BMC Gastroenterol . 2003 Nov 06;3(1):31. Cytotoxic isolates of Helicobacter pylori from peptic ulcer diseases decrease K+-dependent ATPase activity in HeLa cells; Shanjana A et al.; BACKGROUND: Helicobacter pylori is a Gram negative bacterium that plays a central role in the etiology of chronic gastritis and peptic ulcer diseases . However, not all H . pylori positive cases develop advanced disease . This discriminatory behavior has been attributed to the difference in virulence of the bacteria . Among all virulence factors, cytotoxin released by H . pylori is the most important factor . In this work, we studied variation in H . pylori isolates from Indian dyspeptic patients on the basis of cytotoxin production and associated changes in K+-dependent ATPase (one of its targets) enzyme activity in HeLa cells . METHODS: The patients were retrospectively grouped on the basis of endoscopic and histopathological observation as having gastritis or peptic ulcer . The HeLa cells were incubated with the broth culture filtrates (BCFs) of H . pylori isolates from patients of both groups and observed for the cytopathic effects: morphological changes and viability . In addition, the K+-dependent ATPase activity was measured in HeLa cells extracts . RESULTS: The cytotoxin production was observed in 3/7 (gastritis) and 4/4 (peptic ulcer) H . pylori isolates . The BCFs of cytotoxin producing H . pylori strains reduced the ATPase activity of HeLa cells to 40% of that measured with non-cytotoxin producing H . pylori strains (1.33 micromole Pi/mg protein and 3.36 micromole Pi/mg protein, respectively, p < 0.05) . The decreased activity of ATPase enzyme or the release of cytotoxin also correlated with the increased pathogenicity indices of the patients . CONCLUSIONS: Our results suggest that the isolation of cytotoxic H . pylori is more common in severe form of acid peptic diseases (peptic ulcer) than in gastritis patients from India . Also the cytotoxin released by H . pylori impairs the ion-transporting ATPase and is a measure of cytotoxicity. J Biol Chem, 2004 Jan 23, 279(4), 2712 - 8 Epub 2003 Nov 04. Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4; Guillot L et al.; Pulmonary epithelial cells are continuously exposed to microbial challenges as a result of breathing . It is recognized that immune myeloid cells express Toll-like receptors (TLRs), which play a major role in detecting microbes and initiating innate immune responses . In contrast, little is known concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function, and the signaling pathways involved . In this work, we demonstrated by reverse transcription-PCR and/or immunoblot that TLR4 and the accessory molecule MD-2 are constitutively expressed in distinct human alveolar and bronchial epithelial cells . We further characterized by flow cytometry, biotinylation/precipitation, and confocal microscopy the intracellular localization of TLR4 in these cells . Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern . Using respiratory epithelial cells isolated from TLR4 knock-out and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells . Furthermore we showed that this cell response to LPS involves a signaling complex including the kinases interleukin-1 receptor-associated kinase (IRAK), p38, Jnk, and ERK1/2 . Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates . Altogether these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria . The intracellular localization of TLR4 in lung epithelia is expected to play an important role in the prevention of the development of chronic inflammatory disease. J Autoimmun, 2003 Nov, 21(3), 277 - 81 Impaired responses to toll-like receptor 4 and toll-like receptor 3 ligands in human cord blood; De Wit D et al.; Toll-like receptor (TLR)-4 signaling pathway plays an essential role in host defense against gram-negative bacteria while TLR-3-mediated signaling is critically involved in anti-viral immunity . To gain insight into the defects responsible for impaired Th1 responses in human newborns, we investigated the responses of human cord blood cells to lipopolysaccharide, LPS, and to polyinosinic-polycytidylic acid, Poly (I:C), ligands of TLR-4 and TLR-3, respectively . Measurement of cytokine levels revealed a profound defect in IL-12 (p70) synthesis and an increased release of IL-10 in cord blood exposed to LPS or Poly (I:C), as compared to adult blood . Moreover, Poly (I:C)-induced IFN-alpha production was found to be significantly impaired in cord blood . Phenotypic maturation of myeloid DC in response to LPS or Poly (I:C) was next compared in cord and adult blood . We observed that neonatal myeloid DC displayed decreased upregulation of CD40, CD80 whereas CD86 and HLA-DR upregulation did not differ significantly between adults and neonates . Taken together, these findings might be relevant to the increased vulnerability of human newborns to intracellular pathogens and to their inability to develop efficient Th1-type responses. Am J Surg, 2003 Nov, 186(5), 526 - 30 Endotoxin potentiates lung injury in cerulein-induced pancreatitis; Gray KD et al.; BACKGROUND: In this study we examine the effect of endotoxin (lipopolysaccharide) on lung injury in the setting of acute pancreatitis (AP) . METHODS: Twelve hourly injections of cerulein (50 microg/kg/h) were used to induce pancreatitis in mice . Intraperitoneal lipopolysaccharide (LPS {6 mg/kg}) was administered 24 hours after the initial cerulein injection . Twenty-four hours after LPS injection, myeloperoxidase (MPO) activity, nuclear factor (NF)-kappaB activation, and tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and chemokines MIP-2 and KC levels were measured in pancreas, liver, and lung tissues . Four groups of mice were studied: cerulein-LPS, cerulein-saline, saline-LPS, and saline-saline treated mice . RESULTS: Elevated serum lipase confirmed pancreatitis in cerulein treated mice . Lung MPO activity was significantly increased in the cerulein-LPS group . NF-kappaB was activated in the liver but not in pancreas and lung tissue . Chemokines MIP-2 and KC were elevated in pancreatic tissue only . CONCLUSIONS: These findings suggest that gram-negative infections may be an important predisposition for the development of adult respiratory distress syndrome in the setting of AP and that hepatic NF-kappaB may mediate multisystem injury. Proteomics, 2003 Nov, 3(11), 2249 - 57 Proteomics of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1, using a matrix-assisted laser desorption/ionization-tandem-time of flight mass spectrometer; Vanrobaeys F et al.; Shewanella oneidensis MR-1 is a gram-negative facultative aerobic bacterium living at oxic-anoxic interfaces in nature . The plasticity of terminal electron-acceptors used under anaerobic conditions is huge, but the adaptation to these different environmental conditions remains unclear . In this work, we used a proteomic approach to study the protein content when the organism is grown under anaerobic respiration conditions on insoluble ferric oxide . By analysis of two-dimensional gel patterns of soluble protein extracts, we discovered 20 differentially displayed proteins . The protein spots were further analyzed by mass spectrometry for which we used, in addition to nano-high-performance liquid chromatography coupled to an electrospray ionization-quadrupole-time of flight instrument, a recently introduced matrix-assisted laser desorption/ionization (MALDI) tandem-time of flight mass spectrometer . The instrument allows the acquisition of high quality spectra, in both the mass spectrometry and tandem mass spectrometry mode, and is therefore able to identify protein spots unambiguously . Advantageous to electrospray ionization is a minimised sample handling, inherent to MALDI ionization, and the presence of high energy fragmentation ions, generating sequence information that also can differentiate isobaric amino acids . With this strategy, we could point out a regulatory protein that is up-regulated under iron(III) respiration . This protein, the aerobic respiration control protein (ArcA), has been reported as being a regulator during anaerobiosis in other species . To our knowledge, this is the first report of the possible involvement of ArcA from S . oneidensis MR-1 in the reduction of ferric oxide. Med Microbiol Immunol (Berl), 2004 Nov, 193(4), 163 - 71 Epub 2004 Nov. Expression and localization of type III secretion-related proteins of Chlamydia pneumoniae; Lugert R et al.; The entire developmental cycle of the obligate intracellular bacteria Chlamydia pneumoniae takes place within the inclusion body . As many gram negative bacteria, Chlamydia possess a type III-secretion system (TTSS), which allows them to target effector molecules into the host cell . The expression and localization of several proteins constituting the TTSS apparatus and of proteins supposed to be secreted by the TTSS have been investigated . For the TTSS-constituting proteins, we selected representatives such as YscN (ATPase), LcrE (putative "lid" of the TTSS) and LcrH1 (postulated to be a chaperone) . Furthermore, we focused on the putative effector proteins IncA, IncB, IncC, Cpn0809 and Cpn1020 . Expression of these proteins was detected by reverse transcriptase-PCR followed by immunoblot analysis using antisera that were generated against the corresponding recombinant proteins . Thereby, expression could be detected on the RNA and/or protein level . Intracellular localization of proteins under investigation was determined by immunofluorescence assays using the respective antisera . YscN was shown to be distributed equally throughout the inclusion body, whereas LcrE gave a more prominent staining of the inclusion membrane . IncA was detected mainly on the membrane of the inclusion body, whereas IncB and IncC were shown to be located within the inclusion . Immunofluorescence assays with antisera raised against Cpn0809 and Cpn1020 showed completely different labeling . Signals corresponding to Cpn0809 and Cpn1020 were distributed within the host cell rather than inside the inclusions . Taken together, the different localization patterns of the effector proteins indicate differences in function and interplay with the host cell. Hautarzt, 2003 Nov, 54(11), 1080 - 2 {Necrotizing otitis externa caused by Stenotrophomonas maltophilia}; Borner D et al.; The gram-negative bacterium Stenotrophomonas maltophilia is feared, especially in intensive care wards, as a nosocomial opportunistic pathogen causing fulminant septicemia and organ failure in immunosuppressed patients . A patient with chronic lymphocytic leukemia developed a cutaneous infection with the clinical pattern of necrotizing otitis externa. Biochem Biophys Res Commun, 2003 Nov 14, 311(2), 465 - 72 Rapid up-regulation of IRAK-M expression following a second endotoxin challenge in human monocytes and in monocytes isolated from septic patients; Escoll P et al.; The exposure of human monocytes to the gram-negative endotoxin LPS provokes them to enter a transient state in which they are refractory to further stimulation by LPS . This phenomenon is known as 'endotoxin tolerance' (ET) and it is characterized by a decrease in leukocyte proinflammatory cytokine production in response to LPS . In the present study, we have analyzed the expression of IRAK-M mRNA and protein in a human model of ET using human monocytes isolated from peripheral blood . In these monocyte cultures, IRAK-M mRNA was expressed 6h after stimulation with different doses of LPS . However, endotoxin pretreatment induced a more immediate up-regulation of IRAK-M gene expression, transcripts appearing only one hour after a second LPS-challenge, and the production of high levels of IRAK-M protein in these tolerant monocytes . We also analyzed the response of monocytes isolated from septic patients within a temporal tolerance timeframe when stimulated ex vivo with LPS . In contrast to monocytes from healthy volunteers and patients outside of the tolerance timeframe, monocytes from septic patients rapidly expressed IRAK-M mRNA when stimulated with LPS ex vivo . Moreover, the expression of IRAK-M mRNA was more rapidly induced in the presence of a PI3K inhibitor, suggesting a connection between these two kinases . Thus, our data indicate that IRAK-M could play a pivotal role in the process of ET in human monocytes and provide evidence that PI3K is involved in regulating its expression. Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2059 - 67 Characteristics of an anaerobic, syntrophic, butyrate-degrading bacterium in paddy field soil; Zou BZ et al.; The number of syntrophic butyrate-degrading bacteria in a flooded paddy field soil was 1.7 x 10(3) MPN/g dry soil . Butyrate was degraded to acetate and methane when paddy soils were incubated anaerobically with the addition of butyrate . However, butyrate degradation was completely suppressed by the addition of the specific inhibitor of methanogenesis, 2-bromoethanesulfonate (BES) to the soil . A hydrogen-using methanogen, strain TM-8, was isolated from flooded paddy field soil . Strain TM-8 was identified as Methanobacterium formicicum based on its physiology and phylogeny . Syntrophic butyrate-degrading bacteria were enumerated and isolated using strain TM-8 . A syntrophic butyrate-degrading bacterium, strain TB-6, was isolated in coculture with strain TM-8 from paddy soil . The strain was Gram-negative, had curved rods, and grew on crotonate . Sulfate was not used as an electron acceptor . Strain TB-6 was closely related to S . wolfei subsp . wolfei . The relation between strain TB-6 and the members of Syntrophomonas are discussed. Curr Opin Chem Biol, 2003 Oct, 7(5), 570 - 9 Mechanisms of drug/H+ antiport: complete cysteine-scanning mutagenesis and the protein engineering approach; Tamura N et al.; The notorious difficulty of elucidating structures of membrane transporters by crystallography has long prevented our understanding of active transport mechanism coupled with ion/proton transport . The determination of the first crystal structure of the drug/H+ antiporter AcrB was a breakthrough for structure-based understanding of drug/H+ antiport . However, although AcrB is a major multidrug exporter in Gram-negative organisms, the majority of bacterial drug exporters are major facilitator superfamily (MFS) drug transporters . As no crystal structures have been solved for MFS transporters, the alternative protein-engineering methods are still very useful for estimating structures and functions of drug/H+ antiporters . This review describes this alternative approach for investigating the structure and function of tetracycline/H+ antiporters. J Endotoxin Res, 2003, 9(5), 308 - 12 Role of interferons in LPS hypersensitivity; Freudenberg MA et al.; The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component . Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e . the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens . LPS sensitivity increases following treatment of mice with live or killed micro-organisms . Two types of sensitization have been recognized, strong, IFN-gamma-dependent and moderate IFN-gamma-independent . IL-12 and IL-18 are intimately involved in the induction of IFN-gamma by bacteria . We showed that Gram-negative bacteria induce IFN-gamma in mice also by an IFN-beta-dependent pathway that requires IL-18 and is independent of IL-12 signaling . This pathway is STAT4 dependent, the activation of which is directly linked to IFN-beta . Further, IFN-beta can be replaced by IFN-alpha . While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-beta . Therefore, the IFN-beta pathway of IFN-gamma induction, unlike the IL-12 pathway, proceeds only in LPS responder mice . The IFN-alpha/beta-dependent pathway is expected to play a role whenever IFN-alpha or IFN-beta, and IL-18 are produced concomitantly during infection. J Endotoxin Res, 2003, 9(5), 281 - 91 Inhibition of LPS-responses by synthetic peptides derived from LBP associates with the ability of the peptides to block LBP-LPS interaction; Arana Mde J et al.; The ability of LPS-binding protein (LBP) to greatly potentiate cell responses to lipopolysaccharide (LPS) may largely contribute to LPS toxicity in sepsis . The study of agents with the capacity to block the interaction between LBP and LPS might improve the understanding of the role of LBP in Gram-negative infections as well as offering new therapeutic tools for septic disorders . Here we confirm the ability of synthetic peptides comprising the human LBP amino acid region 86-108 to interfere with the LBP-LPS interaction . The analysis of selected alanine mutants of a blocking peptide corresponding to the LBP region 86-99 suggests the importance of peptide amphipathicity for the inhibitory activity . The potency of the native peptide and a selected analogue at inhibiting in vitro and in vivo LPS-induced responses was associated with their relative activity in blocking LBP-LPS interaction . It was remarkable that these peptides were at least 500-fold more active in vivo than in vitro . Also, the inhibitory activity of peptides LBP86-99 and LBPK95A seems to be independent of LBP concentrations, a behavior that may be relevant for the potential use of these peptides in septic disorders where LBP serum concentrations are considerably elevated. Infect Immun, 2003 Nov, 71(11), 6426 - 34 Identification of a 3-deoxy-D-manno-octulosonic acid biosynthetic operon in Moraxella catarrhalis and analysis of a KdsA-deficient isogenic mutant; Luke NR et al.; Lipooligosaccharide (LOS), a predominant surface-exposed component of the outer membrane, has been implicated as a virulence factor in the pathogenesis of Moraxella catarrhalis infections . However, the critical steps involved in the biosynthesis and assembly of M . catarrhalis LOS currently remain undefined . In this study, we used random transposon mutagenesis to identify a 3-deoxy-D-manno-octulosonic acid (KDO) biosynthetic operon in M . catarrhalis with the gene order pyrG-kdsA-eno . The lipid A-KDO molecule serves as the acceptor onto which a variety of glycosyl transferases sequentially add the core and branch oligosaccharide extensions for the LOS molecule . KdsA, the KDO-8-phosphate synthase, catalyzes the first step of KDO biosynthesis and is an essential enzyme in gram-negative enteric bacteria for maintenance of bacterial viability . We report the construction of an isogenic M . catarrhalis kdsA mutant in strain 7169 by allelic exchange . Our data indicate that an LOS molecule consisting only of lipid A and lacking KDO glycosylation is sufficient to sustain M . catarrhalis survival in vitro . In addition, comparative growth and susceptibility assays were performed to assess the sensitivity of 7169kdsA11 compared to that of the parental strain . The results of these studies demonstrate that the native LOS molecule is an important factor in maintaining the integrity of the outer membrane and suggest that LOS is a critical component involved in the ability of M . catarrhalis to resist the bactericidal activity of human sera. Arch Mal Coeur Vaiss, 2003 Sep, 96(9), 923 - 6 {Cardiobacterium hominis endocarditis . A case report}; Balcou-Leroy E et al.; We report the case of a Cardiobacterium hominis endocarditis causing an acute mitral insufficiency complicated of left heart failure . The patient has been treated after a few days by surgical valvuloplasty . Cardiobacterium hominis is a bacteria of the HACCEK group, bacille gram-negative, sometimes anaerobic, difficult to isolate . Recently, Polymerase Chain Reaction analysis appears to be effective for the the diagnosis in the identification of fastidious micro-organisms like Cardiobacterium hominis . We have reviewed in the literature 71 cases of Cardiobacterium hominis endocarditis; clinical presentation is often sub-acute, the bacteriological diagnosis is based on hemocultures for which the culture is slow and require enriched environments . Hemodynamic and thrombo-embolic complications are frequent because of the high pathogenicity of the bacteria which provides big and friable vegetations . Despite a high sensibility to antibiotherapy, surgical intervention is often required. J Immunol, 2003 Nov 1, 171(9), 4792 - 800 Novel and detrimental effects of lipopolysaccharide on in vitro generation of immature dendritic cells: involvement of mitogen-activated protein kinase p38; Xie J et al.; Dendritic cells (DCs) are recognized as major players in the regulation of immune responses to a variety of Ags, including bacterial agents . LPS, a Gram-negative bacterial cell wall component, has been shown to fully activate DCs both in vitro and in vivo . LPS-induced DC maturation involves activation of p38, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases, and NF-kappaB . Blocking p38 inhibits LPS-induced maturation of DCs . In this study we investigated the role of LPS in the in vitro generation of immature DCs . We report here that in contrast to the observed beneficial effects on DCs, the presence of LPS in monocyte culture retarded the generation of immature DCs . LPS not only impaired the morphology and reduced the yields of the cultured cells, but also inhibited the up-regulation of surface expression of CD1a, costimulatory and adhesion molecules . Furthermore, LPS up-regulated the secretion of IL-1beta, IL-6, IL-8, IL-10, and TNF-alpha; reduced Ag presentation capacity; and inhibited phosphorylation of ERK, but activated p38, leading to a reduced NF-kappaB activity in treated cells . Neutralizing Ab against IL-10, but not other cytokines, partially blocked the effects of LPS . Inhibiting p38 (by inhibitor SB203580) restored the morphology, phenotype, and Ag presentation capacity of LPS-treated cells . SB203580 also inhibited LPS-induced production of IL-1beta, IL-10, and TNF-alpha; enhanced IL-12 production; and recovered the activity of ERK and NF-kappaB . Thus, our study reveals that LPS has dual effects on DCs that are biologically important: activating existing DCs to initiate an immune response, and inhibiting the generation of new DCs to limit such a response. Mar Biotechnol (NY), 2003 Nov-Dec, 5(6), 579 - 83 Epub 2003 Oct 20. Submersed culture production of extracellular wax esters by the marine bacterium Fundibacter jadensis; Bredemeier R et al.; Fundibacter jadensis strain T9, a marine gram-negative bacterium, was isolated from the intertidal sediment of the German North Sea coast by our group . The cells were able to produce considerable amounts of extracellular wax esters when cultivated with n-alkanes (hexadecane or tetradecane) as a carbon source . The dependence of wax ester production and the composition of the purified wax on different culture conditions (C:N:P ratio and dissolved oxygen tension) were tested . Our results show that wax ester production was not directly growth-linked . The C:N:P ratio had no significant influence on the yield of alkane-free purified wax . The dissolved oxygen tension affected the produced amount of the alkane-free purified wax and the composition of the purified wax; when lower than 2% it decreased the yield of purified wax and led to an altered wax ester composition . Tetradecane as a carbon source enhanced the spectrum of the wax ester composition. Curr Eye Res, 2003 Oct, 27(4), 227 - 35 The effect of protein-coated contact lenses on the adhesion and viability of gram negative bacteria; Williams TJ et al.; PURPOSE: Gram negative bacterial adhesion to contact lenses can cause adverse responses . During contact lens wear, components of the tear film adsorb to the contact lens . This study aimed to investigate the effect of this conditioning film on the viability of bacteria . METHODS: Bacteria adhered to contact lenses which were either unworn, worn for daily-, extended- or overnight-wear or coated with lactoferrin or lysozyme . Numbers of viable and total cells were estimated . RESULTS: The number of viable attached cells was found to be significantly lower than the total number of cells on worn (50% for strain Paer1 on daily-wear lenses) or lactoferrin-coated lenses (56% for strain Paer1) . Lysozyme-coated lenses no statistically significant effect on adhesion . DISCUSSION: The conditioning film gained through wear may not inhibit bacterial adhesion, but may act adversely upon those bacteria that succeed in attaching. Bone Marrow Transplant, 2003 Nov, 32(9), 941 - 5 Procalcitonin and C-reactive protein do not discriminate between febrile reaction to anti-T-lymphocyte antibodies and Gram-negative sepsis; Dornbusch HJ et al.; Treatment with antibodies against T-lymphocytes usually triggers a febrile response potentially mimicking or masking infection . Procalcitonin (PCT) is considered a sensitive and specific marker of systemic bacterial and fungal infection . It was the aim of this study to investigate the characteristics of PCT and C-reactive protein (CRP) during treatment with polyclonal or monoclonal anti-T-cell antibodies, in order to examine the ability of these parameters to distinguish between systemic bacterial infection and reaction to antibody treatment . Thus, 15 consecutive febrile episodes after T-cell antibody infusion without clinical signs of infection were compared with nine episodes of Gram-negative sepsis . After T-cell antibody infusion PCT and CRP serum levels increased to a similar extent as in Gram-negative sepsis . Therefore, during T-cell antibody treatment neither PCT nor CRP are adequate for differentiating between fever due to infection or to unspecific cytokine release. Curr Drug Targets Inflamm Allergy, 2002 Mar, 1(1), 53 - 63 Endocannabinoid degradation, endotoxic shock and inflammation; Maccarrone M et al.; Endocannabinoids are an emerging class of lipid mediators, which include amides and esters of long chain polyunsaturated fatty acids . Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors . Endotoxic shock is a potentially lethal failure of multiple organs that can be initiated by the inflammatory agent lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria . LPS has been recently shown to stimulate the production of AEA in rat macrophages, and of 2-AG in rat platelets . The mechanism responsible for this effect has not been elucidated . On the other hand, mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play an essential role in inflammation . As yet, little is known about endogenous modulators and mechanisms of mast cell activation . Here, we review recent literature on the role of endocannabinoids in endotoxic shock and inflammation, and report our recent research on the effects of LPS on the production of AEA and 2-AG in human lymphocytes, and on AEA degradation by a specific AEA membrane transporter (AMT) and an AEA-degrading enzyme (fatty acid amide hydrolase, FAAH) . We also report the ability of the HMC-1 human mast cells to degrade AEA through a nitric oxide-sensitive AMT and a FAAH . The role of endocannabinoids in HMC-1 degranulation is discussed as well . Taken together, it can be suggested that human lymphocytes and mast cells take part in regulating the peripheral endocannabinoid system, which can affect some activities of these cells. Arch Pediatr Adolesc Med, 2003 Oct, 157(10), 1016 - 21 Lake-associated outbreak of Escherichia coli O157:H7 in Clark County, Washington, August 1999; Bruce MG et al.; CONTEXT: Escherichia coli O157:H7, one of hundreds of strains of the gram-negative bacterium E coli, has been implicated in numerous lake-borne outbreaks of infection during the past decade . In August 1999, several children who later became ill with E coli O157:H7 infection reported swimming in a lake in Clark County, Washington . The lake was closed and an investigation begun . OBJECTIVES: To identify the source of the outbreak and determine risk factors for infection with E coli O157:H7.Design, Setting, and Patients Two case-control studies were performed among residents of and visitors to Clark County in August 1999 by using community and campground-registrant control subjects.Main Outcome Measure Risk factors for infection with E coli O157:H7 among Clark County residents or visitors . RESULTS: We identified 37 case patients (including 29 primary-case patients) with a median age of 5 years (age range, 1-14 years for primary-case patients) . Eight children were hospitalized, 3 with hemolytic uremic syndrome; none died . With analysis restricted to primary-case patients, illness was strongly associated with swimming in the lake (18 of 18 case patients vs 1 of 18 neighborhood-matched and age-matched control subjects; matched odds ratio undefined; P<.001) . All primary-case patients were children younger than 15 years who swam in the lake . Illness was associated with placing the head underwater, getting lake water in the mouth, or swallowing lake water (26 of 27 case patients vs 43 of 62 control subjects; matched odds ratio = 11.5; P =.005) . Cultures of lake water yielded E coli O157:H7 that matched the outbreak strain according to results of pulsed-field gel electrophoresis . CONCLUSIONS: To date, this is one of the largest documented outbreaks of E coli O157:H7 infection associated with unchlorinated recreational water and represents the first outbreak in which the strain was isolated from lake water . Guidelines are needed to decrease the risk of enteric illness associated with swimming in recreational lakes. Vet Res, 2003 Sep-Oct, 34(5), 597 - 627 The bovine neutrophil: Structure and function in blood and milk; Paape MJ et al.; Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens . Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection . While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis . In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland . This will result in permanent scarring and reduced numbers of milk secretory cells . The life span of PMN is limited by the onset of apoptosis . To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis . Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue . The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria . One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14 . Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-alpha and sepsis . Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection . Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants . Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis. Vet Res, 2003 Sep-Oct, 34(5), 507 - 19 Coliform mastitis; Hogan J et al.; Gram-negative bacteria that commonly cause bovine mastitis are classified as environmental pathogens . The point sources of coliform bacteria that cause infections include bedding materials, soil, manure and other organic matter in the environment of cows . Rates of coliform mastitis increase during climatic periods that maximize populations in the environment . The portal of entry into the mammary gland for Gram-negative bacteria is the teat canal . Once in the gland, bacteria must utilize available substrates in the mammary secretion to replicate and evade host defenses . Rates of coliform mastitis are greater during the transitional phases of the non- lactating period than during lactation . The ability to infect the non-lactating gland is directly related to the ability of bacteria to acquire iron from the mammary secretion . The primary host defense against coliform mastitis during lactation is the elimination of bacteria by neutrophils migrating into the gland in response to inflammation . Damage to the host is mediated by the release of endotoxin . The severity and duration of clinical signs associated with coliform mastitis are reduced by the use of core-antigen bacterins. J Biol Chem, 2003 Dec 26, 278(52), 52901 - 8 Epub 2003 Oct 09. Lipopolysaccharides indirectly stimulate apoptosis and global induction of apoptotic genes in fibroblasts; Alikhani M et al.; Following Gram-negative bacterial infection there is a reduction in matrix-producing cells . The goal of the present study was to examine the apoptotic effects of lipopolysaccharide (LPS) on fibroblastic cells and to investigate the role that the host response plays in this reaction . This was accomplished in vivo by subcutaneous inoculation of LPS in wild type and TNFR1(-/-)R2(-/-) mice . The direct effects of LPS on fibroblast apoptosis was studied in vitro with normal diploid human fibroblasts . The results indicate that LPS in vivo induces apoptosis of fibroblasts . By RNA profiling we demonstrated that LPS stimulates global expression of apoptotic genes and down-regulates anti-apoptotic genes . Fluorometric studies demonstrated that LPS in vivo significantly increased caspase-8 and caspase-3 activity and by use of specific inhibitors, the activation of caspase-3 was shown to be initiated by caspase-8 with no contribution from caspase-9 . In vitro studies demonstrated that LPS did not induce apoptosis of fibroblasts, whereas tumor necrosis factor (TNF) did . In addition, the pattern of apoptotic gene expression induced by TNF in vitro was nearly identical to that induced by LPS in vivo, as measured by RNase protection assay . Moreover, pre-treatment of cells with TNF greatly enhanced apoptosis induced by a second stimulation with TNF 24 h later, suggesting that the global induction of pro-apoptotic genes was functionally significant . Thus, LPS acts to modulate the expression of a large number of genes that favor apoptosis of fibroblastic cells that is dependent upon activation of caspase-8 and is largely mediated by TNF. FEBS Lett, 2003 Oct 9, 553(1-2), 135 - 40 Endotoxin-neutralizing effects of histidine-rich peptides; Bosshart H et al.; Inflammatory responses of human peripheral blood monocytes to the Gram-negative endotoxin lipopolysaccharide (LPS) are enhanced by structurally diverse substances, such as anionic polysaccharides or cationic polypeptides . Only a few substances are known to effectively blunt LPS-induced monocyte activation . We now show that synthetic poly-L-histidine (Hn) binds to LPS and abrogates the release of the proinflammatory cytokine interleukin-8 (IL-8) in LPS-stimulated human whole blood . LPS-induced stimulation of monocytes was strictly pH-dependent with only minor amounts of IL-8 secreted in acidic blood . Maximum levels of IL-8 secretion occurred at a strongly basic pH . Hn inhibition of the release of IL-8 from LPS-stimulated monocytes was observed under acidic, neutral and physiological conditions . With increasing alkalosis, the effectiveness of Hn was gradually lost, suggesting that protonated, but not deprotonated, Hn was effective in inhibiting LPS-induced monocyte responses . Histidine-rich protein 2 from the malaria parasite, Plasmodium falciparum, inhibited the ability of LPS to evoke an inflammatory response in CD14-transfected THP-1 cells . Further, a short synthetic peptide derived from human histidine- and proline-rich glycoprotein also exhibited LPS-inhibitory effects in CD14 transfectants . Taken together, these observations demonstrate the capacity of histidine-rich peptides, irrespective of their origin, to neutralize LPS-induced proinflammatory host responses. Cell, 2003 Oct 3, 115(1), 2 - 3 Bacterial vesicle formation as a mechanism of protein transfer to animals; Miller SI et al.; Gram-negative bacterial vesicle formation is a mechanism for specific secretion and transfer of a protein toxin to animals . This discovery should stimulate work on the mechanism of protein sorting into vesicles and the role of vesicles in bacterial pathogenesis. J Am Chem Soc, 2003 Oct 15, 125(41), 12475 - 83 Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail; Wilson KA et al.; Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria . Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-) . On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold . MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8 . The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail. Crit Care Med, 2003 Oct, 31(10), 2421 - 8 Cell therapy with a tissue-engineered kidney reduces the multiple-organ consequences of septic shock; Humes HD et al.; OBJECTIVE: Gram-negative septic shock has a clinical mortality rate approaching 50% . The cause of death is secondary to a systemic inflammatory response syndrome with resulting cardiovascular collapse, ischemic damage to vital organs, and multiple-organ systems failure . Renal tubule cell injury occurs early in septic shock but is not clinically appreciated . Since renal tubule cells appear to play a critical role in the immunoregulation of stress states, renal cell therapy during septic shock may alter the detrimental multiple-organ consequences of systemic Gram-negative infection . The development of a tissue-engineered bioartificial kidney consisting of a conventional hemofiltration cartridge in series with a renal tubule assist device (RAD) containing 109 renal proximal tubule cells may be a new therapeutic approach to this clinical disorder . DESIGN: Laboratory study . SETTING: University medical school . SUBJECTS: Pigs weighing 30-35 kg . INTERVENTIONS: To assess the effect of the bioartificial kidney and the RAD in septic shock, pigs were administered 30 x 10(10) bacteria/kg body weight of Escherichia coli into the peritoneal cavity and within 1 hr were immediately placed in a continuous venovenous hemofiltration extracorporeal circuit with either a sham RAD without cells or a RAD with cells . MEASUREMENTS AND MAIN RESULTS: In this animal model, septic shock resulted within hours in acute tubule necrosis in the kidneys of all animals . Renal cell therapy resulted in significantly higher cardiac outputs and renal blood flow rates in treated animals compared with sham controls . RAD treatment also was associated with significantly lower plasma circulating concentrations of interleukin-6 and interferon-gamma compared with sham-treated animals . IL-6 release rates from peripheral blood mononuclear cells isolated from RAD-treated animals were significantly higher after endotoxin stimulation than those isolated from control animals . These physiologic and molecular alterations were associated with nearly a doubling of the average survival time in the RAD-treated group compared with the sham control group . CONCLUSION: These results demonstrate that renal cell therapy ameliorates cardiac and vascular dysfunction, alters systemic cytokine abnormalities, and improves survival time in a large animal model of Gram-negative septic shock . A cell therapeutic approach with a tissue-engineered bioartificial kidney may be a new treatment modality for this current unmet medical need. Psychoneuroendocrinology, 2003 Nov, 28(8), 970 - 91 The influence of photoperiod and sex on lipopolysaccharide-induced hypoactivity and behavioral tolerance development in meadow voles (Microtus pennsylvanicus); Engeland CG et al.; Lipopolysaccharide (LPS), the minimal immunogenic component of Gram-negative bacteria, is released during infection and causes a variety of sickness behaviors including decreased locomotor activity . This study considered how photoperiod and sex influence the effects of LPS in the meadow vole, Microtus pennsylvanicus . Male and female voles were housed under either reproductively stimulatory (long day: 16 h) or inhibitory (short day: 8 h) photoperiods . On Days 1 and 8, voles were injected with LPS (200 microg/kg, i.p.) or saline vehicle and locomotor activity was assessed 2 h later in an automated open field for 1 h . The first exposure to LPS caused significant decrements in locomotor activity in all LPS-treated groups, regardless of photoperiod or sex . On Day 8, both short day males and females exhibited behavioral tolerance to LPS, no longer displaying significant activity decrements . In contrast, long day females reinjected with LPS on Day 8 still exhibited significant hypoactivity on all locomotor measures . Similarly, long day males also appeared to exhibit a sustained expression of sickness behaviors on Day 8 . In long day females, higher circulating progesterone levels were associated with an attenuated rate of tolerance formation to LPS . The present findings support the winter immunoenhancement hypothesis, which states that small mammals which undergo severe seasonal fluctuations undergo compromised immune functioning during the breeding season, and further indicate a potential role for progesterone in modulating these seasonal immune fluctuations in females. Pharmacol Res, 2003 Dec, 48(6), 585 - 91 Prophylaxis against lipopolysaccharide-induced liver injuries by lipoic acid in rats; Suntres ZE; Lipopolysaccharide (LPS) is a major cell wall molecule of Gram-negative bacteria known to stimulate the synthesis and secretion of several metabolites, such as reactive oxygen species, from phagocytes that play an important role in the pathogenesis of tissue injuries . In this study, the prophylactic effect of the antioxidant lipoic acid was evaluated in an animal acute organ injury model . Animals were pre-treated intraperitoneally with lipoic acid (50 mg kg(-1) body weight) or saline; 3 h later, pretreated animals were challenged intravenously with LPS (Escherichia coli 0111:B4, 1.0 mg kg(-1) body weight) or saline and killed 21 h later . Saline-pretreated animals challenged with LPS were extensively damaged in the liver, as evidenced by an increase in plasma alanine and aspartate aminotransferase activities . Also, LPS injection to saline-pretreated animals resulted in significant increases in plasma tumour necrosis factor-alpha (TNFalpha) and nitric oxide (NO) concentrations, suggestive of activation of the proinflammatory response . The LPS challenge to saline-pretreated animals also increased hepatic myeloperoxidase activity as well as protease and chloramine levels, suggestive of neutrophil infiltration and activation of the inflammatory response . In addition, the involvement of oxidative stress was evident, because a significant increase in lipid peroxidation was observed in the livers of saline-pretreated animals challenged with LPS . The administration of lipoic acid prior to LPS challenge resulted in a significant alleviation of liver injuries, evidenced by a general reversal of the altered biochemical indices toward normal among treated animals . These results indicate that lipoic acid may serve as a potentially effective prophylactic pharmacological agent in alleviating LPS-induced tissue injuries. Biotechnol Prog, 2003 Sep-Oct, 19(5), 1519 - 23 Supercritical fluid disruption of Ralstonia eutropha for poly(beta-hydroxybutyrate) recovery; Hejazi P et al.; A new method for disruption of Gram-negative bacterium Ralstonia eutropha by supercritical CO(2) for poly(beta-hydroxybutyrate) (PHB) recovery is proposed . The effects of different parameters such as exposure time, pressure, temperature, volume of methanol as a modifier, and culture history on cell disruption efficiency were investigated using Taguchi's statistical approach to determine optimum conditions . The optimum conditions for cell disruption and PHB recovery were as follows: exposure time, 100 min; pressure, 200 atm; temperature, 40 degrees C; volume of methanol, 0.2 mL . The cell culture time was less significant . At optimum conditions, the maximum efficiency of PHB recovery was found to be 89% . The proposed method is comparable with other recovery methods in terms of the percentage of PHB recovery, while it is environmentally more benign. Postepy Hig Med Dosw, 2003, 57(3), 251 - 62 {Therapeutic strategies in cases of septicemia caused by gram-negative bacteria}; Lukasiewicz J et al.; Septic shock is the most severe manifestation of infection and appears to be increasingly common, especially in the intensive care units . Lipopolysaccharides (LPS, endotoxin) are known to be responsible for the initiation of septic shock, therefore they can be targets for new preventive and therapeutic strategies . This review focuses on endotoxin-based molecular strategies for the prevention and treatment of Gram-negative sepsis and septic shock. AIHA J (Fairfax, Va), 2003 Sep-Oct, 64(5), 684 - 9 The performance of culture-based methods and microscopy for quantification of noninfectious airborne microorganisms in epidemiological studies of highly contaminated work environments; Eduard W; Airborne levels of microorganisms traditionally have been measured by culture-based methods . Culture-based methods are suitable for the detection of infectious agents, but their suitability for the detection of microorganisms with toxic and allergic effects is less clear, because these effects do not depend on viability of the organisms . During the last 15 years several noncultural methods have been developed for the quantification of airborne microorganisms, including microscopic methods . Microscopy may be expected to provide more valid exposure estimates of microorganisms than culture-based methods, because live and dead microorganisms can be detected . However, their validity may also depend on the ability to differentiate between species . The literature was searched for epidemiological studies in which exposure-response analyses were carried out using culture-based methods and/or microscopy . The influence of several factors on exposure-response associations were considered: design; population size; analytical method; sampling method; exposure levels; outcome; and confounder adjustment . Thirteen studies were found, including a total of 49 exposure-response analyses, and 45% of the analyses showed associations . It was found that the potential of microscopic methods to uncover exposure-response associations was only marginally better than that of culture-based methods (47 and 44%, respectively) . Exposure-response associations were more often found with fungi (70%) than with gram-negative bacteria (50%) or total bacteria (22%), perhaps because fungal exposure is more strongly associated to respiratory outcomes than exposure to bacteria . But the shortcomings of the measurement methods may also be important . Further development of measurement methods for bacteria is therefore needed . The complex composition of bioaerosols in many work environments necessitate the assessment of exposure to multiple agents and multivariate statistical analysis of exposure-response associations. J Exp Med, 2003 Oct 6, 198(7), 1035 - 42 Epub 2003 Sep 29. Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14; Akashi S et al.; Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified . LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4 . Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2 . Here, we demonstrate cell surface LPS-TLR4-MD-2 complexes . CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, but is not coprecipitated with LPS-TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2 . A tentative dissociation constant (Kd) for LPS-TLR4-MD-2 complexes was approximately 3 nM, which is approximately 10-20 times lower than the reported Kd for LPS-MD-2 or LPS-CD14 . The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2 . E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14 . These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14. South Med J, 2003 Aug, 96(8), 815 - 7 Bacteremia due to Comamonas species possibly associated with exposure to tropical fish; Smith MD et al.; Comamonas species are environmental gram-negative rods that grow forming pink-pigmented colonies . Despite their common occurrence in nature, they rarely cause human infection . We present a case of Comamonas bacteremia that we think may have been related to tropical fish exposure . The patient was treated successfully with levofloxacin. Rapid Commun Mass Spectrom, 2003, 17(19), 2226 - 32 Determination of phosphorylation sites in lipooligosaccharides from Pseudoalteromonas haloplanktis TAC 125 grown at 15 degrees C and 25 degrees C by nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometry; Ummarino S et al.; Lipooligosaccharides (LOSs) are macromolecules present on the external cellular membrane of Gram-negative bacteria, structurally made of two distinct regions, lipid A and Core . By varying their growth temperature, bacteria such as psychrophiles change the phosphorylation distribution of the LOSs produced . The level of phosphorylation and the phosphate group positions in LOSs produced by the extremophile psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125, grown at 15 degrees C and 25 degrees C, were investigated by nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) and tandem mass spectrometry (MS/MS) . The samples, obtained by phenol/chloroform/petroleum ether (PCP) extraction of dried cells, were treated with hydrazine at 37 degrees C in order to reduce the heterogeneity by removal of the ester-linked fatty acid moieties . The molecular ion distributions in these LOS fractions were investigated in negative ion mode . Based on these data it was postulated that the sample grown at 25 degrees C contained four phosphate groups while that at 15 degrees C contained three . In order to determine phosphorylation sites in sugar chains, the samples were submitted to low collision energy MS/MS for sequencing . In the sample with three phosphates, one was found to be linked to the tetrasaccharide Core region, more precisely to position C-4 of the Kdo unit . The two remaining phosphate groups were both linked to the 2-acylamide-2-deoxy-D-glucopyranose of the lipid A moiety, and two possible distributions could be postulated on the basis of the fragmentation pattern obtained; in the first case both phosphate groups are linked as a pyrophosphate moiety to position C-1 of the proximal glucosamine (reducing residue), while in the second case one phosphate is linked to position C-1 of the proximal glucosamine and the other to position C-4' of the distal glucosamine (non-reducing residue) . This distribution was also found in the lipid A moiety of the tetraphosphorylated sample grown at 25 degrees C, which bears two phosphate groups on the Core region, one on position C-4 of the Kdo and the other on position C-7 or C-8 of the same residue . The phosphate locations were derived from the intra-ring cleavage ions of sugar moieties in the LOSs obtained by an optimized CID procedure using negative ion QTOF-MS/MS . Eur J Immunol, 2003 Oct, 33(10), 2717 - 26 Inhibition of T helper 2-type responses, IgE production and eosinophilia by synthetic lipopeptides; Akdis CA et al.; In allergy and asthma, the fine balance between the T helper (Th) 1, Th2 and T regulatory cytokine responses appears to be shifted towards Th2 . Here, we report that synthetic lipopeptides which contain the typical lipid part of the lipoprotein of gram-negative bacteria stimulate a distinct regulatory cytokine pattern and inhibit several Th2 cell-related phenomena . The most potent analogue of synthetic lipopeptides, lipopeptide CGP 40774 (LP40) was not active in MyD88-deficient mice and stimulated Toll-like receptor (TLR)-2, but not TLR-4 . LP40 potentiated the production of IFN-gamma and IL-10, but not IL-4 and IL-5 by human T cells . In addition, triggering of TLR-2 by lipopeptides promoted the in vitro differentiation of naive T cells towards IL-10- and IFN-gamma-producing T cells and suppressed IL-4 production by Th2 cells . Accordingly, LP40 inhibited IgE production induced by allergen, anti-IgD antibody, Nippostrongylus brasiliensis or murine acquired immunodeficiency virus . Furthermore, ovalbumin-induced lung eosinophilic inflammation was abolished and Schistosoma mansoni egg-induced granuloma size and eosinophil counts were suppressed in mice by LP40 . These results demonstrate that stimulation of TLR-2 by lipopeptides represents a novel way for possible treatment of allergy and asthma by regulating the disrupted cytokine balance. Med Oncol, 2003, 20(3), 291 - 4 Paclitaxel, carboplatin, and oral etoposide in advanced gastric adenocarcinoma: association with severe myelotoxicity; Bar-Sela G et al.; The prognosis of locally advanced or metastatic adenocarcinoma of the stomach is poor . In an attempt to improve therapeutic results, we undertook a phase II trial to investigate a combination of paclitaxel, carboplatin, and oral etoposide, all active drugs in this malignancy and with a synergistic effect in combination . Fourteen patients with advanced gastric adenocarcinoma were treated with paclitaxel 200 mg/m2 iv, carboplatin AUC-6 iv on d 1, and oral etoposide 50 mg/d alternating with 100 mg/d on d 1-10 . Cycles were repeated every 3 wk . Of the 14 patients treated, partial response was observed in 3/12 (25%) evaluable patients . Median survival for the entire group was 7 mo . The treatment was associated with severe myelotoxicity . Neutropenic fever that required hospitalization developed in 7/14 (50%) of patients, and symptomatic anemia that required red blood cell transfusion was noted in 8/14 (57%) . There was one drug-related death associated with neutropenic fever, Gram negative sepsis, grade 4 thrombocytopenia, and gastrointestinal bleeding . Nonhematological toxicity was moderate . We conclude that the current regimen of paclitaxel, carboplatin, and oral etoposide is not recommended in advanced gastric carcinoma owing to unacceptable myelotoxicity. Cell Mol Life Sci, 2003 Aug, 60(8), 1547 - 58 Membrane protein folding on the example of outer membrane protein A of Escherichia coli; Kleinschmidt JH; The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA) . This review de-scribes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example . OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded beta-barrel transmembrane domain and a 154-residue periplasmic domain . OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm . In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp . After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer . Insertion and folding of the beta-barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e . the formation of large amounts of beta-sheet secondary structure and beta-barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane . In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i . e . into bilayers of lipids of different headgroup structures and hydrophobic chain lengths . Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers . Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal . Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of beta-barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates. Biochem Biophys Res Commun, 2003 Oct 10, 310(1), 94 - 7 Characterisation of plasmids purified from Acetobacter pasteurianus 2374; Krahulec J et al.; Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses . All plasmids were carrying the kanamycin resistance gene . Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells . Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid . All plasmids are capable to coexist with each other in Acetobacter cells . On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli . The nucleotide sequence of replication regions showed significant homology . The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained. Nat Rev Mol Cell Biol, 2003 Sep, 4(9), 738 - 43 Priming virulence factors for delivery into the host; Stebbins CE et al.; Several medically important Gram-negative bacterial pathogens inject virulence factors into host cells through a type III secretion system and specialized bacterial chaperones are required for their effective delivery . Recent structural work shows that these chaperones maintain virulence factors in a partially non-globular conformation that is primed for unfolding and translocation through the 'injectisome'. Blood, 2004 Jan 1, 103(1), 93 - 9 Epub 2003 Sep 22. Protection from endotoxemia by adenoviral-mediated gene transfer of human bactericidal/permeability-increasing protein; Alexander S et al.; Sepsis represents a growing concern in high-risk patients and there has been a lack of effective preventives and therapies . Bacterial/permeability increasing protein (BPI) is a human neutrophil granule-associated defense molecule specific for Gram-negative bacteria and their products . To develop a BPI-transgene-based prophylactic or therapeutic modality, we have developed a recombinant, replication-deficient adenoviral vector expressing full-length human BPI protein (AdhBPI) . The expression of BPI is under control of a murine cytomegalovirus (CMV) promoter . Using in vitro and in vivo systems, AdhBPI-mediated gene transfer led to extracellular secretion of BPI protein, which effectively neutralized endotoxin (lipopolysaccharide {LPS}) and markedly reduced the production of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) by freshly isolated murine alveolar macrophages . By using a mouse model of nonlethal sepsis elicited with LPS, we demonstrated that in vivo gene transfer of BPI was able to markedly inhibit the effect of a large dose of LPS on cytokine responses when injected intraperitoneally . Furthermore, such in vivo BPI gene transfer also improved the survival of mice suffering from lethal septic shock elicited by intraperitoneal injection of d-galactosamine and LPS . Thus, our results suggest that human BPI gene transfer vector has the potential to be used as a therapeutic agent for septic conditions. Hematol J, 2003, 4(5), 295 - 302 Disseminated intravascular coagulation; Slofstra SH et al.; Disseminated Intravascular Coagulation (DIC) is an acquired syndrome representing a hypercoagulable state, haemorrhagic symptoms and multiple organ failure . The clinical relevance of this syndrome is complicated since there is no established way of diagnosing DIC and it is difficult to distinguish whether clinical features are attributable to the underlying disease or DIC . Experimental studies, based on models of gram-negative sepsis and the Generalized Shwartzman Reaction, show that DIC is characterized by strongly enhanced inflammatory activity, activated coagulation and impaired fibrinolysis . In this review we propose that activated neutrophils play a pivotal role in the pathophysiology of DIC, particularly by contributing to inflammation and vascular injury . Additionally, a distinct role for granulocytes in fibrinolysis has also been suggested . Although the underlying procoagulant pathways of DIC and the important role of tissue factor have been unravelled, therapeutic interventions counteracting the mediators of these pathways proved mainly unsuccessful (with the positive exception of activated protein C) . Dissecting the molecular interactions at the onset and progression of DIC might therefore help to elucidate the fundamental consequences of DIC, possibly contributing to better diagnostic tools and more effective therapeutic options. J Mol Biol, 2003 Oct 3, 332(5), 999 - 1014 The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation; Chimento DP et al.; Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes . The primary components of each transport system are an outer membrane transporter and the energy-coupling protein TonB . In Escherichiacoli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane . Cobalamins bind to BtuB with nanomolar affinity . Previous studies implicated calcium in high-affinity binding of cyanocobalamin (CN-Cbl) to BtuB . We previously solved four structures of BtuB or BtuB complexes: an apo-structure of a methionine-substitution mutant (used to obtain experimental phases by selenomethionine single-wavelength anomalous diffraction studies); an apo-structure of wild-type BtuB; a binary complex of calcium and wild-type BtuB; and a ternary complex of calcium, CN-Cbl and wild-type BtuB . We present an analysis of the binding of calcium in the binary and ternary complexes, and show that calcium coordination changes upon substrate binding . High-affinity CN-Cbl binding and calcium coordination are coupled . We also analyze the binding mode of CN-Cbl to BtuB, and compare and contrast this binding to that observed in other proteins that bind Cbl . BtuB binds CN-Cbl in a manner very different from Cbl-utilizing enzymes and the periplasmic Cbl binding protein BtuF . Homology searches of bacterial genomes, structural annotation based on the presence of conserved Cbl-binding residues identified by analysis of our BtuB structure, and detection of homologs of the periplasmic Cbl-binding binding protein BtuF enable identification of putative BtuB orthologs in enteric and non-enteric bacterial species. Bone, 2003 Sep, 33(3), 419 - 25 Human chaperonin 60 (Hsp60) stimulates bone resorption: structure/function relationships; Meghji S et al.; It is established that the molecular chaperone, chaperonin 60, from various bacteria and from Homo sapiens has cell-cell signalling activity and is able to induce proinflammatory cytokine synthesis . We previously reported that chaperonin 60 proteins from Gram-negative bacteria, but not mycobacteria, have the capacity to resorb cultured murine calvarial bone . We now report that lipopolysaccharide-low human recombinant chaperonin 60 (Hsp60) is a relatively weak cytokine-inducing agonist but is a potent stimulator of murine calvarial bone resorption . The osteolytic activity of Hsp60 was significantly inhibited by indomethacin, interleukin-1 receptor antagonist, and osteoprotegerin, but 5-lipoxygenase inhibitors were less effective . Analysis of Hsp60 truncation mutants revealed that N-terminal mutants (Delta1-137, Delta1-358, and Delta1-465) retained bone resorbing activity . In contrast, a C-terminal truncation mutant (Delta1-26 + Delta466-573) was inactive . This suggests that the active domain in this protein is found within residues 466-573 . It is now established that Hsp60 is present in the blood of the majority of the population with the normal range encompassing levels able to activate bone cells . The possibility exists that this protein could play a role in bone remodelling. Antioxid Redox Signal, 2003 Aug, 5(4), 359 - 66 The influence of His94 and Pro149 in modulating the activity of V . cholerae DsbA; Blank J et al.; DsbA is the primary catalyst of disulfide bond formation in the periplasm of gram-negative bacteria . Numerous theoretical and experimental studies have been undertaken to determine the molecular mechanisms by which DsbA acts as a potent oxidant, whereas the homologous cytoplasmic protein, thioredoxin, acts as a reductant . Many of these studies have focused on the nature of the two residues that lie between the active-site cysteines . Although these are clearly important, they are not solely responsible for the differences in activity between these thiol-disulfide oxidoreductases . Q97 in the helical domain of E . coli DsbA has been implicated in influencing the redox potential of E . coli DsbA . In V . cholerae DsbA, the analogous residue is H94 . In this study, the effect of H94 on the oxidase activity of DsbA is examined, along with the role of the conserved cis-proline residue P149 . The DsbA mutant H94L shows a nearly fourfold increase in activity over the wild-type enzyme . To our knowledge, this is the first time an increase in the normal activity of a thiol-disulfide oxidoreductase has been reported . Potential reasons for this increase in activity are discussed. Antibiot Khimioter, 2003, 48(4), 3 - 6 {Ivermectin inhibits activation of Kupffer cells induced by lipopolysaccharide toxin}; Viktorov AV; Lipopolysaccharide-stimulated liver macrophages (Kupffer cells) secrete many physiologically active substances responsible for inflammatory reaction of the organism . The mechanism by which ivermectin, a macrocyclic lactone possessing a broad antiparasitic activity, modulates basic effects elicited by lipopolysaccharide in the primary culture of rat Kupffer cells was studied . It was found that ivermectin in the absence of endotoxin did not affect a functional state of the Kupffer cells . Preincubation of Kupffer cells with ivermectin (1 mM), however, significantly blocked response to the subsequent administration of lipopolysaccharide (1 mg/ml) . In particular, secretion of tumor necrosis factor TNF alpha, nitric oxide NO and prostaglandin E2 was suppressed . Also, an LPS-triggered rise in the intracellular concentration of calcium ions was less pronounced . Removal of chloride anions from the extracellular medium completely abolished inhibitory effects of ivermectin . It is suggested that invermectin exerts its action via binding to the glycine-gated chloride receptors/channels of the Kupffer cells, which may reduce toxic reactions manifestations observed under infections caused by Gram-negative bacteria. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1471 - 7 Shewanella waksmanii sp . nov., isolated from a sipuncula (Phascolosoma japonicum); Ivanova EP et al.; Two marine bacterial strains, KMM 3823(T) and KMM 3836, isolated from a sipuncula (Phascolosoma japonicum), a common inhabitant of Troitsa Bay in the Gulf of Peter the Great (Sea of Japan), were studied . Comparative 16S rRNA gene sequence-based phylogenetic analysis placed these bacteria into a separate branch of the 'Gammaproteobacteria' within members of the genus SHEWANELLA: KMM 3823(T) showed the highest similarity (96.6 %) with Shewanella fidelis . The DNA G+C contents of the two strains studied were 43.0 mol% . The level of DNA homology between these two strains was conspecific (93 %), indicating that they represent a single genospecies . These organisms were greenish-brown, Gram-negative, polarly flagellated, facultatively anaerobic, mesophilic (temperature range 4-30 degrees C), neutrophilic, haemolytic and were able to degrade elastin, gelatin and DNA . They were susceptible to ampicillin, carbenicillin, gentamicin and kanamycin . The predominant fatty acids were characteristic for shewanellas: 13 : 0-i, 15 : 0-i and 16 : 1(n-7); up to 6.7 % of eicosapentaenoic fatty acid, 20 : 5(n-3), was produced during growth at 28 degrees C . Phylogenetic evidence, confirmed by DNA hybridization and phenotypic characteristics revealed that the two bacteria studied constitute a new species, Shewanella waksmanii sp . nov., the type strain of which is KMM 3823(T) (=CIP 107701(T)=ATCC BAA-643(T)). Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1261 - 9 Silicibacter pomeroyi sp . nov . and Roseovarius nubinhibens sp . nov., dimethylsulfoniopropionate-demethylating bacteria from marine environments; Gonzalez JM et al.; Three Gram-negative, rod-shaped, aerobic bacteria that were capable of degrading dimethylsulfoniopropionate (DMSP) were isolated from marine waters . These isolates (DSS-3(T), DSS-10 and ISM(T)) exhibited the ability to demethylate and cleave DMSP, as well as to degrade other sulfur compounds related to DMSP that are cycled in marine environments . Intracellular poly-beta-hydroxybutyrate inclusions, surface blebs and one polar, complex flagellum that rotated exclusively in the clockwise direction were observed for DSS-3(T) . The outer membrane of ISM(T) was separated from the cytoplasm at the poles in a toga-like morphology . The primary fatty acid in both strains was C(18 : 1)omega7c . DNA G+C contents for the isolates were 68.0+/-0.1, 68.1+/-0.1 and 66.0+/-0.2 mol% for DSS-3(T), DSS-10 and ISM(T), respectively . 16S rRNA gene sequence analyses placed these organisms within the Roseobacter lineage of the alpha-PROTEOBACTERIA: Closely related species were Silicibacter lacuscaerulensis and Ruegeria atlantica (DSS-3(T) and DSS-10) and Roseovarius tolerans (ISM(T)) . Neither DSS-3(T) nor ISM(T) exhibited 16S rRNA similarity >97 % or DNA-DNA hybridization values >45 % to their nearest described relatives . Genotypic and phenotypic analyses support the creation of two novel species: Silicibacter pomeroyi sp . nov . with strain DSS-3(T) (=ATCC 700808(T)=DSM 15171(T)) as the type strain, and Roseovarius nubinhibens sp . nov . with strain ISM(T) (=ATCC BAA-591(T)=DSM 15170(T)) as the type strain. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1253 - 60 Sphingomonas aurantiaca sp . nov., Sphingomonas aerolata sp . nov . and Sphingomonas faeni sp . nov., air- and dustborne and Antarctic, orange-pigmented, psychrotolerant bacteria, and emended description of the genus Sphingomonas; Busse HJ et al.; Seven psychrotolerant, Gram-negative bacterial strains, five dust- and airborne isolates (MA101b(T), MA306a, MA405/90, MA-olki(T) and NW12(T)) and two from the Antarctic (Ant 20 and M3C203B-B), were subjected to a polyphasic characterization to determine their taxonomic position . High 16S rDNA sequences similarities (99.3-100.0 %) demonstrated that they were closely related to each other . Phylogenetic evaluation of their 16S rDNA sequences revealed that they are members of the genus Sphingomonas sensu stricto, encompassing a separate branch within this genus . They shared 94.4-96.6 % 16S rDNA sequence similarity with species of this genus . All Sphingomonas-specific signature nucleotides were also detected . The presence of the major ubiquinone Q-10, sym-homospermidine as the predominant polyamine, Sphingomonadaceae-specific sphingoglycolipid in the polar lipid patterns and a fatty acid profile containing C(14 : 0) 2-OH and lacking 3-OH fatty acids were in agreement with identification of these strains as members of the genus Sphingomonas sensu stricto . Results from DNA-DNA hybridizations and comparison of protein patterns indicated that the seven strains are members of three distinct species . One species is represented by strains MA101b(T), MA306a and MA405/90, the second by strains NW12(T), Ant 20 and M3C203B-B and the third by one strain, MA-olki(T) . Their distinction at the species level was also supported by results of biochemical characterization and partly supported by riboprints and genomic fingerprints . On the basis of these results, three novel species of the genus Sphingomonas are proposed: Sphingomonas aurantiaca sp . nov., consisting of strains MA101b(T) (=DSM 14748(T)=LMG 21377(T)), MA306a and MA405/90 (=DSM 14749=LMG 21378), Sphingomonas faeni sp . nov . MA-olki(T) (=DSM 14747(T)=LMG 21379(T)) and Sphingomonas aerolata sp . nov., represented by strains NW12(T) (=DSM 14746(T)=LMG 21376(T)), Ant 20 (=ICMP 13599) and M3C203B-B (=SMCC M3C203B-B). Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1231 - 9 Methylocella silvestris sp . nov., a novel methanotroph isolated from an acidic forest cambisol; Dunfield PF et al.; Two strains of Gram-negative, aerobic, non-pigmented, non-motile, rod-shaped, methane-oxidizing bacteria were isolated from an acidic forest cambisol near Marburg, Germany, and were designated as strains BL2(T) and A1 . These bacteria were morphologically and phenotypically similar to Methylocella palustris K(T) . The cells possess a highly specific bipolar appearance . They lack the intracytoplasmic membranes common to all methane-oxidizing bacteria except Methylocella, but contain a vesicular membrane system connected to the cytoplasmic membrane . A soluble methane monooxygenase was present, but no particulate methane monooxygenase could be detected . These bacteria utilize the serine pathway for carbon assimilation . Strains BL2(T) and A1 are moderately acidophilic, mesophilic organisms capable of growth at pH values between 4.5 and 7 (with an optimum at pH 5.5) and at temperatures between 4 and 30 degrees C . Compared with Methylocella palustris K(T), these strains have greater tolerance of cold temperatures, dissolved salts and methanol . On the basis of 16S rRNA gene sequence identity, of species with validly published names, strain BL2(T) is most closely related to Methylocella palustris K(T) (97.3 % identity), Beijerinckia indica subsp . indica ATCC 9039(T) (97.1 %) and Methylocapsa acidiphila B2(T) (96.2 %) . The DNA G+C content is 60 mol% and the major phospholipid fatty acid is 18 : 1omega7 . Strain BL2(T) showed only 21-22 % DNA-DNA hybridization with Methylocella palustris K(T) . The data therefore suggest that strains BL2(T) and A1 represent a novel species of Methylocella; the name Methylocella silvestris sp . nov . is proposed, with strain BL2(T) (=DSM 15510(T)=NCIMB 13906(T)) as the type strain. FEMS Microbiol Lett, 2003 Sep 12, 226(1), 57 - 64 Eukaryotic cell signaling and transcriptional activation induced by bacterial porins; Galdiero M et al.; The protein composition of the outer membrane of Gram-negative bacteria consists of about 20 immunochemically distinct proteins, termed outer membrane proteins (OMPs) . Apart from their structural role, OMPs have been shown to have other functions, particularly with regard to transport, and have been classified as permeases and porins . Porins, during their interaction with the host, are immunogenic and also directly stimulate several cellular functions . Porins work both as molecules present on the bacterial surface and as molecules released by bacteria . Lipopolysaccharide and OMPs, the major structural macromolecular constituents of the outer membrane, carry out a fundamental role in the pathogenesis of Gram-negative infections . This brief review describes the multiple facets of the biological activities of porins both in vitro and in vivo, particularly focusing on their ability to induce the expression of cytokines and other factors that modulate cellular activities with either pathological or adaptive consequences . This brief discussion will focus on the signal transmission mechanisms induced by bacterial porins. Anasthesiol Intensivmed Notfallmed Schmerzther, 2003 Sep, 38(9), 573 - 6 {Extended spectrum beta-lactamse (ESBL)-induced antibiotics resistance in gram-negative agents: what should be watched in intensive care medicine?}; Kola A et al.; Third-generation cephalosporins have an important part in the therapy of infections due to gram-negative bacteria . Extended spectrum beta-lactamases (ESBL) are able to hydrolize these antibiotics . As the number of ESBL-producing bacteria is increasing worldwide, detection of ESBL-producers is important for the both of prevention and therapy. J Struct Biol, 2003 Aug, 143(2), 145 - 52 Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti--a combined molecular biology and force spectroscopy investigation; Bartels FW et al.; Specific protein-DNA interaction is fundamental for all aspects of gene transcription . We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants . The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis . ExpG is a transcriptional activator of exp gene expression . We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM) . Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28 bp conserved region was found . AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165 pN in a logarithmic dependence from the loading rates of 70-79000 pN/s . Two different regimes of loading rate-dependent behaviour were identified . Thermal off-rates in the range of k(off)=(1.2+/-1.0) x 10(-3)s(-1) were derived from the lower loading rate regime for all promoter regions . In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism. Protein Eng, 2003 Aug, 16(8), 629 - 35 Tandem repeats of Sushi3 peptide with enhanced LPS-binding and -neutralizing activities; Li C et al.; Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection . Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, was previously shown to bind and neutralize LPS activity . For large-scale production of this peptide and to mimick other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in Escherichia coli . The recombinant tetramer of S3 (rS3-4mer) was purified by anion exchange and digested into monomers (rS3-1mer) . Both the rS3-4mer and rS3-1mer were functionally analyzed for their ability to bind LPS by an ELISA-based method and surface plasmon resonance . The LAL inhibition and TNFalpha-release test showed that rS3-1 mer can neutralize the LPS activity as effectively as the synthetic S3 peptide, while rS3-4mer displays an enhanced inhibitory effect on LPS-induced activities . Both recombinant peptides exhibited low cytotoxicity and no haemolytic activity on human cells . This evidence suggests that the recombinant sushi peptides have potential use for the detection, removal of endotoxin and/or anti-endotoxin strategies. J Plant Physiol, 2003 Aug, 160(8), 945 - 52 Transformation of peanut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana; Yang H et al.; In order to test an alternative selectable marker system for the production of transgenic peanut plants (Arachis hypogaea), the bacterial mercuric ion reductase gene, merA, was introduced into embryogenic cultures via microprojectile bombardment . MerA reduces toxic Hg(II) to the volatile and less toxic metallic mercury molecule, Hg(0), and renders its source Gram-negative bacterium mercury resistant . A codon-modified version of the merA gene, MerApe9, was cloned into a plant expression cassette containing the ACT2 promoter from Arabidopsis thaliana and the NOS terminator . The expression cassette also was inserted into a second vector containing the hygromycin resistance gene driven by the UBI3 promoter from potato . Stable transgenic plants were recovered through hygromycin-based selection from somatic embryo tissues bombarded with the plasmid containing both genes . However, no transgenic somatic embryos were recovered from selection on 50-100 micromol/L HgCl2 . Expression of merA as mRNA was detected by Northern blot analysis in leaf tissues of transgenic peanut, but not in somatic embryos . Western blot analysis showed the production of the mercuric ion reductase protein in leaf tissues . Differential responses to HgCl2 of embryo-derived explants from segregating R1 seeds of one transgenic line also were observed. J Fish Dis, 2003 May, 26(5), 251 - 62 Piscirickettsia salmonis: a Gram-negative intracellular bacterial pathogen of fish; Fryer JL et al.; Piscirickettsia salmonis is the first Gram-negative, intracellular bacterial pathogen isolated from fish and is a significant cause of mortality in salmonid fish . Recent reports of P . salmonis or P . salmonis-like organisms from new fish hosts and geographic regions have increased the interest in the bacterium . In this review, the important characteristics of the bacterium including recent taxonomic changes, features of the disease caused by the bacterium including transmission, hosts, reservoirs, diagnostic procedures, and current approaches for prevention and treatment have been discussed . The reader is also directed to other reviews concerning the bacterium and the disease it causes (Fryer & Lannan 1994, 1996; Almendras & Fuentealba 1997; Lannan, Bartholomew & Fryer 1999; House & Fryer 2002; Mauel & Miller 2002). J Leukoc Biol, 2003 Oct, 74(4), 507 - 13 Epub 2003 Jul 15. Expression of glucocorticoid resistance following social stress requires a second signal; Avitsur R et al.; Stimulation of splenocytes from socially stressed mice {social disruption (SDR)} with Gram-negative bacterial lipopolysaccharide (LPS) revealed a state of functional glucocorticoid (GC) resistance . LPS-stimulated splenocytes were less sensitive to the inhibitory effects of corticosterone . This study demonstrated that activation signals were required for the expression of splenic GC resistance . The results demonstrated that six cycles of SDR induced splenomegaly and increased the number of CD11b-positive monocytes . SDR also increased the viability of cultured, nonstimulated splenocytes, and addition of corticosterone reduced the viability of these cells in a dose-dependent manner . However, following stimulation with LPS, the sensitivity of SDR splenocytes to GC was reduced . Similar results were obtained using lipid A, a fraction of the LPS molecule that binds to Toll-like receptor (TLR)4 . Furthermore, C3H/HeJ mice that do not possess a functional TLR4 molecule responded to SDR with an increased number of CD11b-positive monocytes in the spleen and increased viability of nonstimulated splenocytes . However, neither LPS nor lipid A stimulation resulted in the expression of GC resistance . Together, these findings suggest that the expression of GC resistance in response to SDR requires a second signal that can be provided by ligation of TLR4. Am J Physiol Lung Cell Mol Physiol, 2004 Jan, 286(1), L129 - 36 Epub 2003 Sep 05. Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway; Alcorn JF et al.; The lung collectin surfactant protein A (SP-A) has both anti-inflammatory and prophagocytic activities . We and others previously showed that SP-A inhibits the macrophage production of tumor necrosis factor (TNF)-alpha stimulated by the gram-negative bacterial component LPS . We propose that SP-A decreases the production of proinflammatory cytokines by alveolar macrophages via a CD14-independent mechanism . SP-A inhibited LPS-simulated TNF-alpha production in rat and mouse macrophages in the presence and absence of serum (72% and 42% inhibition, respectively) . In addition, SP-A inhibited LPS-induced mRNA levels for TNF-alpha, IL-1 alpha, and IL-1 beta as well as NF-kappa B DNA binding activity . SP-A also diminished ultrapure LPS-stimulated TNF-alpha produced by wild-type and CD14-null mouse alveolar macrophages by 58% and 88%, respectively . Additionally, SP-A inhibited TNF-alpha stimulated by PMA in both wild-type and TLR4-mutant macrophages . These data suggest that SP-A inhibits inflammatory cytokine production in a CD14-independent manner and also by mechanisms independent of the LPS signaling pathway. Pneumonol Alergol Pol, 2003, 71(1-2), 43 - 50 {Results of treatment for chronic pleural empyema}; Rzyman W et al.; The aim of the study was to assess the results of the treatment in 97 patients with chronic pleural empyema treated in the department of thoracic surgery between 1988 and 1997 . The majority of patients were between 30 and 50 years old . Most of the group were men and more than a half had a concomitant disease, which may predispose to empyema development . Nevertheless all the empyemas were in the chronic phase 1/3 of patients were successfully treated only with closed chest tube drainage and the remaining group with lung decortication . The Gram-negative bacterial flora dominated in the culture from empyema sac . Spirometric values and blood gas analysis showed significant reduction of lung function before the treatment . We found the relation between an early institution of closed tube drainage and the shorter stay at the hospital . Moreover in a significant proportion of patients pleural drainage was a sufficient way of treatment . CONCLUSIONS: Drainage of the empyema should be performed at early phase of the disease . It should be recommended that pleural drainage precede the surgical management of empyema . Delate of surgical intervention is the main cause of the high mortality rate in empyema following esophageal perforation. Appl Environ Microbiol, 2003 Sep, 69(9), 5243 - 7 Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots; Dodd CC et al.; Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7 . TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E . coli O157 in inoculated feeds . Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E . coli O157 in cattle feeds . TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample . All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite . Identification of E . coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin . E . coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin . E . coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods . There was no correlation between E . coli O157 prevalence and generic coliform counts in feeds . The prevalence of E . coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E . coli O157 in order to develop strategies to prevent food-borne disease in humans. Front Biosci, 2003 Sep 01, 8, s1422 - 36 Three paradoxes of ferric enterobactin uptake; Klebba PE; Bacteria elaborate iron chelators that scavenge iron from the environment, including their human and animal hosts, and iron acquisition is a determinant of pathogenicity . One such iron chelate, the siderophore ferric enterobactin, enters Gram-negative bacteria through the FepA protein of the outer membrane . The ferric enterobactin transport process is a high-affinity, multi-specific, multi-component, energy dependent reaction, that is a paradigm of ligand-gated transport: FeEnt binding activates FepA to transport competency . On the basis of the FepA, FhuA, FecA and BtuB crystal structures, and in light of recent molecular biological, biochemical, and biophysical findings, this review considers the mechanism of ferric enterobactin uptake . The discussion focuses on three preeminent questions about the transport reaction: the function of the N-terminal globular domain that resides within the FepA channel, the mechanistic contributions of TonB to the activities of ligand-gated porins, and the energy dependence of metal transport reactions through the OM bilayer . Available data points to the idea that the N-terminal globular domains of these receptor proteins dynamically exit their pores during transport, creating a suction-force that pulls ligands through the surface loops into the periplasm . The functions of TonB and energy in these processes remain unknown. Arch Immunol Ther Exp (Warsz), 2003, 51(4), 231 - 6 Are NKT cells essential for endotoxic shock? Emoto M. Endotoxic shock is a major health threat caused by gram-negative bacteria and their unique cell wall component, lipopolysaccharide, which induces exaggerated production of proinflammatory cytokines . Although macrophages play a central role in the pathogenesis of endotoxic shock, natural killer (NK)1+ cells are also involved in this mechanism . NK1+ cells comprise two major populations, namely NK cells and NKT cells . It remains, however, elusive whether NK cells, NKT cells or both are involved in the induction of endotoxic shock . This review will focus on the relative contribution of these NK1+ cells to the pathogenesis of endotoxic shock. J Biol Chem, 2003 Nov 14, 278(46), 45753 - 62 Epub 2003 Sep 02. Escherichia coli K-1 interaction with human brain micro-vascular endothelial cells triggers phospholipase C-gamma1 activation downstream of phosphatidylinositol 3-kinase; Sukumaran SK et al.; Escherichia coli, the most common Gram-negative bacterium that causes meningitis in neonates, invades human brain microvascular endothelial cells (HBMEC) by rearranging host cell actin via the activation of phosphatidylinositol 3-kinase (PI3K) and PKC-alpha . Here, further, we show that phospholipase (PLC)-gamma1 is phosphorylated on tyrosine 783 and condenses at the HBMEC membrane beneath the E . coli entry site . Overexpression of a dominant negative (DN) form of PLC-gamma, the PLC-z fragment, in HBMEC inhibits PLC-gamma1 activation and significantly blocks E . coli invasion . PI3K activation is not affected in PLC-z/HBMEC upon infection, whereas PKC-alpha phosphorylation is completely abolished, indicating that PLC-gamma1 is downstream of PI3K . Concomitantly, the phosphorylation of PLC-gamma1 is blocked in HBMEC overexpressing a dominant negative form of the p85 subunit of PI3K but not in HBMEC overexpressing a dominant negative form of PKC-alpha . In addition, the recruitment of PLC-gamma1 to the cell membrane in both PLC-z/HBMEC and DN-p85/HBMEC is inhibited . Activation of PI3K is associated with the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 1,4,5-trisphosphate (PIP3), which in turn recruits PLC-gamma1 to the cell membrane via its interaction with pleckstrin homology domain of PLC-gamma1 . Utilizing the pleckstrin homology domains of PKC-delta and Btk proteins fused to green fluorescent protein (GFP), which specifically interact with PIP2 and PIP3, respectively, we show herein that E . coli invasion induces the breakdown of PIP2 at the plasma membrane near the site of E . coli interaction . PIP3, on the other hand, recruits the GFPBkt to the cell membrane beneath the sites of E . coli attachment . Our studies further show that E . coli invasion induces the release of Ca2+ from intracellular pools as well as the influx of Ca2+ from the extracellular medium . This elevation in Ca2+ levels is completely blocked both in PLC-z/HBMEC and DN-p85/HBMEC, but not in DN-PKC/HBMEC . Taken together, these results suggest that E . coli infection of HBMEC induces PLC-gamma1 activation in a PI3K-dependent manner to increase Ca2+ levels in HBMEC . This is the first report demonstrating the recruitment of activated PLC-gamma1 to the sites of bacterial entry. Vet Pathol, 2003 Sep, 40(5), 579 - 82 Left ventricular outflow tract-right atrial communication (Gerbode type defect) associated with bacterial endocarditis in a dog; Ramirez GA et al.; Left ventricular (LV) outflow tract-right atrial (RA) communication associated with bacterial endocarditis is described in a 6-year-old intact male Great Pyrenees dog with a 4- to 5-day history of fever, lethargy, weight loss, severe regenerative anemia, and asplenia . Typical vegetative mural endocardial lesions were observed grossly . Histologic evaluation revealed small gram-negative coccobacilli that were consistent with Bordetella avium-like organisms . These bacteria were associated with severe endocardial inflammation characterized by neutrophilic infiltration, extensive necrosis of endocardium, and fibrin deposition . LV-RA shunt (Gerbode defect) is a rare cardiac defect in humans that can be either congenital or, more rarely, secondary to septic endocarditis, valve replacement procedures, or thoracic trauma . B . avium-like organisms causing septicemia and endocarditis in immunocompromised and asplenic human patients have been described . To our knowledge, no previous descriptions of Gerbode defect associated with bacterial endocarditis in domestic animals have been reported in veterinary literature. J Biol Chem, 2003 Dec 5, 278(49), 49428 - 37 Epub 2003 Aug 29. Transcriptional regulation of limulus factor C: repression of an NFkappaB motif modulates its responsiveness to bacterial lipopolysaccharide; Wang L et al.; Serine proteases play fundamental roles in invertebrate development, hemostasis, and innate immunity . This is exemplified by the limulus Factor C, which is a serine protease that binds a pathogen-associated molecule, lipopolysaccharide (LPS) to trigger a blood coagulation cascade . As a central molecule in the limulus innate immunity and hemostasis, Factor C gene expression has been detected in two major immune defense tissues, the amebocytes and hepatopancreas . Infection of the limulus with live Gram-negative bacteria induces a 2-3-fold increase in mRNA transcripts in both tissues . However, in vitro studies in Drosophila cell lines using Factor C promoter-reporter chimera DNA constructs, and site-directed mutagenesis of the promoter demonstrated that a proximal kappaB binding site, aided by an adjacent dorsal-like binding motif responds dramatically to LPS and dorsal transcription factor overexpression . Electrophoretic mobility shift assay further confirmed a strong interaction of the limulus kappaB motif with Rel proteins . However, deletion constructs of the Factor C promoter harboring different numbers of dorsal-like binding sites upstream of the kappaB motif as well as the electrophoretic mobility shift assay of these motifs with Rel proteins strongly suggest that the up-regulation of Factor C gene expression is attenuated during microbial challenge . The repression of the dramatic activation of this pathogen-responsive gene by LPS is probably effected via competition between the dorsal-like motifs over the proximal LPS-responsive kappaB unit, or through inhibition from the upstream repressive element(s), which accounts for the gene expression pattern observed in vivo . Our findings demonstrate that blood coagulation and innate immune response are integrated at the transcriptional level in this ancient organism, and that this LPS-responsive serine protease is controlled by an evolutionarily conserved NFkappaB pathway. Curr Opin Struct Biol, 2003 Aug, 13(4), 443 - 52 Multidrug-exporting secondary transporters; Murakami S et al.; The major cause of intrinsic drug resistance in Gram-negative bacteria is a resistance nodulation division type multidrug exporter, which couples with an outer membrane channel and a membrane fusion protein and exports drugs out of the cell, bypassing the periplasm; this process is driven by proton motive force . A recent crystal structure determination of a major resistance nodulation division type multidrug exporter, AcrB in Escherichia coli, greatly advances our understanding of the multidrug export mechanism . The most striking feature of the AcrB trimer is the presence of three vestibules open to the periplasm at the boundary between the periplasmic headpiece and the transmembrane region . Substrates can gain access to the central cavity from the periplasmic surface of the cytoplasmic membrane and are then actively transported through the extramembrane pore into the outer membrane channel TolC, via the funnel at the top of the AcrB headpiece. Curr Opin Struct Biol, 2003 Aug, 13(4), 404 - 11 The versatile beta-barrel membrane protein; Wimley WC; The beta-barrel membrane protein is found in the outer membranes of bacteria, mitochondria and chloroplasts . Approximately 2-3% of the genes in Gram-negative bacterial genomes encode beta-barrels . Whereas there are fewer than 20 known three-dimensional beta-barrel structures, genomic databases currently contain thousands of beta-barrels belonging to dozens of families . New research is revealing the variety of beta-barrel structures and the variety of functions performed by these versatile proteins. J Biol Chem, 2003 Nov 14, 278(46), 45713 - 9 Epub 2003 Aug 26. The SufE protein and the SufBCD complex enhance SufS cysteine desulfurase activity as part of a sulfur transfer pathway for Fe-S cluster assembly in Escherichia coli; Outten FW et al.; The sufABCDSE operon of the Gram-negative bacterium Escherichia coli is induced by oxidative stress and iron deprivation . To examine the biochemical roles of the Suf proteins, we purified all of the proteins and assayed their effect on SufS cysteine desulfurase activity . Here we report that the SufE protein can stimulate the cysteine desulfurase activity of the SufS enzyme up to 8-fold and accepts sulfane sulfur from SufS . This sulfur transfer process from SufS to SufE is sheltered from the environment based on its resistance to added reductants and on the analysis of available crystal structures of the proteins . We also found that the SufB, SufC, and SufD proteins associate in a stable complex and that, in the presence of SufE, the SufBCD complex further stimulates SufS activity up to 32-fold . Thus, the SufE protein and the SufBCD complex act synergistically to modulate the cysteine desulfurase activity of SufS . We propose that this sulfur transfer mechanism may be important for limiting sulfide release during oxidative stress conditions in vivo. Mol Microbiol, 2003 Sep, 49(5), 1253 - 66 A bacterial conjugation machinery recruited for pathogenesis; Seubert A et al.; Type IV secretion systems (T4SS) are multicomponent transporters of Gram-negative bacteria adapted to functions as diverse as DNA transfer in bacterial conjugation or the delivery of effector proteins into eukaryotic target cells in pathogenesis . The generally modest sequence conservation between T4SS may reflect their evolutionary distance and/or functional divergence . Here, we show that the establishment of intraerythrocytic parasitism by Bartonella tribocorum requires a putative T4SS, which shares an unprecedented level of sequence identity with the Trw conjugation machinery of the broad-host-range antibiotic resistance plasmid R388 (up to 80% amino acid identity for individual T4SS components) . The highly conserved T4SS loci are collinear except for the presence of numerous tandem gene duplications in B . tribocorum, which mostly encode variant forms of presumed surface-exposed pilus subunits . Conservation is not only structural, but also functional: R388 mutated in either trwD or trwH encoding essential T4SS components could be trans-complemented for conjugation by the homologues of the B . tribocorum system . Conservation also includes the transcription regulatory circuit: both T4SS loci encode a highly homologous and interchangeable KorA/KorB repressor system that negatively regulates the expression of all T4SS components . This striking example of adaptive evolution reveals the capacity of T4SS to assume dedicated functions in either DNA transfer or pathogenesis over rather short evolutionary distance and implies a novel role for the conjugation systems of widespread broad-host-range plasmids in the evolution of bacterial pathogens. Mol Microbiol, 2003 Sep, 49(5), 1167 - 77 Mycobacterial porins--new channel proteins in unique outer membranes; Niederweis M; Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant to many antibiotics . Pore proteins spanning the outer membrane mediate the diffusion of hydrophilic nutrients . Mycobacterium tuberculosis possesses at least two porins in addition to the low activity channel protein OmpATb . OmpATb is essential for adaptation of M . tuberculosis to low pH and survival in macrophages and mice . The channel activity of OmpATb is likely to play a major role in the defence of M . tuberculosis against acidification within the phagosome of macrophages . MspA is the main porin of Mycobacterium smegmatis . It forms a tetrameric complex with a single central pore of 10 nm length and a cone-like structure . This structure differs clearly from that of the trimeric porins of Gram-negative bacteria, which form one 4 nm long pore per monomer . The 45-fold lower number of porins compared to Gram-negative bacteria and the exceptional length of the pores are two major determinants of the low permeability of the outer membrane of M . smegmatis for hydrophilic solutes . The importance of the synergism between slow transport through the porins and drug efflux or inactivation for the development of drugs against M . tuberculosis is discussed. FEBS Lett, 2003 Aug 28, 550(1-3), 168 - 74 Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein; Pang A et al.; Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs) . Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms . Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding . Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region . Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein . Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain . The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs. Infect Immun, 2003 Sep, 71(9), 5324 - 31 Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival; Lin M et al.; Ehrlichia chaffeensis and Anaplasma phagocytophilum are agents of human monocytic and granulocytic ehrlichioses, respectively . They are extremely sensitive to mechanical stress and are pleomorphic gram-negative bacteria . Membrane incorporation of cholesterol from the eukaryotic host is known to be essential for other fragile and pleomorphic bacteria and mycoplasmas that lack a cell wall . Thus, we tested whether cholesterol is required for E . chaffeensis and A . phagocytophilum . Using a freeze fracture technique and biochemical analysis, these bacteria were found to contain significant levels of membrane cholesterol . These bacteria lack genes for cholesterol biosynthesis or modification . However, host cell-free bacteria had the ability to take up directly exogenous cholesterol or NBD-cholesterol, a fluorescent cholesterol derivative . Treatment of the bacteria with cholesterol extraction reagent methyl-beta-cyclodextrin caused their ultrastructural changes . Furthermore, pretreatment of the bacteria with methyl-beta-cyclodextrin or NBD-cholesterol deprived these bacteria of the ability to infect leukocytes, thus killing these obligate intracellular bacteria . Analysis of E . chaffeensis and A . phagocytophilum genome sequences revealed that these bacteria lack all genes for the biosynthesis of lipid A and most genes for the biosynthesis of peptidoglycan, which confer structural strength to gram-negative bacteria . Taken together, these results suggest that human ehrlichiosis agents became cholesterol dependent due to the loss of these genes . As the first report of gram-negative bacteria incorporating cholesterol for survival, these findings offer insight into the unique nature of their parasitism and imply that cholesterol is important in the control of human ehrlichioses. Chem Commun (Camb), 2003 Aug 7, (15), 1966 - 7 Using biofunctional magnetic nanoparticles to capture gram-negative bacteria at an ultra-low concentration; Gu H et al.; After conjugation to vancomycin (Van), chemically stable and highly magnetic anisotropic FePt magnetic nanoparticles (approximately 4 nm) become water-soluble and capture E . coli at 15 cfu mL(-1). J Exp Med, 2003 Aug 18, 198(4), 521 - 31 Distinct mutations in IRAK-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections; Medvedev AE et al.; We identified previously a patient with recurrent bacterial infections who failed to respond to gram-negative LPS in vivo, and whose leukocytes were profoundly hyporesponsive to LPS and IL-1 in vitro . We now demonstrate that this patient also exhibits deficient responses in a skin blister model of aseptic inflammation . A lack of IL-18 responsiveness, coupled with diminished LPS and/or IL-1-induced nuclear factor-kappaB and activator protein-1 translocation, p38 phosphorylation, gene expression, and dysregulated IL-1R-associated kinase (IRAK)-1 activity in vitro support the hypothesis that the defect lies within the signaling pathway common to toll-like receptor 4, IL-1R, and IL-18R . This patient expresses a "compound heterozygous" genotype, with a point mutation (C877T in cDNA) and a two-nucleotide, AC deletion (620-621del in cDNA) encoded by distinct alleles of the IRAK-4 gene (GenBank/EMBL/DDBJ accession nos . AF445802 and AY186092) . Both mutations encode proteins with an intact death domain, but a truncated kinase domain, thereby precluding expression of full-length IRAK-4 (i.e., a recessive phenotype) . When overexpressed in HEK293T cells, neither truncated form augmented endogenous IRAK-1 kinase activity, and both inhibited endogenous IRAK-1 activity modestly . Thus, IRAK-4 is pivotal in the development of a normal inflammatory response initiated by bacterial or nonbacterial insults. J Bacteriol, 2003 Sep, 185(17), 5287 - 9 Efflux of cytoplasmically acting antibiotics from gram-negative bacteria: periplasmic substrate capture by multicomponent efflux pumps inferred from their cooperative action with single-component transporters; Palmer M; In gram-negative bacteria, coexpression of single- and multicomponent efflux pumps may result in multiplicative enhancement of the level of resistance against cytoplasmically acting antibiotics . Here, a simple model is presented to show that this cooperative effect can be accounted for only if substrate capture by the multicomponent efflux transporter occurs in the periplasm but not the cytosol. Ther Apher Dial, 2003 Feb, 7(1), 108 - 14 Extracorporeal endotoxin removal for the treatment of sepsis: endotoxin adsorption cartridge (Toraymyxin); Shoji H; Toray Industries Inc . has developed an endotoxin removal cartridge (Toraymyxin) composed of a polymyxin B immobilized, fibrous adsorbent . Toraymyxin has been listed as a blood purification medical device for endotoxin removal to be reimbursed by the Japanese national health insurance since 1994 . Toraymyxin can be applied to patients with endotoxemia or suspected gram-negative infection, which fulfilll the conditions of Systemic Inflammatory Response Syndrome (SIRS) and have septic shock demanding vasopressor infusion . Since 1994, over 30,000 patients have received this treatment . The safety of this device has been confirmed and improvement of hemodynamic dysfunction has been shown to be a major benefit . Infection resulting from an acute abdominal condition requiring surgery has been shown to be one of the good indications for Toraymyxin . Further studies are now ongoing to establish Toraymyxin treatment as one of the options to treat sepsis and septic shock patients and to clarify the mechanisms involved in this therapy. Pediatr Nephrol, 2003 Oct, 18(10), 1066 - 8 Epub 2003 Aug 12. Decrease of thrombomodulin contributes to the procoagulant state of endothelium in hemolytic uremic syndrome; Fernandez GC et al.; The typical form of hemolytic uremic syndrome (D+HUS) is a thrombotic microangiopathy that causes acute renal failure in children . The etiology of this disease is a toxin called Shiga-like toxin (Stx), present in certain strains of gram-negative bacteria . Vascular endothelial cell (EC) injury appears to be central in the pathogenesis of D+HUS . Thrombomodulin (TM) is a glycoprotein present in EC with anti-thrombogenic properties . The objective of this study was to investigate the effects of Stx on the surface expression of TM in EC using an in vitro culture of human glomerular microvascular endothelial cells . We also evaluated other inflammatory mediators {tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide}, which are known to increase Stx receptor expression and are potentially involved in the pathogenesis of D+HUS . Stx2 induced a significant decrease of TM expression in this cell type after pre-incubation with TNF-alpha . This decrease could not be attributed to the inhibition of protein synthesis only, as cycloheximide, another inhibitor of protein synthesis, did not affect TM surface expression . These results suggest that the Stx2-induced decrease of TM expression in glomerular EC might contribute to the local procoagulant state present in D+HUS. Am J Respir Cell Mol Biol, 2004 Feb, 30(2), 228 - 32 Epub 2003 Aug 14. Surfactant blocks lipopolysaccharide signaling by inhibiting both mitogen-activated protein and IkappaB kinases in human alveolar macrophages; Raychaudhuri B et al.; Surfactant plays an important role in lung homeostasis and is also involved in maintaining innate immunity within the lung . Lipopolysaccharide (LPS) from gram-negative bacteria is known to elicit acute proinflammatory responses in lung diseases such as acute respiratory distress syndrome and pneumonia, among others . Our previous studies demonstrated that the clinically used, natural surfactant product Survanta inhibited proinflammatory cytokine secretion from LPS-stimulated human alveolar macrophages . Here we investigated the effect of Survanta on mitogen-activated protein (MAP) and IkappaB kinases . Survanta blocked LPS-induced activation of nuclear factor-kappaB, a key regulatory transcription factor involved in cytokine production, by preventing phosphorylation of IkappaBalpha, and its subsequent degradation . IkappaB is phosphorylated by specific kinases (IKK) before degradation . Survanta inhibited activity of both alpha and beta subunits of IKK, thereby delaying the phosphorylation of IkappaB . Interestingly, IKK-alpha is predominant in alveolar macrophages, whereas IKK-beta predominates in monocytes . Survanta also inhibited extracellular signal-regulated kinase and p38 MAP kinase activity induced by LPS . Data are the first to show that surfactant may regulate lung homeostasis in part by inhibiting proinflammatory cytokine production through reduction of IKK and MAP kinase activity. Biomol Eng, 2003 Jul, 20(4-6), 317 - 24 Antarctic marine bacterium Pseudoalteromonas sp . 22b as a source of cold-adapted beta-galactosidase; Turkiewicz M et al.; The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified a