AIMS: Elicitation of an innate immune response to bacterial products is mediated through pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs) and the NODs . The recently characterised Asp299Gly polymorphism in the lipopolysaccharide (LPS) receptor TLR4 is associated with impaired LPS signalling and increased susceptibility to Gram negative infections . We sought to determine whether this polymorphism was associated with Crohn's disease (CD) and/or ulcerative colitis (UC) . METHODS: Allele frequencies of the TLR4 Asp299Gly polymorphism and the three NOD2/CARD15 polymorphisms (Arg702Trp, Gly908Arg, and Leu1007fsinsC) were assessed in two independent cohorts of CD patients (cohort 1, n = 334; cohort 2, n = 114), in 163 UC patients, and in 140 controls . A transmission disequilibrium test (TDT) was then performed on 318 inflammatory bowel disease (IBD) trios . RESULTS: The allele frequency of the TLR4 Asp299Gly polymorphism was significantly higher in CD (cohort 1: 11% v 5%, odds ratio (OR) 2.31 (95% confidence interval (CI) 1.28-4.17), p = 0.004; and cohort 2: 12% v 5%, OR 2.45 (95% CI 1.24-4.81), p = 0.007) and UC patients (10% v 5%, OR 2.05 (95% CI 1.07-3.93), p = 0.027) compared with the control population . A TDT on 318 IBD trios demonstrated preferential transmission of the TLR4 Asp299Gly polymorphism from heterozygous parents to affected children (T/U: 68/34, p = 0.01) . Carrying polymorphisms in both TLR4 and NOD2 was associated with a genotype relative risk (RR) of 4.7 compared with a RR of 2.6 and 2.5 for TLR4 and NOD2 variants separately . CONCLUSION: We have reported on a novel association of the TLR4 Asp299Gly polymorphism with both CD and UC . This finding further supports the genetic influence of PRRs in triggering IBD.

Apoptosis, 2004 Jul, 9(4), 467 - 74
LPS-induced apoptosis is dependent upon mitochondrial dysfunction; Kuwabara T et al.; Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN) . Apoptosis occurs in a manner that is independent of bacterial virulence proteins . In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells . LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases . While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential . Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP . These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells .

Harefuah, 2004 May, 143(5), 364 - 7, 390
{Q fever pericarditis}; Edelstein S et al.; Q fever is a zoonotic disease caused by Coxiella burnetii--an obligate, gram negative, intracellular bacteria . The term Q (Query) was first used because at the time the disease was named its etiology was unknown . Q fever is divided into acute and chronic infections characterized by different evolution, serological profiles and treatment . Pericarditis, as a manifestation of Q fever is rare and difficult to diagnose . This is due to the following: firstly, the clinical presentation of acute Q fever is pleomorphic, nonspecific and self limited, and secondly, the diagnosis relies on the physician's interest and the presence of a reliable diagnostic laboratory . The objective of that review is to increase the physician's awareness of the clinical presentation of Q fever, to discuss the importance of the diagnosis and laboratory tests and to guide the physician as to when to provide treatment and the relevant patient population to be treated.

Annu Rev Biochem, 2004, 73, 107 - 46
Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins; Tugarinov V et al.; Recent developments in NMR spectroscopy, which include new experiments that increase the lifetimes of NMR signals or that precisely define the orientation of internuclear bond vectors with respect to a common molecular frame, have significantly increased the size of proteins for which quantitative structural and dynamic information can be obtained . These experiments have, in turn, benefited from new labeling strategies that continue to drive the field . The utility of the new methodology is illustrated by considering applications to malate synthase G, a 723 residue enzyme, which is the largest single polypeptide chain for which chemical shift assignments have been obtained to date . New experiments developed specifically to address the complexity and low sensitivity of spectra recorded on this protein are presented . A discussion of the chemical information that is readily available from studies of systems in the 100 kDa mol wt range is included . Prospects for membrane protein structure determination are discussed briefly in the context of an application to an Escherichia coli enzyme, PagP, localized to the outer membrane of gram-negative bacteria.

Proteomics, 2004 May, 4(5), 1280 - 92
Proteome analysis of Madrid E strain of Rickettsia prowazekii; Chao CC et al.; Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus . The threat of typhus as a biological weapon lies in its stability in the dried louse feces and in its infection by inhalation of an aerosol . Consequently, it is listed as a select agent and warrants more research to understand its pathogenesis . Although the genomic DNA sequence of strain Madrid E has been completed, the actual expression of the individual protein has not been investigated . In order to provide a global view of the expressed protein profile, the whole cell lysate of purified rickettsia (Madrid E strain) was reduced, alkylated, and digested with trypsin . The total digest was characterized by a two-dimensional liquid chromatography mass spectrometry system and analyzed with a modified version of the ProteomeX workstation . A total of 252 proteins out of 834 predicted protein-coding genes were identified, 238 proteins were identified by the detection of at least two unique peptides . Only 14 proteins were identified by the detection of one unique peptide in all three separate analyses . Among the 238 proteins identified by multiple unique peptides, 230 proteins were found in at least two of three separate analyses . The reproducible and convenient methodology and the information described here have provided a foundation for future proteome study of various R . prowazekii strains with different virulence.

Chembiochem, 2004 Apr 2, 5(4), 491 - 9
Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars; Jose J et al.; Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups . In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed . For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay . Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway . Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions . The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS) . The activity of whole cells displaying SDH at the surface was determined in an optical test . Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose . The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

J Biol Chem, 2004 Aug 20, 279(34), 35709 - 18 Epub 2004 Jun 07.
Nonreducing terminal modifications determine the chain length of polymannose O antigens of Escherichia coli and couple chain termination to polymer export via an ATP-binding cassette transporter; Clarke BR et al.; The chain length of bacterial lipopolysaccharide O antigens is regulated to give a modal distribution that is critical for pathogenesis . This paper describes the process of chain length determination in the ATP-binding cassette (ABC) transporter-dependent pathway, a pathway that is widespread among Gram-negative bacteria . Escherichia coli O8 and O9/O9a polymannans are synthesized in the cytoplasm, and an ABC transporter exports the nascent polymer across the inner membrane prior to completion of the LPS molecule . The polymannan O antigens have nonreducing terminal methyl groups . The 3-O-methyl group in serotype O8 is transferred from S-adenosylmethionine by the WbdD(O8) enzyme, and this modification terminates polymerization . Methyl groups are added to the O9a polymannan in a reaction dependent on preceding phosphorylation . The bifunctional WbdD(O9a) catalyzes both reactions, but only the kinase activity controls chain length . Chain termination occurs in a mutant lacking the ABC transporter, indicating that it precedes export . An E . coli wbdD(O9a) mutant accumulated O9a polymannan in the cytoplasm, indicating that WbdD activity coordinates polymannan chain termination with export across the inner membrane.

Appl Environ Microbiol, 2004 Jun, 70(6), 3687 - 94
Identification of differential gene expression in bacteria associated with coral black band disease by using RNA-arbitrarily primed PCR; Frias-Lopez J et al.; RNA-arbitrarily primed PCR techniques have been applied for the first time to identify differential gene expression in black band disease (BBD), a virulent coral infection that affects reef ecosystems worldwide . The gene activity for the BBD mat on infected surfaces of the brain coral Diploria strigosa was compared with that for portions of the BBD mat that were removed from the coral and suspended nearby in the seawater column . The results obtained indicate that three genes (DD 95-2, DD 95-4, and DD 99-9) were up-regulated in the BBD bacterial mat on the coral surface compared to the transcript base levels observed in the BBD mat suspended in seawater . Clone DD 95-4 has homology with known amino acid ABC transporter systems in bacteria, while clone DD 99-9 exhibits homology with chlorophyll A apoprotein A1 in cyanobacteria . This protein is essential in the final conformation of photosystem I P700 . DD 95-2, the only gene that was fully repressed in the BBD mat samples suspended in seawater, exhibited homology with the AraC-type DNA binding domain-containing proteins . These transcriptional activators coordinate the expression of genes essential for virulence in many species of gram-negative bacteria.

Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2004 Jun, 16(6), 358 - 60
{Anti-endotoxin core glycolipid antibody: the preparation of immune serum of E . Coli J5}; Xu XN et al.; OBJECTIVE: To prepare high titer anti-endotoxin core glycolipid (J5) antibody (CGL) for the treatment of Gram-negative bacteremia and septic shock . METHODS: Nontoxic bacterial vaccine (50 x 10(12)U/L) against E.Coli O111:B4 mutant strain J5 was prepared . J5 bacterial vaccine was injected into rabbits through ear marginal vein (saline as control preparation), one time pre three days, totally five times . Injected doses were as following: 0.1 ml, 0.2 ml, 0.4 ml, 0.6 ml, and 0.8 ml . One week after fifth injection, blood samples from heart were collected and immune serum was isolated . Indirect clotting test was used to determine the titer of antibody and cross reaction . RESULTS: Among 12 immunized rabbits, titers of antibody against E . Coli J5 were exceeding 1:1 024 in 6 rabbits, and they had cross reaction with various kinds of Gram-negative bacterial endotoxins . CONCLUSION: The titer of anti-endotoxin core glycolipid (E.Coli J5) antibody prepared by us appears to be high, and it can combine with various kinds of Gram-negative bacterial endotoxins.

Scand J Immunol, 2004 Jun, 59(6), 553 - 8
Anti-inflammatory cytokines induce lipopolysaccharide tolerance in human monocytes without modifying toll-like receptor 4 membrane expression; Moreno C et al.; Toll-like receptor 4 (TLR4) participates in innate immunity by detecting lipopolysaccharides (LPS) of Gram-negative bacterial cell walls . TLR4 macrophage expression in mice is modulated by LPS . This fact constitutes, at least partially, the molecular basis for LPS tolerance . Very recently, the effect of interferon-gamma (IFN-gamma), a pro-inflammatory cytokine, has been described on TLR4 membrane expression of human monocytes . IFN-gamma up-regulates TLR4 expression and antagonizes the LPS-induced TLR4 down-regulation . These data prompted us to study the expression of membrane TLR4 in human mono- cytes in which LPS tolerance was induced by LPS and by anti-inflammatory cytokines {interleukin-10 (IL-10) and transforming growth factor beta1 (TGFbeta1)} . Data concerning this latter model, and more specifically, the effect of anti-inflammatory cytokines over TLR4 expression, are not available at present . We show here that membrane TLR4 expression in human monocytes falls after LPS exposure . The effect was prolonged for 12 h, but then expression returned to normal levels . The incubation of human monocytes with IL-10, TGFbeta1 or a mixture of both induces no alterations in membrane TLR4 expression . However, these cytokines are able to substitute the tolerizing LPS exposure in order to induce LPS tolerance . Our data help to achieve a better understanding of the way cytokines control the cellular expression of TLR.

J Biol Chem, 2004 Aug 13, 279(33), 34833 - 9 Epub 2004 Jun 04.
Solution structure of Cox11, a novel type of beta-immunoglobulin-like fold involved in CuB site formation of cytochrome c oxidase; Banci L et al.; Cytochrome c oxidase assembly process involves many accessory proteins including Cox11, which is a copper-binding protein required for Cu incorporation into the Cu(B) site of cytochrome c oxidase . In a genome wide search, a number of Cox11 homologs are found in all of the eukaryotes with complete genomes and in several Gram-negative bacteria . All of them possess a highly homologous soluble domain and contain an N-terminal fragment that anchors the protein to the membrane . An anchor-free construct of 164 amino acids was obtained from Sinorhizobium meliloti, and the first structure of this class of proteins is reported here . The apoform has an immunoglobulin-like fold with a novel type of beta-strand organization . The copper binding motif composed of two highly conserved cysteines is located on one side of the beta-barrel structure . The apoprotein is monomeric in the presence of dithiothreitol, whereas it dimerizes in the absence of the reductant . When copper(I) binds, NMR and extended x-ray absorption fine structure (EXAFS) data indicate a dimeric protein state with two thiolates bridging two copper(I) ions . The present results advance the knowledge on the poorly understood molecular aspects of cytochrome c oxidase assembly.

J Bacteriol, 2004 Jun, 186(12), 3903 - 10
Structural basis for iron binding and release by a novel class of periplasmic iron-binding proteins found in gram-negative pathogens; Shouldice SR et al.; We have determined the 1.35- and 1.45-A structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A . M . haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever . The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host . The ferric (Fe(3+)) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion . The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion . Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm.

J Bacteriol, 2004 Jun, 186(12), 3814 - 25
Regulation of hypercompetence in Legionella pneumophila; Sexton JA et al.; Although many bacteria are known to be naturally competent for DNA uptake, this ability varies dramatically between species and even within a single species, some isolates display high levels of competence while others seem to be completely nontransformable . Surprisingly, many nontransformable bacterial strains appear to encode components necessary for DNA uptake . We believe that many such strains are actually competent but that this ability has been overlooked because standard laboratory conditions are inappropriate for competence induction . For example, most strains of the gram-negative bacterium Legionella pneumophila are not competent under normal laboratory conditions of aerobic growth at 37 degrees C . However, it was previously reported that microaerophilic growth at 37 degrees C allows L . pneumophila serogroup 1 strain AA100 to be naturally transformed . Here we report that another L . pneumophila serogroup 1 strain, Lp02, can also be transformed under these conditions . Moreover, Lp02 can be induced to high levels of competence by a second set of conditions, aerobic growth at 30 degrees C . In contrast to Lp02, AA100 is only minimally transformable at 30 degrees C, indicating that Lp02 is hypercompetent under these conditions . To identify potential causes of hypercompetence, we isolated mutants of AA100 that exhibited enhanced DNA uptake . Characterization of these mutants revealed two genes, proQ and comR, that are involved in regulating competence in L . pneumophila . This approach, involving the isolation of hypercompetent mutants, shows great promise as a method for identifying natural transformation in bacterial species previously thought to be nontransformable.

J Bacteriol, 2004 Jun, 186(12), 3712 - 20
The type II protein secretion system of Legionella pneumophila promotes growth at low temperatures; Soderberg MA et al.; The gram-negative bacterium Legionella pneumophila grows in both natural and man-made water systems and in the mammalian lung as a facultative intracellular parasite . The PilD prepilin peptidase of L . pneumophila promotes type IV pilus biogenesis and type II protein secretion . Whereas pili enhance adherence, Legionella type II secretion is critical for intracellular growth and virulence . Previously, we observed that pilD transcript levels are greater in legionellae grown at 30 versus 37 degrees C . Using a new pilD::lacZ fusion strain, we now show that pilD transcriptional initiation increases progressively as L . pneumophila is grown at 30, 25, and 17 degrees C . Legionella pilD mutants also had a dramatically reduced ability to grow in broth and to form colonies on agar at the lower temperatures . Whereas strains specifically lacking type IV pili were not defective for low-temperature growth, mutations in type II secretion (lsp) genes greatly impaired the capacity of L . pneumophila to form colonies at 25, 17, and 12 degrees C . Indeed, the lsp mutants were completely unable to grow at 12 degrees C . The growth defect of the pilD and lsp mutants was complemented by reintroduction of the corresponding intact gene . Interestingly, the lsp mutants displayed improved growth at 25 degrees C when plated next to a streak of wild-type but not mutant bacteria, implying that a secreted, diffusible factor promotes low-temperature growth . Mutants lacking either the known secreted acid phosphatases, lipases, phospholipase C, lysophospholipase A, or protease grew normally at 25 degrees C, suggesting the existence of a critical, yet-to-be-defined exoprotein(s) . In summary, these data document, for the first time, that L . pneumophila replicates at temperatures below 20 degrees C and that a bacterial type II protein secretion system facilitates growth at low temperatures.

Proteomics, 2004 Jun, 4(6), 1597 - 613
Fine-tuning the prediction of sequences cleaved by signal peptidase II: a curated set of proven and predicted lipoproteins of Escherichia coli K-12; Gonnet P et al.; A curated set of 81 proven and 44 predicted lipoproteins of Escherichia coli K-12 was defined with the combined use of a literature survey, a variety of predictive tools and human expertise . The well-documented Gram-negative proteome of E . coli K-12 was chosen to assess how the different approaches complement each other and to ensure a stable definition of a consistent set of lipoproteins . The results of detailed analysis of such proteins at the level of a single proteome are presented, corroborated and rationalized.

J Fr Ophtalmol, 2004 Apr, 27(4), 420 - 3
{Rules and regulations concerning contact lens-related infection}; Feys J; Contact lens-related infectious keratitis is a potentially sight-threatening complication . Bacterial keratitis, mostly due to Gram-negative bacteria, is associated with poor lens hygiene, overnight wear, and contaminated lens care solutions . Contamination of the lens storage case may cause fungal keratitis . Acanthamoeba infection is related to the use of tap water or swimming while wearing soft lenses . Viruses are of less concern among contact lens wearers . Possible transmission of Creutzfeldt-Jakob disease by multi-patient trial lenses must be taken in account . To minimize these risk factors, regulations are applied at various levels: CE marking of contact lenses and care products as they are medical devices; contact lens fitting only by health care professionals; distribution of contact lenses by opticians and lens care solutions by opticians and pharmacists; hygienic management of trial lenses following official recommendations . Contact lens-related keratitis must be reported to health care Authorities.

Chem Phys Lipids, 2004 Jul, 130(2), 83 - 98
The 2004 Biophysical Society-Avanti Award in Lipids address: roles of bilayer structure and elastic properties in peptide localization in membranes; McIntosh TJ; This review details how bilayer structural/elastic properties impact three distinct areas of biological significance . First, the partitioning of melittin into bilayers and melittin-induced bilayer leakage depended strongly on bilayer composition . The incorporation of cholesterol into phosphatidylcholine bilayers decreased melittin-induced leakage from 73 to 3%, and bilayers composed of lipopolysaccharide (LPS), the main lipid on the surface of Gram-negative bacteria, also had low (3%) melittin-induced leakage . Second, transbilayer peptides of different hydrophobic lengths were largely excluded from bilayer microdomains ("rafts") enriched in sphingomyelin (SM) and cholesterol, even when the length of the transbilayer peptide domain matched the hydrocarbon thickness of the raft bilayer . This is likely due to the large area compressibility modulus of SM:cholesterol bilayers . Third, the major water barrier of skin, the extracellular lamellae of the stratum corneum, was found to contain tightly packed asymmetric lipid bilayers with cholesterol located preferentially on one side of the bilayer and a unique skin ceramide containing an unsaturated acyl chain on the opposite side . We argue that, in each of these three areas, key factors are differences in lipid hydrocarbon chain packing for different lipids, particularly the tight hydrocarbon chain packing caused by cholesterol's strong interaction with saturated chains.

Biosci Biotechnol Biochem, 2004 May, 68(5), 1146 - 8
Isolation of an exopolysaccharide-producing bacterium, Sphingomonas sp . CS101, which forms an unusual type of sphingan; Seo EJ et al.; An exopolysaccharide-producing Gram negative bacterium was isolated and determined to be a Sphingomonas sp . (CS101) . A sugar composition analysis of an exopolysaccharide indicated that the Sphingomonas sp . CS101 secreted an exopolysaccharide composed of glucose, mannose, fucose, and rhamnose in the ratio of 2.1:1.1:1.0:0.1, suggesting that this exoplysaccharide is an unusual type of sphingan family . The mean molecular weight of the exopolysaccharide was determined to be 4.2x10(5) Da by size exclusion chromatography coupled with multi-angle laser-light scattering (SEC/MALLS) analysis . An exopolysaccharide was produced up to 17 g/l (pH 7; 30 degrees C) with the optimal medium condition over 4 days of cultivation.

Shock, 2004 Jun, 21(6), 566 - 71
Increased lymphoid tissue apoptosis in baboons with bacteremic shock; Efron PA et al.; The molecular mechanisms of immune cell apoptosis during sepsis remain unclear . Two young adult baboons (Papio sp.) received a lethal dose of live Escherichia coli and were sacrificed at either 16 (for animal welfare concerns) or 24 h post-septic shock . An additional baboon, which received no bacteria, served as a control . Necropsy was performed immediately with subsequent immunohistochemical staining of lymphoid tissue . Immunohistologic analysis of tissues from the septic baboons revealed marked systemic lymphocyte apoptosis occurring in all lymphoid tissues examined . Focally, pyknotic and karyorrhectic lymphocytes demonstrated activation of a mitochondrial-dependent cell death pathway (active caspase 9 and apoptosis-inducing factor) . Other regions demonstrated apoptotic lymphocytes with activation of a death receptor-dependent cell pathway (Fas ligand) . Thus, we have demonstrated for the first time in primates that overwhelming gram-negative bacteremia produces an early and profound lymphocyte death that occurs through multiple cell death pathways . Bacteremic shock in the baboon may be an appropriate model for studying experimental therapies aimed at blocking lymphocyte apoptosis because their response appears comparable to humans dying from sepsis.

Can J Microbiol, 2003 Dec, 49(12), 733 - 40
Controlled induction of the RpoS regulon in Escherichia coli, using an RpoS-expressing plasmid; Chen G et al.; RpoS, an alternative sigma factor produced by many gram-negative bacteria, primarily controls genes that are expressed in stationary phase in response to nutrient deprivation . To test the idea that induction of RpoS in the exponential phase, when RpoS is not normally expressed, increases RpoS-dependent gene expression, we constructed a plasmid carrying the rpoS gene under the control of an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible T7lac promoter . Northern and Western analyses revealed that levels of RpoS mRNA and protein, respectively, increased in response to the inducer IPTG . Assays of changes in RpoS-dependent functions (catalase activity and glycogen accumulation), confirmed that induced RpoS was functional in exponential phase and was sufficient for the expression of RpoS-dependent functions . Controlled expression of RpoS and RpoS-dependent genes by plasmid-encoded rpoS may thus offer a useful tool for the study of RpoS-dependent gene expression.

Invest Ophthalmol Vis Sci, 2004 Jun, 45(6), 1871 - 8
Expression of toll-like receptor 4 and its associated lipopolysaccharide receptor complex by resident antigen-presenting cells in the human uvea; Chang JH et al.; PURPOSE: To investigate the in vivo expression of toll-like receptor 4 (TLR4) and its associated lipopolysaccharide (LPS) receptor complex in the human eye . METHODS: Normal human ocular tissues were evaluated for in vivo TLR4, MD-2, and CD14 mRNA and protein expression by RT-PCR and immunohistochemistry, respectively . The distribution patterns and phenotypes of the cells expressing these proteins were further characterized by confocal microscopy and double-label immunofluorescence studies . RESULTS: Normal human uvea, retina, sclera, and conjunctiva constitutively expressed TLR4, MD-2, and CD14 mRNA . The protein expression of these molecules was restricted, however, to resident antigen-presenting cells (APCs) in the normal human uvea, consisting mainly of HLA-DR(+) dendritic cells (DCs) . These APCs endowed with the complete LPS receptor complex appeared to be strategically positioned in perivascular and subepithelial locations for surveying blood-borne or intraocular LPS . In contrast, other cell types of the normal human cornea, conjunctiva, retina, and sclera did not express TLR4/MD-2 protein in vivo as detectable by immunohistochemistry . CONCLUSIONS: The present study demonstrates for the first time that resident APCs in the normal human uvea express TLR4 and its associated LPS receptor complex . This has significant implications for the understanding of normal ocular immunity as well as unraveling the potential role of Gram-negative bacteria in the pathogenesis of acute anterior uveitis (AAU).

Med Pr, 2004, 55(1), 31 - 40
{Occupational bio hazards: current issues}; Dutkiewicz J; Over the last decade, there was noted a large advancement of knowledge on living organisms and their products posing a potential occupational risk . Novel risk factors, often new to science, were identified, the role and significance of already known factors better comprehended, and occupational groups endangered by biological hazards more thoroughly recognized . Novel viruses and prions, emerging in different parts of the world, may pose a particular threat to health and life of health care workers, agriculture workers and veterinarians . A new coronavirus (SCoV) that evoked a rapid outbreak of disease described as severe acute respiratory syndrome (SARS) in the first half of 2003 may serve as an example . The disease was particularly common among health care workers . Previously discovered zoonotic viruses, Nipah virus in pigs and Hendra virus in horses, may be a cause of fatal encephalitis in animal farmers . Hantaviruses (Puumala, Hantaan, Sin Nombre and others) infecting field rodents may be a cause of hemorrhagic fever with renal syndrome (HFRS) or pulmonary syndrome (HPS) in farmers and laboratory workers . Prions responsible for inducing a zoonotic variant of Creutzfeldt-Jakob disease (vCJD) are considered to be a potential cause of work-related infections in agricultural and health care workers, however, this assumption has not as yet been supported by any conclusive evidence . In many countries, blood-borne occupational infections with hepatitis C virus (HCV) is the major epidemiological problem among health care workers, mostly because no vaccine against this virus has been produced to date . Vaccinations effectively restricted the number of occupational infections with hepatitis B virus (HBV), and work-related infections with human immunodeficiency virus (HIV) causing acquired immunodeficiency syndrome (AIDS) are very rare . Hazardous bioserosols, occurring in many work environments, pose an occupational health hazard of particular importance . Many new biological factors present in organic dusts that may induce work-related allergic and immunotoxic diseases among farmers and workers of the agricultural and wood industries have been identified . Droplet aerosols, which are generated from water, oils, oil-water emulsions and other liquids in various work environments, may contain infectious agents (Legionella spp.) as well as allergic and/or toxic agents . It has been shown that allergens and endotoxins produced by Gram-negative bacteria occurring in oil mist from metalworking fluids may cause occupational respiratory diseases in workers of the metallurgic industry.

Clin Infect Dis, 2004 Jun 1, 38(11), 1579 - 84 Epub 2004 May 07.
Clinical significance of Roseomonas species isolated from catheter and blood samples: analysis of 36 cases in patients with cancer; De I et al.; This report analyzes 36 cases of bacteremia or catheter-related infection caused by Roseomonas species, a group of pink, slimy, waterborne, gram-negative coccobacilli . The causative species included the newly described Roseomonas mucosa (22 cases {61%}) and Roseomonas gilardii subspecies rosea (8 cases {22%}) and known species R . gilardii subspecies gilardii (5 cases {14%}) and Roseomonas genomospecies 4 (1 case {3%}) . Twenty-nine (81%) of the cases were symptomatic, with fever being the most common symptom (in 27 {75%} of the cases) . Twenty (56%) of the infections were monomicrobic . Six cases (17%) involved persistent catheter colonization, and 5 of these cases required removal of the catheter to clear the infection . All infections resolved, most with empirical antibiotic treatment . A summary of the antibiotic susceptibility pattern of these strains and other reported series show that Roseomonas species are consistently susceptible to amikacin and imipenem and frequently susceptible to ciprofloxacin and ticarcillin, but essentially nonsusceptible to ceftazidime and cefepime . This result may guide future therapy for infections due to Roseomonas species.

J Biol Chem, 2004 Jul 30, 279(31), 32116 - 24 Epub 2004 May 20.
AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex; Tikhonova EB et al.; Many transporters of Gram-negative bacteria involved in the extracellular secretion of proteins and the efflux of toxic molecules operate by forming intermembrane complexes . These complexes are proposed to span both inner and outer membranes and create a bridge across the periplasm . In this study, we analyzed interactions between the inner and outer membrane components of the tri-partite multidrug efflux pump AcrAB-TolC from Escherichia coli . We found that, once assembled, the intermembrane AcrAB-TolC complex is stable during the separation of the inner and outer membranes and subsequent purification . All three components of the complex co-purify when the affinity tag is attached to either of the proteins suggesting bi-partite interactions between AcrA, AcrB, and TolC . We show that antibiotics, the substrates of AcrAB-TolC, stabilize interactions within the complex . However, the formation of the AcrAB-TolC complex does not require an input of energy.

Infect Immun, 2004 Jun, 72(6), 3350 - 8
Suppression of serum antibody responses by pertussis toxin after respiratory tract colonization by Bordetella pertussis and identification of an immunodominant lipoprotein; Carbonetti NH et al.; Pertussis toxin (PT), a virulence factor secreted by Bordetella pertussis, contributes to respiratory tract infection and disease caused by this pathogen . By comparing a wild-type (WT) B . pertussis strain to a mutant strain with an in-frame deletion of the ptx genes encoding PT (DeltaPT), we recently found that the lack of PT confers a significant defect in respiratory tract colonization in mice after intranasal inoculation . In this study, we analyzed serum antibody responses in mice infected with the WT or DeltaPT strain and found that infection with the DeltaPT strain elicited greater responses to several B . pertussis antigens than did infection with the WT, despite the lower colonization level achieved by the DeltaPT strain . The same enhanced antibody response was observed after infection with a strain expressing an enzymatically inactive PT; but this response was not observed after infection with B . pertussis mutant strains lacking filamentous hemagglutinin or adenylate cyclase toxin, nor when purified PT was administered with the DeltaPT inoculum, indicating a specific role for PT activity in this immunosuppressive effect . In particular, there were consistent strong serum antibody responses to one or more low-molecular-weight antigens after infection with the DeltaPT strain . These antigens were Bvg independent, membrane localized, and also expressed by the closely related pathogens Bordetella parapertussis and Bordetella bronchiseptica . Two-dimensional gel electrophoresis and mass spectrometry were used to identify one of the immunodominant low-molecular-weight antigens as a protein with significant sequence homology to peptidoglycan-associated lipoprotein in several other gram-negative bacterial species . However, a serum antibody response to this protein alone did not protect mice against respiratory tract infection by B . pertussis.

Infect Immun, 2004 Jun, 72(6), 3260 - 6
Lipopolysaccharide protects primary B lymphocytes from apoptosis by preventing mitochondrial dysfunction and bax translocation to mitochondria; Souvannavong V et al.; Mature B lymphocytes undergo apoptosis when they are cultured in the absence of survival factors . Gram-negative bacterial lipopolysaccharide (LPS) prevents this spontaneous apoptosis . This study aimed to better define the signaling pathway(s) involved in the antiapoptotic activity of this endotoxin . We report here that, in addition to its effects on spontaneous apoptosis, LPS protects B cells from apoptosis induced by the broad-spectrum protein kinase inhibitor staurosporine . LPS increased cell viability and concomitantly maintained the mitochondrial transmembrane potential (DeltaPsim) and high glutathione levels . Moreover, LPS inhibited cytosolic cytochrome c release and decreased caspase-9 activation . Unlike staurosporine, LPS induced the retention of Bax, a proapoptotic protein of the Bcl-2 family, in the cytosol by preventing its translocation to mitochondria . These results suggest that Bax relocalization from the cytosol to the mitochondria is an important step of mature B-cell apoptosis and that the antiapoptotic activity of LPS occurs upstream of mitochondrial events.

Infect Immun, 2004 Jun, 72(6), 3252 - 9
Therapeutic vaccination against Helicobacter pylori in the beagle dog experimental model: safety, immunogenicity, and efficacy; Rossi G et al.; Helicobacter pylori is a gram-negative bacterium that colonizes the human gastric mucosa causing gastritis and peptic ulcer and increasing the risk of gastric cancer . The efficacy of current antibiotic-based therapies can be limited by problems of patient compliance and increasing antibiotic resistance; the vaccine approach can overcome these limits . The present study describes the therapeutic vaccination of experimentally H . pylori-infected beagle dogs, an animal model that reproduces several aspects of the human infection with H . pylori . The vaccine consisted of three recombinant H . pylori antigens, CagA, VacA, and NAP, formulated at different doses (10, 25, or 50 microg each) with alum and administered intramuscularly either weekly or monthly . No adverse effects were observed after vaccination and a good immunoglobulin G response was generated against each of the three antigens . Bacterial colonization and gastritis were decreased after the completion of the vaccination cycle, especially in the case of the monthly immunization schedule . In conclusion, therapeutic vaccination in the beagle dog model was safe and immunogenic and was able to limit H . pylori colonization and the related gastric pathology.

J Mol Microbiol Biotechnol, 2003, 6(3-4), 164 - 73
Analysis of fruE, a novel developmental gene of Myxococcus xanthus; Akiyama T et al.; Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation . In the present study, a TnV insertion developmental mutation, Omega773, of M . xanthus was analyzed . The TnV Omega773 insertion was found to be located within a novel developmental gene, fruE . The FruE protein is composed of 140 amino acid residues and bears an N-terminal signal peptide . The amino acid sequence of FruE shared no significant similarity with any other known protein in the databases . The fruE mutant displayed a development-delayed phenotype . The formation of tightly aggregated mounds in the fruE mutant was slower than that in the wild-type strain . The initiation of spore production in the fruE mutant was delayed by 12 h in comparison to the wild-type strain, and the process of spore formation was more asynchronous than that of the wild-type strain . The transcription initiation sites of the fruE gene were located 81 bp (P1) and 57 bp (P2) upstream of the fruE initiation codon . Although both promoters were active during vegetative growth and development, the P1 promoter was more active during development and the P2 promoter was more active during vegetative growth . The expression of the fruE gene increased to a peak at 6 h poststarvation and then decreased . The decrease in fruE expression was not observed in the D and E signal mutants .

J Comput Aided Mol Des, 2004 Jan, 18(1), 1 - 11
Theoretical study of selective methylation in the synthesis of azithromycin; Duran D et al.; Azithromycin is a 15-membered macrolide antibiotic which is active in vitro against clinically important gram-negative bacteria . In this study, the selectivity of the methylation mechanism was analyzed computationally on the 2'-OCbz-3'-NMeCbz derivative of azithromycin in vacuum and in DMF . We have shown that the methylation of the hydroxy group on C-6 is energetically unfavorable compared to the other hydroxy groups in vacuum; the softness values further showed that the C-6 anion is not reactive towards CH3I in the methylation mechanism . To understand the effect of the solvent on the methylation process, detailed molecular dynamics simulations were performed in DMF using the anions at the C-4", C-6, C-11 and C-12 positions . We find the conformations of the anions not to be affected by the presence of the solvent . The radial distribution functions of the solvent molecules around the O- of the anions demonstrate that DMF molecules cluster around the C-6 anion . The relative strength of the anion-solvent interactions reveal that the solvent molecules provide the largest stabilization to the C-6 anion and prevent the methylation at this position . The latter descriptor was found to be an important factor in explaining the experimentally observed selectivity towards the methylation of the C-4", C-6, C-11 and C-12 anions.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 877 - 83
Prevotella shahii sp . nov . and Prevotella salivae sp . nov., isolated from the human oral cavity; Sakamoto M et al.; Two bacterial strains, EHS11(T) and EPSA11(T), which were isolated from the human oral cavity, were characterized in terms of phenotypic and biochemical characteristics, cellular fatty acid profiles and phylogenetic position based on 16S rRNA gene sequence analysis . 16S rRNA gene sequence analysis showed that each of the isolates belonged to a novel species of the genus Prevotella . Strain EHS11(T) was related to Prevotella loescheii (about 95 % similarity), whereas strain EPSA11(T) was related to Prevotella oris (about 94 % similarity) . Both strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods . The cellular fatty acid composition of strain EPSA11(T) was very similar to that of P . oris JCM 8540(T) . On the other hand, the cellular fatty acid composition of strain EHS11(T) was significantly different from those of other Prevotella species . The predominant fatty acids in strain EHS11(T) are C(18 : 1)omega9c, C(16 : 0) and C(16 : 0) 3-OH, whereas other Prevotella species, except for P . loescheii JCM 8530(T), possess anteiso-C(15 : 0), iso-C(17 : 0) 3-OH and C(18 : 1)omega9c . The predominant fatty acids in P . loescheii JCM 8530(T) are anteiso-C(15 : 0), C(16 : 0) and C(18 : 1)omega9c . DNA-DNA hybridization experiments revealed a genomic distinction of strains EHS11(T) and EPSA11(T) from P . loescheii JCM 8530(T) and P . oris JCM 8540(T) . On the basis of these data, two novel Prevotella species are proposed: Prevotella shahii sp . nov . and Prevotella salivae sp . nov . The type strains of P . shahii and P . salivae are EHS11(T) (=JCM 12083(T)=DSM 15611(T)) and EPSA11(T) (=JCM 12084(T)=DSM 15606(T)), respectively.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 847 - 50
Pseudomonas lutea sp . nov., a novel phosphate-solubilizing bacterium isolated from the rhizosphere of grasses; Peix A et al.; A phosphate-solubilizing bacterial strain designated OK2(T) was isolated from rhizospheric soil of grasses growing spontaneously in a soil from Spain . Cells of the strain were Gram-negative, strictly aerobic, rod-shaped and motile . Phylogenetic analysis of the 16S rRNA gene indicated that this bacterium belongs to the gamma-subclass of Proteobacteria within the genus Pseudomonas and that the closest related species is Pseudomonas graminis . The strain produced catalase but not oxidase . Cellulose, casein, starch, gelatin and urea were not hydrolysed . Aesculin was hydrolysed . Growth was observed with many carbohydrates as carbon sources . The main non-polar fatty acids detected were hexadecenoic acid (16 : 1), hexadecanoic acid (16 : 0) and octadecenoic acid (18 : 1) . The hydroxy fatty acids detected were 3-hydroxydecanoic acid (3-OH 10 : 0), 3-hydroxydodecanoic acid (3-OH 12 : 0) and 2-hydroxydodecanoic acid (2-OH 12 : 0) . The G+C DNA content determined was 59.3 mol% . DNA-DNA hybridization showed 48.7 % relatedness between strain OK2(T) and P . graminis DSM 11363(T) and 26.2 % with respect to Pseudomonas rhizosphaerae LMG 21640(T) . Therefore, these results indicate that strain OK2(T) (=LMG 21974(T)=CECT 5822(T)) belongs to a novel species of the genus Pseudomonas, and the name Pseudomonas lutea sp . nov . is proposed.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 699 - 703
Legionella drancourtii sp . nov., a strictly intracellular amoebal pathogen; La Scola B et al.; A Legionella-like amoebal pathogen (LLAP), formerly named LLAP12(T), was characterized on the basis of microscopic appearance, staining characteristics, growth in Acanthamoeba polyphaga at different temperatures, DNA G+C content, serological cross-reactivity and 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analysis . LLAP12(T) was found to be a motile, Gram-negative bacterium that grew within cytoplasmic vacuoles in infected amoebae . The infecting bacteria induced lysis of their amoebal hosts and time taken to do so was dependent on incubation temperature . Recovery of LLAP12(T) from amoebae onto axenic media could not be achieved . Phylogenetic analysis of LLAP12(T), based on 16S rRNA and mip gene sequence analysis, indicated that it lay within the radiation of the Legionellaceae and that it clustered specifically with Legionella lytica and Legionella rowbothamii . The divergence observed between LLAP12(T) and these two species was of a degree equal to, or greater than, that observed between other members of the family . In support of this delineation, LLAP12(T) was found not to cross-react serologically with any other Legionella species . The mip and 16S rRNA gene sequence-based analyses also indicated that LLAP12(T) was related very closely to two other previously identified LLAP isolates, LLAP4 and LLAP11 . Taken together, these results support the proposal of LLAP12(T) as the type strain of Legionella drancourtii sp . nov.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 693 - 7
Agarivorans albus gen . nov., sp . nov., a gamma-proteobacterium isolated from marine animals; Kurahashi M et al.; Six bacterial strains were isolated from healthy marine organisms that were collected from the coast of the Kanto area in Japan . Phylogenetic analysis based on 16S rDNA sequence similarity showed that the six isolates formed a separate cluster in the gamma-Proteobacteria and were related to the genera Alteromonas and Glaciecola (<91.6 % similarity) . The isolates were related closely to each other (DNA-DNA reassociation values of 74-93 %) . The isolates had a polar flagellum and were Gram-negative, mesophilic, strictly aerobic rods that required salt for growth . Distinct phenotypic features of this group included the ability to hydrolyse agar and white pigmentation of colonies . The DNA G+C content of the isolates was 48-50 mol% . The major quinone was Q-8 . Phenotypic characteristics of the isolates differed from those of members of the genera Alteromonas and GLACIECOLA: The name Agarivorans albus gen . nov., sp . nov . is proposed for the six isolates; the type strain is MKT 106(T) (=IAM 14998(T)=LMG 21761(T)).

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 669 - 73
Reinekea marinisedimentorum gen . nov., sp . nov., a novel gammaproteobacterium from marine coastal sediments; Romanenko LA et al.; A Gram-negative, oxidase- and catalase-positive, rod-shaped bacterium, designated strain KMM 3655(T), was isolated from a coastal marine sediment sample . The novel bacterium required sodium ions for growth and grew between 0.5 and 5 % NaCl and at 4-37 degrees C, but not at 40 degrees C . It reduced nitrate, formed acids from glucose under aerobic and anaerobic conditions, utilized a limited spectrum of organic substrates and did not produce gelatinase, caseinase, amylase or chitinase . The major isoprenoid quinone was Q8 . Polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unknown phospholipid . Fatty acid analysis of strain KMM 3655(T) revealed C(16 : 0), C(16 : 1)omega7c and C(18 : 1)omega7c as predominant components . The G+C content of the DNA was 51.1 mol% . Phylogenetic analysis of the 16S rDNA sequence placed the new isolate within the gamma-Proteobacteria as a separate deep branch, with about 90 % sequence similarity to representatives of the genus Oceanospirillum and other remotely related genera . Combined phylogenetic and physiological data show that the new marine sediment isolate, KMM 3655(T), represents a novel genus and species, for which the name Reinekea marinisedimentorum gen . nov., sp . nov . is proposed . The type strain is KMM 3655(T) (=DSM 15388(T)).

Environ Microbiol, 2004 Jun, 6(6), 611 - 21
Lentisphaera araneosa gen . nov., sp . nov, a transparent exopolymer producing marine bacterium, and the description of a novel bacterial phylum, Lentisphaerae; Cho JC et al.; Two phylogenetically distinct marine strains producing transparent exopolymers (TEP), designated HTCC2155(T) and HTCC2160, were cultivated from Oregon coast seawater by dilution to extinction in a high throughput culturing format . When cultured in low-nutrient seawater media, these strains copiously produced Alcian Blue-stainable viscous TEP . Growing cells were attached to each other by the TEP in a three dimensional network . Polymerase chain reaction employing 16S rDNA primers specific for the novel isolates indicated that they are indigenous to the water column of the Atlantic and Pacific oceans . The abundance of the isolates as determined by 16S rRNA dot blots, however, indicated that they are less than 1% of the total bacterial community . In phylogenetic analyses, the strains consistently formed a new phylum-level lineage within the domain Bacteria, together with members of the candidate phylum VadinBE97, which consists of Victivallis, the first cultured genus in the candidate phylum, and 16S rRNA gene clones from DNA extracted from marine or anaerobic terrestrial habitats . Five putative subgroups were delineated within this phylum-level lineage, including a marine group and an anaerobic group . The isolates are Gram negative, strictly aerobic, chemoheterotrophic, and facultatively oligotrophic sphere-shaped bacteria . The DNA G+C content of strain HTCC2155(T) was 48.3 mol% and the genome size was 2.9 mb . It is proposed from these observations that the strains be placed into a new genus and a new species named Lentisphaera araneosa (type strain HTCC2155(T) = ATCC BAA-859(T) = KCTC 12141(T)) gen . nov., sp . nov., the cultured marine representative of the Lentisphaerae phyl . nov., and the phylum be divided into two novel orders named the Lentisphaerales ord . nov . and the Victivallales ord . nov.

Mol Plant Microbe Interact, 2004 May, 17(5), 447 - 55
The HopPtoF locus of Pseudomonas syringae pv . tomato DC3000 encodes a type III chaperone and a cognate effector; Shan L et al.; Type III secretion systems are highly conserved among gram-negative plant and animal pathogenic bacteria . Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells . Analysis of the whole genome sequence of Pseudomonas syringae pv . tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P . syringae pv . phaseolicola . The HopPtoF locus harbors two genes, ShcF(Pto) and HopF(Pto), that are preceded by a single hrp box promoter . We present evidence here to show that ShcF(Pto) and HopF(Pto) encode a type III chaperone and a cognate effector, respectively . ShcF(Pto) interacts with and stabilizes the HopF(Pto) protein in the bacterial cell . Translation of HopF(Pto) starts at a rare initiation codon ATA that limits the synthesis of the HopF(Pto) protein to a low level in bacterial cells.

Nucleic Acids Res, 2004 May 11, 32(8), 2566 - 77 Print 2004.
Predicting transmembrane beta-barrels in proteomes; Bigelow HR et al.; Very few methods address the problem of predicting beta-barrel membrane proteins directly from sequence . One reason is that only very few high-resolution structures for transmembrane beta-barrel (TMB) proteins have been determined thus far . Here we introduced the design, statistics and results of a novel profile-based hidden Markov model for the prediction and discrimination of TMBs . The method carefully attempts to avoid over-fitting the sparse experimental data . While our model training and scoring procedures were very similar to a recently published work, the architecture and structure-based labelling were significantly different . In particular, we introduced a new definition of beta- hairpin motifs, explicit state modelling of transmembrane strands, and a log-odds whole-protein discrimination score . The resulting method reached an overall four-state (up-, down-strand, periplasmic-, outer-loop) accuracy as high as 86% . Furthermore, accurately discriminated TMB from non-TMB proteins (45% coverage at 100% accuracy) . This high precision enabled the application to 72 entirely sequenced Gram-negative bacteria . We found over 164 previously uncharacterized TMB proteins at high confidence . Database searches did not implicate any of these proteins with membranes . We challenge that the vast majority of our 164 predictions will eventually be verified experimentally . All proteome predictions and the PROFtmb prediction method are available at services/PROFtmb/.

Cell, 2004 May 14, 117(4), 483 - 94
Crystal structure of the DegS stress sensor: How a PDZ domain recognizes misfolded protein and activates a protease; Wilken C et al.; Gram-negative bacteria respond to misfolded proteins in the cell envelope with the sigmaE-driven expression of periplasmic proteases/chaperones . Activation of sigmaE is controlled by a proteolytic cascade that is initiated by the DegS protease . DegS senses misfolded protein in the periplasm, undergoes autoactivation, and cleaves the antisigma factor RseA . Here, we present the crystal structures of three distinct states of DegS from E . coli . DegS alone exists in a catalytically inactive form . Binding of stress-signaling peptides to its PDZ domain induces a series of conformational changes that activates protease function . Backsoaking of crystals containing the DegS-activator complex revealed the presence of an active/inactive hybrid structure and demonstrated the reversibility of activation . Taken together, the structural data illustrate in molecular detail how DegS acts as a periplasmic stress sensor . Our results suggest a novel regulatory role for PDZ domains and unveil a novel mechanism of reversible protease activation.

J Am Chem Soc, 2004 May 19, 126(19), 6005 - 16
Spectroscopic description of the two nitrosyl-iron complexes responsible for fur inhibition by nitric oxide; D'Autreaux B et al.; Ferric uptake regulation protein (Fur) is a global regulator, ubiquitous in Gram negative bacteria, that acts as a transcriptional repressor when it binds ferrous ion . Fur is involved in responses to several types of stress related to iron metabolism, such as stress induced by nitric oxide (NO) generated by macrophages against bacterial invasion . NO was recently shown to react with Fe(2+) ions in FeFur (iron substituted Fur protein) leading to an Fur bound iron-nitrosyl complex, unable to bind DNA, and characterized by a g = 2.03 EPR signal, associated with an S = (1)/(2) ground state . This electronic configuration could arise from either a mononitrosyl-iron {Fe(NO)}(7) or a dinitrosyl-iron {Fe(NO)(2)}(9) complex . The use of several spectroscopic tools such as EPR, ENDOR, FTIR, Mossbauer, and UV-visible spectroscopies as well as mass spectrometry analysis was necessary to characterize the iron-nitrosyl species in Fur . Furthermore, changes of C132 and C137 into glycines by site directed mutagenesis reveal that neither of the two cysteines is required for the formation of the g = 2.03 signal . Altogether, we found that two species are responsible for Fur inhibition in NO stress conditions: the major species, S(1/2), is an {Fe(NO)(2)}(9) (S = (1)/(2)) complex without bound thiolate and the minor species is probably a diamagnetic {Fe(NO)(2)}(8) (S = 0) complex . This is the first characterization of these physiologically relevant species potentially linking iron metabolism and the response to NO toxicity in bacteria.

FEMS Microbiol Lett, 2004 May 15, 234(2), 333 - 41
NF-kappaB activation suppresses host cell apoptosis during Rickettsia rickettsii infection via regulatory effects on intracellular localization or levels of apoptogenic and anti-apoptotic proteins; Joshi SG et al.; Rickettsia rickettsii, a gram-negative and obligate intracellular bacterium, is the causative agent of Rocky Mountain spotted fever . In human infections, the primary target of R . rickettsii infection is vascular endothelium . Our laboratory has shown that activation of nuclear transcription factor-kappa B (NF-kappaB) during R . rickettsii infection of cultured human endothelial cells protects against apoptosis by preventing the activation of apical caspases-8 and -9, and the effector caspase-3 . To understand upstream signaling mechanisms, we have determined the effect of NF-kappaB blockade on the status of different Bcl-2 (B-cell lymphoma 2) proteins in this study . Quantitative analysis following TUNEL and Hoechst staining confirmed that infection of endothelial cells with R . rickettsii for 6 h in the presence of a specific NF-kappaB inhibitor, MG132, resulted in induction of apoptosis . Infection-induced apoptosis of EC was associated with decreased level of Bid and accumulation of Bad, while cytosolic level of Bax remained relatively unchanged . Further, the cellular levels of apoptosis antagonist Bcl-2 were found to be down-regulated and apoptogenic mitochondrial proteins Smac and cytochrome c were released into cytoplasm . These results implicate an important regulatory role for NF-kappaB in controlling the intracellular levels and/or localization of pro- as well as anti-apoptotic proteins of Bcl-2 family, the intricate balance of which is a critical determinant of downstream signaling mechanisms governing cell fate during intracellular infection .

Comb Chem High Throughput Screen, 2004 May, 7(3), 239 - 49
Anti-endotoxin agents . 1 . Development of a fluorescent probe displacement method optimized for the rapid identification of lipopolysaccharide-binding agents; Wood SJ et al.; Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria . Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient . Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time . We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS, and have shown using animal models of sepsis that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy . Assays reported previously in the literature do not lend themselves well to the rapid screening of large numbers of structurally diverse compounds . In this report, we describe a highly sensitive and robust fluorescent displacement assay using BODIPY TR cadaverine (BC), which binds specifically to the toxic center of LPS, lipid A, and is competitively displaced by compounds displaying an affinity for lipid A . The assay clearly discriminates subtle differences in the binding of polymyxin B, and its nonapeptide derivative, with LPS . The spectral properties of the BODIPY fluorophore are ideally suited for screening diverse structural classes of compounds, including those with conjugated aromatic groups, or with chromophores in the 260-500 nm range . The fluorescent probe: LPS complex is stable under physiologically relevant salt concentrations, resulting in the rapid rejection of spurious binders interacting via non-specific electrostatic interactions, and, therefore, in greatly improved dispersion of ED(50)values.

Biochemistry, 2004 May 18, 43(19), 5811 - 9
Kinetics and mechanism of iron(III) complexation by ferric binding protein: the role of phosphate; Gabricevic M et al.; Iron transport across the periplasmic space to the cytoplasmic membrane of certain Gram-negative bacteria is mediated by a ferric binding protein (Fbp) . This requires Fe(3+) loading of Fbp at the inner leaflet of the outer membrane . A synergistic anion is required for tight Fe(3+) sequestration by Fbp . Although phosphate fills this role in the protein isolated from bacterial cell lysates, nitrilotriacetate anion (NTA) can also satisfy this requirement in vitro . Here, we report the kinetics and mechanism of Fe(3+) loading of Fbp from Fe(NTA)(aq) in the presence of phosphate at pH 6.5 . The reaction proceeds in four kinetically distinguishable steps to produce Fe(3+)Fbp(PO(4)) as a final product . The first three steps exhibit half-lives ranging from ca . 20 ms to 0.5 min, depending on the concentrations, and produce Fe(3+)Fbp(NTA) as an intermediate product of significant stability . The rate for the first step is accelerated with an increasing phosphate concentration, while that of the third step is retarded by phosphate . Conversion of Fe(3+)Fbp(NTA) to Fe(3+)Fbp(PO(4)) in the fourth step is a slow process (half-life approximately 2 h) and is facilitated by free phosphate . A mechanism for the Fe(3+)-loading process is proposed in which the synergistic anions, phosphate and NTA, play key roles . These data suggest that not only is a synergistic anion required for tight Fe(3+) sequestration by Fbp, but also the synergistic anion plays a critical role in the process of inserting Fe(3+) into the Fbp binding site.

Microbiology, 2004 May, 150(Pt 5), 1475 - 83
Molecular and functional characterization of type I signal peptidase from Legionella pneumophila; Lammertyn E et al.; Legionella pneumophila is a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia . Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated . As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) of L . pneumophila was of particular interest . This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane . This paper reports the cloning and the transcriptional analysis of the L . pneumophila lepB gene encoding the type I signal peptidase (SPase) . Reverse transcription PCR experiments showed clear lepB expression when L . pneumophila was grown both in culture medium, and also intracellularly in Acanthamoeba castellanii, a natural eukaryotic host of L . pneumophila . In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e . a LepA homologue and a ribonuclease III homologue . SPase activity of the LepB protein was demonstrated by in vivo complementation analysis in a temperature-sensitive Escherichia coli lepB mutant . Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens . Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition . Finally it was shown by in vivo experiments that L . pneumophila LepB is a target for (5S,6S)-6-{(R)-acetoxyethyl}-penem-3-carboxylate, a specific inhibitor of type I SPases.

Biomacromolecules, 2004 May-Jun, 5(3), 903 - 13
Synthesis, characterization, and bioavailability of mannosylated shell cross-linked nanoparticles; Joralemon MJ et al.; Saccharide-functionalized shell cross-linked (SCK) polymer micelles designed as polyvalent nanoscaffolds for selective interactions with receptors on Gram negative bacteria were constructed from mixed micelles composed of poly(acrylic acid-b-methyl acrylate) and mannosylated poly(acrylic acid-b-methyl acrylate) . The mannose unit was conjugated to the hydrophilic chain terminus of the amphiphilic diblock copolymer precursor, from which the SCK nanoparticles were derived, by the growth of the diblock copolymer from a mannoside functionalized atom transfer radical polymerization (ATRP) initiator . Mixed micelle formation between the amphiphilic diblock copolymer and mannosylated amphiphilic diblock copolymer was followed by condensation-based cross-linking between the acrylic acid residues present in the periphery of the polymer micelles to afford SCK nanoparticles . SCKs presenting variable numbers of mannose functionalities were prepared from mixed micelles of controlled stoichiometric ratios of mannosylated and nonmannosylated diblock copolymers . The polymer micelles and SCKs were characterized by dynamic light scattering (DLS), electrophoretic light scattering, atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU) . Surface availability and bioactivity of the mannose units were evaluated by interactions of the nanostructures with the model lectin Concanavalin A via DLS studies, with red blood cells (rabbit) via agglutination inhibition assays and with bacterial cells (E . coli) via TEM imaging.

Appl Environ Microbiol, 2004 May, 70(5), 2741 - 7
Dissimilatory arsenate reduction with sulfide as electron donor: experiments with mono lake water and Isolation of strain MLMS-1, a chemoautotrophic arsenate respirer; Hoeft SE et al.; Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors . Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction . We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water . Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide . No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide . The rate of arsenate reduction in lake water was dependent on the amount of available arsenate . We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution . The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite . Chemoautotrophy was confirmed by the incorporation of H(14)CO(3)(-) into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products . Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the delta-Proteobacteria, located near sulfate reducers like Desulfobulbus sp . (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake . However, strain MLMS-1 does not grow with sulfate as its electron acceptor.

J Biomed Mater Res, 2004 Jun 1, 69A(3), 561 - 6
Titanium implants enhance pulmonary nitric oxide production and lung injury in rats exposed to endotoxin; Yang RS et al.; An increase in levels of elemental Ti in the blood and lung of rats with a Ti alloy implant has been demonstrated . However, the pathophysiological role of the elevated elemental Ti level in the circulation remains unclear . Rats were implanted with Ti alloy discs for 4 weeks . The levels of elemental Ti in the blood and lung were especially increased compared with other tissues . The Ti alloy implant enhanced lung injury related to endotoxin from Gram-negative bacteria (lipopolysaccharide, LPS), which was characterized by lung edema and other histological changes such as recruitment of neutrophils, interstitial edema, and alveolar hemorrhage in the lung . In the presence of endotoxin, an increase of nitrite production was shown in the plasma and bronchoalveolar lavage fluid of rats implanted with a Ti alloy . Moreover, the Ti alloy implant further enhanced the induction of inducible nitric oxide (NO) synthase (iNOS) protein expression induced by LPS in the lung . These endotoxin-related responses in the presence or absence of the Ti alloy implant could be inhibited by aminoguanidine (an iNOS inhibitor) . These results provide the first experimental evidence that circulating Ti released from Ti alloy implants has an ability to affect pulmonary iNOS protein expression, and enhance the pathogenesis of acute lung injury during endotoxemia .

Adv Dent Res, 2003 Dec, 17, 95 - 9
Genetic and molecular characterization of a dental pathogen using genome-wide approaches; Actis LA et al.; Actinobacillus actinomycetemcomitans causes periodontitis, a costly chronic infection that affects a large number of patients . The pathogenesis of this dental infection is a multifactorial process that results in a serious degenerative disease of the periodontium . Although significant progress has been achieved after the identification of this Gram-negative bacterium as the etiological agent of this infection, much remains to be done to understand in detail the bacterial factors and host-pathogen interactions involved in the pathogenesis of this disease . Classic research approaches have resulted in the identification of important virulence factors and cellular processes, although they have provided a rather narrow picture of some of the steps of this complex process . In contrast, a much wider picture could be obtained with the application of tools such as bioinformatics and genomics . These tools will provide global information regarding the differential expression of genes encoding factors and processes that lead to the pathogenesis of this disease . Furthermore, comparative genomics has the potential of helping us to understand the emergence and evolution of this human pathogen . This genome-wide approach should provide a more complete picture of the pathogenesis process of this disease, and will facilitate the development of efficient diagnostic, preventive, and therapeutic measures for this disease.

J Endotoxin Res, 2004, 10(2), 97 - 106
rBPI(10-193) is secreted by CHO cells and retains the activity of rBPI21; Horwitz AH et al.; rBPI23, a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills Gram-negative bacteria and neutralizes endotoxin . rBPI21, a variant in which cysteine 132 is changed to alanine, retains the activities of rBPI23 . Analysis of certain purified rBPI21 preparations revealed that some of the molecules had lost nine amino acids from the amino terminus . To explore the effect of this modification on structure and activity, we cloned and expressed a variant of rBPI21, designated rBPI(10-193), which lacks the first nine amino acids . A monoclonal antibody believed to recognize the amino terminus of rBPI21 cross-reacted with rBPI21, but not with rBPI(10-193) or full length recombinant BPI (rBPI) . These results demonstrated that the antibody recognizes the first nine amino acids of rBPI21 and that this region of the holoprotein (rBPI) is inaccessible to the antibody (as suggested by the known 3-D structure) . Purified rBPI(10-193) and rBPI21 were similarly potent in in vitro assays measuring bactericidal, endotoxin binding and neutralization activities . In a mouse model of lethal bacteremia, rBPI(10-193) and rBPI21 were similarly potent whereas in a mouse endotoxin challenge model, rBPI(10-193) appeared to be at least 2-fold more potent than rBPI21 . In conscious rats, a rapid bolus dose of 40 mg/kg of rBPI21 caused a significant transient decrease in blood pressure while the same dose of rBPI(10-193) caused no blood pressure decrease . We conclude from these studies that the first nine amino acids of rBPI21 are not essential for the anti-infective and endotoxin-neutralizing activities of BPI.

J Endotoxin Res, 2004, 10(2), 85 - 95
A new method for removing endotoxin from plasma using hemocompatible affinity chromatography technology, applicable for extracorporeal treatment of septic patients; Amoureux MC et al.; The pathogenesis of sepsis begins with the proliferation of micro-organisms at a site of infection, followed by invasion of the bloodstream and other organs . Gram-negative bacteria account for a large part of sepsis cases . The structural component of Gram-negative bacteria, endotoxin or lipopolysaccharide (LPS), induces the synthesis and release of endogenous mediators of sepsis . A growing number of investigations of the molecular mechanisms occurring in sepsis, point to endotoxin as a central mediator leading to multi-organ failure and death . In numerous clinical trials, attempts to target molecules downstream of endotoxin have been made, but have not been associated with improved survival . We describe an affinity-based system for the selective removal of endotoxin from plasma . The small-scale device, a 1.5 ml cartridge, contains beads that bind endotoxin with high specificity and efficiency . In addition, evidence is presented that this device does not affect plasma hemostasis, nor does it activate the complement system . Taken together, these results represent a proof of principle for endotoxin removal from plasma, which may be of clinical value to treat sepsis by extracorporeal circulation of the blood through a scaled-up version of this endotoxin-removing device.

Curr Issues Mol Biol, 2004 Jul, 6(2), 111 - 24
Type V protein secretion: simplicity gone awry?
Desvaux M, Parham NJ, Henderson IR.
Since its discovery in the late 1980's, the family of secreted proteins termed the autotransporters has been expanding continuously to become the largest group of secreted proteins in Gram-negative bacteria . The type V secretion pathway, which includes the autotransporters (type Va) together with the two-partner secretion system (type Vb) and the Oca family (type Vc), can be defined by secreted proteins that are (i) translocated across the outer membrane via a transmembrane pore formed by a beta-barrel and (ii) contain all the information required for translocation through the cell envelope . In the light of new discoveries and controversies in this research field, the secretion process of autotransporters, or the type Va secretion system, will be discussed here and placed in the context of the more general field of bacterial protein translocation.

Eur J Immunol, 2004 May, 34(5), 1441 - 50
Inhibition of hepatic transcriptional induction of lipopolysaccharide-binding protein by transforming-growth-factor beta 1; Hallatschek W et al.; LPS-binding protein (LBP) is an acute-phase protein with the ability to bind and transfer LPS of Gram-negative bacteria, as well as cell wall compounds of other pathogenic bacteria . This soluble pattern-recognition molecule is present in high concentrations in serum and represents an important defense mechanism of the host . Regulation of the hepatic acute-phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host organism . We show here that TGF-beta 1, in a dose-dependent fashion, is able to inhibit LBP transcript accumulation and LBP protein synthesis induced by IL-6, IL-1 beta and dexamethasone in hepatoma cell lines . These data were confirmed employing primary human hepatocytes, where TGF-beta 1 also inhibited LBP protein synthesis . We identified and analyzed several Smad-binding sites (Smads are major regulatory elements of TGF-beta 1) within the LBP promoter, and found that one of them was active . We furthermore identified an AP-1-binding site clearly conferring inhibitory effects of TGF-beta 1 towards LBP promoter activity, shown by gel shift and promoter mutagenesis experiments . Further elucidating the mechanism of transcriptional regulation of proteins involved in innate immune responses may potentially help to develop novel intervention strategies for the acute-phase response, sepsis, and septic shock.

FEMS Microbiol Lett, 2004 May 1, 234(1), 81 - 6
Actinobacillus pleuropneumoniae metalloprotease: cloning and in vivo expression; Garcia Gonzalez O et al.; The complete amino acid and nucleotide sequence of a secreted metalloprotease produced by Actinobacillus pleuropneumoniae serotype 1 is reported . A clone showing proteolytic activity in cell-free culture media was selected from a genomic library of A . pleuropneumoniae serotype 1 in pUC 19 . The sequence obtained contained an open reading frame encoding a protein with 869 amino acids . This protein was identified as a zinc neutral-metalloprotease belonging to the aminopeptidase family, with a predicted molecular weight of approximately 101 kDa . This sequence showed high homology with other predicted or sequenced aminopeptidases reported for different Gram-negative bacteria . Expression of the protease was observed in lung tissue from pigs that died of porcine pleuropneumonia suggesting a role in pathogenesis.

OMICS, 2004 Spring, 8(1), 43 - 55
Genomic insights into gene regulation of Desulfovibrio vulgaris Hildenborough; Hemme CL et al.; Traditional laboratory studies of the sulfate-reducing bacteria have focused primarily on the biochemistry of the organisms . As genomic sequences of sulfate-reducing species have become available, insights have been gained into the metabolic and regulatory networks of these organisms . A computational analysis is reported of the transcriptional regulatory networks of Desulfovibrio vulgaris Hildenborough, the first mesophilic gram-negative sulfate-reducing bacterium for which a genome sequence is available . A set of conserved DNA motifs were derived from libraries of potential promoter regions of putative D . vulgaris regulons with the AlignACE program suite . Although one motif showed similarity to the Escherichia coli GlpR binding site, most of the motifs returned were apparently unique . A number of expected orthologs for regulatory proteins have not yet been recognized in D . vulgaris.

Trends Immunol, 2004 Feb, 25(2), 53 - 5
Meet the relatives: a family of BPI- and LBP-related proteins; Bingle CD et al.; Until recently, two key members of the innate immune response to Gram negative bacteria, bactericidal permeability-increasing protein (BPI) and lipopolysaccharide (LPS)-binding protein, have been considered to be members of a small family of lipid-binding proteins that also contains cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) . A recent paper has characterised three related proteins that are expressed in the mouth, nose and upper airways . Taken together with other recent data, it is clear that a large family of such proteins exists and these additional members might also function in the innate immune response.

Biotechnol Lett, 2004 Mar, 26(5), 379 - 83
Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate; Morono Y et al.; Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell . However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell . Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells . We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining . We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

J Mass Spectrom, 2004 Apr, 39(4), 378 - 83
Determination of fatty acid positions in native lipid A by positive and negative electrospray ionization mass spectrometry; Sforza S et al.; Lipid A is the endotoxic principle of the lipopolysaccharide fraction from Gram-negative bacteria . It is involved in the elicitation of cytokine production that leads to massive inflammation and to septic shock as a lethal consequence . For this reason, the structural elucidation of lipopolysaccharides from toxic Gram-negative bacteria is an important and complicated task, mainly owing to its natural heterogeneity . Here, a new methodology to infer the distribution of the primary and secondary acyl residues is described, based on electrospray ionization mass spectrometry (ESI-MS) of intact lipid A under high cone voltage in order to achieve in-source fragmentation . Under these conditions, acyl fragmentation is induced and a different regioselective cleavage of secondary fatty acids is observed in positive and negative ESI-MS, allowing the rapid identification of the lipid A structure .

Infect Immun, 2004 May, 72(5), 3042 - 7
Presence of pili on the surface of Francisella tularensis; Gil H et al.; Francisella tularensis is a highly infectious gram-negative bacterium with potential for use as a bioweapon . Analysis of the F . tularensis live vaccine strain (LVS) ultrastructure by electron microscopy revealed the presence of long, thin fibers, similar in appearance to type 4 pili . The highly virulent F . tularensis Schu S4 strain was found to contain type 4 pilus genes, and we confirmed that these genes are present and expressed in the LVS.

Infect Immun, 2004 May, 72(5), 2837 - 42
pagP is required for resistance to antibody-mediated complement lysis during Bordetella bronchiseptica respiratory infection; Pilione MR et al.; To efficiently colonize and persist in the lower respiratory tract, bacteria must survive multiple host immune mechanisms . Bordetella bronchiseptica is a gram-negative respiratory pathogen that naturally infects mice and persists in the lower respiratory tract for up to 49 days postinoculation . In this work, we examined the effect of mutation of the pagP gene on the persistence of B . bronchiseptica in the lower respiratory tract of mice . The pagP gene encodes a palmitoyl transferase that is responsible for the addition of a palmitoyl group to the lipid A region of B . bronchiseptica lipopolysaccharide . Data presented here confirm that a B . bronchiseptica deltapagP mutant demonstrates defective persistence in the lower respiratory tract of wild-type mice . We hypothesized that the defective persistence of the B . bronchiseptica deltapagP mutant was due to an increased susceptibility of this mutant to a host immune response . In vivo data indicate that both B cells and the complement component C3 are required for the reduced bacterial numbers of the deltapagP mutant on day 14 postinoculation . In addition, an in vitro complement killing assay demonstrated that B . bronchiseptica exhibits pagP-dependent resistance to antibody-mediated complement killing at low concentrations of immune serum . Taken together, these results suggest that pagP is required for B . bronchiseptica to resist antibody-mediated complement lysis during respiratory infection.

Biol Chem, 2004 Feb, 385(2), 137 - 43
The periplasmic E . coli chaperone Skp is a trimer in solution: biophysical and preliminary crystallographic characterization; Schlapschy M et al.; The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria . Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins . Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution . Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions . Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found . A first data set was collected to 2.60 A resolution.

Hokkaido Igaku Zasshi, 2004 Mar, 79(2), 111 - 5
{What is Helicobacter pylori?: associated diseases in human}; Sugiyama T et al.; Helicobacter pylori (H . pylori) is a spiral gram-negative bacterium, characterized by positive urease, catalase and oxidase activity . The complete resolution of the full genome has elucidated the biological characteristics, the pathogenicity, and the bacterial evolution . The infection is acquired in childhood by oral-oral transmission in developed countries, probably an intra-familiar transmission . The infection has an association with chronic histological gastritis, peptic ulcer, gastric cancer and MALT lymphoma in the stomach . All patients with H . pylori infection have histological gastritis and most of them remain asymptomatic for life . Only a minority of the infected individuals occurs ulceration or gastric cancer . Cure of the infection prevents recurrence of peptic ulcer without persistent treatments against ulceration . Concerning gastric cancer, WHO concluded in 1994 that H . pylori is a definite carcinogen in humans on the basis of epidemiological studies . Evidences that the infection leads to the development of gastric cancer have been accumulated in animal models as well as in vitro experimental studies . Such clinical diversities are caused by variations of H . pylori pathogenicity, host susceptibility, environmental factors including foods and those interactions . At present, an eradication treatment of H . pylori, combined with a proton-pump inhibitor and two antibiotics (clarithromycin and amoxicillin) is restrictedly approved in the patients with gastric or duodenal ulcer by Japanese health care system.

Genet Mol Res, 2004 Mar 31, 3(1), 181 - 94
Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification; Carepo MS et al.; Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems . The complete genome sequence of C . violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use . We examined the biotechnological potential of C . violaceum for environmental detoxification . Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail . Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins.

Genet Mol Res, 2004 Mar 31, 3(1), 148 - 61
Chromobacterium violaceum genome: molecular mechanisms associated with pathogenicity; Brito CF et al.; Chromobacterium violaceum is a versatile, Gram-negative beta-protebacterium that grows in a variety of ecosystems in tropical and subtropical areas, such as the water and borders of the Negro River, in the Amazon region of Brazil . Although it is a saprophyte and is generally considered non-pathogenic, sporadic cases of human infection have been described, mainly in young children and in immunodeficient individuals . Although rare, infections with C . violaceum are characterized by rapid dissemination and high mortality . With the complete genome sequence of C . violaceum now available, a detailed description of the molecular arsenal required for this bacterium's remarkable versatility has been revealed . Most importantly, a more detailed picture of its biotechnological properties, including the characteristic violacein pigment, has emerged . The complete genome sequence also enabled us to make a thorough examination of the repertoire of genes encoding probable virulence factors, which determine the potential for pathogenesis . We described a number of genes involved in infectious processes, such as host cell adhesion, "contact-dependent secretion" of factors that promote cell invasion, as well as other virulence factors, such as cytolytic proteins . We also described genes involved with the synthesis of lipopolysaccharides and proteoglycan, known to elicit the synthesis of pro-inflammatory cytokines and involved in the detoxification process, which may contribute to the evasion of the bacteria from the host immune response.

Genet Mol Res, 2004 Mar 31, 3(1), 92 - 101
Chemotaxis and flagellar genes of Chromobacterium violaceum; Pereira M et al.; The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism . This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C . violaceum . C . violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome . We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C . violaceum . A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins . The chemoreceptors found in C . violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C . violaceum genome . Also, the C . violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system . Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C . violaceum genome.

Protein Sci, 2004 May, 13(5), 1402 - 6
Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions; Yu CS et al.; Gram-negative bacteria have five major subcellular localization sites: the cytoplasm, the periplasm, the inner membrane, the outer membrane, and the extracellular space . The subcellular location of a protein can provide valuable information about its function . With the rapid increase of sequenced genomic data, the need for an automated and accurate tool to predict subcellular localization becomes increasingly important . We present an approach to predict subcellular localization for Gram-negative bacteria . This method uses the support vector machines trained by multiple feature vectors based on n-peptide compositions . For a standard data set comprising 1443 proteins, the overall prediction accuracy reaches 89%, which, to the best of our knowledge, is the highest prediction rate ever reported . Our prediction is 14% higher than that of the recently developed multimodular PSORT-B . Because of its simplicity, this approach can be easily extended to other organisms and should be a useful tool for the high-throughput and large-scale analysis of proteomic and genomic data.

Curr Opin Struct Biol, 2004 Apr, 14(2), 161 - 70
Structural biology of bacterial pathogenesis; Remaut H et al.; Recent years have seen a rapid increase in structural information on proteins implicated in bacterial pathogenesis . The different modes by which bacteria establish contact with their host tissues are exemplified by the structures of bacterial adhesins in complex with their cognate host receptor . A more detailed structural understanding of the various Gram-negative secretion systems has emerged with the determination of the structures of type I and type IV secretion system components, and with the elucidation of the mechanism of fibre formation in the chaperone-usher pathway of pilus biogenesis . Finally, the structures of complexes of secreted virulence factors bound to their host targets have unravelled the mechanisms by which bacterial pathogens exploit cellular processes to their advantage.

Crit Care Med, 2004 Mar, 32(3), 700 - 7
Comparison of Cox and Gray's survival models in severe sepsis; Kasal J et al.; BACKGROUND: Although survival is traditionally modeled using Cox proportional hazards modeling, this approach may be inappropriate in sepsis, in which the proportional hazards assumption does not hold . Newer, more flexible models, such as Gray's model, may be more appropriate . OBJECTIVES: To construct and compare Gray's model and two different Cox models in a large sepsis cohort . To determine whether hazards for death after sepsis were nonproportional . To explore how well the different survival modeling approaches describe these data . DESIGN: Analysis of combined data from the treatment and placebo arms of a large, negative, sepsis trial . SETTING: Intensive care units at 136 U.S . medical centers . SUBJECTS: A total of 1090 adults aged 18 yrs or older with signs and symptoms of severe sepsis and documented or probable Gram-negative infection . MEASUREMENTS: We considered 27 potential baseline risk factors and modeled survival over the 28 days after the onset of sepsis . We tested proportionality in single-variable Cox analysis using Schoenfeld residuals and log-log plots . We constructed a traditional multivariable Cox model, a multivariable Cox model with time-varying covariates, and a multivariable Gray's model . RESULTS: In single-variable analyses, 20 of the 27 potential factors were significantly associated with mortality, and 10 of 20 had nonproportional hazards . In multivariate analysis, all three models retained a very similar set of significant covariates (two models retained the identical set of nine variables, and the third differed only in that it retained the same nine plus a tenth variable) . Four of the nine common covariates had nonproportional hazards . Of the three models, Gray's model best captured these changing hazard ratios over time . CONCLUSION: We confirm that many of the important predictors of mortality in severe sepsis are nonproportional and find that Gray's model seems best suited for modeling survival in this condition.

J Bacteriol, 2004 May, 186(9), 2774 - 80
The theta subunit of Escherichia coli DNA polymerase III: a role in stabilizing the epsilon proofreading subunit; Taft-Benz SA et al.; The function of the theta subunit of Escherichia coli DNA polymerase III holoenzyme is not well established . theta is a tightly bound component of the DNA polymerase III core, which contains the alpha subunit (polymerase), the epsilon subunit (3'-->5' exonuclease), and the theta subunit, in the linear order alpha-epsilon-theta . Previous studies have shown that the theta subunit is not essential, as strains carrying a deletion of the holE gene (which encodes theta) proved fully viable . No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates . On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of epsilon to be modestly enhanced by the presence of theta . Here, we report a series of genetic experiments that suggest that theta has a stabilizing role for the epsilon proofreading subunit . The observations include (i) defined DeltaholE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong DeltaholE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of alpha-epsilon interactions by the presence of theta . theta appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of theta might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.

Biometals, 2004 Apr, 17(2), 99 - 104
Microbial growth promotion studies of exochelin MN and analogues thereof; Dong L et al.; The ability of exochelin MN and three synthetic analogues to promote the growth of various strains of mycobacteria and Gram-negative bacteria was investigated . The results indicated that growth promotion ability of these compounds depends either on ligand exchange with mycobactin or on the exochelin permease . Despite stronger iron complexing capacity, the structural analogues showed weaker growth promotion ability than exochelin MN, which further supported our hypothesis of pH-dependent iron(III)-release of exochelin MN.

Cancer Res, 2004 Apr 15, 64(8), 2918 - 22
Sequence variants of toll-like receptor 4 are associated with prostate cancer risk: results from the CAncer Prostate in Sweden Study; Zheng SL et al.; Inflammation has been implicated as an etiological factor in several human cancers . Growing evidence suggests that chronic inflammation may also play a role in the etiology of prostate cancer . Considering that genetic susceptibility is a major risk factor for this disease, we hypothesize that sequence variants in genes that regulate inflammation may modify individual susceptibility to prostate cancer . The lipopolysaccharide receptor Toll-like receptor 4 (TLR4) is a central player in the signaling pathways of the innate immune response to infection by Gram-negative bacteria and is an important candidate inflammatory gene . We performed a systematic genetic analysis of TLR4 sequence variants by evaluating eight single-nucleotide polymorphisms that span the entire gene among 1383 newly diagnosed prostate cancer patients and 780 age- and residence-matched controls in Sweden . We found an association between a sequence variant (11381G/C) in the 3'-untranslated region of the TLR4 gene and prostate cancer risk . The frequency of the variant genotypes (CG or CC) was significantly higher in the patients (24.1%) than in the controls (19.7%; P = 0.02) . The frequency of risk genotypes among patients diagnosed before the age of 65 years was even higher (26.3%) . Compared with men who had the wild-type genotype of this single-nucleotide polymorphism (GG), those with GC or CC genotypes had a 26% increased risk for prostate cancer (odds ratio, 1.26; 95% confidence interval, 1.01-1.57) and 39% increased risk increased risk for early onset prostate cancer (before age 65 years; odds ratio, 1.39; 95% confidence interval, 1.02-1.91) . The risk attributable to this variant for prostate cancer in Sweden was estimated to be 4.9% . Although the biological mechanism of the observed association remains to be elucidated, our finding supports a role for a bacteria-associated response pathway, possibly acting via inflammation, in the development of prostate cancer.

Clin Exp Immunol, 2004 May, 136(2), 224 - 31
Reproducibility of a novel model of murine asthma-like pulmonary inflammation; McKinley L et al.; Sensitization to cockroach allergens (CRA) has been implicated as a major cause of asthma, especially among inner-city populations . Endotoxin from Gram-negative bacteria has also been investigated for its role in attenuating or exacerbating the asthmatic response . We have created a novel model utilizing house dust extract (HDE) containing high levels of both CRA and endotoxin to induce pulmonary inflammation (PI) and airway hyperresponsiveness (AHR) . A potential drawback of this model is that the HDE is in limited supply and preparation of new HDE will not contain the exact components of the HDE used to define our model system . The present study involved testing HDEs collected from various homes for their ability to cause PI and AHR . Dust collected from five homes was extracted in phosphate buffered saline overnight . The levels of CRA and endotoxin in the supernatants varied from 7.1 to 49.5 mg/ml of CRA and 1.7-6 micro g/ml of endotoxin in the HDEs . Following immunization and two pulmonary exposures to HDE all five HDEs induced AHR, PI and plasma IgE levels substantially higher than normal mice . This study shows that HDE containing high levels of cockroach allergens and endotoxin collected from different sources can induce an asthma-like response in our murine model.

Acta Cytol, 2004 Mar-Apr, 48(2), 211 - 4
Diagnosis of Strongyloides stercoralis in a peritoneal effusion from an HIV-seropositive man . A case report; Hong IS et al.; BACKGROUND: Strongyloides stercoralis, a nematode parasite in humans with free-living and autoinfective cycles, is often an asymptomatic infection of the upper small intestine . If the host becomes immunocompromised, autoinfection may increase the intestinal worm burden and lead to disseminated strongyloidiasis . The parthenogenetic adult female larvae can remain embedded in the mucosa of the small intestine for years, producing eggs that develop into either rhabditiform, noninfective larvae or filariform, infective larvae . Manifestations of dissemination occur when the filariform larvae penetrate the intestinal wall and migrate into the blood . Pulmonary involvement is common, and the central nervous system may be affected . Blood eosinophilia is typical, and gram-negative sepsis from enteric bacteria may occur . Much less commonly described is invasion of the peritoneal cavity with peritoneal effusion . CASE: A 49-year-old man who came to the United States from Liberia 4 years earlier presented with sudden onset of severe abdominal distention, generalized weakness and marked pedal edema . Diagnostic paracentesis showed numerous filariform larvae of S stercoralis . Stool examination confirmed the presence of both rhabditiform and filariform larvae . Subsequently the patient was found to be HIV seropositive, with a CD4 lymphocyte count of 59 . CONCLUSION: Early detection of S stercoralis may alter the often-fatal course of infection . The present case is the second reported one in the English-language literature of the diagnosis of S stercoralis in ascitic fluid.

J Korean Med Sci, 2004 Apr, 19(2), 302 - 4
Staged surgery for chronic primary aortoduodenal fistula in a septic patient; Cho YP et al.; Aortoenteric fistula is one of the most challenging problems that confront the vascular surgeons . Controversy remains over the optimal treatment because of the continued publication of series with high mortality, amputation, and aortic disruption rates . A positive preoperative blood culture is the best predictor of mortality with increased amputation rates due to infection of the extra-anatomic bypass . Therefore, in selected cases with sepsis, a prudent management protocol is required . We report a 68-yr-old male presenting with a chronic primary aortoduodenal fistula extensively involving the duodenum and Gram-negative sepsis . We planned a staged operation . Initially, an emergency laparotomy and control of the aorta allowed stabilization of the patient, identification of the fistula, and direct in situ placement of the prosthetic graft followed by an en bloc resection of the aneurysm and the surrounding structures . After he recovered from sepsis and had been stabilized, a staged extra-anatomic bypass followed by transabdominal removal of the temporarily placed graft was done . This management plan will allow the highest success rate and may be a prudent management protocol for these difficult cases.

Am J Clin Pathol, 2004 Apr, 121(4), 581 - 7
Moraxella osloensis blood and catheter infections during anticancer chemotherapy: clinical and microbiologic studies of 10 cases; Han XY et al.; Moraxella osloensis, a gram-negative bacterium that is saprophytic on skin and mucosa, rarely causes infections . Moreover, infections in patients with cancer have not been reported . We describe 10 cases of M . osloensis blood or catheter infections that occurred during anticancer chemotherapy with or without preexisting neutropenia . The organism was identified definitively by sequencing analysis of the 16S ribosomal RNA gene . Fever (up to 39.7 degrees C) with substantial neutrophilia characterized these infections . The infections were monomicrobic for 3 patients and polymicrobic for 7 patients . Nine patients acquired the infection through central venous catheter colonization . The likely sources of the organism were sinusitis (3 cases), bronchitis (1 case), presumed subclinical mucositis from anticancer therapy (4 cases), and cutaneous graft-vs-host disease (2 cases) . The infections resolved, without catheter removal, after antibiotic therapy with cell wall-active agents, to which all strains were shown to be susceptible . The M . osloensis strains exhibited significant morphologic variations on gram stain, and sheep blood agar was the preferred culture medium for 9 strains.

Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 6194 - 9 Epub 2004 Apr 12.
The crystal structure of filamentous hemagglutinin secretion domain and its implications for the two-partner secretion pathway; Clantin B et al.; Filamentous hemagglutinin (FHA), the major 230-kDa adhesin of the whooping cough agent Bordetella pertussis, is one of the most efficiently secreted proteins in Gram-negative bacteria . FHA is secreted by means of the two-partner secretion (TPS) pathway . Several important human, animal, and plant pathogens also secrete adhesins and other virulence factors by using this mode of secretion . A TPS system is composed of two separate proteins, with TpsA the secreted protein and TpsB its associated specific outermembrane transporter . All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain . We report here the 1.7- A structure of a functionally secreted 30-kDa N-terminal fragment of FHA . It reveals that the TPS domain folds into a beta-helix, with three extrahelical motifs, a beta-hairpin, a four-stranded beta-sheet, and an N-terminal capping, mostly formed by the nonconserved regions of the TPS domain . The structure thus explains why the TPS domain is able to initiate folding of the beta-helical motifs that form the central domain of the adhesin, because it is itself a beta-helical scaffold . It also contains less conserved extrahelical regions most likely involved in specific properties, such as the recognition of the outer-membrane transporter . This structure is representative of the TPS domains found so far in >100 secreted proteins from pathogenic bacteria . It also provides a mechanistic insight into how protein folding may be linked to secretion in the TPS pathway.

J Clin Microbiol, 2004 Apr, 42(4), 1590 - 5
Endocarditis with ruptured cerebral aneurysm caused by Cardiobacterium valvarum sp . nov; Han XY et al.; A fastidious gram-negative bacterium was isolated from the blood of a 37-year-old man who had insidious endocarditis with a sudden rupture of a cerebral aneurysm . Characterization of the organism through phylogenetic and phenotypic analyses revealed a novel species of Cardiobacterium, for which the name Cardiobacterium valvarum sp . nov . is proposed . C . valvarum will supplement the current sole species Cardiobacterium hominis, a known cause of endocarditis . Surgeries and antibiotic treatment cured the patient's infection and associated complications . During cardiac surgery, a congenital bicuspid aortic valve was found to be the predisposing factor for his endocarditis.

BMC Bioinformatics . 2004 Mar 15;5(1):29.
A Hidden Markov Model method, capable of predicting and discriminating beta-barrel outer membrane proteins; Bagos PG et al.; BACKGROUND: Integral membrane proteins constitute about 20-30% of all proteins in the fully sequenced genomes . They come in two structural classes, the alpha-helical and the beta-barrel membrane proteins, demonstrating different physicochemical characteristics, structure and localization . While transmembrane segment prediction for the alpha-helical integral membrane proteins appears to be an easy task nowadays, the same is much more difficult for the beta-barrel membrane proteins . We developed a method, based on a Hidden Markov Model, capable of predicting the transmembrane beta-strands of the outer membrane proteins of gram-negative bacteria, and discriminating those from water-soluble proteins in large datasets . The model is trained in a discriminative manner, aiming at maximizing the probability of correct predictions rather than the likelihood of the sequences . RESULTS: The training has been performed on a non-redundant database of 14 outer membrane proteins with structures known at atomic resolution; it has been tested with a jacknife procedure, yielding a per residue accuracy of 84.2% and a correlation coefficient of 0.72, whereas for the self-consistency test the per residue accuracy was 88.1% and the correlation coefficient 0.824 . The total number of correctly predicted topologies is 10 out of 14 in the self-consistency test, and 9 out of 14 in the jacknife . Furthermore, the model is capable of discriminating outer membrane from water-soluble proteins in large-scale applications, with a success rate of 88.8% and 89.2% for the correct classification of outer membrane and water-soluble proteins respectively, the highest rates obtained in the literature . That test has been performed independently on a set of known outer membrane proteins with low sequence identity with each other and also with the proteins of the training set . CONCLUSION: Based on the above, we developed a strategy, that enabled us to screen the entire proteome of E . coli for outer membrane proteins . The results were satisfactory, thus the method presented here appears to be suitable for screening entire proteomes for the discovery of novel outer membrane proteins . A web interface available for non-commercial users is located at: and it is the only freely available HMM-based predictor for beta-barrel outer membrane protein topology.

Med Sci (Paris), 2004 Mar, 20(3), 346 - 51
{Bacterial porin and antibiotic susceptibility}; Pages JM; In Gram negative bacteria, hydrophilic antibiotics such as beta-lactams and fluoroquinolons used the bacterial porin channel during their entry . The balance of the porin expression level and the molecular parameters which govern the molecule diffusion through the pore are important physiological points . Acquired in vivo beta-lactam resistance is often associated with porin loss, and recently clinical resistant strains synthetizing mutated porin have been described . These data highlight both the importance of the channel characteristics and the amino acid residues involved in the drug diffusion process . In addition, several mechanisms, including various repressors or activators as well as molecules inhibiting the pore synthesis or activity, argue for the complexity and plasticity of the bacterial control of porin function . All these aspects play a key role in both membrane permeability and efficiency of the antibiotic resistance process.

Appl Environ Microbiol, 2004 Apr, 70(4), 2391 - 7
Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723; Dai M et al.; Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil . However, degradation of PCP is slow and S . chlorophenolicum cannot tolerate high levels of PCP . We have used genome shuffling to improve the degradation of PCP by S . chlorophenolicum . We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain . Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM . Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain . Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.

Genes Cells, 2004 Apr, 9(4), 271 - 8
RMF inactivates ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel; Yoshida H et al.; In gram-negative bacteria such as Escherichia coli, protein synthesis is suppressed by the formation of 100S ribosomes under stress conditions . The 100S ribosome, a dimer of 70S ribosomes, is formed by ribosome modulation factor (RMF) binding to the 70S ribosomes . During the stationary phase, most of the 70S ribosomes turn to 100S ribosomes, which have lost translational activity . This 100S formation is called the hibernation process in the ribosome cycle of the stationary phase . If stationary phase cells are transferred to fresh medium, the 100S ribosomes immediately go back to active 70S ribosomes, showing that inactive 100S <--> active 70S interconversion is a major system regulating translation activity in stationary phase cells . To elucidate the mechanisms of translational inactivation, the binding sites of RMF on 23S rRNA in 100S ribosome of E . coli were examined by a chemical probing method using dimethyl sulphate (DMS) . As the results, the nine bases in 23S rRNA were protected from DMS modifications and the modification of one base was enhanced . Interestingly A2451 is included among the protected bases, which is thought to be directly involved in peptidyl transferase activity . We conclude that RMF inactivates ribosomes by covering the peptidyl transferase (PTase) centre and the entrance of peptide exit tunnel . It is surprising that the cell itself produces a protein that seems to inhibit protein synthesis in a similar manner to antibiotics and that it can reversibly bind to and release from the ribosome in response to environmental conditions.

Extremophiles, 2004 Apr, 8(2), 125 - 32 Epub 2004 Jan 21.
Promoters from a cold-adapted bacterium: definition of a consensus motif and molecular characterization of UP regulative elements; Duilio A et al.; Although low-temperature tolerant micro-organisms were discovered long ago, limited information is still available on the transcription machinery in cold-adapted bacteria . This knowledge represents a necessary background for the investigation of the adaptation of gene-expression mechanisms at low temperatures . The recent development of a shuttle genetic system for the transformation of the cold-adapted Gram-negative bacterium Pseudoalteromonas haloplanktis strain TAC125 has made possible the isolation of the psychrophilic promoters described in this paper . TAC125 genomic DNA fragments were cloned in the shuttle vector and the promoter-containing recombinant clones were selected for their ability to express a promoter-less lacZ gene . The nucleotide sequence of several selected inserts and the transcription start points of the transcribed m-RNAs were determined . A promoter consensus sequence for Pseudoalteromonas haloplanktis TAC125 was proposed on the basis of a sequence comparison between the various active promoters . Furthermore, the identification and the functional characterization of two UP elements from this cold-adapted bacterium are also reported.

Methods Mol Med, 2004, 98, 199 - 206
Animal models of endotoxic shock; Villa P et al.; Endotoxin/lipopolysaccharide (LPS) is the major mediator that triggers the cellular and humoral responses of the shock induced by Gram-negative bacteria . The toxic responses of LPS are mediated by various factors and mainly by tumor necrosis factor alpha (TNFalpha) . To study the role of TNF and to identify anti-TNF molecules in endotoxic/septic shock, numerous animal models have been utilized . The models described here are among the most widely used and are represented by LPS given at high dose, or at low dose in D-galactosamine-sensitized mice . The endpoints of these models are the survival, the organ toxicity, or the regulation of cytokines, and in particular of TNFalpha . An additional and more complex model of endotoxic/septic shock, the polymicrobial model of cecal ligation and puncture, where a synergistic interaction of several mediators occurs, is then described.

Am J Physiol Lung Cell Mol Physiol, 2004 Jul, 287(1), L239 - 49 Epub 2004 Apr 02.
Lipopolysaccharide induces expression of fibronectin alpha 5 beta 1-integrin receptors in human monocytic cells in a protein kinase C-dependent fashion; Roman J et al.; LPS is an outer-membrane glycolipid component of gram-negative bacteria known for its fervent ability to activate monocytic cells and for its potent proinflammatory capabilities . In addition, LPS triggers the release of cytokines and chemokines as well as cell-cell adhesion molecules . We postulate that LPS may also affect the expression of matrix-binding integrin receptors, thereby modulating cell-adhesive functions in monocytic cells . To test this hypothesis, we investigated the effects of LPS on the expression of the integrin alpha(5)beta(1), a fibronectin receptor, in a human monocytic cell line (U937) as well as in isolated human peripheral blood mononuclear cells (PBMCs) . We found that LPS increased the expression of alpha(5)beta(1) receptors and enhanced the adherence of U937 cells and PBMCs to fibronectin-coated surfaces; this was blocked by anti-alpha(5)beta(1) antibodies . LPS increased alpha(5)-subunit mRNA accumulation in a dose- and time-dependent manner . The induction by LPS occurred, at least in part, at the level of gene transcription as indicated by experiments using alpha(5) intact and deletion promoter constructs . LPS-induced alpha(5) gene transcription was associated with rapid induction of conventional PKC-alpha protein and activity, was blocked by PKC inhibitors, and was mimicked by lipid A . Finally, we found that an anti-CD14 antibody was able to inhibit the LPS response . Overall, the data suggest that LPS stimulates alpha(5) gene transcription via CD14 and PKC-dependent signals to enhance the expression of functional alpha(5)beta(1) receptors in monocytic cells . This process may help stimulate monocytic cell activation and facilitate their migration into fibronectin-containing tissues during infection.

Lancet, 2004 Apr 3, 363(9415), 1110 - 5
WHO guidelines for management of severe malnutrition in rural South African hospitals: effect on case fatality and the influence of operational factors; Ashworth A et al.; BACKGROUND: WHO case-management guidelines for severe malnutrition aim to improve the quality of hospital care and reduce mortality . We aimed to assess whether these guidelines are feasible and effective in under-resourced hospitals . METHODS: All children admitted with a diagnosis of severe malnutrition to two rural hospitals in Eastern Cape Province from April, 2000 to April, 2001, were studied and their case-fatality rates were compared with the rates in a period before guidelines were implemented (March, 1997 to February, 1998) . Quality of care was assessed by observation of medical and nursing practices, review of medical records, and interviews with carers and staff . A mortality audit was used to identify cause of death and avoidable contributory factors . FINDINGS: At Mary Theresa Hospital, case-fatality rates fell from 46% before implementation to 21% after implementation . At Sipetu Hospital, the rates fell from 25% preimplementation to 18% during 2000, but then rose to 38% during 2001, when inexperienced doctors who were not trained in the treatment of malnutrition were deployed . This rise coincided with less frequent prescribing of potassium (13% vs 77%, p<0.0001), antibiotics with gram-negative cover (15% vs 46%, p=0.0003), and vitamin A (76% vs 91%, p=0.018) . Most deaths were attributed to sepsis . For the two hospitals combined, 50% of deaths in 2000-01 were due to doctor error and 28% to nurse error . Weaknesses within the health system--especially doctor training, and nurse supervision and support--compromised quality of care . INTERPRETATION: Quality of care improved with implementation of the WHO guidelines and case-fatality rates fell . Although major changes in medical and nursing practice were achieved in these under-resourced hospitals, not all tasks were done with adequate care and errors led to unnecessary deaths.

Curr Opin Microbiol, 2004 Apr, 7(2), 109 - 14
Structure of the histone-like protein H-NS and its role in regulation and genome superstructure; Rimsky S; H-NS belongs to the group of histone-like proteins in Gram-negative bacteria and is also a pleiotropic regulator of genes implicated in many responses to environmental changes . It plays a dual role in structuring DNA and in regulating transcription . Recent advances have been made in elucidating the structure and oligomerisation properties of this protein, thus aiding in the understanding of the molecular relationship between its two major functions.

FEBS Lett, 2004 Apr 9, 563(1-3), 165 - 9
CcmF(C) involved in cytochrome c maturation is present in a large sized complex in wheat mitochondria; Giege P et al.; In land plant mitochondria, c-type cytochromes are assembled via a mechanism similar to that found in Gram-negative bacteria and different from that used by mitochondria from other eukaryotes . The wheat mitochondrial genome encodes CCM (for cytochrome c maturation) proteins, among them CcmF(C), a protein similar to the C-terminal part of the bacterial CcmF . The gene is transcribed into a 1.7 kb transcript at steady state . However, the 3' termini of the transcript were found to occur upstream of the deduced gene termination codon . This discrepancy was solved by RNA editing that introduces a novel termination codon, thus shortening the reading frame by 27 codons . The processed transcript is translated into a protein integrated in the mitochondrial inner membrane . We also show that the protein is part of a large (700 kDa) protein complex, that possibly represents a cytochrome c assembly complex.

Structure (Camb), 2004 Apr, 12(4), 623 - 32
Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose; Allouch J et al.; Agarose is a gel-forming polysaccharide with an alpha-L(1,4)-3,6-anhydro-galactose, beta-D(1,3)-galactose repeat unit, from the cell walls of marine red algae . beta-agarase A, from the Gram-negative bacterium Zobellia galactanivorans, is secreted to the external medium and degrades agarose with an endo-mechanism . The structure of the inactive mutant beta-agarase A-E147S in complex with agaro-octaose has been solved at 1.7 A resolution . Two oligosaccharide chains are bound to the protein . The first one resides in the active site channel, spanning subsites -4 to -1 . A second oligosaccharide binding site, on the opposite side of the protein, was filled with eight sugar units, parallel to the active site . The crystal structure of the beta-agarase A with agaro-octaose provides detailed information on agarose recognition in the catalytic site . The presence of the second, parallel, binding site suggests that the enzyme might be able to unwind the double-helical structure of agarose prior to the catalytic cleavage.

Am J Physiol Regul Integr Comp Physiol, 2004 Aug, 287(2), R437 - 45 Epub 2004 Apr 01.
Hypoxic suppression of E . coli-induced NF-kappa B and AP-1 transactivation by oxyradical signaling; Matuschak GM et al.; Transactivation of the DNA-binding proteins nuclear factor-kappa B (NF-kappa B) and activator protein (AP)-1 by de novo oxyradical generation is a stereotypic redox-sensitive process during hypoxic stress of the liver . Systemic trauma is associated with splanchnic hypoxia-reoxygenation (H/R) followed by intraportal gram-negative bacteremia, which collectively have been implicated in posttraumatic liver dysfunction and multiple organ damage . We hypothesized that hypoxic stress of the liver before stimulation by Escherichia coli serotype O55:B5 (EC) amplifies oxyradical-mediated transactivation of NF-kappa B and AP-1 as well as cytokine production compared with noninfectious H/R or gram-negative sepsis without prior hypoxia . Livers from Sprague-Dawley rats underwent perfusion for 180 min with or without 0.5 h of hypoxia (perfusate PO(2), 40 +/- 5 mmHg) followed by reoxygenation and infection with 10(9) EC or 0.9% NaCl infusion . In H/R + EC livers, nuclear translocation of NF-kappa B and AP-1 was unexpectedly reduced in gel shift assays vs . normoxic EC controls, as were perfusate TNF-alpha and IL-1 beta levels . Preceding hypoxic stress paradoxically increased postbacteremic reduced-to-oxidized glutathione ratios plus nuclear localization of I kappa B alpha and phospho-I kappa B alpha, but not JunB/FosB profiles . Notably, xanthine oxidase inhibition increased transactivation as well as cytokine production in H/R + EC livers . Thus brief hypoxic stress of the liver before intraportal gram-negative bacteremia potently suppresses activation of canonical redox-sensitive transcription factors and production of inflammatory cytokines by mechanisms including xanthine oxidase-induced oxyradicals functioning in an anti-inflammatory signaling role . These results suggest a novel multifunctionality of oxyradicals in decoupling hepatic transcriptional activity and cytokine biosynthesis early in the posttraumatic milieu.

Curr Microbiol, 2004 Feb, 48(2), 159 - 63
Acidianus tengchongensis sp . nov., a new species of acidothermophilic archaeon isolated from an acidothermal spring; He ZG et al.; A new thermoacidophilic, obligately chemolithotrophic, facultatively aerobic archaeon Acidianus S5(T), was isolated from a Tengchong acidothermal spring in southwestern China . It is a Gram-negative, nonmotile, irregular coccoid organism with a cell diameter of 1.2 microm . The optimal pH and temperature for growth are 2.5 and 70 degrees C, respectively . Under anaerobic conditions, the organism reduces elemental sulfur with molecular hydrogen, producing hydrogen sulfide . Under aerobic conditions, it oxidizes elemental sulfur and produces sulfuric acid . No growth occurs when it is cultivated in an iron medium, indicating that ferrous iron cannot serve as an energy source . The G+C content is 38% (mol/mol), which is much different from that of other Acidianus species (31%-32.7%) . The phylogenetic distances, based on 16S rDNA sequences, to A . brierleyi, A . infernus, and A . ambivalens were 0.2, 2.6, and 2.5%, respectively . DNA-DNA hybridization rates of strain S5(T) to A . brierleyi, A . infernus, and A . ambivalens are 44, 22, and 23%, respectively . Thus, a new name, Acidianus tengchongensis sp . nov., is proposed for this strain S5(T).

Genes Immun, 2004 Jun, 5(4), 283 - 8
A coding mutation within the first exon of the human MD-2 gene results in decreased lipopolysaccharide-induced signaling; Hamann L et al.; MD-2 is an accessory protein of the Toll-like receptor (TLR)-4, necessary for assembling a receptor complex to sense low quantities of lipopolysaccharide in order to subsequently trigger innate immune responses . MD-2 and TLR-4 are expressed on a variety of immunocompetent cells . Mutations within the TLR-4 gene have been shown to attenuate immune responses against lipopolysaccharide in mice . In humans, a TLR-4 polymorphism has been associated with a higher risk for developing severe Gram-negative sepsis and with a lower risk for atherosclerosis . Since MD-2 is an essential part of the lipopolysaccharide receptor complex, we screened 20 patients that underwent surgical cancer therapy for novel MD-2 mutations by a single-strand conformation polymorphism technique . In one patient we found an A --> G substitution at position 103, resulting in an amino-acid exchange from Thr 35 to Ala . Reporter gene assays revealed that this mutation resulted in a reduced lipopolysaccharide-induced signaling . The patient displayed an uneventful postoperative course, with the exception of slightly decreased TNF-alpha levels after in vitro stimulation with LPS as compared to wt patients . Genotyping of a further 41 patients by a newly developed Lightcycler/FRET method failed to detect any additional polymorphism carriers, indicating that this is a rare mutation.

Lipids, 2004 Jan, 39(1), 75 - 9
Incorporation of 1-chlorooctadecane into FA and beta-hydroxy acids of Marinobacter hydrocarbonoclasticus; Aubert E et al.; The lipids of the gram-negative marine bacterium Marinobacter hydrocarbonoclasticus, cultivated in synthetic seawater supplemented with 1-chlorooctadecane as sole source of carbon, were isolated, purified, and their structures determined . Three pools of lipids were isolated according to the sequential procedure used: unbound lipids extracted by organic solvents, ester-bound lipids released under alkaline conditions, and amide-bound lipids released by acid hydrolysis . FA isolated from the unbound lipids included omega-chlorinated (21%, w/w, of this fraction; C16 predominant) and nonchlorinated compounds (22%, w/w; C18 predominant) . These acids were accompanied by a high proportion of omega-chloro-C18 alcohols (43%, w/w) and a lower amount of omega-chloro-beta-hydroxy-C18, -C16, and -C14 acids (5%, w/w) . These data, together with the isolation from the culture medium of gamma-butyrolactone, suggested a metabolism of 1-chlorooctadecane through oxidation into omega-chloro acid and then the classic beta-oxidation pathway . The analysis of the ester-bound and amide-bound lipids revealed that significant amounts of omega-chloro-beta-hydroxy C10-C12 acids were incorporated into the lipopolysaccharides of the bacterium . Incorporation of these omega-chloro-beta-hydroxy acids into the lipopolysaccharides represents a novel route for chloroalkane assimilation in hydrocarbonoclastic gram-negative bacteria . The formation of chlorinated hydroxy acids, like the omega-chloro FA in the cellular lipids, could account for an incomplete mineralization of chloroparaffins in the environment.

Clin Exp Pharmacol Physiol, 2004 Apr, 31(4), 226 - 30
A novel water-soluble vitamin E derivative prevents acute lung injury by bacterial endotoxin; Uchiyama K et al.; 1 . Various chemokines, such as keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-1alpha and macrophage chemoattractant protein (MCP)-1, are involved in the pathogenesis of acute lung injury induced by bacterial endotoxin (lipopolysaccharide; LPS) . Oxidative stress is an important regulator of the expression of these chemokines, whereas vitamin E protects against LPS-induced insults . In the present study, we determined the effects of 2-(alpha-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), a novel water-soluble vitamin E derivative with excellent anti-oxidant activity, on acute lung injury induced by intratracheal instillation of LPS (125 micro g/kg) in mice . 2 . When TMG was administered intratracheally and intravenously (0.1, 1.0 or 10 mg/kg), it dose-dependently decreased the infiltration of neutrophils into bronchoalveolar lavage fluid after LPS challenge . 3 . Histological examination showed that treatment with TMG ameliorated the LPS-induced infiltration of neutrophils into the lungs . Furthermore, TMG attenuated the LPS-induced increase in pulmonary expression of KC, MIP-1alpha and MCP-1 at both the transcriptional and translational levels . 4 . These results indicate that TMG is a possible treatment for acute lung injury, especially that caused by Gram-negative bacteria . The therapeutic effect of TMG may be mediated, at least in part, by suppression of the local expression of chemokines, possibly through its strong anti-oxidant activity.

Syst Appl Microbiol, 2004 Feb, 27(1), 43 - 9
Chitinimonas taiwanensis gen . nov., sp . nov., a novel chitinolytic bacterium isolated from a freshwater pond for shrimp culture; Chang SC et al.; A bacterial strain, designated cfT was isolated from surface water of a freshwater pond for shrimp (Macrobrachium rosenbergii) culture at Ping-Tung (Southern Taiwan) . Cells of this organism were Gram-negative, slightly curved rods which were motile by means of a single polar flagellum . Strain cfT utilized chitin as the exclusive carbon, nitrogen, and energy source for growth, both under aerobic and anaerobic conditions . Optimum conditions for growth were between 25 and 37 degrees C, 0 and 1% NaCl and pH 6 to 8 . Strain cfT secreted two chitinolytic enzymes with approximate molecular weight 52 and 64 kDa, which hydrolyzed chitin to produce chitotriose as major product . Sequence comparison of an almost complete 16S rDNA gene showed less than 92% sequence similarity with known bacterial species . Phylogenetic analysis based on the neighbour-joining and other methods indicated that the organism formed a distinct lineage within the beta-subclass of Proteobacteria . The predominant cellular fatty acids of strain cfT were hexadecanoic acid (about 29%), octadecenoic acid (about 12%) and summed feature 3 (16:1 omega7c or 15 iso 2-OH or both {about 49%}) . Its DNA base ratio was 62.8 mol% G+C . We propose to classify strain cfT (= CCRC 17210T = LMG 22011T) as Chitinimonas taiwanensis gen . nov., sp . nov.

Biochem Cell Biol, 2004 Feb, 82(1), 71 - 86
Biosynthesis, transport, and modification of lipid A; Trent MS; Lipopolysaccharide (LPS) is the major surface molecule of Gram-negative bacteria and consists of three distinct structural domains: O-antigen, core, and lipid A . The lipid A (endotoxin) domain of LPS is a unique, glucosamine-based phospholipid that serves as the hydrophobic anchor of LPS and is the bioactive component of the molecule that is associated with Gram-negative septic shock . The structural genes encoding the enzymes required for the biosynthesis of Escherchia coli lipid A have been identified and characterized . Lipid A is often viewed as a constitutively synthesized structural molecule . However, determination of the exact chemical structures of lipid A from diverse Gram-negative bacteria shows that the molecule can be further modified in response to environmental stimuli . These modifications have been implicated in virulence of pathogenic Gram-negative bacteria and represent one of the molecular mechanisms of microbial surface remodeling used by bacteria to help evade the innate immune response . The intent of this review is to discuss the enzymatic machinery involved in the biosynthesis of lipid A, transport of the molecule, and finally, those enzymes involved in the modification of its structure in response to environmental stimuli.

Trends Microbiol, 2004 Apr, 12(4), 186 - 92
Innate recognition of lipopolysaccharide by Toll-like receptor 4-MD-2; Miyake K; Toll-like receptors (TLRs) are pathogen recognition molecules that activate the immune system as part of the innate immune response . Microbial recognition by TLRs plays a crucial role in the host immune system's decision to respond or not to a particular microbial infection . Lipopolysaccharide (LPS), a membrane glycolipid of Gram-negative bacteria, exhibits strong immunostimulating activity among TLR ligands and has been studied in great detail . Recent studies have shown that cell surface TLR4-MD-2 physically interacts with LPS and triggers the release of an LPS signal, revealing a host-pathogen interaction mediated by TLR.

Mol Microbiol, 2004 Apr, 52(1), 67 - 79
The inner membrane subassembly of the enteropathogenic Escherichia coli bundle-forming pilus machine; Crowther LJ et al.; Type IV pili (Tfps) are filamentous surface appendages expressed by Gram-negative microorganisms and play numerous roles in bacterial cell biology . Tfp biogenesis machineries are highly conserved and resemble protein secretion and DNA uptake systems . Although components of Tfp biogenesis systems have been identified, it is not known how they interact to form these machineries . Using the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli as a model Tfp system, we provide evidence of a cytoplasmic membrane subassembly of the Tfp assembly machine composed of putative cytoplasmic nucleotide-binding and cytoplasmic membrane proteins . A combination of genetic, biochemical and biophysical approaches revealed interactions among putative cytoplasmic nucleotide-binding proteins BfpD and BfpF and cytoplasmic membrane proteins BfpC and BfpE of the BFP biogenesis machine . The polytopic membrane protein BfpE appears to be a central component of this subassembly as it interacts with BfpC, BfpD and BfpF . We report that BFP biogenesis probably requires interactions among BfpC, BfpD and BfpE, whereas BFP retraction requires interaction of the PilT-like putative ATPase BfpF with a conserved domain of BfpE . BfpE is the first protein that is not a member of the PilT family to be implicated in Tfp retraction . Furthermore, we found that the putative ATPases BfpD and BfpF play antagonistic roles in BFP biogenesis and retraction, respectively, by interacting with distinct domains of the BFP biogenesis machine.

Ann Ig, 2003 Nov-Dec, 15(6), 817 - 24
{The discharge form: advantages and limits legionellosis cases individuation}; Trerotoli P et al.; Despite legionellosis surveillance is active in Italy since many years, the disease notification appears still undervalued . A multicentric survey was carried out among 5 big Italian hospitals . It examined 11,435 discharge forms (1999-2001), reporting pneumonia diagnosis . Legionellosis (II class of notify system for infectious disease) was studied among pneumonia diagnosis by discharge forms . According to the ICD9-CM, there's no specific code for legionellosis (this disease is included among "others gram-negative pneumonia") . So the presumed pneumonia imputable to Legionella spp were the 2.7% of the whole number of analyzed discharge forms . Besides, the data regarding the other pneumonia showed that the etiological agent was specified only in the 11.2% of the case . This situation could be rectified both introducing adequate discharge forms codes and promoting the etiological diagnosis during the hospital stay.

Antimicrob Agents Chemother, 2004 Apr, 48(4), 1235 - 41
Involvement of enterobactin synthesis pathway in production of microcin H47; Azpiroz MF et al.; Microcin H47 (MccH47) is a gene-encoded peptide antibiotic produced by an Escherichia coli clinical isolate which is active on strains of gram-negative bacteria . Its uptake by E . coli K-12-susceptible cells depends on the presence of any of the outer membrane proteins Cir, Fiu, and FepA, the three catechol receptors of this organism . The nucleotide sequence of a portion of the MccH47 genetic system that had not yet been studied was elucidated . Five open reading frames were identified, three of which corresponded to genes encoding functions related to catechol-type siderophores: mchA and mchS1 are iroB and iroD homologues, respectively, and mchS4 was found to promote the production of the catecholate siderophore enterobactin . The possible relationship between enterobactin synthesis and MccH47 production was studied . Enterobactin-deficient strains failed to produce MccH47 when transformed with the antibiotic genetic determinants and upon introduction of the ent genetic cluster, the production of both the siderophore and MccH47 was restored . Further studies demonstrated that at least the enterobactin nonribosomal peptide synthase EntF is necessary for MccH47 synthesis . The relationship found between MccH47 and catecholate siderophore production is discussed, and a model outlining MccH47 synthesis is proposed.

Syst Appl Microbiol, 2004 Mar, 27(2), 139 - 45
Balneomonas flocculans gen . nov., sp . nov., a new cellulose-producing member of the alpha-2 subclass of Proteobacteria; Takeda M et al.; A new bacterial strain capable of producing cellulose was isolated from a hot spring . The isolate was Gram-negative, aerobic, and rod-shaped . The optimum temperature for growth was 40-45 degrees C . Methanol, glucose and other common carbohydrates were not utilized as sole growth substrates . Thiosulfate was not oxidized . The G+C content of the DNA was determined to be 64.0 mol% . Comparative 16S rDNA analysis indicated that Bosea thiooxidans and some strains of the genus Methylobacterium were the nearest relatives . The isolate can be distinguished from these relatives by its defectiveness in methanol utilization and thiosulfate oxidation . On the basis of its phenotypic properties and phylogeny, it is proposed that the isolate be designated Balneomonas flocculans gen . nov., sp . nov . The type strain is TFBT (= JCM 11936T, = KCTC 12101T, = IAM 15034T, = ATCC BAA-817T).

J Biol Chem, 2004 Jun 11, 279(24), 25400 - 10 Epub 2004 Mar 24.
Oxidation and transamination of the 3"-position of UDP-N-acetylglucosamine by enzymes from Acidithiobacillus ferrooxidans . Role in the formation of lipid a molecules with four amide-linked acyl chains; Sweet CR et al.; Lipid A, a major component of the outer membranes of Escherichia coli and other Gram-negative bacteria, is usually constructed around a beta-1',6-linked glucosamine disaccharide backbone . However, in organisms like Acidithiobacillus ferrooxidans, Leptospira interrogans, Mesorhizobium loti, and Legionella pneumophila, one or both glucosamine residues are replaced with the sugar 2,3-diamino-2,3-dideoxy-d-glucopyranose . We now report the identification of two proteins, designated GnnA and GnnB, involved in the formation of the 2,3-diamino-2,3-dideoxy-d-glucopyranose moiety . The genes encoding these proteins were recognized because of their location between lpxA and lpxB in A . ferrooxidans . Based upon their sequences, the 313-residue GnnA protein was proposed to catalyze the NAD(+)-dependent oxidation of the glucosamine 3-OH of UDP-GlcNAc, and the 369-residue GnnB protein was proposed to catalyze the subsequent transamination to form UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-d-glucopyranose (UDP-GlcNAc3N) . Both gnnA and gnnB were cloned and expressed in E . coli using pET23c+ . In the presence of l-glutamate and NAD(+), both proteins were required for the conversion of {alpha-(32)P}UDP-GlcNAc to a novel, less negatively charged sugar nucleotide shown to be {alpha-(32)P}UDP-GlcNAc3N . The latter contained a free amine, as judged by modification with acetic anhydride . Using recombinant GnnA and GnnB, approximately 0.4 mg of the presumptive UDP-GlcNAc3N was synthesized . The product was purified and subjected to NMR analysis to confirm the replacement of the GlcNAc 3-OH group with an equatorial NH(2) . As shown in the accompanying papers, UDP-GlcNAc3N is selectively acylated by LpxAs of A . ferrooxidans, L . interrogans, and M . loti . UDP-GlcNAc3N may be useful as a substrate analog for diverse enzymes that utilize UDP-GlcNAc.

MMWR Morb Mortal Wkly Rep, 2004 Mar 26, 53(11), 241 - 3
Osteomyelitis/septic arthritis caused by Kingella kingae among day care attendees--Minnesota, 2003; Centers for Disease Control and Prevention (CDC); Kingella kingae is a fastidious gram-negative coccobacillus that colonizes the respiratory and oropharyngeal tract in children . K . kingae occasionally causes invasive disease, primarily osteomyelitis/septic arthritis in young children, bacteremia in infants, and endocarditis in school-aged children and adults . Although diagnosis of this organism frequently is missed, invasive disease is uncommon . Only sporadic, non-epidemiologically linked cases have been reported previously . In October 2003, the Minnesota Department of Health (MDH) investigated a cluster of two confirmed cases and one probable case of osteomyelitis/septic arthritis caused by K . kingae among children aged 17-21 months attending the same toddler classroom in a day care center . All reported within the same week with onset of fever, preceding or concurrent upper respiratory illness (URI), and refusal to bear weight on the affected limb . This report summarizes these cases and describes the epidemiologic investigation of the day care center . The findings underscore the need for clinicians and laboratorians to consider K . kingae infection in young children with Gram stain--negative or culture-negative skeletal infections.

Prostaglandins Leukot Essent Fatty Acids, 2004 Apr, 70(4), 391 - 7
Eicosapentaenoic acid and gamma-linolenic acid increase hippocampal concentrations of IL-4 and IL-10 and abrogate lipopolysaccharide-induced inhibition of long-term potentiation; Kavanagh T et al.; Inflammatory changes in brain exert a negative impact on cognitive function and in animal studies, these changes are associated with impairment in hippocampal-dependent learning paradigms and in long-term potentiation (LTP), which is a putative biological substrate for learning and/or memory . Lipopolysaccharide (LPS), a component of the cell wall of gram negative bacteria, induces inflammatory changes in the brain and leads to impairment of LTP . Since eicosapentaenoic acid (EPA) inhibits LPS-induced changes in vitro, we assessed the possibility that treatment of rats with EPA, alone or in combination with gamma-linolenic acid (GLA) might inhibit LPS-induced changes in vivo . The data presented indicate that the LPS-induced inhibition of LTP and decrease in hippocampal concentration of anti-inflammatory cytokines IL-10 and IL-4 are blocked in rats treated with EPA, GLA or both . The evidence suggests that these effects may be coupled with fatty acid-induced up-regulation of peroxisome proliferator-activated receptor-gamma which possesses known anti-inflammatory effects.

Nat Rev Microbiol, 2003 Oct, 1(1), 45 - 54
Coupling cell movement to multicellular development in myxobacteria; Kaiser D; The myxobacteria are Gram-negative organisms that are capable of multicellular, social behaviour . In the presence of nutrients, swarms of myxobacteria feed cooperatively by sharing extracellular digestive enzymes, and can prey on other bacteria . When the food supply runs low, they initiate a complex developmental programme that culminates in the production of a fruiting body . Myxobacteria move by gliding and have two, polarly positioned engines to control their motility . The two engines undergo coordinated reversals, and changes in the reversal frequency and speed are responsible for the different patterns of movement that are seen during development . The myxobacteria communicate with each other and coordinate their movements through a cell-contact-dependent signal . Here, the cell movements that culminate in the development of the multicellular fruiting body are reviewed.

Tidsskr Nor Laegeforen, 2004 Mar 18, 124(6), 776 - 8
{Plasmapheresis in the treatment of septic shock}; Busund R; BACKGROUND: Sepsis is an increasingly common cause of morbidity and mortality and the leading cause of death in intensive care units . In recent years many new therapies for sepsis have been tested in randomised clinical trials, but most of them have failed to reduce mortality . Plasmapheresis is a nonselective method by which plasma is separated from the blood and replaced with donor plasma and/or albumin . The theoretical rationale is that plasmapheresis removes the harmful mediators and replenishes the consumed plasma factors, thus restoring the homeostatic milieu . MATERIALS AND METHODS: This article is based upon own research and a review of the current literature found in PubMed . RESULTS: Over the last 30 years, several experimental and clinical studies have reported a beneficial effect of plasmapheresis in the treatment of patients with severe sepsis and septic shock . Most of the studies have focused on Gram-negative sepsis . Only a few of the clinical studies are prospective, randomised trials with appropriate control groups . INTERPRETATION: There is not sufficient evidence to justify the use of plasmapheresis as standard treatment of patients with severe sepsis and septic shock . However, plasmapheresis should be considered for fulminant Gram-negative septic shock . If applied, it should be started urgently and repeated in unresponsive patients and when a patient's clinical condition is deteriorating.

Infect Immun, 2004 Apr, 72(4), 2123 - 30
Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-kappa B and interleukin-8 expression; Ryan KA et al.; Toll-like receptor 4 (TLR4) has been identified as a transmembrane protein involved in the host innate immune response to gram-negative bacterial lipopolysaccharide (LPS) . Upon activation by LPS recognition, the TIR domain of TLR4 signals through MyD88 to activate the nuclear factor kappa B (NF-kappa B) pathway, a critical regulator of many proinflammatory genes, including interleukin-8 (IL-8) . Emerging evidence suggests that reactive oxygen species (ROS) can contribute to diverse signaling pathways, including the LPS-induced cascade . In the present study we investigated the role of ROS in TLR-mediated signaling . Purified Escherichia coli LPS, a highly specific TLR4 agonist, elicited an oxidative burst in the monocyte-like cell line THP-1 in a time- and dose-dependent manner . This oxidative burst was shown to be dependent on the presence of TLR4 through transfection studies in HEK cells, which do not normally express this protein, and with bone marrow-derived macrophages from C3H/HeJ mice, which express a mutated TLR4 protein . LPS-stimulated IL-8 expression could be blocked by the antioxidants N-acetyl-L-cysteine and dimethyl sulfoxide at both the protein and mRNA levels . These antioxidants also blocked LPS-induced IL-8 promoter transactivation as well as the nuclear translocation of NF-kappa B . These data provide evidence that ROS regulate immune signaling through TLR4 via their effects on NF-kappa B activation.

Genes Dev, 2004 Mar 1, 18(5), 584 - 94
Targeting of TAK1 by the NF-kappa B protein Relish regulates the JNK-mediated immune response in Drosophila; Park JM et al.; The molecular circuitry underlying innate immunity is constructed of multiple, evolutionarily conserved signaling modules with distinct regulatory targets . The MAP kinases and the IKK-NF-kappa B molecules play important roles in the initiation of immune effector responses . We have found that the Drosophila NF-kappa B protein Relish plays a crucial role in limiting the duration of JNK activation and output in response to Gram-negative infections . Relish activation is linked to proteasomal degradation of TAK1, the upstream MAP kinase kinase kinase required for JNK activation . Degradation of TAK1 leads to a rapid termination of JNK signaling, resulting in a transient JNK-dependent response that precedes the sustained induction of Relish-dependent innate immune loci . Because the IKK-NF-kappa B module also negatively regulates JNK activation in mammals, thereby controlling inflammation-induced apoptosis, the regulatory cross-talk between the JNK and NF-kappa B pathways appears to be broadly conserved.

Biochem Biophys Res Commun, 2004 Apr 9, 316(3), 795 - 801
Periplasmic proteins of Escherichia coli are highly resistant to aggregation: reappraisal for roles of molecular chaperones in periplasm; Liu Y et al.; Periplasmic proteins of Gram-negative bacteria like Escherichia coli are subjected to immediate affect of environmental fluctuation that may unfold proteins, due to the permeability of the outer membrane to small molecules . They are thus supposedly protected by certain molecular chaperones . Nevertheless, no homologues of typical molecular chaperones have so far been found in periplasm, and the recently reported chaperone activities of periplasmic protein disulfide isomerase (PDI) and peptidyl prolyl isomerase (PPI) seem to be too weak to satisfy such assumed needs . In an attempt to reveal whether periplasmic proteins exhibit certain unusual properties, we discovered that such proteins as a whole are highly resistant to aggregation under a wide variety of denaturing conditions . Furthermore, in an effort to unveil the nature behind this phenomenon we purified and examined four prominent periplasmic proteins . Our results demonstrate that these proteins unfold at rather mild denaturing conditions and expose hydrophobic surfaces during such unfolding process, but hardly form complexes with a typical molecular chaperone . Based on these observations, we propose that the periplasmic proteins have been evolved to resist the formation of aggregates when subjected to various denaturing conditions and molecular chaperones may thus not be needed in periplasm.

Respiration, 2004 Mar-Apr, 71(2), 199 - 201
Shewanella putrefaciens isolated in a case of ventilator-associated pneumonia; Jorens PG et al.; We report the isolation of the aquatic gram-negative bacterium Shewanella putrefaciens from the bronchoalveolar lavage in a case of ventilator-associated pneumonia . To our knowledge, this species has never been isolated from the lower respiratory tract before as a potential pathogen .

Genomics, 2004 Apr, 83(4), 658 - 66
Cloning and expression of a mouse member of the PLUNC protein family exclusively expressed in tongue epithelium; LeClair EE et al.; Palate, lung, and nasal epithelium clone (Plunc, now renamed Splunc1) is a small secreted protein expressed in the oropharynx and upper airways of humans, mice, rats, and cows . This protein is structurally homologous to known mediators of host defense against gram-negative bacteria . We have characterized the genomic sequence and expression of a novel but closely related gene from rodents, which we call splunc5 . Mouse Splunc5 sequence is 60% identical to mouse Splunc1 . The genes also share highly conserved genomic elements including intron-exon structure and intronic sequence . Strikingly, splunc5 is expressed exclusively in the interpapillary epithelium of the tongue's dorsal surface . By comparing the expression profiles of splunc5, splunc1, and a third related sequence, lplunc1, in mice, we show that these genes are expressed in unique domains along a continuous corridor of oral, lingual, pharyngeal, and respiratory epithelia . This expression pattern is consistent with the hypothesis that these proteins protect epithelial surfaces colonized by potentially pathogenic microorganisms.

Antioxid Redox Signal, 2004 Apr, 6(2), 311 - 20
Apoptotic cells as sources for biologically active oxidized phospholipids; Kadl A et al.; Acute inflammation is characterized by an accumulation of polymorphonuclear cells (PMNs), generation of reactive oxygen species, subsequent apoptosis of PMNs, and finally phagocytosis of apoptotic cells by macrophages . Recently, it has been demonstrated that during apoptosis oxidation of membrane phospholipids, especially phosphatidylserine, occurs . Moreover, we have shown that membrane vesicles released from apoptotic cells contain biologically active oxidized phospholipids . The involvement of oxidized phospholipids in the development of atherosclerosis, which is described as a chronic inflammatory disease, is increasingly recognized . These oxidized phospholipids were shown to induce several proinflammatory genes, such as monocyte chemoattractant protein 1 or interleukin-8, and it is hypothesized that lipid oxidation products also play a role in other chronic inflammatory disorders . On the other hand, oxidized phospholipids were shown to exert antiendotoxin effects by inhibiting lipopolysaccharide-induced signaling, representing a possible feedback loop during gram-negative infection . Additionally, it has been described that oxidized phospholipids are capable of inducing genes such as heme oxygenase-1 that are important for the resolution of acute inflammation . Moreover, oxidized phospholipids serve as recognition signals on apoptotic cells facilitating phagocytosis . In this review, we discuss the hypothesis that oxidized phospholipids generated in apoptotic cells (a) propagate chronic inflammation and (b) contribute to the resolution of acute inflammation.

J Endotoxin Res, 2004, 10(1), 55 - 65
Endotoxin induces leukocyte transmigration and changes in permeability of the airway epithelium via protein-kinase C and extracellular regulated kinase activation; Serikov VB et al.; Lipopolysaccharide (LPS) endotoxin of Gram-negative bacteria compromises the integrity of the airway epithelial barrier and initiates migration of leukocytes across the epithelium . The goal of the present study was to identify the role of extracellular regulated kinase (ERK1/2) transduction pathways in these processes . The first aim was to determine whether LPS induces ERK1/2 activation and changes in epithelial permeability in epithelial cells alone or only in the presence of immune cells . The second aim was to determine whether the changes in the epithelial permeability were diminished by ERK1/2 blockade . The third aim was to investigate the role of protein kinase C (PKC) activation as an upstream event in activation of ERK1/2 . In vitro 20 microg/ml LPS challenge reduced epithelial barrier function, and induced ERK1/2 phosphorylation in primary cultures of bovine tracheal epithelium and in the transformed human airway epithelial cell line, Calu-3 . LPS initiated migration of neutrophil-like and monocyte-like transformed HL-60 cell across sheets of Calu-3 cells . The migration rate and the associated changes in the electrical resistance, permeability to albumin, and ERK1/2 phosphorylation were all blocked by calphostin C, the specific blocker of PKC and by PD98059 (2'-amino-3'-methoxyflavone), a selective cell-permeable inhibitor of MAP kinase kinase . In rats, in vivo perfusion of the lumen of an isolated segment of trachea with LPS (0.1 mg/ml) initiated migration of neutrophils and increased the permeability to albumin . Again, these effects were markedly inhibited by PD98059 and calphostin C (by > 50%) . We conclude that epithelial ERK1/2 is activated by endotoxin via PKC and is an important pathway in regulation of epithelial permeability.

J Endotoxin Res, 2004, 10(1), 3 - 13
The plasma kallikrein/kinin and renin angiotensin systems in blood pressure regulation in sepsis; Shariat-Madar Z et al.; The hemodynamics of septic shock after endotoxinemia is influenced by the plasma kallikrein/kinin and the renin angiotensin systems . In recent years, new information has improved understanding of the protein/biologically active peptide interactions between these two systems . The plasma kallikrein/kinin system, more commonly known as the contact system, has undergone a re-evaluation as to how it assembles on cell membranes for physiological and pathophysiological activation and as to its role in Gram-negative sepsis . It has been proposed that it counterbalances the plasma renin angiotensin system . Furthermore, more knowledge about the renin angiotensin system has become available on how it either opposes the actions of the kallikrein/kinin system or, in some cases, summates with it . Understanding the interactions between these two systems may lead to development of better pharmacological treatments for endotoxin-induced shock.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 475 - 80
Sulfitobacter delicatus sp . nov . and Sulfitobacter dubius sp . nov., respectively from a starfish (Stellaster equestris) and sea grass (Zostera marina); Ivanova EP et al.; On the basis of data from phenotypic and genotypic characterization and analysis of 16S rRNA gene sequences, two novel species belonging to the genus Sulfitobacter are described . Strains KMM 3584(T), a pale-yellowish, non-motile strain isolated from a starfish (Stellaster equestris), and KMM 3554(T), which is motile by means of a single subpolar flagellum and was isolated from sea grass (Zostera marina), are marine, Gram-negative, aerobic, rod-shaped organisms . Both strains have the ability to degrade gelatin, but not casein, chitin, agar, DNA, Tween 80 or starch . Strain KMM 3584(T) decomposed alginate and grew at NaCl concentrations of 1-8 % and temperatures of 12-37 degrees C, whereas strain KMM 3554(T) grew in 1-12 % NaCl and at temperatures of 10-30 degrees C . The predominant fatty acid was 18 : 1omega7, amounting to up to 80 % of the total fatty acids . The other characteristic feature was the presence of 18 : 2 isomers . The DNA G+C contents of KMM 3584(T) and KMM 3554(T) were respectively 60.0 and 63.7 mol% . The level of DNA similarity between the two strains was 33 % . DNA from KMM 3584(T) and KMM 3554(T) had hybridization values of 5-24 % and 10-41 %, respectively, with DNA from the type strains of Sulfitobacter pontiacus, Sulfitobacter brevis, Sulfitobacter mediterraneus and Staleya guttiformis . It is proposed that strains KMM 3584(T) (=LMG 20554(T)=ATCC BAA-321(T)) and KMM 3554(T) (=LMG 20555(T)=ATCC BAA-320(T)) represent two novel species, Sulfitobacter delicatus sp . nov . and Sulfitobacter dubius sp . nov., respectively.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 457 - 61
Deinococcus indicus sp . nov., an arsenic-resistant bacterium from an aquifer in West Bengal, India; Suresh K et al.; An arsenic- and radiation-resistant bacterium, strain Wt/1a(T), was isolated from water from an arsenic-contaminated aquifer located in the Chakdah district of West Bengal, India . The bacterium stains Gram-negative and is rod-shaped, non-motile, non-sporulating and red-pigmented . Cell-wall peptidoglycan contains ornithine as the diamino acid, MK-8 is the major menaquinone, C(15 : 1) and C(16 : 1) are the major fatty acids and the DNA G+C content of the organism is 65.8 mol% . Based on these phenotypic and chemotaxonomic characteristics, strain Wt/1a(T) was identified as a member of the genus Deinococcus . Strain Wt/1a(T) exhibited maximum 16S rRNA gene sequence similarity (95 %) with Deinococcus grandis; however, strain Wt/1a(T) exhibited only 14 % similarity to D . grandis IAM 13005(T) at the DNA-DNA level . Furthermore, strain Wt/1a(T) (compared to D . grandis IAM 13005(T)) is more resistant to arsenate and arsenite, is positive for arginine dihydrolase, utilizes a number of carbon sources and exhibits quantitative differences in fatty acid composition and qualitative differences in lipid composition . Strain Wt/1a(T) is identified as a novel species of the genus Deinococcus, for which the name Deinococcus indicus sp . nov . is proposed . The type strain of Deinococcus indicus is Wt/1a(T) (=MTCC 4913(T)=DSM 15307(T)).

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 329 - 35
Aeropyrum camini sp . nov., a strictly aerobic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney; Nakagawa S et al.; A novel hyperthermophilic archaeon, designated strain SY1(T), was isolated from a deep-sea hydrothermal vent chimney sample collected from the Suiyo Seamount in the Izu-Bonin Arc, Japan, at a depth of 1385 m . The cells were irregular cocci (1.2 to 2.1 micro m in diameter), occurring singly or in pairs, and stained Gram-negative . Growth was observed between 70 and 97 degrees C (optimum, 85 degrees C; 220 min doubling time), pH 6.5 and 8.8 (optimum, pH 8.0), and salinity of 2.2 and 5.3 % (optimum, 3.5 %) . It was a strictly aerobic heterotroph capable of growing on complex proteinaceous substrates such as yeast extract and tryptone . The G+C content of the genomic DNA was 54.4 mol% . Phylogenetic analysis based on the 16S rDNA sequence of the isolate indicated that the isolate was closely related to Aeropyrum pernix strain K1(T) . However, no significant genetic relatedness was observed between them by DNA-DNA hybridization . On the basis of the molecular and physiological traits of the new isolate, the name Aeropyrum camini sp . nov . is proposed, with the type strain SY1(T) (=JCM 12091(T)=ATCC BAA-758(T)).

Curr Opin Allergy Clin Immunol, 2004 Apr, 4(2), 113 - 7
Hygiene hypothesis and endotoxin: what is the evidence?
Eder W, von Mutius E.
PURPOSE OF REVIEW: The hygiene hypothesis has gained strong support over the past few years . Exposure to microbial products in early life could be an underlying factor in this hypothesis, but the mechanisms that lead from a less clean and more crowded environment to a lower prevalence of asthma and allergies are not known . Among the variety of potential microbial molecules that may confer protection against the development of asthma and allergies, endotoxin, a component of Gram-negative bacteria, has incited lively as well as controversial discussions . This review focuses on recent studies on endotoxin and its role in the context of the hygiene hypothesis . RECENT FINDINGS: Results from cross-sectional surveys, prospective cohorts, and experimental studies in vitro and in rodents suggest that exposure to house dust endotoxin in early life protects from atopic sensitization and IgE-mediated diseases, but is a risk factor for wheezing in infancy . SUMMARY: Numerous studies have supported the hygiene hypothesis, but whether endotoxin by itself confers the protection or whether it acts as a marker for another environmental exposure is still unclear . The challenge for the future will be to identify those factors that confer the protection proposed by the hygiene hypothesis, and to find strategies to modify the environment without causing harm to susceptible individuals.

J Neuroimmunol, 2004 Apr, 149(1-2), 1 - 9
Differential expression of chemokines and chemokine receptors during microglial activation and inhibition; Kremlev SG et al.; Intrauterine infection produces an inflammatory response in the fetus characterized by increased inflammatory cytokines in the fetal brain and activation of brain microglial cells . Intrauterine infection can release bacterial cell wall products into the fetal circulation . Lipopolysaccharides (LPS) are derived from the cell walls of gram negative organisms . The degree of microglial cell activation may influence the extent of brain injury following an inflammatory stimulus . Chemokines, which are released by activated microglia, regulate the influx of inflammatory cells to the brain . Accordingly, therapeutic strategies that reduce the extent of chemokine expression in microglial cells may prove neuroprotective . Minocycline (MN), a semisynthetic tetracycline derivative, protects brain against global and focal ischemia in rodents and inhibits microglial cell activation . To determine if minocycline can reduce the production of chemokines and chemokine receptors in response to LPS, microglial-like BV-2 and HAPI cells were cultured in the presence or absence of 100 ng/ml of LPS . Enzyme-linked immunosorbent assay (ELISA) and semi-quantitative RT-PCR were used to examine changes in inflammatory chemokines (macrophage inflammatory protein-1 (MIP-1alpha), regulated upon activation, normal T cell expressed and secreted (RANTES), and inducible protein-10 (IP-10)) and chemokine receptor (C-C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 3 (CXCR3)) production, respectively . We found that in both cell lines chemokine release after 4-, 8-, and 16-h exposure to LPS was significantly higher compared to non-exposed cells for all the chemokines measured, P<0.001 . Minocycline inhibited chemokine release of LPS-stimulated BV-2 cells . There was even greater inhibition (up to 50%) of mRNA expression after exposure to LPS (P<0.001) . We conclude that endotoxin enhanced the expression of chemokines and chemokine receptors in microglial-like cell lines . Modulation of this expression was achieved with minocycline . Recognition of the mechanisms whereby minocycline exerts its anti-inflammatory effect on microglia may uncover specific targets for pharmacologic intervention.

J Appl Microbiol, 2004, 96(4), 878 - 86
Molecular and phenotypic characterization of nonmotile Gram-negative bacteria associated with spoilage of freshwater fish; Garcia-Lopez I et al.; AIMS: Characterization of nonmotile bacteria associated with freshwater fish spoilage and that phenotypically resembled Psychrobacter spp . METHODS AND RESULTS: A population of 44 nonmotile Gram-negative rods could not be assigned to the genus Psychrobacter on the basis of a definitive test (transformation assay) . Conventional and commercial phenotypic systems did not help in identification . A second extensive phenotypic analysis using different temperatures and media confirmed these isolates as nonmotile although electron microscopic examination showed that all but two had one to four polar flagella and other appendages . On the basis of numerical taxonomy, this population was divided into six clusters, one of them consisting of five fluorescent strains . Sequencing of fluorescent and non fluorescent representative strains from each cluster demonstrated that strains from five clusters had between 97.8 and 98.8% sequence homology with Pseudomonas fragi IFO 3458 . This and an unknown strain from deep sea were the closest organisms (80.9% sequence homology) to one aflagellated representative strain of the remaining cluster . CONCLUSIONS: Oxidase-positive, nonmotile, nonfermenter Gram-negative rods isolated from freshwater fish can be wrongly ascribed to the genus Psychrobacter . SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular methods are necessary for the identification of environmental isolates and species with an incomplete phenotypic description . This work emphasizes the need for a sound description of Ps . fragi based on molecular and phenotypic characterization.

Clin Lab Sci, 2004 Winter, 17(1), 35 - 9
Francisella tularensis: possible agent in bioterrorism; Gallagher-Smith M et al.; Francisella tularensis, the causative agent of tularemia, is a highly infectious gram-negative coccobacillus . Due to its high infectivity it is of major concern to public health officials as a possible biological weapon . Although accidental exposure can occur through arthropod bites, handling infected animals, or breathing in aerosols, cases are usually isolated and contained . In the event of an intentional exposure such as in a bioterrorist attack, inhalation of aerosols can result in devastating consequences with much causality . Although a vaccine is available, sufficient quantities may not be readily accessible in an actual attack . Therefore, it is very important for both medical professionals and public health officials to be prepared to contain and control the situation should it actually occur.

Proc Natl Acad Sci U S A, 2004 Mar 23, 101(12), 4186 - 91 Epub 2004 Mar 09.
Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations; Gioannini TL et al.; Host proinflammatory responses to minute amounts of endotoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2 . Optimal sensitivity to endotoxin requires an ordered series of endotoxin-protein and protein-protein interactions . At substoichiometric concentrations, LBP facilitates delivery of endotoxin aggregates to soluble CD14 (sCD14) to form monomeric endotoxin-sCD14 complexes . Subsequent interactions of endotoxin-sCD14 with TLR4 and/or MD-2 have not been specifically defined . This study reports the purification of a stable, monomeric, bioactive endotoxin-MD-2 complex generated by treatment of endotoxin-sCD14 with recombinant MD-2 . Efficient generation of this complex occurred at picomolar concentrations of endotoxin and nanogram per milliliter doses of MD-2 and required presentation of endotoxin to MD-2 as a monomeric endotoxin-CD14 complex . TLR4-dependent delivery of endotoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of endotoxin occurred with the purified endotoxin-MD-2 complex, but not with purified endotoxin aggregates with or without LBP and/or sCD14 . The presence of excess MD-2 inhibited delivery of endotoxin-MD-2 to HEK/TLR4 cells and cell activation . These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of endotoxin occurs by sequential interaction and transfer of endotoxin to LBP, CD14, and MD-2 and simultaneous engagement of endotoxin and TLR4 by MD-2.

Ginecol Obstet Mex, 2003 Oct, 71, 515 - 21
{Bacteriological study of surgical infected wounds in elective surgery . Bacteriology of surgical wound infection}; Rembis-Sainz V et al.; OBJECTIVE: To identify the frequency and type of microorganisms isolated from infected surgical wounds at the Instituto Nacional de Perinatologia, and identify the association among the microorganisms isolated and the outcome of the infected patients . METHODOLOGY: Observational, descriptive and cross-sectional study carried out between January 1999 and January 2001 . Postoperative patients of an obstetric or gynecologic procedure, complicated with a surgical wound infection and with a culture and smear of the wound, were included . RESULTS: During the study period 41 surgical wound infections were identified . The general incidence of surgical infections was 3.9 infections per 1,000 surgical procedures . The incidence of infections after abdominal hysterectomy was 12.4 per 1,000, after vaginal hysterectomy 6.5 per 1,000, postcesarean section 5.3 per 1,000 and after episiotomy 1 per 1,000 procedures . The comparison among bacterial isolates in 1988 with 1999-2000 period showed a high frequency of gram negative bacteria isolation in the second period . CONCLUSIONS: In this study the incidence of surgical infections was less than the incidence reported in the medical literature, but the type of bacteria isolated was similar to other studies.

Biol Blood Marrow Transplant, 2004 Mar, 10(3), 204 - 12
Alpha-interferon with very-low-dose donor lymphocyte infusion for hematologic or cytogenetic relapse of chronic myeloid leukemia induces rapid and durable complete remissions and is associated with acceptable graft-versus-host disease; Posthuma EF et al.; Donor lymphocyte infusion (DLI) results in complete cytogenetic remission (CCR) of relapsed chronic-phase chronic myeloid leukemia (CML-CP) after allogeneic stem cell transplantation (SCT) in up to 80% of patients . The main complication of DLI is graft-versus-host disease (GVHD) . Decreasing the dose of DLI is associated with less GVHD but also with a longer interval between treatment and CCR . We postulated that combining alpha-interferon (alpha-IFN) with DLI would enable us to decrease the dose of DLI, thereby limiting GVHD, and at the same time to decrease the interval between DLI and CCR for patients with either a hematologic or cytogenetic relapse . For molecular relapses, we hypothesized that because of a lower tumor load, very low doses of DLI without alpha-IFN could be an effective treatment . Two groups of CML-CP patients treated with DLI at a very low dose of 0.5 to 1.0 x 10(7) mononuclear cells per kilogram, containing 2 to 6 x 10(6) CD3+ T cells per kilogram, were analyzed: 13 patients with a cytogenetic or a hematologic relapse after allogeneic SCT (group A) were treated with additional alpha-IFN therapy at a dose of 3 x 10(6) U 5 d/wk, and 8 patients with a molecular relapse were treated without alpha-IFN (group B) . Twelve patients from group A reached a CCR . The median interval between DLI and CCR was 7 weeks (range, 5-18 weeks) for group A . All patients with a CCR reached complete donor chimerism at a median of 10 weeks after DLI (range, 6-121 weeks) . Eleven patients reached molecular remission at a median of 15 weeks after DLI (range, 8-34 weeks) . In group B, all patients reached a molecular remission at a median of 14 weeks (range, 12-29 weeks) . Five patients from group A developed acute GVHD grade II to IV and extensive chronic GVHD . In group B, 1 patient developed acute GVHD grade II to IV and subsequently developed extensive chronic GVHD . With a median follow-up of 62 months, 10 patients in group A are alive and in continuous CCR . One patient had a molecular relapse, for which she successfully received additional DLI; another patient reached molecular remission only after 5 doses of DLI . Two patients from group A died of a gram-negative sepsis, and 1 died of an acute myocardial infection . In group B, all patients are alive and in molecular remission with a median follow-up of 20 months . One patient's disease progressed but was successfully treated with DLI plus alpha-IFN . In conclusion, very-low-dose DLI in combination with alpha-IFN as treatment for cytogenetic or hematologic relapses of CML-CP after allogeneic SCT reduced the interval to obtain a CCR with acceptable GVHD when compared with the literature . Patients with a CCR also reached complete donor chimerism and complete molecular remissions . For patients with a molecular relapse, very-low-dose DLI alone is sufficient to induce molecular remissions in most patients and is associated with limited GVHD.

Microbiology, 2004 Mar, 150(Pt 3), 649 - 56
Downregulation of the motA gene delays the escape of the obligate predator Bdellovibrio bacteriovorus 109J from bdelloplasts of bacterial prey cells; Flannagan RS et al.; Bdellovibrio bacteriovorus is a Gram-negative bacterium that preys on other Gram-negative bacteria . The lifecycle of B . bacteriovorus alternates between an extracellular flagellated and highly motile non-replicative attack-phase cell and a periplasmic non-flagellated growth-phase cell . The prey bacterium containing periplasmic bdellovibrios becomes spherical but osmotically stable, forming a structure known as the bdelloplast . After completing the growth phase, newly formed bdellovibrios regain their flagellum and escape the bdelloplast into the environment, where they encounter more prey bacteria . The obligate predatory nature of B . bacteriovorus imposes a major difficulty to introducing mutations in genes directly involved in predation, since these mutants could be non-viable . This work reports the cloning of the B . bacteriovorus 109J motAB operon, encoding proteins from the flagellar motor complex, and a genetic approach based on the expression of a motA antisense RNA fragment to downregulate motility . Periplasmic bdellovibrios carrying the plasmid expressing antisense RNA displayed a marked delay in escaping from bdelloplasts, while the released attack-phase cells showed altered motility . These observations suggest that a functionally intact flagellar motor is required for the predatory lifecycle of B . bacteriovorus . Also, the use of antisense RNA expression may be a useful genetic tool to study the Bdellovibrio developmental cycle.

Biochemistry, 2004 Mar 9, 43(9), 2373 - 83
Fluorinated aminoglycosides and their mechanistic implication for aminoglycoside 3'-phosphotransferases from Gram-negative bacteria; Kim C et al.; Aminoglycoside 3'-phosphotransferases {APH(3')s} are important bacterial resistance enzymes for aminoglycoside antibiotics . These enzymes phosphorylate the 3'-hydroxyl of these antibiotics, a reaction that inactivates the drug . A series of experiments were carried out to shed light on the details of the turnover chemistry by these enzymes . Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3') types Ia and IIa with kanamycin A, neamine, and their respective difluorinated analogues 4'-deoxy-4',4'-difluorokanamycin A and 4'-deoxy-4',4'-difluoroneamine were carried out, in conjunction with measurements of thio effect and viscosity studies . The fluorinated analogues were shown to be severely impaired as substrates for these enzymes . The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3')-Ia was studied with nonfluorinated substrates . Residue 198 is the proposed active site base that promotes the aminoglycoside hydroxyl for phosphorylation . These findings collectively argue that the Gram-negative APH(3')s show significant nucleophilic participation in the transition state for the phosphate transfer reaction.

J Cell Biochem, 2004 Mar 1, 91(4), 786 - 95
Effect of LPS on basal and induced apo E secretion by 25-OH chol and 9cRA in differentiated CaCo-2; Ripolles Piquer B et al.; The infection and inflammation process is associated with disturbances in lipid and lipoprotein metabolism . The apolipoprotein E (apo E) plays an important role in the lipoprotein metabolism and has been linked to inflammatory disease such as atherosclerosis and Alzheimer disease . An anti-inflammatory effect has also been suggested . The heterodimer nuclear receptor Liver-X-Receptor(alpha)/Retinoid-X-Receptor (LXR(alpha)/RXR) is considered to be a transcription factor for apo E . The aim of this study was to determine whether lipopolysaccharide (LPS) (principal component of the outer membrane Gram-negative bacteria) has an effect on apo E secretion by intestinal mucosa cells, using the Caco-2 cell line . Differentiated Caco-2 cells grown on filter inserts were incubated apically with LPS and/or 25-hydroxycholesterol (25-OH chol) and 9 cis retinoic acid (9cRA), ligands of LXR and RXR, respectively . The apical and basolateral media were separately collected . Apo E was detected by specific antibodies after protein separation by Two-dimensional nondenaturing gradient gel electrophoresis and apo E secreted in the cell culture media was measured by enzyme linked immunosorbent assay (ELISA) . Apo E mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) . LXR(alpha) and RXR mass was analyzed by Western Blot . We demonstrate here that CaCo-2 cells secrete apo E, by either apical or basolateral sides, associated with a high-density like lipoprotein, with a stoke's diameter comprised between 7.10 and 8.16 nm . We show that only apical secretion is decreased by LPS in a dose and time dependent manner . This is associated with a decrease in apo E gene expression contrasting with an increase of Il-8, a chemokine factor . Moreover, we demonstrate that only basolateral apo E secretion by CaCo-2 is significantly increased by 25-OH chol and 9cRA while apical secretion remains unchanged . LPS does not decrease the 25-OH chol and 9cRA mediated apo E secretion in basolateral compartment, while apical secretion is diminished under these circumstances . Our results provide evidence for the polarized secretion of apo E by intestinal epithelium . They also demonstrate that apo E secretion by CaCo-2 cell line is decreased by LPS through an LXR(alpha)/RXR independent signaling pathway .

J Autoimmun, 2004 Mar, 22(2), 153 - 8
Immune response to lipopolysaccharide in primary biliary cirrhosis and autoimmune diseases; Ballot E et al.; A bacteriological aetiology is suspected to be the triggering factor in primary biliary cirrhosis . We studied lipid A, the toxic and immunogenic moiety of gram-negative bacteria lipopolysaccharide, which accumulates abnormally in Kupffer cells, hepatocytes, and biliary epithelial cells in primary biliary cirrhosis patients . Anti-lipid A antibody levels from serum samples from 36 primary biliary cirrhosis patients, drawn before and after ursodeoxycholic acid treatment, were compared to those from patients with other liver diseases (n=236), non-hepatic diseases (n=249), and healthy subjects (n=75) . In primary biliary cirrhosis patients, the prevalence of IgM anti-lipid A antibodies was higher before than after ursodeoxycholic acid therapy (64% vs 22%, respectively; P<0.001) . Patients with anti-lipid A antibodies had significantly higher IgM levels than those without antibodies (8.7+/-1.1 g/l vs 4.4+/-0.8 g/l, P<0.02) . Total IgM levels were correlated with anti-lipid A antibody levels (r=0.65, P<0.02) . After therapy, the serum IgM levels decreased significantly (P<0.03) . These results indicate that bacterial antigens may participate in the observed increase of serum IgM levels, and support an aetiological role of a gut-derived endotoxin antigen in the pathogenesis of primary biliary cirrhosis.

Pediatr Crit Care Med, 2004 Mar, 5(2), 112 - 5
Terlipressin bolus induces systemic vasoconstriction in septic shock; Peters MJ et al.; OBJECTIVE: To report the use of a synthetic, long-acting, vasopressin analog, terlipressin, as an effective vasoconstrictor in septic shock . DESIGN: Case report . SETTING: A 22-bed pediatric intensive care unit in a tertiary referral center . Patient: An 11-yr-old male with multiple-organism Gram-negative septic shock with high normal cardiac output as assessed by pulse contour analysis and low systemic vascular resistance despite norepinephrine infusion . INTERVENTION: Two peripherally administered doses of terlipressin (0.5 mg) . MEASUREMENTS AND MAIN RESULTS: Each dose of terlipressin was associated with a rapid increase in systemic vascular resistance, despite weaning and discontinuation of norepinephrine infusion from 0.15 microg.kg(-1).min(-1) lasting approximately 6 hrs . CONCLUSION: Terlipressin may be useful for sepsis-induced vasodilation.

Am J Pathol, 2004 Mar, 164(3), 763 - 72
Bacterial chaperone protein, Skp, induces leukocyte chemotaxis via C5a receptor; Shrestha A et al.; C5a receptor has been identified as a leukocyte chemotactic receptor to two intrinsic chemical mediators, C5a and the S19 ribosomal protein dimer, so far . We found an Escherichia coli protein that also induced the chemotactic responses of monocytes and polymorphonuclear leukocytes via the C5a receptor . We identified the E . coli-derived chemoattractant to be Skp by the molecular size and the N-terminal amino acid sequence . Skp is a periplasmic chaperone protein widely present in gram-negative bacterial species . Immunoabsorption experiments indicated that Skp was the major leukocyte chemotactic factor in the E . coli extract . Receptor-antagonizing experiments with analogue peptides of S19 ribosomal protein and of C5a suggested that Skp induced the receptor activation by the two-step binding mechanism as in the cases of the intrinsic mediators, sharing the ligand-binding site of the receptor among them at each binding step . The C5a receptor would play a role in the host defense directly recognizing the bacteria-derived protein, besides identifying the signals of the intrinsic chemical mediators.

South Med J, 2004 Feb, 97(2), 194 - 8
Nanobacteria-caused mitral valve calciphylaxis in a man with diabetic renal failure; Jelic TM et al.; We have found that nanobacteria, recently discovered Gram-negative atypical bacteria, can cause local calciphylaxis on the mitral valve in a setting of high-calcium X phosphorous product in the blood . We present the case of a 33-year-old man with diabetic renal failure on continuous ambulatory peritoneal dialysis who died as a result of multiple brain infarcts due to embolizations from mitral valve vegetations . Systemic calciphylaxis was not present . Spectrometric analysis of the mitral valve vegetations showed that they were composed of calcium phosphate, carbonate apatite form, and fibrin . The electron microscopy of the thrombotic vegetation demonstrated nanobacterium as a nidus for carbonate apatite formation . Investigation for the presence of nanobacteria in the multiple organs involved in systemic calciphylaxis may be of help in elucidating the pathogenesis of this frequently fatal disorder.

Paediatr Respir Rev, 2004, 5 Suppl A, S65 - 71
Something old, something new: indoor endotoxin, allergens and asthma; Liu AH; Endotoxin and allergen exposure have been explored in the context of asthma for more than a century . Building upon a pyramid of knowledge are recent observations that provide new insights to the effect of these exposures on the development of asthma . Some of these studies challenge some previously held concepts of the role of these exposures in asthma inception . Indoor allergens are well established as the basis of inflammation in sensitised asthmatics, contributing to disease severity . Then does greater exposure to indoor allergens cause allergen sensitisation and asthma as well? While risk of sensitisation to house dust mites generally increases with higher levels of exposure, this does not seem to hold for cats, where higher levels of cat allergen exposure are associated with less sensitisation . Indeed, several recent studies suggest that early childhood exposure to animals, as indoor pets or in farming stables, are associated with a lower prevalence of asthma, hay fever, and inhalant allergen sensitisation . Endotoxin in asthma provides a similar paradox . Endotoxin is a potent immune-stimulatory component of the bacterial cell wall of all gram-negative bacteria . As such, endotoxin is ubiquitous in our environment . Endotoxin exposure has been well demonstrated to underlie "Monday Asthma" or byssinosis in cotton workers, and has since emerged as a frequent cause of asthma-like symptoms in a wide range of occupational settings . Asthmatics are particularly sensitive to inhaled endotoxin, and inhalation induces both immediate and sustained airflow obstruction . The paradox of endotoxin exposure is that higher levels of exposure in early life might mitigate the development of allergy and persistent asthma . With endotoxin exposure being significantly higher in homes with animals and in farming households, where allergy and asthma are less likely to develop, endotoxin and other microbial exposures in early life may keep allergen sensitisation and asthma from developing by promoting Th1-type immune development . These observations, consistent with the "Hygiene Hypothesis" of allergy and asthma, are an encouraging glimpse of the potential for early immune modulatory approaches to asthma therapy and prevention.

Trop Gastroenterol, 2003 Jul-Sep, 24(3), 124 - 8
Stress ulcer prophylaxis in patients on ventilator; Darlong V et al.; The objective of the study was to assess the efficacy of the H2-receptor antagonists and sucralfate for the prophylaxis of stress ulcer in patients on a ventilator in an intensive care unit in the general intensive care unit of our institute . A randomized, clinical controlled trial was conducted . Fifty-two critically ill patients, who required mechanical ventilation for more than 24 hours, were randomly divided into 3 groups . Group I received ranitidine 50 mg (intravenous) 8 hourly, group II received tablet sucralfate 1 g 8 hourly through a Ryle's tube, whereas group III was not given any drug . The incidence of upper gastrointestinal bleed, change in gastric pH and growth of gram-negative organisms in the gastric juice and bronchoalveolar lavage (BAL) culture were noted and analysed . The treatment groups were similar with respect to the baseline characteristics . The incidence of upper gastrointestinal bleeding was similar in the ranitidine (12.5%) and sucralfate groups (14.35%) but was high in the control group (57.14%) . The mean gastric pH was significantly low in the control group (mean pH 2.07) compared to the ranitidine (mean pH 5.25) and sucralfate groups(mean pH 3.54)(p < 0.05) . The incidence of positive culture for gram-negative organisms was significantly high in the ranitidine group (75%) in comparison with the sucralfate group (33.33%) (p < 0.002) . However, the incidence of positive growth in the BAL culture was similar in all three groups . We conclude that both ranitidine and sucralfate are equally effective in decreasing the incidence of upper gastrointestinal haemorrhage and other stress- related lesions . Though ranitidine was more effective in increasing the gastric pH, the incidence of gastric colonization was higher in the ranitidine group compared to the sucralfate group.

Infect Immun, 2004 Mar, 72(3), 1487 - 95
Nod1 is an essential signal transducer in intestinal epithelial cells infected with bacteria that avoid recognition by toll-like receptors; Kim JG et al.; The transcription factor NF-kappaB in human intestinal epithelial cells plays a central role in regulating genes that govern the onset of mucosal inflammatory responses following intestinal microbial infection . Nod1 is a cytosolic pattern recognition receptor in mammalian cells that senses components of microbial peptidoglycans and signals the activation of NF-kappaB . The aim of these studies was to assess the functional importance of Nod1 in activating NF-kappaB and NF-kappaB proinflammatory target genes in human intestinal epithelium . Human colon epithelial cells that constitutively express Nod1 were used as a model intestinal epithelium . These cells do not signal through Toll-like receptor 4 (TLR4) or respond to bacterial lipopolysaccharide, but they express functional TLR5 and interleukin 1 (IL-1) receptors that signal the activation of NF-kappaB in response to bacterial flagellin or IL-1 stimulation . Stable expression of dominant negative (DN) Nod1 in colon epithelial cells prevented IkappaB kinase and NF-kappaB activation in response to infection with enteroinvasive Escherichia coli . In contrast, DN Nod1 did not eliminate IL-1 or flagellin-stimulated NF-kappaB activation . Inhibition of NF-kappaB was accompanied by inhibition of NF-kappaB target genes that provide signals for the mucosal influx of neutrophils during intestinal infection . We conclude that signaling through Nod1 is required for activating NF-kappaB in human intestinal epithelial cells infected with gram-negative enteric bacteria that can bypass TLR activation . Signaling through Nod1 provides the intestinal epithelium with a backup mechanism for rapidly activating innate immunity during infection with a group of highly invasive pathogenic gram-negative bacteria.

Infect Immun, 2004 Mar, 72(3), 1223 - 9
Role of MyD88 in diminished tumor necrosis factor alpha production by newborn mononuclear cells in response to lipopolysaccharide; Yan SR et al.; Human newborns are more susceptible than adults to infection by gram-negative bacteria . We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS) . In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-alpha) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation . At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-alpha . However, in the absence of plasma, MNC from newborns secreted significantly less TNF-alpha compared to MNC from adults . Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-alpha secretion was significantly lower for newborn MNC compared to adult MNC . Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry . However, the intensity of the CD14 marker was greater for adult than for newborn cells . Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells . The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IkappaBalpha phosphorylation, as well as IkappaBalpha degradation . Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88) . We found a direct relation between adult and newborn TNF-alpha secretion and MyD88, which was significantly decreased in newborn monocytes . Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-alpha secretion in newborn monocytes.

Biometals, 2004 Feb, 17(1), 53 - 64
Catecholates and mixed catecholate hydroxamates as artificial siderophores for mycobacteria; Wittmann S et al.; Different mono-, bis- or triscatecholates and mixed mono- or biscatecholate hydroxamates were synthesized as potential siderophores for mycobacteria . SiderOphore activity was tested by growth promotion assays using wild type strains and iron transport mutants of mycobacteria as well as Gram-negative bacteria . Some triscatecholates and biscatecholate hydroxamates were active in mutants of Mycobacterium smegmatis deficient in mycobactin and exochelin biosynthesis or exochelin permease, respectively, indicating an uptake route independent of the exochelin/mycobactin pathway . Structure activity relationships were studied . Ampicillin conjugates of some of these compounds were inactive against mycobacteria but active against Gram-negative bacteria.

J Endod, 2004 Feb, 30(2), 88 - 91
Proinflammatory cytokine profiles in pulp fibroblasts stimulated with lipopolysaccharide and methyl mercaptan; Coil J et al.; Pulpal disease is intimately associated with the immune system's response to bacteria products . Clinical pathology is mediated in part by the production of pyrogenic cytokines, especially interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and IL-6 . Methyl mercaptan (CH3SH), a volatile sulfur compound produced by anaerobic Gram-negative bacteria, has been shown to contribute to the production of IL-1 by human mononuclear cells . In this report, we investigated the production of IL-1, TNF-alpha, and IL-6 by human pulp fibroblasts when stimulated for various periods of time with lipopolysaccharide (LPS) with or without the presence of CH3SH . We found that LPS and CH3SH had no effect on the production of IL-1 or TNF-alpha . However, LPS stimulated IL-6 production, and this production was augmented when CH3SH was present . We conclude that the volatile sulfur compound CH3SH plays a role in activation and modulation of the immune response through its role in production of IL-6.

Gynecol Obstet Invest, 2004, 57(4), 224 - 32 Epub 2004 Feb 11.
Gram-negative bacterial endotoxin- induced infertility: a birds eye view; Deb K et al.; Alleviation of infertility on the one hand and development of improved methods of contraception on the other are global concerns to woman's health . The molecular signals that regulate implantation are of clinical relevance since understanding the nature of these signals may lead to strategies to correct implantation failure and to develop novel contraceptive approaches . The other pressing concern is the poor pregnancy rate resulting from in vitro fertilization (IVF) . The pregnancy rate in IVF programs remains about 20-30% in spite of the high rate of successful fertilization . This has led to the proposition that additional uterine factors, critical for the implantation process, must be limiting . Identification of such parameters could help in determining the appropriate physiological state of the uterus for embryo transfer . Several factors are known to have a direct or indirect impact on the ability of the uterus to develop to a functionally receptive state . This would disrupt the normal coordination between embryonic and uterine development even though all molecular players may seem otherwise normal .

J Med Microbiol, 2004 Mar, 53(Pt 3), 245 - 8
Capnocytophaga canimorsus endocarditis; Sandoe JA; Capnocytophaga canimorsus is a fastidious, Gram-negative rod that forms part of the normal oral flora of dogs and cats . Known for its ability to cause fulminant sepsis following dog bites, particularly in asplenic patients or alcoholics, this bacterium is also an uncommon cause of endocarditis . This article reviews 12 cases of endocarditis caused by C . canimorsus . Mean age of patients was 53 years, with 78% of cases occurring in males . Overall, a history of dog-bite was documented in four cases (33%) and a further four (33%) reported contact with dogs . Four (33%) of the endocarditis cases had underlying cardiological risk factors and two abused alcohol, but none had had a previous splenectomy . Subacute presentation, often involving more than one hospital admission, was common, as were initially negative blood cultures . A variety of antibiotics was used, but penicillins were the most common therapy . Three (25%) of the 12 endocarditis patients died.

Circulation, 2004 Feb 17, 109(6), 784 - 9
Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice; Oyama J et al.; BACKGROUND: Myocardial ischemia and reperfusion-induced tissue injury involve a robust inflammatory response, but the proximal events in reperfusion injury remain incompletely defined . Toll-like receptor 4 (TLR4) is a proximal signaling receptor in innate immune responses to lipopolysaccharide of Gram-negative pathogens . TLR4 is also expressed in the heart and vasculature, but a role for TLR4 in the myocardial response to injury separate from microbial pathogens has not been examined . This study assessed the role of TLR4 in myocardial infarction and inflammation in a murine model of ischemia-reperfusion injury . METHODS AND RESULTS: Myocardial ischemia-reperfusion (MIR) was performed on 2 strains of TLR4-deficient mice (C57/BL10 ScCr and C3H/HeJ) and controls (C57/BL10 ScSn and C3H/OuJ) . Mice were subjected to 1 hour of coronary ligation, followed by 24 hours of reperfusion . TLR4-deficient mice sustained significantly smaller infarctions compared with control mice given similar areas at risk . Fewer neutrophils infiltrated the myocardium of TLR4-deficient Cr mice after MIR, indicated by less myeloperoxidase activity and fewer CD45/GR1-positive cells . The myocardium of TLR4-deficient Cr mice contained fewer lipid peroxides and less complement deposition compared with control mice after MIR . Serum levels of interleukin-12, interferon-gamma, and endotoxin were not increased after ischemia-reperfusion . Neutrophil trafficking in the peritoneum was similar in all strains after injection of thioglycollate . CONCLUSIONS: TLR4-deficient mice sustain smaller infarctions and exhibit less inflammation after myocardial ischemia-reperfusion injury . The data suggest that in addition to its role in innate immune responses, TLR4 serves a proinflammatory role in murine myocardial ischemia-reperfusion injury.

J Biol Chem, 2004 Feb 20, 279(8), 6658 - 65 Epub 2003 Dec 05.
Transcriptional regulation of the cyclooxygenase-2 gene in macrophages by PU.1; Joo M et al.; Macrophages are an abundant source of cyclooxygenase-2 (COX-2) enzymatic products, but a specific mechanism for macrophage COX-2 gene expression has not been described . We examined whether PU.1, a myeloid-specific Ets family transcription factor, is involved . Sequence analysis revealed two potential c-Ets binding sites in the COX-2 promoter (COX-2p) which bind to immunoreactive PU.1 . Chromatin immunoprecipitation analysis shows inducible PU.1 binding to these sites in response to lipopolysaccharide, and COX-2 protein production is augmented by ectopic expression of PU.1 but not by PU.1S148A, indicating that PU.1 phosphorylation is likely involved . Interestingly, expression of PU.1 results in acetylation of CCAAT/enhancer-binding protein-beta (C/EBP-beta) and increased production of COX-2 protein . Coimmunoprecipitation experiments suggest a role for p300 in C/EBP-beta acetylation and COX-2 expression . In contrast, E1A inhibits acetylation of C/EBP-beta and is correlated with decreased COX-2 expression . Together, these data suggest that PU.1 is activated by phosphorylation of Ser148 in response to lipopolysaccharide treatment and subsequently binds to sequences in the endogenous COX-2p in a time-dependent manner . Concomitantly, C/EBP-beta becomes acetylated, and expression of the COX-2 gene increases . We speculate that a combinatorial role of PU.1 and C/EBP-beta mediates the robust production of COX-2 products by macrophages which occurs in Gram-negative bacterial sepsis.

J Neurosci, 2004 Feb 11, 24(6), 1340 - 9
Exacerbation of motor neuron disease by chronic stimulation of innate immunity in a mouse model of amyotrophic lateral sclerosis; Nguyen MD et al.; Innate immunity is a specific and organized immunological program engaged by peripheral organs and the CNS to maintain homeostasis after stress and injury . In neurodegenerative disorders, its putative deregulation, featured by inflammation and activation of glial cells resulting from inherited mutations or viral/bacterial infections, likely contributes to neuronal death . However, it remains unclear to what extent environmental factors and innate immunity cooperate to modulate the interactions between the neuronal and non-neuronal elements in the perturbed CNS . In the present study, we addressed the effects of acute and chronic administration of lipopolysaccharide (LPS), a Gram-negative bacterial wall component, in a genetic model of neurodegeneration . Transgenic mice expressing a mutant form of the superoxide dismutase 1 (SOD1(G37R)) linked to familial amyotrophic lateral sclerosis were challenged intraperitoneally with a single nontoxic or repeated injections of LPS (1 mg/kg) . At different ages, SOD1(G37R) mice responded normally to acute endotoxemia . Remarkably, only a chronic challenge with LPS in presymptomatic 6-month-old SOD1(G37R) mice exacerbated disease progression by 3 weeks and motor axon degeneration . Closely associated with the severity of disease is the stronger and restricted upregulation of the receptor of innate immunity Toll-like receptor 2 and proinflammatory cytokines in degenerating regions of the ventral spinal cord and efferent fiber tracts of the brain from the LPS-treated SOD1(G37R) mice . This robust immune response was not accompanied by the establishment of acquired immunity . Our results provide solid evidence that environmental factors and innate immunity can cooperate to influence the course of disease of an inherited neuropathology.

Respiration, 2004 Jan-Feb, 71(1), 66 - 71
Gut decontamination prevents bronchoscopy-induced bacterial translocation . An experimental study in rats; Nayci A et al.; BACKGROUND: Selective gut decontamination is suggested to suppress the gram-negative bacterial overgrowth in the intestine and consequently to reduce bacterial translocation . OBJECTIVE: The purpose of the present study is to examine the effects of gut decontamination on bronchoscopy-induced bacterial translocation, and intestinal mucosal injury . METHODS: Forty-five rats were assigned into three groups . Group 1 served as control (n=15) . Group 2 (n=15) and group 3 (n=15) underwent bronchoscopy . In addition, group 3 underwent gut decontamination . Gut decontamination was performed two days prior to bronchoscopy with erythromycin and neomycin . Twenty-four hours after bronchoscopy, blood, mesenteric lymph nodes, spleen, liver, ileum and cecum were harvested for bacterial determination . The ileum was also assessed and graded histologically according to Chiu's injury scale . RESULTS: In the bronchoscopy group, bacterial translocation to the mesenteric lymph nodes was found in 7/15 rats (46.7%), compared to none of the controls (p=0.01) . These rats also showed significant evidence of intestinal injury, compared to the controls (mean ranks, 32.7 or 8.5, p<0.0001) . On the other hand, gut decontamination prevented bacterial translocation, compared to the bronchoscopy group (p=0.011) . However, gut decontamination provided no beneficial effect on the intestinal mucosal injury, compared to the bronchoscopy group . These animals also revealed significant intestinal injury, compared to the controls (mean ranks, 27.8 or 8.5, p<0.0001) . CONCLUSIONS: Our data shows that despite no amelioration in bronchoscopy-induced intestinal mucosal injury, gut decontamination has a preventive role for bronchoscopy-induced bacterial translocation .

Clin Pharmacokinet, 2004, 43(3), 205 - 10
Bayesian pharmacokinetics of gentamicin in a haemodialysis population; Vercaigne LM et al.; BACKGROUND: Aminoglycosides are commonly used in the haemodialysis population . Standard pharmacokinetic approaches require multiple sampling to describe the parameters of drug distribution and elimination in the intra- and interdialytic periods . OBJECTIVE: To characterise the pharmacokinetics of gentamicin in a haemodialysis population by using Bayesian pharmacokinetic methods and only two plasma concentrations . DESIGN AND PARTICIPANTS: Prospective case series of 13 adult (aged 36-70 years) haemodialysis patients (Fresenius F80 dialysers were used) receiving gentamicin . METHODS: Patients with suspected or confirmed Gram-negative infections were given gentamicin . At 48 hours after receiving the dose (at the next haemodialysis session), patients provided two blood samples, one immediately before the dialysis session and another 1 hour after haemodialysis . Data on dosage, timing and plasma concentrations for all subjects were analysed with PASTRX version 10.6 and Bayesian pharmacokinetic analysis . Volume of distribution (Vd), interdialytic elimination rate constant (k(inter)), interdialytic elimination half-life (t1/2beta, inter)) and interdialytic clearance (CL(inter)) were determined from a single predialysis plasma concentration . Elimination rate constant (k(dial)), elimination half-life (t1/2beta, dial)) and clearance (CL(dial)) during 3.5-4 hours of dialysis were also determined from the pre- and post-plasma concentrations . RESULTS: Pharmacokinetic parameters (mean +/- SD) were: Vd 0.288 +/- 0.002 L/kg, k(inter) 0.015 +/- 0.004h(-1), t1/2beta, inter) 48 +/- 11h, CL(inter) 5.9 +/- 2.4 mL/min, k(dial) 0.25 +/- 0.05 h(-1), t1/2beta, dial) 3.0 +/- 1.0h and CL(dial) 91 +/- 24 mL/min . CONCLUSIONS: The rate of elimination of gentamicin was 17-fold greater (95% CI 13.7-20.7) on haemodialysis with a Fresenius F80 than off haemodialysis . All of the pharmacokinetic parameters of interest were determined using Bayesian pharmacokinetic procedures and only two plasma gentamicin concentrations.

Urol Res . 2004 Feb 6; {Epub ahead of print}
Morphological and immunological characteristics of nanobacteria from human renal stones of a north Indian population; Khullar M et al.; The aim of this study was to detect, isolate and characterize the nanobacteria from human renal stones from a north Indian population, and to determine their role in biomineralization . Renal stones retrieved from the kidneys of 65 patients were processed and subjected to mammalian cell culture conditions . The isolated bacteria were examined using scanning (SEM) and transmission electron microscopy (TEM) . They were characterized for the presence of DNA, proteins and antigenicity . The role of these bacteria in biomineralization was studied by using the (14)C-oxalate based calcium oxalate monohydrate (COM) crystallization assay . We observed the presence of apatite forming, ultrafilterable gram negative, coccoid microorganisms in 62% of the renal stones . SEM studies revealed 60-200 nm sized organisms with a distinct cell wall and a capsule . TEM images showed needle like apatite structures both within and surrounding them . They were heat sensitive, showed antibiotic resistance and accelerated COM crystallization . A potent signal corresponding to the presence of DNA was observed in demineralized nanobacterial cells by flow cytometry . The protein profile showed the presence of several peptide bands of which those of 18 kDa and 39kDa were prominent . Apatite forming nanosized bacteria are present in human renal stones and may play a role in the pathophysiology of renal stone formation by facilitating crystallization and biomineralization . However, further studies are required to establish the exact mechanism by which nanobacteria are involved in the causation of renal stones.

Appl Environ Microbiol, 2004 Feb, 70(2), 712 - 21
Modular broad-host-range expression vectors for single-protein and protein complex purification; Fodor BD et al.; A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created . The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation . They are useful in the purification of proteins and protein complexes from gram-negative bacterial species . The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter . These examples demonstrate the versatility and flexibility of the vectors . The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host . The vectors were useful in purifying different proteins from T . roseopersicina . The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation . These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.

J Immunol, 2004 Feb 15, 172(4), 2039 - 47
Inhibitory effect of Toll-like receptor 4 on fusion between phagosomes and endosomes/lysosomes in macrophages; Shiratsuchi A et al.; Toll-like receptor 4 (TLR4) of macrophages recognizes LPS of Gram-negative bacteria in cooperation with CD14, which is also involved in the recognition of apoptotic cells . In this study we asked whether TLR4 plays a role in the phagocytic clearance of apoptotic cells by macrophages . Macrophages were prepared from peritoneal fluid of thioglycolate-treated mice carrying either a wild-type or a disrupted TLR4-encoding gene and were examined for their ability to phagocytose apoptotic mouse thymocytes, apoptotic Jurkat T cells, Ig-opsonized mouse thymocytes, Ig-opsonized zymosan particles, and latex beads . Both populations of macrophages equally expressed CD14 on their surfaces and showed almost equal activities of binding to and engulfing all these targets . However, apoptotic thymocytes, apoptotic Jurkat cells, and opsonized thymocytes disappeared more rapidly in TLR4-deficient macrophages than in wild-type macrophages, and the fusion between endosomes/lysosomes and phagosomes containing any target cells or particles was accelerated in mutant macrophages . Activation of the transcription factor NF-kappaB appeared not to occur in wild-type macrophages after engulfment, and the rate of apoptotic cell degradation in wild-type macrophages remained the same regardless of the activation of NF-kappaB . Finally, immunohistochemical analyses showed that ectopically expressed TLR4 was associated with phagosomes in a macrophage-derived cell line . All these results collectively indicate that TLR4 negatively regulates the degradation of engulfed cells in macrophages via a pathway independent of NF-kappaB.

Curr Diab Rep, 2004 Feb, 4(1), 46 - 50
Periodontal disease and diabetes mellitus; Pucher J et al.; Infections of the tissue surrounding the teeth (periodontitis) are usually caused by anaerobic gram-negative microorganisms . This infection causes destruction of the supporting alveolar bone and can lead to tooth loss . Removal of these microorganisms can slow or arrest the progression of periodontitis . Diabetes patients are at greater risk of developing periodontitis, may not respond as well to periodontal therapy as nondiabetic patients, and may require more aggressive treatment to manage periodontitis . Microorganisms that cause periodontitis and the host response to these may increase insulin resistance in diabetic patients . Treatment of periodontitis could improve glycemic control . A model is presented in which periodontal pathogens may cause increases in proinflammatory cytokines that mediate increases in insulin resistance, resulting in an increase in blood glucose . Following periodontal therapy, this process may be reversed.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 26 - 31
L-glutamate enhances the expression of Thermus maltogenic amylase in Escherichia coli; Jung HM et al.; Escherichia coli BL21 (DE3) transformed with a thermostable Thermus maltogenic amylase (ThMA), isolated from a Gram-negative bacterium Thermus strain IM6501, grew well and efficiently produced ThMA in a complex medium but not in a chemically defined medium (DM) . By supplementing L-glutamate to DM medium, both the specific growth rate and ThMA expression significantly increased . Alterations in the cellular responses of recombinant E . coli to L-glutamate were analyzed at the protein level by two-dimensional gel electrophoresis and mass spectrometry . The ppGpp synthase (RelA) was significantly reduced in cells grown with L-glutamate and was consistent with the low level of ppGpp, an indicator of stringent response . On the other hand, protein chain elongation factor (EF-Tu) and manganese-containing superoxide dismutase (MnSOD), which protects cells against oxidative damage, was significantly elevated by L-glutamate supplementation . These results indicate that L-glutamate enhances ThMA expression and increases the E . coli growth rate not only by overcoming the stringent response but also by increasing the synthesis of EF-Tu and MnSOD.

Mol Microbiol, 2004 Jan, 51(2), 319 - 34
From the inside out--processing of the Chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells; Wehrl W et al.; Polymorphic membrane protein (Pmp)21 otherwise known as PmpD is the longest of 21 Pmps expressed by Chlamydophila pneumoniae . Recent bioinformatical analyses annotated PmpD as belonging to a family of exported Gram-negative bacterial proteins designated autotransporters . This prediction, however, was never experimentally supported, nor was the function of PmpD known . Here, using 1D and 2D PAGE we demonstrate that PmpD is processed into two parts, N-terminal (N-pmpD), middle (M-pmpD) and presumably third, C-terminal part (C-pmpD) . Based on localization of the external part on the outer membrane as shown by immunofluorescence, immuno-electron microscopy and immunoblotting combined with trypsinization, we demonstrate that N-pmpD translocates to the surface of bacteria where it non-covalently binds other components of the outer membrane . We propose that N-pmpD functions as an adhesin, as antibodies raised against N-pmpD blocked chlamydial infectivity in the epithelial cells . In addition, recombinant N-pmpD activated human monocytes in vitro by upregulating their metabolic activity and by stimulating IL-8 release in a dose-dependent manner . These results demonstrate that N-PmpD is an autotransporter component of chlamydial outer membrane, important for bacterial invasion and host inflammation.

Plant J, 2004 Feb, 37(4), 589 - 602
Activation of a COI1-dependent pathway in Arabidopsis by Pseudomonas syringae type III effectors and coronatine; He P et al.; Gram-negative bacteria use a variety of virulence factors including phytotoxins, exopolysaccharides, effectors secreted by the type III secretion system, and cell-wall-degrading enzymes to promote parasitism in plants . However, little is known about how these virulence factors alter plant cellular responses to promote disease . In this study, we show that virulent Pseudomonas syringae strains activate the transcription of an Arabidopsis ethylene response factor (ERF) gene, RAP2.6, in a coronatine insensitive 1 (COI1)-dependent manner . A highly sensitive RAP2.6 promoter-firefly luciferase (RAP2.6-LUC) reporter line was developed to monitor activities of various bacterial virulence genes . Analyses of P . syringae pv . tomato DC3000 mutants indicated that both type III secretion system and the phytotoxin coronatine are required for RAP2.6 induction . We show that at least five individual type III effectors, avirulence B (AvrB), AvrRpt2, AvrPphB, HopPtoK, and AvrPphEPto, contributed to RAP2.6 induction . Gene-for-gene recognition was not involved in RAP2.6 induction because plants lacking RPM1 and RPS2 responded normally to AvrB and AvrRpt2 in RAP2.6 expression . Interestingly, the role of coronatine in RAP2.6 induction can be partially substituted by the addition of avrB in DC3000, suggesting that AvrB may mimic coronatine . These results suggest that P . syringae type III effectors and coronatine act by augmenting a COI1-dependent pathway to promote parasitism.

Vet Rec, 2004 Jan 17, 154(3), 73 - 80
Endoscopic renal evaluation and biopsy of Chelonia; Hernandez-Divers SJ; Sixty-nine tortoises, turtles and terrapins representing 28 species of the order Chelonia, class Reptilia were evaluated by endoscopy for renal disease . Under general anaesthesia, coelomic and/or extracoelomic endoscopic evaluations and biopsies of the kidney(s) were undertaken . Endoscopic approaches required a 2 to 4 mm skin incision in the prefemoral fossa, and minimal blunt dissection through the subcutaneous tissues . For the coelomic approach the coelomic aponeurosis of the transverse and oblique abdominal muscles was penetrated so that the cranioventral kidney(s) could be examined and biopsied . The extracoelomic approach required the endoscope to be advanced in a caudodorsal direction, between the coelomic aponeurosis and the broad iliacus muscle, so that the dorsolateral kidney(s) could be examined and biopsied . Both techniques were safe and effective for obtaining renal biopsies for histological examination and microbiological culture . Several renal pathologies were identified including glomerulonephrosis, tubulonephrosis, interstitial nephritis, uric acid accumulation, tubulonephritis, glomerulonephritis, renal oedema, glomerulosclerosis, nephrosclerosis, soft tissue mineralisation, and pyelonephritis . Several infectious conditions were identified, including a predominance of Gram-negative bacterial infections, two cases of hexamitiosis, and one case of mycobacteriosis.

Braz J Biol, 2003 Aug, 63(3), 491 - 5 Epub 2004 Jan 20.
Bacterial phagocytosis by macrophage of autogenous splenic implant; Marques RG et al.; Autogenous splenic implant seems to be the only alternative for preservation of splenic tissue after total splenectomy . This work was carried out to analyze the morphologic regeneration of autotransplanted splenic tissue in Wistar rats and to determine the bacterial phagocytic function of their macrophages . We utilized an experimental model with thirty-two rats, of both sexes, submitted to total splenectomy combined with autotransplantation in greater omentum of slices of the whole spleen mass . The animals were divided into two groups: I--young rats weighing 100 to 150 g; and II--adult rats weighing 250 to 300 g . Sixteen weeks later animals were intravenously inoculated with a suspension of Escherichia coli AB1157 . Twenty minutes after inoculation, the animals were sacrificed and the splenic autotransplants were removed for morphological study . There was regeneration of autotransplanted splenic tissue in all animals . A similar morphological aspect among all animals was observed, with splenic tissue showing red and white pulps, lymphoid follicles, and marginal zone, with a moderate architectural disarrangement . Macrophages containing gram-negative bacterial aggregates as well as macrophages with hemosiderin pigments within the cytoplasm were observed . Blood vessels showed preserved walls, with no signs of vasculitis or thrombosis . The present results suggest that autogenous splenic implants in the greater omentum of the rat acquire the macro- and microscopic architecture of a normal spleen, with reduced dimensions, and preserve bacterial phagocyte function.

Roum Arch Microbiol Immunol, 2002 Jul-Sep, 61(3), 217 - 29
Molecular cloning of a functional cis-acting, Bam HI-flanked, 1.6 Kb 'Mob' cassette for demonstrating rapid conversion of col EI origin-DNA cloning vectors into conjugal form; Mukhopadhyaya PN et al.; A 1.6 Kb mobilization (Mob) fragment originating from broad host range IncP plasmid RP4 is effectively cloned into two different Col EI-origin based cloning vectors, pBluescript II SK+ and pT-Adv, to generate pPAR-I and pPAR-II, respectively . The vectors have different genetic markers and were demonstrated to get mobilized at significant frequency into a laboratory and an enteroroxigenic strain of Escherichia coli with all the genetic markers of the recombinant clones expressing efficiently in the recipient host cells . Important restriction endonuclease recognition sequences in the multiple cloning sites of the conjugal vector DNA molecules remained unique . Significance and relevance of the current study with regard to other gene delivery system in gram negative bacteria are discussed.

Shock, 2004 Feb, 21(2), 182 - 8
Modulation of macrophage responsiveness to lipopolysaccharide by IRAK-1 manipulation; Cuschieri J et al.; Local activation of the macrophage by endotoxin is essential for the eradication of invasive gram-negative infections . Circulating endotoxin at lower concentrations results in immune cell activation at distant sites leading to tissue injury . Although the cellular mechanisms involved in these potentially dissimilar events are incomplete, it appears that the proximal kinase IRAK-1 plays a role . Thus, sense and antisense IRAK-1 oligonucleotides were used to determine the role IRAK-1 plays in macrophage activation by systemic (1-100 ng/mL) and local (1000 ng/mL) concentration of lipopolysaccharide (LPS) within THP-1 cells . Within the sense group, 1-1000 ng/mL of LPS within the sense group resulted in cellular activation of ERK-1/2, p38, and JNK/SAPK and the nuclear activation of NF-kappaB and AP-1 . This activation was associated with proinflammatory cytokine production and cellular spreading . Systemic concentrations of LPS within the antisense group were associated with significant attenuation of intracellular signaling, cytokine production, and cellular spreading compared with the sense group . Local concentrations of LPS within the antisense group, however, were associated only with a delay in intracellular signaling, with no effect on cytokine production or cell spreading compared with the sense group . Based on these results, it appears that IRAK-1 is essential to macrophage activation at systemic, but not local, concentrations of LPS . These data suggest that redundant pathways exist that are functional at higher concentrations of LPS . Therefore, IRAK-1 appears to be the central kinase involved in the activation of the macrophage at distant sites during septic shock but is not necessary for activation in areas of local infection.

Shock, 2004 Feb, 21(2), 115 - 20
Antibiotic treatment in a murine model of sepsis: impact on cytokines and endotoxin release; Vianna RC et al.; Experimental and clinical studies in sepsis indicate that antibiotic therapy may induce the release of endotoxin (LPS) from the outer membrane of gram-negative bacteria and therefore may affect the physiologic response and survival . The aim of this study was to evaluate if antibiotics commonly used to treat secondary peritonitis are capable of changing survival rates, proinflammatory and anti-inflammatory cytokine concentrations, and the release of endotoxin in a murine model of sepsis . Sepsis was induced by cecal ligation and puncture (CLP) in Swiss mice using an 18-gauge needle . The animals received injections of saline solution or imipenem or a combination of ciprofloxacin plus clindamycin every 8 h for 3 days . Antibiotic treatment induced an increase in survival rate and decreased plasma and peritoneal fluid levels of TNF-alpha and IL-6 at 6 and 24 h after CLP as compared with saline-treated animals . Antibiotic-treated animals also showed an early (6 h) decrease and a late (24 h) increase in IL-10 concentration in the peritoneal fluid . LPS concentrations were elevated in all groups, but imipenem-treated animals showed higher levels (2.2 EU/mL) than ciprofloxacin plus clindamycin (1.3 EU/mL) and saline-treated (1.5 EU/mL) groups . We conclude that antibiotic-induced endotoxin release is not a major determinant in the inflammatory response and prognosis in murine models of sepsis.

Science, 2004 Jan 30, 303(5658), 689 - 92
A predator unmasked: life cycle of Bdellovibrio bacteriovorus from a genomic perspective; Rendulic S et al.; Predatory bacteria remain molecularly enigmatic, despite their presence in many microbial communities . Here we report the complete genome of Bdellovibrio bacteriovorus HD100, a predatory Gram-negative bacterium that invades and consumes other Gram-negative bacteria . Its surprisingly large genome shows no evidence of recent gene transfer from its prey . A plethora of paralogous gene families coding for enzymes, such as hydrolases and transporters, are used throughout the life cycle of B . bacteriovorus for prey entry, prey killing, and the uptake of complex molecules.

Semin Immunol, 2004 Feb, 16(1), 43 - 53
Sensing infection in Drosophila: Toll and beyond; Ferrandon D et al.; Drosophila has evolved a potent immune system that is somewhat adapted to the nature of infections through the selective activation of either one of two NF-kappa B-like signalling pathways, the Toll and IMD (Immune deficiency) pathways . In contrast to the mammalian system, the Toll receptor does not act as a pattern recognition receptor (PRR) but as a cytokine receptor . The sensing of microbial infections is achieved by at least four PRRs that belong to two distinct families: the peptidoglycan recognition proteins (PGRPs) and the Gram-negative binding proteins (GNBPs)/beta-glucan recognition proteins (beta GRPs).

Semin Immunol, 2004 Feb, 16(1), 11 - 6
Endotoxin recognition molecules, Toll-like receptor 4-MD-2; Miyake K; Toll-like receptors (TLRs) are innate pathogen recognition molecules for microbial products . Lipopolysaccharide (LPS), a membrane constituent of Gram-negative bacteria, is one of the most potent microbial products . LPS is recognized by TLR4 and MD-2 . TLR4 is a transmembrane protein, the extracellular domain of which is composed of a protein motif called leucine-rich repeats (LRR) . MD-2 is an extracellular molecule that is associated with the extracellular LRR of TLR4 . MD-2 has a role in cell surface expression of TLR4 and interaction with LPS . TLR4-MD-2 contributes to containment of infections by Gram-negative bacteria by activating immune responses.

Brain Res, 2004 Feb 20, 998(2), 139 - 47
Effects of lipopolysaccharide treatment on feeding of goldfish: role of appetite-regulating peptides; Volkoff H et al.; The gram-negative bacteria-derived endotoxin lipopolysaccharide (LPS) is known to play an important role in immune and neurological manifestations during bacterial infections . In mammals, peripheral or brain administration of LPS induces anorexia and is thought to exert its effects through activation of pro-inflammatory cytokines . In this study, we investigated the effects of peripheral (intraperitoneal, IP) and central (intracerebroventricular, ICV) injections of LPS on food intake of goldfish . Fish treated IP with 10, 25, 50, 100 or 250 ng/g LPS or ICV with 1, 10 and 100 ng/g LPS showed a significant dose-dependent decrease in food intake, compared to the saline-treated fish . We also examined the brain mRNA expression of several hypothalamic appetite-related neuropeptides in response to the administration of LPS . IP injections of LPS (100 ng/g) induced a decrease in NPY expression and an increase in CCK, CRF and CART expression . These results indicate that LPS is a potent anorexigenic factor in goldfish and that this endotoxin induces a reduction in appetite, at least in part, by influencing gene expression of appetite-related neuropeptides.

J Org Chem, 2004 Feb 6, 69(3), 778 - 84
Synthetic peptidoglycan substrates for penicillin-binding protein 5 of Gram-negative bacteria; Hesek D et al.; The major constituent of the bacterial cell wall, peptidoglycan, is comprised of repeating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) with an appended peptide . Penicillin-binding proteins (PBPs) are involved in the final stages of bacterial cell wall assembly . Two activities for PBPs are the cross-linking of the cell wall, carried out by dd-transpeptidases, and the dd-peptidase activity, that removes the terminal d-Ala residue from peptidoglycan . The dd-peptidase activity moderates the extent of the cell wall cross-linking . There exists a balance between the two activities that is critical for the well-being of bacterial cells . We have cloned and purified PBP5 of Escherichia coli . The membrane anchor of this protein was removed, and the enzyme was obtained as a soluble protein . Two fragments of the polymeric cell wall of Gram-negative bacteria (compounds 5 and 6) were synthesized . These molecules served as substrates for PBP5 . The products of the reactions of PBP5 and compounds 5 and 6 were isolated and were shown to be d-Ala and the fragments of the substrates minus the terminal d-Ala . The kinetic parameters for these enzymic reactions were evaluated . PBP5 would appear to have the potential for turnover of as many as 1.4 million peptidoglycan strands within a single doubling time (i.e., generation) of E . coli.

J Biol Chem, 2004 Apr 2, 279(14), 13809 - 16 Epub 2004 Jan 28.
Role of vav1- and src-related tyrosine kinases in macrophage activation by CpG DNA; Stovall SH et al.; Macrophage activation by CpG DNA requires toll-like receptor 9 and the adaptor protein MyD88 . Gram-negative bacterial lipopolysaccharide also activates macrophages via a toll-like receptor pathway (TLR-4), but we and others have reported that lipopolysaccharide also stimulates tyrosine phosphorylation in macrophages . Herein we report that exposure of RAW 264.7 murine macrophages to CpG DNA (but not non-CpG DNA) provoked the rapid tyrosine phosphorylation of vav1 . PP1, a selective inhibitor of src-related tyrosine kinases, blocked both the CpG DNA-mediated tyrosine phosphorylation of vav1 and the CpG DNA-mediated up-regulation of macrophage tumor necrosis factor secretion and inducible nitric-oxide synthase protein accumulation . Furthermore, we found that the inducible expression of any of three dominant interfering mutants of vav1 (a truncated protein, vavC; a form containing a point mutation in the regulatory tyrosine residue, vavYF174; and a form with an in-frame deletion of six amino acids required for the guanidine nucleotide exchange factor (GEF) activity of vav1 for rac family GTPases, vavGEFmt) consistently inhibited CpG DNA-mediated up-regulation of tumor necrosis factor secretion and inducible nitric-oxide synthase protein accumulation in RAW-TT10 macrophages . Finally, we determined that CpG DNA-mediated up-regulation of NF-kappaB activity (but not mitogen-activated protein kinase activation) was inhibited by preincubation with PP1 or by expression of the truncated vavC mutant . Taken together, our results indicate that the tyrosine phosphorylation of vav1 by a src-related tyrosine kinase or kinases plays an important role in the macrophage response to CpG DNA.

Biophys J, 2004 Feb, 86(2), 913 - 22
Inner field compensation as a tool for the characterization of asymmetric membranes and Peptide-membrane interactions; Hagge SO et al.; Symmetric and asymmetric planar lipid bilayers prepared according to the Montal-Mueller method are a powerful tool to characterize peptide-membrane interactions . Several electrical properties of lipid bilayers such as membrane current, membrane capacitance, and the inner membrane potential differences and their changes can be deduced . The time-resolved determination of peptide-induced changes in membrane capacitance and inner membrane potential difference are of high importance for the characterization of peptide-membrane interactions . Intercalation and accumulation of peptides lead to changes in membrane capacitance, and membrane interaction of charged peptides induces changes in the charge distribution within the membrane and with that to changes in the membrane potential profile . In this study, we establish time-resolved measurements of the capacitance minimization potential DeltaPsi on various asymmetric planar lipid bilayers using the inner field compensation method . The results are compared to the respective ones of inner membrane potential differences DeltaPhi determined from ion carrier transport measurements . Finally, the time courses of membrane capacitances and of DeltaPsi have been used to characterize the interaction of cathelicidins with reconstituted lipid matrices of various Gram-negative bacteria.

Ann Surg, 2004 Feb, 239(2), 251 - 6
Cecropin B enhances betalactams activities in experimental rat models of gram-negative septic shock; Ghiselli R et al.; OBJECTIVE: To investigate the efficacy of imipenem, piperacillin combined with cecropin B in the prevention of lethality in 2 rat models of septic shock . Main outcome measures were bacterial growth in blood and intra-abdominal fluid, endotoxin and TNF-alpha concentrations in plasma, and lethality . BACKGROUND: Sepsis remains a serious clinical problem despite intense efforts to improve survival . It is a major cause of morbidity and mortality in hospitalized patients . The primary cause of Gram-negative shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of Gram-negative bacteria . METHODS: Adult male Wistar rats were given (1) . an intraperitoneal injection of 1 mg of Escherichia coli 0111:B4 LPS or (2) . 2 x 1010 CFU of E . coli ATCC 25922 . All animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/kg cecropin B, 20 mg/kg imipenem, and 120 mg/kg piperacillin alone and combined with 1 mg/kg cecropin B . Each group included 20 animals.RESULTS All compounds reduced the lethality when compared with controls . Piperacillin and imipenem significantly reduced the lethality and the number of E . coli in abdominal fluid compared with saline treatment . On the other hand, each betalactam determined an increase of plasma endotoxin and TNF-alpha concentration . Combination between cecropin B and betalactams showed to be the most effective treatment in reducing all variables measured . CONCLUSION: Cecropin B enhances betalactams activities in Gram-negative sepic shock rat models.

Biosci Biotechnol Biochem, 2004 Jan, 68(1), 1 - 19
Molecular dissection of the Selenomonas ruminantium cell envelope and lysine decarboxylase involved in the biosynthesis of a polyamine covalently linked to the cell wall peptidoglycan layer; Takatsuka Y et al.; The wild type of Selenomonas ruminantium subsp . lactilytica, which is a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, requires one of the normal saturated volatile fatty acids with 3 to 10 carbon atoms for its growth in a glucose medium; however, no such obligate requirement of fatty acid is observed when the cells are grown in a lactate medium . This bacterium is characterized by a unique structure of the cell envelope and a novel lysine decarboxylase and its regulatory protein . In the first part of this article, we will refer to the chemical structure of phospholipid and lipopolysaccharide in the cell membranes of this bacterium compared with that from the general Gram-negative bacteria for understanding their biological functions . S . ruminantium has neither free nor bound forms of Braun lipoprotein which plays an important role of the maintenance of the structural integrity of the cell surface in general Gram-negative bacteria . However, S . ruminantium has cadaverine, which links covalently to the peptidoglycan as a pivotal constituent for the cell division . In the second part of this article, we will refer to the chemical structure of the cadaverine-containing peptidoglycan, its biosynthesis, and the biological function . In the third part of this article, we will depict the molecular cloning of the genes encoding S . ruminanitum lysine decarboxylase (LDC) and its regulatory protein of 22-kDa (22-kDa protein; P22) which has similar characteristics to that of antizyme of ornithine decarboxylase in eukaryotic cells, and the molecular dissection of these proteins for understanding the regulation of cadaverine biosynthesis . Finally, we will illustrate a proposed structure of the cell envelope, a processes of biosynthesis of the cadaverine-containing peptidoglycan layer, and the LDC degradation mechanism in S . ruminantium, on the basis of the analyses of the cell envelope components, the results from the in vitro experiments on the biosynthesis of the peptidoglycan layer, and the current status of the knowledge on LDC and P22 in this organism.

Infect Immun, 2004 Feb, 72(2), 691 - 700
Host cell contact-induced transcription of the type IV fimbria gene cluster of Actinobacillus pleuropneumoniae; Boekema BK et al.; Type IV pili (Tfp) of gram-negative species share many characteristics, including a common architecture and conserved biogenesis pathway . Much less is known about the regulation of Tfp expression in response to changing environmental conditions . We investigated the diversity of Tfp regulatory systems by searching for the molecular basis of the reported variable expression of the Tfp gene cluster of the pathogen Actinobacillus pleuropneumoniae . Despite the presence of an intact Tfp gene cluster consisting of four genes, apfABCD, no Tfp were formed under standard growth conditions . Sequence analysis of the predicted major subunit protein ApfA showed an atypical alanine residue at position -1 from the prepilin peptidase cleavage site in 42 strains . This alanine deviates from the consensus glycine at this position in Tfp from other species . Yet, cloning of the apfABCD genes under a constitutive promoter in A . pleuropneumoniae resulted in pilin and Tfp assembly . Tfp promoter-luxAB reporter gene fusions demonstrated that the Tfp promoter was intact but tightly regulated . Promoter activity varied with bacterial growth phase and was detected only when bacteria were grown in chemically defined medium . Infection experiments with cultured epithelial cells demonstrated that Tfp promoter activity was upregulated upon adherence of the pathogen to primary cultures of lung epithelial cells . Nonadherent bacteria in the culture supernatant exhibited virtually no promoter activity . A similar upregulation of Tfp promoter activity was observed in vivo during experimental infection of pigs . The host cell contact-induced and in vivo-upregulated Tfp promoter activity in A . pleuropneumoniae adds a new dimension to the diversity of Tfp regulation.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 183 - 9
Methanocalculus chunghsingensis sp . nov., isolated from an estuary and a marine fishpond in Taiwan; Lai MC et al.; Three novel halotolerant, hydrogenotrophic methanogens, designated strains K1F9705bT, K1F9705c and O1F9704a, were isolated from an estuary in Eriln Shi, Taiwan, and from a nearby marine water aquaculture fishpond . These isolates were irregular cocci that stained Gram-negative . Strains K1F9705bT and K1F9705c were non-motile, but strain O1F9704a was weakly motile with flagella . They were able to use formate and H2/CO2 to form methane, but they could not catabolize acetate, methanol, trimethylamine or secondary alcohols . Acetate was required for cell growth . Tungsten greatly stimulated the growth of strains K1F9705bT and K1F9705c, but did not affect the growth of strain O1F9704a . Optimal pH and temperature for growth of these three isolates were respectively 7.2 and 37 degrees C . Optimal NaCl concentration for growth was 0.5% for strain O1F9704a and 1.0% for strains K1F9705c and K1F9705bT . Moreover, all strains grew well at up to 8-12% NaCl . Analysis of the 16S rRNA gene revealed that these isolates are members of the genus Methanocalculus, but are distinct from Methanocalculus taiwanensis, Methanocalculus pumilus and Methanocalculus halotolerans, with sequence similarities of 98.4, 98.3 and 98.2%, respectively . In addition, strain K1F9705bT possessed 85, 80, 37, 29 and 10% DNA-DNA relatedness to strain K1F9705c, strain O1F9704a, M . pumilus, M . halotolerans and M . taiwanensis, respectively . Analysis of protein profiles and the Mr of surface (S)-layer glycoprotein subunits showed that these three new isolates are closely related to, but distinct from, known Methanocalculus species . A novel species, Methanocalculus chunghsingensis sp . nov., is proposed for strains K1F9705bT, K1F9705c and O1F9704a . The type strain is K1F9705bT (=OCM 772T=DSM 14646T).

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 151 - 6
Methylocella tundrae sp . nov., a novel methanotrophic bacterium from acidic tundra peatlands; Dedysh SN et al.; A novel species, Methylocella tundrae, is proposed for three methanotrophic strains (T4T, TCh1 and TY1) isolated from acidic Sphagnum tundra peatlands . These strains are aerobic, Gram-negative, non-motile, dinitrogen-fixing rods that possess a soluble methane monooxygenase and utilize the serine pathway for carbon assimilation . Strains T4T, TCh1 and TY1 are moderately acidophilic organisms capable of growth between pH 4.2 and 7.5 (optimum 5.5-6.0) and between 5 and 30 degrees C (optimum 15 degrees C) . The major phospholipid fatty acid is 18:1omega7c . The DNA G+C content of strain T4T is 63.3 mol% . The three strains possess almost identical 16S rRNA gene sequences and are most closely related to two previously identified species of Methylocella, Methylocella palustris (97% similarity) and Methylocella silvestris (97.5% similarity) . DNA-DNA hybridization values of strain T4T with Methylocella palustris KT and Methylocella silvestris BL2T were respectively 27 and 36% . Thus, the tundra strains represent a novel species, for which the name Methylocella tundrae sp . nov . is proposed . Strain T4T (=DSM 15673T=NCIMB 13949T) is the type strain.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 77 - 83
Desulfatibacillum aliphaticivorans gen . nov., sp . nov., an n-alkane- and n-alkene-degrading, sulfate-reducing bacterium; Cravo-Laureau C et al.; A novel marine sulfate-reducing bacterium, strain CV2803T, which is able to oxidize aliphatic hydrocarbons, was isolated from a hydrocarbon-polluted marine sediment (Gulf of Fos, France) . The cells were rod-shaped and slightly curved, measuring 0.6x2.2-5.5 microm . Strain CV2803T stained Gram-negative and was non-motile and non-spore-forming . Optimum growth occurred in the presence of 24 g NaCl l(-1), at pH 7.5 and at a temperature between 28 and 35 degrees C . Strain CV2803T oxidized alkanes (from C13 to C18) and alkenes (from C7 to C23) . The DNA G+C content was 41.4 mol% . Comparative sequence analyses of the 16S rRNA gene and dissimilatory sulfite reductase (dsrAB) gene and those of other sulfate-reducing bacteria, together with its phenotypic properties, indicated that strain CV2803T was a member of a distinct cluster that contained unnamed species . Therefore, strain CV2803T (=DSM 15576T=ATCC BAA-743T) is proposed as the type strain of a novel species in a new genus, Desulfatibacillum aliphaticivorans gen . nov., sp . nov.

Microbes Infect, 2004 Jan, 6(1), 38 - 50
Enteropathogenic Escherichia coli outer membrane proteins induce changes in cadherin junctions of Caco-2 cells through activation of PKCalpha; Malladi V et al.; Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhoea . Despite the advances made in understanding EPEC-host cell interactions, the molecular mechanisms underlying watery diarrhoea have not been understood fully . Loss of transepithelial resistance and increased monolayer permeability by disruption of tight junctions has been implicated in this process . Apart from disruption of tight junctions, an important factor known to regulate monolayer permeability is E-cadherin and its interaction with beta-catenin, both of which constitute the adherens junctions . Our previous studies using HEp-2 cells demonstrated the morphological and cytoskeletal changes caused by cell-free outer membrane preparations (OMPs) of EPEC . In this study, we have shown that EPEC and its OMP induce significant changes in the adherens junctions of Caco-2 monolayers . We also observed significant phosphorylation of protein kinase Calpha (PKCalpha) in cells treated with either whole EPEC or its OMP . Immunoprecipitation of cell lysates with anti-E-cadherin and probing with phospho-PKCalpha monoclonal antibodies and anti-beta-catenins revealed that in these cells, phosphorylated PKCalpha is associated with cadherins, leading to the dissociation of the cadherin/beta-catenin complex . Immunofluorescence showed beta-catenins dissociated from the membrane-bound cadherins and redistributed into the cytoplasm . Expression of dominant negative PKCalpha reversed these effects caused by either whole EPEC or its OMP and also reduced the associated increase in monolayer permeability . It is possible that this mechanism may complement the earlier known pathways for loss of barrier function involving myosin light chain kinase activation and also may play a role in causing host cell death by apoptosis.

Pediatr Transplant, 2003 Oct, 7(5), 413 - 7
Bordetella bronchiseptica infection in pediatric lung transplant recipients; Ner Z et al.; Bordetella bronchiseptica are small, pleomorphic Gram-negative coccobacilli which are commensal organisms in the upper respiratory tract of many wild and domestic animals ('kennel cough' in dogs) . While it is common for health care providers to ask about exposure to ill family/friends, most do not routinely inquire about the health or immunization status of household pets . We report two cases of B . bronchiseptica pneumonia in lung transplant recipients {cystic fibrosis (CF); ages 10 and 15 yr; one male} who contracted B . bronchiseptica from pet dogs . We compared their course and outcome to four children (two CF, one congenital heart disease and one Duchenne's muscular dystrophy; four males, age range 6 months to 14 yr) with B . bronchiseptica cultured from the respiratory tract . Two of the four patients also acquired their illnesses from pet dogs and two from unknown sources . One lung transplant recipient expired from progressive respiratory failure . We conclude that B . bronchiseptica can cause serious infections in both immunosuppressed and immunocompetent children . We speculate that a detailed history of exposure to ill pets (particularly dogs), and the immunization status of all pets should be included in the routine evaluation of all pediatric transplant recipients.

Dis Aquat Organ, 2003 Dec 3, 57(1-2), 1 - 9
Isolation and characterization of novel Helicobacter spp . from the gastric mucosa of harp seals Phoca groenlandica; Harper CG et al.; Since the recent discovery of Helicobacter cetorum in cetaceans and its role in the development of gastritis, speculation has existed as to whether pinnipeds have Helicobacter spp . associated gastritis and peptic ulcer disease . The gastric mucosa of 4 stranded harp seals Phoca groenlandica from the Massachusetts coastline were assessed for Helicobacter spp . by culture and PCR . We cultured 2 novel Helicobacter spp . from the pyloric antrum of 1 of the 4 harp seals studied, and identified these by PCR in 2 of the 4 seals . Both gram-negative bacterial isolates were catalase- and oxidase-positive . However, a fusiform helicobacter with flexispira morphology was urease-positive, and a spiral-shaped helicobacter was urease-negative . Slender, spiral and fusiform-shaped bacteria were detected in the gastric mucosa by the Warthin-Starry stain . Histopathologic analysis revealed mild diffuse lymphoplasmacytic gastritis within the superficial mucosa of the pyloric antrum of both infected seals . The 2 bacterial isolates were classified by 16S rRNA analysis; they clustered with other enteric helicobacters and represent 2 novel Helicobacter spp . The urease-negative bacterial isolate clustered with H . canis and the urease-positive isolate clustered with an isolate from a sea lion and isolates from sea otters . This cluster of pinniped isolates has 97 % similarity to a number of Helicobacter species, but appears to be most closely related to other helicobacters with flexispira morphology . These findings suggest that the novel Helicobacter spp . may play a role in the etiopathogenesis of gastrointestinal diseases in pinnipeds . To our knowledge, this represents the first isolation and characterization of a novel Helicobacter spp . from pinnipeds.

Mar Biotechnol (NY), 2003 Sep-Oct, 5(5), 409 - 16
Isolation and characterization of a fucoidan-degrading marine bacterium; Sakai T et al.; Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from the algal bodies of Cladosiphon okamuranus (class Phaeophyceae, order Chordariales, family Chordariaceae) with a yield of 2.0% of the wet weight of the alga . To obtain enzymes that digest the fucoidan, we screened bacteria in the guy contents of the sea cucumber Stichopus japonicus for their ability to decrease the fucoidan in their culture media, and successfully isolated one bacterial strain that could decrease it . The bacterial strain was gram-negative and possessed menaquinone 7 as the predominant respiratory quinone, and the GC content of its genomic DNA was 52% . The results of the phylogenetic analysis of its 16S ribosomal DNA sequence indicated that the bacterial strain was a member of the division "Verrucomicrobia." However, as the bacterial strain is phylogenetically and phenotypically distinct from verrucomicrobial species described previously, the strain was assumed to be a new member of the division "Verrucomicrobia." When the bacterial strain was cultivated in an algal fucoidan-containing medium, the strain decreased fucoidan from C . okamuranus (44%), Nemacystus decipiens (19%), Laminaria japonica (31%), Kjellmaniella crassifolia (23%), sporophyl of Undaria pinnatifida (22%), Fucus vesiculosus (42%), and Ascophyllum nodosum (61%).

Extremophiles . 2004 Jan 21; {Epub ahead of print}
Promoters from a cold-adapted bacterium: definition of a consensus motif and molecular characterization of UP regulative elements; Duilio A et al.; Although low-temperature tolerant micro-organisms were discovered long ago, limited information is still available on the transcription machinery in cold-adapted bacteria . This knowledge represents a necessary background for the investigation of the adaptation of gene-expression mechanisms at low temperatures . The recent development of a shuttle genetic system for the transformation of the cold-adapted Gram-negative bacterium Pseudoalteromonas haloplanktis strain TAC125 has made possible the isolation of the psychrophilic promoters described in this paper . TAC125 genomic DNA fragments were cloned in the shuttle vector and the promoter-containing recombinant clones were selected for their ability to express a promoter-less lacZ gene . The nucleotide sequence of several selected inserts and the transcription start points of the transcribed m-RNAs were determined . A promoter consensus sequence for Pseudoalteromonas haloplanktis TAC125 was proposed on the basis of a sequence comparison between the various active promoters . Furthermore, the identification and the functional characterization of two UP elements from this cold-adapted bacterium are also reported.

J Endotoxin Res, 2003, 9(6), 341 - 7
Hyperlipoproteinemic low-density lipoprotein receptor-deficient mice are more susceptible to sepsis than corresponding wild-type mice; Lanza-Jacoby S et al.; High circulating concentrations of lipoproteins have been shown to modify the cytokine response and reduce mortality after endotoxin or live bacterial challenge . Sepsis, however, is more complex than endotoxemia, and it is not clear whether elevated plasma lipoproteins will be protective . Previous studies have shown that the low-density-lipoprotein receptor deficient (LDLR-/-) mice with increased circulating LDL are protected against the lethal effects of endotoxemia and Gram-negative infection . We evaluated whether the LDLR-/- mice would be protected against the effects of sepsis induced by cecal ligation and puncture (CLP) . Mortality was greater in LDLR-/-mice than in control C57Bl/6J mice . At 120 h after inducing sepsis, 20% of the control mice survived whereas none of theLDLR-/-mice were alive . Prior to inducing sepsis, serum concentrations of amyloid A protein and lipopolysaccharide binding protein (LBP) were significantly elevated in the LDLR-/-mice in comparison to the C57Bl/6J mice . Protein expression of sCD14 was also greater in the serum from the LDLR-/-mice than the C57Bl/6J mice . The elevated serum concentrations of LBP and CD14 were not associated with increases in the levels of liver CD14 mRNA and LBP mRNA . After inducing sepsis, serum concentration of interleukin (IL)-1beta was also significantly higher in LDLR-/-mice than in the control C57Bl/6J mice . These findings indicate that the LDLR-/-mice were more susceptible to the lethal effects of sepsis induced by CLP . The LDLR-/-mice also had higher serum concentrations of baseline, acute phase response proteins, SAA and LBP, and increased production of IL-1beta in response to CLP.

J Surg Res, 2004 Jan, 116(1), 116 - 20
Simvastatin suppresses LPS-induced Akt phosphorylation in the human monocyte cell line THP-1; Patel TR et al.; BACKGROUND: Activation of the small GTPase, Rac, requires post-translational modification by isoprenylation . Statins interfere with this process by blocking the synthesis of isoprenoid intermediates . The protein kinase Akt is a multifunctional regulator of cell behavior that has been linked to Rac activation . We have shown that lipopolysaccharide (LPS) stimulation leads to Rac activation in THP-1 cells . Therefore, we hypothesized that LPS stimulation would also activate Akt, a downstream effector of Rac, and that this may be blocked by statin pretreatment . MATERIALS AND METHODS: THP-1 cells were maintained in 1% fetal calf serum with or without 20 microM simvastatin for 24 h, followed by LPS stimulation for increasing time . Cytoskeletal changes were observed using Alexa-Phalloidin . Akt was immunoprecipitated from total cell lysate . Activated Akt was detected by immunoblotting with a phospho-Akt antibody and was quantified by image densitometry . RESULTS: LPS stimulation of THP-1 cells results in membrane ruffling and cell polarization . Furthermore, LPS increased Akt activation in THP-1 cells when compared with the nonstimulated controls . Akt phosphorylation peaked after 15 min of LPS stimulation and was suppressed by pretreatment with simvastatin . CONCLUSIONS: These data demonstrate that LPS stimulation leads to increased Akt phosphorylation, which can be suppressed with simvastatin pretreatment . This suggests one possible mechanism through which simvastatin could modulate LPS-induced signaling events in monocytes to improve the host response to Gram-negative infections.

Pediatr Pulmonol, 2004 Feb, 37(2), 158 - 64
BPI-ANCA of pediatric cystic fibrosis patients can impair BPI-mediated killing of E . coli DH5alpha in vitro; Schultz H et al.; Gram-negative bacterial lung infections and chronic bacterial colonization are major threats for pediatric cystic fibrosis (CF) patients . Besides impeded mucociliary clearance, other mechanisms that contribute to increased susceptibility to infections are presumed . The bactericidal/permeability-increasing protein (BPI), which is delivered by neutrophil granulocytes and mucosal epithelial cells, is one of the most potent innate antibiotics against Gram-negative bacteria and endotoxin . Antineutrophil cytoplasmic autoantibodies against BPI (BPI-ANCA) have been found in up to 90% of CF patients, and titers correlated inversely with lung function parameters . As major pulmonary damage is mediated by Gram-negative bacteria and their products, the question was raised as to whether BPI-ANCA can inhibit the antibiotic function of BPI in these patients . Sera of 23 pediatric CF patients were analyzed for the presence of BPI-ANCA by indirect immunofluorescence, ELISA, epitope mapping, and Western blotting . Patients' IgG were tested in a bacterial growth inhibition assay with recombinant BPI (rBPI) and an amino-terminal fragment of BPI (rBPI(21)) that retains antibiotic activity for inhibition of the antibiotic function of BPI against E . coli DH5alpha in vitro . BPI was recognized by 21 of 23 patients' sera in our detection assays . Thirteen of 23 patients' BPI-ANCA (56%) could inhibit the antibiotic function in vitro . Moreover, epitope mapping over the whole BPI sequence revealed that more patients' BPI-ANCA recognize the amino-terminal part of BPI than can be detected by ELISA . Thus, in pediatric CF patients, BPI-ANCA may contribute to diminished bacterial clearance by inhibiting the antibiotic function of BPI .

Rev Med Univ Navarra, 2003 Jul-Sep, 47(3), 29 - 33
{Toll type receptors: molecular bases of the relationship between innate and adaptation responses of the immune system}; Moreno C et al.; Microbes have on their surface molecular patterns that are common among a broad range of pathogens . These patterns are recognized by a wide variety of cellular receptors, the most important of which are a family of transmembrane proteins termed "Toll-like receptors" (TLR) . TLRs are pattern-recognition receptors that have key roles in detecting pathogens and initiating inflammatory responses . The receptor of Gram negative bacterial LPS, TLR4, is the best characterized member of the TLR family . So far, ten mammalian toll-like receptors (TLR1-TLR10) have been identified . Recent studies revealed that the TLR signaling pathway is a critical mediator of sepsis . An understanding of TLRs and their signaling pathway will reveal a therapeutic target in sepsis and other immune mediated diseases.

Biol Cell, 2003 Dec, 95(9), 603 - 13
Tyrosine kinase-mediated cell signalling in the activation of Mytilus hemocytes: possible role of STAT-like proteins; Canesi L et al.; In bivalve molluscs, cell-mediated immunity is carried out by circulating hemocytes, resembling the monocyte/macrophage lineage of vertebrates, that can kill the microbes through phagocytosis and various cytotoxic reactions . Previous data demonstrated that activation of MAPKs (Mitogen Activated Protein Kinases) is involved in the response of mussel hemocytes (Mytilus galloprovincialis Lam.) to bacterial challenge . In this work, the possibility that modulation of components of tyrosine kinase-mediated cell signalling may participate in the activation of mussel hemocytes was investigated . Cell pre-treatment with the macrophage activator IFN gamma significantly increased the bactericidal activity of mussel hemocytes towards E . coli . Human recombinant IFN gamma stimulated tyrosine phosphorylation of different members of STAT-like proteins (Signal Transducers and Activators of Transcription), as evaluated by Western blotting of hemocyte protein extracts with specific anti-phospho-STAT antibodies . A similar increase in phosphorylation of immunoreactive STATs was observed in hemocytes incubated with E . coli, this indicating that tyrosine phosphorylation of STAT-like members represents a physiological step in hemocyte activation . IFN gamma lead to persistent phosphorylation of immunoreactive STAT1, a transcription factor that plays a critical role in innate immunity towards Gram negative bacteria in mammalian systems; moreover, hemocyte pretreatment with IFN gamma significantly increased bacteria-induced STAT1 phosphorylation, whereas IFN alpha did not . IFN gamma also transiently affected the phosphorylation state of different MAPKs . The extent and time course of MAPK phosphorylation induced by IFN gamma were distinct from those elicited by either IFN alpha or bacterial challenge . Overall, the results indicate that the hemocyte function can be modulated by heterologous cytokines and bacterial signals that act in concert through tyrosine kinase-mediated transduction pathways converging on STAT- and MAPK-like members.

Cell, 2004 Jan 9, 116(1), 15 - 24
Metal import through microbial membranes; Ferguson AD et al.; Transport systems of Gram-negative bacteria coordinate the passage of metabolites through the outer membrane, periplasm, and the cytoplasmic membrane without compromising the protective properties of the cell envelope . Active transporters orchestrate the import of metals against concentration gradients . These thermodynamically unfavorable processes are coupled to both an electrochemical proton gradient and the hydrolysis of ATP . Crystallographic structures of transport proteins now define in molecular detail most components of an active metal import pathway from Escherichia coli.

Eur Cytokine Netw, 2003 Oct-Dec, 14(4), 234 - 7
The cytokine synthesis by heterozygous carriers of the Toll-like receptor 4 Asp299Gly polymorphism does not differ from that of wild type homozygotes; Heesen M et al.; Previous studies have found that heterozygosity for the A896G mutation of the endotoxin receptor TLR4 confers susceptibility to Gram-negative infections and septic shock . To evaluate the underlying mechanisms, we studied the association of the TLR4 polymorphism with endotoxin-induced cytokine synthesis in human whole blood . Monocyte CD14 density and monocyte count were also determined . Healthy individuals were genotyped by means of a real-time polymerase chain reaction . Plasma concentrations of TNF-alpha, IL-6, and IL-8 were measured by chemiluminescence . No significant differences in cytokine synthesis were observed between heterozygous individuals and homozygous carriers of the wild type allele . Our study suggests that heterozygosity for this TLR4 mutation is not a major factor determining the cytokine response to endotoxin . Copyright John Libbey Eurotext 2003.

Diagn Microbiol Infect Dis, 2003 Dec, 47(4), 623 - 4
Molecular diagnosis of arthritis due to streptobacillus moniliformis; Wallet F et al.; Streptobacillus moniliformis was identified as etiologic agent of arthritis utilizing a 16S rDNA molecular kit in our clinical laboratory . With the increasing of human contacts with rat as pet, this method would appear suitable to identify fastidious Gram-negative rod involved in arthritis specially when the clinical context is not evocative.

J Immunol, 2004 Jan 15, 172(2), 1266 - 72
Bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury; Yamada M et al.; Tissue repair often occurs in organs damaged by an inflammatory response . Inflammatory stimuli induce a rapid and massive release of inflammatory cells including neutrophils from the bone marrow . Recently, many studies suggested that bone marrow cells have the potential to differentiate into a variety of cell types . However, whether inflammatory stimuli induce release of bone marrow-derived progenitor cells (BMPCs), or how much impact the suppression of BMPCs has on the injured organ is not clear . Here we show that LPS, a component of Gram-negative bacterial cell walls, in the lung airways, induces a rapid mobilization of BMPCs into the circulation in mice . BMPCs accumulate within the inflammatory site and differentiate to become endothelial and epithelial cells . Moreover, the suppression of BMPCs by sublethal irradiation before intrapulmonary LPS leads to disruption of tissue structure and emphysema-like changes . Reconstitution of the bone marrow prevents these changes . These data suggest that BMPCs are important and required for lung repair after LPS-induced lung injury.

Rev Esp Quimioter, 2003 Sep, 16(3), 289 - 94
{Influence of consensual protocols on the use of antibiotics and bacterial resistance in a general hospital . A prospective study 1995-2000}; Gomez J et al.; Reasoned and consensual protocols, by means of diversification of the use of antibiotics, significantly influence their consumption, preventing and reducing the development of bacterial resistance against the Gram-negative microorganisms most frequently isolated in general hospitals . The increase in the consumption of piperacillin-tazobactam was found to be significantly associated with an increased resistance of E . cloacae, but less so in K . pneumoniae and E . coli . Its activity against P . aeruginosa and against P . mirabilis was not affected throughout the study and it maintained a high sensivity to the end against P . aeruginosa and against P . mirabilis and partial recovery of activity against A . baumannii . No statistically significant differences were found between the stable consumption of cefotaxime, ciprofloxacin, ceftazidime and imipenem and bacterial resistance against E . coli, P . aeruginosa, P . mirabilis, K . pneumoniae, E . cloacae and A . baumannii . Imipenem presented greater activity against E . coli, K . pneumoniae, E . cloacae and A . baumannii . Piperacillin-tazobactam showed greater activity against P . aeruginosa, while ciprofloxacin showed the least activity against E . coli and P . mirabilis . Cefotaxime had the least activity against E . cloacae, P . aeruginosa and A . baumannii . Ceftazidime showed stable activity and was similar to piperacillin-tazobactam against E . coli, P . mirabilis, K . pneumoniae, E . cloacae and A . baumannii.

Toxicol Lett, 2004 Feb 28, 147(1), 1 - 13
Lipopolysaccharide enhancement of 12-o-tetradecanoylphorbol 13-acetate-mediated transformation in rat glioma C6, accompanied by induction of inducible nitric oxide synthase; Chen TJ et al.; Lipopolysaccharide (LPS) from Gram-negative bacterial has been identified as an important molecule involved in the inflammatory process through inducing nitric oxide (NO) production . However, the effect of LPS in carcinogenesis is still undefined . In the present study, the biological effect of LPS was examined in 12-o-tetradecanoylphorbol 13-acetate (TPA)-treated rat glioma cells C6 . Results of MTT assay showed that LPS and TPA exhibited no significant cytotoxicity in glioma C6 cells . Interestingly, transformation foci were found in LPS/TPA-treated glioma C6 cells, but not in LPS- or TPA-treated cells . The transformation foci induced by LPS/TPA were also observed in the absence of serum . It indicates that induction of transformation foci formation by LPS and TPA is independent on the serum in glioma C6 cells . Induction of iNOS gene expression and NO production was examined in LPS/TPA-treated cells, but not obvious in LPS- or TPA-treated cells . NO donor sodium nitroprusside (SNP) induces transformation in glioma C6 cells in according with elevating NO production . In addition, LPS/TPA induces metalloproteinase 9 (MMP9) activity by gelatin activity assay in gel . Wogonin and quercetin but not rutin, inhibitors of iNOS gene expression and NO production induced by LPS, showed the significant inhibition on LPS/TPA-induced transformation foci formation, accompanied by inhibiting iNOS gene expression, NO production and MMP9 activity . Results of the present study provide scientific evidences to link the inflammatory responses and carcinogenesis, and suggest that NO derived from inflammation may contribute to the progression of carcinogenesis; natural products with anti-inflammatory effects such as wogonin and quercetin possess the ability to block transformation induced by LPS/TPA.

Pediatr Infect Dis J, 2003 Dec, 22(12), 1138 - 42
Ciprofloxacin in treatment of fever and neutropenia in pediatric cancer patients; Mullen CA; This article reviews clinical trials of outpatient management of fever and neutropenia in pediatric cancer patients . The syndrome of fever and neutropenia is discussed, and strategies of identifying patients at low risk for complex or fatal infections are described . A number of clinical trials in a wide range of clinical settings and countries have demonstrated that low risk pediatric cancer patients with fever and neutropenia can be prospectively identified and safely treated as outpatients . In addition outpatient management has been shown to be less costly than conventional intravenous therapy in hospitalized patients . Oral fluoroquinolones, including ciprofloxacin, have been used as a component of therapy in several trials because of their ease of administration and their activity against the majority of pathogenic bacteria causing illness in this group . The article also discusses the role of antibiotic prophylaxis of fever and neutropenia in certain high risk settings, such as hematopoietic stem cell transplantation . In selected high risk patients, prophylactic use of limited spectrum fluoroquinolones such as ciprofloxacin may reduce the incidence of Gram-negative bacteremias . Use of fluoroquinolone therapy as prophylaxis, however, is controversial because of concerns about an emergence of resistant organisms . Prudent use of fluoroquinolones as therapy and prophylaxis is essential to prolonging the benefits of this class of compounds.

J Immunol, 2004 Jan 1, 172(1), 410 - 7
Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt): evidence that the holotoxin is composed of three subunits: CdtA, CdtB, and CdtC; Shenker BJ et al.; We have shown the Actinobacillus actinomycetemcomitans produces an immunosuppressive factor encoded by the cytolethal distending toxin (cdt)B gene, which is homologous to a family of Cdts expressed by several Gram-negative bacteria . We now report that the capacity for CdtB to induce G(2) arrest in Jurkat cells is greater in the presence of the other Cdt peptides: CdtA and CdtC . Plasmids containing the cdt operon were constructed and expressed in Escherichia coli; each plasmid contained a modified cdt gene that expressed a Cdt peptide containing a C-terminal His tag . All three Cdt peptides copurified with the His-tagged Cdt peptide . Each of the peptides associated with the complex was truncated; N-terminal amino acid analysis of CdtB and CdtC indicated that the truncation corresponds to cleavage of a previously described signal sequence . CdtA was present in two forms in crude extracts, 25 and 18 kDa; only the 18-kDa fragment copurified with the Cdt complexes . Cdt complexes were also immunoprecipitated from A . actinomycetemcomitans extracts using anti-CdtC mAb . Exposure of Jurkat cells to 40 pg resulted in >50% accumulation of G(2) cells . CdtB and CdtC were detected by immunofluorescence on the cell surface after 2-h exposure to the holotoxin . CdtA was not detected by immunofluorescence, but all three peptides were associated with Jurkat cells when analyzed by Western blot . These studies suggest that the active Cdt holotoxin is a heterotrimer composed of truncated CdtA, CdtB, and CdtC, and all three peptides appear to associate with lymphocytes.

Infect Immun, 2004 Jan, 72(1), 310 - 21
Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia; Rossier O et al.; Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase . By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L . pneumophila infection of both amoebae and human macrophages . Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies . Examination of the developing L . pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria . In contrast to lsp mutants, L . pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication . L . pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes . Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays . The lspF mutant was also defective in an in vivo kinetic assay . Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo . Thus, the L . pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.

Clin Microbiol Infect, 2003 Dec, 9(12), 1242 - 4
Bacteremia caused by a novel species of Sphingobacterium; Tronel H et al.; A Gram-negative rod was isolated from the blood cultures of an 84-year-old man with foot cellulitis . The bacterium was first identified as Sphingobacterium spiritivorum on the basis of standard assimilation tests . However, sequencing analysis of its 16S rRNA genes and whole genome hybridization studies with other related bacteria showed that this isolate belongs to a so far undescribed species of Sphingobacterium, close to S . mizutae . This bacterium was susceptible to most of the antibiotics tested, including glycopeptides, but was resistant to aminoglycosides and polymyxins . Treatment with amoxicillin-clavulanate cured the infection.

Environ Microbiol, 2004 Jan, 6(1), 12 - 8
Plastic phenotypic resistance to predation by Bdellovibrio and like organisms in bacterial prey; Shemesh Y et al.; Predation at the lowest trophic level, i.e . between bacteria, is poorly understood, hindering efforts to assess its impact on the structure of bacterial communities . The interaction of Bdellovibrio and Bacteriovorax (Bdellovibrio and like organisms, BLOs), a group of obligate, ubiquitous predatory bacteria, with their Gram-negative bacterial prey results in the multiplication of the predator and in the lysis, but not in the eradication, of the prey . We show that the residual, surviving populations of prey cells exposed to predation stress differ from the populations before exposure, as they exhibit increased resistance to predation . This resistance was demonstrated in a number of Gram-negative prey . Moreover, predation resistance is not specific for the BLO strain experienced by the prey . The phenomenon does not stem from a mutation but is a plastic response associated with a phenotypic change, and it disappears upon removal of the predator . As resistance to predation is not total, this mechanism can ensure survival of both predator and prey.

Nature, 2003 Dec 18, 426(6968), 862 - 6
Evolutionary conservation of biogenesis of beta-barrel membrane proteins; Paschen SA et al.; The outer membranes of mitochondria and chloroplasts are distinguished by the presence of beta-barrel membrane proteins . The outer membrane of Gram-negative bacteria also harbours beta-barrel proteins . In mitochondria these proteins fulfil a variety of functions such as transport of small molecules (porin/VDAC), translocation of proteins (Tom40) and regulation of mitochondrial morphology (Mdm10) . These proteins are encoded by the nucleus, synthesized in the cytosol, targeted to mitochondria as chaperone-bound species, recognized by the translocase of the outer membrane, and then inserted into the outer membrane where they assemble into functional oligomers . Whereas some knowledge has been accumulated on the pathways of insertion of proteins that span cellular membranes with alpha-helical segments, very little is known about how beta-barrel proteins are integrated into lipid bilayers and assembled into oligomeric structures . Here we describe a protein complex that is essential for the topogenesis of mitochondrial outer membrane beta-barrel proteins (TOB) . We present evidence that important elements of the topogenesis of beta-barrel membrane proteins have been conserved during the evolution of mitochondria from endosymbiotic bacterial ancestors.

J Control Release, 2004 Jan 8, 94(1), 63 - 74
Bacterial ghosts as novel advanced drug delivery systems: antiproliferative activity of loaded doxorubicin in human Caco-2 cells; Paukner S et al.; Systemic application of anticancer drugs often causes severe toxic side effects . To reduce the undesired effects, advanced drug delivery systems are needed which are based on specific cell targeting vehicles . In this study, bacterial ghosts from Mannheimia haemolytica were used for site-specific delivery of doxorubicin (DOX) to human colorectal adenocarcinoma cells (Caco-2) . Bacterial ghosts are non-denatured envelopes of Gram-negative bacteria with fully intact surface structures for specific attachment to mammalian cells . The in vitro release profile of DOX-ghosts demonstrated that the loaded drug was non-covalently associated with the bacterial ghosts and that the drug delivery vehicles themselves represent a slow release system . Adherence studies showed that the M . haemolytica ghosts more efficiently than E . coli ghosts targeted the Caco-2 cells and released the loaded DOX within the cells . Cytotoxicity assays revealed that the DOX-ghosts exhibited potent antiproliferative activities on Caco-2 cells as the DOX associated with ghosts was two magnitude of orders more cytotoxic than free DOX provided in the medium at the same concentrations . Notably, a significant reduction in the cell viability was measured with DOX-ghosts at low DOX concentrations, which had no inhibitory effect when applied as free DOX after incubation for 16 h or when applied at higher concentrations for only 10 min to the cells . As the higher antiproliferative effects of DOX on Caco-2 cells were mediated by the specific drug targeting properties of the bacterial ghosts, the bacterial ghost system represents a novel platform for advanced drug delivery.

Curr Protein Pept Sci, 2003 Dec, 4(6), 389 - 95
Molecular genetics of Porphyromonas gingivalis: gingipains and other virulence factors; Nakayama K; Porphyromonas gingivalis is a black-pigmented anaerobic gram-negative bacterium that is a major pathogen of chronic adult periodontitis, an inflammatory disease of tooth-supporting tissues . P . gingivalis possesses a number of potential virulence factors . Among them, cell-surface-associated and secreted proteinases such as Arg-gingipain and Lys-gingipain have received much attention because they can degrade various host proteins and cause inflammation . Molecular genetic analysis is extremely powerful to evaluate the significance of each virulence factor in a pathogenic microorganism . This review will describe the introduction of molecular genetics to analysis of pathogenesis of P . gingivalis and the findings that have been obtained using knockout mutants of various potential virulence factors, especially proteinases.

Shock, 2004 Jan, 21(1), 65 - 71
Pretreatment with high-fat enteral nutrition reduces endotoxin and tumor necrosis factor-alpha and preserves gut barrier function early after hemorrhagic shock; Luyer MD et al.; Gram-negative sepsis is a potentially fatal clinical syndrome characterized by a proinflammatory response (tumor necrosis factor-alpha) to bacterial (endo)toxins and gut barrier function loss . Recently, we found that high-fat enteral nutrition protects against late bacterial translocation in a model of hemorrhagic shock in rats . However, the basis for this protection is unknown . We hypothesized that the observed protection is the result of an early inhibition of endotoxin and the subsequent inflammatory response resulting in a preserved gut barrier function . Sprague-Dawley rats were divided into a group that was starved overnight (HS-S), fed with a low-fat enteral diet (HS-LF) or fed wih a high-fat enteral diet (HS-HF), and subsequently subjected to a nonlethal hemorrhagic shock . Ninety minutes after hemorrhage, arterial endotoxin significantly decreased in HS-HF rats (4.0 +/- 0.6 pg/mL) compared with HS-LF rats (10.7 +/- 0.9 pg/mL, P = 0.002) and HS-S rats (15.2 +/- 2.2 pg/mL P = 0.001) . Interestingly, arterial tumor necrosis factor-alpha was also decreased in HS-HF rats (17.9 +/- 10.4 pg/mL) compared with HS-LF (83.5 +/- 16.7 pg/mL, P < 0.01) and HS-S rats (180.9 +/- 67.9 pg/mL, P < 0.02) . Loss of tight junction structure (ZO-1) observed in ileum and colon of control hemorrhagic shock rats was prevented in HS-HF rats . In parallel, intestinal barrier function was preserved in HS-HF rats, evidenced by a reduced permeability to horseradish peroxidase (P < 0.05), less bacterial invasion, and a 10-fold reduction of bacterial translocation early after hemorrhagic shock . This report describes a new strategy to nutritionally prevent endotoxemia, the subsequent inflammatory response and gut barrier failure following hemorrhagic shock . High-fat enteral nutrition requires further evaluation as an intervention to prevent a potentially fatal systemic inflammatory response in patients at risk for sepsis.

J Mol Biol, 2004 Jan 9, 335(2), 437 - 53
Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors; Belin D et al.; It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes . The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2, PAI-2) . The facultative secretion of PAI-2 is mediated by a signal sequence proposed to be inefficient by design . We show here that the same internal domain that promotes an inefficient translocation of murine PAI-2 in mammalian cells is a weak signal sequence in Escherichia coli . In contrast, the ovalbumin signal sequence is much more efficient, whereas the corresponding sequence elements from LEI, maspin and PI-10 are entirely devoid of signal sequence activity in E.coli . Mutations that improve the activity of the PAI-2 signal sequence and that convert the N-terminal regions of maspin and PI-10 into efficient signal sequences have been characterized . Taken together, these results indicate that several structural features contribute to the weak activity of the PAI-2 signal sequence and provide new insights into the plasticity of the "hydrophobic core" of signal sequences . High-level expression of two chimeric proteins containing the PAI-2 signal sequence is toxic, and the reduced viability is accompanied by a rapid decrease in the membrane proton motive force, in ATP levels and in translation . In unc- cells, which lack the F0F1 ATP-synthase, the chimeric proteins retain their toxicity and their expression only affected the proton motive force . Thus, the properties of these toxic signal sequences offer a new tool to dissect the interactions of signal sequences with the protein export machinery.

Am J Reprod Immunol, 2003 Oct, 50(4), 346 - 50
Searching for links between endotoxin exposure and pregnancy loss: CD14 polymorphism in idiopathic recurrent miscarriage; Karhukorpi J et al.; PROBLEM: Lipopolysaccharide (LPS) (endotoxin) is a well-known inducer of abortions in mice . In addition it has been proposed that gut-derived LPS of gram-negative bacteria may play a role in triggering idiopathic recurrent miscarriage (IRM) in humans . CD14 is one of the key molecules that mediates the effects of LPS . Promoter region polymorphism (-159C/T) in the CD14 gene is functionally important by regulating CD14 levels . High-producing CD14 genotype (TT) associates with deleterious effects of gut-derived LPS in hepatic cirrhosis in humans . It is not known whether women with IRM are genetically more prone to suffer from toxic effects of LPS . METHOD OF STUDY: By using polymerase chain reaction we analyzed the CD14 promoter region polymorphism in 38 women with IRM and in 127 normal controls of Finnish origin . RESULTS: There were no significant differences in the CD14 (-159C/T) allele or the genotype frequencies between the IRM women and the controls . However, there was a trend associating the presence of the T allele with increased odds of miscarriage . CONCLUSIONS: Although we were not able to find a statistically significant association between CD14 genotypes and IRM in our relatively small study population, a further study with a larger sample size is warranted to explore the role of high-producing CD14 genotypes in IRM . Also studies highlighting environmental LPS triggers and other intrinsic mediators of LPS signalling are needed to solve the enigmatic role of LPS in IRM in humans.

Carbohydr Res, 2003 Nov 14, 338(23), 2503 - 19
Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly; Samuel G et al.; The O-antigen is an important component of the outer membrane of Gram-negative bacteria . It is a repeat unit polysaccharide and consists of a number of repeats of an oligosaccharide, the O-unit, which generally has between two and six sugar residues . O-Antigens are extremely variable, the variation lying in the nature, order and linkage of the different sugars within the polysaccharide . The genes involved in O-antigen biosynthesis are generally found on the chromosome as an O-antigen gene cluster, and the structural variation of O-antigens is mirrored by genetic variation seen in these clusters . The genes within the cluster fall into three major groups . The first group is involved in nucleotide sugar biosynthesis . These genes are often found together in the cluster and have a high level of identity . The genes coding for a significant number of nucleotide sugar biosynthesis pathways have been identified and these pathways seem to be conserved in different O-antigen clusters and across a wide range of species . The second group, the glycosyl transferases, is involved in sugar transfer . They are often dispersed throughout the cluster and have low levels of similarity . The third group is the O-antigen processing genes . This review is a summary of the current knowledge on these three groups of genes that comprise the O-antigen gene clusters, focusing on the most extensively studied E . coli and S . enterica gene clusters.

Carbohydr Res, 2003 Nov 14, 338(23), 2449 - 57
Structures of polysaccharides and oligosaccharides of some Gram-negative marine Proteobacteria; Nazarenko EL et al.; The chemical structures of polysaccharides and LPS core oligosaccharides, isolated from various Gram-negative marine bacteria from the genera Pseudoalteromonas and Shewanella belonging to the Alteromonadaceae family and gamma-subclass of Proteobacteria, are reviewed . The polysaccharides are distinguished by the acidic character (e.g., due to the presence of hexuronic and aldulosonic acids and their derivatives) and the occurrence of unusual sugars, including N-acyl derivatives of 6-deoxyamino sugars, such as N-acetyl-D-quinovosamine, N-acetyl-L-fucosamine and N-acetyl-6-deoxy-L-talosamine, and higher sugars like 2,6-dideoxy-2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-D-galactopyranose (shewanellose) . Many constituent sugars have various uncommon non-sugar substituents, such as alanine, formic, lactic and hydroxybutyric acids, sulfate, phosphate, and 2-aminopropane-1,3-diol.

Carbohydr Res, 2003 Nov 14, 338(23), 2431 - 47
Structure of bacterial lipopolysaccharides; Caroff M et al.; Bacterial lipopolysaccharides are the major components of the outer surface of Gram-negative bacteria They are often of interest in medicine for their immunomodulatory properties . In small amounts they can be beneficial, but in larger amounts they may cause endotoxic shock . Although they share a common architecture, their structural details exert a strong influence on their activity . These molecules comprise: a lipid moiety, called lipid A, which is considered to be the endotoxic component, a glycosidic part consisting of a core of approximately 10 monosaccharides and, in "smooth-type" lipopolysaccharides, a third region, named O-chain, consisting of repetitive subunits of one to eight monosaccharides responsible for much of the immunospecificity of the bacterial cell.

Microbes Infect, 2003 Dec, 5(15), 1407 - 14
Richard Pfeiffer and Alexandre Besredka: creators of the concept of endotoxin and anti-endotoxin; Rietschel ET et al.; Richard Pfeiffer, working with Robert Koch in Berlin, intellectually and experimentally conceived the concept of endotoxin as a heat-stable bacterial poison responsible for the pathophysiological consequences of certain infectious diseases . Pfeiffer's definition of endotoxin included the inability to evoke neutralizing antibodies against this bacterial toxin . Alexandre Besredka, Ilya (Elie) Metchnikoff's successor at the Institut Pasteur in Paris, was the first to demonstrate that, in fact, antibodies could be engendered which were capable of suppressing the poisonous effects of endotoxin . Endotoxin and anti-endotoxin antibodies have since then fascinated researchers of many disciplines and continue to do so, particularly in the fields of diagnosis, prevention, and therapy of severe Gram-negative infections.

Curr Microbiol, 2003 Nov, 47(5), 412 - 6
Microbulbifer arenaceous sp . nov., a new endolithic bacterium isolated from the inside of red sandstone; Tanaka T et al.; An endolithic bacterium, strain RSBr-1, was isolated from the inside of a piece of red sandstone from coastal areas of Scotland . RSBr-1 was gram negative, oxidase and catalase positive, and cells were non-motile rods . Sodium was required for growth . The optimum sodium chloride concentration and pH for growth were 4% and pH 8.0, respectively . Eumelanin was produced in marine broth and in BY medium . RSBr-1 hydrolyzes chitin, esculin, gelatin, and starch, but not agar . Nitrate reduction is positive . Taxonomic characterization of this strain indicated that it belongs to the genus Microbulbifer . The difference between the aligned 16S rDNA sequences of RSBr-1 and the closest relative, M . elongata, is greater than the difference between the 16S rDNA sequences of M . hydrolyticus and M . elongata . On the basis of the phenotypic and genotypic comparison of this isolate with the other strains, RSBr-1 is proposed as a new species, Microbulbifer arenaceous, with type strain RSBr-1.

Ginekol Pol, 2003 Oct, 74(10), 1256 - 61
{Nosocomial infections in a neonatology department, 1995-2002}; Rudnicki J et al.; Nosocomial infections in newborns department are common due to number of invasive diagnostic and therapeutic procedures, prolonged hospitalization and development antibiotic resistance culture . Sepsis achieved 1 to 8 newborn infants for 1000 live births . This is still unresolved very important medical, organization, ethical and medical problem . The aim of this study was the estimation on the number, etiology and clinical form of nosocomial infection in Neonatology Department as well as the way of spread . We analyzed nosocomial infection in 8770 newborn infants in Neonatology Department with Intensive Therapy Chair and Clinic Obstetric and Perinatology Pomeranian University of Medicine from 1995 to 2002 . For this retrospective study we used data from Commission for Nosocomial Infection . In analyzed period total percentage of newborn infants with nosocomial infection was under 1%, but in NICU was over 11% . Inborn vertical infection was 26.8% and horizontal strictly nosocomial infection was diagnosed in 73.2% . Etiology was mainly due to Gram negative bacterial infection . Clinically sepsis, pneumonia and meningitis was diagnosed . Clinical manifestation and laboratory tests like CRP, PCT, blood count, leukocyte index and microbiological culture was used for diagnosis . The most often positive bacterial culture was obtained from cock, washstand, bath and medical staff . Nosocomial infections in neonatology department are significant medical problem which need continuous monitoring, systemic prevention and in case of infection early intervention.

Microbiol Mol Biol Rev, 2003 Dec, 67(4), 593 - 656
Molecular basis of bacterial outer membrane permeability revisited; Nikaido H; Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane . Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules . This review summarizes the development in the field since our previous review (H . Nikaido and M . Vaara, Microbiol . Rev . 49:1-32, 1985) was published . With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function . We are now beginning to see how the export of large proteins occurs across the outer membrane . With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

J Clin Microbiol, 2003 Dec, 41(12), 5492 - 9
Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens; Versage JL et al.; Tularemia is the zoonotic disease caused by the gram-negative coccobacillus Francisella tularensis . Its wide distribution in the environment poses a challenge for understanding the transmission, ecology, and epidemiology of the disease . F . tularensis is also considered a potential biological weapon due to its extreme infectivity . We have developed a multitarget real-time TaqMan PCR assay capable of rapidly and accurately detecting F . tularensis in complex specimens . Targeted regions included the ISFtu2 element and the 23kDa, fopA, and tul4 genes . Analysis of the four TaqMan assays demonstrated that three (ISFtu2, 23kDa, and tul4) performed within our established criterion of a detection limit of one organism . The combined use of the three assays was highly specific, displaying no cross-reactivity with the non-Francisella bacteria tested and capable of differentially diagnosing both F . tularensis and Francisella philomiragia . When the multitarget TaqMan assay (ISFtu2, 23kDa, and tul4) was compared to culturing, using environmentally contaminated specimens, the TaqMan PCR assay was significantly more sensitive than culturing (P </= 0.05) . The sensitive and specific nature of this rapid multitarget TaqMan assay provides a valuable new tool that with future evaluations can be used for analyzing clinical specimens, field samples during bioterrorism threat assessment, and samples from outbreaks and for improving our understanding of the ecology and environmental prevalence of F . tularensis.

J Inorg Biochem, 2004 Jan, 98(1), 68 - 72
Study on biological effect of La3+ on Escherichia coli by atomic force microscopy; Liu P et al.; The biological effects of rare-earth metal ions on the organism have been studied using La3+ as a probe ion and Escherichia coli cell as a target organism . Atomic force microscopy (AFM) studies reveal that La3+ substantially changes the structure of the outer cell membrane responsible for the cell permeability . Significant damages of the outer cell membrane are observed using scanning electron microscopy (SEM) after the introduction of La3+ . In result, the cell becomes easily attacked by lysozyme . Moreover, inductively coupled plasma-mass spectrometry (ICP-MS) measurements show considerable amount of Ca2+ and Mg2+ in the supernatant from the La3+ exposed cells . It is proposed that La3+ can replace Ca2+ from the binding sites because of their close ionic radii and similar ligand specificities . Lipopolysaccharide (LPS), which forms the outer membrane of Gram-negative bacteria, could not serve as the cellular envelope steadily after Ca2+ and Mg2+ released from their binding sites on the LPS patches.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 2067 - 72
Pseudomonas rhizosphaerae sp . nov., a novel species that actively solubilizes phosphate in vitro; Peix A et al.; A bacterial strain (designated IH5(T)), isolated from rhizospheric soil of grasses growing spontaneously in Spanish soil, actively solubilized phosphates in vitro when bicalcium phosphate was used as a phosphorus source . This strain was Gram-negative, strictly aerobic, rod-shaped and motile . The strain produced catalase, but not oxidase . Cellulose, casein, starch, gelatin, aesculin and urea were not hydrolysed . Growth was observed with many carbohydrates as the carbon source . The main non-polar fatty acids detected were hexadecenoic acid (C(16 : 1)), hexadecanoic acid (C(16 : 0)) and octadecenoic acid (C(18 : 1)) . The hydroxy fatty acids detected were 3-hydroxydecanoic acid (C(10 : 0) 3-OH), 3-hydroxydodecanoic acid (C(12 : 0) 3-OH) and 2-hydroxydodecanoic acid (C(12 : 0) 2-OH) . Phylogenetic analysis of 16S rRNA indicated that this bacterium belongs to the genus Pseudomonas in the gamma-subclass of the Proteobacteria and that the closest related species is Pseudomonas graminis . The DNA G+C content was 61 mol% . DNA-DNA hybridization showed 23 % relatedness between strain IH5(T) and P . graminis DSM 11363(T) . Therefore, strain IH5(T) belongs to a novel species from the genus Pseudomonas, for which the name Pseudomonas rhizosphaerae sp . nov . is proposed (type strain, IH5(T)=LMG 21640(T)=CECT 5726(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1979 - 83
Herbaspirillum lusitanum sp . nov., a novel nitrogen-fixing bacterium associated with root nodules of Phaseolus vulgaris; Valverde A et al.; Several bacterial strains were isolated from root nodules of Phaseolus vulgaris plants grown in a soil from Portugal . The strains were Gram-negative, aerobic, curved rod-shaped and motile . The isolates were catalase- and oxidase-positive . The TP-RAPD (two-primer randomly amplified polymorphic DNA) patterns of all strains were identical, suggesting that they belong to the same species . The complete 16S rDNA sequence of a representative strain was obtained and phylogenetic analysis based on the neighbour-joining method indicated that this bacterium belongs to the beta-Proteobacteria and that the closest related genus is Herbaspirillum . The DNA G+C content ranged from 57.9 to 61.9 mol% . Growth was observed with many different carbohydrates and organic acids including caprate, malate, citrate and phenylacetate . No growth was observed with maltose, meso-inositol, meso-erythritol or adipate as sole carbon source . According to the phenotypic and genotypic data obtained in this work, the bacterium represents a novel species of the genus Herbaspirillum, and the name Herbaspirillum lusitanum sp . nov . is proposed . The type strain is P6-12(T) (=LMG 21710(T)=CECT 5661(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1937 - 40
Dialister invisus sp . nov., isolated from the human oral cavity; Downes J et al.; Six strains of anaerobic, Gram-negative coccobacilli isolated from the root canals of patients with endodontic infections (five strains) and from a deep periodontal pocket (one strain) were subjected to a comprehensive range of phenotypic and genetic tests and were found to comprise a homogeneous group . Following 16S rRNA gene sequence analysis, they were found to be most closely related to Dialister pneumosintes, with 93 % sequence similarity between the two taxa . A novel species, Dialister invisus sp . nov., is proposed . Biochemically, the species is largely unreactive and is asaccharolytic, with only traces of acetate and propionate detected as metabolic end-products . The G+C content of the DNA of D . invisus strains is 45-46 mol% . The type strain is E7.25(T) (=CCUG 47026(T)=DSM 15470(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1931 - 5
Methanocaldococcus indicus sp . nov., a novel hyperthermophilic methanogen isolated from the Central Indian Ridge; L'Haridon S et al.; An autotrophic, hyperthermophilic methanogen, strain SL43(T), was isolated from a deep-sea hydrothermal chimney sample collected on the Central Indian Ridge at a depth of 2420 m . The coccoid, surface-layer-carrying, Gram-negative-staining cells were heavily flagellated and exhibited a slight tumbling motility . The temperature range for growth at pH 6.5 was 50-86 degrees C, with optimum growth at 85 degrees C . The optimum pH for growth was 6.6 and the optimum NaCl concentration for growth was 30 g l(-1) . The novel isolate used H(2) and CO(2) as the only substrates for growth and produced methane . Selenium and yeast extract stimulated growth significantly . In the presence of CO(2) and H(2), the organism reduced elemental sulfur to hydrogen sulfide . Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin or streptomycin . The G+C content of the genomic DNA was 30.7 mol% . On the basis of 16S rRNA gene sequence analysis, this organism was most closely related to Methanocaldococcus infernus ME(T) (3.2 % distance) . Its phylogenetic distinctiveness was confirmed by RFLP analysis of the 16S rDNA, a reliable tool for differentiating hyperthermophilic methanococci . On the basis of phylogenetic and physiological characteristics, it is proposed that strain SL43(T) (=DSM 15027(T)=JCM 11886(T)) be designated as the type strain of a novel species, Methanocaldococcus indicus sp . nov.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1901 - 6
Oceanicaulis alexandrii gen . nov., sp . nov., a novel stalked bacterium isolated from a culture of the dinoflagellate Alexandrium tamarense (Lebour) Balech; Strompl C et al.; Five bacterial strains were isolated from a non-toxigenic strain of the marine dinoflagellate Alexandrium tamarense (Lebour) Balech CCMP 116 (NEPCC C116), during a survey of the diversity of bacteria associated with paralytic shellfish toxin-producing cultures of Alexandrium spp . (Dinophyta) . All strains were strictly aerobic, Gram-negative, straight or curved rods . Cells were dimorphic, with stalks (or prosthecae) and non-motile or non-stalked and motile, by means of a single polar flagellum . The bacteria grew best at salt concentrations ranging from 2 to 10 % and growth occurred at 10 degrees C, but not at 50 degrees C . The G+C content of the chromosomal DNA of the strains was determined to be 61-62 mol% . Major cellular fatty acids of the bacteria presented a unique profile . 16S rRNA gene sequence analysis showed the five strains to be related to genera of budding bacteria of marine origin in the 'Alphaproteobacteria', namely, Hirschia, Maricaulis and Hyphomonas, although they exhibited substantial differences in morphology, substrate utilization and fatty acid profile to members of these genera . The five strains are proposed to comprise a new species of a new genus, Oceanicaulis alexandrii gen . nov., sp . nov., the type strain of which is C116-18(T) (=DSM 11625(T)=NCIMB 13905(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1807 - 11
Pseudoalteromonas sagamiensis sp . nov., a marine bacterium that produces protease inhibitors; Kobayashi T et al.; A marine bacterium producing protease inhibitors was isolated from neritic sea water and was studied phenotypically, genotypically and phylogenetically . This bacterium (strain B-10-31(T)) produced three types of protease inhibitor, namely, marinostatin, monastatin and leupeptin, which were considerably different in terms of their chemical structure and properties . Strain B-10-31(T) was a rod-shaped, non-spore-forming, Gram-negative, strictly aerobic bacterium that was motile by means of one polar flagellum . The strain required Na(+) for growth and exhibited optimal growth at 27 degrees C, pH 8.0 and 2 % (w/v) NaCl . It utilized various substrates, such as D-glucose, maltose, maltotriose, N-acetylglucosamine, L-threonine, L-serine, L-arginine, L-proline, L-alpha-alanine and L-glutamate, as the sole energy source . Ubiquinone-8 was the major respiratory quinone . The major fatty acids were C(16 : 0), C(16 : 1) omega7c, C(16 : 1) omega9c and C(18 : 1) omega7c . The G+C content of the DNA of strain B-10-31(T) was 42.0 mol% . Phylogenetic analysis, based on 16S rDNA sequences, showed that the strain clustered in the gamma-Proteobacteria . The aerobic marine bacterium Pseudoalteromonas bacteriolytica was the species most closely related to the new isolate (90.4 % 16S rDNA sequence similarity); other described species in the gamma-Proteobacteria cluster showed low levels of sequence similarity with strain B-10-31(T) (<90 %) . Based on the above results, it is proposed that the novel marine bacterium should be classified as a new species, for which the name Pseudoalteromonas sagamiensis (type strain B-10-31(T)=JCM 11461(T)=DSM 14643(T)) is proposed.

Eur Cytokine Netw, 2003 Jul-Sep, 14(3), 128 - 33
Ex vivo lipopolysaccharide (LPS)-induced TNF-alpha, IL-1beta, IL-6 and PGE2 secretion in whole blood from Type 1 diabetes mellitus patients with or without aggressive periodontitis; Araya AV et al.; Several studies have demonstrated that diabetes is a risk factor for developing periodontal disease, increasing its prevalence and severity . Furthermore, periodontitis may impair the metabolic control and adequate treatment of diabetic patients . LPS from Gram-negative bacteria penetrates the periodontal tissues and subsequently recruits and activates immune cells . Progression to severe periodontitis with loss of supporting structures is mediated by several factors, including secretion of a broad spectrum of inflammatory and destructive (PGE2) . mediators such as cytokines (TNF-alpha, IL-1b and IL-6), chemokines (IL-8) and prostaglandin E2 . The aim of this work is to investigate differences in the TNF-a, IL-1b and IL-6 expression and prostaglandin E2 (PGE2) release in blood from diabetic patients with and without aggressive periodontitis (AP) stimulated with lipopolysaccharide (LPS) . For this purpose we recruited 29 Type 1 diabetes mellitus (DM) patients, 14 with AP and 15 without AP . Fourteen healthy individuals formed the control group . For cytokine expression and PGE2 secretion, an ex vivo whole blood culture system was used . Cytokines and PGE2 were detected by commercial immunometric assays . A wide range of inter-individual variability in spontaneous and LPS-induced TNF-alpha, IL-1b and IL-6 levels in patient groups and controls was found . The mean of spontaneous and LPS-induced TNF-alpha and IL-1b levels did not differ significantly (p > 0.5) when patients were compared to control individuals . Although not significant, the spontaneous TNF-alpha, IL-1b and IL-6 levels in the group of Type 1 DM with AP were higher than in controls, while in diabetic patients without AP, these values were depressed in comparison with controls . In both groups of patients, the means of LPS-induced IL-6 levels were higher than the controls but the differences observed were not significant (p = 0.07) . However, the LPS-induced PGE2 levels varied significantly when all groups were compared (p = 0.007) . The means of LPS-induced PGE2 levels for Type 1 diabetic patients with AP (p = 0.0009) and without AP (p = 0.024) were significantly higher than the levels observed for healthy controls . Finally, we conclude that Type 1 diabetic patients with or without AP did not express higher LPS-induced TNF-a, IL-1b and IL-6 levels than controls . However, the PGE2 levels released were significantly higher than those detected in controls.

Comp Med, 2003 Oct, 53(5), 493 - 7
Left ventricular end-diastolic pressure-volume relationship in septic rats with open thorax; Cesar S et al.; To estimate changes in compliance, we evaluated the effects of sepsis on the end-diastolic pressure-volume relationship (EDPVR) in the left ventricle of rats that had undergone an open thorax procedure . Sepsis was induced in male Wistar Hannover rats (n = 7; 240 to 270 g) by intraperitoneal administration of a slurry of cecal contents; control rats (n = 7) were given 5% dextrose only . On the third day after induction of sepsis, left ventricular (LV) pressure and LV dimensions were recorded simultaneously in animals of both groups . Using a micromanometer and ultrasonic crystals, measurements were obtained at baseline and during the increase of afterload . Blood samples were taken for determination of complete blood count, white blood cell differential count, and lactate concentration, and for bacteriologic examination . Septic rats lost weight, and developed changes in body temperature, ascites, and abscesses in the abdominal and thoracic cavities, gram-negative bacteremia, and increase in heart rate . On the third day after induction of sepsis, LV EDPVR decreased, compared with that in the control rats (regression coefficients: control group, 8.41 to 23.95; sepsis group, 3.94 to 7.92) . Myocardial compliance in the left ventricle increased on the third day of sepsis in the open-thorax rat model, as evidenced by the downward shift of LV EDPVR in rats with sepsis, compared with controls.

Biochem Biophys Res Commun, 2003 Dec 26, 312(4), 1278 - 83
Computational differentiation of N-terminal signal peptides and transmembrane helices; Yuan Z et al.; Signal peptides and transmembrane helices both contain a stretch of hydrophobic amino acids . This common feature makes it difficult for signal peptide and transmembrane helix predictors to correctly assign identity to stretches of hydrophobic residues near the N-terminal methionine of a protein sequence . The inability to reliably distinguish between N-terminal transmembrane helix and signal peptide is an error with serious consequences for the prediction of protein secretory status or transmembrane topology . In this study, we report a new method for differentiating protein N-terminal signal peptides and transmembrane helices . Based on the sequence features extracted from hydrophobic regions (amino acid frequency, hydrophobicity, and the start position), we set up discriminant functions and examined them on non-redundant datasets with jackknife tests . This method can incorporate other signal peptide prediction methods and achieve higher prediction accuracy . For Gram-negative bacterial proteins, 95.7% of N-terminal signal peptides and transmembrane helices can be correctly predicted (coefficient 0.90) . Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 99% (coefficient 0.92) . For eukaryotic proteins, 94.2% of N-terminal signal peptides and transmembrane helices can be correctly predicted with coefficient 0.83 . Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 87% (coefficient 0.85) . The method can be used to complement current transmembrane protein prediction and signal peptide prediction methods to improve their prediction accuracies.

J Biotechnol, 2003 Dec 19, 106(2-3), 233 - 53
Myxobacteria: proficient producers of novel natural products with various biological activities--past and future biotechnological aspects with the focus on the genus Sorangium; Gerth K et al.; Myxobacteria are gram-negative bacteria which are most noted for their ability to form fruiting bodies upon starvation . Within the last two decades, they increasingly gained attention as producers of natural products with biological activity . Here, recent and future biotechnological research on certain key myxobacteria and on their ability to produce natural products is reviewed with the focus on the production of myxovirescin, soraphen and epothilone . Aspects of product improvement and yield as well as statistics regarding secondary metabolite formation are discussed . Future research will deal with the exploitation of the biosynthetic potential of the myxobacteria, for example via the isolation of new myxobacterial species with different physiological properties . Additionally, the genetic potential of myxobacteria to form natural products can be exploited by the identification and activation of biosynthetic gene clusters . These can be found frequently within their genomes, which is shown by the analysis of the unfinished genomes of Myxococcus xanthus and Sorangium cellulosum . The current status of the S . cellulosum functional genome project with model strain So ce56 is discussed.

Mol Microbiol, 2004 Jan, 51(1), 63 - 72
Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum; Otto GP et al.; The Gram-negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free-living amoebae and mammalian phagocytes . L . pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes . The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER . The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy-inducing starvation, led to the hypothesis that L . pneumophila infection requires macroautophagy . As L . pneumophila replicates in Dictyostelium discoideum, and macroautophagy genes have been identified and mutated in D . discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L . pneumophila . Mutation of the apg1, apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation . We show that L . pneumophila replicates normally in D . discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild-type D . discoideum . Furthermore, a green fluorescent protein (GFP)-tagged marker of autophagosomes, Apg8, does not systematically co-localize with DsRed-labelled L . pneumophila . We conclude that macroautophagy is dispensable for L . pneumophila intracellular replication in D . discoideum.

Nervenarzt, 2003 Dec, 74(12), 1118 - 21
{Bacterial meningitis as a complication of Fusobacterium necroforum infection in adults}; Spitzer C et al.; Fusobacterium necrophorum, an anaerobic, gram-negative rod, belongs to the physiological flora of the oropharynx . It causes Lemierre's syndrome characterized by oropharyngeal infection, septic thrombophlebitis of the neck, in particular of the internal jugular vein, and metastatic abscesses, predominantly in the lungs . Rarely, and mainly in children, it causes meningitis . Here we report the clinical course of a 25-year-old woman with F . necrophorum meningitis . She presented with incomplete, right third nerve palsy . Within a few days, she developed fever, meningism and progressive reduction of vigilance . Cerebrospinal fluid analysis showed typical signs of bacterial meningitis . After the identification of F . necrophorum, the antibiotic treatment was changed to meropenem, which led to continuous improvement of the clinical symptoms . Due to persistent signs of inflammation in the CSF, metronidazole was added to the antibiotic regime . This case report demonstrates that F . necrophorum should always be considered in the diagnostic workup of bacterial meningitis in adults.

Int J Biochem Cell Biol, 2004 Feb, 36(2), 271 - 80
Mutational analysis of the large periplasmic loop 7-8 of the putrescine transporter PotE in Escherichia coli; Minchin RF et al.; The PotE protein is a putrescine-ornithine antiporter found in many gram-negative bacteria . It is a member of the APA family of transporters and has 12 predicted alpha-helical transmembrane spanning segments (TMS) . While the substrate binding site has previously been mapped to a region near the surface of the cytoplasmic lipid layer, no structural feature within the periplasmic domains of PotE have been shown to be important for function . We examined the role of the only large outer loop, situated between transmembrane spanning segment 7 and 8, in putrescine uptake . Deletion of the highly conserved amino acids in the region closest to transmembrane spanning segment 7 produced a protein with little activity . Glycine-scanning mutagenesis of this region showed that Val(249) and Leu(254) were required for optimal transporter function . The V249G mutant transported putrescine at a lower maximal rate compared to wild-type (WT) but with the same substrate binding affinity . In contrast, the L254G mutant had a higher substrate affinity . A series of Val(249) mutants indicated that the hydrophobicity of this residue, which is located at or near the membrane surface, is important for PotE function . Secondary structure predictions of the large outer loop indicated the presence of a hydrophobic alpha-helix in the centre with a hydrophobic region at each end suggesting that the loop was not entirely exposed to the aqueous periplasmic space . The study shows that loop 7-8 is important for PotE function, possibly by forming a re-entrant loop in the channel of the transporter.

J Struct Biol, 2003 Dec, 144(3), 320 - 6
Projection structure of the bacterial oxalate transporter OxlT at 3.4A resolution; Heymann JA et al.; OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters . It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes . From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously determined the three-dimensional structure of this transporter at 6.5 A resolution . Here, we report conditions to obtain crystalline, two-dimensional sheets of OxlT with diameters exceeding 2 microm . Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV . Computed optical diffraction patterns from the best images display measurable reflections to about 3.4A, and electron diffraction patterns show spots to about 3.2 A resolution in the best cases . As in the case of the tube-like crystals, the new crystalline sheets also belong to the p22(1)2(1) symmetry group . However, the unit cell dimensions of 102.7A x 67.3 A are significantly smaller in one direction than those previously observed with the tube-like crystals that display unit cell dimensions of 100.3A x 79.0 A . Different regions of OxlT are involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell.

Enferm Infecc Microbiol Clin, 2003 Dec, 21(10), 552 - 6
{Timing of bacterial colonization in severe burns: is strict isolation necessary?}; Barret JP; INTRODUCTION: Infection is still one of the main causes of mortality in severe burn patients . Strict isolation has been used for the prevention of infection, but the efficacy of this measure is debatable . The aim of this study was to determine the timing of bacterial colonization in these patients and to ascertain whether strict isolation is indicated . METHODS: Thirty consecutive children with severe burns were studied . Patients were only barrier-nursed during dressing changes . On admission and twice weekly over the entire hospital stay, burn, sputum, gastric aspirates, feces, and blood samples were obtained for culture . All isolates were tested for specific biotypes . Results were studied with linear regression and repeated measures ANOVA to determine the timing of colonization and cross-colonization between patients . RESULTS: On admission, normal cutaneous flora were isolated from burn cultures of all patients . The remaining cultures were negative . After one week, gastric aspirates were found to be colonized by gram-negative bacteria and fungi . This was followed by colonization of feces, burn, and sputum cultures . Biotype identification showed unidirectional colonization from the gastrointestinal tract to burns and upper airway . There were no cross infections between patients . CONCLUSIONS: Microbial colonization in severe burn patients was endogenous in nature and there were no cross infections . Thus, strict isolation is not necessary in burn centers, except during outbreaks of multi-resistant microorganisms.

Biochem Soc Trans, 2003 Dec, 31(Pt 6), 1406 - 8
Bacterial production of methylglyoxal: a survival strategy or death by misadventure?
Booth IR, Ferguson GP, Miller S, Li C, Gunasekera B, Kinghorn S.
The production of MG (methylglyoxal) in bacterial cells must be maintained in balance with the capacity for detoxification and protection against this electrophile . Excessive production of MG leads to cell death . Survival of exposure to MG is best understood in the Gram-negative bacteria . The major mechanism of protection is the spontaneous reaction of MG with GSH to form hemithiolacetal, followed by detoxification by the glyoxalase system leading to the production of D-lactate . The KefB and KefC glutathione-gated K(+) efflux systems are integrated with the activity of the glyoxalase system to regulate the cytoplasmic pH in response to exposure to electrophiles . Bacteria only produce MG when an imbalance occurs in metabolism . Operation of the MG bypass enables cells to adapt, such that balance is restored to metabolism . Excessive production of MG is an adaptive ploy, which, if it fails, has fatal consequences . On this basis one might define MG-induced loss of life as "death by misadventure" rather than suicide!

Biol Neonate, 2004, 85(3), 159 - 66 Epub 2003 Nov 28.
Platelet-activating factor concentration in the stool of human newborns: effects of enteral feeding and neonatal necrotizing enterocolitis; Amer MD et al.; Epidemiologic studies have identified enteral feedings as a risk factor for necrotizing enterocolitis (NEC) . Enteral feedings provide the substrate for colonization of the newborn gut with gram-negative bacteria with endotoxin production, which may trigger the production of endogenous inflammatory mediators, including platelet-activating factor (PAF) . In this prospective study, we examined the effect of enteral feeding on PAF concentration in the stool of preterm and full-term human newborns . The concentration of PAF levels in stool was measured at the following times: at passage of first meconium, within 24 h prior to the onset of feedings, at the 3rd and 14th day of feeding and at any time confirmed NEC developed . Stool samples also were analyzed for levels of acetylhydrolase, the PAF breakdown enzyme . Stool PAF concentration rose significantly following the start of enteral feedings . The mean PAF concentration for day 14 samples was significantly higher than the mean concentration of meconium samples (4.90 +/- 1.03 vs . 1.81 +/- 0.38 ng/g, p < 0.05) and day 0 samples (4.90 +/- 1.03 vs . 1.79 +/- 0.39 ng/g, p < 0.05) . For the 7 patients diagnosed with definite NEC, the mean stool PAF concentration was 12.42 +/- 0.77 ng/g, significantly elevated compared to the mean PAF levels in stool from healthy infants at all sampling times (p < 0.01) . There was no significant change in acetylhydrolase activity at any of the sampling times . Stool PAF concentration increases with the provision of enteral feedings and rises further with the development of NEC . Since stool acetylhydrolase activity remained unchanged, we speculate the increase of PAF in stool likely represents increased PAF production at the local level following the provision of enteral feedings or the development of neonatal necrotizing enterocolitis .

Mol Pharmacol, 2003 Dec, 64(6), 1521 - 9
PC-SPES: a potent inhibitor of nuclear factor-kappa B rescues mice from lipopolysaccharide-induced septic shock; Ikezoe T et al.; Septic shock is the most common cause of death in intensive care units, and no effective treatment is available at present . Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived proinflammatory cytokines, in which activation of nuclear factor-kappaB (NF-kappaB) plays an important role . PC-SPES is an eight-herb mixture active against a variety of malignancies, including prostate cancer and leukemia . In this study, we demonstrated that PC-SPES inhibited the LPS-induced NF-kappaB reporter activity in RAW264.7 macrophages . Electrophoretic mobility shift assay showed that PC-SPES inhibited the binding of NF-kappaB to specific DNA sequences; however, it did not affect either degradation of inhibitory kappaBalpha or nuclear translocation of NF-kappaB . Also, we explored the effect of PCSPES on LPS-induced mitogen-activated protein (MAP) kinase signaling; PC-SPES did not affect LPS-induced phosphorylation of MAP kinases, including c-Jun NH2-terminal kinase, p38, and extracellular signal-regulated kinase 1/2 . Moreover, PC-SPES decreased the production of proinflammatory cytokines and inducible enzymes, such as tumor necrosis factor (TNF) alpha, interleukin (IL)-1beta, IL-6, cyclooxygenase-2, as well as inducible nitric-oxide synthase in RAW264.7 macrophages and peritoneal macrophages from C57BL/6 mice after the cells were stimulated by either LPS or LPS and interferon-gamma . Furthermore, PC-SPES rescued C57BL/6 mice from death caused by LPS-induced septic shock in conjunction with decreased serum levels of TNFalpha and IL-1beta . Together, PC-SPES is a potent inhibitor of NF-kappaB and might be useful for the treatment of sepsis and inflammatory diseases.

Mol Cell Proteomics, 2004 Feb, 3(2), 156 - 66 Epub 2003 Nov 28.
Proteomic analysis of the systemic immune response of Drosophila; Levy F et al.; Improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize proteins involved in a physiological process, such as the immune response of the insect model Drosophila melanogaster . Profiling of the proteins present in the hemolymph (insect blood) of noninfected flies versus flies infected with bacteria or fungi was performed by two-dimensional gel electrophoresis, silver or Coomassie staining, and image analysis . Through this differential analysis, more than 70 out of 160 spots were up- or down-regulated by at least 5-fold after microbial infection . Coomassie staining, in-gel digestion, and database searches yielded the identity of a series of proteins that are directly involved in the Drosophila immune system . This included proteases, protease inhibitors, and recognition molecules such as prophenoloxydase-activating enzymes, serpins, and Gram-negative binding protein-like . Proteins with a potential function in the immune response were also identified, such as an odorant binding protein, peptidylglycine alpha-hydroxylating monooxygenase, and transferrin, affording new candidates for further investigation of innate immune mechanisms . Moreover, several molecules resulting from the cleavage of proteins were detected after the fungal infection . Altogether, this first differential proteomic analysis of the immune response of Drosophila paves the way for the study of proteins affected during innate immunity.

J Leukoc Biol, 2004 Feb, 75(2), 307 - 13 Epub 2003 Nov 21.
Desialylation of glycoconjugates on the surface of monocytes activates the extracellular signal-related kinases ERK 1/2 and results in enhanced production of specific cytokines; Stamatos NM et al.; Modulation of the sialic acid content of cell-surface glycoproteins and glycolipids influences the functional capacity of cells of the immune system . The role of sialidase(s) and the consequent desialylation of cell surface glycoconjugates in the activation of monocytes have not been established . In this study, we show that desialylation of glycoconjugates on the surface of purified monocytes using exogenous neuraminidase (NANase) activated extracellular signal-regulated kinase 1/2 (ERK 1/2), an intermediate in intracellular signaling pathways . Elevated levels of phosphorylated ERK 1/2 were detected in desialylated monocytes after 2 h of NANase treatment, and increased amounts persisted for at least 2 additional hours . Desialylation of cell surface glycoconjugates also led to increased production of interleukin (IL)-6, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta by NANase-treated monocytes that were maintained in culture . Neither increased levels of phosphorylated ERK 1/2 nor enhanced production of cytokines were detected when NANase was heat-inactivated before use, demonstrating the specificity of NANase action . Treatment of monocytes with gram-negative bacterial lipopolysaccharide (LPS) also led to enhanced production of IL-6, MIP-1alpha, and MIP-1beta . The amount of each of these cytokines that was produced was markedly increased when monocytes were desialylated with NANase before exposure to LPS . These results suggest that changes in the sialic acid content of surface glycoconjugates influence the activation of monocytes.

Lett Appl Microbiol, 2003, 37(5), 424 - 8
Biohydrogenation of C18 unsaturated fatty acids to stearic acid by a strain of Butyrivibrio hungatei from the bovine rumen; van de Vossenberg JL et al.; AIMS: To identify a ruminal isolate which transforms oleic, linoleic and linolenic acids to stearic acid and to identify transient intermediates formed during biohydrogenation . METHODS AND RESULTS: The stearic acid-forming bacterium, isolated from the rumen of a grazing cow, was a Gram-negative motile rod which utilized a range of growth substrates including starch and pectin but not cellulose or xylan . From its 16S rRNA gene sequence, the isolate was identified as a strain of Butyrivibrio hungatei . During conversion of linoleic acid, 9,11-conjugated linoleic acid formed as a transient intermediate before trans-vaccenic acid accumulated together with stearic acid . Unlike previously studied ruminal biohydrogenating bacteria, B . hungatei Su6 was able to convert alpha-linolenic acid to stearic acid . Linolenic acid was converted to stearic via conjugated linolenic acid, linoleic acid and trans-vaccenic acid as intermediates . Oleic acid and cis-vaccenic acid were converted to a series of trans monounsaturated isomers as well as stearic acid . An investigation of these isomers indicated that mixed trans positional isomers are intermediate in the biohydrogenation of cis monounsaturated fatty acids to stearic acid . CONCLUSION: This, the first rigorous identification and characterization of a ruminal bacterium which forms stearic acid, shows that B . hungatei plays an important role in unsaturated fatty acid transformations in the rumen . SIGNIFICANCE AND IMPACT OF THE STUDY: Biohydrogenating bacteria which convert C18 unsaturated fatty acids to stearic acid have not been available for study for many years . Access to B . hungatei Su6 now provides a fresh opportunity for understanding biohydrogenation mechanisms and rumen processes which lead to saturated fat in ruminant products.

J Dent Res, 2003 Dec, 82(12), 944 - 50
Four reasons to consider a novel class of innate immune molecules in the oral epithelium; LeClair EE; An expanding number of innate immune molecules occupy the "epithelial frontier" . This review introduces a recently recognized class of mammalian proteins with similarity to PLUNC (palate, lung and nasal epithelium clone), which is itself related to the host defense protein BPI (bactericidal/permeability-increasing protein) . Four emerging lines of evidence unite the PLUNC-like proteins: conserved genetic structure, epithelial expression, three-dimensional protein similarity, and a physiological response to injury or inflammation . By analogy to known proteins of the innate immune system, an emerging hypothesis for this family is that they act as sensors of Gram-negative bacteria in the oral cavity, among other areas.

Dig Dis Sci, 2003 Oct, 48(10), 1960 - 7
Acute acalculous cholecystitis associated with systemic sepsis and visceral arterial hypoperfusion: a case series and review of pathophysiology; McChesney JA et al.; Acute acalculous cholecystitis (AAC) is marked by a very high mortality rate but its relative rarity makes its features obscure to many physicians . This often contributes to a delayed diagnosis . In this study, we review one center's experience, examine the clinical features of the disorder, and describe the progression of pathological events that culminate in AAC . We performed a 10-year retrospective review of cases of AAC reported at our institution between 1988 and 1998 . Fifteen cases of AAC were identified from this period, during which 5804 cardiovascular operations were performed . The mortality rate was 46.6% . Multiple organ failure was present in 12 of the 15 cases, and 9 of the patients were over 60 years of age . Prolonged hypotension occurred in 13 patients, and fever in all 15 . Nine cases of gangrenous gallbladder occurred . Gram-negative septicemia was present in 12 . Visceral arterial hypoperfusion was frequently evident at operation or necropsy . Thirteen patients showed clinical jaundice, a disproportionate elevation of the alkaline phosphatase, or both . Heart failure was found in 9 patients . Open cholecystectomy was most often the definitive intervention . Arterial hypoperfusion of the gut and or sepsis appear central to the pathogenesis of AAC in our series . Gallbladder inflammation and cholestasis result and bacterial invasion of the organ ensues, culminating in AAC, frequently with gangrene . A model of the pathogenesis of AAC is discussed.

Cell Mol Life Sci, 2003 Nov, 60(11), 2371 - 88
Lytic transglycosylases in macromolecular transport systems of Gram-negative bacteria; Koraimann G; The cell wall of Gram-negative bacteria is essential for the integrity of the bacterial cell but also imposes a physical barrier to trans-envelope transport processes in which DNA and/or proteins are taken up or secreted by complex protein assemblies . The presence of genes encoding lytic transglycosylases in macromolecular transport systems (bacteriophage entry, type II secretion and type IV pilus synthesis, type III secretion, type IV secretion) suggests an important role for these specialised cell-wall-degrading enzymes . Such enzymes are capable of locally enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly and anchoring of supramolecular transport complexes in the cell envelope . In this review, current knowledge on the role and distribution of these specialised murein-degrading enzymes in diverse macromolecular transport systems is summarised and discussed.

Trends Cell Biol, 2003 Dec, 13(12), 610 - 4
Detection of peptidoglycans by NOD proteins; Royet J et al.; Mechanisms of innate immune defense are based on the recognition of invariant microbial molecular patterns by specific receptors, followed by the activation of signaling pathways and the expression of effector molecules that will defeat the invading microorganism . Two recent reports add to the growing list of these pattern-recognition receptors by showing that the intracellular nucleotide-binding oligomerization domain 1 (NOD1) protein recognizes a diaminopimelate-containing muropeptide, a cell-wall component of Gram-negative bacteria.

Can J Vet Res, 2003 Oct, 67(4), 271 - 7
Identification and preliminary characterization of a 75-kDa hemin- and hemoglobin-binding outer membrane protein of Actinobacillus pleuropneumoniae serotype 1; Archambault M et al.; The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth . In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm . Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A . pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions . Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein . A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors . Taken together, our data suggest that A . pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm.

Scand J Infect Dis, 2003, 35(9), 568 - 72
Role of TLR4/MD-2 and RP105/MD-1 in innate recognition of lipopolysaccharide; Kimoto M et al.; TLR4 and RP105 are unique members of the Toll-like receptor (TLR) family molecules . They are associated with small molecules called MD-2 and MD-1, respectively, to form heterodimers (TLR4/MD-2 and RP105/MD-1) and function as recognition/signaling molecules of lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria . Analysis of transfectant cell lines and gene-targeted mice revealed that both MD-2 and MD-1 are involved in the recognition of LPS as well as in the regulation of intracellular distribution and the surface expression of TLR4 and RP105, respectively . Since RP105 or MD-1-deficient mice show a reduced but not complete lack of LPS responsiveness, there may be functional associations between TLR4/MD-2 and RP105/MD-1 . In addition, there was an increased frequency of RP105-negative B-lymphocytes in the peripheral blood in several rheumatic diseases, such as systemic lupus erythematosus, suggesting the involvement of RP105 in the pathophysiology of autoimmunity . Further analysis of the structure and function of TLR4/MD-2 and RP105/MD-1 will provide a better understanding of the pathophysiology, and a chance to develop evidence-based treatments for septic shock syndrome and autoimmunity.

Am J Physiol Lung Cell Mol Physiol, 2004 Apr, 286(4), L877 - 85 Epub 2003 Nov 14.
Innate immune responses to LPS in mouse lung are suppressed and reversed by neutralization of GM-CSF via repression of TLR-4; Bozinovski S et al.; The innate immune inflammatory response to lipopolysaccharide (LPS, an endotoxin) is essential for lung host defense against infection by gram-negative bacteria but is also implicated in the pathogenesis of some lung diseases . Studies on genetically altered mice implicate granulocyte-macrophage colony-stimulating factor (GM-CSF) in lung responses to LPS; however, the physiological effects of GM-CSF neutralization are poorly characterized . We performed detailed kinetic and dose-response analyses of the lung inflammation response to LPS in the presence of the specific GM-CSF-neutralizing antibody 22E9 . LPS instilled into the lungs of BALB/c mice induced a dose-dependent inflammation comprised of intense neutrophilia, macrophage infiltration and proliferation, TNF-alpha and matrix metalloproteinase release, and macrophage inflammatory protein-2 induction . The neutralization of anti-GM-CSF in a dose-dependent fashion suppressed these inflammatory indexes by 85% when given before or after LPS or after repeat LPS challenges . Here we report for the first time that the physiological expression of Toll-like receptor-4 in lung is reduced by anti-GM-CSF . We observed that lower Toll-like receptor-4 expression correlated with a similar decline in peak TNF- levels in response to endotoxin . Consequently, sustained expression of key inflammatory mediators over 24 h was reduced . These data expand the understanding of the contribution of GM-CSF to innate immune responses in lung and suggest that blocking GM-CSF might benefit some lung diseases where LPS has been implicated in etiology.

Pharmacol Ther, 2003 Nov, 100(2), 171 - 94
Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex; Fujihara M et al.; Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent activator of macrophages . Activated macrophages produce a variety of inflammatory cytokines . Excessive production of cytokines in response to LPS is regarded as the cause of septic shock . On the other hand, macrophages exposed to suboptimal doses of LPS are rendered tolerant to subsequent exposure to LPS and manifest a profoundly altered response to LPS . Increasing evidence suggests that monocytic cells from patients with sepsis and septic shock survivors have characteristics of LPS tolerance . Thus, an understanding of the molecular mechanisms underlying activation and deactivation of macrophages in response to LPS is important for the development of therapeutics for septic shock and the treatment of septic shock survivors . Over the past several years, significant progress has been made in identifying and characterizing several key molecules and signal pathways involved in the regulation of macrophage functions by LPS . In this paper, we summarize the current findings of the functions of the LPS receptor complex, which is composed of CD14, Toll-like receptor 4 (TLR4), and myeloid differentiation protein-2 (MD-2), and the signal pathways of this LPS receptor complex with regard to both activation and deactivation of macrophages by LPS . In addition, recent therapeutic approaches for septic shock targeting the LPS receptor complex are described.

Trends Microbiol, 2003 Nov, 11(11), 511 - 8
H-NS in Gram-negative bacteria: a family of multifaceted proteins; Tendeng C et al.; DNA-packaging and the control of gene expression constitute a major challenge for bacteria to survive and adapt to environmental changes . The use of multiple strategies to solve these problems could explain the presence of various nucleoid-associated proteins in bacteria . H-NS, one of these proteins, has been extensively studied in Escherichia coli, and a variety of phenotypes have been associated with a mutation in its structural gene . However, the role of H-NS in bacterial physiology and its mechanism of action are still a matter of debate . The expanding number of H-NS-related proteins identified in Gram-negative bacteria reveals interesting clues about their structure-function-evolution relationship.

Crit Care Med, 2003 Nov, 31(11), 2627 - 33
Reassessing the value of short-term mortality in sepsis: comparing conventional approaches to modeling; Clermont G et al.; OBJECTIVE: Clinical trials of therapies for sepsis have been mostly unsuccessful in impacting mortality . This may be partly due to the use of insensitive mortality end points . We explored whether modeling survival was more sensitive than traditional end points in detecting mortality differences in cohorts of patients with sepsis . DESIGN: Patients were stratified into seven a priori defined paired subgroups that reflected high and low mortality risk according to known clinical risk factors . We fitted an exponential survival model to the high- and low-risk cohort of each subgroup, providing estimates of the rate of dying, long-term survival, and excess day 1 mortality . Mortality in the high- and low-risk cohorts in each subgroup was compared using model parameters, fixed-point mortality, and Kaplan-Meier survival analysis . SETTING: Eight intensive care units within a university teaching institution . PATIENTS: One hundred thirty patients with severe sepsis or suspected Gram-negative bacteremia . INTERVENTIONS: None . MEASUREMENTS AND MAIN RESULTS: Overall mortality of the cohort was 58.5% at 28 days . The survival of the entire cohort was well described by an exponential model (r2 =.99) . Modeling identified differences in high- and low-risk cohorts in five of the seven paired subgroups, while conventional end-points only detected differences in 2 . CONCLUSIONS: Modeling survival was more sensitive than conventional end-points in identifying survival differences between high- and low-risk subgroups . We encourage further evaluation of modeling in the search for more sensitive mortality end points.

J Clin Microbiol, 2003 Nov, 41(11), 5327 - 32
Isolation of Bartonella washoensis from a dog with mitral valve endocarditis; Chomel BB et al.; We report the first documented case of Bartonella washoensis bacteremia in a dog with mitral valve endocarditis . B . washoensis was isolated in 1995 from a human patient with cardiac disease . The main reservoir species appears to be ground squirrels (Spermophilus beecheyi) in the western United States . Based on echocardiographic findings, a diagnosis of infective vegetative valvular mitral endocarditis was made in a spayed 12-year-old female Doberman pinscher . A year prior to presentation, the referring veterinarian had detected a heart murmur, which led to progressive dyspnea and a diagnosis of congestive heart failure the week before examination . One month after initial presentation, symptoms worsened . An emergency therapy for congestive heart failure was unsuccessfully implemented, and necropsy evaluation of the dog was not permitted . Indirect immunofluorescence tests showed that the dog was strongly seropositive (titer of 1:4,096) for several Bartonella antigens (B . vinsonii subsp . berkhoffii, B . clarridgeiae, and B . henselae), highly suggestive of Bartonella endocarditis . Standard aerobic and aerobic-anaerobic cultures were negative . However, a specific blood culture for Bartonella isolation grew a fastidious, gram-negative organism 7 days after being plated . Phenotypic and genotypic characterizations of the isolate, including partial sequencing of the citrate synthase (gltA), groEL, and 16S rRNA genes, indicated that this organism was identical to B . washoensis . The dog was seronegative for all tick-borne pathogens tested (Anaplasma phagocytophilum, Ehrlichia canis, and Rickettsia rickettsii), but the sample was highly positive for B . washoensis (titer of 1:8,192) and, according to indirect immunofluorescent-antibody assay, weakly positive for phase II Coxiella burnetii infectionPublication Types:

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