Gram negative

ALTEX, 1994, 11(5), 24 - 29
{Investigation of the endotoxin content of veterinary vaccines}; Cussler K et al.; Vaccines composed of gram-negative bacteria contain endotoxin in considerable amounts . This may result in adverse effects after vaccination of sensitive animals . For reasons of safety and animal welfare the endotoxin level of veterinary vaccines should be limited . The limulus amebocyte lysate test is suitable to check the endotoxin content in most vaccines . Safety tests in animals should only be performed if the results of the limulus test are satisfactory.

Curr Infect Dis Rep, 2001 Feb, 3(1), 13 - 19
What's New in beta-lactamases?
Bradford PA.
beta-lactamases continue to be the leading cause of resistance to beta-lactam antibiotics, among gram-negative bacteria . In recent years, both the incidence and the prevalence of extended-spectrum beta-lactamases, inhibitor-resistant beta-lactamases, AmpC-type enzymes, and both metallo- carbapenemases and nonmetallo-carbapenemases have increased . These beta-lactamases provide resistance to oximino-cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, cephamycins, and carbapenems, respectively . Strains expressing these beta-lactamases will generate a host of therapeutic challenges as we begin the 21st century.

Bull Exp Biol Med, 2000 Oct, 130(10), 997 - 1000
A single endotoxin aggression causes dose-dependent reversible activation of rat liver Ito cells without their transdifferentiation into myofibroblasts; Salakhov IM et al.; The effect of Gram-negative bacterial lipopolysaccharide on rat hepatocytes and sinusoidal cells was studied . The damage and regeneration potential of the liver were evaluated by activation of perisinusoidal Ito cells and proliferative activity of liver cells . Compensatory and repair reactions in the liver induced by lipopolysaccharide manifested by proliferation of liver cells and reversible activation of Ito cells without their transdifferentiation into myofibroblasts.

Ann Med Interne (Paris), 2000 Dec, 151(8), 624 - 8
{Main complications of diabetes mellitus in Africa}; Sidibe EH; In Africa, a rise in complications of diabetes mellitus has gone in hand with the growing disease prevalence, clearly demonstrating the importance of assessing complications . Diabetes mellitus constitutes a major financial burden in developing countries in Africa with relatively limited resources . Ketoacidosis is observed in 24% of juvenile diabetes and is the inaugural sign in 76% of all cases, progressing to coma in 34% . Even in type 2 diabetes, acidoketosis occurs in 34% of the cases . Infection is particularly frequent and is often fatal in tropical Africa because of the involvement of Staphyococcus and Gram-negative microorganisms . Hyperleukocytosis and anemia are correlated with ineffective antibiotic therapy . Pulmonary tuberculosis is the ninth most frequent complication of diabetes . Overall mortality is 14.9 per 1000 person-years of diabetes . Mean age at death is 51.6 years for women and 57.6 years for men after a mean 12.5 year disease duration . Thirty percent of all deaths result from acute metabolic complications, infections and stroke . More than half of the patients with insulin-dependent-diabetes have retinopathy . Differences observed in patients with different ethnic origins is linked basically to unfavorable social and economic conditions that worsen the risk of poor blood glucose control . Retinopathy accounts for 32% of all ocular complications, similar to other African data and more generally in ophthalmology centers . The rate of neuropathy is high, reaching 70% in patients with microangiopathy . Impotence concerns 48.7% of the diabetic population with a mean age of 41.4+/-15.5 years . Coronary artery disease had a recognized influence on hemoglobin diseases, particularly when the coronarography is normal . Lower limb arteriopathy is observed in 18% of the diabetic patients.

Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 323 - 5
Crystallization and preliminary crystallographic study of the periplasmic domain of the Escherichia coli TolR protein; Abergel C et al.; The TolR protein from Escherichia coli is part of the Tol-Pal multiprotein complex used by group A colicins to penetrate and kill cells . All genes of the Tol-Pal system are conserved in Gram-negative bacteria and this system is thought to play a role in the maintenance of the bacterial envelope integrity, although its exact function is not known . The TolR protein comprises 142 amino acids . The periplasmic domain of the TolR protein has been expressed, purified and crystallized . The crystals belong to the tetragonal space group P4(1)22, with unit-cell parameters a = 46.3, c = 178.0 A . There are one or two molecules in the asymmetric unit . Frozen crystals diffract to at least 3.2 A resolution using synchrotron radiation . Selenomethionine-substituted periplasmic TolR protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing.

Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 317 - 9
Crystallization and preliminary crystallographic study of the peptidoglycan-associated lipoprotein from Escherichia coli; Abergel C et al.; The peptidoglycan-associated lipoprotein (Pal) from Escherichia coli is part of the Tol--Pal multiprotein complex used by group A colicins to penetrate and kill cells . Pal homologues are found in many Gram-negative bacteria and the Tol--Pal system is thought to play a role in bacterial envelope integrity . The Pal protein comprises 152 amino acids . Crystals of the C-terminal 109-amino-acid fragment of the Pal protein have been produced . The crystals belong to the tetragonal space group I4(1), with unit-cell parameters a = b = 89.3, c = 67.2 A . There are two molecules in the asymmetric unit . Frozen crystals diffract to at least 2.8 A resolution using synchrotron radiation . Selenomethionine-substituted truncated Pal protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing.

Clin Infect Dis, 2001 Jan 15, 32(2), 220 - 7 Epub 2000 Dec 22.
Molecular typing demonstrating transmission of gram-negative rods in a neonatal intensive care unit in the absence of a recognized epidemic; Almuneef MA et al.; Molecular typing techniques have been used in outbreak investigations . In this study, molecular typing techniques were used to track the spread of gram-negative rods (GNRs) in a neonatal intensive care unit (NICU) in the absence of an outbreak . Stool or rectal swab cultures for GNRs were obtained from all infants on admission, weekly, and on discharge . GNRs were tested for gentamicin susceptibility and were typed by contour-clamped homogeneous electric field electrophoresis . Transmission of identical strains of GNRs among infants was noted . Shared strains were more gentamicin resistant compared with unique strains (53% vs . 10%; P=.0001) . Infants first colonized when they were >1 week of age had more total days of antibiotic treatment and had a higher rate of acquiring a shared and gentamicin-resistant strain, compared with infants colonized earlier . Antibiotic use increases colonization of infants in the NICU with resistant and shared strains of GNRs.

Br J Haematol, 2000 Dec, 111(4), 1093 - 102
Procalcitonin in paediatric cancer patients: its diagnostic relevance is superior to that of C-reactive protein, interleukin 6, interleukin 8, soluble interleukin 2 receptor and soluble tumour necrosis factor receptor II; Fleischhack G et al.; Sensitive parameters of inflammation are rare in neutropenic cancer patients . In this study, procalcitonin (PCT), C-reactive protein (CRP), interleukin 6 (IL-6), IL-8, the soluble IL-2 receptor (sIL-2R) and the soluble tumour necrosis factor receptor II (sTNFRII) were evaluated for their diagnostic relevance in febrile episodes of cancer patients . Plasma or serum levels of these parameters were determined in neutropenic children with febrile episodes (n = 122) classified according to both the kind of infection {60 cases of fever of unknown origin (FUO), 28 cases of localized infection, 13 cases of pneumonia, 20 cases of bacteraemia, one case of fungaemia} and the World Health Organization (WHO) score of chemotherapy-induced mucositis . At baseline and during the febrile episodes, the highest levels of all parameters were observed in cases of gram-negative bacteraemia . However, in FUO and localized infections, low or only slightly elevated median levels of all parameters were documented . The degree of chemotherapy-induced mucositis did not influence the value of any parameter . In comparison with the other inflammatory parameters, PCT (optimum cut-off level 0.5 microg/l) was a more sensitive and more specific parameter in the diagnosis of high-risk (gram-negative bacteraemia) and low-risk (FUO) episodes, as well as in the sequential assessment of all febrile neutropenic episodes.

Anaesthesia, 2001 Feb, 56(2), 130 - 44
The haemodynamics of human septic shock; MacKenzie IM; In the time it has taken medicine to develop the techniques to describe the circulatory changes of severe infections, both pattern and process have been profoundly influenced by the use of intravenous fluids, vasopressors, antibiotics, steroids, mechanical ventilation and haemoflltration . Constant features of severe sepsis include a reduction in peripheral vascular tone on both the arterial and venous sides of the circulation, a defect in oxygen utilisation resulting in lactic acidosis, and varying degrees of myocardial dysfunction . These events have a temporal progression, the precise pattern observed depending on the tempo of the infection, the influence of therapeutic manoeuvres, the age and comorbidities of the patient, and the time the observations are made in the course of events . Early sepsis is accompanied by a decrease in systemic vascular resistance and a metabolic acidosis . The clinical picture includes fever, tachycardia, tachypnoea, respiratory alkalosis and an increased cardiac output with warm, dry peripheries and a bounding pulse . Advanced sepsis involves varying degrees of venous and myocardial contractile failure, and is characterised by progressive acidaemia, respiratory failure and marked sympathetic adrenergic activation . In the absence of vigorous fluid resuscitation, the cardiac output is decreased and the patients are cold, clammy peripherally shut down, and frequently confused, obtunded or comatose . In infections with a silent primary focus (predominantly involving Gram-negative organisms), this stage is frequently the first to attract the attention of attending staff . Late sepsis is characterised by profound acidaemia, vascular hypo-responsiveness, multiple organ failure and death.

Insect Biochem Mol Biol, 2001 Mar 1, 31(3), 219 - 29
Innate immune response of Aedes aegypti; Lowenberger C; Insects are able to protect themselves from invasion by pathogens by a rapid and potent arsenal of inducible immune peptides . This fast, extremely effective response is part of the innate immunity exhibited by all insects and many invertebrates, and shows striking similarities with the innate immune response of vertebrates . In Aedes aegypti invasion of the hemocoel by bacteria elicits the production of defensins, cecropins, a peptide active only against Gram-negative bacteria, and several other peptides that we are now characterizing . However, not all insects utilize the same peptides in the same concentrations, which may reflect the pathogens to which they may have been exposed through evolutionary time . These protective measures we see in mosquitoes are the current state of the evolution of a rapid immune response that has contributed to the success of insects in inhabiting essentially every niche on earth . The molecules involved in the response of Aedes aegypti to pathogens, and the potential role of these peptides against eukaryotic parasites ingested and transmitted by mosquitoes are discussed.

J Insect Physiol, 2001 Apr, 47(4-5), 475 - 83
Suppression of pederin biosynthesis through antibiotic elimination of endosymbionts in Paederus sabaeus; Kellner RL; Biosynthesis of the unique defensive compound pederin is confined to female rove beetles of the genus Paederus (Coleoptera: Staphylinidae) . These (+)-females endow their eggs with toxin whereas (-)-females, which occur both naturally and in laboratory reared specimens, do not . The latter are aposymbionts lacking biosynthetic capabilities because of endosymbiotic deficiency . They can, however, be induced to accumulate pederin if fed with (+)-eggs during larval development . The endosymbionts can thus be transmitted by ingestion of (+)-Eggs . (+)-eggs treated with benzylpenicillin, erythromycin, oxytetracycline or streptomycin show that the induction of pederin accumulation depends on the antibiotic's spectrum of efficaciousness, its dosage and duration of the treatment . Certain bacteria, probably belonging to the gram-negative type, must be transmitted to produce (+)-females.

Neuroscience, 2001, 102(2), 273 - 80
The effects of the bacterial endotoxin lipopolysaccharide on synaptic transmission and plasticity in the CA1-subiculum pathway in vivo; Commins S et al.; Lipopolysaccharide is derived from the cell wall of gram-negative bacteria and is a potent endotoxin which causes the release of cytokines in the CNS . We examined the effect of lipopolysaccharide on synaptic transmission and synaptic plasticity in the hippocampal area CA1-subicular pathway in vivo . We found that lipopolysaccharide did not affect baseline synaptic transmission in this pathway; it did, however, reduce the magnitude of paired-pulse facilitation, a form of short-term plasticity thought to be primarily presynaptic in origin . We then examined the interaction between lipopolysaccharide and two common models for the biological basis of memory: high-frequency stimulation induced long-term potentiation and low-frequency stimulation induced long-term depression of synaptic transmission . We found that lipopolysaccharide blocked long-term potentiation following high-frequency stimulation and also induced potentiation of synaptic transmission after low-frequency stimulation . Lipolysaccharide blocked paired-pulse facilitation selectively at short rather than longer interstimulus intervals . Thus, lipopolysaccharide has different effects on synaptic transmission in this pathway depending on the frequency and length of stimulation.These results provide new insights into the action of lipopolysaccharide on various forms of plasticity in the hippocampus, an area known to play a vital role in learning and memory.

Trends Biochem Sci, 2001 Jan, 26(1), 3 - 6
Type I secretion and multidrug efflux: transport through the TolC channel-tunnel; Buchanan SK; The crystal structure of TolC from Escherichia coli was recently determined to 2.1-A resolution and shows a unique type of channel architecture: a 12-stranded beta-barrel spans the outer membrane and is attached to a long alpha-helical channel that penetrates far into the periplasm . The structure suggests a mechanism for its role in secretion of proteins and in efflux of toxic small molecules . The TolC export pathway is compared with several import pathways of gram-negative bacteria where the outer membrane protein structures are also known.

Mech Ageing Dev, 2000 Dec 20, 121(1-3), 89 - 100
Chemokine production by peripheral blood mononuclear cells in elderly subjects; Pulsatelli L et al.; The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections . In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Ialpha, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin . Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Ialpha and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lalpha . In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in 'specific' immune response during ageing . The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly.

Nucleic Acids Res, 2001 Feb 15, 29(4), 863 - 71
Polymorphism in the 3'-untranslated region of TNFalpha mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFalpha mRNA; Di Marco S et al.; Tumor necrosis factor alpha (TNFalpha) acts as a beneficial mediator in the process of host defence . In recent years major interest has focused on the AU-rich elements (AREs) present in the 3'-untranslated region (3'-UTR) of TNFalpha mRNA as this region plays a pivotal role in post-transcriptional control of TNFalpha production . Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFalpha mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFalpha . In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3'-UTR of TNFalpha mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFalpha protein . We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFalpha 3'-UTR . One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs . Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFalpha mRNA, these results suggest that mutations in the ARE of TNFalpha mRNA decrease the production of TNFalpha protein in macrophages by hindering the binding of HuR to the ARE.

Microbiology, 2001 Jan, 147(Pt 1), 11 - 9
Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages; Lutke-Eversloh T et al.; This is the first report on the biosynthesis of a hitherto unknown, sulfur-containing polyester and also the first report on a bacterial polymer containing sulfur in the backbone . The Gram-negative polyhydroxyalkanoate (PHA)-accumulating bacterium Ralstonia eutropha synthesized a copolymer of 3-hydroxybutyrate and 3-mercaptopropionate, poly(3HB-co-3MP), when 3-mercaptopropionic acid or 3,3'-thiodipropionic acid was provided as carbon source in addition to fructose or gluconic acid under nitrogen-limited growth conditions . The peculiarity of this polymer was the occurrence of thioester linkages derived from the thiol groups of 3MP and the carboxyl groups of 3MP or 3HB, respectively, which occurred in addition to the common oxoester bonds of PHAs . Depending on the cultivation conditions and the feeding regime, poly(3HB-co-3MP) contributed up to 19% of the cellular dry weight, with a molar fraction of 3MP of up to 43% . The chemical structure of poly(3HB-co-3MP) was confirmed by GC/MS, IR spectroscopy, (1)H- and (13)C-NMR spectroscopy, and elemental sulfur analysis . The identification of this novel biopolymer reveals a new quality regarding the substrate range of PHA synthases and their capability for the synthesis of technically interesting polymers.

Antimicrob Agents Chemother, 2001 Feb, 45(2), 413 - 9
Plasmid location and molecular heterogeneity of the L1 and L2 beta-lactamase genes of Stenotrophomonas maltophilia; Avison MB et al.; An approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia . This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 beta-lactamase genes . The location of L1 and L2 on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems . Sequence analysis of the 10 L1 genes revealed three novel genes, L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1 (L1a), respectively . The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lower k(cat)/K(m) ratios respectively) . L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a . Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences . Three novel L2 genes (L2b, L2c, and L2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275 L2 (L2a) by 4, 9, and 25%, respectively . Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found . It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.

JAMA, 2000 Apr 5, 283(13), 1723 - 30
E5 murine monoclonal antiendotoxin antibody in gram-negative sepsis: a randomized controlled trial . E5 Study Investigators; Angus DC et al.; CONTEXT: Knowledge and understanding of gram-negative sepsis have grown over the past 20 years, but the ability to treat severe sepsis successfully has not . OBJECTIVE: To assess the efficacy and safety of E5 in the treatment of patients with severe gram-negative sepsis . DESIGN: A multicenter, double-blind, randomized, placebo-controlled trial conducted at 136 US medical centers from April 1993 to April 1997, designed with 90% power to detect a 25% relative risk reduction, incorporating 2 planned interim analyses . SETTING: Intensive care units at university medical centers, Veterans Affairs medical centers, and community hospitals . PATIENTS: Adults aged 18 years or older, with signs and symptoms consistent with severe sepsis and documented or probable gram-negative infection . INTERVENTION: Patients were assigned to receive 2 doses of either E5, a murine monoclonal antibody directed against endotoxin (n = 550; 2 mg/kg per day by intravenous infusion 24 hours apart) or placebo (n = 552) . MAIN OUTCOME MEASURES: The primary end point was mortality at day 14; secondary end points were mortality at day 28, adverse event rates, and 14-day and 28-day mortality in the subgroup without shock at presentation . RESULTS: The trial was stopped after the second interim analysis . A total of 1090 patients received study medication and 915 had gram-negative infection confirmed by culture . There were no statistically significant differences in mortality between the E5 and placebo groups at either day 14 (29.7% vs 31.1%; P = .67) or day 28 (38.5% vs 40.3%; P = .56) . Patients presenting without shock had a slightly lower mortality when treated with E5 but the difference was not significant (28.9% vs 33.0% for the E5 and placebo groups, respectively, at day 28; P = .32) . There was a similar profile of adverse event rates between E5 and placebo . CONCLUSIONS: Despite adequate sample size and high enrollment of patients with confirmed gram-negative sepsis, E5 did not improve short-term survival . Current study rationale and designs should be carefully reviewed before further large-scale studies of patients with sepsis are conducted.

J Immunol, 2000 Apr 15, 164(8), 4301 - 6
IL-1 receptor-associated kinase modulates host responsiveness to endotoxin; Swantek JL et al.; Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock . Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer . The IL-1 intracellular signaling pathway has been implicated in Toll-like receptor signal transduction . LPS-induced activation of the IL-1 receptor-associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells . We demonstrate that LPS activates IRAK in murine macrophages . IRAK-deficient macrophages, in contrast, are resistant to LPS . Deletion of IRAK disrupts several endotoxin-triggered signaling cascades . Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK-deficient mice withstand the lethal effects of LPS . These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection.

APMIS, 2000 Mar, 108(3), 201 - 8
The bactericidal/permeability-increasing protein (BPI) is membrane-associated in azurophil granules of human neutrophils, and relocation occurs upon cellular activation; Calafat J et al.; Neutrophilic granulocytes contain the 55 kDa bactericidal/permeability-increasing protein (BPI) . BPI binds to lipopolysaccharides (LPS), and exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species . We have investigated the subcellular location of BPI in immature and mature neutrophils using cryotechnique for immunoelectron microscopy . BPI was found to colocate with myeloperoxidase (MPO), a marker for azurophil granules, and it also showed the same pattern of distribution as CD63, a transmembrane-anchored protein . This suggests that BPI is membrane-associated in the azurophil granules in neutrophils . Its presence in azurophil granules was further confirmed by the finding of BPI in the azurophil granules of neutrophil promyelocytes of the bone marrow . Induction of selective release of azurophilic granules by the Na-ionophore monensin resulted in fusion of endosomes with azurophil granules, leading to the formation of large vacuoles containing MPO, CD63, and BPI . After phagocytosis of serum-treated zymosan (STZ), BPI was detected in phagosomes, both in association with membranes as well as in the lumen, suggesting the release of BPI into activated compartments . The results show that BPI is present in azurophil granules, is probably primarily membrane-associated, and is relocated after activation, following the same route as MPO and CD63.

J Biol Chem, 2001 Mar 23, 276(12), 8820 - 4 Epub 2001 Jan 02.
Apolipoprotein E protects against bacterial lipopolysaccharide-induced lethality . A new therapeutic approach to treat gram-negative sepsis; Van Oosten M et al.; Septic shock is the most common cause of death in intensive care units and no effective treatment is available at present . Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived cytokines . Previously, we showed that apolipoprotein E (apoE), an established modulator of lipid metabolism, can bind LPS, thereby redirecting LPS from macrophages to hepatocytes in vivo . We now report that intravenously administered LPS strongly increases the serum levels of apoE . In addition, apoE can prevent the LPS-induced production of cytokines and subsequent death in rodents . Finally, apoE-deficient mice show a significantly higher sensitivity toward LPS than control wild-type mice . These findings indicate that apoE may have a physiological role in the protection against sepsis, and recombinant apoE may be used therapeutically to protect against LPS-induced endotoxemia.

Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2151 - 6
Roseigium denhamense gen . nov., sp . nov . and Roseibium hemelinense sp . nov., aerobic bacteriochlorophyll-containing bacteria isolated from the east and west coasts of Australia; Suzuki T et al.; Phenotypic and phylogenetic studies were performed with 10 strains of bacteriochlorophyll-containing bacteria isolated from a variety of marine environments (surface of Rhodophyta, sand and algal sand mat) on the east and west coasts of Australia . The strains were aerobic, chemoheterotrophic, Gram-negative, motile rods with peritrichous flagella . Bacteriochlorophyll a was synthesized under aerobic conditions . Catalase, nitrate reductase, oxidase and phosphatase were produced . ONPG reaction was positive . The strains have been divided into genotype group 1 (seven strains) and genotype group 2 (three strains) according to previously described DNA-DNA hybridization data . Strains OCh 254T and OCh 368T have been included in genotype groups 1 and 2, respectively . The results of 165 rRNA gene sequence comparisons revealed that strains OCh 254T and OCh 368T formed a new cluster within the alpha-2 group of the alpha subclass of the Proteobacteria . The similarity value of the 16S rRNA gene sequences between strain OCh 254T and the most closely related species, Stappia aggregata, was 95.6 % . The sequence similarity value between strains OCh 254T and OCh 368T was 97.1% . It was concluded that these two strains should be placed into a new genus, Roseibium gen . nov., as Roseibium denhamense sp . nov . and Roseibium hamelinense sp . nov . The type species of the genus is Roseibium denhamense . The type strains of Roseibium denhamense and Roseibium hamelinense are OCh 254T (= JCM 10543T) and OCh 368T (= JCM 10544T), respectively.

Minerva Med, 2000 Jul-Aug, 91(7-8), 161 - 7
{Infections and cardiovascular diseases on an ischemic basis . The case of Helicobacter pylori}; Pellicano R et al.; Cardiac and cerebrovascular diseases are an important cause of mortality in industrialized countries . "Classical" risk factors cannot fully explain epidemiological variations of these diseases . From several years infections have been linked to ischemic vascular events and recent publications pointed to the role of Helicobacter pylori, a Gram negative bacterium, involved in the pathogenesis of gastritis and peptic ulcer . Results on the association between this bacterium and acute myocardial infarction or stroke are controversial, due to the degree of studies heterogeneity . There is the need for extensive prospectic studies to evaluate the incidence of these diseases in relation to the presence of Helicobacter pylori infection . Interventional randomized studies employing an antibiotic treatment for patients affected by ischemic vascular diseases will rapidly answer the question of wheather Helicobacter pylori has a causal role in the pathogenesis of acute myocardial infarction and ischemic stroke.

Toxicology, 2000 Nov 30, 155(1-3), 37 - 44
Pivotal role of nitric oxide in delayed pharmacological preconditioning against myocardial infarction; Xi L et al.; The phenomenon of 'ischemic preconditioning' (IP) has been vigorously investigated during the past 15 years . As our knowledge on the possible protective mechanisms of IP has been increasingly expanded, novel approaches based on preconditioning with pharmacological agents have recently emerged . Two drugs have been used to induce delayed preconditioning against myocardial infarction caused by ischemia/reperfusion . One of the drugs was monophosphoryl lipid A (MLA)--a detoxified derivative of lipopolysaccharide from gram-negative strains; and another drug was RC552--a novel synthetic glycolipid that mimics the chemical structure of MLA . We have shown that pretreatment of adult mice with MLA or RC552 (350 microg/kg) 24 h prior to the global ischemia and reperfusion in the isolated perfused heart attenuated myocardial injury . Infarct size was significantly reduced in MLA or RC552-treated groups as compared with the vehicle-treated group . The delayed cardioprotection was associated with a moderate but significant increase of nitric oxide level in the ischemic myocardium . Treatment with S-methylisothiourea (3 mg/kg), a selective inhibitor of inducible nitric oxide synthase (iNOS) abolished MLA or RC552-induced delayed protection . In addition, neither MLA nor RC552 reduced infarct size in iNOS knockout mice . Our findings suggest that both MLA and RC552 are able to induce delayed myocardial preconditioning via iNOS-dependent pathway.

Oral Microbiol Immunol, 2000 Dec, 15(6), 383 - 7
Identification of an antigenic protein Pga30 from Porphyromonas gingivalis W50; Hendtlass A et al.; Porphyromonas gingivalis is a black-pigmented, gram-negative bacterium that has been implicated as a major pathogen in the development of adult periodontitis . In an approach to identify a P . gingivalis antigen uniquely seroreactive with healthy subjects, we produced a surface and periplasmic extract of P . gingivalis, separated that extract into 36 fractions using anion-exchange chromatography and screened each fraction for reactivity in an enzyme-linked immunosorbent assay (ELISA) using sera from eight periodontitis patients and eight age- and sex-matched healthy controls . All of the diseased subjects harboured subgingival P . gingivalis by DNA probe analysis and exhibited similar seroreactivity profiles to the anion exchange fractions . However, only two of the healthy subjects (C10 and C15) were seroreactive with the fractions . The highest reactivity for all the seropositive subjects was with the same anion-exchange fractions 13-15 . The anion exchange fraction (14) with the highest seroreactivity was subjected to gel filtration chromatography, and fraction 22 from this chromatography exhibited the highest reactivity with all the seropositive subjects . However, fraction 27 was found to be uniquely seroreactive with healthy subject C10, as it was not recognized by sera from any of the diseased or the other healthy subjects . This fraction was further purified by reversed-phase high-pressure liquid chromatography and shown to contain a 30-kDa protein as determined by SDS-PAGE . Control subject C10 had no pocket depths greater than 3 mm and no sites that bled on gentle probing; however, P . gingivalis was detected in subgingival plaque samples at a level of 10(5)-10(6) cells per site in two of the ten sites sampled . This subject was also unusual in that he exhibited a seroreactivity profile similar to that of diseased subjects, which was not characteristic of the healthy control subjects . The unique reactivity of the 30-kDa antigen, designated Pga30, with subject C10 serum was confirmed in a Western blot with the purified antigen . N-Terminal sequence analysis of Pga30 produced a single, unambiguous protein sequence confirming the purity of the protein . A search of the database using the N-terminal sequence obtained did not reveal any significant sequence similarity . In conclusion, we have identified a P . gingivalis antigen that was uniquely reactive in an ELISA and Western blot with serum from a subject with no clinical signs of periodontitis who harbored P . gingivalis in subgingival plaque.

Oral Microbiol Immunol, 2000 Dec, 15(6), 378 - 82
Histatin 5 inhibits inflammatory cytokine induction from human gingival fibroblasts by Porphyromonas gingivalis; Imatani T et al.; Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease . It has been demonstrated that outer-membrane proteins as well as lipopolysaccharides from P . gingivalis ATCC 53977 can induce interleukin 6 (IL-6) and IL-8 from the cells of the periodontium in vitro . But, they cannot induce IL-1 and tumor necrosis factor-alpha from the cells . In the present study, we studied the effects of salivary protein on cytokine induction from human gingival fibroblasts by P . gingivalis outer-membrane protein . Histatin 5 suppressed the IL-6 and IL-8 induction by P . gingivalis outer-membrane protein . This activity was more effective when outer-membrane protein was incubated with histatin 5 before addition to the cell culture . The present study indicates that histatin 5 restrains induction of inflammatory cytokines by periodontal pathogens and that histatin is one of the salivary proteins responsible for this activity.

Oral Microbiol Immunol, 2000 Aug, 15(4), 226 - 31
Sensitivity of Actinobacillus actinomycetemcomitans and Capnocytophaga spp . to the bactericidal action of LL-37: a cathelicidin found in human leukocytes and epithelium; Tanaka D et al.; The bactericidal activity of synthetic LL-37, a cathelicidin, was assessed against Actinobacillus actinomycetemcomitans (three strains) and Capnocytophaga spp . (three strains) . All strains were sensitive to LL-37, and exhibited 99% effective dose of 7.5-to-11.6 micrograms/ml . An amidated form of LL-37, pentamide-37, killed with about the same efficacy as LL-37 . Partial inhibition of killing was noted at physiologic concentrations of NaCl, and complete inhibition was observed at 400 mM NaCl . At approximately the 99% effective dose--i.e., 10 micrograms/ml--LL-37 also lost activity against A . actinomycetemcomitans in the presence of native or heat-inactivated 10-15% normal human AB serum . Pentamide-37 was less sensitive to serum inhibition than LL-37 . In conclusion, certain oral, gram-negative bacteria are sensitive to the bactericidal activity of LL-37 at low concentrations of serum and salt, a condition likely to be found within the membrane-delimited phagolysosome . Modified forms of LL-37, such as pentamide-37, may be more suitable for future therapeutic application in the presence of serum.

FASEB J, 2001 Jan, 15(1), 155 - 163
Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components; Laflamme N et al.; The recent characterization of human homologues of Toll may be the missing link for the transduction events leading to NF-kappaB activity and proinflammatory gene transcription during innate immune response . Indeed, CD14 is not thought to participate directly in the cell signaling, but rather one or more of the mammalian Toll-like receptors (TLRs) acts in concert with the lipopolysaccharide (LPS) receptor to discriminate between microbial pathogens or their products and initiate transmembrane signaling . Mammalian cells may express as many as 10 distinct TLRs, although the importance of TLR4 in response to gram-negative bacteria and LPS is now supported by the fact that TLR4-mutated mice are LPS resistant . We investigated the expression of TLR4 across the rat brain under basal conditions and in response to systemic LPS and IL-1beta injection . We first cloned the rat TLR4 cDNA via RNA isolation and polymerase chain reaction (PCR) amplification with a proofreading polymerase . Total RNA was isolated from the rat liver tissue using Tri-Reagent and reverse transcribed into cDNA using Superscript II reverse transcriptase and an oligonucleotide primer with a degenerate 3' end of sequence 5'-T12(GAC)N-3' . Positive hybridization signal was found in the leptomeninges, choroid plexus (chp), subfornical organ, organum vasculosum of the lamina terminalis, median eminence, and area postrema . Scattered small cells also displayed a convincing hybridization signal within the brain parenchyma . Few well-defined nuclei exhibited positive TLR4 transcript: the supramamillary nucleus, cochlear nucleus, and the lateral reticular nucleus . The circumventricular organs, the leptomeninges, and chp also exhibited constitutive expression of the LPS receptor mCD14 . In contrast to the strong up-regulation of the gene encoding mCD14 during endotoxemia, neither LPS nor IL-1beta caused a convincing increase in the TLR4 mRNA levels across the CNS . A down-regulation of the gene encoding TLR4 was found in the cerebral tissue of immune-challenged animals . The constitutive expression of both mCD14 and TLR4 may explain the innate immune response in the brain, which originates from the structures devoid of blood-brain barrier in presence of circulating LPS.

Hepatogastroenterology, 2000 Nov-Dec, 47(36), 1555 - 9
Application of peroral cholangioscopy in an endemic area with high prevalence of hepatocellular carcinoma and choledocholithiasis; Wang HP et al.; BACKGROUND/AIMS: Peroral cholangioscopy with a mother-baby scope system has been introduced for two decades . The paper presents the experience of peroral cholangioscopy at a university hospital in Taiwan where the prevalence of hepatocellular carcinoma and choledocholithiasis was high . METHODOLOGY: A total of 27 sessions of peroral cholangioscopy were performed in 26 patients during a period of 4 years . Of them, 20 patients were for diagnosis and the rest 6 for removing the retained biliary stones . RESULTS: The overall successful rate was 96.3% . The post-procedure complication rate was 11.5% with 2 cholangitis and 1 gram-negative septicemia . There were a total of 19 successful diagnostic sessions . These resulted in definite histological diagnosis in 5 patients and more precise diagnoses subsequently confirmed by surgery in 5 patients . In the remaining 9 patients with tentative diagnoses, 5 confirmed their diagnoses but 4 patients changed their diagnoses after peroral cholangioscopy . By this procedure, hepatocellular carcinoma and choledocholithiasis can be well identified and differentiated . CONCLUSIONS: Peroral cholangioscopy is a safe and valuable modality in diagnosing and treating difficult biliary tract disease when handled with care . It is particularly useful in an endemic area with high prevalence of hepatocellular carcinoma and choledocholithiasis.

Biochemistry, 2001 Jan 16, 40(2), 514 - 23
Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) . Identification of the zinc binding site; Jackman JE et al.; UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A in Gram-negative bacteria . Compounds targeting this enzyme are proposed to chelate the single, essential zinc ion bound to LpxC and have been demonstrated to stop the growth of Escherichia coli . A comparison of LpxC sequences from diverse bacteria identified 10 conserved His, Asp, and Glu residues that might play catalytic roles . Each amino acid was altered in both E . coli and Aquifex aeolicus LpxC and the catalytic activities of the variants were determined . Three His and one Asp residues (H79, H238, D246, and H265) are essential for catalysis based on the low activities (<0.1% of wild-type LpxC) of mutants with alanine substitutions at these positions . H79 and H238 likely coordinate zinc; the Zn(2+) content of the purified variant proteins is low and the specific activity is enhanced by the addition of Zn(2+) . The third side chain to coordinate zinc is likely either H265 or D246 and a fourth ligand is likely a water molecule, as indicated by the hydroxamate inhibition, suggesting a His(3)H(2)O or His(2)AspH(2)O Zn(2+)-polyhedron in LpxC . The decreased zinc inhibition of LpxC mutants at E78 suggests that this side chain may coordinate a second, inhibitory Zn(2+) ion . Given the absence of any known Zn(2+) binding motifs, the active site of LpxC may have evolved differently than other well-studied zinc metalloamidases, a feature that should aid in the design of safe antibiotics.

Arterioscler Thromb Vasc Biol, 2001 Jan, 21(1), E1 - 8
Chlamydia pneumoniae proteins induce secretion of the 92-kDa gelatinase by human monocyte- derived macrophages; Vehmaan-Kreula P et al.; Chlamydia pneumoniae, an intracellular Gram-negative respiratory bacterium, and macrophages are present in inflammatory tissue sites such as atherosclerotic lesions, where abnormal degradation of the extracellular matrix takes place . To evaluate the potential of C pneumoniae for participation in matrix destruction, we studied the effect of this bacterium on the production of 3 matrix-degrading metalloproteinases, 92-kDa gelatinase, interstitial collagenase-1, and stromelysin-1, and their natural inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) by human monocyte-derived macrophages differentiated in vitro . Spontaneous production of collagenase and stromelysin by these cells was minimal and was not influenced by C pneumoniae . In contrast, the cells secreted substantial basal quantities of 92-kDa gelatinase, the secretion of which was stimulated (on average, 2.5-fold) by C pneumoniae . C pneumoniae regulated the expression of 92-kDa gelatinase by macrophages at the pretranslational level . Macrophages secreted only small quantities of TIMP-1 . The chlamydial proteins Omp2, MOMP, and HSP60 were also found to participate in the induction of 92-kDa gelatinase by C pneumoniae . Denaturation of chlamydial proteins by boiling reduced 92-kDa gelatinase secretion only partially (by 35%), suggesting that the heat-stabile lipopolysaccharide molecules also stimulate secretion of the enzyme . The results show that production of 92-kDa gelatinase by human macrophages is selectively upregulated by C pneumoniae, which suggests that these bacteria, when present in a macrophage-containing inflammatory environment, actively participate in the destruction of the extracellular matrix.

J Immunol, 2001 Jan 15, 166(2), 1075 - 8
Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccharide in CD14-deficient and Toll-like receptor 4-deficient mice; Haziot A et al.; Despite the lack of a proinflammatory response to LPS, CD14-deficient mice clear Gram-negative bacteria (Escherichia coli 0111) at least 10 times more efficiently than normal mice . In this study, we show that this is due to an early and intense recruitment of neutrophils following the injection of Gram-negative bacteria or LPS in CD14-deficient mice; in contrast, neutrophil infiltration is delayed by 24 h in normal mice . Similar results of early LPS-induced PMN infiltration and enhanced clearance of E . coli were seen in Toll-like receptor (TLR) 4-deficient mice . Furthermore, the lipid A moiety of LPS induced early neutrophil infiltration not only in CD14-deficient and TLR-4-deficient mice, but also in normal mice . In conclusion, the lipid A component of LPS stimulates a unique and critical pathway of innate immune responses that is independent of CD14 and TLR4 and results in early neutrophil infiltration and enhanced bacterial clearance.

J Immunol, 2001 Jan 15, 166(2), 982 - 8
Viral infection causes rapid sensitization to lipopolysaccharide: central role of IFN-alpha beta; Nansen A et al.; LPS is the major active agent in the pathogenesis of Gram-negative septic shock . In this report we have studied the influence of concurrent viral infection on the outcome of LPS-induced shock . We find that infection with vesicular stomatitis virus sensitizes mice to LPS at an early time point following infection . Treatment of mice with the chemical IFN inducer, polyinosinic:polycytidylic acid, has a similar effect . This hypersensitivity to LPS correlated with hyperproduction of TNF-alpha in vivo . The cellular and molecular mechanisms underlying this phenomenon were investigated using Ab-depleted and gene-targeted mice . Our results revealed that while NK cell depletion and elimination of IFN-gamma partially protected against the sensitizing effects of vesicular stomatitis virus and polyinosinic:polycytidylic acid, the most striking effect was observed in IFN-alphabetaR-deficient mice . Thus hyperproduction of TNF-alpha was completely abrogated in IFN-alphabetaR-deficient mice, indicating that the principal mechanism underlying rapid virus-induced sensitization to LPS is an IFN-alphabeta-mediated priming of mice for an augmented production of TNF-alpha in response to LPS . This conclusion was further supported by the finding that pretreatment of mice with rIFN-alphabeta mimicked the effect of viral infection . In conclusion, our results reveal a previously unrecognized proinflammatory effect of IFN-alphabeta and point to a new pathway through which viral infection may influence the outcome of concurrent bacterial infection.

Genetica, 2000, 108(1), 19 - 24
Transposome insertional mutagenesis and direct sequencing of microbial genomes; Hoffman LM et al.; Preformed transposase-transposon complexes called 'Transposomes' have been electroporated into bacterial cells . The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo . The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0 x 10(4) and 1.0 x 10(7) kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species . Transposon integration sites were examined by direct genome sequencing of chromosomal DNA . Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon . The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences . Mutant phenotypes were rapidly correlated with genomic insertions sites.

Clin, Eye Vis . Care . 2000 Dec, 12(3-4), 155 - 159
Neuroretinitis: a clinical syndrome of cat-scratch disease; Rost Monahan S; Cat-scratch disease is usually a benign self-limited illness, characterized by regional lymphadenopathy lasting between 3 and 6 weeks . The causative organism is Bartonella henselae, a small gram-negative rod . Between 1 and 2% of patients who contract the illness experience blurred vision, metamorphopsia and scotomas as a result of neuroretinitis, an associated clinical syndrome . The classical clinical findings in cat-scratch neuroretinitis include disc edema and a stellate pattern of exudates in the macula . However, a myriad of other signs has been documented, suggesting a much wider spectrum of intra-ocular disease . The following case report presents a young patient with neuroretinitis, and a history of lymphadenopathy secondary to cat-scratch disease.

Br J Surg, 2001 Jan, 88(1), 22 - 30
Experimental models of gram-negative sepsis; Parker SJ et al.; BACKGROUND: The mortality rate from sepsis has improved little over the past two decades . One reason for this has been the use of flawed or inappropriate experimental models in preclinical sepsis studies . METHODS: A literature review of animal models of sepsis was performed following a Medline search based on the following medical subject headings: disease models, endotoxin, inflammation, peritonitis and sepsis . Additional references were identified from the papers identified in the search . RESULTS AND CONCLUSION: Many animal models of sepsis have been described but none has proved to be superior . Extrapolation of results from endotoxicosis or bacterial infusion models should be regarded with caution . Peritonitis models should be accepted as the 'gold standard' but the use of appropriate virulent bacterial species needs to be ensured . A standardized panel of animal models for the preclinical assessment of immunomodulatory agents should be established, including at least one immuno- suppressed model to simulate the immunocompromised patient with sepsis . A uniform and valid definition of sepsis applicable to both small and large animal species is required.

Mol Cell Biol, 2001 Jan, 21(2), 438 - 48
Lipopolysaccharide-induced activation of beta2-integrin function in macrophages requires Irak kinase activity, p38 mitogen- activated protein kinase, and the Rap1 GTPase; Schmidt A et al.; Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, is a potent activator of macrophages . Besides inducing many transcriptional pathways, LPS also elicits rapid morphological changes such as cell spreading . Here we have investigated the signaling pathway that controls macrophage beta2-integrin-dependent spreading in response to LPS . We show that inhibition of the adapter protein MyD88, the interleukin-1 receptor-associated kinase Irak, the p38 mitogen-activated protein kinase, or the Ras-like GTPase Rap1 blocks LPS-induced spreading . In addition, Irak activates p38 and stimulates p38-dependent spreading . The activation of p38 by Irak requires Irak's kinase activity . We find that p38 controls spreading independently of its role in transcription but rather through activation of Rap1 . Together, our results suggest that beta2-integrin-dependent spreading of macrophages in response to LPS is controlled by a linear signaling pathway via MyD88, Irak, p38, and Rap1.

J Bacteriol, 2001 Jan, 183(2), 725 - 35
Temporal regulation of genes encoding the flagellar proximal rod in Caulobacter crescentus; Boyd CH et al.; The gram-negative bacterium Caulobacter crescentus has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase . The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program . The flagellar regulon is arranged into a rigid trans-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum . The flgBC-fliE genes were identified as a result of the C . crescentus genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function . Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with sigma(70) is responsible for regulating transcription of other early flagellar genes in C . crescentus . Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, and flgB deletions do not prevent class III gene expression . This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing fliP, which encodes a member of the flagellum-specific protein export apparatus.

J Bacteriol, 2001 Jan, 183(2), 557 - 69
ihfA gene of the bacterium Myxococcus xanthus and its role in activation of carotenoid genes by blue light; Moreno AJ et al.; Myxococcus xanthus responds to blue light by producing carotenoids . Several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes . We had already reported the isolation of a carotenoid-less, Tn5-induced strain (MR508), whose mutant site was unlinked to the indicated regulatory genes . Here, we show that OmegaMR508::Tn5 affects all known light-inducible promoters in different ways . It blocks the activation of two of them by light but makes the activity of a third one light independent . The OmegaMR508 locus has been cloned and sequenced . The mutation had occurred at the promoter of a gene we propose is the M . xanthus ortholog of ihfA . This encodes the alpha subunit of the histone-like integration host factor protein . An in-frame deletion within ihfA causes the same effects as the OmegaMR508::Tn5 insertion . Like other IhfA proteins, the deduced amino acid sequence of M . xanthus IhfA shows much similarity to HU, another histone-like protein . Sequence comparison data, however, and the finding that the M . xanthus gene is preceded by gene pheT, as happens in other gram-negative bacteria, strongly argue for the proposed orthology relationship . The M . xanthus ihfA gene shows some unusual features, both from structural and physiological points of view . In particular, the protein is predicted to have a unique, long acidic extension at the carboxyl terminus, and it appears to be necessary for normal cell growth and even vital for a certain wild-type strain of M . xanthus.

J Appl Physiol, 2001 Jan, 90(1), 261 - 8
PAF increases vascular permeability without increasing pulmonary arterial pressure in the rat; Clavijo LC et al.; In vivo pulmonary arterial catheterization was used to determine the mechanism by which platelet-activating factor (PAF) produces pulmonary edema in rats . PAF induces pulmonary edema by increasing pulmonary microvascular permeability (PMP) without changing the pulmonary pressure gradient . Rats were cannulated for measurement of pulmonary arterial pressure (Ppa) and mean arterial pressure . PMP was determined by using either in vivo fluorescent videomicroscopy or the ex vivo Evans blue dye technique . WEB 2086 was administered intravenously (IV) to antagonize specific PAF effects . Three experiments were performed: 1) IV PAF, 2) topical PAF, and 3) Escherichia coli bacteremia . IV PAF induced systemic hypotension with a decrease in Ppa . PMP increased after IV PAF in a dose-related manner . Topical PAF increased PMP but decreased Ppa only at high doses . Both PMP (88 +/- 5%) and Ppa (50 +/- 3%) increased during E . coli bacteremia . PAF-receptor blockade prevents changes in Ppa and PMP after both topical PAF and E . coli bacteremia . PAF, which has been shown to mediate pulmonary edema in prior studies, appears to act in the lung by primarily increasing microvascular permeability . The presence of PAF might be prerequisite for pulmonary vascular constriction during gram-negative bacteremia.

Appl Environ Microbiol, 2001 Jan, 67(1), 155 - 61
Exposure of workers to airborne microorganisms in open-air swine houses; Chang CW et al.; This study quantified the levels of airborne microorganisms in six swine farms with more than 10,000 pigs in subtropical Taiwan . We evaluated breeding, growing, and finishing stalls, which were primarily open-air buildings, as well as partially enclosed farrowing and nursery piggeries . Airborne culturable bacteria, gram-negative bacteria, and fungi were placed on appropriate media by using an all-glass impinger or single-stage Andersen microbial sampler . Results showed that mean concentrations of culturable bacteria and gram-negative bacteria were 3.3 x 10(5) and 143.7 CFU/m(3), respectively . The concentration of airborne culturable fungi was about 10(3) CFU/m(3), with Cladosporium the predominant genus . The highest airborne levels of culturable bacteria and gram-negative bacteria were identified in the finishing units . The air of the nursery stalls was the least contaminated with culturable and gram-negative bacteria . Irregular and infrequent cleaning, high pig density, no separation of wastes from pen floors, and accumulation of water as a result of the processes for cleaning and reducing pig temperature possibly compromise the benefits of the open characteristic of the finishing units with respect to airborne bacterial concentration.

Appl Environ Microbiol, 2001 Jan, 67(1), 89 - 99
Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia; Entcheva P et al.; Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes . We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia . Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes . DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb . The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms . These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB) . Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons . Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied . Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit . Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria . In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

Schweiz Med Wochenschr, 2000 Dec 2, 130(48), 1837 - 44
{Evaluation of antibiotic prophylaxis in neutropenic patients with hematologic malignancies}; Delarive P et al.; The benefits of oral prophylaxis for neutropenia have remained controversial up to now . We evaluated retrospectively the effect of antibiotic prophylaxis with ciprofloxacin and penicillin on the prevention of bacterial infections in 112 cases of prolonged neutropenia in adult patients treated for haematological malignancies . 41 patients received prophylaxis between December 1993 and November 1994 while 71 patients did not receive prophylaxis between December 1994 and November 1995 . There were no significant differences between groups in age, sex, type or stage of haemopathy, type of chemotherapy and duration of neutropenia . The antibiotic prophylaxis reduced the number of overall infections (p = 0.05) and the number of gram-negative bacteraemias (p = 0.02) . The median time to the onset of fever, the duration of fever, the duration of antibiotic treatment, the duration of hospitalization or admission to the intensive care unit, the number of serious complications or death were not influenced by antibiotic prophylaxis . The prophylaxis did not reduce the overall incidence of bacteraemia, of clinically documented infections or of fever of unknown origin . This retrospective study confirms that oral prophylaxis with ciprofloxacin and penicillin decreases the incidence of infections and, in particular, of gram-negative bacteraemia, but does not modify the overall morbidity and mortality in our patients . In view of the risk of emergence of bacterial resistance, these data do not support the routine use of oral antibiotic prophylaxis in neutropenic patients with haematological malignancies.

J Pediatr Hematol Oncol, 2000 Nov-Dec, 22(6), 552 - 7
Clinical manifestation of beta-thalassemia/hemoglobin E disease; Fucharoen S et al.; PURPOSE: To review the clinical manifestation and changes in hematologic parameters of patients with beta-thalassemia/hemoglobin (Hb) E . MATERIALS AND METHODS: Retrospective analysis of the clinical manifestation of 378 patients with beta-thalassemia/Hb E attending the hematology clinic at Siriraj Hospital between 1957 and 1982 . RESULTS: A wide spectrum of clinical phenotypes has been observed . Most patients show clinical symptoms by 10 years of age . The majority of patients survive with or without occasional transfusion . Splenectomy was performed in 26.5% of patients . Patients come to the hospital because of anemia, fever, abdominal mass, and jaundice . Gastrointestinal tract disturbances are the most common presenting symptoms (34.6%), especially abdominal pain (10%) and cholecystitis (5.1%) . Respiratory tract infections were found in 21.8% of patients and cardiovascular complications, including congestive heart failure, occurred in 11.9% . Other less common symptoms and complications included bone pain, chronic leg ulcers, paraplegia and hypertension-associated convulsions, and cerebral hemorrhage after multiple blood transfusion . Patients usually die between 20 to 40 years of age (67%), mainly from congestive heart failure and septicemia . Septicemia was often caused by Gram-negative bacteria . CONCLUSION: These clinical features observed in patients with beta-thalassemia/Hb E are probably the results of chronic anemia and iron overload . The study of the life history and clinical courses of patients with beta-thalassemia/Hb E should provide important information for the better management of these patients.

Shock, 2000 Dec, 14(6), 599 - 604
Biliary obstruction exacerbates the hepatic microvascular inflammatory response to endotoxin; Ito Y et al.; Gram-negative sepsis is a serious complication for patients with obstructive jaundice . The present study was conducted to elucidate the response of hepatic microcirculation to endotoxin 2 weeks after bile duct ligation (BDL) or sham-operation in rats . Two hours after lipopolysaccharide (LPS) injection (1, 10, or 100 microg/kg, iv.), the hepatic microvasculature was examined using in vivo microscopy . BDL elicited increases in leukocytes adhering to the sinusoidal wall, swelling of sinusoidal endothelial cells as well as phagocytic activity of hepatic macrophages and a decrease in the numbers of perfused sinusoids . LPS (1, 10, 100 microg/kg) further increased leukocyte adhesion and reduced the numbers of perfused sinusoids in a dose-dependent manner . Leukocyte adhesion in response to LPS (1, 10, 100 microg/kg) in BDL rats was increased 6.1-fold, 5.9-fold, and 3.3-fold, respectively when compared with sham-operated rats . The numbers of perfused sinusoids in response to LPS (1, 10, 100 microg/kg) in BDL rats were decreased by 42%, 36%, and 45% . While 1 and 10 microg/kg LPS also elicited an increase in phagocytic activity in BDL rats when compared with sham-operated rats, the response to 100 microg/kg LPS was suppressed . LPS did not affect the numbers of swollen endothelial cell in BDL rats . The present study demonstrated that chronic biliary obstruction enhanced the hepatic microvascular response to low doses of endotoxin . This observation suggests that exaggerated hepatic microcirculatory dysfunction during sepsis contributes to the development of liver injury and a high incidence of morbidity and mortality in biliary obstruction.

Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2059 - 67
Isolation and some properties of a mesophilic and mixotrophic iron-oxidizing bacterium, OKM-9; Inoue T et al.; An iron-oxidizing bacterium strain, OKM-9, isolated from mud obtained from the bottom of a pond, Minamikata Ohike, in Okayama prefecture, Japan, grew well in an FeSO4 x 7H2O (3%)-medium (pH 2.5) with 0.03% yeast extract . However, the strain could not grow either in an FeSO4 x 7H2O (3%)-medium without yeast extract or in a yeast extract (0.03%)-medium (pH 2.5) without Fe2+ . The strain did not use elemental sulfur as an energy source and did not have the activity to fix carbon dioxide . Strain OKM-9 could grow in an FeSO4 x 7H2O (3%)-medium with twenty different L-amino acids instead of yeast extract . Incorporation of {U-14C} glutamic acid into the cells was dependent on the energy produced by the oxidation of Fe2+ . Strain OKM-9 did not grow heterotrophically using amino acids and hexoses as a sole energy and carbon source . The results that strain OKM-9 absolutely required ferrous iron (Fe2+) as a sole energy source and yeast extract or L-amino acids as a carbon source for growth strongly suggest that the strain is a mixotrophic iron-oxidizing bacterium . Strain OKM-9 was a gram-negative and rod-shaped bacterium (0.4-0.6 x 1.6-2.2 microm) and the mean G + C content of the DNA of the bacterium was 59.6 mol% . The optimum temperature and pH for growth were 30 degrees C and 2.1, respectively . However, the strain could not grow at temperatures above 45 degrees C . Iron-oxidizing activities of strain OKM-9 measured with intact cells and the plasma membrane were 14.3 and 5.7 microl O2 uptake/mg protein/min, respectively . The pyridine ferrohemochromes prepared from the plasma membrane of this strain showed absorption peaks characteristic of alpha-bands of heme a and b, but not heme c, at 587 and 557 nm, respectively . The results suggest that the cytochromes composing an iron-oxidation system of strain OKM-9 are different from those of the well-known mesophilic iron-oxidizing bacteria Thiobacillus ferrooxidans and Leptospirillum ferrooxidans.

Eur Cytokine Netw, 2000 Dec, 11(4), 597 - 601
Differential response of a(2)-macroglobulin-deficient mice in models of lethal TNF-induced inflammation; Hochepied T et al.; Tumor necrosis factor (TNF) is an essential mediator in the pathogenesis of Gram-negative septic shock . Injection of TNF into normal mice leads to systemic, lethal inflammation, which is indistinguishable from lipopolysaccharide (LPS)-induced lethal inflammation . alpha(2)-macroglobulin (A2M) is a major positive acute phase protein with broad-spectrum protease-inhibitory activity . Mouse A2M-deficient (MAM-/-) mice were significantly protected against lethal systemic inflammation induced by TNF . The protection is not due to faster clearance of the injected TNF . The induction of tolerance to TNF-induced lethality by repetitive administration of small doses of human TNF for five consecutive days was equally efficient in both mutant mice compared to wild-type mice . In D-(+)-galactosamine (GalN)-sensitized mice, TNF induces lethal inflammatory hepatitis . MAM(-/-) mice are equally sensitive to the lethal combination of TNF/GalN . Furthermore, interleukin-1-induced desensitization to TNF/GalN was not impaired in MAM(-/-) mice . We conclude that MAM plays a mediating role in TNF-induced lethal shock and that MAM deficiency does not reduce changes in efficiency of tolerance and desensitization to TNF and TNF/GalN-induced lethality, respectively.

Mol Microbiol, 2001 Jan, 39(1), 1 - 11
Emerging strategies in microbial haem capture; Genco CA et al.; Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins . These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins . Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell . Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell . This review describes recent work on the discovery and characterization of these systems . Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded) . Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.

J Pharmacol Exp Ther, 2001 Jan, 296(1), 71 - 6
A metabolic fragment of bradykinin, Arg-Pro-Pro-Gly-Phe, protects against the deleterious effects of lipopolysaccharide in rats; Morinelli TA et al.; Extensive research has provided few therapeutic agents for the treatment of septicemia . Bradykinin, an endogenous vasodepressor hormone, is a key mediator in the hypotension seen with septicemia . The present investigation shows that a stable metabolic fragment of bradykinin, arginine-proline-proline-glycine-phenylalanine (RPPGF), prevents the deleterious effects of endotoxin {lipopolysaccharide (LPS); a component of the membrane of Gram negative bacteria}, the signaling agent responsible for the effects of septicemia, in both anesthetized rats and in isolated rat aortic segments . Survival time of rats treated with LPS (12 mg/kg) was significantly (p < 0.05) prolonged by pretreatment with RPPGF {140.3 +/- 16 min (n = 10)} compared with rats receiving saline and LPS {93.2 +/- 8 min (n = 39)} . Prolongation of survival was not seen when rats were pretreated with either bradykinin or with PRGFP (proline-arginine-glycine-phenylalanine-proline) . Isolated aortic segments treated with LPS (30 microg/ml) showed a significantly reduced ability to contract in response to phenylephrine compared with control segments not receiving LPS . Pretreatment of the segments with RPPGF significantly reversed the LPS-induced reduction in contractile response of the segments . Removal of the endothelial layer did not alter the protection provided by RPPGF . These results demonstrate the ability of a stable metabolic fragment of bradykinin, RPPGF, to protect against the deleterious effects produced by LPS . The findings presented here may provide the basis for a new developmental area for novel therapeutic agents in the treatment of septicemia.

J Immunol, 2001 Jan 1, 166(1), 574 - 81
CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression; Perera PY et al.; Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock . Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages . Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins . The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock . Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14 . In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required . Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages correlated with a marked inhibition of NF-kappa B nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappa B nuclear translocation in response to LPS . These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage.

Curr Rheumatol Rep, 2000 Aug, 2(4), 297 - 305
T-cell studies in the spondyloarthropathies; Marker-Hermann E et al.; A growing body of evidence suggests that T lymphocytes play an important role in initiating and maintaining the inflammatory process characteristic of the human leukocyte antigen (HLA)-B27-associated spondyloarthropathies . T cells seem to be involved in the primary defense reaction against arthritis-triggering gram-negative bacteria at the site of extra-articular infection, in determining the systemic cytokine pattern, in the recirculation process between gut mucosa and the joint, and in mediating secondary autoimmune joint inflammation . The factors involved in disease chronicity (namely in ankylosing spondylitis and psoriatic arthritis) are still unknown . Autoreactive T cells may contribute to this process by recognition of cross-reactive self-epitopes (ie, molecular mimicry between bacterial and self-antigens) . Autoreactive T cells may as well be inappropriately upregulated by bacterial superantigens, or by local inflammatory reactions leading to the uncovering of former cryptic self-epitopes . In this paper, we review recent studies on peripheral blood and synovial T cells in patients with reactive arthritis, enteropathic spondyloarthropathy, psoriatic arthritis, and ankylosing spondylitis.

Infect Immun, 2001 Jan, 69(1), 543 - 6
Carboxy-terminal proteolytic processing of Helicobacter pylori vacuolating toxin; Nguyen VQ et al.; The vacA gene of Helicobacter pylori strain 60190 encodes a 1, 287-amino-acid protoxin, which undergoes cleavage of a 33-amino-acid amino-terminal signal sequence and carboxy-terminal proteolytic processing to yield a mature secreted toxin . Several features of VacA suggest that it belongs to the autotransporter family of gram-negative bacterial secreted proteins . Based on matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis, we calculate that the mature toxin has a mass of 88.2+/-0.2 kDa and consists of approximately 821 amino acids.

Infect Immun, 2001 Jan, 69(1), 486 - 93
Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications; Naroeni A et al.; Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals . These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells . Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells . However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown . We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes . The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells . We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell . By in vitro reconstitution experiments, we observed a specific association between killed B . suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature . This association was observed with killed bacteria but not with live bacteria . Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.

Infect Immun, 2001 Jan, 69(1), 479 - 85
Synthesis and surface expression of CD14 by human endothelial cells; Jersmann HP et al.; Previous studies have reported that human vascular endothelial cells lack the membrane-bound lipopolysaccharide (LPS) receptor, CD14 (mCD14) . By optimizing assay conditions, including the selection of anti-CD14 monoclonal antibody, we now demonstrate that human umbilical vein endothelial cells (HUVEC) express CD14 on the cell surface . Single-passage HUVEC showed approximately 20 times less expression of CD14 than monocytes . Interestingly, there was significant loss of surface CD14 expression with increasing numbers of culture passages . Evidence for synthesis of CD14 by HUVEC was provided by the finding that L-{(35)S}methionine was incorporated into CD14 . In addition, the expression of CD14 on HUVEC was upregulated by LPS, lysophosphatidic acid, and tissue culture supplements, and this upregulation was dependent on protein synthesis . Furthermore, the results imply that mCD14 is required for LPS-induced activation of endothelial cells in the absence of serum and that it acts in concert with serum factors (soluble CD14) . Our results provide evidence that CD14 is expressed by endothelial cells and suggest that the previous inability to observe expression of this molecule has been due to culture and staining conditions . This finding has important implications for the understanding of the mechanisms by which LPS stimulates endothelial cells and the management of sepsis caused by gram-negative bacteria.

Clin Sci (Lond), 2001 Jan, 100(1), 101 - 10
Protein and energy metabolism in chronic bacterial infection: studies in melioidosis; Paton NI et al.; Chronic infection is often accompanied by a wasting process, the metabolic basis of which is not fully understood . The aims of the present study were to measure protein and energy metabolism in patients with melioidosis (a serious and antibiotic-refractory Gram-negative bacterial infection which is endemic in South-East Asia) in order to define the metabolic abnormalities that might contribute to wasting . Whole-body protein turnover was measured using the {(13)C}leucine technique, both in the fasted state and while consuming a high-energy meal . Resting energy expenditure was measured by indirect calorimetry, and total energy expenditure by the bicarbonate/urea method . Results were normalized for fat-free mass, as estimated from skinfold thickness . Protein turnover was increased in melioidosis patients compared with healthy controls during fasting (170.9 compared with 124.1 micromol x kg(-1) x h(-1); P=0.04), but the net rate of catabolism (22.2 compared with 20.5 micromol x kg(-1) x h(-1); P=0.77) and the anabolic response to feeding were similar in the two groups . Resting energy expenditure was higher in melioidosis patients compared with controls (191.4 and 157.3 kJ x kg(-1) x day(-1) respectively; P=0.04), but total energy expenditure (measured in a separate group of eight patients with melioidosis) was low (192.1 kJ x kg(-1) x day(-1)) . In conclusion, this study found no evidence of metabolic causative factors, such as accelerated net protein catabolism during fasting, a blunted anabolic response to feeding or increased daily energy expenditure, and therefore suggests that reduced energy intake is the prime cause of wasting . The observed normal response to feeding should encourage nutritional approaches to prevent wasting.

Mol Microbiol, 2000 Nov, 38(4), 805 - 16
SdiA, an Escherichia coli homologue of quorum-sensing regulators, controls the expression of virulence factors in enterohaemorrhagic Escherichia coli O157:H7; Kanamaru K et al.; The quorum-sensing system in bacteria is a well-known regulatory system that controls gene expression in a cell density-dependent manner . A transcriptional regulator (LuxR homologue), signal synthase (LuxI homologue) and autoinducer (acyl homoserine lactone) are indispensable for this system in most Gram-negative bacteria . In this study, we found that SdiA, an Escherichia coli LuxR homologue, is a negative regulator of the expression of virulence factors EspD and intimin in enterohaemorrhagic E . coli (EHEC) O157:H7 . The expression of EspD and intimin was inhibited at the RNA level upon SdiA overexpression . SdiA has a DNA-binding motif in its C-terminal part and can bind to the promoter regions of the esp and eae genes in vitro . Extracellular factors, which accumulate in culture supernatants of O157:H7 at the stationary phase of growth and inhibit EspD and intimin synthesis, bind to the N-terminal part of SdiA in vivo and in vitro . O157:H7 overproducing the N-terminal part of SdiA exhibited hypertranscription of EspD and intimin, suggesting that the overproduced N-terminal part had inhibited the activity of intact SdiA through titration of the extracellular factors . These results indicate that a quorum-sensing system including the SdiA protein controls colonization by O157:H7.

Proc Natl Acad Sci U S A, 2001 Jan 2, 98(1), 283 - 8
A secondary drug resistance mutation of TEM-1 beta-lactamase that suppresses misfolding and aggregation; Sideraki V et al.; In Gram-negative bacteria, TEM-1 beta-lactamase provides the major mechanism of plasmid-mediated beta-lactam resistance . Natural variants of TEM-1 with increased antibiotic resistance have appeared in response to the use of extended-spectrum beta-lactam antibiotics (e.g., ceftazidime) and beta-lactamase inhibitors (e.g., clavulanic acid) . Some of the variant enzymes are more efficient at catalyzing beta-lactam hydrolysis, whereas others are more resistant to inhibitors . M182T is a substitution observed in both types of variant TEM-1 beta-lactamases . This mutation is found only in combination with other amino acid substitutions, suggesting that it may correct defects introduced by other mutations that alter the specificity . An engineered core mutation, L76N, which diminishes the periplasmic beta-lactamase activity by 100-fold, was used as a model to understand the mechanism of suppression of the M182T mutation . Biochemical studies of the L76N enzyme alone and in combination with the M182T mutation indicate that the M182T substitution acts at the level of folding but does not affect the thermodynamic stability of TEM-1 beta-lactamase . Thus, the M182T substitution is an example of a naturally occurring mutation that has evolved to alter the folding pathway of a protein and confer a selective advantage during the evolution of drug resistance.

Eur J Intern Med, 2000 Dec 20, 11(6), 343 - 344
A rare manifestation of Kingella kingae infection; Reekmans A et al.; Kingella kingae is an aerobic gram-negative coccobacillus that has been associated predominantly with bone and joint infection but also with septicemia and endocarditis . Until now, only four cases of proven K . kingae meningitis have been reported . We describe a case of a K . kingae meningitis in a male adolescent who presented with a history of fever of unclear origin.

Int J Med Microbiol, 2000 Oct, 290(4-5), 301 - 5
Genomics of Bordetella pertussis toxins; Antoine R et al.; Bordetella pertussis, the etiologic agent of whooping cough, produces numerous toxins including pertussis toxin (PTX), adenylate cyclase toxin (AC), dermonecrotic toxin (DNT) and tracheal cytotoxin (TCT) . PTX is composed of five different subunits organised in a typical A-B type structure of which the A part possesses an enzymatic ADP-ribosyltransferase activity and the B moiety expresses receptor-binding activity . The secretion of this toxin requires nine other genes (ptl) organised in an operon together with the five structural genes of PTX . To further characterise the genetic locus of this major virulence factor, we analysed the ptx/ptl upstream and downstream sequences . Comparison of these regions between three species of Bordetella (B . pertussis, Bordetella parapertussis and Bordetella bronchiseptica) revealed differences in the upstream region . Analysis of two strains of B . bronchiseptica naturally lacking the ptx genes showed that only the ptx/ptl genes were deleted in these strains, and that the upstream and downstream regions were conserved . Upstream of the PTX structural genes and the promoter, an open reading frame (bugT) was identified, the product of which is homologous with putative proteins from several other Gram-negative organisms . Detailed analysis of the genome of B . pertussis which is currently sequenced at the Sanger Centre revealed the presence of 90 genes coding for proteins homologous to BugT, which qualifies the bug gene family as the most populated one of Bordetella . These bug genes are located in various genetic environments, including the proximities of genes coding for other toxins, such as DNT and AC . The Bug proteins are highly conserved in terms of size and periodicity of predicted secondary structure elements, but have also a high variability in their amino acid composition reflected in their wide range of isoelectric points . The function of these genes which is currently unknown is under investigation . To characterise the expression and regulation of these genes, as well as of novel putative B . pertussis virulence factors, we designed a transcriptional fusion vector to be inserted in precise locations of the B . pertussis chromosome by homologous recombination . The reporter gene present in this vector allowed us to show that at least some of the bug genes are expressed.

J Air Waste Manag Assoc, 2000 Nov, 50(11), 1957 - 67
Investigation of the concentration of bacteria and their cell envelope components in indoor air in two elementary schools; Liu LJ et al.; Bacterial cell envelope components are widely distributed in airborne dust, where they act as inflammatory agents causing respiratory symptoms . Measurements of these agents and other environmental factors are assessed in two elementary schools in a southeastern city in the United States . Muramic acid (MA) was used as a marker for bacterial peptidoglycan (PG), and 3-hydroxy fatty acids (3-OH FAs) were used as markers for Gram-negative bacterial lipopolysaccharide (LPS) . Culturable bacteria were collected using an Andersen sampler with three different culture media . In addition, temperature (T), relative humidity (RH), and CO2 were continuously monitored . Concentrations of airborne MA and 3-OH FAs were correlated with total suspended particulate (TSP) levels . Outdoor MA (mean = 0.78-1.15 ng/m3) and 3-OH FA levels (mean = 2.19-2.18 ng/m3) were similar at the two schools . Indoor concentrations of airborne MA and 3-OH FAs differed significantly between schools (MA: 1.44 vs . 2.84 ng/m3; 3-OH FAs: 2.96 vs . 4.57 ng/m3) . Although indoor MA levels were low, they were significantly related to teachers' perception of the severity of indoor air quality (IAQ) problems in their classrooms . Concentrations of CO2 correlated significantly with all bacteria measurements . Because CO2 levels were related to the number of occupants and the ventilation rates, these findings are consistent with the hypothesis that the children and teachers are sources of bacterial contamination . Many culturable bacteria present in indoor air are opportunistic organisms that can be infectious for compromised individuals, while both culturable and nonculturable bacterial remnants act as environmental toxins for both healthy and compromised individuals . Measuring the "total bacteria load" would be most accurate in assessing the biotoxicity of indoor air . Chemical analysis of MA and 3-OH FAs, when coupled with the conventional culture method, provides complementary information for assessing biocontamination of indoor air.

Int Microbiol, 1999 Mar, 2(1), 29 - 31
Construction of plasmid vectors bearing a NotI-expression cassette based on the lac promoter; Jaenecke S et al.; We have constructed two plasmid vectors for cloning and expression of DNA fragments controlled by the lac promoter as a NotI-expression cassette . Whereas plasmid pSJ33 allows mobilization of the expression cassette into a wide variety of Gram-negative bacteria by RP4-mediated conjugation, the low-copy-number plasmid pSJP18Not facilitates cloning and expression in Escherichia coli when high gene dosage may be detrimental . In addition to their suitable cloning features (e.g . multiple cloning site, lacZ alpha fragment, compatible with ColE1-derived vectors), these plasmids are particularly useful as auxiliary vectors for cloning of the expression cassettes at the NotI site of mini-transposon elements {1, 2} and their eventual stable insertion into the host chromosome.

Int Microbiol, 1998 Mar, 1(1), 19 - 26
Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis; Moriyon I et al.; The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance . This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B . melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes . On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids . This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.

J Electron Microsc (Tokyo), 2000, 49(3), 459 - 62
Deformation of the envelope of a spherical gram-negative bacterium during the atomic force microscopic measurements; Boulbitch A; A theoretical approach for the description of the deformation of the envelope of a spherical Gram-negative bacteria is presented . It is shown that the force-displacement relation taken on top of bacteria is accurately approximated by a linear dependence . The bacterial rigidity is shown to be controlled mainly by its turgor pressure, while the lateral rigidity of the bacterial wall determines the distance from the tip at which the displacement vanishes.

ALTEX, 1999, 16(3), 146 - 149
New Applications of the Human Whole Blood Pyrogen Assay (PyroCheck); Fennrich S et al.; The absence of pyrogens in injectable drugs is an indispensable safety control because contaminants causing fever pose a life-threatening risk to the patient resulting in the worst case in death by shock . When fever- inducing agents, i.e.pyrogens, come into contact with the immunocompetent cells in blood, these cells release mediators which transmit the fever signal to the thermoregulatory centre of the brain . The Phamocopoeia lists currently two test systems for pyrogenicity: 1 . The in vivo rabbit pyrogen test which measures the fever reaction following injection of the sample to the animals . 2 . The in vitro Limulus Amebocyte Lysate assay (LAL) which measures the coagulation in a lysate prepared from the blood of the horseshoe crab specifically initiated by endotoxins, i.e . cell wall components from Gram-negative bacteria . The new test presented here (PyroCheck) exploits the reaction of monocytes/macrophages for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample in any form, be it a solution, a powder or even solid material . Pyrogenic contaminations initiate the release of the "endogenous pyrogen" Interleukin-1beta determined by ELISA after a fixed incubation time . The technology presently listed in the Pharmacopoeia is limited to parenteralia (rabbit test: biologicals and pharmaceuticals, LAL: predominantly pharmaceuticals) . In the EU Medical Devices Directive from 1995 the rabbit pyrogen test for medical products is in some cases requested . (in some cases LAL of an eluate from the device) . However, pyrogen-testing needs to cover also innovative high-tech products such as medical devices (implants, medical plastic materials, dialysis machines), cellular therapies and species-specific agents (e.g . recombinant proteins) . Here we report that the human blood test PyroCheck is suitable for testing filters in air quality control as well as for assessing medical devices and biocompatibility (dialysate fluid), i.e . that it can be extended to a wide spectrum of applications.

Am J Respir Cell Mol Biol, 2000 Dec, 23(6), 780 - 7
CD14(+) cells are necessary for increased survival of eosinophils in response to lipopolysaccharide; Meerschaert J et al.; There has been considerable interest in the effect that gram-negative bacterial endotoxin (lipopolysaccharide {LPS}) can have in asthma, given that inhalation of LPS has been shown to cause bronchial hyperresponsiveness . Further, there is evidence that the endotoxin-binding protein CD14 may be a marker for asthma . Inhaled LPS has been shown to cause an influx of eosinophils into the nasal airway and to increase the survival of CD16-negatively selected eosinophils in vitro . In this study, we compared survival of eosinophils isolated via CD16-negative selection with eosinophils that were isolated using both CD16- and CD14-negative selection criteria . Survival of CD16-negatively selected eosinophils was enhanced by LPS in a dose-dependent manner and was inhibited by the endotoxin antagonists polymyxin B or lipid X . In contrast, depletion of CD14(+) cells within the eosinophil preparations (CD14/CD16-negatively selected eosinophils) decreased the effect of LPS on survival . Preincubation of CD16-negatively selected eosinophils with antibody 60bd, which blocks LPS binding to CD14, prevented the survival-enhancing effect of LPS . However, CD14 was not detected on eosinophils by flow cytometry, even after incubation with LPS for up to 24 h . These results suggest that the survival-enhancing effect of LPS on eosinophils requires the presence of CD14(+) cells in the population . It is our hypothesis that enhanced eosinophil survival with LPS involves the contribution of another cell type.

J Surg Res, 2000 Dec, 94(2), 159 - 66
Lipopolysaccharide-binding protein accelerates and augments Escherichia coli phagocytosis by alveolar macrophages; Klein RD et al.; BACKGROUND: The first step in bacterial clearance by leukocytes is attachment and phagocytosis . Although lipopolysaccharide-binding protein (LBP) is best known for potentiating LPS-induced cytokine production through a CD14-dependent pathway, recent studies suggest that LBP plays a critical role in clearance of gram-negative bacteria and is essential for survival after bacterial challenge . We therefore sought to examine LBP's effect on Escherichia coli phagocytosis by alveolar macrophages (AMs) and to determine if this effect is mediated through CD14 . MATERIALS AND METHODS: Phosphatidylinositol-specific phospholipase C (PIPLC)-treated and untreated rat AMs were incubated in the presence of increasing doses of recombinant LBP or negative control protein (choramphenicol acetyltransferase) prior to E . coli-FITC (Ec-F) BioParticle challenge . Phagocytosed bacteria were assayed by fluorescence measurement . A time course study was also performed . RESULTS: LBP potentiated phagocytosis of Ec-F BioParticles by AMs in a dose-dependent fashion . Kinetic studies showed that LBP augmented Ec-F phagocytosis by 76% at 30 min . Treatment of AMs with PIPLC to remove CD14 resulted in only a partial decrease in LBP-mediated enhancement of phagocytosis . CONCLUSION: These results clearly demonstrate that LBP plays an important role in enhancing Ec-F binding and phagocytosis in a time- and dose-dependent manner . This observed increase may not require the presence of CD14 as significant potentiation of phagocytosis still occurred after PIPLC treatment . We postulate that the LBP-mediated increase in Ec-F phagocytosis can occur in the absence of CD14 through the presence of another receptor .

Braz J Infect Dis, 1997 Aug, 1(4), 182 - 185
Central Venous Catheter-Related Infections in Intensive Care Units; Arruda E et al.; Objective: To determine the rates of colonization and infection related to central venous catheter (CVC), the causative microorganisms, and the influence of various factors . Methods: From June to August 1993, all CVC in 4 Intensive Care Units were evaluated from their insertion to removaL Data were collected by 3 nurses . Blood and catheter tips were cultured . Results: Of 84 catheters, 29.8% were colonized, 9.5% of patients showed evidence of local infection, and 4.8% had primary bloodstream infections . The internal jugular vein was the most common site for catheter insertion (81%) . Causes of removal were: end of need (48.3%), suspected infection (23.3%), malfunction (20%), routine change (8.3%) . Among removals because of suspected infection, 50% presented evidence of local infection, 43% were colonized(>15cfu), but there were no bloodstream infections . The average time of catheter use for those which became colonized was longer than for catheters that did not become colonized (p=0.008) . The average time of catheter use associated with removal for infection (local and bloodstream) was longer than for removal for other reasons (p=0.042) . Among colonized catheters, 16% developed bloodstream infection and 20% local infection . Immunosupressive drugs, cancer, diabetes mellitus, HIV-infection, and neutropenia were not associated with infection or colonization . The most common microorganisms were gram-negative rods and S.aureus . Conclusions: The duration of venous catheter use increased the risk of colonization and infection . This observation suggests that physicians must strive for the shortest time of use of venous catheters, but it does not indicate a need for routine central venous catheter removal.

Curr Opin Chem Biol, 2000 Dec, 4(6), 653 - 60
Bacterial adhesins: structural studies reveal chaperone function and pilus biogenesis; Knight SD et al.; During the past year, remarkable progress has been made in understanding how periplasmic chaperones fold and protect protein modules that are destined for assembly into adhesive pili in Gram-negative bacteria . The first two three-dimensional structures of complexes of periplasmic chaperones with substrate pilus subunits have revealed much about the structural basis for chaperone-mediated folding and aggregation prevention, and have provided insight into the structure of adhesive pili.

FEMS Microbiol Ecol, 2000 Dec 1, 34(2), 129 - 137
Isolation of new fastidious alpha Proteobacteria and Afipia felis from hospital water supplies by direct plating and amoebal co-culture procedures; La Scola B et al.; As water is a source of nosocomial infections in hospitals, the presence of fastidious Gram-negative bacteria in water samples taken in a university hospital was investigated . Water samples were inoculated onto agar plates and into amoebal microplates for co-culture . Sixty-eight alpha proteobacteria isolates were obtained and characterized using phenotypic methods and 16S rRNA gene sequence comparison . The latter approach divided the strains into seven clusters . Of these, one corresponded to previously recognized Afipia felis and it is likely that six were closely related new species . As these bacteria are fastidious and can not be cultivated on standard microbiological media, their possible role in hospital-acquired human infections should be investigated.

Microbiology, 2000 Dec, 146 Pt 12, 3251 - 7
A functional water channel protein in the pathogenic bacterium Brucella abortus; Rodriguez MC et al.; The gene for a new bacterial aquaporin, AqpX, was cloned from the pathogenic Gram-negative bacterium BRUCELLA: abortus . The gene was mapped on the large chromosome of B . abortus . It is flanked by one upstream and two downstream copies of the BRUCELLA: repeated sequence Bru-RS . Prediction from the nucleotide sequence indicated that the protein is a member of the MIP family, which comprises channels for water and/or solute transport . Expression in XENOPUS: oocytes and cryoelectron microscopy of Escherichia coli cells transformed with the aqpX gene confirmed that the protein is an efficient water channel . Glycerol uptake experiments in E . coli also showed that the protein is not able to transport glycerol.

J Am Soc Nephrol, 2000 Dec, 11(12), 2199 - 211
Basic fibroblast growth factor selectively enhances TNF-alpha-induced apoptotic cell death in glomerular endothelial cells: effects on apoptotic signaling pathways; Messmer UK et al.; Endothelial cell damage of glomeruli and kidney arterioles seems to play a pivotal role in several pathologic situations, such as Gram-negative sepsis, glomerulonephritis, and acute renal failure . Bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) have been identified as potent inducers of apoptotic cell death in bovine glomerular endothelial cells . Both agents elicited apoptotic DNA laddering within 12 to 24 h . Basic fibroblast growth factor (bFGF) was generally described as a protective factor for endothelial cells against radiation-, TNF-alpha-, and UV-light-induced programmed cell death . Therefore, whether bFGF also affects apoptosis of microvascular endothelial cells was questioned . Surprising was that simultaneous treatment of glomerular endothelial cells with bFGF and either LPS or TNF-alpha left LPS-induced death unaffected, whereas TNF-alpha-induced death induction was potentiated, amounting to 48.9+/-6.3% versus 22.4+/-4.3% DNA degradation with TNF-alpha alone . Comparably, acidic FGF also selectively potentiated TNF-alpha-induced apoptosis . In mechanistic terms, bFGF synergistically increased TNF-alpha-induced mitochondrial permeability transition, the release of cytochrome c from mitochondria to the cytosol, and upregulation of the proapoptotic protein Bak and significantly enhanced activation of caspase-8 protease activity . In contrast, stress-activated protein kinase and nuclear factor kappaB activation, which represent primary signals of TNF/TNF receptor interaction, downregulation of the antiapoptotic protein Bcl-x(L), and caspase-3-like protease activation, were unaffected . As bFGF did not affect LPS-induced apoptotic cell death, bFGF also left LPS-induced Bak upregulation and Bcl-x(L) downregulation unaffected . The results point to a selective bFGF-mediated enhancement of distinct proapoptotic pathways induced by TNF-alpha in glomerular endothelial cells.

FEMS Microbiol Lett, 2000 Dec 1, 193(1), 25 - 30
The Bvg accessory factor (Baf) enhances pertussis toxin expression in Escherichia coli and is essential for Bordetella pertussis viability; Wood GE et al.; Pertussis toxin expression in the Gram-negative respiratory pathogen, Bordetella pertussis, is regulated by the BvgAS two-component system . Previous studies suggested that an additional gene encoding a Bvg accessory factor (Baf) was required, along with BvgAS, for expression of a ptx-lacZ fusion in Escherichia coli grown in rich medium . However, other studies showed that BvgAS is sufficient for ptx-lacZ expression in minimal medium . Here we show that Baf acts with BvgAS to further increase ptx-lacZ expression in E . coli grown in minimal media and this is concomitant with a two-fold increase in BvgA protein levels . Gene replacement experiments show that baf is essential for viability of B . pertussis, suggesting that Baf affects the expression of other genes in addition to ptx.

J Biol Chem, 2001 Feb 23, 276(8), 5577 - 83 Epub 2000 Nov 28.
Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T; Kneidinger B et al.; The glycan repeats of the surface layer glycoprotein of Aneurinibacillus thermoaerophilus L420-91T contain d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose, both of which are also constituents of lipopolysaccharides of Gram-negative plant and human pathogenic bacteria . The two genes required for biosynthesis of the nucleotide-activated precursor GDP-d-rhamnose, gmd and rmd, were cloned, sequenced, and overexpressed in Escherichia coli . The corresponding enzymes Gmd and Rmd were purified to homogeneity, and functional studies were performed . GDP-d-mannose dehydratase (Gmd) converted GDP-d-mannose to GDP-6-deoxy-d-lyxo-4-hexulose, with NADP+ as cofactor . The reductase Rmd catalyzed the second step in the pathway, namely the reduction of the keto-intermediate to the final product GDP-d-rhamnose using both NADH and NADPH as hydride donor . The elution behavior of the intermediate and end product was analyzed by high performance liquid chromatography . Nuclear magnetic resonance spectroscopy was used to identify the structure of the final product of the reaction sequence as GDP-alpha-d-rhamnose . This is the first characterization of a GDP-6-deoxy-d-lyxo-4-hexulose reductase . In addition, Gmd has been shown to be a bifunctional enzyme with both dehydratase and reductase activities . So far, no enzyme catalyzing these two types of reactions has been identified . Both Gmd and Rmd are members of the SDR (short chain dehydrogenase/reductase) protein family.

Curr Infect Dis Rep, 2000 Apr, 2(2), 168 - 173
Lemierre's Syndrome; Armstrong AW et al.; Lemierre's syndrome is characterized by an oropharyngeal infection followed by internal jugular vein septic thrombophlebitis and metastatic emboli, most often to the lungs and joints . The syndrome is most commonly associated with the anaerobic gram-negative rod Fusobacterium necrophorum . Diagnosis is established with evidence of metastatic infection and internal jugular vein thrombophlebitis . CT is considered the diagnostic procedure of choice . Treatment should include an extended course of a beta-lactamase-resistant antibiotic and surgical drainage of any purulent fluid collection . Anticoagulation remains controversial, and ligation of the internal jugular vein is reserved for patients with persistent sepsis and recurrent emboli . With appropriate therapy, mortality is 4% to 12%; but mortality is increased when therapy is delayed.

Curr Infect Dis Rep, 2000 Apr, 2(2), 141 - 146
Recent Advances in Diagnosis and Treatment of Cat Scratch Disease; Margileth AM; The cause of cat scratch disease (CSD), first described in France in 1950 and in the United States in 1951, was unknown until 1983 when the bacterium in lymph nodes was detected using a Warthin-Starry silver stain . Afipia felis has been an infrequent cause of CSD since1988, when this gram-negative bacterium was first isolated from 10 patients with CSD . In 1992 Bartonella organisms were isolated from immunocompetent and immunocompromised patients . An indirect fluorescent antibody test to detect bartonella-specific serum immunoglobulins was developed in 1992 . Since then multiple studies have shown that three Bartonella species may produce either CSD in humans, usually Bartonella henselae or Bartonella clarridgeiae, or bacteremia in healthy cats . Also, these two bacteria and Bartonella quintana cause bacillary angiomatosis, bacillary peliosis, or relapsing bacteremia in humans . Cats are healthy carriers of Bartonella organisms and may be bacteremic for months to years . Cat-to-cat transmission of Bartonella organisms involves the cat flea in absence of direct contact transmission . CSD is the most common cause of regional lymphadenitis in children and adolescents . Present knowledge on the etiology, clinical features, epidemiology, pathogenesis, diagnosis, and management of CSD are presented . Also, brief comments about the etiology, clinical presentation, and treatment of bacillary angiomatosis and bacillary peliosis are provided.

Toxicology, 2000 Nov 2, 152(1-3), 37 - 45
Induction of proliferation and cytokine production in human T lymphocytes by lipopolysaccharide (LPS); Ulmer AJ et al.; Lipopolysaccharide (LPS), also known as endotoxin, is a compound of the cell wall of Gram-negative bacteria, which has been demonstrated to induce inflammatory reactions in vitro as well as in vivo, including lethal shock . A great number of different cells have been documented to be reactive to LPS, e.g . monocytes/macrophages, vascular cells, polymorphonuclear cells, and even B lymphocytes . We have now established that T lymphocytes could also contribute to an inflammatory reaction to LPS . LPS is a potent inducer of human T-lymphocyte proliferation and cytokine production . The activation of T lymphocytes by LPS requires direct cell-to-cell contact with viable accessory monocytes . This interaction was found to be MHC-unrestricted, but strongly dependent on costimulatory signals provided by B7/CD28 interactions . The frequency of responding T lymphocytes is less than 1:1000 . A very exciting finding was that not only monocytes, but also CD34+ hematopoietic stem cells, which circulate in peripheral blood in very low frequency, exert essential accessory cell activity during stimulation of T lymphocytes by LPS . In contrast, the response of T lymphocytes to conventional recall antigens is not controlled by blood stem cells . These conclusions are based on the observation that depletion of CD34-positive blood stem cells resulted in a complete loss of LPS-induced T-lymphocyte stimulation . Addition of CD34-enriched blood stem cells led to a recovery of reactivity of T lymphocyte to LPS . The characteristics of T-lymphocyte activation indicate that LPS is neither active as a mitogen, or as a superantigen, or as a classical antigen, but may activate T lymphocyte through a new, so far undescribed, mechanism . Furthermore, the involvement of hematopoietic blood stem cells in the activation of T lymphocytes by LPS demonstrates a role of these cells in inflammatory and immunological events.

Intensive Care Med, 2000 Sep, 26(9), 1248 - 51
Bactericidal/permeability-increasing protein (BPI) in sepsis correlates with the severity of sepsis and the outcome; Rintala E et al.; OBJECTIVE: To compare the prognostic significance of bactericidal/permeability-increasing protein (BPI), group II phospholipase A2 (PLA2-II), C-reactive protein (CRP), tumour necrosis factor-alpha (TNF), interleukin-8 (IL-8) and interferon-gamma (IFN) in terms of predicting severity of sepsis and outcome . DESIGN: A prospective study . SETTING: Medical intensive care unit (ICU) of a university hospital . PATIENTS: Thirty-four patients with severe sepsis requiring ICU treatment . MEASUREMENTS AND RESULTS: The levels of BPI, PLA2-II, CRP, TNF, IL-8 and IFN were measured in these 34 patients . High levels of BPI were associated particularly with Gram-negative sepsis . BPI and BPI/neutrophil ratios correlated positively with PLA2-II, CRP, TNF and IL-8 and negatively with blood pressure . At 24 h, BPI/neutrophil ratios, IL-8 and Simplified Acute Physiology Scores II (SAPS II) scores were higher in non-survivors than in survivors . No such associations were noted in the levels of CRP, PLA2-II, TNF or IFN . The areas under the curve (AUC(ROC)s) of SAPS II scores and IL-8 were higher than AUC(ROC) of BPI/neutrophil ratio . CONCLUSION: The BPI and BPI/neutrophil ratios may serve as adjunctive tools to illustrate the severity of sepsis . However, their predictive power for sepsis-related death was not comparable to that of SAPS II scores and IL-8.

J Biol Chem, 2001 Mar 16, 276(11), 8111 - 7 Epub 2000 Nov 21.
Conserved residues Ser(16) and His(20) and their relative positioning are essential for TonB activity, cross-linking of TonB with ExbB, and the ability of TonB to respond to proton motive force; Larsen RA et al.; The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria . The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane protein ExbB are essential to this process . The TonB signal anchor is predicted to form an alpha-helix, with a conserved face comprised of residues Ser(16), His(20), Leu(27), and Ser(31) . Deletion of either Ser(16) or His(20) or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation . In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished . Replacement of residues 17-19 by three consecutive alanines produced a wild type TonB allele, indicating that the intervening residues (Val, Cys, and Ile) contributed only to spacing . These data indicated that the spatial relationship of Ser(16) to His(20) was essential to function and suggested that the motif HXXXS defines the minimal requirement for the coupling of TonB to the cytoplasmic membrane electrochemical gradient . Deletion of Trp(11) resulted in a TonB that remained active yet was unable to cross-link with ExbB . Because Trp(11) was demonstrably not involved in the actual cross-linking, these results suggest that the TonB/ExbB interaction detected by cross-linking occurred at a step in the energy transduction cycle distinct from the coupling of TonB to the electrochemical gradient.

Microb Comp Genomics, 2000, 5(2), 103 - 20
A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium; Inouye S et al.; Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation . Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features . All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases . The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues . These regulatory domains share no significant sequence similarities . The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases . From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases . Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases . Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases . We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.

Cytometry, 2000 Dec 1, 41(4), 316 - 20
Lipopolysaccharide (LPS) labeled with Alexa 488 hydrazide as a novel probe for LPS binding studies; Triantafilou K et al.; BACKGROUND: Lipopolysaccharide (LPS) comprises the outer cell wall of all gram-negative bacteria . It consists of an oligosaccharide core and lipid A . All LPS-induced biological responses are lipid A-dependent . Once released, LPS triggers a host systemic inflammatory response that leads to septic shock . Binding studies have helped to reveal some of the molecular interactions behind septic shock . Such studies have employed methods of labeling bacterial LPS with either radiochemicals or fluorescent dyes . Poor labeling of the LPS has resulted in the use of high concentrations of LPS in order to detect its binding . METHODS: In this study, we have devised a new methodology for labeling LPS, using hydrazide and galactose oxidase in order to oxidize galactose residues to aldehyde groups in the oligosaccharide core of the LPS . RESULTS: We have managed to generate a conjugate that is highly fluorescent (LPS-to-Alexa 488 labeling ratio of 1:5) and biologically active . CONCLUSIONS: For the first time, this probe has enabled us to detect LPS binding even at pg/ml concentrations . Using this methodology, any Alexa-hydrazide dye can be conjugated to LPS, providing us with novel probes for imaging studies .

Infect Immun, 2000 Dec, 68(12), 6720 - 8
Multiple roles for Bordetella lipopolysaccharide molecules during respiratory tract infection; Harvill ET et al.; Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors . Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide (B . pertussis), a trisaccharide plus an O-antigen-like repeat (B . bronchiseptica), or an altered trisaccharide plus an O-antigen-like repeat (B . parapertussis) . Deletion of the wlb locus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligosaccharide . We have used wlb deletion (Deltawlb) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae . Each mutant was defective compared to its parent strain in colonization of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed significantly . Although the Deltawlb mutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5(-/-) mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufficient to explain the in vivo defects . B . pertussis and B . parapertussis Deltawlb mutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity . Interestingly, the B . bronchiseptica Deltawlb strain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell- and T-cell-deficient mice, suggesting that wlb-dependent LPS modifications in B . bronchiseptica modulate interactions with adaptive immunity . These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice . In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria.

Res Microbiol, 2000 Oct, 151(8), 655 - 68
Genetic organisation of the lipopolysaccharide O-antigen biosynthesis region of brucella melitensis 16M (wbk); Godfroid F et al.; Brucella spp . are Gram-negative, facultative intracellular bacteria that cause a zoonotic world-wide disease . As in other Gram-negative bacteria, its S-LPS (smooth lipopolysaccharide) is a major determinant of virulence . The Brucella melitensis 16M LPS O-antigen is a homopolymer of 4-formamido-4,6, dideoxymannose . In this study, the previously cloned 14-kb wbk gene cluster was sequenced, and seven open reading frames (ORFs) as well as four insertion sequences were identified . Six of the seven ORFs are homologous to LPS biosynthesis genes from other organisms . The gmd, per and wbkC gene products are predicted to be involved in 4-formamido-4,6,dideoxymannose synthesis . By deletion experiments, we demonstrated that the putative formyltransferase WbkC is absolutely required for the O-side-chain production . The wbkA gene product is similar to several mannosyltransferases and is probably involved in the polymerisation of the B . melitensis O-side-chain . We also identified two genes (wzm and wzt) encoding proteins with high similarity to several two-component ABC (ATP-binding cassette) transporters . Their implication in O-antigen translocation across the inner membrane was confirmed by gene replacement . Finally, no function has been assigned to the wbkB gene either by homology search or functionally, because deletion of wbkB did not interfere with the O-antigen structure . The seven ORFs have a low G + C content, indicating that they might have been acquired by lateral transfer from a progenitor with more A + T rich DNA.

Biochim Biophys Acta, 2000 Oct 31, 1488(1-2), 91 - 101
Bacterial phospholipase A: structure and function of an integral membrane phospholipase; Snijder HJ et al.; Within the large family of lipolytic enzymes, phospholipases constitute a very diverse subgroup with physiological functions such as digestion and signal transduction . Most phospholipases may associate with membranes at the lipid-water interface . However, in many Gram-negative bacteria, a phospholipase is present which is located integrally in the bacterial outer membrane . This phospholipase (outer membrane phospholipase A or OMPLA) is involved in transport across the bacterial outer membrane and has been implicated in bacterial virulence . OMPLA is calcium dependent and its activity is strictly regulated by reversible dimerisation . Recently the crystal structure of this integral membrane enzyme has been elucidated . In this review, we summarise the implications of these structural data for the understanding of the function and regulation of OMPLA, and discuss a mechanism for phospholipase dependent colicin release in Escherichia coli.

Comp Biochem Physiol B Biochem Mol Biol, 2000 Oct, 127(2), 243 - 50
Chemical characterization of the lectin from the freshwater prawn Macrobrachium rosenbergii (De Man) by MALDI-TOF; Zenteno R et al.; The serum of the freshwater prawn contains a sialic acid specific lectin (MrL) that agglutinates erythrocytes from rat and rabbit, as well as some Gram negative and positive bacterial strains . In this work, we performed the chemical characterization of the MrL purified by affinity chromatography on stroma from rat erythrocytes and by ion exchange chromatography . In its active form, MRL is a dimeric glycoprotein with 9.5 kDa per subunit . The amino acid sequence of the lectin was deduced from peptides obtained after trypsin treatment by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis (MALDI-TOF) . The predicted amino acid sequence of the lectin showed 54% homology with the hyperglycemic hormone from Macrobrachium rosenbergii . It also showed homology with the variable region of the human immunoglobulin kappa (22%) and lambda (27%) light chains . The lectin is a glycoprotein with 11% (w/w) carbohydrate content and is constituted by Gal, Man, GlcNAc, GalNAc and NeuAc in a molar ratio of 4:3:2:1:0.6 . The primary structure of the carbohydrate chains of the lectin from the freshwater prawn was determined by affinity chromatography of MrL-glycopeptides on Con A and LCA lectin columns, which indicated that the main carbohydrate chains conforming the lectin are N-glycosidically linked . Man3 GlcNAc2.1 oligosaccharides were the most abundant structures with 57%) followed by Gal1.3 Man3 GlcNAc2.8 with 24% . Our results suggest that the freshwater prawn possess a lectin in the hemolymph plasma, related to those from the immunoglobulin superfamily.

Dtsch Med Wochenschr, 2000 Oct 6, 125(40), 1186 - 91
{Adjuvant antibiotic therapy as a 2-step treatment concept in early-onset periodontitis: a strategy for eradication of bacteria that cause periodontitis}; Sigusch B et al.; BACKGROUND AND OBJECTIVE: The periodontal region is a source of gram-negative bacterial infection . The pathogens involved have recently also been demonstrated in atheromatous plaques . They may increase the risk of myocardial infarction . In this study a strategy for eradicating periodontal bacteria and thus healing in patients with periodontal pockets and advanced destruction of alveolar bone was examined . PATIENTS AND METHODS: Initial periodontal status was documented in 36 patients with periodontitis (24 women, 12 men) who were then randomly assigned to one of three groups . Those in two of the groups were given either metronidazole or doxycycline orally as adjuvant treatment, while the third group received no antibiotics . Patients in all groups were treated according to a two-step procedure . In step 1, extensive supra- and subgingival plaques and concrements were removed . In step 2, root debridement and/or closed curettage of all pockets was undertaken in one visit, and the antibiotic given to the patients in groups 1 and 2 . Results were assessed 3 weeks after the first step and 6 months, 2 and 4 years after step 2 . RESULTS: The initial examination at 3 weeks revealed significant changes in all three groups with regard to the incidence of plaque and sulcus bleeding, but not regarding probing depth and attachment level . However, there were significant changes in probing depth and attachment level in all groups after step 2 . Periodontal attachment was significantly improved at 2 and 4 years in the patients on metronidazole, but not those on doxycycline or no antibiotics . The greatest decrease in bleeding tendency was recorded in the metronidazole group . This group also had the greatest gain of new alveolar bone compared with the other two groups . Complete eradication of Porphyromonas gingivalis and Actinobacillus (Hemophilus) actinomycetemcomitans, important pathogenic bacteria that may have an atherogenic action, was obtained only in patients on metronidazole . CONCLUSION: The tissue-sparing two-step procedure brought about good clinical and radiological results, which can be significantly bettered by the addition of metronidazole, achieving eradication of pathogens involved in periodontal disease.

J Bacteriol, 2000 Dec, 182(23), 6614 - 21
Developmental aggregation of Myxococcus xanthus requires frgA, an frz-related gene; Cho K et al.; Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle that includes multicellular fruiting body formation . Frizzy mutants are characterized by the formation of tangled filaments instead of hemispherical fruiting bodies on fruiting agar . Mutations in the frz genes have been shown to cause defects in directed motility, which is essential for both vegetative swarming and fruiting body formation . In this paper, we report the discovery of a new gene, called frgA (for frz-related gene), which confers a subset of the frizzy phenotype when mutated . The frgA null mutant showed reduced swarming and the formation of frizzy aggregates on fruiting agar . However, this mutant still displayed directed motility in a spatial chemotaxis assay, whereas the majority of frz mutants fail to show directed movements in this assay . Furthermore, the frizzy phenotype of the frgA mutant could be complemented extracellularly by wild-type cells or strains carrying non-frz mutations . The phenotype of the frgA mutant is similar to that of the abcA mutant and suggests that both of these mutants could be defective in the production or export of extracellular signals required for fruiting body formation rather than in the sensing of such extracellular signals . The frgA gene encodes a large protein of 883 amino acids which lacks homologues in the databases . The frgA gene is part of an operon which includes two additional genes, frgB and frgC . The frgB gene encodes a putative histidine protein kinase, and the frgC gene encodes a putative response regulator . The frgB and frgC null mutants, however, formed wild-type fruiting bodies.

J Leukoc Biol, 2000 Nov, 68(5), 723 - 8
Morphine enhances interleukin-12 and the production of other pro-inflammatory cytokines in mouse peritoneal macrophages; Peng X et al.; In this study we investigated the capacity of morphine to modulate expression of cytokines in peritoneal macrophages . Mice were implanted subcutaneously with a 75-mg morphine slow-release pellet, and 48 h later resident peritoneal macrophages were harvested . Control groups received placebo pellets, naltrexone pellets, or morphine plus naltrexone pellets . Adherent cells were stimulated with lipopolysaccharide (LPS: 10 microg/mL) plus interferon-gamma (IFN-gamma: 100 units/mL) to induce cytokine production . After 24 h RNA was extracted for analysis of cytokine mRNA levels by reverse transcriptase-polymerase chain reaction, or supernatants were collected after 48 h for determination of cytokine production by enzyme-linked immunosorbent assay (ELISA) . Morphine enhanced mRNA expression of interleukin (IL)-12 p40 and tumor necrosis factor alpha (TNF-alpha) compared with controls, whereas IL-10 levels were unchanged by drug treatment . ELISA data showed that both IL-12 p40 and p70 were increased by morphine . The enhancement of IL-12 at both the mRNA and protein levels was antagonized by naltrexone, indicating that the modulation of this cytokine by morphine is via a classic opioid receptor . These results are particularly interesting in light of our previous observation that 48 h after morphine pellet implantation, the peritoneal cavity is colonized with gram-negative and other enteric bacteria . The enhancement of IL-12 by morphine might be related to morphine-induced sepsis.

J Leukoc Biol, 2000 Nov, 68(5), 669 - 78
Structural requirements for intracellular processing and sorting of bactericidal/permeability-increasing protein (BPI): comparison with lipopolysaccharide-binding protein; Bulow E et al.; The bactericidal/permeability-increasing protein (BPI), which is stored in the azurophil granules of neutrophils, and the circulating lipopolysaccharide-binding protein (LBP) share the same structure . Both bind lipopolysaccharide of gram-negative bacteria through their amino-terminal domains . The carboxy-terminal domain of BPI promotes bacterial attachment to phagocytes, whereas the corresponding domain of LBP delivers lipopolysaccharide to monocytes/macrophages . Our aim was to investigate the role of the amino-and carboxy-terminal domains of BPI and LBP for sorting and storage in myeloid cells after transfection of cDNA to two rodent hematopoietic cell lines . Full-length BPI and LBP were both targeted for storage in these cells . Deletion of the carboxy-terminal half of BPI resulted in storage followed by degradation while the reciprocal deletion of the amino-terminal half led to retention in the endoplasmic reticulum for proteasomal degradation . Chimeras between halves of BPI and LBP were also targeted for storage, but those containing carboxy-terminal BPI had the highest stability, again indicating a role for the carboxy-terminal domain of BPI in protection against degradation . Therefore, we propose a critical stability function for the hydrophobic carboxy-terminal domain of BPI during intracellular sorting for storage while the amino-terminal domain may confer targeting for storage.

J Vet Med Sci, 2000 Oct, 62(10), 1105 - 7
Anaerobic orbital abscess/cellulitis in a Yorkshire Terrier dog; Homma K et al.; A retrobulbar abscess/cellulitis occurred in a Yorkshire Terrier dog . The clinical signs were exophthalmos, prolapsed nictitating membrane and purulent ocular discharge . Ultrasonography showed a marked soft tissue swelling of the retrobulbar tissues as well as echogenic parallel lines between the globe and the medial orbital rim . Surgical exploration of the orbit was performed and no foreign body was found . The pterygopalatine fossa was incised and therapeutic retrobulbar drainage attempted . A drain was placed to encourage ventral drainage of the abscess . Anaerobic cultures revealed heavy growth of gram negative rods (prevotella bivia and prevotella buccae were isolated) . Recovery was successful but subsequent treatment for keratoconjunctivitis sicca was necessary . A full recovery of tear production occurred after several weeks.

Mol Microbiol, 2000 Nov, 38(3), 543 - 51
Identification and characterization of in vivo attenuated mutants of Brucella melitensis; Lestrate P et al.; Brucella melitensis 16M is a Gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans . This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes . Signature-tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella . A library of transposon mutants was screened in a murine infection model . Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice . The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild-type strain . The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells . The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone-like protein and proteins of unknown function . The mutants selected in this study provide new insights into the molecular basis of Brucella virulence.

FEMS Microbiol Lett, 2000 Nov 15, 192(2), 211 - 5
Phenotypic analyses of frz and dif double mutants of Myxococcus xanthus; Shi W et al.; Myxococcus xanthus is a Gram-negative gliding bacterium that aggregates and develops into multicellular fruiting bodies in response to starvation . Two chemosensory systems (frz and dif), both of which are homologous to known chemotaxis proteins, were previously identified through characterization of various developmental mutants . This study aims to examine the interaction between these two systems since both of them are required for fruiting body formation of M . xanthus . Through detailed phenotypic analyses of frz and dif double mutants, we found that both frz and dif are involved in cellular reversal and social motility; however, the frz genes are epistatic in controlling cellular reversal, whereas the dif genes are epistatic in controlling social motility . The study suggests that the integration of these two chemotaxis systems may play a central role in controlling the complicated social behaviors of M . xanthus.

Biochem Biophys Res Commun, 2000 Nov 2, 277(3), 617 - 21
Endotoxin and NO induce MIP-1gamma gene transcription in ANA-1 murine macrophages; Guo HT et al.; The host response to gram-negative endotoxin is characterized by an influx of inflammatory cells into host tissues, mediated in part by localized production of chemokines . In this study, using subtractive suppression hybridization analysis, we demonstrate that ANA-1 murine macrophages produce the CC chemokine, MIP-1gamma, in response to LPS-mediated NO production . Gene transcription and protein translation are upregulated in the setting of LPS-induced NO synthesis . However, NO alone is a necessary but insufficient cofactor for induction of MIP-1gamma protein expression; an NO-independent LPS signalling pathway is also required . This study suggests a novel mechanism by which NO modulates the host inflammatory response to endotoxin .

New Microbiol, 2000 Oct, 23(4), 449 - 56
Pathogenic mechanisms of Bartonella quintana; Liberto MC et al.; Bartonella quintana is an epi- and intracellular gram-negative rod responsible for both acute and chronic clinical manifestations . We review the literature about pathogenic mechanisms of B . quintana and discuss our data . Our efforts to clarify Bartonella quintana pathogenesis run on two parallel tracks . The first one concerns interactions between Bartonella quintana and endothelial cells by evaluation and modulation of apoptosis, signal transduction pathways and inflammation . The second one concerns some biological activities of Bartonella quintana endotoxin on human whole blood and endothelium . The elucidation of the mechanisms regulating the inflammatory/proliferative pattern of chronic clinical manifestations of Bartonella quintana infections may offer a contribution for addressing the pathogenesis of intracellular bacterial persistence.

Expert Opin Investig Drugs, 2000 Jun, 9(6), 1363 - 72
Novel agents in the therapy of endotoxic shock; Howe LM; Endotoxic shock, or Gram-negative septic shock, can occur as a component of Gram-negative sepsis and is characterised by hypotension, poor tissue perfusion and multi-organ dysfunction . Despite antibiotic therapy and intensive care management, the morbidity and mortality rates of Gram-negative septic shock remain high . Endotoxin mediates its effects through interaction with receptors on the surface of a variety of host cells . These interactions result in the production and release of numerous biochemical mediators including nitric oxide, cytokines, prostaglandins and leukotrienes and toxic oxygen radicals . It is these biochemical mediators that exert toxic effects during endotoxic shock and which are often the target of novel treatment strategies . Several of these pharmacological agents are currently being investigated for use in Gram-negative septic shock and include inhibitors of the enzyme responsible for nitric oxide production, scavengers of the nitric oxide molecule and cytokine modulators . Although many agents have been studied for potential use as modulators of cytokine levels, this study will focus on pentoxifylline and the 21-aminosteroids, or lazaroids . Examination of the literature regarding pharmacological agents used to treat endotoxic shock often yields confusing and contradictory results . The reasons for these mixed results include differences in models, drug dosages, dosing methods and intervals and timing of administration relative to disease duration and severity . However, despite mixed results, several of the drugs discussed in this paper offer promise in the therapy of an often frustrating and lethal condition.

Expert Opin Investig Drugs, 2000 Feb, 9(2), 237 - 46
Novel oral cephalosporins; Cazzola M; The therapeutic use of oral cephalosporins to treat infectious diseases continues to challenge clinicians; many attempts have been made over recent years to improve the efficacy and spectrum of these anti-infectives . Many oral cephalosporins are in development and include cefdinir, cefprozil, cefetamet pivoxil, cefcapene pivoxil, cefcanel daloxate hydrochloride in Phase II trials, S-1090 in Phase III trials and the novel compounds E1100, E1101 and BRL-57347 . Differences between these drugs are sometimes subtle and the improvement over existing compounds modest, although recent cephalosporins have shown greater activity against Gram-negative bacterial infections . A better knowledge of the pharmacodynamic and pharmacokinetic interrelationships of existing oral cephalosporins is demanded for a more rational use of these compounds and to avoid the subsequent development of resistance . Perhaps with such an approach, the perceived need for new cephalosporins will diminish.

Rev Esp Cardiol, 2000 Oct, 53(10), 1311 - 7
{Inflammation, infection and coronary artery disease: myths and realities . Special XXXV Conference of the National Congress of the Spanish Society of Cardiology}; Kaski JC; In the past decades it has become apparent that inflammation plays a role in atherogenesis and rapid coronary artery disease progression . Active, or vulnerable, atheromatous plaques are responsible for acute coronary events and contain high concentrations of inflammatory cells as well as molecules involved in the inflammatory process, such as cytokines, adhesion molecules and growth factors . From a clinical perspective, early detection of these plaques may prevent the occurrence of serious coronary events . Unfortunately, current diagnostic techniques -i.e . angiography- do not allow the characterization of events taking place in the arterial wall . Therefore, these diagnostic tools cannot identify vulnerable plaques . Recent studies have suggested that markers of systemic inflammation may help in the detection of high risk patients . Although the role of inflammation in the pathogenesis of atherosclerosis is established, it is not known what triggers inflammation in this context . Infectious agents such as viruses and Gram negative bacteria -i.e . Chlamydia pneumoniae- have been postulated to play a role . Several mechanisms, involving inflammation and immunological processes, have been suggested to explain how chronic infections may cause atherosclerosis . Small pilot studies have also been carried out which suggest a causal role of infection in coronary artery disease . These results, however, await confirmation by other large, currently ongoing, studies . The infectious hypothesis of atherosclerosis is still a matter of debate; however, this theory has contributed to the rapid advance of our knowledge regarding the pathogenesis of coronary artery disease in the past few years . Moreover, the notion that coronary artery disease can be considered to be an inflammatory condition in its own right has opened new and challenging avenues for research.

Biochim Biophys Acta, 2000 Oct 18, 1482(1-2), 73 - 83
The bacterial lipocalins; Bishop RE; The lipocalins were once regarded as a eukaryotic protein family, but new members have been recently discovered in bacteria . The first bacterial lipocalin (Blc) was identified in Escherichia coli as an outer membrane lipoprotein expressed under conditions of environmental stress . Blc is distinguished from most lipocalins by the absence of intramolecular disulfide bonds, but the presence of a membrane anchor is shared with two of its closest homologues, apolipoprotein D and lazarillo . Several common features of the membrane-anchored lipocalins suggest that each may play an important role in membrane biogenesis and repair . Additionally, Blc proteins are implicated in the dissemination of antibiotic resistance genes and in the activation of immunity . Recent genome sequencing efforts reveal the existence of at least 20 bacterial lipocalins . The lipocalins appear to have originated in Gram-negative bacteria and were probably transferred horizontally to eukaryotes from the endosymbiotic alpha-proteobacterial ancestor of the mitochondrion . The genome sequences also reveal that some bacterial lipocalins exhibit disulfide bonds and alternative modes of subcellular localization, which include targeting to the periplasmic space, the cytoplasmic membrane, and the cytosol . The relationships between bacterial lipocalin structure and function further illuminate the common biochemistry of bacterial and eukaryotic cells.

Appl Environ Microbiol, 2000 Nov, 66(11), 4751 - 7
Isolation and characterization of a Helicobacter sp . from the gastric mucosa of dolphins, Lagenorhynchus acutus and Delphinus delphis; Harper CM et al.; Gastric ulcerations in dolphins have been reported for decades . Some of these lesions were associated with parasitic infections . However, cases of nonparasitic gastric ulcers with no clearly defined etiology also have been reported in wild and captive dolphins . Considerable speculation exists as to whether dolphins have Helicobacter-associated gastritis and peptic ulcer disease . The stomachs of seven stranded Atlantic white-sided dolphins, Lagenorhynchus acutus, and 1 common dolphin, Delphinus delphis, were assessed for the presence of Helicobacter species . Novel Helicobacter species were identified by culture in the gastric mucosa of two of the eight dolphins studied and by PCR in seven of the eight dolphins . The gram-negative organisms were urease, catalase, and oxidase positive . Spiral to fusiform bacteria were detected in gastric mucosa by Warthin Starry staining . Histopathology revealed mild to moderate diffuse lymphoplasmacytic gastritis within the superficial mucosa of the main stomach . The pyloric stomach was less inflamed, and bacteria did not extend deep into the glands . The lesions parallel those observed in Helicobacter pylori-infected humans . Bacteria from two dolphins classified by 16S rRNA analysis clustered with gastric helicobacters and represent a novel Helicobacter sp . most closely related to H . pylori . These findings suggest that a novel Helicobacter sp . may play a role in the etiopathogenesis of gastritis and gastric ulcers in dolphins . To our knowledge this represents the first isolation and characterization of a novel Helicobacter sp . from a marine mammal and emphasizes the wide host distribution and pathogenic potential of this increasingly important genus.

J Interferon Cytokine Res, 2000 Oct, 20(10), 915 - 21
Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes; Matsumura T et al.; Toll-like receptor (TLR) 2 and TLR4 are members of the interleukin-1 receptor (IL-1R) family and transduce similar signals as IL-1R in response to bacteria and bacterial components . In this study, we investigated the regulation of their gene expression in murine tissues, especially in the liver and hepatocytes . When mice were administered lipopolysaccharide (LPS), TLR2 mRNA was upregulated in the brain, heart, lung, liver, and kidney . In contrast, it was downregulated in the spleen . TLR4 mRNA was decreased in the brain . In the heart and lung, it increased, and it was not affected in the liver, kidney, and spleen . TLR mRNA was further analyzed in the liver and hepatocytes . Like LPS treatment, administration of IL-1, IL-6, or tumor necrosis factor (TNF) upregulated TLR2 mRNA . However, none of them affected the TLR4 mRNA level . In primary cultured hepatocytes, TLR2 mRNA was upregulated by LPS, IL-1, or TNF but not by IL-6 or dexamethasone . None of them affected TLR4 mRNA expression . Similar responses were observed in the murine hepatoma cell line Hepa 1-6 . These results suggest that in infection with gram-negative bacteria, LPS and proinflammatory cytokines differentially regulate gene expression of TLR2 and TLR4 in murine hepatocytes, which may lead to pathologic and host defense reactions in the liver.

J Am Soc Nephrol, 2000 Nov, 11(11), 2122 - 7
Tunneled-cuffed catheter associated infections in hemodialysis patients who are seropositive for the human immunodeficiency virus; Mokrzycki MH et al.; Infection rates in tunneled-cuffed catheters (TCC) are reported to be higher in immunocompromised patients . The purpose of this study was to evaluate TCC-associated infection rates in patients with HIV infection (HIV+) . Data were collected in 40 HIV + patients and 41 controls (C), and in 118 TCC (HIV+, 58; C, 60) for 28,146 catheter days (HIV+, 16,227; C, 11,919) . There were no significant differences in the TCC bacteremia rates (HIV+, 2.23 versus C, 2.53 per 1000 TCC days, P: = NS) or in the TCC exit site infection rates (HIV+, 2.20 versus C, 2.24 per 1000 TCC days, P: = NS) between the groups . The number of TCC removed due to infection was also similar, (HIV+ versus C: 17 versus 15%, P: = NS) . In the HIV+ group, the association of hepatitis B surface antigenemia with TCC exit site infection was dependent on the history of injection drug use . Black race was a significant risk factor for higher TCC exit site infection rates, whereas prophylactic antibiotic use and high CD4 count were significantly associated with lower TCC exit site infection rates . None of the factors significantly predicted bacteremia rate in either group (HIV+ or C) . In comparison to controls, HIV+ patients had a fivefold increased risk of having a Gramnegative organism (P: = 0.02) and a sevenfold increased risk of a fungal isolate (P: = 0.08), although the latter finding was not statistically significant . HIV infection is not a significant risk factor for TCC-associated infection but is associated with a higher prevalence of Gram-negative and fungal species.

Am J Physiol Lung Cell Mol Physiol, 2000 Nov, 279(5), L878 - 83
Free radical-mediated transgene inactivation of macrophages by endotoxin; Dokka S et al.; Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations . The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process . Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent . The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested . This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin . Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity . Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers . The ROS scavenger N-t-butyl-alpha-phenylnitrone, the H(2)O(2) scavenger catalase, and the.OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O(2)(-) scavenger superoxide dismutase had lesser effects . These results indicate that multiple oxidative species are involved in the transfection inactivation process and that.OH formed by H(2)O(2)-dependent, metal-catalyzed Fenton reaction play a major role in this process.

Prog Nucleic Acid Res Mol Biol, 2001, 66, 107 - 57
Protein traffic in bacteria: multiple routes from the ribosome to and across the membrane; Muller M et al.; Bacteria use several routes to target their exported proteins to the plasma membrane . The majority are exported through pores formed by SecY and SecE . Two different molecular machineries are used to target proteins to the SecYE translocon . Translocated proteins, synthesized as precursors with cleavable signal sequences, require cytoplasmic chaperones, such as SecB, to remain competent for posttranslational transport . In concert with SecB, SecA targets the precursors to SecY and energizes their translocation by its ATPase activity . The latter function involves a partial insertion of SecA itself into the SecYE translocon, a process that is strongly assisted by a couple of membrane proteins, SecG, SecD, SecF, YajC, and the proton gradient across the membrane . Integral membrane proteins, however, are specifically recognized by a direct interaction between their noncleaved signal anchor sequences and the bacterial signal recognition particle (SRP) consisting of Ffh and 4.5S RNA . Recognition occurs during synthesis at the ribosome and leads to a cotranslational targeting to SecYE that is mediated by FtsY and the hydrolysis of GTP . No other Sec protein is required for integration unless the membrane protein also contains long translocated domains that engage the SecA machinery . Discrimination between SecA/SecB- and SRP-dependent targeting involves the specificity of SRP for hydrophobic signal anchor sequences and the exclusion of SRP from nascent chains of translocated proteins by trigger factor, a ribosome-associated chaperone . The SecYE pore accepts only unfolded proteins . In contrast, a class of redox factor-containing proteins leaves the cell only as completely folded proteins . They are distinguished by a twin arginine motif of their signal sequences that by an unknown mechanism targets them to specific pores . A few membrane proteins insert spontaneously into the bacterial plasma membrane without the need for targeting factors and SecYE . Insertion depends only on hydrophobic interactions between their transmembrane segments and the lipid bilayer and on the transmembrane potential . Finally, outer membrane proteins of Gram-negative bacteria after having crossed the plasma membrane are released into the periplasm, where they undergo distinct folding events until they insert as trimers into the outer membrane . These folding processes require distinct molecular chaperones of the periplasm, such as Skp, SurA, and PpiD.

Arch Biochem Biophys, 2000 Oct 1, 382(1), 152 - 9
Ectonucleotide diphosphohydrolase activities in hemocytes of larval Manduca sexta; Meyer-Fernandes JR et al.; In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP . In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells) . The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells . Both activities were linear with cell density and with time for at least 90 min . The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner . At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2 . This stimulatory activity was not observed when Ca2+ replaced Mg2+ . The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively . The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable . However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH . These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole . To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases . These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents . Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.

J Immunol, 2000 Nov 1, 165(9), 5202 - 10
Brucella abortus lipopolysaccharide in murine peritoneal macrophages acts as a down-regulator of T cell activation; Forestier C et al.; Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs . We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway . LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4(+) T cells but not those of OVA to specific CD8(+) T cells . This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL . In addition, B . abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides . At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4(+) T cell activation . This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.

Adv Perit Dial, 2000, 16, 267 - 70
A prospective multicenter comparison of peritonitis in peritoneal dialysis patients aged above and below 65 years . Levante PD Multicenter Group; Perez-Contreras J et al.; Various authors have found peritonitis rates in older peritoneal dialysis (PD) patients to be higher, similar, or even lower when compared to rates in younger populations . We prospectively analyzed all episodes of peritonitis registered in the 381 patients (219 males, 162 females; mean age, 55.5 +/- 17.0 years) who were treated with PD during four years (1993-1996) in our multicenter group . Patients were distributed into two groups . Group A included 138 patients aged 65 years or over (mean age: 72.3 +/- 5.1 years); group B included 243 patients aged below 65 years (mean age: 46.0 +/- 13.6 years) . No differences were seen in general PD characteristics . Normalized protein catabolic rate (nPCR) was higher in younger patients (p = 0.001) . Patients in group A experienced more peritonitis (135 episodes; 1 episode per 16.7 patient-months) than patients in group B (198 episodes; 1 episode per 21.7 patient-months; p = 0.01) . Although no differences were seen in the general characteristics of the peritonitis episodes, gram-negative peritonitis and peritonitis not achieving a cure were more commonly secondary to enteric bacteria in group A (p = 0.03) . We conclude that PD patients aged 65 years or over are at higher risk of peritonitis . Also, in this age group, gram-negative peritonitis and peritonitis with any evolution except cure are more likely to be due to enteric bacteria.

Trends Microbiol, 2000 Oct, 8(10), 478 - 80
Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions; Solomon JM et al.; Legionella pneumophila, the Gram-negative bacterium that causes Legionnaires' disease, can be cultured in the laboratory in a variety of fresh-water amoebae and macrophage-like cell lines . None of these hosts, however, is amenable to genetic analysis, which has limited the ability of researchers to analyse the host factors essential for L . pneumophila growth . In this article, we describe a novel method in which L . pneumophila is grown within the soil amoeba Dictyostelium discoideum and how D . discoideum genetics is being used to analyse the host cell factors involved in L . pneumophila pathogenesis.

Int J Med Microbiol, 2000 Mar, 290(1), 37 - 49
Cloning and functional characterization of a 30 kb gene locus required for lipopolysaccharide biosynthesis in Legionella pneumophila; Luneberg E et al.; The spontaneous Legionella pneumophila lipopolysaccharide (LPS) mutant 137, which did not bind the LPS-specific mAb 2625, was complemented with a genomic library from the parental wild-type strain . Transformants were screened for reconstitution of the wild-type LPS phenotype, able to bind mAb 2625 . By this strategy, a 32,661 bp region comprising 30 open reading frames (Orfs) was identified . Orfs with significant homologies to genes encoding enzymes required for LPS or capsule biosynthesis of Gram-negative bacteria were located on the gene locus . The mutation of strain 137 could be assigned to a deletion of a cytosine residue in Orf 8 . The protein encoded by Orf 8 exhibited homology to bacterial methyl-transferases . The L . pneumophila LPS gene locus included genes with deduced products likely to be involved in LPS core oligosaccharide biosynthesis (rmlA-D, rhamnosyl-transferases, acetyl-transferase) as well as LPS O-chain biosynthesis and translocation (mnaA, neuB, neuA, wecA, wzt, wzm) . The neuA (Orf 25) and neuB (Orf 24) gene products were functionally characterized by complementation of the capsule negative E . coli K1 mutants EV5 and EV24, respectively . By introduction of the L . pneumophila neuA gene into E . coli EV5 and the neuB gene into EV24, expression of the K1 polysialic acid capsule could be restored . We, therefore, conclude that the biosynthesis pathway of legionaminic acid, the structural unit of the L . pneumophila Sg1 O-antigen, might be similar to the biosynthesis of sialic acid . Southern blot analysis indicated the entire gene locus to be present in L . pneumophila serogroup (Sg)1 strains, whereas only parts of the DNA stretch hybridized to DNA from Sg2 to Sg14 strains.

J Biol Chem, 2001 Jan 12, 276(2), 1146 - 51
Disulfide-dependent folding and export of Escherichia coli DsbC; Liu X et al.; DsbC, a member of the Dsb family in the periplasm of Gram-negative bacteria, is not only a disulfide isomerase but also a chaperone . Five DsbC mutants with Cys in the active site sequence of Cys(98)-Gly-Tyr-Cys(101) and the nonactive site disulfide Cys(141)-Cys(163) replaced by Ser have been studied . The results show that the active site Cys residues are necessary for enzyme activities but not required for chaperone activity, while the lack of the nonactive site disulfide results in a decreased chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase but has no effect on enzyme activities . Wild-type DsbC was overexpressed and correctly processed as a soluble periplasmic protein . Mutation in one of these Cys residues results in aggregation or extracellular/membrane locations, but does not affect the proper processing . DsbC mutated in either Cys residue of nonactive site disulfide shows higher sensitivity to unfolding by guanidine hydrochloride and slower refolding compared with wild-type DsbC and the active site Cys mutants . The above results provide experimental evidence for structural role of the nonactive site disulfide in folding and biological activities of DsbC.

Curr Microbiol, 2000 Mar, 40(3), 185 - 9
Variations in the ability of ruminal gram-negative Prevotella species to resist monensin; Callaway TR et al.; Gram-negative, ruminal Prevotella strains (n = 15) differed greatly in their sensitivity to the feed additive monensin . Strains that were repeatedly transferred with sublethal doses tolerated more monensin than those that were unadapted, but growth experiments indicated that the sensitivity range was as great as 2000-fold . Prevotella bryantii B(1)4 grew with monensin concentrations as high as 20 microM, but P . ruminicola H15a, D31d, 20-63, E40a, and D42f never initiated growth if monensin was greater than 0.01 microM . Washed cell preparations that were energized with glucose lost intracellular potassium when monensin was added, and potassium depletion could also be used as an index of monensin sensitivity . Adapted cells of P . bryantii B(1)4 had a half-maximal potassium depletion constant (K(d)) of 3.2 microM, but the K(d) values of P . ruminicola strains H15a, D31d, 20-63, E40a, and D42f were less than 0.04 microM . Maximal potassium depletion (K(max)) values range from 90% to 40%, and monensin-adapted cells always had lower K(max) values than unadapted cells . A linear regression of log K(d)/K(max) versus percentage decrease in optical density divided by monensin concentration had an r(2) of 0.75, and this regression indicated that potassium depletion from washed cells closely correlated with growth inhibition . P . bryantii B(1)4 had a K(d)/K(max) ratio that was sevenfold greater than other Prevotella strains, and this result indicated that P . bryantii may be unusual in its ability to grow with very high concentrations of monensin.

J Immunol, 2000 Oct 15, 165(8), 4272 - 80
Divergent response to LPS and bacteria in CD14-deficient murine macrophages; Moore KJ et al.; Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock . The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS . In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted . As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways . The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages . However, in contrast to the findings with LPS, whole E . coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell . This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type . Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor . An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E . coli . These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1781 - 7
'Flexispira rappini' strains represent at least 10 Helicobacter taxa; Dewhirst FE et al.; 'Flexispira rappini' is a provisional name given to gram-negative, microaerophilic, motile, spindle-shaped micro-organisms with spiral periplasmic fibres and bipolar tufts of sheathed flagella . Several investigators, including Kirkbride, Romero, and Archer isolated strains possessing this morphology . Previously, the phylogenetic position of three 'Flexispira rappini' strains was determined by 165 rRNA sequencing, which indicated that flexispira were members of the genus Helicobacter . As more organisms with 'F . rappini' morphology were isolated, it became apparent that there were multiple Helicobacter taxa with this distinctive morphology . The purpose of this study was to examine a collection of 36 'F . rappini' strains from diverse habitats by using 165 rRNA sequence analysis . The strains fell into 10 taxa, each possibly representing a novel Helicobacter species . Two of these flexispira taxa were previously named, by us, Helicobacter bilis and Helicobacter trogontum . Currently, none of the flexispira taxa contains enough phenotypically and genotypically characterized strains to be formally named 'Helicobacter rappinii'.

Eur J Surg, 2000 Sep, 166(9), 728 - 33
Influence of glutathione metabolising enzymes in rats with gram-negative sepsis; Hansson L et al.; OBJECTIVE: To evaluate the role of glutathione metabolising enzymes in rats with Gram-negative sepsis . SETTING: University hospital, Sweden . ANIMALS: 61 male Sprague Dawley rats . INTERVENTIONS: Animals were divided into two groups, one of which was given tert-butyl-4-hydroxyanisole (BHA), a powerful inducer of glutathione metabolising enzymes . A gelatine capsule containing bacteria and adjuvant substances was placed in the abdomen . In one group it contained only adjuvant substances, and the controls underwent sham laparotomy . MAIN OUTCOME MEASURES: Activities of glutathione metabolising enzymes, and histological effect on pulmonary tissue . RESULTS: Activities of glutathione metabolising enzymes were reduced in lung tissue after the induction of sepsis . Pre-treatment with BHA increased enzyme activity and reduced the histological changes . CONCLUSION: Glutathione metabolising enzymes may have a role in sepsis, and pre-treatment with BHA seems to prevent histological changes in pulmonary tissue in septic rats.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 273 - 8
Detection and quantification of tetracyclines by whole cell biosensors; Hansen LH et al.; Three different mini-Tn5 plasmids, containing a tetracycline-inducible promoter, Ptet and a regulatory gene, tetR, in operon fusions with a reporter gene system (lacZYA, luxCDABE or gfp), were constructed . These biosensor constructs responded to low levels of tetracyclines by producing beta-galactosidase, light or green fluorescent protein . They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism . All three constructs were transferred successfully to different gram-negative bacteria by conjugation . An Escherichia coli strain containing the Ptet-lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors.

FASEB J, 2000 Dec, 14(15), 2393 - 5
Heat shock enhances transcriptional activation of the murine-inducible nitric oxide synthase gene; Goldring CE et al.; There is considerable interest in determining the conditions leading to enhanced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) biosynthesis . Using in vivo footprinting, we demonstrate that heat shock of murine macrophages concurrent with lipopolysaccharide (LPS) treatment stimulated changes in guanine methylation sensitivity at ?898/9, at a putative partial heat shock element (HSE) and at -893/4, a site bordering an E-box, within the iNOS gene enhancer, suggesting inducible occupation by transcription factors at these regions . LPS treatment accompanied by heat shock provoked increased iNOS gene transcription, increased levels of iNOS protein, and increased production of NO compared with LPS treatment alone . Electrophoretic mobility shift analysis revealed low constitutive levels of specific binding to an E-box and a partial HSE within the iNOS enhancer . Binding to the E-box was increased by LPS treatment or by heat shock, achieving a greater increase by a combination of both treatments . The proteins occupying this site were identified as belonging to the USF family of transcription factors . Heat shock or LPS increased binding to the HSE, and the factor responsible for this interaction was identified as heeat shock factor-1 (HSF-1) . Mutations at the HSE revealed the importance of HSF-1 in the induction of iNOS by LPS . Thus, our data reveal two novel regulatory sites in the murine iNOS gene, one of which is implicated in enhancing iNOS expression via LPS stimulation, and provide the first evidence that heat shock enhances transcription of the iNOS gene . These results could have implications in the host response mechanism to fever-associated gram-negative infection.

Rev Prat, 2000 Sep 1, 50(13), 1409 - 13
{Bacteriology and pathogenicity of Helicobacter pylori}; Skouloubris S et al.; Helicobacter pylori is the prototype of bacteria belonging to a new genus, the Helicobacter genus . It is a gram-negative, highly motile and microaerophilic bacterium, with a spiral shape, that colonizes the human gastric mucosa and causes several gastroduodenal diseases . Pathogenicity of H . pylori relies upon its capacity to adapt to a hostile environment and to escape the host response . Resistance to acidity, motility, adhesion, molecular mimicry, resistance to phagocytosis, synthesis of a cytotoxin, induction of an inflammatory response are the major strategies developed by H . pylori to colonize persistently and damage gastric tissue.

Annu Rev Microbiol, 2000, 54, 615 - 40
The disease spectrum of Helicobacter pylori: the immunopathogenesis of gastroduodenal ulcer and gastric cancer; Ernst PB et al.; Helicobacter pylori is a gram-negative bacterium that resides under microaerobic conditions in a neutral microenvironment between the mucus and the superficial epithelium of the stomach . From this site, it stimulates cytokine production by epithelial cells that recruit and activate immune and inflammatory cells in the underlying lamina propria, causing chronic, active gastritis . Although epidemiological evidence shows that infection generally occurs in children, the inflammatory changes progress throughout life . H . pylori has also been recognized as a pathogen that causes gastroduodenal ulcers and gastric cancer . These more severe manifestations of the infection usually occur later in life and in a minority of infected subjects . To intervene and protect those who might be at greatest risk of the more severe disease outcomes, it is of great interest to determine whether bacterial, host, or environmental factors can be used to predict these events . To date, several epidemiological studies have attempted to define the factors affecting the transmission of H . pylori and the expression of gastroduodenal disease caused by this infection . Many other laboratories have focused on identifying bacterial factors that explain the variable expression of clinical disease associated with this infection . An alternative hypothesis is that microorganisms that cause lifelong infections can ill afford to express virulence factors that directly cause disease, because the risk of losing the host is too great . Rather, we propose that gastroduodenal disease associated with H . pylori infection is predominantly a result of inappropriately regulated gastric immune responses to the infection . In this model, the interactions between the immune/inflammatory response, gastric physiology, and host repair mechanisms would dictate the disease outcome in response to infection.

Annu Rev Microbiol, 2000, 54, 567 - 613
Legionella pneumophila pathogesesis: a fateful journey from amoebae to macrophages; Swanson MS et al.; Legionella pneumophila first commanded attention in 1976, when investigators from the Centers for Disease Control and Prevention identified it as the culprit in a massive outbreak of pneumonia that struck individuals attending an American Legion convention () . It is now clear that this gram-negative bacterium flourishes naturally in fresh water as a parasite of amoebae, but it can also replicate within alveolar macrophages . L . pneumophila pathogenesis is discussed using the following model as a framework . When ingested by phagocytes, stationary-phase L . pneumophila bacteria establish phagosomes which are completely isolated from the endosomal pathway but are surrounded by endoplasmic reticulum . Within this protected vacuole, L . pneumophila converts to a replicative form that is acid tolerant but no longer expresses several virulence traits, including factors that block membrane fusion . As a consequence, the pathogen vacuoles merge with lysosomes, which provide a nutrient-rich replication niche . Once the amino acid supply is depleted, progeny accumulate the second messenger guanosine 3',5'-bispyrophosphate (ppGpp), which coordinates entry into the stationary phase with expression of traits that promote transmission to a new phagocyte . A number of factors contribute to L . pneumophila virulence, including type II and type IV secretion systems, a pore-forming toxin, type IV pili, flagella, and numerous other factors currently under investigation . Because of its resemblance to certain aspects of Mycobacterium, Toxoplasma, Leishmania, and Coxiella pathogenesis, a detailed description of the mechanism used by L . pneumophila to manipulate and exploit phagocyte membrane traffic may suggest novel strategies for treating a variety of infectious diseases . Knowledge of L . pneumophila ecology may also inform efforts to combat the emergence of new opportunistic macrophage pathogens.

Clin Infect Dis, 2000 Sep, 31(3), 839 - 40
Veillonella parvula meningitis: case report and review of Veillonella infections; Bhatti MA et al.; Veillonella parvula is a small, nonfermentative anaerobic gram-negative coccus that is part of the normal flora of the mouth, gastrointestinal tract, and vagina in humans . When isolated from clinical specimens, V . parvula is often regarded as a contaminant or commensal, but it has been implicated as a pathogen in infections of the sinuses, lungs, heart, bone, and central nervous system . Meningitis, however, is extremely rare; to our knowledge, only 2 cases have been previously described in the literature . We report a case of V . parvula meningitis and review the literature on Veillonella infections.

J Clin Microbiol, 2000 Oct, 38(10), 3870 - 1
Isolation of Moraxella canis from an ulcerated metastatic lymph node; Vaneechoutte M et al.; Moraxella canis was isolated in large numbers from an ulcerated supraclavicular lymph node of a terminal patient, who died a few days later . Although the patient presented with septic symptoms and with a heavy growth of gram-negative diplococci in the lymph node, blood cultures remained negative . M . canis is an upper-airway commensal from dogs and cats and is considered nonpathogenic for humans, although this is the third reported human isolate of this species.

Immunology, 2000 Oct, 101(2), 254 - 61
Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms; Bergamini A et al.; Granulocyte-macrophage colony-stimulating factor (GM-CSF) has multiple effects on the antigen phenotype and function of macrophages . In this study we investigated the effect of GM-CSF on cytokine production by macrophages . We found that GM-CSF may modify the tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) response to lipopolysaccharide (LPS) through two different mechanisms . Relatively early in culture, GM-CSF increases the amount of cytokines synthesized by responding cells; this effect appears to be unrelated to modulation of CD14 expression and LPS-binding capacity . After prolonged incubation, GM-CSF up-regulates both CD14 expression and LPS-binding capacity, and the frequency of cytokine-producing cells . Release of CD14 in the culture supernatant was decreased in the presence of GM-CSF, suggesting that a reduced shedding was responsible for the effect of GM-CSF on CD14 expression . Enhancement of cytokine production was also observed in GM-CSF-treated macrophages after stimulation by phorbol 12-myristate 13-acetate (PMA), thus indicating that GM-CSF affects both CD14-dependent and -independent cytokine production . Finally, GM-CSF did not modulate the LPS- and PMA-induced production of IL-10 and IL-12 . We conclude that GM-CSF may play a role in manipulating the activation-induced expression of pro-inflammatory cytokines by macrophages . Enhanced production of these cytokines could play an important role in the pathogenesis of Gram-negative septic shock syndrome and in defence against infectious agents.

Appl Environ Microbiol, 2000 Oct, 66(10), 4528 - 31
Cometabolic degradation of dibenzofuran by biphenyl-cultivated Ralstonia sp . strain SBUG 290; Becher D et al.; Cells of the gram-negative bacterium Ralstonia sp . strain SBUG 290 grown in the presence of biphenyl are able to cooxidize dibenzofuran which has been 1,2-hydroxylated . Meta cleavage of the 1, 2-dihydroxydibenzofuran between carbon atoms 1 and 9b produced 2-hydroxy-4-(3'-oxo-3'H-benzofuran-2'-yliden)but-2-enoic acid, which was degraded completely via salicylic acid . The presence of these intermediates indicates a degradation mechanism for dibenzofuran via lateral dioxygenation by Ralstonia sp . strain SBUG 290.

Pediatr Radiol, 2000 Sep, 30(9), 640 - 3
Regional lymphadenopathy in cat-scratch disease: ultrasonographic findings; Garcia CJ et al.; BACKGROUND: Cat-scratch disease (CSD) is considered to be an emerging disease worldwide and is caused by Bartonella henselae, a gram-negative bacterium introduced by a scratch or bite of a cat . The most common clinical manifestation is regional lymphadenopathy, but clinical recognition may be difficult, as atypical manifestations may occur . The diagnosis is confirmed with serologic testing and histology is rarely needed . This paper is based on our experience with the use of ultrasonography in the diagnosis of CSD . OBJECTIVE: The aim of this study was to describe the sonographic and color Doppler appearances of regional lymphadenopathy in CSD, as this has not widely reported in the literature . MATERIALS AND METHODS: Forty-seven patients (average 9.4 years) were included who all had serologically and/or histologically proven CSD and had been studied using US early in the clinical course . All had a positive history of exposure to cats and exhibited regional lymphadenopathy . RESULTS: US showed large hypoechoic adenopathy with some transmission enhancement and high vascularization on color-flow Doppler imaging . In 30 patients, abdominal US was also performed and splenic and/or hepatic granulomata were found in 10 . CONCLUSIONS: In our experience, sonography and especially color-Doppler and power-Doppler sonography was helpful in the diagnosis of CSD . We believe it should be used in the initial study of children with regional lymphadenopathy, and serologic testing should be performed when CSD is suspected.

Mikrobiologiia, 2000 Jul-Aug, 69(4), 506 - 10
{Features of phospholipid composition of marine proteobacteria of Pseudoalteromonas species}; Frolova GM et al.; The study of the phospholipid composition of 14 type strains of marine proteobacteria of the genus Pseudoalteromonas showed that phospholipids are the main polar lipid constituents of membranes in these proteobacteria . The phospholipid patterns of the strains studied were found to be similar and involved five phospholipids typical of gram-negative bacteria, namely, phosphatidylethanolamine, phosphatidylglycerol, bisphosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid . The major phospholipids were phosphatidylethanolamine and phosphatidylglycerol, which add up to 89-97% of total phospholipids; bisphosphatidic acid was dominant among minor phospholipids . The prevalence of phosphatidylethanolamine (62-77% of total phospholipids) and the absence of diphosphatidylglycerol are the characteristic features of most bacteria of this genus . As in Escherichia coli, the phospholipid composition of the marine proteobacteria depended on the presence of magnesium in the medium.

Microbes Infect, 2000 Aug, 2(10), 1225 - 35
Version 2000: the new beta-lactamases of Gram-negative bacteria at the dawn of the new millennium; Thomson KS et al.; beta-lactamases of Gram-negative bacteria are evolving dynamically . New developments include the production of enzymes with novel substrate profiles, reduced susceptibility to beta-lactamase inhibitors, and the simultaneous production of multiple types of beta-lactamases . The changes represent evolutionary upgrades which provide modern pathogens with a greater potential to resist beta-lactam antibiotics and cause formidable therapeutic, infection control, and diagnostic challenges . This review is a clinically oriented outline of recent developments in the beta-lactamase production of Gram-negative bacteria.

Microbes Infect, 2000 Aug, 2(10), 1181 - 92
The role of immune responses in bone loss during periodontal disease; Baker PJ; A network of cytokines and other soluble mediators unites the immune system and bone; bacterial infections induce immune responses which may perturb this network . Periodontal diseases are Gram-negative infections resulting in bone loss in the jaw . Evidence is presented that immune responses to these infections produces net resorption of bone.

Microbes Infect, 2000 Aug, 2(10), 1147 - 57
Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes; Brattig NW et al.; The majority of Onchocerca volvulus-infected persons show signs of cellular anergy, and long-time survival of adult and larval parasites in subcutaneous tissue is observed . The mechanisms leading to immunological hyporesponsiveness are poorly understood . Monocytes/macrophages represent a link between the innate and acquired immune system and are candidate cells to promote inflammatory and antiinflammatory processes . In the present study we have shown that products of microfilarial (O . volvulus) and adult (O . volvulus and O . ochengi) parasites affect monocytes in vitro . An early production of TNF-alpha by exposed monocytes was followed by the production of IL-10 and a reduced expression of HLA-DR and the costimulatory molecules B7-1 and B7-2, while other adhesion receptors remained unaffected . Downregulation of the functional membrane receptors failed to occur after treatment of the cells with anti-IL-10 antibodies . The engagement of CD14, a dominant membrane receptor on monocytes and major binding protein for lipopolysaccharides, was indicated by partial blocking of monocyte modulation by neutralizing antibodies to CD14 and by the antagonistic lipid A analog compound 406 . Lipopolysaccharide-like molecules were detected in sterile products of O . volvulus stages which could originate from Wolbachia bacteria related to Gram-negative Rickettsiales, known to be abundant in the hypodermis and the female reproductive organs of O . volvulus . The present results indicate that the monocyte/macrophage may be a major target cell for immunomodulatory parasite-derived and intraparasitic, bacteria-derived molecules, thereby contributing to the host's cellular hyporesponsiveness.

Int Arch Occup Environ Health, 2000 Aug, 73(6), 369 - 75
Exposure and acute exposure-effects before and after modification of a contaminated humidification system in a synthetic-fibre plant; Pal TM et al.; OBJECTIVE: Follow-up study of exposure and acute exposure-effects after modification to steam humidification of a contaminated cold water system which had caused an outbreak of humidifier fever in a synthetic-fibre plant . METHODS: Before and after modification of the system aerobiological measurements were performed . Concentrations of fungi and bacteria, in colony forming units (cfu) per m3, were measured by stationary air sampling with an Andersen sampler . Endotoxin levels (pg/m3) were determined by Limulus Amoebocyte Lysate (LAL) assay in pooled dust from personal air sampling . An indication of exposure levels of oil-mist was obtained by monitoring with a direct reading optical photometer . Changes as acute exposure-effects in spirometry and white blood cell count, during an afternoon shift were compared in exposed and non-exposed workers before and after modification . RESULTS: Measured levels of fungi, total bacteria, Gram-negative bacteria and endotoxins both before and after modification were below levels which would be expected to be associated with the exposure-effects . However, after modification, we found that the statistically significant differences in levels of bacteria and endotoxins with a department without humidification no longer existed . Mean oil-mist concentrations were below 1 mg/m3, with short-time peak exposure during certain tasks of up to 5 mg/m3 . Before modification, in exposed workers there was significantly more decline of spirometry, and more increase of white blood cell count during the first afternoon shift, compared with non-exposed workers . In exposed workers, the white blood cell count increase was positively associated with decline of spirometry . After modification, differences between exposed and non-exposed workers no longer existed . CONCLUSION: Follow-up investigation of acute exposure-effects demonstrated the effectiveness of remedial actions taken against a contaminated humidification system . Follow-up of exposure-effects in particular is recommended when there is doubt about the interpretation of exposure measurements.

Crit Care Med, 2000 Sep, 28(9 Suppl), S9 - 11
Pathophysiology of disseminated intravascular coagulation in sepsis; ten Cate H; Disseminated intravascular coagulation (DIC) is an acquired syndrome characterized by intravascular fibrin formation occurring in the course of a variety of severe diseases . In gram-negative sepsis, endotoxin is the bacterial component eliciting a cascade of tissue factor dependent hypercoagulable reactions mediated by cytokines, including tumor necrosis factor-alpha and interleukin-6 . Fibrinolysis is activated in this process by the action of tumor necrosis factor-alpha, but its activity is impaired by the predominant inhibitory effect of plasminogen activator inhibitor-1 . Natural inhibitory mechanisms include antithrombin, the protein C system, and tissue factor pathway inhibitor . Each of these defense systems counteracts the harmful effects of DIC, and its acquired deficiency is associated with increased mortality in observational studies . The generation of several proteases in DIC, including factor Xa and thrombin, has potential interactions with inflammatory pathways that may potentiate the systemic inflammatory syndrome that often accompanies DIC . Experimental studies support the notion that defects in the protein C pathway modulate the inflammatory response, and illustrate that coagulation and inflammation are coupled systems in DIC.

Avian Dis, 2000 Jul-Sep, 44(3), 725 - 9
Ornithobacterium rhinotracheale infection in commercial laying-type chickens; Sprenger SJ et al.; Ornithobacterium rhinotracheale is a gram-negative, rod-shaped, pleomorphic bacterium that has been isolated from flocks of turkeys and broilers from around the world . Infections cause respiratory disease, mortality, and growth suppression, or clinical signs of infection may be absent . In layers, there have been few reports of disease caused by O . rhinotracheale . This is the first report of O . rhinotracheale infection in United States layer flocks.

Invest Ophthalmol Vis Sci, 2000 Oct, 41(11), 3474 - 9
PCR-based evidence of bacterial involvement in eyes with suspected intraocular infection; Okhravi N et al.; PURPOSE: To assess the usefulness of polymerase chain reaction (PCR) in detection of bacteria in ocular samples . METHODS: Thirty-seven samples (aqueous and vitreous) were collected from 25 eyes showing typical symptoms and clinical signs of bacterial endophthalmitis . Ocular samples were also collected from 38 eyes that underwent routine surgery and from 15 eyes with intraocular inflammation due to nonbacterial causes . Panbacterial PCR was performed with a nested pair of 16S rRNA gene primers . Subsequent bacterial identification was completed for 18 paired samples (nine eyes) using restriction fragment length polymorphism (RFLP) and DNA sequencing . RESULTS: A 100% concordance was obtained between PCR and culture-positive samples . A PCR product was amplified from all 37 intraocular samples from eyes with suspected infection, whereas only 15 of 22 vitreous samples and 5 of 15 aqueous samples were culture positive . Culture-negative PCR-positive samples contained a preponderance of gram-negative bacterial sequences . Cloning and DNA analysis revealed 30 DNA sequences and included eight bacterial 16S rDNA, which currently remain unidentifiable . The presence of bacterial DNA was associated with an inflammatory response suggestive of infection and not colonization . All 15 samples from inflamed eyes with diverse uveitis diagnoses were PCR negative . The false-positive rate, due to contamination during sampling, was 5% . CONCLUSIONS: Bacterial DNA was detected in all patients with typical clinical signs of endophthalmitis . Gram-negative organisms seem to play a much more important role in the pathogenesis of this disease than previously thought . PCR-based techniques have great value in the confirmation of the diagnosis of bacterial endophthalmitis especially in culture-negative eyes.

Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 316 - 24
Haem-polypeptide interactions during cytochrome c maturation; Thony-Meyer L; Cytochrome c maturation involves the translocation of a polypeptide, the apocytochrome, and its cofactor, haem, through a membrane, before the two molecules are ligated covalently . This review article focuses on the current knowledge on the journey of haem during this process, which is known best in the Gram-negative bacterium Escherichia coli . As haem always occurs bound to protein, its passage across the cytoplasmic membrane and incorporation into the apocytochrome appears to be mediated by a set of proteinaceous maturation factors, the Ccm (cytochrome c maturation) proteins . At least three of them, CcmC, CcmE and CcmF, are thought to interact directly with haem . CcmE binds haem covalently, thus representing an intermediate of the haem trafficking pathway . CcmC is required for binding of haem to CcmE, and CcmF for releasing it from CcmE and transferring it onto the apocytochrome . The mechanism by which haem crosses the cytoplasmic membrane is currently unknown.

Biochemistry, 2000 Sep 26, 39(38), 11777 - 87
The lipopolysaccharide barrier: correlation of antibiotic susceptibility with antibiotic permeability and fluorescent probe binding kinetics; Snyder DS et al.; Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a permeability barrier, making the outer membrane relatively impermeable to hydrophobic antibiotics, detergents, and host proteins . Mutations in the LPS biosynthetic apparatus increase bacterial susceptibility to such agents . To determine how this increased susceptibility is mediated, we have correlated antibiotic susceptibilities of rough (antibiotic resistant) and deep rough (antibiotic susceptible) bacterial strains with antibiotic permeabilities and fluorescent probe binding kinetics for bilayers composed of LPS purified from the same strains . Bilayer permeabilities of two hydrophobic beta-lactam antibiotics were measured by encapsulating the appropriate beta-lactamases in large unilamellar vesicles . In the presence of MgCl(2), permeabilities of LPS bilayers from rough and deep rough bacteria were similar and significantly lower than those of bacterial phospholipids (BPL) . Addition of BPL to the LPS bilayers increased their antibiotic permeability to approximately the level of the BPL bilayers . Binding rates of the fluorescent probe bis-aminonaphthylsulfonic acid (BANS) were 2 orders of magnitude slower for both rough and deep rough LPS bilayers compared to that of bilayers composed of BPL or mixtures of LPS and BPL . On the basis of these results and the observation that deep rough bacteria have higher levels of phospholipid on their surface than do rough bacteria (Kamio, Y., and Nikaido, H . (1976) Biochemistry 15, 2561-2569), we argue that the high susceptibility of deep rough bacteria is due to the presence of phospholipids on their surface . Experiments with phospholipid bilayers showed that the addition of PEG-lipids (containing covalently attached hydrophilic polymers) had little effect on permeability and binding rates, whereas the addition of cholesterol reduced permeability and slowed binding to levels approaching those of LPS . Therefore, we argue that the barrier provided by LPS is primarily due to its tight hydrocarbon chain packing (Snyder et al., (1999) Biochemistry 38, 10758-10767) rather than to its polysaccharide headgroup.

Infect Immun, 2000 Oct, 68(10), 5864 - 8
Genetic control of susceptibility to Porphyromonas gingivalis-induced alveolar bone loss in mice; Baker PJ et al.; Periodontal disease affects a large percentage of the human population . Resorption of the alveolar bone of the jaw is a pivotal sequela of periodontal disease, because this bone is the attachment site for the periodontal ligaments that anchor the teeth . Using a murine model in which alveolar bone loss is induced by oral infection with Porphyromonas gingivalis, a gram-negative bacterium associated with human adult periodontal disease, we provide evidence suggesting that susceptibility to such bone loss is a genetically determined trait . AKR/J, DBA/2J, and BALB/cByJ or BALB/cJ mice were highly susceptible, while A/J, A/HeJ, 129/J, SJL/J, and C57BL/6J mice were much more resistant . When susceptible BALB/cJ and BALB/cByJ mice were crossed to resistant strains, two patterns were observed . (BALBc/ByJ x C57BL/6J)F(1) offspring were susceptible, suggesting C57BL/6J has recessive resistance alleles, while (BALB/cJ x A/J)F(1) mice were all resistant, suggesting that A/J mice have dominant resistance alleles . These results suggest a tractable genetic basis for P . gingivalis-induced alveolar bone loss and open the possibility of exploiting the mouse model to identify loci important for host susceptibility and resistance to periodontal disease.

Clin Infect Dis, 2000 Aug, 31(2), 524 - 32 Epub 2000 Sep 14.
Human necrobacillosis, with emphasis on Lemierre's syndrome; Hagelskjaer Kristensen L et al.; Lemierre's syndrome is the classical presentation of human necrobacillosis . It is characterized by a primary infection in the head in a young, previously healthy person who subsequently develops persistent high fever and disseminated metastatic abscesses, frequently including a septic thrombophlebitis of the internal jugular vein . The main pathogen is Fusobacterium necrophorum, an obligate anaerobic, pleomorphic, gram-negative rod . Clinical microbiologists have a key role in alerting clinicians and advising proper antibiotic treatment when the characteristic microscopic morphology of the pleomorphic F . necrophorum is seen in Gram stains from positive anaerobic cultures of blood and pus . Early diagnosis and prolonged appropriate antibiotic treatment with good anaerobic coverage are crucial to reduce morbidity and mortality . F . necrophorum also causes human necrobacillosis with foci caudal to the head, mainly in elderly patients with high mortality related to age and predisposing diseases, such as cancers of the primary focus.

Structure Fold Des, 2000 Sep 15, 8(9), R171 - 5
Bacterial export takes its Tol; Wiener MC; The recent crystal structure of TolC elegantly indicates its function and provides insight into its mechanism for export of a wide range of molecules across the periplasmic space and outer membrane of Gram-negative bacteria . The structure is compared to those of other proteins that are embedded in bacterial outer membranes or that traverse the periplasmic space.

J Biol Chem, 2000 Dec 15, 275(50), 39608 - 16
Cytochrome c nitrite reductase from Wolinella succinogenes . Structure at 1.6 A resolution, inhibitor binding, and heme-packing motifs; Einsle O et al.; Cytochrome c nitrite reductase catalyzes the 6-electron reduction of nitrite to ammonia . This second part of the respiratory pathway of nitrate ammonification is a key step in the biological nitrogen cycle . The x-ray structure of the enzyme from the epsilon-proteobacterium Wolinella succinogenes has been solved to a resolution of 1.6 A . It is a pentaheme c-type cytochrome whose heme groups are packed in characteristic motifs that also occur in other multiheme cytochromes . Structures of W . succinogenes nitrite reductase have been obtained with water bound to the active site heme iron as well as complexes with two inhibitors, sulfate and azide, whose binding modes and inhibitory functions differ significantly . Cytochrome c nitrite reductase is part of a highly optimized respiratory system found in a wide range of Gram-negative bacteria . It reduces both anionic and neutral substrates at the distal side of a lysine-coordinated high-spin heme group, which is accessible through two different channels, allowing for a guided flow of reaction educt and product . Based on sequence comparison and secondary structure prediction, we have demonstrated that cytochrome c nitrite reductases constitute a protein family of high structural similarity.

Curr Gastroenterol Rep, 1999 Aug, 1(4), 308 - 13
Helicobacter pylori infection in pediatric patients; Tolia V; Helicobacter pylori is a gram-negative, spiral organism that colonizes the gastric mucosa and causes primary antral gastritis and peptic ulcer disease . Recent studies indicate that H . pylori infection acquired in childhood may cause gastric cancer in later life . H . pylori is present in both asymptomatic and symptomatic children as determined by various epidemiologic studies . The natural history of H . pylori infection is still an enigma . Its association with varied symptoms in children is considered controversial because it is frequently associated with nonulcerative gastritis in this age group . This article focuses on some of the newer developments addressing the epidemiology, transmission, pathophysiology, clinical presentation, and treatment of H . pylori infection in the pediatric population.

Vet Immunol Immunopathol, 2000 Aug 31, 76(1-2), 151 - 6
Effect of isoproterenol and dexamethasone on the lipopolysaccharide induced expression of CD11b on bovine neutrophils; Diez-Fraile A et al.; The present experiments investigate the changes in expression of CD11b on bovine neutrophils and its modulation by isopropylnoradrenaline (IPN, isoproterenol), dexamethasone (DX), phenylephrine (alpha-agonist) and clenbuterol (beta-agonist) . Both IPN and DX caused a dose-dependent inhibition of LPS-induced CD11b expression . A combination of IPN and DX elicited a synergistical decrease of the CD11b expression . Clenbuterol mimicked the effect of IPN, whereas phenylephrine did not . The effect of IPN and DX could at least partly be mediated through a decreased TNF-alpha production by monocytes since tumor necrosis factor-alpha (TNF-alpha) is shown to mediate a dose-dependent CD11b up-regulation . Stimulation of stress hormone receptors partly immuno-suppresses neutrophil functions by inhibition of CD11b expression on the neutrophil surface upon LPS stimulation . This inhibition is probably related to a decrease in TNF-alpha production . A similar mechanism of immuno-suppression could contribute to the higher susceptibility of cattle to Gram-negative bacterial infections of the udder and lung during periods of stress.

Pathol Int, 2000 Aug, 50(8), 660 - 6
Sclerosing encapsulating peritonitis and non-occlusive mesenteric infarction found at autopsy in a man who had undergone continuous ambulatory peritoneal dialysis: a histochemical and immunohistochemical study; Ohmori T et al.; This is a report of a post-mortem histological, histochemical, and immunohistochemical examination of a rare case of sclerosing encapsulating peritonitis (SEP) and non-occlusive mesenteric infarction (NOMI), two serious complications of continuous ambulatory peritoneal dialysis (CAPD), with which a man suffering hepatitis C virus (HCV)-induced liver cirrhosis for 7 years and trauma-induced paraplegia for 50 years had been treated for 1 year . The direct cause of death was encephalopathy caused by extreme hyperammonemia (11 250 microg/dL in serum) . The autopsy revealed that the SEP had drastically reduced the length of the small intestine to 210 cm, 180 cm of which presented acute ischemic enteritis with Gram-negative bacterial infection . Histological examination of the SEP revealed that the exterior was composed of normal serosal elastic lamina, but with a cocoon-like appearance remarkably thickened by fibrosis to 3-8 times that of the normal subserosal layer and consisting of spindle cells and blood vessels, with some infiltration of mast cells and lymphocytes . The immunohistochemical examination of the spindle cells revealed few AE1/AE3(+) cells, HHF35(+) cells, and CD34(+) cells, many CD117(+) cells with slight proliferative activity based on MIB-1 positivity (proliferation index <1%), but no CD44(+) cells . It was concluded that either the few CD34(+) and/or the many CD117(+) cells were mesenteric stem cells that had originated from the serosa, proliferated, then differentiated into myofibroblasts or fibroblasts, producing collagen and hyaluronic acid in the matrix, leading to the gradual formation of the SEP, which was induced by the continual irritation of CAPD.

Extremophiles, 2000 Aug, 4(4), 237 - 45
A new facultatively autotrophic hydrogen- and sulfur-oxidizing bacterium from an alkaline environment; Sorokin DY et al.; An alkaliphilic bacterium, strain AHO 1, was isolated from an enrichment culture with hydrogen at pH 10 inoculated with a composite sample of sediments from five highly alkaline soda lakes (Kenya) . This bacterium is a gram-negative, nonmotile, rod-shaped, obligately aerobic, and facultatively autotrophic hydrogen-oxidizing organism . It was able to oxidize reduced sulfur compounds to sulfate during heterotrophic growth . It utilized a wide range of organic compounds as carbon and energy sources and grew mixotrophically with hydrogen and acetate . With sulfur compounds, mixotrophic growth was observed only in acetate-limited continuous culture . The normal pH range for autotrophic growth with hydrogen was pH 8.0-10.25, with a pH optimum at 9-9.5 . Growth at pH values lower than 8.0 was extremely slow . Heterotrophic growth with acetate was optimal at pH 10.0 . The hydrogen-oxidizing activity of whole cells was maximal at pH 9.0 and still substantial up to pH 11 . NAD-dependent hydrogenase activity was found in the soluble fraction of the cell-free extract, but no methylene blue-dependent activity in either the soluble or membrane fractions was observed . On the basis of its pH profile, the soluble hydrogenase of strain AHO 1 was a typical pH-neutral enzyme . Phylogenetic analysis revealed that strain AHO 1 belongs to the alpha-3 subgroup of the Proteobacteria with a closest relation to a recently described alkaliphilic aerobic bacteriochlorophyll a-containing bacterium "Roseinatronobacter thiooxidans."

Med J Malaysia, 1999 Mar, 54(1), 52 - 7
Nosocomial bacterial sepsis in babies weighing 1000-1499 g in Kelantan; Halder D et al.; From January to December 1992, 92 babies weighing 1000-1499 gm here to referred as very low birth weight (VLBW) were admitted to NICU (Neonatal Intensive Care Unit), Hospital University Sains Malaysia (HUSM) . Sixty babies were inborn giving a VLBW rate of 7.5 per 1000 live births . Incidence of nosocomial sepsis was 32.6% (30/92) of whom 43.3% (13/30) died . Eighty percent (24/30) of the septic babies had blood culture positive for gram negative organisms of which 40% (12/30) were sensitive only to imipenem . Ventilator support within 24 hours of life was required in 41.3% (38/94) babies of whom 42% (16/38) babies developed nosocomial sepsis . Delayed initiation of feeding was significantly associated with nosocomial sepsis . A strict asepsis policy and early feeding of the VLBW infant are essential components of any strategy to prevent of sepsis due to nosocomial infection.

Clin Exp Allergy, 2000 Sep, 30(9), 1230 - 4
Exposure to endotoxin or other bacterial components might protect against the development of atopy; von Mutius E et al.; BACKGROUND: Several recent studies have shown that growing up on a farm confers significant protection against the development of atopy . These findings point particularly towards the importance of exposure to stable dust and farm animals . It has furthermore been reported that endotoxin, an intrinsic part of the outer membrane of gram negative bacteria, is abundant in environments where livestock and poultry is kept . The aim of this study was therefore to measure the level of environmental endotoxin exposure in homes of farmers' children, children with regular contact to livestock and control children with no contact to farm animals . METHODS: Eighty-four farming and nonfarming families were identified in rural areas in Southern Germany and Switzerland . Samples of settled and airborne dust were collected in stables, and of settled dust indoors from kitchen floors and the children's mattresses . Endotoxin concentrations were determined by a kinetic Limulus assay . RESULTS: Endotoxin concentrations were highest in stables of farming families, but were also significantly higher indoors in dust from kitchen floors (143 EU/mg vs 39 EU/mg, P < 0.001) and children's mattresses (49479 EU/m2 vs 9383 EU/m2, P < 0.001) as compared to control children from nonfarming families . In addition, endotoxin levels were also significantly higher in mattresses and dust from kitchen floors in households where children had regular contact to farm animals (38.6 EU/mg and 23340 EU/m2, respectively) as compared to control subjects . CONCLUSION: We propose that the level of environmental exposure to endotoxin and other bacterial wall components is an important protective determinant for the development of atopic diseases in childhood.

J Clin Microbiol, 2000 Sep, 38(9), 3394 - 8
Multilaboratory validation of rapid spot tests for identification of Escherichia coli; York MK et al.; To validate the accuracy of rapid tests for identification of Escherichia coli, five laboratories sequentially collected 1,064 fresh, clinically significant strains with core criteria of indole-positive, oxidase-negative, nonspreading organisms on sheep blood agar plates (BAP), having typical gram-negative rod plate morphology, defined as good growth on gram-negative rod-selective media . An algorithm using beta-hemolysis on BAP, lactose reaction on eosin-methylene blue or MacConkey agar, L-pyrrolidonyl-beta-naphthylamide (PYR), and 4-methylumbelliferyl-beta-D-glucuronide (MUG) was evaluated . Identifications using the algorithm were compared to those obtained using commercial kit system identifications . One thousand strains were E . coli and 64 were not E . coli by kit identifications, which were supplemented with conventional biochemical testing of low probability profiles . Of the 1,064 isolates meeting the core criteria, 294 were beta-hemolytic and did not require further testing to be identified as E . coli . None of the 64 non-E . coli strains were hemolytic, although other indole-positive, lactose-negative species were found to be hemolytic when further strains were examined in a follow-up study . Of the remaining strains, 628 were identified as E . coli by a lactose-positive and PYR-negative reaction . For nonhemolytic, lactose-negative E . coli, PYR was not helpful, but a positive MUG reaction identified 65 of 78 isolates as E . coli . The remaining 13 E . coli strains required kit identifications . This scheme for E . coli identification misidentified three non-E . coli strains as E . coli, for an error rate of 0.3% . A total of 13 kit identifications, 657 PYR tests, and 113 MUG tests were needed to identify 1,000 E . coli strains with the algorithm . The use of this rapid system saves laboratory resources, provides timely identifications, and yields rare misidentifications.

Bone Marrow Transplant, 2000 Aug, 26(3), 251 - 5
High rate of secondary viral and bacterial infections in patients undergoing allogeneic bone marrow mini-transplantation; Mohty M et al.; New approaches using nonmyeloablative-conditioning regimens have been developed to cause minimal procedure-related toxicity . Such novel therapeutic options are being explored with good preliminary results concerning feasibility and engraftment . However many aspects remain under-evaluated, and few data are available about viral and nonviral infections after these highly immunosuppressive regimens . We present our preliminary data on 21 patients receiving a highly immunosuppressive conditioning strategy, focusing on early infectious complications . Early viral infections before day 45, especially CMV, occurred at a high rate (65%) . Furthermore, 33% of patients presented with late bacterial infections (predominately gram negative) although they were not neutropenic compared to conventional conditioning regimens . Although there is presently real interest in these new conditioning regimens which result in reduced immediate transplant-related mortality, it is important that investigators be aware of these pitfalls which may secondarily increase transplant toxicity . Further studies are needed to confirm these findings.

Toxicology, 2000 Aug 21, 149(2-3), 75 - 87
Potentiation of mercury-induced nephrotoxicity by endotoxin in the Sprague-Dawley rat; Rumbeiha WK et al.; Endotoxin (lipopolysaccharide; LPS) and mercury are nephrotoxic compounds of food safety concern . Endotoxin is a product of cell walls of gram negative bacteria . Humans are constantly exposed to LPS through food, water and air . Food is the main source of mercury exposure for humans . Endotoxin potentiates the toxicity of a number of xenobiotics, but its interaction with nephrotoxic heavy metals has not been investigated . We tested the hypothesis that endotoxin enhances mercury-induced nephrotoxicity . Thirty-two, 41-43-day-old, male Sprague-Dawley rats were allocated randomly to four groups of eight rats each as follows: group I received 0.9% sodium chloride, group II received 2.0 mg of Escherichia coli 0128:B12 LPS kg(-1) once, group III received 0.5 mg mercuric chloride kg(-1) once, and group IV received 2.0 mg E . Coli 0128:B12 LPS kg(-1) once 4 h before receiving 0.5 mg mercury chloride kg(-1) once . Mercury, LPS and 0.9% sodium chloride were all injected IV through the tail vein . Rats were monitored for 48 h after mercury injection . Serum creatinine, urea nitrogen, and polyuria were significantly increased in rats given LPS plus mercury relative to those given either agent alone or saline (P</=0.05) . The most severe morphologic lesions were found in rats given LPS plus mercury, which also had significantly greater renal mercury concentration than those given mercury alone (P < or = 0 . 05) . In conclusion, LPS potentiated mercury-induced nephrotoxicity.

Microbes Infect, 2000 Jul, 2(9), 1061 - 72
Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story; Stathopoulos C et al.; Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane . Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host . Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers . This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.

Appl Environ Microbiol, 2000 Sep, 66(9), 3778 - 83
A serine protease-encoding gene (aprII) of Alteromonas sp . Strain O-7 is regulated by the iron uptake regulator (Fur) protein; Tsujibo H et al.; The ferric uptake regulator (Fur) box-like sequence was located upstream of the serine protease-encoding gene (aprII) from a marine bacterium, Alteromonas sp . strain O-7 . To clarify whether the production of AprII (the gene product of aprII) is regulated by the environmental iron concentrations, this strain was cultured under iron-depleted or iron-rich conditions and the level of AprII in the culture supernatant was analyzed by Western blotting . The production of AprII was significantly repressed under iron-rich conditions . Northern hybridization analysis demonstrated that AprII biosynthesis was regulated by iron through the control of transcription . These results indicate that aprII is a new member of the iron regulon and plays an important role in the iron acquisition system of the strain . Furthermore, the gene encoding Fur was cloned and sequenced . The deduced amino acid sequence of the cloned Fur showed high sequence similarity with that from gram-negative bacteria.

Cell, 2000 Aug 18, 102(4), 487 - 97
Translation/secretion coupling by type III secretion systems; Karlinsey JE et al.; Type III secretion systems mediate export of virulence proteins and flagellar assembly subunits in Gram-negative bacteria . Chaperones specific to each class of secreted protein are believed to prevent degradation of the secreted substrates . We show that an additional role of chaperones may be to regulate translation of secreted proteins . We show that the chaperone FIgN is required for translation of the flgM gene transcribed from one mRNA transcript (a flagellar class 3 transcript), but not from another (a flagellar class 2 transcript) . FIgM translated from the class 3 transcript is primarily secreted whereas FIgM translated from the class 2 transcript is primarily retained in the cytoplasm . These results suggest FIgM and other type III secretion substrates possess both mRNA and amino acid secretion signals, and supports a new role for type III chaperones in translation/secretion coupling.

Neuroimmunomodulation, 2000, 8(2), 91 - 7
Lipopolysaccharide-induced leptin release is not mediated by nitric oxide, but is blocked by dexamethasone; Mastronardi CA et al.; The adipocyte hormone, leptin, has homology with tumor necrosis factor-alpha (TNF-alpha) and the leptin receptor (OB-Rb) has homology with the interleukin(IL)-6 receptor . Lipopolysaccharide (LPS) from gram-negative bacteria stimulates the release of many pro-inflammatory cytokines, such as TNF-alpha, IL-1 and IL-6, among others . To test the hypothesis that LPS would also stimulate the release of leptin, LPS (0.6 mg/kg) was injected intravenously into conscious male rats bearing external jugular venous catheters . Vehicle (0.9% NaCl) was injected into control animals . Blood samples (0.3 ml) were drawn immediately before injection and every 10 min afterwards for 120 min . Plasma leptin concentrations increased gradually in the LPS-treated rats, reaching a peak at 120 min of nearly twice the starting level . To determine if this release was mediated by nitric oxide (NO), nitroarginine methyl ester (NAME), an inhibitor of NO synthase, was injected at doses of 11 or 23 mg/kg (i . v.) . The lower dose decreased plasma leptin slightly, whereas the higher dose of NAME increased plasma leptin at 110 min but had no effect on LPS-induced leptin release . On the other hand, dexamethasone (DEX) (0.85 mg/kg), a synthetic glucocorticoid, injected 15 min before LPS, decreased LPS-induced leptin release by 80% (p < 0.001) . The results indicate that LPS is a stimulant of leptin release, that NO has little control over basal or LPS-induced leptin release, but that glucocorticoids, epitomized by DEX, largely block LPS-induced leptin release . Increased release of leptin during infection may contribute to the decreased feeding, altered hormonal release and metabolic changes that occur during infection .

J Biochem (Tokyo), 2000 Sep, 128(3), 371 - 5
Purification and characterization of cytochrome c-553 from Helicobacter pylori; Koyanagi S et al.; Helicobacter pylori, a microaerophilic Gram-negative spiral bacterium residing in the human stomach, contains a small size soluble cytochrome c . This cytochrome c was purified from the soluble fraction of H . pylori by conventional chromatographies involving octyl-cellulose and CM-Toyopearl . Its reduced form gave an alpha absorption band at 553 nm, and thus the cytochrome was named H . pylori cytochrome c-553 . The cytochrome, giving a band below 10,000 Da upon SDS-PAGE, was determined to have a mass of 8,998 by time of flight mass spectroscopy . Its N-terminal peptide sequence was TDVKALAKS---, indicating that the nascent polypeptide was cleaved to produce a signal peptide of 19 amino acid residues and a mature protein composed of 77 amino acid residues . The cb-type cytochrome c oxidase oxidized ferrocytochrome c-553 of this bacterium actively (V(max) of about 250 s(-1)) with a small K(m) (0.9 microM) . Analysis of the effect of the salt concentration on the oxidase activity indicated that oxidation of cytochrome c-553 is highly inhibited under high ionic conditions . The amino acid sequence of H . pylori cytochrome c-553 showed the closest similarity to that of Desulfovibrio vulgaris cytochrome c-553, and these sequences showed a weak relationship to that of the cytochrome c(8)-group among class I cytochromes c.

Plasmid, 2000 Sep, 44(2), 201 - 7
A broad-host-range plasmid for isolating mobile genetic elements in gram-negative bacteria; Schneider D et al.; Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria . The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance . In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons . A new insertion sequence (IS) element was identified in Ralstonia eutropha .

Plasmid, 2000 Sep, 44(2), 152 - 62
Transcription reporters that shuttle cloned DNA between high-copy Escherichia coli plasmids and low-copy broad-host-range plasmids; Ouimet MC et al.; We describe and apply lacZ transcription reporter plasmids designed for both biochemical analyses requiring high DNA yield and physiological studies requiring low gene dosage . Standard DNA ligations are performed at seven unique restriction sites 5' to the lacZ gene on high-copy ColE1 plasmids suitable for double- or single-strand DNA sequencing . A divergent gusA transcription reporter is included and serves as an internal control . Rec(+) Escherichia coli cells readily shuttle DNA placed between gusA and lacZ by allelic exchange with pRK290-based plasmids that subsequently conjugate and replicate in most gram-negative bacteria . We applied this system to study Caulobacter crescentus cell cycle promoters directed by the CtrA response-regulator protein . Synthetic oligonucleotides were ligated to create altered CtrA binding sites and corresponding promoters with varied transcription strength . We also document the phenomenon of long-range promoter interference . A strong promoter can repress up to twofold the transcription from a divergent promoter located 100 bp away . However, the cell cycle timing of both promoters is not changed . Additional applications of our system and theoretical aspects of promoter organization are discussed .

Microvasc Res, 2000 Sep, 60(2), 121 - 30
Morphine attenuates leukocyte/endothelial interactions; Ni X et al.; Gram-negative sepsis and subsequent endotoxic shock after surgery remain problematic in the United States and throughout the world . While morphine is widely prescribed for postoperative trauma pain management, there are reports that morphine may compromise the immune system and contribute to postoperative sepsis . The current study tested the hypothesis that morphine attenuates leukocyte rolling and sticking in both arterioles and venules via nitric oxide production . Nude mice implanted with slow-release morphine pellets were used in this study . The dorsal skinfold chamber model for intravital fluorescence microscopy on awake mice was used . Leukocyte/endothelial interactions were evaluated after bolus injection of oxidized low density lipoprotein . Morphine was found to significantly attenuate leukocyte rolling and sticking in both the arterial and venular side of the microcirculation . This attenuation was reversed by simultaneous implantation of naloxone pellets . The mechanisms of this attenuation were further investigated by administration of the nitric oxide synthase inhibitors NG-nitro-l-arginine (NOLA) and aminoguanidine (AG) in drinking water . NOLA was found to significantly reverse this morphine-induced attenuation of leukocyte rolling and sticking in both arterioles and venules . However, AG did not have the same effect . The results indicate that morphine interferes with leukocyte/endothelial cell interactions via stimulation of nitric oxide production .

Allergy, 2000 Aug, 55(8), 705 - 11
Association of asthma symptoms and severity with indoor bioaerosols; Ross MA et al.; BACKGROUND: In this study, repeated measurements were made of levels of mold spores, bacteria, and dust-mite allergens over a 7-month period in the homes of asthmatics, and relationships with measures of asthma severity were evaluated . METHODS: A sample of 57 asthmatic individuals, living in 44 homes in East Moline, Illinois, and nearby communities, participated in a panel study . The homes were visited up to nine times during the study to collect air and dust samples . Asthma severity indicators were derived from questionnaire data and from the daily health records from the panel study . Associations between indoor levels of mold spores, bacteria, and dust-mite allergens were tested with several asthma severity indicators . RESULTS: There was evidence of associations between all asthma severity measures and levels of total and gram-negative bacteria, but mold-spore abundance was associated only with emergency room (ER) visits for asthma . No significant associations were found with house-dust-mite allergen and any of the asthma severity indicators, but the levels of dust-mite allergen were low, with median concentrations of 0.18 microg/g dust Der f 1 and 0.19 microg/g dust Der p 1 . CONCLUSIONS: Some evidence was found for associations of increased concentrations of gram-negative bacteria and mold spores with asthma severity, particularly with ER visits . No association was found between house-dust-mite allergen and asthma severity indicators; however, the mite-allergen levels in the study homes were generally well below the proposed threshold level of 2 microg/g dust.

Nucleic Acids Res, 2000 Sep 1, 28(17), 3216 - 23
Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome; Kong H et al.; Helicobacter pylori is a gram-negative bacterium, which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases . The genomic sequences of two H.pylori strains, 26695 and J99, have been published recently . About two dozen potential restriction-modification (R-M) systems have been annotated in both genomes, which is far above the average number of R-M systems in other sequenced genomes . Here we describe a functional analysis of the 16 putative Type II R-M systems in the H . pylori J99 genome . To express potentially toxic endonuclease genes, a unique vector was constructed, which features repression and antisense transcription as dual control elements . To determine the methylation activities of putative DNA methyltransferases, we developed polyclonal antibodies able to detect DNA containing N6-methyladenine or N4-methylcytosine . We found that <30% of the potential Type II R-M systems in H.pylori J99 strain were fully functional, displaying both endonuclease and methyltransferase activities . Helicobacter pylori may maintain a variety of functional R-M systems, which are believed to be a primitive bacterial 'immune' system, by alternatively turning on/off a subset of numerous R-M systems.

J Infect Dis, 2000 Sep, 182(3), 983 - 7 Epub 2000 Aug 17.
Effect of thalidomide on chemokine production by human microglia; Lokensgard JR et al.; Thalidomide, a psychoactive drug that readily crosses the blood-brain barrier, has been shown to possess immunomodulatory attributes, including the inhibition of cytokine production by monocytes and microglia . In this study, we investigated the effect of thalidomide on chemokine production by human microglial cells . Microglial cells were stimulated with lipopolysaccharide, a key cell-wall component of gram-negative bacteria responsible for meningitis, and production of chemokines (regulated upon activation normally T cell expressed and secreted {RANTES}, monocyte chemoattractant protein {MCP}-1, macrophage inflammatory protein {MIP}-1beta, and interleukin {IL}-8) was examined by ELISA . Thalidomide treatment was found to cause potent and selective inhibition of IL-8 production in a dose-responsive manner . This inhibition was associated with decreased intracellular IL-8 staining as well as reduced transcription of IL-8 mRNA . In addition, thalidomide treatment of lipopolysaccharide-stimulated microglia inhibited the activation of protein NF-kappaB, a transcription factor known to be important for IL-8 production . These results suggest thalidomide could have a therapeutic role in acute bacterial meningitis through inhibition of IL-8-mediated neutrophil chemotaxis.

Infect Immun, 2000 Sep, 68(9), 5056 - 61
Application of signature-tagged mutagenesis for identification of escherichia coli K1 genes that contribute to invasion of human brain microvascular endothelial cells; Badger JL et al.; Escherichia coli K1 is the leading cause of gram-negative bacterial meningitis in neonates . It is principally due to our limited understanding of the pathogenesis of this disease that the morbidity and mortality rates remain unacceptably high . To identify genes required for E . coli K1 penetration of the blood-brain barrier (BBB), we used the negative selection strategy of signature-tagged transposon mutagenesis (STM) to screen mutants for loss or decreased invasion of human brain microvascular endothelial cells (HBMEC) which comprise the BBB . A total of 3,360 insertion mutants of E . coli K1 were screened, and potential HBMEC invasion mutants were subjected to a secondary invasion screen . Those mutants that failed to pass the serial invasion screens were then tested individually . Seven prototrophic mutants were found to exhibit significantly decreased invasive ability in HBMEC . We identified traJ and five previously uncharacterized loci whose gene products are necessary for HBMEC invasion by E . coli K1 . In addition, cnf1, a gene previously shown to play a role in bacterial invasion, was identified . More importantly, a traJ mutant was attenuated in penetration of the BBB in the neonatal rat model of experimental hematogenous meningitis . This is the first in vivo demonstration that traJ is involved in the pathogenesis of E . coli K1 meningitis.

Science, 2000 Aug 18, 289(5482), 1185 - 8
Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase; Belaaouaj A et al.; In determining the mechanism of neutrophil elastase (NE)-mediated killing of Escherichia coli, we found that NE degraded outer membrane protein A (OmpA), localized on the surface of Gram-negative bacteria . NE killed wild-type, but not OmpA-deficient, E . coli . Also, whereas NE-deficient mice had impaired survival in response to E . coli sepsis, as compared to wild-type mice, the presence or absence of NE had no influence on survival in response to sepsis that had been induced with OmpA-deficient E . coli . These findings define a mechanism of nonoxidative bacterial killing by NE and point to OmpA as a bacterial target in host defense.

Angew Chem Int Ed Engl, 2000 Jun 16, 39(12), 2042 - 2052
The Tetracycline Repressor-A Paradigm for a Biological Switch; Saenger W et al.; The excessive use of antibiotics has enabled bacteria to develop resistance through a variety of mechanisms . The most common bacteriostatic action of the broad-spectrum antibiotic tetracycline (Tc) is by the inactivation of the bacterial ribosome so that the protein biosynthesis is interrupted and the bacteria die . The most common mechanism of resistance in gram-negative bacteria against Tc is associated with the membrane-intrinsic protein TetA, which exports invaded Tc out of the bacterial cell before it can attack its target, the ribosome . The expression of TetA is tightly regulated by the homodimeric Tet repressor (TetR)(2), which binds specifically with two helix-turn-helix motifs of operator DNA (tetO; K(ass) approximately 10(11) M(-1)) located upstream from the tetA gene on a plasmid or transposon . When Tc diffuses into the cell it chelates Mg(2+) and the complex {MgTc}(+) binds to (TetR)(2) to form the induced complex (TetR small middle dot{MgTc}(+))(2) . This process is associated with conformational changes, which sharply reduce the affinity of (TetR)(2) to tetO, so that expression of TetA can take place, thus conferring resistance to the bacteria cells against Tc . Crystallographic studies show sequence-specific protein-nucleic acid interactions in the (TetR)(2) small middle dottetO complex and how the binding of two {MgTc}(+) to (TetR)(2) enforces conformational changes that are stabilized by cooperative binding of two chains of eight water molecules each so that the formed (TetR small middle dot{MgTc}(+))(2) is no longer able to recognize and bind to tetO . Since the switching mechanisms of the TetR/{MgTc}(+) system is so tight, it has proven very useful in the regulation of eukaryotic gene expression and may also be applicable in gene therapy.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1539 - 46
Acidisphaera rubrifaciens gen . nov., sp . nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments; Hiraishi A et al.; Four strains of aerobic, mesophilic, acidophilic bacteria that produced bacteriochlorophyll (BChl) a were isolated from acidic hot springs and mine drainage . The characteristics of the four isolates were almost identical . The isolates were strictly aerobic and chemo-organotrophic . They were gram-negative, non-motile cocci and coccobacilli, formed salmon-pink colonies on solidified media and produced BChl a and carotenoids only under aerobic growth conditions . The cells also produced small amounts of zinc-substituted BChl a when grown in the presence of 1 mM zinc sulfate . Anaerobic growth in the light was not found, but aerobic growth was stimulated by continuous incandescent illumination . The isolates grew in a pH range of 3.5-6.0, with pH optima of 4.5-5.0 . A phylogenetic analysis based on 16S rDNA sequences showed that the isolates clustered in the major acidophilic group of the class Proteobacteria, which includes species of the genera Acidiphilium and Rhodopila . The anaerobic phototrophic bacterium Rhodopila globiformis was the closest relative to the new isolates (95% level of sequence similarity) . The G+C content of the genomic DNA of the isolates was 69.1-69.8 mol% . On the basis of these results, it was concluded that the four isolates should be classified into a new genus and a new species, for which the name Acidisphaera rubrifaciens is proposed . The type strain is strain HS-AP3T (= JCM 10600T).

J Mol Microbiol Biotechnol, 2000 Apr, 2(2), 195 - 8
A novel ubiquitous family of putative efflux transporters; Harley KT et al.; We describe a novel family of putative efflux transporters (PET) found in bacteria, yeast and plants . None of the members of the PET family has been functionally characterized . The bacterial and yeast proteins display a duplicated internal repeat element consisting of an N-terminal hydrophobic sequence of about 170 residues, exhibiting six putative transmembrane alpha-helical spanners (TMSs), followed by a large (230 residue), C-terminal, hydrophilic, cytoplasmic domain . The plant proteins exhibit only one such unit, but they have a larger C-terminal cytoplasmic domain . Arabidopsis thaliana encodes at least seven paralogues of the PET family . The gram-negative bacterial proteins are sometimes encoded by genes that are found in operons that also contain genes that encode membrane fusion proteins . This fact strongly suggests that PET family proteins are efflux pumps . The sequence, topological and phylogenetic characteristics of these proteins as well as the operonic structures of their encoded genes when relevant are described.

J Mol Microbiol Biotechnol, 2000 Apr, 2(2), 125 - 44
Phylogenetic analyses of the constituents of Type III protein secretion systems; Nguyen L et al.; Multicomponent Type III protein secretion systems transfer gram-negative bacterial virulence factors directly from the bacterial cytoplasm to the cytoplasm of a host eukaryotic cell in a process that may involve a single energy-coupled step . Extensive evidence supports the conclusion that the genetic apparatuses that encode these systems have been acquired independently by different gram-negative bacteria, presumably by lateral transfer . In this paper we conduct phylogenetic analyses of currently sequenced constituents of these systems and their homologues . The results reveal the relative relatedness of these systems and show that they evolved with little or no exchange of constituents between systems . This fact suggests that horizontal transmission of the genes encoding these systems always occurred as a unit without the formation of hybrid gene clusters . Moreover, homologous flagellar proteins show phylogenetic clustering that suggests that the flagellar systems and Type III protein secretory systems diverged from each other following very early duplication of a gene cluster sharing many (but not all) genes . Phylogenies of most or all of the flagellar proteins follow those of the source organisms with little or no lateral gene transfer suggesting that homologous flagellar proteins are true orthologues . We suggest that the flagellar apparatus was the evolutionary precursor of Type III protein secretion systems.

APMIS, 2000 May, 108(5), 349 - 56
Lipid profiles of Helicobacter pylori colony variants; Tannaes T et al.; A phase variation in Helicobacter pylori has been previously described . In one phase the bacterium had a cell wall lipid content typical for gram-negative bacteria (HpL), whereas in the other phase the bacterium was found to have a cell wall with increased amounts of lysophospholipids (HpS) . The conversion is spontaneous, but could also be induced by acid (HpS(ind)) and was associated with in vitro release of Vac A and urease . The purpose of the present study was to determine the full phospholipid content of the cell wall to indicate a molecular mechanism of the colony variation . There were no appreciable differences between the lipid profiles of HpS and HpS(ind), while there were major differences between HpL and the S-variant . In the S-variant, lysophospholipids constituted about 50% of the total phospholipids, as compared to less than 2% in the L-variant . The proportion of total and individual cholesteryl glucosides also showed considerable changes . HpL was dominated by the phosphate-linked cholesteryl glucoside (72%) while the acylated cholesteryl glucoside was the main cholesteryl glucoside of the S-variant (65%) . Our results demonstrate a dramatic change in cell wall properties after acid induction and spontaneously in vitro, and suggest some molecular mechanisms for this variation from an in vitro non-virulent to a virulent variant.

Biotechnol Prog, 2000 Jul-Aug, 16(4), 557 - 63
Expression of single chain antibodies (ScFvs) for c-myc oncoprotein in recombinant Escherichia coli membranes by using the ice-nucleation protein of Pseudomonas syringae; Bassi AS et al.; The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in certain Gram-negative bacteria . In this study, the INP from Pseudomonas syringae was applied as a fusion partner with the single chain antibody fragment (ScFv) against the human oncoprotein c-myc . Two new plasmids pNinaZ-myc and pNinaZScFv-BsaA1 were constructed and cloned into Escherichia coli JM109 . The expression of the fusion protein was successfully demonstrated in the cloned cells . The fusion proteins had no effect on the viability of the host cells . Ice nucleation activity measurements and flow cytometry studies were followed to investigate the membrane expression of the fusion protein.

Mol Microbiol, 2000 Jul, 37(2), 239 - 53
Structure and function of bacterial outer membrane proteins: barrels in a nutshell; Koebnik R et al.; The outer membrane protects Gram-negative bacteria against a harsh environment . At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction . Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane alpha-helices, but instead fold into antiparallel beta-barrels . Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined . They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA . These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins . Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.

Syst Appl Microbiol, 2000 Jun, 23(2), 210 - 8
Methylopila helvetica sp . nov . and Methylobacterium dichloromethanicum sp . nov.--novel aerobic facultatively methylotrophic bacteria utilizing dichloromethane; Doronina NV et al.; Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described . These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source . The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1 . The main quinone of all the strains is Q-10 . The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization . All strains belong to the alpha-subgroup of the alpha-Proteobacteria . 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov . with the type strain DM9 (=VKM B-2189) . The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence . Strain DM4 is named Methylobacterium dichloromethanicum sp . nov . (VKM B-2191 = DSMZ 6343).

Toxicol Pathol, 2000 Jul-Aug, 28(4), 588 - 600
The prophylactic effects of natural water-soluble antioxidant from spinach and apocynin in a rabbit model of lipopolysaccharide-induced endotoxemia; Lomnitski L et al.; Radical-scavenging antioxidants, as part of the cellular defense system, function to inhibit the formation and propagation of free radicals and active oxygen species formation . In previous studies we demonstrated that endotoxin lipopolysaccharide (LPS) promotes oxidative stress and associated pathological changes in a rat model and that use of selected antioxidants was effective in reducing LPS-related lipid peroxidation product formation in the liver, as well as LPS-related pathological changes in different organs . In this study, several toxicological parameters (ie, clinical signs, blood chemistry, and histopathological changes) were compared among groups of male New Zealand rabbits injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group injected with LPS without pretreatment with antioxidants, groups injected with either of the 2 antioxidants only, and an untreated control group . The antioxidants used were a water-soluble natural antioxidant (NAO) from spinach and the NADPH oxidase inhibitor, apocynin . Exposure to LPS alone was associated clinically with depression, tachypnea, outer ear vasodilation, and iris congestion; biochemically with a significant increase in blood total bilirubin, transaminase activity, and glucose, total cholesterol, and triglyceride levels; macroscopically with multiple whitish areas in the liver; and histologically with hepatocellular focal necrosis and acute inflammation, thymic and splenic lymphoid necrosis and depletion, acute uveitis and hemorrhages in the ciliary processes, and decreased adrenal cortical cytoplasmic vacuolation considered consistent with depletion of steroidal hormone contents . The NAO had more effective prophylactic capacities than the apocynin . The protective effects were obvious in all investigated parameters . The results indicate the possible therapeutic efficacy of NAO in the treatment of clinical endotoxemia associated with gram-negative bacterial sepsis that is known to be associated with oxidative stress.

Nucleic Acids Res, 2000 Aug 15, 28(16), 3100 - 4
Mapping analysis of the Xylella fastidiosa genome; Frohme M et al.; A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping . Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes . The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure . Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process . A subset of the eventual clone coverage was distributed to the Brazilian X.FASTIDIOSA: sequencing network . Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design . Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.

Glycobiology, 2000 Aug, 10(8), 761 - 71
The human gastric colonizer Helicobacter pylori: a challenge for host-parasite glycobiology; Karlsson KA; The Gram-negative bacterium Helicobacter pylori was first described in 1983 and currently represents one of the most active single research topics in biomedicine . It is specific for the human stomach and chronically colonizes a majority of the global population, which results in a symptom-free local inflammation . In 10-20% of carriers, gastroduodenal disease develops, including gastric or duodenal ulcer, and atrophic gastritis, which is a precondition to gastric cancer . A probable long coevolution of microbe and homo sapiens in a restricted niche has apparently generated a complex and sophisticated interplay . Access to complete bacterial genome sequences assists in a comparative functional characterization . A dynamic glycosylation of both microbe and host cells is of growing interest to analyze . Several glycoforms of bacterial surface lipopolysaccharides show advanced molecular mimicry of host epitopes and a distinct phase variation . An unusually large family of 32 outer membrane proteins probably reflects the complex interrelationship with the host . The unique diversity found for carbohydrate-binding specificities may be mediated by these surface proteins, of which the Lewis b-binding adhesin is the only known example so far, and these binding activities are subject to phase variation . The host mucosa glycosylation may also vary with different conditions, allowing a modulated crosstalk between microbe and host . The bacterium actively stimulates the host inflammatory response, apparently for nutritional purposes, and there is no evidence for a spontaneous elimination of the microbe . Colonization appears to be preventive for upper stomach and esophageal diseases . Current antibiotic treatment eradicates the microbe and cures ulcer disease . Alternative approaches must, however, be developed for a potential global prevention of disease.

Br J Pharmacol, 2000 Aug, 130(7), 1646 - 54
Involvement of interleukin-1 in the inflammatory actions of aminobisphosphonates in mice; Yamaguchi K et al.; Aminobisphosphonates (aminoBPs) are potent inhibitors of bone resorption . However, they cause undesirable inflammatory reactions, including fever, in humans . Intraperitoneal injection of aminoBPs into mice also induces inflammatory reactions, including a prolonged elevation of the activity of the histamine-forming enzyme, histidine decarboxylase (HDC) . Because interleukin-1 (IL-1) is a typical pyrogen and a strong inducer of HDC, we examined whether aminoBPs induce inflammatory reactions in mice deficient in genes for both IL-1alpha and IL-1beta (IL-1-KO mice) . In control mice, aminoBPs induced an elevation of HDC activity and other inflammatory reactions (enlargement of the spleen, atrophy of the thymus, exudate in the thorax and increase in granulocytic cells in the peritoneal cavity) . These responses were all weak or undetectable in IL-1-KO mice . We have previously shown that lipopolysaccharides (LPSs) from Escherichia coli and Prevotella intermedia (a prevalent gram-negative bacterium both in periodontitis and endodontal infections) are capable of inducing HDC activity in various tissues in mice . In control mice treated with an aminoBP, the LPS-induced elevations of serum IL-1 (alpha and beta) and tissue HDC activity were both markedly augmented . However, such an augmentation of HDC activity was small or undetectable in IL-1-KO mice . These results, taken together with our previous findings (i) suggest that IL-1 is involved in the aminoBP-induced inflammatory reactions and (ii) lead us to think that under some conditions, inflammatory reactions induced by gram-negative bacteria might be augmented in patients treated with an aminoBP . In this study, we also obtained a result suggesting that IL-1-deficiency might be compensated by a second, unidentified, mechanism serving to induce HDC in response to LPS when IL-1 is lacking.

Gene, 2000 Jul 25, 253(1), 95 - 105
Structural similarities and evolutionary relationships in chloride-dependent alpha-amylases; D'Amico S et al.; The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion . This search provides 38 alpha-amylases potentially binding a chloride ion . All belong to animals, including mammals, birds, insects, acari, nematodes, molluscs, crustaceans and are also found in three extremophilic Gram-negative bacteria . An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species . On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca(2+) and Cl(-) binding sites, a protease-like catalytic triad and disulfide bonds . The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dependent alpha-amylases.

Curr Microbiol, 2000 Jul, 41(1), 15 - 20
Methanosarcina mazei strain O1M9704, methanogen with novel tubule isolated from estuarine environment; Lai MC et al.; A new methanogenic isolate, designated as strain O1M9704 (=OCM 667), was isolated from the sediment of the estuarine environment in Eriln Shi, Taiwan . This strain grew on trimethylamine and methanol, but it did not catabolize H2-CO2, acetate, or formate . Cells grew optimally at 37 degrees C with 0.5% NaCl in neutral pH . The cells were stained Gram-negative, nonmotile, irregular coccus 0.3-0.6 microm in diameter . A comparison of 16S rDNA sequences phylogenetically related strain O1M9704 to Methanosarcina mazei . Gas vacuoles were observed both under phase contrast microscope and in thin sections in the electron microscope . Negative stain of electron micrographs showed a novel character of strain O1M9704 . with tubule structure extended out of the cells . The tubule structure and gas vacuoles may benefit the adaptation of methanoarchaea in estuarine environment.

Dis Aquat Organ, 2000 Jun 19, 41(2), 115 - 22
Infectivity, transmission and 16S rRNA sequencing of a rickettsia, Coxiella cheraxi sp . nov., from the freshwater crayfish Cherax quadricarinatus; Tan CK et al.; A rickettsia-like organism isolated from infected, farm-reared Cherax quadricarinatus was cultured in the yolk sac of developing chicken eggs, but could not be cultured in 3 continuous cell lines, bluegill fry (BF-2), fathead minnow (FHM), and Spodoptera frugiperda (Sf-9) . The organism was confirmed by fulfilling Koch's postulates as the aetiological agent of mortalities amongst C . quadricarinatus . When C . quadricarinatus was inoculated with the organism, mortality was 100% at 28 degrees C and 80% at an ambient temperature of 24 degrees C . Horizontal transmission with food and via the waterborne route was demonstrated, but mortalities were lower at 30 and 10% respectively over a 4 wk period . The 16S rRNA sequence of 1325 base pairs of the Gram-negative, obligate intracellular organism was 95.6% homologous to Coxiella burnetii . Of 18 species compared to this rickettsia, the next most closely related bacterium was Legionella pneumophila at 86.7% . The suggested classification of this organism is Order Rickettsiales, family Rickettsiaceae, tribe Rickettsieae, within the genus Coxiella . We suggest it should be named Coxiella cheraxi sp . nov.

South Med J, 2000 Jul, 93(7), 708 - 9
Bacteremia and possible endocarditis caused by Moraxella phenylpyruvica; Guttigoli A et al.; Moraxella phenylpyruvica, a gram-negative coccobacillus, is usually considered a nonpathogenic bacterium . Only a few cases of invasive disease caused by this organism were reported in the 1970s . We report a well-documented case of bacteremia and possible endocarditis due to M phenylpyruvica in a previously healthy man.

Intensive Care Med, 2000 May, 26(5), 538 - 44
Endotoxaemia in patients with severe sepsis or septic shock; Venet C et al.; OBJECTIVE: To examine the incidence and the bacteriological and clinical significance of endotoxaemia in ICU patients with severe sepsis or septic shock . DESIGN: Prospective review . SETTING: A 15-bed general ICU in a university hospital . PATIENTS: One hundred sixteen patients hospitalised in our ICU fulfilling Bone's criteria for severe sepsis or septic shock and with an available early endotoxin assay (chromogenic limulus assay) . INTERVENTIONS: None . MEASUREMENTS AND RESULTS: The clinical characteristics of the population were: age 63.6 +/- 11.4 years; SAPS II: 45.4 +/- 15.6; mechanical ventilation: 72.4%; septic shock: 51.7% (n = 60); bacteraemia: 28.4% (n = 33); gram-negative bacteria (GNB) infection 47.4% (n = 55); ICU mortality: 39.6% (n = 46) . Detectable endotoxin occurred in 61 patients (51.2%; mean level: 310 +/- 810 pg/ml) . There was no relationship between detectable endotoxin and severity of infection at the moment of the assay . Endotoxaemia was associated with a higher incidence of bacteraemia (39.3% vs 16.3%; p = 0.01) . There was a trend (p = 0.09) towards an association between positive endotoxin and gram-negative bacteraemia or GNB infection but this was non-significant . This relationship became significant only in the case of bacteraemia associated with GNB infection irrespective of the site of infection . CONCLUSION: Early detection of endotoxaemia appeared to be associated with GNB infection only in cases of bacteraemic GNB infection . Early endotoxaemia correlated neither to occurrence of organ dysfunction nor mortality in patients with severe sepsis or septic shock . This study suggests that the use of endotoxaemia as a diagnostic or a prognostic marker in daily practice remains difficult.

J Clin Microbiol, 2000 Aug, 38(8), 3043 - 7
Molecular identification of Dialister pneumosintes in subgingival plaque of humans; Doan N et al.; Dialister pneumosintes is a nonfermentative, anaerobic, gram-negative rod that grows with small, circular, transparent, shiny, smooth colonies on blood agar . Even though D . pneumosintes has been recovered from deep periodontal pockets, little is known about the relationship between the organism and destructive periodontal disease . This study describes a rapid PCR method to identify D . pneumosintes in periodontal samples . The PCR identification method detected as little as 10 pg of D . pneumosintes DNA or about 1 to 10 cells without nonspecific amplification of various periodontopathic bacteria . Twelve of 22 subgingival samples from adult periodontitis lesions yielded D . pneumosintes either by culture or by PCR identification . In culture-positive samples, D . pneumosintes averaged 3.9% (0.001 to 10.8%) of total isolates . Studies are needed to delineate virulence factors of D . pneumosintes pertinent to periodontal disease.

Mikrobiologiia, 2000 May-Jun, 69(3), 389 - 95
{Characteristics of morphology and ultrastructure of bacteria of the genus Dethiosulfovibrio}; Surkov AV et al.; Cell morphology and fine structure were studied in two strains of rod-shaped, strictly anaerobic, gram-negative sulfidogenic bacteria: strain SR12T (DSM 12538) and strain WS100 (DSM 12537) belonging to "Dethiosulfovibrio starorussensis." Cells of both strains, as well as cells of the type species of the genus Dethiosulfovibrio, D . peptidovorans, were found to possess multiple intracellular incomplete cross septa in the stationary growth phase.

Biochem Biophys Res Commun, 2000 Aug 2, 274(2), 488 - 95
A rolling-circle plasmid from Psychrobacter sp . TA144: evidence for a novel rep subfamily; Tutino ML et al.; In this paper we report the cloning and sequencing of two small plasmids, pTAUp and pTADw, from the Antarctic Gram-negative Psychrobacter sp strain TA144 . The observation that pTAUp contains a putative Rep-coding gene (Psyrep) suggested that its duplication occurs via a rolling-circle replication mechanism . This hypothesis was confirmed by the identification of the pTAUp single-stranded DNA form . The putative pTAUp plus origin of replication was found at the 3' end of the Psyrep by using an in vivo complementation assay . Structural similarities at the level of (i) gene organization, (ii) protein sequence, and (iii) nick site sequences strongly suggest that the psychrophilic enzyme belongs to a new subfamily of replication enzymes .

Acta Paediatr Taiwan, 1999 Jan-Feb, 40(1), 9 - 12
Shock in the pediatric emergency service: five years' experience; Chang P et al.; We undertook a retrospective study of the risk factors determining outcome of nontraumatic patients with shock in the pediatric emergency service . From October 1992 through September 1997, 22 patients with the diagnosis of shock were identified, including 11 with septic shock (50%), 7 with hypovolemic shock (32%) and 4 with cardiogenic shock (18%) . Their age ranged from 2 months to 19 years old . Among the cases, 14 patients (64%) had other underlying diseases . Gram-negative bacterial sepsis (6/11, 55%), dilated cardiomyopathy (2/4, 50%) and acute gastroenteritis (7/7, 100%) were the most frequent causes of septic, cardiogenic and hypovolemic shock, respectively . In total, 12 patients (55%) died . The mortality rate was high in septic shock (9/11, 82%) and cardiogenic shock patients (3/4, 75%), but low in hypovolemic shock patients (0/7, 0%) . The risk factors of poor outcome in patients with shock included thrombocytopenia, prolonged prothrombin time and partial thromboplastin time . Patients with leukopenia, a higher level of C-reactive protein, or under 2 years of age tended to have poor outcome.

Harefuah, 1998 Jul, 135(1-2), 3 - 7, 88
{Brucellosis in pregnancy}; Hackmon R et al.; Brucellosis is rare in pregnancy . Recently, an increase in the incidence of this disease has been observed in our area . We present 7 cases of brucellosis in pregnancy and review the literature on the effects of brucellosis on the outcome of pregnancy . Brucellosis is rare in the Middle East and Africa and the most common source of infection is unpasteurized milk products . Brucella is a coccobacillus, gram-negative bacterium, whose hosts are mostly animals . There is controversy about the relationship between brucellosis and the outcome of pregnancy . There is some evidence that there is a higher rate of complications such as abortion, premature rupture of membranes and preterm delivery in infected animals . Reasons for this difference in the impact of brucella in animals and man include the absence of the carbohydrate erythritol in the human placenta, which appears to be a preferential medium and growth factor for brucella in the placentas of animals . There is uncertainty regarding effects of brucella in early pregnancy and no evidence of its transplacental passage in later pregnancy, causing adverse obstetrical outcome, although recently there has been a single report of Brucella abortus (biotype 2) . We present 7 cases of brucellosis in pregnant women found between 1977-1988 . Its incidence among the women who delivered here is 0.007% (7/92, 768 deliveries) . Our first case was complicated by preterm premature rupture of membranes and preterm delivery in the 20th week of gestation . In 2 other cases there was preterm delivery with 1 developing clinical chorioamnionitis . The 4 remaining women delivered at term, although 1 had preterm premature rupture of membranes and intra-uterine growth retardation, and 2 had postpartum endometritis.

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 9240 - 5
Snapshots of usher-mediated protein secretion and ordered pilus assembly; Saulino ET et al.; Type 1 pilus biogenesis was used as a paradigm to investigate ordered macromolecular assembly at the outer cell membrane . The ability of Gram-negative bacteria to secrete proteins across their outer membrane and to assemble adhesive macromolecular structures on their surface is a defining event in pathogenesis . We elucidated genetic, biochemical, and biophysical requirements for assembly of functional type 1 pili . We discovered that the minor pilus protein FimG plays a critical role in nucleating the formation of the adhesive tip fibrillum . Genetic methods were used to trap pilus subunits during their translocation through the outer membrane usher protein, providing data on the structural interactions that occur between subunit components during type 1 pilus formation . Electron microscopic and biochemical analyses of these stepwise assembly intermediates demonstrated that translocation of pilus subunits occurs linearly through the usher's central channel, with formation of the pilus helix occurring extracellularly . Specialized pilin subunits play unique roles both in this multimerization and in the final ultrastructure of the adhesive pilus.

Protein Eng, 2000 Jul, 13(7), 491 - 500
Effects of pore mutations and permeant ion concentration on the spontaneous gating activity of OmpC porin; Liu N et al.; Porins are trimers of beta-barrels that form channels for ions and other hydrophilic solutes in the outer membrane of Gram-negative bacteria . The X-ray structures of OmpF and PhoE show that each monomeric pore is constricted by an extracellular loop that folds into the channel vestibule, a motif that is highly conserved among bacterial porins . Electrostatic calculations have suggested that the distribution of ionizable groups at the constriction zone (or eyelet) may establish an intrinsic transverse electrostatic field across the pore, that is perpendicular to the pore axis . In order to study the role that electrostatic interactions between pore residues may have in porin function, we used spontaneous mutants and engineered site-directed mutants that have an altered charge distribution at the eyelet and compared their electrophysiological behavior with that of wild-type OmpC . We found that some mutations lead to changes in the spontaneous gating activity of OmpC porin channels . Changes in the concentration of permeant ions also altered this activity . These results suggest that the ionic interactions that exist between charged residues at the constriction zone of porin may play a role in the transitions between the channel's closed and open states.

Toxicol Appl Pharmacol, 2000 Aug 1, 166(3), 173 - 85
Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline; Yee SB et al.; Individuals are commonly exposed to bacterial endotoxin (lipopolysaccharide {LPS}) through gram-negative bacterial infection and from its translocation from the gastrointestinal lumen into the circulation . Inasmuch as noninjurious doses of LPS augment the hepatotoxicity of certain xenobiotic agents, exposure to small amounts of LPS may be an important determinant of susceptibility to chemical intoxication . Monocrotaline (MCT) is a pyrrolizidine alkaloid phytotoxin that at large doses produces centrilobular liver lesions in rats . In the present study, MCT was coadministered with LPS to determine whether LPS would enhance its hepatotoxicity . Doses of MCT (100 mg/kg, ip) and LPS (7.4 x 10(6) EU/kg, iv), which were nonhepatotoxic when administered separately, produced significant liver injury in male, Sprague-Dawley rats when given in combination . Within 18 h after MCT administration, this cotreatment resulted in enhanced plasma alanine aminotransferase and aspartate aminotransferase activities, two markers of liver injury . Histologically, overt hemorrhage and necrosis appeared between 12 and 18 h . The lesions were centrilobular and midzonal and exhibited characteristics similar to lesions associated with larger doses of MCT and LPS, respectively . In the presence of LPS, the threshold for MCT toxicity was reduced to 13-33% of the dose required for toxicity with MCT alone . A study in isolated, hepatic parenchymal cells revealed no interaction between MCT and LPS in producing cytotoxicity . In summary, coexposure of rats to noninjurious doses of MCT and LPS resulted in pronounced liver injury . Results in vitro suggest that the enhanced toxicity does not result from a direct interaction of MCT and LPS with hepatic parenchymal cells . These results provide additional evidence that exposure to small amounts of LPS may be a determinant of susceptibility to food-borne hepatotoxins .

J Biol Chem, 2000 Sep 29, 275(39), 30202 - 10
Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin; Guedin S et al.; Many pathogenic Gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu . The secretion of filamentous hemagglutinin (FHA) by Bordetella pertussis depends on the pore-forming outer membrane protein FhaC, which belongs to a growing family of protein transporters . Protein alignment and secondary structure predictions indicated that FhaC is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface loops and short periplasmic turns . The membrane topology of FhaC was investigated by random insertion of the c-Myc epitope and the tobacco etch virus protease-specific cleavage sequence . FhaC was fairly permissive to short linker insertions . Furthermore, FhaC appeared to undergo conformational changes upon FHA secretion . Surface detection of the inserted sequences indicated that several predicted loops in the C-terminal moiety as well as the N terminus of the protein are exposed . However, a large surface-predicted region in the N-terminal moiety of FhaC was inaccessible from the surface . In addition, the activity and the stability of the protein were affected by insertions in that region, indicating that it may have important structural and/or functional roles . The surface exposure of the N terminus and the presence of an odd number of beta-strands are novel features for beta-barrel outer membrane proteins.

Arq Bras Cardiol, 2000 Feb, 74(2), 119 - 28
Chlamydia pneumoniae and atherosclerosis . Identification of bacterial DNA in the arterial wall; Coutinho MS et al.; OBJECTIVE: The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis . The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification . METHODS: Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed . From these patients, 39 were males and the mean age was 65+/-6 years . The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae . The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls . RESULTS: Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae . CONCLUSION: DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis . This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

Clin Microbiol Rev, 2000 Jul, 13(3), 428 - 38
Bartonella infection in animals: carriership, reservoir potential, pathogenicity, and zoonotic potential for human infection; Breitschwerdt EB et al.; Recent observations have begun to support a role for Bartonella spp . as animal as well as human pathogens . Bartonella spp . are vector-transmitted, blood-borne, intracellular, gram-negative bacteria that can induce prolonged infection in the host . Persistent infections in domestic and wild animals result in a substantial reservoir of Bartonella organisms in nature that can serve as a source for inadvertent human infection . The prevalence of bacteremia can range from 50 to 95% in selected rodent, cat, deer, and cattle populations . Dogs infected with Bartonella spp . can develop lameness, endocarditis, granulomatous lymphadenitis, and peliosis hepatis, lesions that have also been reported in association with human infection . Understanding the role of Bartonella spp . as pathogens in cats and other wild or domestic animals awaits the results of additional studies . Considering the extensive animal reservoirs and the large number of insects that have been implicated in the transmission of Bartonella spp., both animal and human exposure to these organisms may be more substantial than is currently believed.

Digestion, 2000, 62(1), 1 - 13
Protective action of lipopolysaccharidesin rat caerulein-induced pancreatitis: role of nitric oxide; Jaworek J et al.; Lipopolysaccharides (LPS), the component of the cell wall of gram-negative bacteria, have been implicated in the pathogenesis of acute pancreatitis, but the mechanism of their action on the pancreas has not been fully explored . The aim of this study was to investigate the effects of various doses of LPS on the integrity of intact pancreas and that involved in acute caerulein-induced pancreatitis (CIP) in the rat and to compare these effects with those of nitric oxide (NO) donor, S-nitrose-acetylpenicillamine (SNAP) . The expression of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) mRNA was also examined in the isolated pancreatic acini obtained from the inflamed pancreas of rats treated with LPS . CIP was produced by subcutaneous (s.c.) infusion of caerulein (5 microg/kg.h for 5 h) to conscious rats . Bolus injections of various doses of LPS (0.1, 1, 10, 20 or 40 mg/kg) or SNAP (1.5, 3 or 6 mg/kg) were made intraperitoneally (i.p.) either alone or 30 min prior to s.c . infusion of caerulein to induce CIP . Infusion of caerulein produced acute pancreatitis confirmed by histological examination and manifested by an increase of pancreatic mass (by about 200%) . Blood levels of amylase and lipase were augmented by 400 and 800% respectively, whereas the pancreatic blood flow (PBF) was decreased by 50% in rats with CIP . Injection of low doses of LPS (0.1-1 mg/kg i.p.) or SNAP (1.5-3 mg/kg i.p.) 30 min prior to caerulein infusion reversed the harmful effects of pancreatic overstimulation with caerulein and reduced significantly the histological manifestations of CIP such as edema, neutrophil infiltration and vacuolization of the acinar cells . These protective effects of low doses of LPS pretreatment on the pancreas were completely antagonized by the suppression of the activity of NO synthase (NOS) with N(G)-nitro-L-arginine (L-NNA) applied (20 mg/kg i.p.) 15 min prior to the LPS injection . Combination of L-arginine (100 mg/kg i.p.), a substrate for NOS, with L-NNA given prior to low doses of LPS, restored the LPS-induced protection of the pancreas in rats with CIP . In contrast, higher doses of LPS (20-40 mg/kg i.p.) or SNAP (6 mg/kg i.p.), which produced a significant fall of the PBF, did not protect the pancreas against CIP . Administration of various doses of LPS to rats with CIP resulted in significant and dose-dependent stimulation of NO biosynthesis in the isolated acini obtained from the pancreas of these animals . LPS enhanced the expression of both cNOS and iNOS in the pancreatic acini obtained from rats subjected to CIP . The signal for cNOS mRNA was detected in all samples, reaching peak at the protective dose of LPS (1 mg/kg i . p.), while iNOS was overexpressed only at the highest doses of LPS that failed to exhibit the protective activity . We conclude that the pretreatment with low doses of LPS protects the pancreas against the damage provoked by CIP and this effect could be attributed, at least in part, to the activation of L-arginine-NO system in the pancreas .

Eur J Immunol, 2000 Jun, 30(6), 1562 - 7
Differential desensitization of lipopolysaccharide-inducible chemokine gene expression in human monocytes and macrophages; Kaufmann A et al.; Bacterial lipopolysaccharide (LPS) has been shown to induce a wide variety of pro-inflammatory cytokines and chemokines . An initial challenge with minute amounts of LPS causes tolerance to later LPS effects which is characterized by a much lower or abrogated release of pro-inflammatory cytokines . To explore the relationship between the production of chemokines and the induction of LPS tolerance, we pretreated human monocytes with increasing LPS doses and thereafter restimulated with LPS . The re-expression of the CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES was substantially suppressed after pre-incubation with low LPS doses . In striking contrast, the re-expression of neutrophil-attracting IL-8 and melanoma growth stimulatory activity-alpha and of the monocyte-attracting monocyte chemotactic protein-1 remained high and was, in part, initially increased after restimulation with LPS . The corresponding gene expression pattern as determined by Northern blot analyses correlated closely with the release of chemokines and cytokines . Thus, a basic set of chemotactic mediators that are still produced by otherwise LPS-desensitized monocytes/macrophages may ensure the continuing recruitment of monocytes and neutrophils into an inflammatory process caused by gram-negative bacteria.

Am J Physiol Regul Integr Comp Physiol, 2000 Jul, 279(1), R99 - R108
Brief hypoxic stress suppresses postbacteremic NF-kappaB activation and TNF-alpha bioactivity in perfused liver; Loftis LL et al.; Reductions in hepatic O(2) delivery are common early after gram-negative bacteremic sepsis owing to cardiopulmonary dysfunction and derangements in sinusoidal perfusion . Although gram-negative endotoxin and cellular hypoxia independently enhance activation of nuclear factor-kappaB (NF-kappaB) via generation of reactive O(2) species (ROS), the combination of these stimuli downregulates hepatic TNF-alpha gene expression . Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF-alpha gene expression is transcriptionally mediated by reduced activation of NF-kappaB . Buffer-perfused rat livers (n = 52) were studied over 180 min after intraportal infection at t = 0 with 10(9) live Escherichia coli (EC), serotype O55:B5, or 0.9% NaCl controls under normoxic conditions, compared with 0.5 h of constant-flow hypoxia (PO(2) approximately 41 +/- 7 Torr) beginning at t = 30 min, followed by 120 min of reoxygenation . In parallel studies, tissue was obtained at peak hypoxia (t = 60 min) . To determine the role of xanthine oxidase (XO)-induced ROS in modulating NF-kappaB activity after hypoxia/reoxygenation (H/R), livers were pretreated with the XO inhibitor allopurinol, with results confirmed in organs of tungstate-fed animals . Electrophoretic mobility shift assays were performed on nuclear extracts of whole liver lysates using (32)P-labeled oligonucleotides specific for NF-kappaB . Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-kappaB nuclear translocation and TNF-alpha bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O(2) consumption . XO inhibition reversed the hypoxic suppression of NF-kappaB nuclear translocation and ameliorated decreases in cell-associated TNF-alpha . Thus decreases in hepatic O(2) delivery reduce postbacteremic nuclear translocation of NF-kappaB and hepatic TNF-alpha biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.

Arch Microbiol, 2000 May-Jun, 173(5-6), 373 - 82
Effect of metal complexation on the bioavailability of nitrilotriacetic acid to Chelatobacter heintzii ATCC 29600; White VE et al.; Many polluted sites contain a mixture of organics and heavy metals . Nitrilotriacetic acid has been chosen as a model organic compound to study the effect of metal binding on organic bioavailability and degradation of organics . The effect of varying the ratio of metal to nitrilotriacetic acid on its utilisation has been examined using the gram-negative bacterium Chelatobacter heintzii ATCC 29600 . The following parameters of substrate utilisation were examined: growth, degradation, respiration, mineralisation and nitrilotriacetic acid uptake . Complexation of nitrilotriacetic acid by Cu(II), Ni(II), Co(II) and Zn(II) prevented utilisation of nitrilotriacetic acid by C . heintzii; complexation to Fe(III) or Mn(II) did not . The pattern of inhibition was consistent with a 1:1 stoichiometry of metal binding to nitrilotriacetic acid . Inhibition was not due to metal ion toxicity, but was a result of metal-nitrilotriacetic acid complexes being recalcitrant to degradation . In addition, the effect of complexing (phosphate) and non-complexing (PIPES) buffers on bioavailability was examined: Co and Zn prevented degradation of nitrilotriacetic acid in PIPES buffer, but not in phosphate buffer . This was due to the removal of Co and Zn from solution by phosphate precipitation, leaving nitrilotriacetic acid uncomplexed . The results demonstrated that metal-organic complexation can alter the bioavailability of organic pollutants and may also modulate the toxicity of heavy metals.

Oral Microbiol Immunol, 1999 Dec, 14(6), 339 - 47
Isolation and characterization of the rml gene homologs from Porphyromonas gingivalis; Shibata Y et al.; We cloned four genes from the Porphyromonas gingivalis chromosome, the gene products of which catalyze the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate when they were expressed in Escherichia coli . The amino acid sequences deduced from these genes showed significant homology to proteins encoded by the rml genes involved in dTDP-L-rhamnose biosynthesis in other gram-negative bacteria . Reverse transcriptase polymerase chain reaction analysis revealed that these four genes are expressed as a single transcript in P . gingivalis . To clarify the role of the rml gene homologs in this organism, construction of mutants defective in the rml gene homologs was attempted by allelic exchange . Unexpectedly, any mutants defective in the rml gene homologs were unable to be isolated, and the allelic exchange was possible only if the wild-type rml gene homologs were present on the chromosome . These results suggest that the rml gene homologs might be essential for the viability of P . gingivalis under the culture conditions used in this study.

Pediatr Allergy Immunol, 2000 May, 11(2), 64 - 70
Anti-neutrophil cytoplasmic antibodies directed against the bactericidal/permeability-increasing protein (BPI) in pediatric cystic fibrosis patients do not recognize N-terminal regions important for the anti-microbial and lipopolysaccharide-binding activity of BPI; Schultz H et al.; This study was performed to examine the prevalence and clinical correlates of bactericidal/permeability-increasing protein anti-neutrophil cytoplasmic antibodies (BPI-ANCA) in pediatric cystic fibrosis (CF) patients and to elucidate their possible role in CF pulmonary pathology . Sera of 27 CF patients were tested for ANCA by indirect immunofluorescence (IFT) and by enzyme-linked immunosorbent assay (ELISA) for ANCA sub-specificities . BPI-ANCA were examined by using standard ELISA for BPI, lipopolysaccharide-binding protein (LBP), and BPI/LBP fusion proteins to epitope map the main binding sites and look for cross-reactivity with LBP . Pulmonary function and serum concentrations of total immunoglobulin G (IgG) were measured and infections were diagnosed . In addition, release of reactive oxygen species (ROS) by neutrophil granulocytes was measured after stimulation with monoclonal BPI-ANCA . Using IFT, two patients showed atypical ANCA staining, six patients exhibited perinuclear ANCA staining, and no cytoplasmic ANCA staining was detected . Of 27 patients, 13 (48%) were BPI-ANCA (IgG) positive, and three were also immunoglobulin A (IgA) BPI-ANCA positive; one patient had ANCA against lactoferrin; and no proteinase 3 ANCA was detected in any of the patients . All BPI-ANCA bound to the C-terminal region of the molecule; none bound to the N-terminus or to LBP . There was no significant correlation between clinical data and the occurrence of BPI-ANCA in this cross-sectional study . Release of ROS from granulocytes was induced by monoclonal BPI-ANCA . Activation of neutrophils and possible modulation of BPI-mediated opsonophagocytosis and disposal of Gram-negative bacteria and lipopolysaccharides by BPI-ANCA raise the possibility that they contribute to pulmonary pathology in pediatric CF patients but intervention longitudinal studies in large groups of patients are needed to establish a causative association.

Analyst, 2000 Apr, 125(4), 651 - 6
Rapid isolation method for lipopolysaccharide and lipid A from gram-negative bacteria; Yi EC et al.; A fast, convenient extraction method for lipopolysaccharide (LPS), using a commercial RNA isolating reagent, allows the isolation of LPS or lipid A from low milligram (dry weight) quantities of bacterial cells . The method avoids the use of specialized equipment and has been used for processing relatively large numbers of samples . The major components of the commercial RNA isolating reagent, Tri-Reagent, are phenol and guanidinium thiocyanate in aqueous solution . The bacterial cell membranes are disrupted with guanidinium thiocyanate, which eliminates the need for mechanical cell disruption (e.g . French press) or heating . LPS and its degradation products, with particular attention paid to its bioactive lipid A portion, were measured and compared with those from the most common conventional extraction method, hot phenol-water . Negative ion quadrupole ion trap and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid composition analysis by capillary gas chromatography, total and free phosphate by UV spectrophotometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that LPS and lipid A isolated using the Tri-Reagent approach were cleaner and suffered less degradation through loss of phosphate and (or) fatty acyl side chains from lipid A . The Tri-Reagent extraction method generated low free phosphate contamination, 11% of the total phosphate concentration, whereas the hot phenol-water extraction method gave approximately 58% as free, inorganic phosphate . Similar results were observed for the degradation of fatty acyl side chains . The time required by the new method is considerably shorter (two or three days) than that required by conventional hot phenol-water extraction (about two weeks).

Microbios, 2000, 102(402), 69 - 77
H107, a new aminoglycoside anti-Pseudomonas antibiotic produced by a new strain of Spirillospora; Hacene H et al.; Spirillospora spp . (strain 719) has been the source of several antibiotics . One of these designated H107 is produced as a trace . Compared with other antibiotics produced by the same strain, it was obtained only from the broth filtrate after precipitation with acetic acid followed by extraction with n-butanol . It was a water soluble metabolite active against Gram-negative bacteria and especially Pseudomonas spp., and was identified as an aminoglycoside compound . This is the first report of aminoglycoside anti-Pseudomonas production by Spirillospora.

J Infect Dis, 2000 Jul, 182(1), 191 - 9 Epub 2000 Jun 30.
Targeting lipopolysaccharides by the nontoxic polymyxin B nonapeptide sensitizes resistant Escherichia coli to the bactericidal effect of human neutrophils; Rose F et al.; The nonapeptide of polymyxin B (PMBN) has been reported to sensitize various pathogenic gram-negative bacteria to the direct bactericidal effect of human serum . To investigate the impact of PMBN on human neutrophil-effected killing of the serum- and phagocytosis-resistant Escherichia coli strains C14 and O111, serum was coapplied with PMBN or with neutrophils, but this did not result in decreased numbers of viable bacteria . In contrast, the most potent bacterial killing occurred in the presence of neutrophils plus serum components plus PMBN . The effect of this on E . coli C14 was the appearance of inositol phosphates, diacylglycerol, respiratory burst, elastase liberation, and generation of lipid mediators (leukotriene B(4), 5-HETE, and platelet-activating factor) . Strong neutrophil activation required early, but not late, complement components and was blocked by inhibition of phagocytosis with cytochalasin D . PMBN seems to cause dramatic support of natural host defense by complement-dependent sensitization of E . coli to the bactericidal efficacy of human neutrophils.

J Exp Med, 2000 Jul 3, 192(1), 23 - 9
The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells; Ogata H et al.; The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS) . The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies . Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4 . Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans . The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS . Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor kappaB activation . These data suggest the existence of the TLR4-RP105 signaling module in the LPS-induced B cell activation.

Clin Infect Dis, 2000 Jun, 30(6), 936 - 8
Methylobacterium mesophilicum infection: case report and literature review of an unusual opportunistic pathogen; Sanders JW et al.; Methylobacterium mesophilicum is a methylotrophic, pink pigmented, gram-negative rod that was initially isolated from environmental sources that is being increasingly reported as a cause of opportunistic infections in immunocompromised hosts . We present the case of an immunocompromised woman who developed a central catheter infection with M . mesophilicum and review the other 29 cases reported in the literature, noting that it is frequently resistant to beta-lactam agents but is generally susceptible to aminoglycosides and quinolones.

J Immunol, 2000 Jul 15, 165(2), 1053 - 8
Pro-carboxypeptidase R is an acute phase protein in the mouse, whereas carboxypeptidase N is not; Sato T et al.; Carboxypeptidase R (EC 3.4.17.20; CPR) and carboxypeptidase N (EC 3 . 4.17.3; CPN) cleave carboxyl-terminal arginine and lysine residues from biologically active peptides such as kinins and anaphylatoxins, resulting in regulation of their biological activity . Human proCPR, also known as thrombin-activatable fibrinolysis inhibitor, plasma pro-carboxypeptidase B, and pro-carboxypeptidase U, is a plasma zymogen activated during coagulation . CPN, however, previously termed kininase I and anaphylatoxin inactivator, is present in a stable active form in plasma . We report here the isolation of mouse proCPR and CPN cDNA clones that can induce their respective enzymatic activities in culture supernatants of transiently transfected cells . Potato carboxypeptidase inhibitor can inhibit carboxypeptidase activity in culture medium of mouse proCPR-transfected cells . The expression of proCPR mRNA in murine liver is greatly enhanced following LPS injection, whereas CPN mRNA expression remains unaffected . Furthermore, the CPR activity in plasma increased 2-fold at 24 h after LPS treatment . Therefore, proCPR can be considered a type of acute phase protein, whereas CPN is not . An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators generated at the site of Gram-negative bacterial infection and may consequently prevent septic shock . In view of the ability of proCPR to also inhibit fibrinolysis, an excess of proCPR induced by LPS may contribute to hypofibrinolysis in patients suffering from disseminated intravascular coagulation caused by sepsis.

Appl Environ Microbiol, 2000 Jul, 66(7), 3037 - 43
Whole-cell versus total RNA extraction for analysis of microbial community structure with 16S rRNA-targeted oligonucleotide probes in salt marsh sediments; Frischer ME et al.; rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult . Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments . Resulting cell extracts are amenable to membrane blotting and hybridization protocols . Using this procedure, the efficiency of cell extraction was high (95.7% +/- 3.7% {mean +/- standard deviation}) relative to direct DAPI (4',6'-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments . To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system . A suite of six 16S rRNA-targeted oligonucleotide probes were utilized . The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r(2) = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust . However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts . The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.

J Chemother, 2000 Jun, 12(3), 232 - 9
Management of fever in neutropenic patients with acute leukemia: current role of ceftazidime plus amikacin as empiric therapy; Fanci R et al.; To evaluate the current role of ceftazidime plus amikacin as empiric therapy in the management of fever in neutropenic patients with acute leukemia, we examined 172 febrile episodes in 106 patients enrolled during 1996-98 . The overall success rate (survival of neutropenia, both with and without protocol modification) was 90%: 39% without modification and 51% with modification . We documented a significant difference in documented infections (DI) and fever of undetermined origin (FUO): success without modification was lower in DI and higher in FUO . Failure (death due to documented or presumed infection) was recorded in 10% of all episodes . Episodes with severe neutropenia were treated in 48% of cases without modification and in 41% with modification . No significant difference was observed in the status of underlying disease . 33% of gram-negative bacteria responsible for bloodstream infections were resistant to ceftazidime, of which 21% were multiresistant strains . We conclude that initial chemotherapy with ceftazidime plus amikacin remains a reasonable option for treating febrile and prolonged neutropenia, although patients with DI are likely to require additional or modified treatment . The emergence of resistant strains is an increasingly important issue.

Structure Fold Des, 2000 Jun 15, 8(6), 585 - 92
A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins; Ferguson AD et al.; BACKGROUND: Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response . High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome . The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated . RESULTS: The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported . It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance . Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA . Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins . CONCLUSIONS: These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response . Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.

Biochem Biophys Res Commun, 2000 Jun 24, 273(1), 22 - 6
S-nitrosothiol formation in blood of lipopolysaccharide-treated rats; Jourd'heuil D et al.; The administration of the gram-negative bacterial cell wall component lipopolysaccharide (LPS) to experimental animals results in the dramatic up-regulation of the inducible form of nitric oxide synthase (iNOS) . The resulting sustained overproduction of nitric oxide (NO) is thought to contribute to the septic shock-like state in these animals . Numerous studies have characterized the kinetics and magnitude of expression of iNOS as well as the production of NO-derived nitrite and nitrate . However, little is known regarding the ability of iNOS-derived NO to interact with physiological substrates such as thiols to yield biologically active S-nitrosothiols during endotoxemia . It has been hypothesized that these relatively stable, vaso-active compounds may serve as a storage system for NO and they may thus play an important role in the pathophysiology associated with endotoxemia . In the present study, we demonstrate that 5 h after i.p . administration of LPS in rats, circulating S-nitrosoalbumin was increased by approximately 3 . 4-fold over control . S-nitrosohemoglobin was increased by approximately 25-fold over controls and by threefold over S-nitrosoalbumin . No increase in low molecular weight S-nitrosothiols (i.e., S-nitrosoglutathione and S-nitrosocysteine) could be detected under our experimental conditions . Taken together these data demonstrate that endotoxemia dramatically enhances circulating S-nitrosothiol formation .

J Pediatr Surg, 2000 Jun, 35(6), 852 - 4; discussion 855
The role of anti-tumor necrosis factor-alpha and interleukin-10 in protecting murine neonates from Escherichia coli sepsis; Lally KP et al.; BACKGROUND/PURPOSE: The neonate is at much higher risk for septic complications and death than the adult . Although some aspects of the infant's immune response are immature, others are fully functional . Many models of septic death are caused by an overexpression of proinflammatory cytokines . If there were inadequate down regulatory mechanisms, this could lead to an overexpression of proinflammatory cytokines . The authors hypothesized that the high mortality rate of the newborn was caused by overexpression of tumor necrosis factor (TNF-alpha) and that interleukin-10 (IL-10) would attenuate this response . The aim of this study was to determine if TNF-alpha plays an important role in early death from Escherichia coli sepsis in the newborn animal and if blocking TNF improves survival . METHODS: A dose response curve was determined for 1 day old C3H/HEN mice using 10(5) intraperitoneal E coli resulting in a 30% to 50% mortality rate . Litters of newborn (1 day old) C3H/HEN mice received a subcutaneous injection of either 25 or 50 ng of murine IL-10 or 20 microL of anti-TNF-alpha 4 hours before a bacterial challenge . Control animals received nothing . Animals were observed for 5 to 7 days . At least 6 litters (18 pups per group) were used for each regimen . RESULTS: Anti-TNF-alpha resulted in a significant improvement in survival rate compared with controls (100% v 53%, P < .001) . In separate experiments, IL-10 at a dose of 25 ng failed to produce any improvement in survival; however, a 50-ng dose resulted in a significant improvement in treated animals compared with controls (95% v 65%, P < .01) . CONCLUSIONS: TNF-alpha plays an important role in neonatal sepsis, suggesting that the newborn mouse is capable of mounting a significant proinflammatory response to gram-negative bacteria . Newborn mice may respond to bacterial challenge with an overexpression of proinflammatory cytokines or an underproduction of downregulating cytokines . Future attempts at immunomodulation in human infants must be undertaken with caution until the inflammatory response is better defined.

Eur J Epidemiol, 2000 Mar, 16(3), 277 - 9
Haemorrhagic papular rash associated to Flavimonas oryzihabitans bacteraemia in a child; Kansouzidou A et al.; Flavimonas oryzihabitans is a gram-negative rod that has rarely been implicated in human infections . The involvement of this organism has been documented in serious infections, the majority of which were cases of bacteraemia or peritonitis . We report the first isolation of the organism in Greece, from a case of bacteraemia, associated with haemorrhagic papular rash, in a paediatric patient and describe the phenotypic characteristics of the strain.

Arch Oral Biol, 2000 Sep, 45(9), 787 - 95
Elevation of histidine decarboxylase activity in the mandible of mice by Prevotella intermedia lipopolysaccharide and its augmentation by an aminobisphosphonate; Funayama H et al.; Lipopolysaccharide (LPS) produced by Gram-negative bacteria is an important cause of inflammation . Aminobisphosphonates are potent inhibitors of bone resorption but have inflammatory side-effects . Here, the effects of LPS from Prevotella intermedia (a prevalent Gram-negative bacterium both in periodontitis and endodontal infections) and alendronate (an aminobisphosphonate) on the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), were examined in mouse mandible . Intravenous injection of P . intermedia LPS increased HDC activity in the mandible, maximal activity being induced within 3-6 h of the injection . The elevation of HDC activity was dependent on the dose of LPS, 10 microg/kg (0.25 microg/mouse) producing a significant elevation in enzyme activity . Intraperitoneal injection of alendronate (40 micromol/kg) also produced an increase in HDC activity . Moreover, the elevation of HDC activity induced by P . intermedia LPS was markedly augmented in mice given alendronate 3 days before the LPS injection . These results (i) suggest that P . intermedia LPS may stimulate the synthesis of histamine in the mandible and that the newly formed histamine may make at least some contribution to the development of inflammation (apical periodontitis and/or osteomyelitis); (ii) should encourage the clinical testing of antihistaminergic agents against inflammation; and (iii) confirm that care needs to be taken when administering aminobisphosphonates to patients.

J Bacteriol, 2000 Jul, 182(14), 3998 - 4004
A novel bacterial ATP-binding cassette transporter system that allows uptake of macromolecules; Momma K et al.; A gram-negative bacterium, Sphingomonas sp . strain A1, isolated as a producer of alginate lyase, has a characteristic cell envelope structure and forms a mouth-like pit on its surface . The pit is produced only when the cells have to incorporate and assimilate alginate . An alginate uptake-deficient mutant was derived from cells of strain A1 . One open reading frame, algS (1,089 bp), exhibiting homology to the bacterial ATP-binding domain of an ABC transporter, was cloned as a fragment complementing the mutation . algS was followed by two open reading frames, algM1 (972 bp) and algM2 (879 bp), which exhibit homology with the transmembrane permeases of ABC transporters . Disruption of algS of strain A1 resulted in the failure to incorporate alginate and to form a pit . Hexahistidine-tagged AlgS protein (AlgS(His6)) overexpressed in Escherichia coli and purified by Ni(2+) affinity column chromatography showed ATPase activity . Based on these results, we propose the occurrence of a novel pit-dependent ABC transporter system that allows the uptake of macromolecules.

Clin Immunol, 2000 Jun, 95(3), 227 - 34
Shiga toxin-2 induces neutrophilia and neutrophil activation in a murine model of hemolytic uremic syndrome; Fernandez GC et al.; It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the hemolytic uremic syndrome (HUS) . Although it is recognized that Stx damage the glomerular endothelium, clinical and experimental evidence suggests that the inflammatory response is able to potentiate Stx toxicity . Lipopolysaccharides (LPS) and neutrophils (PMN) represent two central components of inflammation during a gram-negative infection . In this regard, patients with high peripheral PMN counts at presentation have a poor prognosis . Since the murine model has been used to study LPS-Stx interactions, we analyzed the effects of Stx alone or in combination with LPS on the kinetics of neutrophil production and activation and their participation in renal damage . We observed a sustained neutrophilia after Stx2 injection . Moreover, these neutrophils showed increased expression of CD11b, enhanced cytotoxic capacity, and greater adhesive properties . Regarding the cooperative effects of LPS on Stx2 action, we demonstrated potentiation of neutrophilia and CD11b induction at early times by pretreatment with LPS . Finally, a positive correlation between neutrophil percentage and renal damage (assayed as plasmatic urea) firmly suggests a role for PMN in the pathogenesis of HUS.

FEMS Immunol Med Microbiol, 2000 Jul, 28(3), 247 - 56
Protein phosphorylation pathways involved during lipopolysaccharide-induced expression of CD14 in mouse bone marrow granulocytes; Pedron T et al.; Lipopolysaccharide (LPS) of Gram-negative bacteria interacts with a CD14-independent receptor of mouse bone marrow granulocytes (BMC), and triggers in these cells the expression of CD14, an inducible type of LPS receptor (iLpsR) . This particular response of BMC to LPS required the activation of protein tyrosine kinase and p38 MAP kinase . The inhibition of the LPS effect by the MEK inhibitor PD-98059 suggested that the ERK pathway was also involved . Unexpectedly, protein kinase C, myosin light chain kinase, cAMP-, cGMP-, and Ca(2+)/calmodulin-dependent kinases, as well as ecto-protein kinases, were not required for iLpsR expression . However, other yet unidentified serine/threonine protein kinase(s) were implied since the BMC response to LPS was markedly reduced after exposure to three inhibitors of such kinases (K-252a, H-7, and KT-5823) . The atypical kinase requirements observed in this study may be due either to a novel signaling LPS receptor complex present in BMC, or to the particular events involved in CD14 biosynthesis.

J Cell Biochem, 2000 Jun 12, 78(4), 519 - 32
Human monocyte/neutrophil elastase inhibitor (MNEI) is regulated by PU.1/Spi-1, Sp1, and NF-kappaB; Zeng W et al.; Human monocyte/neutrophil elastase inhibitor (MNEI) is a specific inhibitor of the neutrophil azurophil granule proteases including elastase . To understand the physiological mechanisms that regulate expression of MNEI, we dissected a 1.0 kb region upstream of exon 1 . On transient transfection, promoter activity of MNEI-luciferase constructs was highest in U937 myeloid cells, followed by K562 hematopoietic cells, followed by HeLa cervical carcinoma cells, indicating that the MNEI promoter is most active in myeloid cells and is also active in non-myeloid cells . Three transcription factor binding elements, which confer the majority of activity, are located within the first 180 base pairs of the promoter, one of which, located at -128, was active in U937 and K562 cells but inactive in non-myeloid HeLa cells . The three proximal elements were identified by transient transfection, mutation, gel shift and competition assays as Sp1 at -170, PU.1/Spi-1 at -128, and Sp1 at -66 . The trans-acting factors that bind and control these elements were detected, and their identity confirmed by antibody supershift assays . Further upstream at -821, an additional regulatory element was identified controlled by NF-kappaB, which supports the highest levels of MNEI transcriptional activity . In U937 cells, reporter gene expression by the MNEI-luciferase construct that included the NF-kappaB element was two- to three-fold greater than the construct without the element . In addition, treatment of myeloid cells with lipopolysaccharide, a complex glycolipid of gram-negative bacteria, activated NF-kappaB to bind the -821 element, together suggesting that enhancement of expression of the anti-inflammatory MNEI gene is linked to innate immune responses to bacterial infection.

Blood Purif, 2000, 18(3), 214 - 23
Chronic inflammation in hemodialysis: the role of contaminated dialysate; Lonnemann G; Routine sodium bicarbonate-buffered dialysate is contaminated with predominantly gram-negative micro-organisms . These bacteria release pyrogenic substances such as endotoxins, peptidoglycans, exotoxins and fragments thereof . Pyrogens derived from contaminated dialysate either alone or in costimulation with activated complement components are the most important activators of circulating mononuclear cells in patients on chronic intermittent hemodialysis . Activated mononuclear cells release proinflammatory cytokines which are key mediators in acute and chronic inflammatory diseases associated with long-term hemodialysis therapy . Recent experimental and clinical data suggest that the use of pyrogen-free dialysate prevents activation of mononuclear cells and improves the state of chronic inflammation, as indicated by decreased plasma levels of C-reactive protein in chronic hemodialysis patients . Future clinical studies have to prove whether the use of pyrogen-free dialysate in combination with biocompatible dialyzer membranes and tubings reduces the incidence and severity of chronic inflammatory diseases (beta(2)-microglobulin amyloidosis, muscle protein wasting, atherosclerosis) in long-term hemodialysis patients .

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7709 - 14
PapD-like chaperones provide the missing information for folding of pilin proteins; Barnhart MM et al.; A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures . The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly . The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange . Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence . Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding . Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold . This mechanism of small periplasmic chaperone function described here deviates from classical hsp60 chaperone-assisted folding.

Antimicrob Agents Chemother, 2000 Jul, 44(7), 1878 - 86
Molecular and biochemical heterogeneity of class B carbapenem-hydrolyzing beta-lactamases in Chryseobacterium meningosepticum; Bellais S et al.; Although the carbapenem-hydrolyzing beta-lactamase (CHbetaL) BlaB-1 is known to be in Chryseobacterium meningosepticum NCTC 10585, a second CHbetaL gene, bla(GOB-1), was cloned from another C . meningosepticum clinical isolate (PINT) . The G+C content of bla(GOB-1) (36%) indicated the likely chromosomal origin of this gene . Its expression in Escherichia coli DH10B yields a mature CHbetaL with a pI of 8.7 and a relative molecular mass of 28.2 kDa . In E . coli, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems . GOB-1 had a broad-spectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam) . The catalytic efficiency for meropenem was higher than for imipenem . GOB-1 had low amino acid identity with the class B CHbetaLs, sharing 18% with the closest, L-1 from Stenotrophomonas maltophilia, and only 11% with BlaB-1 . Most of the conserved amino acids that may be involved in the active site of CHbetaLs (His-101, Asp-103, His-162, and His-225) were identified in GOB-1 . Sequence heterogeneity was found for GOB-1-like and BlaB-1-like beta-lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated C . meningosepticum isolates . Each isolate had a GOB-1-like and a BlaB-1-like gene . The same combination of GOB-1-like and BlaB-1-like beta-lactamases was not found in two different isolates . C . meningosepticum is a bacterial species with two types of unrelated chromosome-borne class B CHbetaLs that can be expressed in E . coli and, thus, may represent a clinical threat if spread in gram-negative aerobes.

Infect Immun, 2000 Jul, 68(7), 4055 - 63
RgpA-Kgp peptide-based immunogens provide protection against Porphyromonas gingivalis challenge in a murine lesion model; O'Brien-Simpson NM et al.; Porphyromonas gingivalis, a gram-negative bacterium, has been linked to the onset and progression of periodontitis, a chronic inflammatory disease of the supporting tissues of the teeth . A major virulence factor of P . gingivalis is an extracellular complex of Arg- and Lys-specific proteinases and adhesins designated the RgpA-Kgp complex (formerly the PrtR-PrtK complex) . In this study we show that the RgpA-Kgp complex, when used as an immunogen with incomplete Freund adjuvant (IFA), protects against challenge with invasive and noninvasive strains of P . gingivalis in the murine lesion model . We identified a variety of peptide vaccine candidates from the RgpA and Kgp polyprotein sequences that involved the putative active site histidine of both proteinases and five repeat motifs in the adhesin domains of both polyproteins implicated in aggregation and binding to host substrates, designated adhesin-binding motif (ABM) peptides . These peptides were synthesized using standard, solid-phase protocols for 9-fluorenylmethoxy carbonyl chemistry with S-acetylmercaptoacetic acid (SAMA) as the N-terminal residue . The SAMA-peptides were then conjugated to diphtheria toxoid and used with IFA to immunize BALB/c mice . Both active-site peptides and three of the five ABM peptides gave protection (P < 0.005) against challenge with P . gingivalis in the murine lesion model . The three ABM peptide sequences that conferred protection exist within a 100-residue span in the RgpA44 and Kgp39 adhesins of the RgpA-Kgp complex . Protective anti-RgpA-Kgp complex mouse antisera recognized the RgpA27, Kgp39, and RgpA44 adhesins in an immunoblot . Epitope mapping of the RgpA27 adhesin using the protective anti-RgpA-Kgp antisera identified a major protective epitope that mapped immediately N terminal to one of the protective ABM peptides in the 100-residue span in RgpA44 and Kgp39 . This identified protective epitope contains clusters of basic residues spatially surrounded by hydrophobic amino acids, a finding which is characteristic of a heparin binding motif.

Infect Immun, 2000 Jul, 68(7), 3971 - 82
Identification and subcellular localization of the Legionella pneumophila IcmX protein: a factor essential for establishment of a replicative organelle in eukaryotic host cells; Matthews M et al.; The gram-negative respiratory pathogen Legionella pneumophila infects and grows within mammalian macrophages and protozoan host cells . Upon uptake into macrophages, L . pneumophila establishes a replicative organelle that avoids fusion with endocytic vesicles . There are 24 dot/icm genes on the L . pneumophila chromosome required for biogenesis of this vacuole . Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems . We have been investigating the function of L . pneumophila dot/icm gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis . The icmX gene product falls into this category . To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an L . pneumophila icmX deletion mutant and murine bone marrow-derived macrophages . These data demonstrate that icmX is required for biogenesis of the L . pneumophila replicative organelle . Immunoblot analysis indicates that the icmX gene product is a polypeptide with an estimated molecular mass of 50 kDa . The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal sec-dependent leader peptide . A truncated IcmX product was secreted into culture supernatants by wild-type L . pneumophila growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected . Proteins similar in molecular weight to IcmX were identified in other Legionella species by immunoblot analysis using a monoclonal antibody specific for L . pneumophila IcmX protein . From these data, we conclude that the IcmX protein is an essential component of the dot/icm secretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the Legionellaceae family.

J Leukoc Biol, 2000 Jun, 67(6), 894 - 901
cAMP induces CD14 expression in murine macrophages via increased transcription; Liu S et al.; CD14, a glycoprotein that binds bacterial lipopolysaccharide, plays a critical role in the inflammatory response to infection by gram-negative bacteria . Studies were undertaken to determine whether cyclic adenosine monophosphate (cAMP) regulates CD14 expression in macrophages . Incubation of RAW 264.7 cells with 8-Br-cAMP resulted in a significant increase in steady-state CD14 mRNA levels . The increase in mRNA levels was also associated with both cell-associated and soluble CD14 protein . H89 completely blocked the 8-Br-cAMP-induced CD14 mRNA up-regulation . There was no change in CD 14 mRNA half-life in the presence of 8-Br-cAMP . The CD14 gene transcription rate was increased about twofold after exposure to 8-Br-cAMP . cAMP-dependent increases in CD14 mRNA were also observed in rat peritoneal macrophages, demonstrating that this is an authentic response of mature macrophages . This study provides evidence that cAMP and protein kinase A are important regulators of CD14 expression in macrophages.

J Leukoc Biol, 2000 Jun, 67(6), 841 - 6
Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis; Malaviya R et al.; Stimulated mast cells release a variety of chemotactic factors such as tumor necrosis factor alpha (TNF-alpha) and leukotriene B4 . Recent studies have shown that mast cell-derived TNF-alpha plays a critical role in host defense against Gram negative bacterial infections by the recruitment of neutrophils to the sites of infection . In the present study, we sought to investigate if mast cells release leukotriene (LT) B4 in response to bacteria and, if so, to establish its in vivo relevance . We show that mast cells release significant amounts of LTB4 and LTC4 in response to exposure to FimH-expressing type 1 fimbriated Escherichia coli in vitro . To test the functional significance of mast cell-derived LTs during an E . coli infection in vivo, we examined the effect of a LT-synthesis inhibitor, A-63162, on bacterial clearance and neutrophil influx in an infectious peritonitis model in mast cell-deficient mice (WBB6F1-W/WV) and their normal congenic control (WBB6F1-+/+) mice . Our results show that a treatment with A-63162 reduced neutrophil influx and bacterial clearance in the peritoneal cavities of mast cell-sufficient but not -deficient mice . Thus, mast cell-derived LTs contribute to host defense by mediating early neutrophil influx and bacterial clearance at sites of infection.

J Biol Chem, 2000 Aug 25, 275(34), 26252 - 8
Lipopolysaccharide inhibits long term potentiation in the rat dentate gyrus by activating caspase-1; Vereker E et al.; Lipopolysaccharide, a component of the cell wall of Gram-negative bacteria, may be responsible for at least some of the pathophysiological sequelae of bacterial infections, probably by inducing an increase in interleukin-1beta (IL-1beta) concentration . We report that intraperitoneal injection of lipopolysaccharide increased hippocampal caspase-1 activity and IL-1beta concentration; these changes were associated with increased activity of the stress-activated kinase c-Jun NH(2)-terminal kinase, decreased glutamate release, and impaired long term potentiation . The degenerative changes in hippocampus and entorhinal cortical neurones were consistent with apoptosis because translocation of cytochrome c and poly(ADP-ribose) polymerase cleavage were increased . Inhibition of caspase-1 blocked these changes, suggesting that IL-1beta mediated the lipopolysaccharide-induced changes.

Int J Colorectal Dis, 2000 Apr, 15(2), 59 - 82
Gut-liver axis; Zeuzem S; The gut and the liver are the key organs in nutrient absorption and metabolism . Bile acids, drugs, and toxins undergo extensive enterohepatic circulation . Bile acids play a major role in several hepatic and intestinal diseases . Endotoxins deriving from intestinal Gram-negative bacteria are important in the pathogenesis of liver and systemic diseases . Chronic liver diseases can influence gastrointestinal motility, which together with other factors may contribute to bacterial overgrowth and in patients with ascites to an increased risk of spontaneous bacterial peritonitis . Patients with end-stage liver disease frequently develop portal hypertension leading to varices, gastric vascular ectasia, and portal hypertensive gastroenteropathy . Several liver and biliary abnormalities are observed in patients with inflammatory bowel disease (primary sclerosing cholangitis, autoimmune hepatitis, cholelithiasis) . The primary defect in hemochromatosis is located in the intestine, causing an inappropriate increase in iron absorption, and the liver is the site of earliest and heaviest iron deposition . Elevated transaminases are observed in many patients with celiac disease, and steatohepatitis frequently develops in patients with jejunoileal bypass and short bowel syndrome . Furthermore, the liver is the primary organ for metastasis of intestinal cancer . Many viral, bacterial, fungal, and parasitic diseases affect the intestine as well as the liver and the biliary tract.

Am J Respir Crit Care Med, 2000 Jun, 161(6), 1942 - 8
Multicenter prospective study of ventilator-associated pneumonia during acute respiratory distress syndrome . Incidence, prognosis, and risk factors . ARDS Study Group; Markowicz P et al.; We investigated the incidence, risk factors for, and outcome of ventilator-associated pneumonia (VAP) in patients with acute respiratory distress syndrome (ARDS) . We compared 134 patients with ARDS with 744 patients without ARDS on mechanical ventilation . Fiberoptic bronchoscopic examination and quantitative bacterial cultures (protected brush or catheter sampling {threshold: 10(3) cfu/ml}, or bronchoalveolar lavage {threshold: 10(4) cfu/ml}) were used to diagnose pneumonia . VAP occurred in 49 patients (36.5%) . The incidence of pneumonia was 23% (173 of 744 patients) among patients without ARDS (p < 0.002) . Nonfermenting gram-negative rods caused significantly more pneumonia in ARDS patients . Mortality rates were identical in ARDS patients with (28 of 49 patients, 57%) and without (50 of 85 patients, 59%) pulmonary infection (p = 0.8) . VAP resulted in a considerable increase in attributable time on mechanical ventilation of both the overall population of ARDS patients and of survivors . Both the use of sucralfate (adjusted odds ratio {OR}: 4 . 42; 95% confidence interval {CI}: 2.01 to 9.7, p = 0.0002) and the duration of exposure to sucralfate (adjusted OR: 1.206; 95% CI: 1 . 095 to 1.328, p = 0.0002) were associated with an increased risk of VAP during ARDS . VAP considerably prolongs the time on mechanical ventilation without affecting survival . Patients given sucralfate may be at greater risk of developing pulmonary infection during ARDS.

Cancer Biother Radiopharm, 1997 Oct, 12(5), 333 - 40
Expression of inflammatory cytokines by murine macrophages activated with a new synthetic lipopeptide JT3002; Kumar R et al.; The present study was undertaken to investigate the effect of JT3002, a new synthetic analogue of a lipoprotein from the outer wall of a gram-negative bacterium on the production of cytokines by mouse peritoneal macrophages . Multilamellar liposomes containing different concentrations of JT3002 induced production of the inflammatory cytokines tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-6 by macrophages in dose- and time-dependent manners . The presence of interferon-gamma enhanced production of tumor necrosis factor-alpha by macrophages exposed to lower concentrations of JT3002 and induced the release of nitric oxide, a potent cytolytic molecule of activated macrophages . Unlike lipopolysaccharide, JT3002 activated macrophages independently of serum, but like lipopolysaccharide, it required protein tyrosine kinase.

J Bacteriol, 2000 Jul, 182(13), 3726 - 33
Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter; Lattemann CT et al.; Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria . Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins . Here we report on the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme beta-lactamase, leading to efficient expression of the fusion protein in E . coli . The beta-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located beta-lactamase was shown to be enzymatically active . Enzymatic activity was completely removed by protease treatment, indicating that surface display of beta-lactamase was almost quantitative . The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of E . coli cells expressing the beta-lactamase AIDA-I fusion protein remained physiologically intact.

Am J Kidney Dis . 2000 Jun;35(6):E30.
Ochrobactrum anthropi bacteremia in a patient on hemodialysis; Chertow GM; Although newer tunneled dialysis catheters offer improved capacity for blood flow and efficiency of dialysis, catheter-associated bacteremia remains an extremely important complication of this access strategy . This is a report of a case of catheter-associated bacteremia with Ochrobactrum anthropi, a water-borne gram-negative rod with an unusual pattern of antibiotic resistance . Given the organism's hydrophilic property and the frequency of catheter use in debilitated individuals with end-stage renal disease, Ochrobactrum anthropi infection should be considered in the differential diagnosis of a hemodialysis patient with unexplained fever.

J Gastroenterol Hepatol, 2000 May, 15(5), 473 - 9
Acute hepatic failure in India: a perspective from the East; Acharya SK et al.; Acute hepatic failure (AHF) in India almost always presents with encephalopathy within 4 weeks of the onset of acute hepatitis . Further subclassification of AHF into hyperacute, acute and subacute forms may not be necessary in this geographical area, where the rapidity of onset of encephalopathy does not seem to influence survival . Viral hepatitis is the cause in approximately 95-100% of patients, who therefore constitute a more homogeneous population than AHF patients in the West . In India, hepatitis E (HEV) and hepatitis B (HBV) viruses are the most important causes of AHF; approximately 60% of cases are caused by to these viruses . Hepatitis B virus core mutants are very important agents in cases where hepatitis B results in AHF in this country . Half of the patients with AHF admitted to our centre are female, one-quarter of whom are pregnant . Therefore, pregnant females who contract viral hepatitis constitute a high-risk group for the development of AHF . However, the outcome of AHF in this group is similar to that in non-pregnant women and men . No association with any particular virus has been identified among sporadic cases of AHF . In our centre, approximately one-third of AHF patients survive with aggressive conservative therapy, whereas two-thirds of deaths occur within 72 h of hospitalization . Cerebral oedema and sepsis are the major fatal complications . Both fungal and gram-negative bacteria are major causes of sepsis . Among patients with AHF, despite the presence of sepsis, its overt clinical features (i.e . fever, leucocytosis) may be absent and objective documentation of the presence of sepsis in such patients is achieved by repeated culture of various body fluids . It should be possible to develop simple, clinical prognostic markers for AHF in this geographical region, in order to identify patients suitable for liver transplantation.

APMIS, 2000 Apr, 108(4), 293 - 5
Vacuolating cytotoxic activity of 40 Helicobacter pylori strains isolated from Turkish patients; Ozsan M et al.; In this study we examined the in vitro vacuolating cytotoxic activity of Helicobacter pylori, which is a gram-negative microaerophilic curved bacterium and a causative agent of gastritis, peptic ulcer and gastric ulcer . A vacuolating cytotoxin assay was performed to assess the vacuolating activity of 40 strains (20 gastritis, 11 gastric ulcer, and 9 duodenal ulcer), which were obtained from patients undergoing upper gastrointestinal endoscopy . The Vero cell line was used in the cytotoxic assay . Of the 40 isolates, 24 (12 gastritis, 6 gastric ulcer, 6 duodenal ulcer) were cytotoxic for the Vero cell line at 1:4 and 1:8 dilutions . Thus, vacuolating cytotoxin of H . pylori affects the Vero cell line, but it seems there is no correlation between the positivity of the strains and the risk of any particular H . pylori disease.

Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 955 - 69
Methylocella palustris gen . nov., sp . nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs; Dedysh SN et al.; A new genus, Methylocella, and a new species, Methylocella palustris, are proposed for three strains of methane-oxidizing bacteria isolated from acidic Sphagnum peat bogs . These bacteria are aerobic, Gram-negative, colourless, non-motile, straight and curved rods that utilize the serine pathway for carbon assimilation, multiply by normal cell division and contain intracellular poly-beta-hydroxybutyrate granules (one at each pole) . These strains use methane and methanol as sole sources of carbon and energy and are moderately acidophilic organisms with growth between pH 4.5 and pH 7.0, the optimum being at pH 5.0-5.5 . The temperature range for growth is 10-28 degrees C with the optimum at 15-20 degrees C . The intracytoplasmic membrane system is different from those of type I and II methanotrophs . Cells contain an extensive periplasmic space and a vesicular membrane system connected to the cytoplasmic membrane . The strains grew only on media with a low salt content (0.2-0.5 g l(-1)) . All three strains were found to possess soluble methane monooxygenase and are able to fix atmospheric nitrogen via an oxygen-sensitive nitrogenase . No products were observed in a PCR with particulate methane monooxygenase-targeted primers; hybridization with a pmoA probe was also negative . The major phospholipid fatty acids are 18:1 acids . The G+C content of the DNA is 61.2 mol% . The three strains share identical 16S rRNA gene sequences and represent a novel lineage of methane-oxidizing bacteria within the alpha-subclass of the class Proteobacteria and are only moderately related to type II methanotrophs of the Methylocystis-Methylosinus group . The three strains are most closely related to the acidophilic heterotrophic bacterium Beijerinckia indica subsp . indica (96.5% 16S rDNA sequence similarity) . Collectively, these strains comprise a new species and genus Methylocella palustris gen . nov., sp . nov.; strain KT (= ATCC 700799T) is the type strain.

Nature, 2000 May 25, 405(6785), 458 - 62
Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin; Borovikova LV et al.; Vertebrates achieve internal homeostasis during infection or injury by balancing the activities of proinflammatory and anti-inflammatory pathways . Endotoxin (lipopolysaccharide), produced by all gram-negative bacteria, activates macrophages to release cytokines that are potentially lethal . The central nervous system regulates systemic inflammatory responses to endotoxin through humoral mechanisms . Activation of afferent vagus nerve fibres by endotoxin or cytokines stimulates hypothalamic-pituitary-adrenal anti-inflammatory responses . However, comparatively little is known about the role of efferent vagus nerve signalling in modulating inflammation . Here, we describe a previously unrecognized, parasympathetic anti-inflammatory pathway by which the brain modulates systemic inflammatory responses to endotoxin . Acetylcholine, the principle vagal neurotransmitter, significantly attenuated the release of cytokines (tumour necrosis factor (TNF), interleukin (IL)-1beta, IL-6 and IL-18), but not the anti-inflammatory cytokine IL-10, in lipopolysaccharide-stimulated human macrophage cultures . Direct electrical stimulation of the peripheral vagus nerve in vivo during lethal endotoxaemia in rats inhibited TNF synthesis in liver, attenuated peak serum TNF amounts, and prevented the development of shock.

Wien Med Wochenschr, 2000, 150(5), 98 - 100
{Bad breath--etiological, diagnostic and therapeutic problems}; Reiss M et al.; Oral malodor has many etiologies and is a clinical problem for many people . This paper reviews the causes and management of oral malador . In the majority of cases the problem has been shown to originate in the oral cavity . Oral malodor, a generic descriptor term for foul smells emanating from the mouth, encompasses ozostomia, stomatodysodia, halitosis (both pathological halitosis and physiological halitosis) and fetor oris or fetor ex ore . These latter terms, in turn, denote different sources of oral malodor . All conditions that favour the retention of anaerobic, mainly gram-negative, bacteria will predispose for the development of bad breath . In addition to periodontal pockets, the most important retention site is the dorsum of the tongue with its numerous papillae . During the night and between meals the conditions are optimal for odour production . Systemic pathological states, such as diabetes mellitus, uremia and hepatic diseases, induce metabolic products that are detectable as oral smells . It is always easy to recognize halitosis, but identifying the exact cause is more complex . The clinical labelling and interpretation of different oral malodors both contribute to the diagnosis and treatment of underlying disease . Treatment is directed at the underlying cause.

Am J Physiol Lung Cell Mol Physiol, 2000 Jun, 278(6), L1289 - 96
Brief hypoxia differentially regulates LPS-induced IL-1beta and TNF-alpha gene transcription in RAW 264.7 cells; Ndengele MM et al.; Episodes of tissue hypoxia and reoxygenation frequently occur during gram-negative bacteremia that progresses to septic shock . However, few studies have evaluated modulation by hypoxia and reoxygenation of the proinflammatory cytokine gene expression that is normally induced by gram-negative bacteremia or endotoxemia . In buffer-perfused organs, hypoxia downregulates Escherichia coli-induced expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the liver but upregulates these cytokines in the lungs . To identify molecular mechanisms underlying these events, we investigated the effects of brief (1.5-h) hypoxia on TNF-alpha and IL-1beta expression in cultured RAW 264.7 cells during their continuous exposure to lipopolysaccharide (LPS) endotoxin derived from E . coli (serotype 055:B5) for up to 24 h . IL-1beta and TNF-alpha concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis . LPS-induced IL-1beta synthesis was downregulated by hypoxia at both the protein and mRNA levels despite no change in cellular redox status as measured by levels of GSH . In contrast, LPS-induced TNF-alpha production was unaffected by hypoxia as assessed by cell lysate mRNA and lysate and supernatant protein levels . Nuclear runoff analysis showed that downregulation of IL-1beta gene expression by hypoxia occurred transcriptionally . Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1beta expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species . Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1beta expression did not require de novo protein synthesis.

Klin Med (Mosk), 2000, 78(4), 19 - 22
{On possible prognostic values of anti-endotoxin immunological parameters in acute myocardial infarction}; Ipatov AI et al.; Endotoxin is a biologically active substance that has a lipopolysaccharide structure . It is found in the cell walls of microorganisms, principally gram-negative bacteria . By contacting with the cell, endotoxin enhances immunity . The findings suggest that acute myocardial infarction (AMI) occurs due to low immunity that remains the same at week 3 of the disease, thus the levels of antiendotoxin agents may be used in predicting AMI.

Microbiol Immunol, 2000, 44(4), 279 - 82
Identification of a two-component signal transduction system involved in fimbriation of Porphyromonas gingivalis; Hayashi J et al.; Porphyromonas gingivalis, a periodontopathogen, is an oral anaerobic gram-negative bacterium with numerous fimbriae on the cell surface . Fimbriae have been considered to be an important virulence factor in this organism . We analyzed the genomic DNA of transposon-induced, fimbria-deficient mutants derived from ATCC 33277 and found that seven independent mutants had transposon insertions within the same restriction fragment . Cloning and sequencing of the disrupted region from one of the mutants revealed two adjacent open reading frames (ORFs) which seemed to encode a two-component signal transduction system . We also found that six of the mutants had insertions in a gene, fimS, a homologue of the genes encoding sensor kinase, and that the insertion in the remaining one disrupted the gene immediately downstream, fimR, a homologue of the response regulator genes in other bacteria . These findings suggest that this two-component regulatory system is involved in fimbriation of P . gingivalis.

Appl Environ Microbiol, 2000 Jun, 66(6), 2613 - 9
Phylogenetic diversity among geographically dispersed Chlamydiales endosymbionts recovered from clinical and environmental isolates of Acanthamoeba spp; Fritsche TR et al.; The recently proposed reorganization of the order Chlamydiales and description of new taxa are broadening our perception of this once narrowly defined taxon . We have recovered four strains of gram-negative cocci endosymbiotic in Acanthamoeba spp., representing 5% of the Acanthamoeba sp . isolates examined, which displayed developmental life cycles typical of members of the Chlamydiales . One of these endosymbiont strains was found stably infecting an amoebic isolate recovered from a case of amoebic keratitis in North America, with three others found in acanthamoebae recovered from environmental sources in North America (two isolates) and Europe (one isolate) . Analyses of nearly full-length 16S rRNA gene sequences of these isolates by neighbor joining, parsimony, and distance matrix methods revealed their clustering with other members of the Chlamydiales but in a lineage separate from those of the genera Chlamydia, Chlamydophila, Simkania, and Waddlia (sequence similarities, <88%) and including the recently described species Parachlamydia acanthamoebae (sequence similarities, 91.2 to 93.1%) . With sequence similarities to each other of 91.4 to 99.4%, these four isolates of intra-amoebal endosymbionts may represent three distinct species and, perhaps, new genera within the recently proposed family Parachlamydiaceae . Fluorescently labeled oligonucleotide probes targeted to 16S rRNA signature regions were able to readily differentiate two groups of intra-amoebal endosymbionts which corresponded to two phylogenetic lineages . These results reveal significant phylogenetic diversity occurring among the Chlamydiales in nontraditional host species and supports the existence of a large environmental reservoir of related species . Considering that all described species of Chlamydiales are known to be pathogenic, further investigation of intra-amoebal parachlamydiae as disease-producing agents is warranted.

Appl Environ Microbiol, 2000 Jun, 66(6), 2408 - 13
Isolation and characterization of Desulfovibrio dechloracetivorans sp . nov., a marine dechlorinating bacterium growing by coupling the oxidation of acetate to the reductive dechlorination of 2-chlorophenol; Sun B et al.; Strain SF3, a gram-negative, anaerobic, motile, short curved rod that grows by coupling the reductive dechlorination of 2-chlorophenol (2-CP) to the oxidation of acetate, was isolated from San Francisco Bay sediment . Strain SF3 grew at concentrations of NaCl ranging from 0.16 to 2.5%, but concentrations of KCl above 0 . 32% inhibited growth . The isolate used acetate, fumarate, lactate, propionate, pyruvate, alanine, and ethanol as electron donors for growth coupled to reductive dechlorination . Among the halogenated aromatic compounds tested, only the ortho position of chlorophenols was reductively dechlorinated, and additional chlorines at other positions blocked ortho dechlorination . Sulfate, sulfite, thiosulfate, and nitrate were also used as electron acceptors for growth . The optimal temperature for growth was 30 degrees C, and no growth or dechlorination activity was observed at 37 degrees C . Growth by reductive dechlorination was revealed by a growth yield of about 1 g of protein per mol of 2-CP dechlorinated, and about 2.7 g of protein per mole of 2,6-dichlorophenol dechlorinated . The physiological features and 16S ribosomal DNA sequence suggest that the organism is a novel species of the genus Desulfovibrio and which we have designated Desulfovibrio dechloracetivorans . The unusual physiological feature of this strain is that it uses acetate as an electron donor and carbon source for growth with 2-CP but not with sulfate.

Mol Plant Microbe Interact, 2000 Jun, 13(6), 637 - 48
Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria; Teplitski M et al.; In gram-negative bacteria, many important changes in gene expression and behavior are regulated in a population density-dependent fashion by N-acyl homoserine lactone (AHL) signal molecules . Exudates from pea (Pisum sativum) seedlings were found to contain several separable activities that mimicked AHL signals in well-characterized bacterial reporter strains, stimulating AHL-regulated behaviors in some strains while inhibiting such behaviors in others . The chemical nature of the active mimic compounds is currently unknown, but all extracted differently into organic solvents than common bacterial AHLs . Various species of higher plants in addition to pea were found to secrete AHL mimic activities . The AHL signal-mimic compounds could prove to be important in determining the outcome of interactions between higher plants and a diversity of pathogenic, symbiotic, and saprophytic bacteria.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 303 - 13
Staleya guttiformis gen . nov., sp . nov . and Sulfitobacter brevis sp . nov., alpha-3-Proteobacteria from hypersaline, heliothermal and meromictic antarctic Ekho Lake; Labrenz M et al.; Two Gram-negative, aerobic, pointed and budding bacteria were isolated from various depths of hypersaline, heliothermal and meromictic Ekho Lake (Vestfold Hills, East Antarctica) . 16S rRNA gene sequence comparisons show the isolates to be phylogenetically close to the genera Sulfitobacter and Roseobacter . Cells can be motile and contain storage granules . Sulfite addition does not stimulate growth . Isolate EL-38T can produce bacteriochlorophyll a and has a weak requirement for sodium ions; polar lipids include phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and an unidentified amino lipid, but not diphosphatidylgycerol . The dominant fatty acid is 18:1omega7c; other characteristic fatty acids are 3-OH 10:0, 3-OH 14:1, 16:0, 18:0, 18:2 and 19:1 . The DNA base composition is 55.0-56.3 mol% G+C . Isolate EL-162T has an absolute requirement for sodium ions . Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and an unidentified amino lipid are present in the polar lipids . Dominant fatty acids of this isolate are 18:1omega7c and 18:1omega9c as well as 18:2 which is present as two isomers . Other characteristic fatty acids are 3-OH 10:0, 3-OH 14:1, 16:0 and 18:0 . The G+C content is 57.9-58.1 mol% . Morphological, physiological and genotypic differences from related, thus far known genera support the description of Staleya guttiformis gen . nov . and sp . nov . with EL-38T (= DSM 11458T) as the type strain and of Sulfitobacter brevis sp . nov . with the type strain EL-162T (= DSM 11443T).

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 219 - 24
A proposal for the reclassification of Bdellovibrio stolpii and Bdellovibrio starrii into a new genus, Bacteriovorax gen . nov . as Bacteriovorax stolpii comb . nov . and Bacteriovorax starrii comb . nov., respectively; Baer ML et al.; Bdellovibrios are unique bacteria with the ability to prey upon a wide variety of susceptible Gram-negative bacteria . Micro-organisms exhibiting this trait have been included in the genus Bdellovibrio despite their isolation from diverse habitats and relatively unstudied taxonomic relatedness . In this study, 16S rDNA sequences were compared from known terrestrial Bdellovibrio species, Bdellovibrio bacteriovorus 100T, Bdellovibrio stolpii Uki2T and Bdellovibrio starrii A3.12T in order to study their phylogenetic relationship . The two sequences from B . stolpii Uki2T and B . starrii A3.12T were 90.0% similar to each other but exhibited only 81.7% and 81.2% similarity, respectively to B . bacteriovorus 100T . Phylogenetic analysis indicated that B . bacteriovorus 100T clustered in a separate clade from B . starrii A3.12T and B . stolpii Uki2T, demonstrating only a distant relationship between B . bacteriovorus 100T and the other two recognized type species . DNA-DNA hybridization experiments also demonstrated <4% hybridization between these three species . On the basis of the results obtained from the phylogenetic analysis and DNA-DNA hybridization studies, it is proposed that B . stolpii Uki2T and B . starrii A3.12T should be transferred to a new genus, Bacteriovorax gen . nov . as Bacteriovorax stolpii comb . nov . and Bacteriovorax starrii comb . nov., respectively . It is also proposed that the type species for the new genus Bacteriovorax should be Bacteriovorax stolpii comb . nov.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 63 - 72
'Candidatus Odyssella thessalonicensis' gen . nov., sp . nov., an obligate intracellular parasite of Acanthamoeba species; Birtles RJ et al.; An intracellular bacterium, strain L13, was observed infecting an environmental isolate of an Acanthamoeba species . The bacterium could not be recovered on axenic medium but was recovered and cultivated in vitro using cultures of Acanthamoeba polyphaga . The 16S rRNA gene sequence of L13 was found to be new, sharing less than 84% similarity with other sequences in the GenBank/EMBL database . L13 was found to be a member of the alpha-Proteobacteria, sharing an evolutionary line of descent with a group of uniquely obligate intracellular organisms comprised of Caedibacter and Holospora species and the NHP bacterium . Viable bacteria appeared to be highly motile within amoebae . Ultrastructural analysis of the bacterium demonstrated that it is rod-shaped and possesses a typical Gram-negative cell wall, but has no other outstanding features except small vesicle-like structures often associated with the outer surface of each bacterium . The host range of L13 was found to be limited to the genus Acanthamoeba . In A . polyphaga, L13 infection was slow to manifest when cultures were incubated below 30 degrees C, but at higher temperatures bacteria multiplied prolifically and induced host cell lysis . The protein profile of the bacterium purified from the amoebae was assessed by SDS-PAGE and its G+C content was estimated to be 41 mol% . Although these results support the proposal of L13 as a new species, its obligate intracellular nature prevented isolation of a definitive type strain . L13 is therefore proposed as 'Candidatus Odyssella thessalonicensis' gen . nov., sp . nov.

J Mol Evol, 2000 May, 50(5), 424 - 42
Conserved structural features in class I major fimbrial subunits (Pilin) in gram-negative bacteria . Molecular basis of classification in seven subfamilies and identification of intrasubfamily sequence signature motifs which might Be implicated in quaternary structure; Girardeau JP et al.; Type 1 and P-pili are prototype members of Class I fimbriae produced by Gram-negative bacteria . Despite common structural characteristics, the low level of amino acid sequence conservation among the Class I major fimbrial subunits (pilins) indicates considerable evolutionary distance between members of this superfamily . We highlight here structural relatedness between Class I pilins from their two-dimensional sequence analysis using hydrophobic cluster analysis (HCA) and secondary structure predictions (PHD program) . We present evidence that all members of the Class I pilin family have clear structural relatedness and suggest that classification based on phylogenetic analysis of Class I pilins into seven subfamilies correlates with differences in structural properties of the amino acid sequences . Using a sensitive alignment process (HCA), we identified 29 residues in topohydrophobic positions which probably play a prominent role in folding . The most striking aspects that distinguish the different pilin subfamilies are (i) large variation in the length of the loops connecting the structurally conserved regions and (ii) intrasubfamily sequence signature motifs located on regions predicted to be in the beta-conformation . We suggest that these "intrasubfamily sequence signature motifs" are part of interactive surfaces which participate in subunit-subunit interactions . These motifs prove highly useful in characterizing and classifying new Class I fimbriae that have not yet been described and whose sequence diverges appreciably from those of characterized groups . (After the submission of our manuscript, the experimental structure of Class I pilus subunits was published . In light of these actual pilin structures, a comparison has been made between the predicted results and the crystal structure in the Note Added in Proof.)

Infect Immun, 2000 Jun, 68(6), 3349 - 51
A model of infected burn wounds using Escherichia coli O18:K1:H7 for the study of gram-negative bacteremia and sepsis; Busch NA et al.; A difficulty that has emerged in the development and preclinical evaluation of adjuvant therapies for gram-negative sepsis is the lack of easily studied animal models that closely mimic human infection . An objective of this study was to adapt a previously described model of infection in burned mice to rats with a defined bacterial strain of Escherichia coli . Challenge with two colonies of live E . coli O18:K1:H7 bacteria into an 8% full-thickness burn of the dorsal skin surface of rats produced predictable bacteremia at 24 to 48 h and 80 to 100% mortality at 3 to 4 days . E . coli O18:K1:H7 was approximately 10-million-fold more virulent than several other gram-negative bacterial strains . The model should be a useful tool in studying the pathogenicity of burn wound infections and in evaluating the efficacy of novel adjuvant therapies for gram-negative sepsis.

Schweiz Rundsch Med Prax, 2000 Mar 30, 89(14), 566 - 70
{Rosacea, acne and other diseases of the seborrheic spectrum}; Boni R; Diseases of seborrhoic origin include rosacea, acne, gram-negative folliculitis, demodex-folliculorum, perioral dermatitis as well as seborrhoic dermatitis . An important prerequisite for adequate therapy is the knowledge of these different diseases . Unfortunately, topic steroids are often applied, resulting in well known corticosteroid side-effects like skin atrophy and teleangiectasias . The different clinical diseases of seborrhoic origin are reviewed here and a treatment guide is presented.

J Wildl Dis, 2000 Apr, 36(2), 248 - 56
Necrobacillosis in free-living male European bison in Poland; Jakob W et al.; In the Bialowieza Primeval Forest (Poland) a chronic disease of the external genital organs has been observed in free-living male European bison (Bison bonasus) since 1980 . Investigations on this disease started in the late 1980s . The most striking findings are necrotic and ulcerative lesions of the prepuce and penis of bison aged from 6 mo to >10 yr . Histologic examination of tissue samples from the prepuce of six bison (9-mo- to 8-yr-old), and from the penis of two bison (3- and 8-yr-old), were characteristic of necrobacillosis . Masses of slender, Gram-negative, rod-like or filamentous bacteria occurred in necrotic tissue . At the periphery of necrotic tissue filamentous bacteria were often arranged in large clusters and strands that advanced towards healthy tissue . Immunolabeling and electron microscopy also suggest that these organisms are Fusobacterium sp.

Vaccine, 2000 Jun 15, 18(25), 2867 - 77
A genetically-defined riboflavin auxotroph of Actinobacillus pleuropneumoniae as a live attenuated vaccine; Fuller TE et al.; Actinobacillus pleuropneumoniae is a gram negative pleiomorphic rod that is the causative agent of a severe, highly infectious and often fatal pleuropneumonia in swine . We have previously reported the construction of genetically-defined stable riboflavin auxotrophs by replacement of a portion of the APP riboflavin biosynthetic operon (ribGBAH) with an antibiotic cassette encoding resistance to kanamycin, and have demonstrated that such riboflavin auxotrophs are avirulent . In this study, we evaluated riboflavin auxotrophs of A . pleuropneumoniae for their ability to stimulate protective immunity against pleuropneumonia . An initial challenge experiment demonstrated that intramuscular vaccination with a live attenuated serotype 1A rib mutant, in a vaccine formulation that included a limiting amount of exogenous riboflavin, provided better protection against challenge with virulent A . pleuropneumoniae than either intratracheal immunization or intramuscular immunization with live bacteria in the absence of exogenous riboflavin . Subsequent studies in which the vaccine inoculating dose, concentration of exogenous riboflavin, and serotype of the vaccine strain were varied demonstrated that immunization with live avirulent riboflavin auxotrophs could elicit significant protection against experimental challenge with both homologous and heterologous virulent serotypes of A . pleuropneumoniae.

J Wildl Dis, 2000 Apr, 36(2), 272 - 83
Pathology of upper respiratory tract disease of gopher tortoises in Florida; McLaughlin GS et al.; Between August 1993 and September 1995, 24 gopher tortoises (Gopherus polyphemus) were received for pathological evaluations from various locations in Florida (USA) . All tortoises were examined for clinical signs of upper respiratory tract disease (URTD) including nasal and ocular discharge, palpebral edema, and conjunctivitis . Of the 24 tortoises, 10 had current or previously observed clinical signs of URTD and 14 did not . A blood sample was drawn for detection of anti-mycoplasma antibodies by ELISA, and nasal lavage samples were collected for culture and detection of Mycoplasma agassizii gene sequences by polymerase chain reaction (PCR) . Of the 14 clinically healthy tortoises, eight were sero-, culture- and PCR-negative, and six were seropositive for antibodies against M . agassizii . Of those six, five were culture- and/or PCR-positive for M . agassizii, and one was culture- and PCR-negative . Of the 10 ill tortoises, nine were seropositive by the ELISA and one was in the suspect range . Nine of the ill tortoises, including the suspect tortoise, were culture- and/or PCR-positive for M . agassizii, and one was culture- and PCR-negative . For histologic evaluation and discussion, the eight sero-, culture-, and PCR-negative tortoises were designated URTD-negative, and the other 16 were classified as URTD-positive . Histologic evaluation of the upper respiratory tract (URT) indicated the presence of mild to severe inflammatory, hyperplastic, or dysplastic changes in 14 URTD-positive tortoises . Seven of eight URTD-negative tortoises had normal appearing nasal cavities; one had mild inflammatory changes . Transmission electron microscopy revealed an organism consistent with Mycoplasma spp . on the nasal mucosal surface of tortoises with clinical signs and lesions of URTD . Additionally, gram-negative bacteria were isolated more frequently from the nasal cavities of URTD-positive tortoises than URTD-negative tortoises . Because clinical signs of URTD were never observed in six of the URTD-positive tortoises, we also conclude that subclinical URTD can occur in gopher tortoises.

Crit Care Med, 2000 Apr, 28(4), 935 - 40
Nosocomial pneumonia in patients undergoing heart surgery; Leal-Noval SR et al.; OBJECTIVE: To determine the risk factors related to the presence of postsurgical nosocomial pneumonia (NP) in patients who had undergone cardiac surgery . DESIGN: A case-control study . SETTING: Postcardiac surgical intensive care unit at a university center . PATIENTS: A total of 45 patients with NP and 90 control patients collected during a 4-yr period . INTERVENTIONS: Pre-, intra-, and postoperative factors were collected and compared between two groups of patients (cases vs . controls) to determine their influence on the development of NP . The diagnosis of NP was always microbiologically confirmed as pulmonary specimen brush culture of > or =10(3) colony-forming units/mL or positive blood culture/pleural fluid culture by the growth of identical microorganisms isolated at the lung . For each patient diagnosed with NP, we selected control cases at a ratio of 1:2 . MEASUREMENTS AND MAIN RESULTS: The incidence of NP was 6.5% . Multivariate analysis found a probable association of the following variables with a greater risk for the development of NP: reintubation (adjusted odds ratio {AOR}, 62.5; 95% confidence interval {CI}, 8.1-480; p = .01); nasogastric tube (AOR, 19.7; 95% CI, 3.5-109; p = .01), transfusion of > or =4 units of blood derivatives (AOR, 12.8; 95% CI, 2-82; p = .01) and empirical treatment with broad-spectrum antibiotics (AOR, 6.6; 95% CI, 1.2-36.8; p = .02) . Culture results showed 13.3% of the NP to be of polymicrobial origin, whereas 77.3% of the microorganisms isolated were Gram-negative bacteria . The mortality (51 vs . 6.7%, p < .01) and the length of stay in the intensive care unit (25+/-14.8 days vs . 5+/-5 days, p < .01) were both greater in patients with NP . CONCLUSIONS: We conclude that the surgical risk factors, except the transfusion of blood derivatives, have little effect on the development of NP . Reintubation, nasogastric tubing, previous therapy with broad-spectrum antibiotics, and blood transfusion are factors most likely associated with NP acquisition.

Eur J Biochem, 2000 May, 267(10), 3032 - 9
Intrinsic conformation of lipid A is responsible for agonistic and antagonistic activity; Seydel U et al.; Lipopolysaccharides (LPS, endotoxin) represent a major virulence factor of Gram-negative bacteria, which can cause septic shock in mammals, including man . The lipid anchor of LPS to the bacterial outer membrane, lipid A, exhibits a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is also responsible for the expression of pathophysiological effects . Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against endotoxically active LPS . By applying orientation measurements with attenuated total reflectance (ATR) infrared spectroscopy on hydrated lipid A samples, we show here that these different biological activities are directly correlated to the intrinsic conformation of lipid A . Bisphosphoryl-hexaacyl lipid A molecules with an asymmetric (4/2) distribution of the acyl chains linked to the diglucosamine backbone have a large tilt angle (> 45 degrees ) of the diglucosamine backbone with respect to the membrane surface, a conical molecular shape (larger cross-section of the hydrophobic than the hydrophilic moiety), and are endotoxically highly active . Monophosphoryl hexaacyl lipid A has a smaller tilt angle, and the conical shape is less expressed in favour of a more cylindrical shape . This correlates with decreasing endotoxic activity . Penta- and tetraacyl lipid A or hexaacyl lipid A with a symmetric acyl chain distribution (3/3) have a small tilt angle (< 25 degrees ) and a cylindrical shape and are endotoxically inactive, but may be antagonistic.

Eur J Biochem, 2000 May, 267(10), 2871 - 81
Proteomic analysis of the Escherichia coli outer membrane; Molloy MP et al.; Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment . Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs . Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram-negative bacterium . In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs . We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein identification using mass spectrometry . Applying this method to examine E . coli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 kDa . Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed . Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two-dimensional electrophoresis . Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated . Furthermore, we show that like the E . coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport . In an additional demonstration we show the value of parallel protein analysis to document changes in E . coli OMP expression as influenced by culture temperature.

Rinsho Byori, 2000 Jan, Suppl 111, 109 - 16
{Gene examination methods (detection and genotyping of resistant genes)--metallo-beta-lactamase}; Shibata N et al.; Metallo-beta-lactamases have a hydrolysing activity against various beta-lactams including carbapenems, and beta-lactamase inhibitors such as clavulanic acid and sulbactum . Infections caused by metallo-beta-lactamase producing gram-negative bacteria are becoming a serious problem in Japanese clinical settings despite the recent progress in medical technology . Current screening methods can not distinguish metallo-beta-lactamase producers from those producing AmpC beta-lactamase . Here we show a convenient method of detecting metallo-beta-lactamase producers using a thiol compound(2-mercaptopropionic acid) as well as a PCR method with metallo-beta-lactamase gene specific primers.

Microbiol Immunol, 2000, 44(2), 123 - 33
Efficient induction of peptide-specific cytotoxic T lymphocytes by LPS-activated spleen cells; Kakugawa K et al.; Lipopolysaccharides of gram-negative bacteria are potent activators of B cells, dendritic cells and monocytes/macrophages . We have investigated the use of LPS-activated spleen cells as antigen-presenting cells to induce CD8+ cytotoxic T lymphocytes in vivo that are reactive to MHC class I binding peptides . Compared with resting spleen cells, CTL induction was more efficient and less variable for different peptides with LPS-activated spleen cells . Cytotoxic responses were specific for the immunized peptides and contained high affinity CD8+ T cells . The removal of dendritic cells and monocytes/macrophages by Sephadex G10 column did not show profound effects on CTL induction, indicating that B-cell blasts were largely responsible . This easily accessible method should facilitate the screening of MHC class I binding peptides to determine whether or not the host's T-cell repertoire contains reactive T cells.

J Immunol, 2000 May 15, 164(10), 5403 - 8
Prostaglandin E2 stimulates AP-1-mediated CD14 expression in mouse macrophages via cyclic AMP-dependent protein kinase A; Iwahashi H et al.; PGs play a functional role in the early stage of Gram-negative bacterial infections, because this prostanoid is produced rapidly by epithelial cells after a bacterial infection . CD14, one of the LPS receptors, is a key molecule in triggering the response to bacterial LPS in association with a Toll-like molecule . Therefore, in this study, we investigated the effect of PG on CD14 expression in mouse macrophages . PGE1, PGE2, and PGA1 among the PGs tested strongly stimulated the expression of the CD14 gene in the cells . The stimulatory action also was observed by Western blot analysis . cAMP-elevating agents stimulated expression of CD14 gene as well . Protein kinase A inhibitor, N-{2-(p-bromocinnamylamino)ethyl}-5-isoquinolinesulfonamide (H-89), but not protein kinase C inhibitor 3-(1-{3-(dimethylamino)propyl}-1H-indol-3-yl)-4-(1H-indol-3-yl)-1H-py rrole-2,5-dione (GF109203X), abolished the stimulated expression of CD14 . A run-on assay showed that PGE2 stimulated the CD14 gene expression at the transcriptional level via protein kinase A . PGE2 also stimulated activation of AP-1, a heterodimer of c-Jun and c-Fos, because the prostanoid increased specific binding of nuclear proteins to the AP-1 consensus sequence and stimulated AP-1-promoted luciferase activity . PGE2-stimulated expression of CD14 was inhibited by antisense c-fos and c-jun oligonucleotides, but not by their sense oligonucleotides . Finally, PGE2 pretreatment synergistically stimulated LPS-induced expression of IL-1beta and IL-6 genes in mouse macrophages . Therefore, the present study demonstrates that PGE2 has the ability to stimulate AP-1-mediated expression of CD14 in mouse macrophages via cAMP-dependent protein kinase A.

J Vestib Res, 2000, 10(1), 1 - 5
Are vestibular hair cells excited to death by aminoglycoside antibiotics?
Smith PF.
Aminoglycoside antibiotics are commonly prescribed throughout the world for the treatment of serious gram-negative bacterial infections . However, their use is limited by nephrotoxicity and ototoxicity . Although the traditional explanation for their ototoxicity in cochlear and vestibular hair cells has been the inhibition of mitochondrial protein synthesis, evidence is accumulating to suggest that many aminoglycosides cause excitotoxicity in hair cells as a result of their agonist action at the polyamine site on the N- methyl-D-aspartate (NMDA) receptor . The aim of this minireview is to summarise and critically evaluate this evidence and to consider the therapeutic implications of this hypothesis.

Indian J Pediatr, 1999 Sep-Oct, 66(5), 725 - 33
Pediatric gastritis and peptic ulcer disease; Blecker U et al.; Inflammation of the gastric and duodenal mucosa is the end result of an imbalance between mucosal defensive and aggressive factors . The degree of inflammation and imbalance between defensive and aggressive factors can then result in varying degrees of gastritis and/or mucosal ulceration . Gastritis and ulcers of the duodenum or stomach can be classified as primary or secondary . The majority of children with chronic gastritis and ulcers in the stomach or duodenum have secondary inflammation or mucosal ulceration . These secondary ulcers generally occur due to a systemic condition like head trauma or overwhelming sepsis, or as sequelae to drug ingestion (i.e . non-steroidal anti-inflammatory agents), but secondary gastroduodenal ulcers can also occur in specific disease conditions such as Zollinger-Ellison syndrome or Crohn's disease . In almost all children with primary duodenal or gastric ulcers mucosal inflammation and, less frequently, ulceration is caused by a spiral shaped, gram-negative, microaerobic rod Helicobacter pylori . Recent epidemiological evidence has linked chronic H . pylori infection with the development of gastric carcinomas.

Scand J Immunol, 2000 May, 51(5), 472 - 8
The early immune response in the liver of BALB/c mice infected with S . typhimurium; Barreiros AP et al.; Gram-negative bacteria acquired through gastrointestinal infection can be a serious cause for the development of septic shock especially in immunosuppressed patients . Thus, the aim of this study was to examine the early events of the immune reaction against S . typhimurium . Bacteria were injected into mice at different concentrations . Four animals from each group were killed at five different points of time . Liver cytokine mRNA expression was determined by semiquantitative rt-PCR and liver histology was examined . Serum cytokine levels of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-4 and IL-10 were determined . intravenous (i.v.) infection with 109 bacteria led to lethal septic shock within 24 h . A delayed production of IFN-gamma, TNF-alpha, IL-18 and IL-10 and milder histological alterations in the liver were observed in these animals . The highest expression of cytokines in the liver and the strongest histological alterations were seen after infection with 107 bacteria . Here, an increased mRNA expression of all proinflammatory cytokines began 1 h after infection . Animals infected with 1 x 102 bacteria had the highest detectable serum levels of IL-6 and IL-10 . These data indicate that the immediate events in the immune reaction within the liver after infection with S . typhimurium are associated with the outcome of the subsequent sepsis.

Mol Microbiol, 2000 Apr, 36(2), 249 - 60
Ralstonia solanacearum produces hrp-dependent pili that are required for PopA secretion but not for attachment of bacteria to plant cells; Van Gijsegem F et al.; As in many other Gram-negative plant pathogenic bacteria, the Ralstonia solanacearum hrp genes are involved in the production of a type III secretion apparatus that allows the translocation of PopA protein to the external medium . Here, we show that hrp genes are also involved in the biogenesis of pili that are mainly composed of the HrpY protein . These pili are produced at one pole of the bacterium and are also released into the external medium where they can form very long straight bundles . An hrpY mutant is defective in pilus production, impaired in interactions with plants and in secretion of the PopA protein but not in attachment to plant cells.

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